Carol Davila University of Medicine and Pharmacy, Bucharest 2 Content Section I – Original contributions I.1. Significance of hyperglyc emia and… [602520]

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HABILITATION THESIS

Ionel Octavian Savu, MD, PhD

Carol Davila University of Medicine and Pharmacy, Bucharest

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Content

Section I – Original contributions

I.1. Significance of hyperglyc emia and hypoxia in the
pathophysiology of chronic complications of diabetes

I.2. Evaluation of the antioxidant status and oxidative st ress in the
serum and erythrocytes of patients with different stages of diabetes
mellitus, especially at the first medical onset

I.3. Involvement of the erythrocytes in L -arginine dependent nitric
oxide metabolism in diabetes

I.4. The impact of basal insulin analogues on glucose variability in
patients with type 2 diabetes undergoing renal replacement therapy
for end stage renal disease

I.5. Concluding remarks

Section II – Perspectives

Section III – References

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Section I – Original Contributions

I.1. Significance of hyperglyc emia and hypoxia in the pat hophysiology of chronic
complications of diabetes

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I.1.1. Significance of hyperglyc emia and hypoxia in the pat hophysiology of
chronic complications of diabetes – the state of the art

I.1.1. 1. The burden of diabetes and its specific chronic complications

Nowadays it is well known that diabetes mellitus (DM) is a serious health problem,
either worldwide or in our country, affecting developed countries mainly. Increasing
prevalence, severe specific complications, and major economical concern are essential
contributors to this scenario. Thus , a recent report published by the World Health
Organization (WHO) estimates that the global prevalence of this disease is expected
to grow from 171 million in 200 0 to 366 million by 2030 (Shaw, Sicree et al.) . From
this perspective, it is notable that the incidence of patients with type 1 diabetes
increased worldwide from 2% to 5% over last 20 years (Maahs, West et al.) . The
same tendency was confirmed by very rece nt data obtained from PREDATORR
cross -sectional epidemiologic study estimating the actual prevalence of diabetes
mellitus, chronic renal disease and associated comorbidities in romanian population
(http://predatorr -study.com ). It has been therefore shown that more than 1.5 milion
romanian adults between 20 and 79 years of age are suffering from diabetes mellitus.
The corresponding disease prevalence is 11.6%, accordingly. Furthermore, the
prevalence for glucose metabolism disturbances (i.e. impaired fasting glucose – IFG
or impaired glucose tolerance – IGT) in the same study is even higher (18.4%). All
together, this epidemiological data support the epidemic burden of diabe tes disease.
Both type 1 and type 2 diabetes markedly increase the risk of microvascular and
macrovascular complications. Microvascular complications are characterized by
wide -range dysfunction in the capillary bed of tissues , and include the retinopathy,
nephropathy, and neuropathy – that eventually affect s nearly all patients with DM ,
since early stages of the disease . Diabet ic retinopathy (DR) is the leading cause of
blindness in adults from many different geographic areas (Klein, Klein et al. 1992) .
On the other hand, d iabetic neurop athy (DN) is the most common cause of non –
traumatic amputations (Feldman 2003) and diabetic nephropathy (DNf) is the major
cause of end -stage renal disease (Bo, Ciccone et al. 2005) .
Atherosclerosis process occurs earlier, a nd foll ows a more aggressive course in
diabetes (Haffner 2000) . Macrovascular complications originate from accelerated

5 atherosclerosis and remain the major cause of death in diabetic patients (Haffner
2000) . Hence, major vascular events such as m yocardial infarction, stroke, and
peripheral vascular disease are two to four times more prevalen t in these patients.
Diabetic environment induces the same rate of cardiovascular events as documented
coronary artery disease (Schulman, Hartmann et al.) in people with out diabetes
(Haffner 2000) . The mortality following myocardial infarction is higher in patients
with diabetes compared with subjects without diabetes (Haffner 2000) .
Premenopausal cardio -protection is lost in w omen with diabetes who therefore
become as vulnerable to CAD as men (Hu, Stampfer et al. 2001) . Foot ulcers
represent a major chronic complication in patients with diabetes. The global burden of
diabetic foot disease is reflected by the fact that on every 30 seconds one diabetic foot
is lost in the world by amputation (Boulton, Vileikyte et al. 2005) .
Thus, these data represent strong evidence attesting that c hronic complications of
diabetes represent a major medical and economical concern.

I.1.1.2. Pathogenic mechanisms involved in chronic compli cations of diabetes
mellitus

The potential pathogenic mechanisms have been proposed to be responsible for the
development of long -term complications of diabetes include: increased polyol
pathway flux, increased advanced glycation end products, activation of proteinkinase
C, increased hexosamine pathway flux) (Brownlee 1995; Lee, Chung et al. 1995;
Sayeski and Kudlow 1996; Koya and King 1998) .

I.1.1.2.a. The polyol pathw ay converts glucose to fructose (Vincent, Russell et al.
2004) . Glucose is first reduc ed to sorbitol by aldose reductase (AR), and then sorbitol
is oxidized to fructose by sorbitol dehydrogenase (SDH). When glucose is in excess,
both enzymes are overexpressed mainly in tissues prone to diabetic complications.
Sorbitol oxidation to fructose is a chemical reaction that is dependent of NADPH
consumption. NADPH pool is also required for maintaining the reduced form of the
antioxidant glutathione . Thus, these depletive mechanisms increase the cell
susceptibility to oxidative stress (Vincent, Russell et al. 2004) .

6 I.1.1.2.b. The production of advanced glycosylated end products (AGEs) involves
non-enzymatic reactions between reactive dicarbonyls and various intracellular or
extracellular substrates such as proteins, DNA or lipids (Ahmed 2005; Toth, Rong et
al. 2008) . Three compounds are mainly generated: glyoxal (by glucose oxidation),
Amadori products (by degradat ion of fructose -lysine adducts); and methylglyoxal
(through the ab normal metabolism of glycolytic intermediates ). AGEs exert their
deleterious effects mainly due to two different mechanisms : structural modifications
and impaired activity of various intracellular or extracellular proteins (Miyazawa,
Nakagawa et al. 2012) ; and extracellular activation of a specific cell -surface receptor
(Hartter, Svoboda et al.) , which is responsible for the secretion of cytok ine and
growth factors and inflammation mediated vasculature damage (Ahmed 2005; Toth,
Rong et al. 2008) . Moreover , it has been shown that high glucose enhances RAGE
expres sion (Yao and Brownlee 2010) .

I.1.1.2.c. Hyperglycemia excessively increases the synthesis of diacylglycerol
(Sarkar, Dash et al.) which over -stimulates the PKC pathway (Koya and King 1998) .
Consecutively, vaso -active proteins are over -expressed such as vascular en dothelial
growth factor (VEGF), PAI -1, NF -κB, and TGF -β, supporting the role of this pathway
in angiogenesis and vascular permeability , vascular occlusion or inflammatory
processes (Koya and King 1998) .

I.1.1.2.d. T he hexosamine biosynthetic pathway is essential for (1) synthesis of
glycosaminoglycans, glycolipids or glycoproteins, and (2) regulation of the protein
transcription and translation (Kolm -Litty, Sauer et al. 1998) . In this pathway, the
glycolysis intermediate fructose -6 phosphate is first conv erted to the active form of
glucosamine – uridine diphosphate -N-acetyl glucosamine (UDP -GlcNAc) (Kolm –
Litty, Sauer et al. 1998) . N-acetyl glucosamine further serves as substrate for O-linked
glycosylation of various proteins to serine or threonine residues within the cytosol and
nucleus . As with polyol pathway, when intracellular glucose concentration is high, the
UDP -GlcNAc is excessively produced (Kolm -Litty, Sauer et al. 1998) . The excess of
sugar residues can compete with phosphate groups altering gene expression of various
proteins functioning as transcription factors, such as SP1, TGF -α and TGF -β (Kolm –
Litty, Sauer et al. 1998; Brownlee 2001) .

7 I.1.1.2.e. It has been shown that hyperglycaemia in diabetes is responsible for the
excessive production of reactive oxygen species (ROS) by the mitochondrial electron –
transport chain and has been suggested to be a common mechanism for all the others
mentioned above (Nishikawa, Edelstein et al. 2000) . Mitochondrial superoxide
overproduction further induces a reve rsible decrease in glyceraldehyde phosphate
dehy drogenase (GAPDH) activity. The s uperoxide exerts this inhibitory effect either
direct (Du, Edelstein et al. 2000) or indirect, as a consequence of poly(ADP -ribose)
polymerase (PARP) activation by oxidative l esions of mitochondrial DNA (Garcia
Soriano, Virag et al. 2001; Du, Matsumura et al. 2003) . Irrespective how modulation
is performed , superoxide GADPH inhibition induces the above -mentioned
mechanisms.

I.1.1.3. Mitochondrial respiratory chain as source o f ROS in diabetes

It is a general agreement that excessively ROS (i.e. superoxide) originate in diabetes
from two major sources in mitochondrial respiratory chain: complex I
(NADH:ubiquinone oxidoreductase or NADH/FADH 2 dehydrogenase) and complex
III (ubiquinol:cytochrome c oxidoreductase) (Turrens and Boveris 1980; Beyer 1992;
Turko, Li et al. 2003; Murphy 2009) .

Mitochondrial oxidative phosphorylation is a process where electrons from reducing
substrates (i.e. NADH or FADH 2/succinate) are transferred to molecular oxygen (O 2)
in a step -by-step manner involving respiratory chain complexes I – IV (Murphy
2009) . These complexes couples the electrons transfer with the transfer of protons
(H+) establish ing a hydrogen gradient across the inner mitochondrial membrane. It is
therefore created an electrochemical proton gradient that drives ATP synthesis, or the
generation of chemical energy in the form of adenosine triphosphate (ATP ) by ATP
synthase (complex V). Four membrane -bound complexes have been identified in
mitochondria. The NADH –ubiquinone oxidoreductase (complex I) accepts electrons
from the Krebs cycle electron carrier NADH and transfers them to ubiquinone
(coenzyme Q) pool. In the complex II ( succinate dehydrogenase) additional electrons
originating f rom succinate are transferred via FAD into the coenzyme Q pool and
used to oxidize succinate to fumarate. When compared to complex I, complex II is a
parallel electron transport pathway to c omplex I where no protons are transported to

8 the int er-membrane sp ace. Hence, the pathway through complex II generates no
electrochemical proton gradient and therefore contributes less energy to the overall
electron transport chain process. The electrons transferred to the coenzyme Q pool are
further passed to ubiquinol – cytochrome c oxidoreductase (complex III), and
subsequently to cyt ochrome c oxidase (complex IV), both processes contributing to
the electrochemical proton gradient. Finally, the electrons are used to reduce
molecular oxygen to water. The majority of mo lecular oxygen is reduced at complex
IV to water via the mitochondrial respiratory chain. Though, 1 –4% of the oxygen is
incompletely reduced to superoxide (O 2-).

I.1.1.4. The cross talking between pathogenic factors in diabetic wounds

Among chronic comp lications of diabetes, diabetic foot ulcers potentially induce
major health disability and hospitalization costs (Boulton, Vileikyte et al. 2005) .
There is a combination of multiple mechanisms that contribute to pathophysiology of
this condition (Shaw and Boulton 1997; Ricco, Thanh Phong et al. 2013) , which
includes sensory, motor and autonomic neuropathy, peripheral vascular disease,
microangiopathy, impaired wound healing related to reduced blood supply, and
chronic hyperglycemia. Repetitive mechanical st resses (i.e. poorly fitting footwear or
high foot pressures), which the patient applies on a wound -prone foot, are also
essential for the occurrence and persistence of the ulcerative lesion (Murray and
Boulton 1995) .

I.1.1.5. The importance of hypoxia in the chronic complications of diabetes

Accumulating data suggest a role for hypoxia, in the development of chronic
complications of diabetes (Botusan, Sunkari et al. 2008; Curtis, Gardiner et al. 2009;
Regazzetti, Peraldi et al. 2009; Villaret, Galitzky e t al. 2010) .

Thus, in spite that endothelial hypoxia has b een recognized as the major initiator of
the atherosclerotic plaque in animal models more than twenty years ago (Boxen
1985) , a direct evidence of hypoxia in hum an atherosclerosis has only recently
emerged (Sluimer, Gasc et al. 2008) . It has been shown that the combination of
increased oxygen demands with insufficient oxygen supply, particularly in

9 macrophage -rich zones from atherosclerotic lesions , generates hypoxia (Bjornheden,
Levin et a l. 1999) . Accumulating data suggest hypoxia as a key factor in the
development and progression of atherosclerosis through several potential
mechanisms: lipid accumulation, inflammation, ATP depletion and angiogenesis
(Hulten and Levin 2009) . Promoting angi ogenesis within the plaque has a dual effect:
compensates for low tissue oxygenation and ATP levels (Hulten and Levin 2009) , and
may also induce lesion instability (Hulten and Levin 2009) . Recently, it has been
proposed that blood flow abnormalities and hy poxia in the inner layer of the media
reflect alterations into the architecture of the vasa vasorum (Jarvilehto and Tuohimaa
2009) . In accordance, t he development of atherosclerosis initiates from the opposite
direction, i.e. the end arteries of vasa vasor um.

Increasing evidences suggest that chronic hypoxia of the tubulo interstitium has an
important pathogenic role not only in early stages of chronic renal diseases, but also is
a common pathway to end -stage renal failure. Thus, it seems that hypoxia per se is a
fibrogenic stimulus for tubular epithelial cells, interstitial fibroblasts and renal
endothelial cells leading to apoptosis or epithelial -mesenchymal trans -differentiation
since early stage of renal disease, even before the development of structura l
tubulointerstitial injury (Fine, Bandyopadhay et al. 2000; Nangaku 2006) . A primary
glomerular lesion further reduces the amount of fun ctional peritubular capillaries due
to decreased capillary flow, increased vasoconstriction and decreased vasodilation
from affected glomeruli , thus leading to tubular dysfunction and fibrosis (Nangaku
2006) . In the late stages, renal fibrosis is the hallmark of ch ronic kidney diseases in
spite of the etiology, and therefore glomerulosclerosis and tubulointerstitial fibros is
are the best indicators for progression to end -stage disease (Nangaku 2006; Fine and
Norman 2008) .

Chronic hypoxia seems to be involved in the pathogenesis of early diabetic
retinopathy. The lost of the trophic effect of the Muller cells under hypoxic conditions
(Tretiach, Madigan et al. 2005) suggests a potential role of chronic hypoxia in
neuroglial dysfunction which recently has been shown to occur at very early stages of
the disease (Antonetti, Barber et al. 2006) . The direct evidence that chronic h ypoxia
could be related to retinal hypoperfusion are controversial (Pournaras, Rungger –
Brandle et al. 2008; Curtis, Gardiner et al. 2009) . Though, lower vitreal P O2 was

10 reported on diabetic patients undergoing vitrectomy (Holekamp, Shui et al. 2006) .
The i ndirect evidences seem to be more consistent. Thus, it has been reported that
patients with very incipient lesions of retinopathy improved ocular function under
pure oxygen atmosphere (Curtis, Gardiner et al. 2009) . Furthermore, intermittent
chronic hypoxi a seems to be also associated with retinopathy since sleep apnea
syndrome has been related with diabetic macular edema and higher levels of
circulating retina -specific rhodopsin mRNA were observed in obese type 2 diabetic
patients with more than 5 dips/hr (Wong, Merritt et al. 2008) . Chronic hypoxia may
be either involved in shifting from hypo to hyperperfusion in diabetic retinopathy
since a certain threshold of hypoxia may overcome the early retinal vas oconstriction
in diabetes (Curtis, Gardiner et al. 20 09). The implications of chronic hypoxia in the
occurrence and progression of advanced retinopathy are consistent and originates
from both in vitro and in vivo studies using growth factors, especially VEGF
(Chiarelli, Santilli et al. 2000; Arjamaa and Nikinmaa 2006; Crawford, Alfaro et al.
2009) . Thus, it has been recently shown that intravitreous VEGF is increased and
correlates with the severity of retinopathy and angiogenesis in diabetic patients with
proliferative diabetic retinopathy (Wang, Wang et al. 2009) .

Hypoxia and reduc ed nerve blood flow are early observed in both humans and
animals with diabetic polyneuropathy (Cameron, Eaton et al. 2001; Sima 2008) . It
could be also speculated that hypoxia may be involved as trigger for e ndoneurial
microa ngiopathy , which may further exacerbate hypoxia by reducing the oxygen
diffusion (Malik, Tesfaye et al. 1993; Cameron, Eaton et al. 2001) . The beneficial
effects of vasodilator treatment on nerve conduction velocity (NCV) or blood flow in
humans or animals (Cameron, Eaton et al. 2001; Sheetz and King 2002) are also
indirect proofs for the role of chronic hypoxia in the development of diabetic
neuropathy.

I.1.1.6. Hypoxia -inducible factor -1 as main mediator of the adaptive responses of
cells to hypoxia

Hypoxia refers to the condition when the oxygen delivery does not meet the oxygen
demands of the tissues. The partial pressure of the oxygen at tissue level varies from
10 to 110 mmHg (1 to 10%). Hypoxia defines any condition characterized by levels

11 of oxyge n lower than above mentioned. Adap tive responses of cells to hypoxia are
mediated by the hypoxia -inducible factor -1 (HIF-1).

HIF is a heterodimeric complex belong ing to the PER -ARNT -SIM (PAS) subfamily
of the basic -helix -loop-helix (bHLH ) family of transcription factors (Crews and Fan
1999) , which are proteins essential for development and homeostasis .

HIF structure is complex, consisting of two subunits: α-subunit and β-subunit (Wang
and Semenza 1995) . The oxygen regulates the α-subuni t only, whereas the β-subunit
is oxygen independent and is known as aryl receptor nuclear translocator (ARNT)
(Hoffman, Reyes et al. 1991; Wang, Jiang et al. 1995) . All three isoforms of the α-
subunit and only two isoforms of the β-subunit (ARNT and ARNT2) are thought to be
involved in the in vivo response to hypoxia (Brahimi -Horn and Pouyssegur 2009) .
Whereas HIF -1α is ubiquitously expressed, HIF -2α and HIF -3α expression is more
restricted (Berchner -Pfannschmidt, Frede et al. 2008) .

Different HIF members have specific particularities but also share some common
characteristics (Berchner -Pfannschmidt, Frede et al. 2008; Brahimi -Horn and
Pouyssegur 2009) .
The N -terminal part is similar to all HIF members and contains bHLH/PAS domain
which mediate DNA binding and induces dimerization.
The C -termina l region confers specificity for HIF members; it is present only in HIF –
1α and HIF -2α and is involved in transactivation of HIF complex by two specific
domains (N -TAD of C -TAD, transactivation domains).
N-TAD domain contains the oxygen -dependent degradation domain (ODDD) and the
two proline residues (P402 and P564) involved in HIF -α degradation in normoxia.
HIF-β and HIF -3α lack C -TAD region.
As a common characteristic for all HIF members, bHLH motif and C-terminal contain
nuclear localization signals (NLS). When stable, NLS allows HIF to translocate into
nucleus (Kallio, Okamoto et al. 1998) .

HIF degradation is essentially proteosomal dependent, involves prolyl hydroxylase
domain -containing proteins (PHD s) – von Hippel Lindau protein complex (pVHL)
pathway, and exerts in normoxia.

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Thus, when the oxygen is present (i.e. normoxia) HIF protein degradation is mainly
initiated by the hydroxylation of the ODDD at two critically proline residues (P402,
from N -terminal region and P564 from C -terminal region) , a reaction which is
catalyzed by PHDs (Ke and Costa 2006; Brahimi -Horn and Pouyssegur 2009) . Their
activity is dependent on substrates (oxygen and 2 – oxoglutarate, 2 -OG) and cofactors
(Fe2+ and ascorbate). The “hypoxia mimetics” are chemical substances, which inhibit
PHDs and stabilize HIF by different mechanisms (iron chelators, such is
desferoxamine – DFX; iron competitors, such is transition metals – cobalt; or
oxoglutarate analogs, such is dymethyloxaly lglycine – DMOG). It is important to note
that PHDs are not able to catalyze HIF proline hydroxylation (Jaakkola, Mole et al.
2001) . There are three PHDs isoforms described in humans, but PHD2 only seems to
be the key limiting enzyme for HIF α stability (Berra, Benizri et al. 2003) , and is able
to shuttle between the cytoplasm and the nucleus, contributing to HIF -1α degradation
in both compartments (Ke and Costa 2006) .

It is important to note that HIF α inactivation in normoxia implies not only a
modulati on at the protein levels, but also a post -translational regulation of
transactional domains. Thus, additional hydroxylation of asparagines 803 (N803)
located at the end of C -TAD result s in the inhibition of HIF activation by
transcriptional co -activators, such as p300, steroid receptor coactivator -1 (SRC -1) or
CBP (CREB -binding protein) (Ke and Costa 2006; Brahimi -Horn and Pouyssegur
2009) . The reaction is catalyzed by Factor Inhibiting HIF -1 (FIH -1), which belongs to
the dyoxygenase family of proteins. As PHDs, FIH -1 needs 2 -OG and oxygen as
substrates and Fe2+ as cofactor, but the k m for oxygen is lower. Therefore, the enz yme
is more sensitive to oxygen gradients. In other words, a certain oxygen level may by
low enough to limit the transactivation but not enough to promote HIF degradation.
The adaptive response is also modulated by the availability of the co -activators that
limits HIF transactivation .

After ODD -hydroxylation, HIF protein is further degraded by ubiquitination. The
process implies its interaction with the von Hippel Lindau protein complex (pVHL), a
component of an E3 multiprotein ubiquitin -ligase complex known as VHL/elongin

13 B/elongin C (VBC) (Ivan and Kaelin 2001) . When polyubiquitinilated, HIF protein is
further directed to 26S proteasome for degradation.

VHL -dependent HIF protein degradation may by accelerated in normoxia by different
mechanisms. Thus, the Arest Defective Protein -1 (ARD -1) acetyl ation of lysine
located in the ODDD (Jeong, Bae et al. 2002) increases the affinity of HIF for pVHL,
that accelerates its proteosomal degradation.

HIF protein degradation can occur also through pathways that are PHDs independent
and pVHL dependent.

It has been reported that HIF α degradation can occur also through other pathways
which are pVHL independent (Ke and Costa 2006) . Thus, SUMOylation may
represent a possible alternative for this pattern of HIF degradation. SUMOylation is a
system which is very similar to ubiquitin pathway (Sarge and Park -Sarge 2009) ,
where the small ubiquitin -like modifier (Tsumoto, Ra et al.) is the most known
component. The SUMOylated proteins are specifically degraded by a de –
SUMOylation system consisting of different cystei ne proteases (SUMO specific
proteases – SENP, 1 -6 in humans). The effects of SUMOylation – de-SUMOylation
on HIF activity are still debated (Bae, Jeong et al. 2004; Berta, Mazure et al. 2007;
Huang, Han et al. 2009) .

HIF degradation may also occur in a p VHL independent manner, either in hypoxic
conditions or in cells defective of VHL (VHL -/- cells) (Kim, Safran et al. 2006) .

HIF-1α may also be degraded independent of proteosomal degradation, in the
lysosomes through chaperone -mediated autophagy (Hubbi, Hu et al. 2013) .

HIF is stabilized under hypoxic conditions . PHDs and FIH are both inactive, HIF -α
translocates into nucleus, dimerizes with HIF -β, and binds to a specific region of the
hypoxia response elements (HREs) that is located in the promoter gene of the target
genes (Lisy and Peet 2008) .

14 Stabilized HIF -1 regulates the expression of many genes trough direct or indirect
mechanisms (Semenza 2011) . Thus, by direct binding to HRE and recruiting of co –
activator proteins mor e than 60 well -defined target -genes involved in different
processes (i.e . angiogenesis, tumor development, erytropoies is, metabolism,
inflammation ) are either up -regulated (Semenza 2003) or repressed (Manalo, Rowan
et al. 2005) , thus mediating the adaptive responses to hypoxia. Indirectly, there are
several ways through HIF -1 can modulate gene expression: (a) by functioning as co –
activator or co -repressor of specific genes; (b) by activation of target genes encoding
microRNAs; (c) by transcription factors, that are either direct activated or inhibited or
that are encoded by the target -genes activated in the first line; (d) and by the
activation of the transcription of genes encoding proteins with histone -demethylase
(HDM) activity, thus modulating the activi ty of a second line of target -genes by
altering their chromatine structure. Opposite to HIF -1α or HIF -2α, the HIF -3α
isoform has been reported to have a dominant negative activity (Makino, Cao et al.
2001; Brahimi -Horn and Pouyssegur 2009) . HIF target gene expression is also
conditioned by modulation of C-TAD and N -TAD activity (Lisy and Peet 2008;
Brahimi -Horn and Pouyssegur 2009) . Thus, N-TAD confers specificity for HIF-targe t
gene expression . Furthermore, both TAD domains are sequential activated in
gradients of hypoxia . Hence, i n mild hypoxia HIF stability is rescued but only N -TAD
activity is preserved, whereas in full hypoxia both N -TAD and C -TAD activities are
present. The gr adual activation is due to higher affinity for oxygen of FIH when
compared wi th PHDs.

NEDD8 is an ubiquitin -like protein implicated in cell cycle progression and
cytoskeletal regulation (Kamitani, Kito et al. 1997) . It has been also shown that it
mediates an altern ative mechanism for HIF stabiliz ation in a ROS -dependent –
pVHL -independent manner (Ryu, Li et al. 2011) . This alternative pathway of HIF
regulation in hypoxia seems to be important since approximately 30% of HIF
stabilization in hypoxia is NEDD8 -dependent (Ryu, Li et al. 2011) .

HIF-1α stabilization may also occur independent of oxygen regulation (Ke and Costa
2006) . This manner of HIF activity modulation has been described for several
hormones (i.e. insulin) and growth factors (i.e. IGF -I, IGF -II, EGF) (Zelzer, Levy et
al. 1998; Feldser, Agani et al. 1999; Treins, Giorgetti -Peraldi et al. 2005) .

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I.1.1.7. HIF -1 is essential for the control of mitochondrial ROS production under
hypoxic condition

Several reports have shown an increase in oxidative stress when different cells and
tissues are exposed to hypoxia (Poyton, Ball et al. 2009; Poyton, Castello et al. 2009) .
It seems that increased ROS production in mitochondria originates from complex II
and III (Chandel, McClintock et al. 2000; Paddenberg, Ishaq et al. 2003) . The
mechanism responsible for the increased ox idative stress when the oxygen tension
decreases is not clear but it might be a conseque nce of a slower rate of electron
transport across inner membrane of mitochondria. However, HIF -1 is essential for
the control of mitochondrial ROS production under hyp oxic condition (Semenza
2011) . The mechanisms behind are complex, and imply the regulation of the balance
between oxidative and glycolytic metabolism of the cell (Semenza 2011) . The end –
point of these metabolic responses seems to be to reduce the mitochond rial mass and
to increase the glucose shift from aerobic to anaerobic glycolysis.

A very recent paper discusses the mechanisms by which hyperglycaemia contributes
to HIF repression in diabetes, also providing a comprehensive analysis of the
functional relevance of how hypoxia and HIF contribute to the development of
chronic diabetes complications (Catrina 2014) .

I.1.1.8. Mito chondrial DNA (mtDNA) and ROS

I.1.1.8.1. MtDNA as particular target to oxidative damage

Once generated, ROS can interact with different macromolecules including DNA.
When compared with nuclear DNA (nDNA) the mitochondrial DNA (mtDNA) is
particularly susceptible to oxidative damage. The close association with the inner
mitochondrial membrane and the specific hist one deficiency represent the main
explanations behind the mtDNA sensitivity to ROS aggression (Yakes and Van
Houten 1997) .

16 Human mtDNA is a highly conserved double -stranded circular molecule of about
16,5 kb located into mitochondrion (Fernandez -Silva, E nriquez et al. 2003) . The name
of its two strands – i.e. the guanine -rich, heavy (H) and cytosine -rich, light (L) ,
originates from the differences in guanine content, which allow the separation on a
cesium chloride gradient. mtDNA is packaged by proteins . The most important one
seems to be TFAM, a transcription factor suggested to regulate the mtDNA copy
number in mammals (Ekstrand, Falkenberg et al. 2004) . In each normal cell, a
mixture of wild-type (normal ) and mutated mtDNA can coexist. This particular
condition for the mitochondrial genome is called heteroplasmy (Wallace 1992) . The
gene sequence in mtDNA contains no introns, and only two noncoding (control)
regions (Fernandez -Silva, Enriquez et al. 2003) . The largest non -coding segment in
human mtDNA is called the displacement loop (D -loop), reaches about 1 kb length,
and it has a triple -stranded structure, in which a nascent H -strand DNA segment of
500-700 nucleotides remains annealed to the parental L -strand.

In humans, mtDNA is the smallest chromosome, coding for only 37 genes and
containing approximately 16,600 base pairs (Fernandez -Silva, Enriquez et al. 2003) .
Mammalian mtDNA genes encode for a small number of polypeptides that are
subunits of the respiratory chain. The rest of different mitochondrial proteins are
encoded by nDNA. Furthermore, nDNA is responsible for the mtDNA maintenance
and expression.

In spite of some exceptions (Schwartz and Vissing 2002) the inheritance of mtDNA is
almost e xclusively maternal. Though, the mutations transmitted to the offsprings by
mtDNA occur more rapid as a result of the fact that only a small numbers of mtDNA
molecules are transmitted to the offsprings (Marchington, Macaulay et al. 1998) . This
is recognized as bottleneck phenom enon. Hence, the level of expression of mtDNA
mutations may considerably differ in different cells or tissues (Clayton 1982) . There
are several explanations for this fact. First, de novo mutations may occur in one
molecule of mtDNA that can increase the am ount of mutated mtDNA to homoplasmy,
i.e. presen ce of only mutated mtDNA (Trifunovic 2006; Trifunovic and Larsson
2008) . Second, in case of mtDNA the replication is not coupled to cell cycle meaning
that one of the daughter cells may have an unbalanced loa d of mutated mtDNA
(Clayton 1982) . Third, a certain threshold level of mtDNA mutation load must be

17 overcome in order to cause mutation expression (89). Fourth, there is a variation in
the distribut ion of mutated mtDNA either in different cells, tissues and organs (due to
heteroplasmy), in different cells of the same organ (due to different intracellular
distribution of mtDNA), or in mitochondria located in different intracellular sites (due
to a dyn amic compartimentalization of mtDNA within the mitochondria) (Trifunovic
2006; Trifunovic and Larsson 2008) .

I.1.1.8 .2. Mt DNA oxidative damage and ageing

The close proximity of mtDNA to the mitochondrial inner membrane where most of
ROS are generated (Yakes and Van Houten 1997) explains the reason why the
alterations of mtDNA may be essential contributors to the ageing process. Therefore,
it has been suggested (Harman 1972) that accumulation of somatic mutations in
human mtDNA might produce cells with a reduced oxidative capacity which can be
responsible for agein g. Thus, the damage to mtDNA is associated with either
increased number of cytochrome c oxidase (COX) deficient cells or a imbalanced
activity of different complexes in mitochondrial respirator y chain in various tissues
(i.e. skeletal muscle, liver, heart and brain) leading to increased ROS production
(Harman 1972) .

Several extensive reviews discuss the implications of somatic mtDNA mutations in
ageing (Trifunovic, Wredenberg et al. 2004; Kujoth, Hiona et al. 2005; Trifunovic
2006; Trifunovic and Larsson 2008; Vermulst, Wanagat et al. 2008; Kukat and
Trifunovic 2009) .

Animal studies performed on genetically -modified (knock -in) mice expressing a
proofreading deficient form of the nuclear -encoded mtDNA polymerase (Polg)
(Trifunovic, Wredenberg et al. 2004; Kujoth, Hiona et al. 2005; Vermulst, Wanagat et
al. 2008) have directly shown that somatic mtDNA mutations may induce a variety of
ageing phenotypes in mammals . Thus, the Polg express a pr ofoundly reduced
exonucle ase activity with a normal DNA p olymerase activity . Therefore, mtDNA
mutator mice express rapid -progressive altered phenotypes with age. They are
completely normal in phenotype expression at birth, show signs of premature ageing
in early adolescence (about 25 weeks of age), and die prematurely before age of about

18 60 weeks. Their genotype is also severe altered. Hence, mtDNA point mutations and
deletions accumulate progressively and randomly i n different levels and various
tissues. It is not yet clear how any of these specific mutations contribute to accelerate
aging process in these animals (Kukat and Trifunovic 2009) .

The majority of the mtDNA deletions observed in human aging tissues are similar
with the ones detected in patients suffering from mitochondrial diseases (Kukat and
Trifunovic 2009) . Most of them are located in the major arc between the two
proposed origins of replication (O H and O L; Mitomap), and are mainly flanked by
short direct repeats.

Thus, one of t he most frequent deletion observed in human tissues is a 4,977
nucleotides deletion that occurs at a del etion “hot spot” between two 13-bp direct
repeats at positions 13447 -13459 and 8470 -8482 in human mtDNA, and is often
associated with other two deletion s (mtDNA7436 and mtDNA10422) (Kukat and
Trifunovic 2009) . Due to the fact that is the common cause of several mitochondrial
diseases (such as chronic progre ssive external ophthalmoplegia – CPEO) , the deletion
is called the “common” deletion. Moreover, it a lso accumulates in different levels and
various tissues during aging, therefore being used as biomarker of mtDNA damage
(Kukat and Trifunovic 2009) .

The a nalysis of human different single cells revealed also a significant proportion of
different deletion s in mtDNA with age. Thus, various COX deficient cells (i.e.
skeletal muscle fibers, cardiomyocytes, or substantia nigra neurons ) isolated from
aged individuals carried a significant proportion of different mtDNA deletions or even
often no detectable full -length mtDNA (Kukat and Trifunovic 2009) . Though, in spite
that the amount of the mutations in the cells can b e substantial, the overall tissue level
may be low (Kukat and Trifunovic 2009) .

The accumulation of mtDNA point mutation with age in human tissu es is
controversial (Kukat and Trifunovic 2009) , mainly for technical reasons . Several
reports describe A to G transitions (i.e. A8344G, A3243G) and transversions (T414G)
accumulating with age in different tissues , but not in others . Though, one mutation
(A13167G) declined significantly with age in muscles of infants 1 hour to 5 weeks

19 old (Zhang, Liu et al. 1998) . Murdock et al. (Murdock, Christacos et al. 2000) , also
found by a different approach no age -dependent accumulation of A3243G and
A8344G mutations in skeletal muscle and brain. Notably though, there is clear data on
mtDNA point mutations accumulation with age in studies performed in different
human single cells, even in human stem cells (Kukat and Trifunovic 2009) .

I.1.1.8.3. MtDNA protection against oxidative damage

Irrespective how are generated, ROS are responsible for various damages on mtDNA
in humans (Yakes and Van Houten 1997) . Hence, ROS can induce many types of
DNA lesions, i.e. abasic (apurinic -apyrimidinic) sites (AP), o xidized DNA bases or
sugars, or DNA strand breaks (single -strand breaks – SSBs, double -strand breaks –
DSBs) (Maynard, Schurman et al. 2009) . Therefore, it can be assumed that mtDNA
protection against oxidative damage may involve beside antioxidant enzymes an
efficient repair system.

The base excision repair (BER) pathway repairs the vast majority of the mtDNA
lesions , those induced by ROS being also included (Maynard, Schurman et al. 2009) .
Briefly, the process involves several steps, as follows (Maynard , Schurman et al.
2009) .
1. Excision of the damaged base by a DNA glycosylase , due to its specific
(glycosylase) activity. An abasic site (AP) is consecutively generated . AP sites re sult
also from ROS attack. T heir re moval is critical because of highly mut agenicity.
Several glycosylases are designated to repair certain lesions. One specific glycosylase
may repair more than one type of damage. Mono -functional glycosylases ha ve only
glycosylase but not lyase activity, meaning that for the 3` incision at AP si te they
need an AP -endonuclease (APE) providing the lyase activity. The left over 5` –
deoxyribose (dRP) residue is further removed by lyase activity of mtDNA polymerase
γ. Bi -functional glycosylases have glycosylase and lyase activity , meaning that they
are able to perform both base excision and incision to the AP site. In this case, t he
leftover is a 3´ unsaturated aldehyde (UA) derived from deoxyribose, or a phosphate
(P), which is subsequently removed by lyase activity of APE.
2. The cleavage of AP sit e and 5’ – leftovers is indirectly able to generate a SSB.
SSBs may be produced also directly by ROS. The direct ROS attack result s in an AP

20 site and a 3´ leftover (phosphoglycolate – PG), which is removed specifically by APE
lyase activity . Irrespective how is generated, t he SSB is further replaced by mtDNA
polymerase γ (Polγ), the only polymerase existing in mitochondria. Presence of both
polymerase and lyase activities make this enzyme the most important for the repair
synthesis step in BER (Maynard, Sc hurman et al. 2009) .
3. The final step is the ligation of the remaining nick by a specific ligase complex.
This is the sh ort-patch BER pathway, where only one nucleotide is incorporated
instead of the damaged base.
4. If the polymerase cannot access the 5´ end and the leftover residue is resistant to its
lyase activity, the process of removing the blocking fragment requires incorporation
of several (from 2 to 6) nucleotides. This is long -patch BER pathway an d involves
several new enzymes, i.e. FEN 1 (flap en donuclease 1) which is a 5´ exo/endonuclease
recently isolated i n mitochondrial extracts from human cells (Liu, Qian et al. 2008;
Szczesny, Tann et al. 2008) .

I.1.1.8.4. MtDNA mutations in diabetes

Data referring to the significance of mtDNA mutations i n diabetes are relatively
limited. Thus , it has been shown that specific mtDNA mutations are associated with
diabetes (Ballinger, Shoffner et al. 1992; Ballinger, Shoffner et al. 1994) , and one
particular mtDNA point mutation causes the disease in 0.5-1% o f the patients with
diabetes (Kadowaki, Kadowaki et al. 1994) . The same mtDNA somatic mutation has
been found to accumulate with age in patients with diabetes (Nomiyama, Tanaka et al.
2004) , and its incidence correlated with the duration of diabetes (Liang , Hughes et al.
1997) . The same ageing association and accumulation in diabetes has been reported
for a 4977 bp deletion in mtDNA (Suzuki, Hinokio et al. 1999) . However, the data
available are still limited, originating from cross sectional studies (Kamiya and Aoki
2003; Garcia -Ramirez, Francisco et al. 2008) , on the unevenly somatic point
mutations and deletions of mtDNA (usually associated with excessive ROS) in
diabetes (Khaidakov, Heflich et al. 2003; Trifunovic, Wredenberg et al. 2004) , or
from periphe ral blood mononuclear cells (PBMC) (Kamiya and Aoki 2003; Garcia –
Ramirez, Francisco et al. 2008) that might not reflect the changes in the tissues that
develop chronic complications.

21 I.1.2. Significance of hyperglyc emia and hypoxia in the pat hophysiology of
chronic complications of diabetes – original contribution beyond the state of the
art

Thus, one of my i mportant research interest has been the study of the significance of
hyperglycaemia and hypoxia in the patophysiology of chronic complications of
diabetes, in order to identify new therapeutic strategies designated to improve their
actual poor outcome. Hence, I have first investigated the e ffect of hyperglycemia and
hypoxia on the stability and function of 1 -alpha subunit of the HIF in either primary
fibroblasts cultures and diabetic wounds collected before developing diabetes and
after 24 weeks of diabetes from a well validated animal model of type 2 diabetes
(db/db mouse). Our animal model has though the advantage of the homogenous
genetic background and of determined diabetes duration. I have shown that
hyperglycaemia has a repre ssive effect on both HIF -1α stability and function, by a
comp lex mechanism involving either the von Hipple Lindau dependent degradation
and transactivation. Then, I have shown that the negative regulatory effect of the
hyperglycaemia is re versible by blocking HIF -1α hydroxylation through chemical
inhibition. Moreover, by local adenovirus -mediated transfer of two stable HIF
constructs, I have demonstrated t hat stabilization of HIF -1α is necessary and
sufficient for promoting wound healing in a diabetes.

Then, by usin g primary human dermal fibroblasts and several diabetes -targeted
organs collected from the above -mentioned animal model, I have investigated the
stability of mtDNA against hyperglycaemia and hypoxia -mediated ROS
overproduction in diabetic condition. I was able to show that mtDNA damage
accumulates only in cells exposed to both hyperglycaemia and hypoxia – hence,
confirming the pathogenic role of the combination of these factors in diabetes, and
that mtDNA was fully protected in cells treated with specific i nhibitors of the
mitochondrial respiratory chain. I therefore here made the first observation that
pathogenic mechanisms relevant in diabetes (i.e., hyperglycaemia and hypoxia) can
induce mtDNA lesions through mitochondrially produced ROS. I have also show n
that mtDNA accumulates in vivo less damage in diabetes, in spite of increased levels
of ROS. This was explained by an increase in the antioxidant capacity (i.e.,
expression of Mn Superoxide Dismutase – SOD 2, and Catalase – CAT) and of the

22 mtBER activ ity (i.e., the activity of APE) in the targeted organs of the diabetic
animals.

Hypergly cemia represses HIF-1α stability

The hyperglycemia repression on HIF-1α stability was first documented in db/db
mouse p rimary fibroblasts (Fig. 1A) and then confirmed in diabetic wounds of the
db/db mice (Fig. 3A). The two different approaches of blocking p -VHL machinery
demonstrated that t he effect of glucose was dependent on the VHL -mediated
degradation mechanism . Thus, HIF-1α stability was not modulated by hyperglycemi a
in renal carcinoma cells that constitutionally lack functional pVHL (Fig. 1B Left) .
VEGF expression – one of the most important target gene for HIF – was also not
influenced by hyperglycemia in human dermal fibroblasts where VHL expression was
down -regulated by treatment with siRNA (Fig. 1B Right).

Hyperglycemia inhibits HIF-1α function

The negative effect of hyperglycemia on HIF -1α function was first demonstrated in
vitro . Both CTAD and NT AD activity were repressed (Fig. 1C) and the expression of
several HIF -1α target genes essential for wound healing (Fig. S1) was down regulated
by hyperglycemia . The same repressive effect of hyperglycemia on HIF -1α function
was then confirmed in vivo . Db/db wounds expressed significant lower HIF-1α (Fig.
3A), in spite more pr onounced hypoxia (Fig. 3B). The expression of HIF-1 target
genes essential for cell motility (i.e., HSP -90), angiogenesis (i.e., VEGFA and VEGF –
R1), and recruitment of CAG (i. e., SDF -1α, SCF, and Tie -2) was also repressed in the
diabetic wounds (Fig. 3C).

HIF stabilization counteracts the repressive effect of hyperglycemia on its
stability and function

The stability and function of HIF-1α is essentially regulated by the hydroxylation of
N-TAD – ODDD located prolyl and C-TAD located asparaginyl residues (Ivan,
Kondo et al. 2001; Jaakkola, Mole et al. 2001; Lando, Peet et al. 2002) . The influence
of the inhibition of hydroxylase activity on the negative regulatory effect of

23 hyperglycemi a was therefore studied. Two structurally different hydroxylase
inhibitors were used , i.e., dimethyloxalylglycine (DMOG) and deferoxamine (DFX).
DMOG is a 2-oxoglutarate analogue while DMOG is an iron chelator, therefore
stabilizing HIF-1 by different mechanisms (Hirota and Semenza 2005) . Both
inhibitors stabilized the HIF -1α to similar levels in hyperglycemia and normoxia
compared to hypoxia in normal glucose concentrations in primary db/db mouse
fibroblasts (Fig. 2A). The expression of HIF target genes was also induced (Fig. 2B)
even in the cells exposed to high glucose concentrations, or in hypoxia as illustrated
for VEGF (Fig. S2). Both treatments improved healing process in diabetic wounds
(Fig. 4 A and B), and the effect persist s in spite of persistent chronic hyperglycemia,
without interfering with the animal’s weight. Both inhibitors were used as the highest
efficient doses as titrated in pilot experiments . Though, DMOG was at least equally as
efficient as DFX, if not more effec tive (Fig. 4E). Different other proprieties may
count for these differences. Hence, in spite of its antioxidant proprieties (Emerit,
Beaumont et al. 2001) , which could be of relative importance considering the
essential role of the ROS in the pathogenesis of chronic complications of diabetes
(Brownlee 2001) , DFX has anti -proliferative effects (Dayani, Bishop et al. 2004) , also
observed in vitro (data not shown). Present data represent strong evidence showing
that the hyperglycemia -induced HIF-1 destabilizat ion can be satisfactorily overcome
by blocking the prolyl hydroxylation . This is in spite of a clear mechanism behind
(Ivan, Kondo et al. 2001; Jaakkola, Mole et al. 2001; Jeong, Bae et al. 2002; Pereira,
Zheng et al. 2003; Cheng, Kang et al. 2007) .

Defec tive wound healing in diabetic mice is critically improved by HIF
stabilization

In order to proof a direct evidence for the central role of HIF in wound healing in
diabetes a stable mutated HIF -1α variant was cloned by homologous recombination
into a spe cific serotype of adenovirus plasmid. Two different point mutations in
which both the critical proline residues (P402A/P563A) have been substituted with
alanine generated two constitutively stable forms of HIF -1α (V -N and V -NC). In the
V-NC construct CTAD was additionally substituted with the potent VP16 TAD (Fig.
S2). These structural modifications of HIF molecule are gain-of-function mutations
changing the gene product such that it g ains an amplified function . The same positive

24 effect on wound healing in diabetic animals as pharmacological inhibitors (Fig. 4C)
was obtained . The V -NC construct had no additive effects compared with the V -N
construct (Fig. 4D) . The result is a strong argument showing that CTAD repression is
not critical for wound healing. The central pathogenic role for the NTAD without any
additional regulatory effect from the CTAD has also been reported for HIF -2α-driven
renal carcinogenesis (Yan, Bartz et al. 2007) . The lack of superiority observed for V-
NC construct compared with VN may al so be explained by the squelching effect (Gill
and Ptashne 1988) of a highly active transactivation activity classically described for
VP16. The data provided by the present experiments were though of highly relevance
for future development of hydroxylase inhibitors as potential therapy.

A stable HIF-1α activates several processes involved in wound healing

In order to evaluate the treatment efficacy with a stable HIF molecule a h istological
analysis of diabetic wounds was performed on deparaffinized, reh ydrated and
hematoxylin and eosin stained samples by two independent observers unaware of the
identity of th e biopsy. A semiquantitative score to evaluate vascularity, granulation,
and dermal and epidermal regenerat ion was used . The micro -vessel density was
measured using semi -quantitative – double blind analysis of the specific binding of
GS-1 isolectin to microvascular structures. All essential steps of wound healing were
improved by the treatment with either hydroxylase inhibi tor or inoculation of the
stable over-expressed form of HIF -1α (Fig. 5A and B).
Furthermore, the histological changes observed here were sustained by the increased
expression of HIF -α target genes encoding angiogenic cytokines (Fig. S4) also
previously re ported by others (Bosch -Marce, Okuyama et al. 2007; Li, Li et al. 2007) .
It is important to note that edema was not observed following the induced
angiogenesis. This observation is in agreement with the phenotype of transgenic mice
over-expressing HIF (Elson, Thurston et al. 2001) and in contrast to the mice over –
expressing VEGF (Thurston, Suri et al. 1999) in the skin. The recruitment of
angiogenic cells is delayed in diabetic wounds (Gallagher, Liu et al. 2007) . Though, it
is not yet known if this is the result of a decreased mobilization or homing processes
at the wound level. The direct detection of angiogenic cells is limited by small
numbers usually present in tissues (Bosch -Marce, Okuyama et al. 2007) . Therefore,
an indirect method to evaluate the rec ruitment of angiogenic cells to the wounds was

25 used. T he levels of the cytokine receptor expression typically present on these cells
were measured. HIF-1α stabilization was followed by an increase at the wound level
of these cytokine receptors (i.e., CXCR4 , C-Kit, Tie -2) (Fig. 5C).

Hence, the key point of present research is that hyperglycemia -induced destabilization
and inhibition of HIF -1α is a central pathogenic mechanism for delayed wound
healing in diabetes. HIF stabilization is essential and sufficient for improving wound
healing in diabetes.

26

Figure 1. Hyperglycemia destabilizes and inhibits HIF -1. (A) Immunoblots
detecting HIF -1 in db/db mouse primary fibroblasts cultured for 48 h in different
glucose concentrations (5.5 and 30 mM) an d then exposed for 6 h in either normoxia
(21% O 2) or hypoxia (1% O 2). (B Left) Immunoblot detecting HIF -1 in renal
carcinoma cells (SKRC7) expressing functionally inactive VHL cells (SKRC7). (B
Right) Relative expression of VEGF in human dermal fibroblast cells exposed to
different glucose concentrations after transfection with VHL -specific siRNA or
scrambled siRNA. (C) Relative luciferase activity in the extract of 3T3 cells exposed
to different oxygen and glucose concentrations after co -transfection with CTAD
(GAL4/mHIF -1 722-822) or NTAD (GAL4/mHIF -1 531-584) and GAL –
responsive reporter gene plasmid (*, p <0.05, 5.5 mM glucose vs. 30 mM glucose).

27

Figure 2. Inhibition of HIF hydroxylases can reverse the glucose inhibition of
HIF-1. (A) Immunoblots detecting HIF -1 in mouse db/db skin fibroblasts show
destabilization of HIF -1 in high glucose concentratio ns (30 mM). This effect is
overcome by treatment with hydroxylase inhibitors (DMOG [2 mM] or DFX [100
M]. (B) Hydroxylase inhibitors induce expression of HIF -1 target genes essential
for wound healing (DMOG [2 mM] or DFX [100 M]) even in presence of hig h
glucose levels (*, p<0.05, treatment vs. control). (No, nomoxia (21% O 2); Hy,
hypoxia (1% O 2); G, glucose).

28

Figure 3. HIF -1 function is negatively regulated in diabetic wounds. (A)
Wounds in diabetic mice are more hypoxic than in normog lycemic control mice as
evaluated by pimonidazole adduct formation; note the detailed granulation tissue
(Inset). (B) Immunoblot detecting HIF -1 in a whole -cell extract from the wound of
diabetic or normoglycemic mice. (C) The expression of HIF target gen es involved in
wound healing is down -regulated in wounds of db/db mice (*, p<0.05 expression in
db/db mice vs. normoglycemic litter -mates).

29

Figure 4. Local stabilization and activation of HIF1 - by hydroxylase inhibitors
of direct transfer of stabilized HIF improve wound healing in diabetic mice.
(Top) The healing rate of full -thickness wounds in db/db mice is promoted by local
treatment with DMOG (2 mM; A1), DFX (1 mM; B1) or by adenovirus -transferred of
stable forms of HIF -1 (V-N; C1), (V -NC; D1) compared with placebo or empty
virus (LacZ), respectively (values are means  SEM; *P<0.05 db/db treated vs. db/db
placebo or LacZ ). C1, Inset: virus expression at the edge of the wound as revealed by
-galactosidase staining. (Lower) Representative examples showing wound healing in
db/db mice treated as in Upper. (D). Immunoblot detecting HIF -1 in whole -cell
protein extracts from w ounds. Note that inhibition of hydroxylase activity by local
treatment with either DMOG (2 mM) or DFX (1 mM) overcomes hyperglycemia –
dependent negative regulation of HIF -1.

30

31
Figure 5. Stabilization and activation of HIF -1 in diabetic wounds is followed
by activation of granulation angiogenesis and recruitment of CAG. (A)
Hematoxylin and eosin staining shows improvement in the granulation tissue and
visualization of the wounds in db/db mice locally treated with DMOG (2 mM) or
DFX (1 mM) or by adenovirus -mediated expression of a stable HIF -1 (V-N; original
magnification X25). (Right) Semiquantitative evaluation for granulation,
angiogenesis, and dermal and epidermal regeneration. Graphs represent means 
SEM. Vehicle -treated or empty virus treated (LacZ) normoglycemic heterozygous
diabetic db/db mice (gray bars), and DMOG, DFX, or HIF V -N treated homozygous
db mice (white bars) are shown (*, P<0.05). (B) Left: vascular density in wounds
evaluated by Griffonia simplicifolia 1 (GS-1) lectin staining is increased in the db/db
diabetic animals after treatment with DMOG (2 mM), DFX (1 mM), or V -N. (Right)
The semiquantitative evaluation of vessel density evaluated by GS -1 lectin staining.
Vehicle -treated or LacZ -treated homozygous diabetic mice (gray bars), and DMOG,
DFX, or V -N-treated homozygous diabetic mice (white bars) are shown. Graphs
represent mean  SEM. (*, P<0.05). (C) Local treatment with DMOG, DFX, or V -N
in wounds of diabetic mice increases mRNA expression of cytokine receptors
typically present on CAG (*, P<0.05 treated vs. placebo or LacZ).

32

Figure S1. Repression of expression of HIF -1 target genes essential for wound
healing in db/db fibroblasts by high glucose levels . (#, P< 0.05 hypoxia vs.
normoxia in 5.5 mM glucose, *, P<0.05 30 mM glucose vs. 5.5 mM glucose in
hypoxia).

Figure S2. Hydroxylase inhibitors (DMOG or DFX) are able to counteract the
hyperglycemia -dependent VEGF repression even in hypoxia (*, P<0.05 vs.
hypox ia 30 mM). No, normoxia (21% O 2); Hy, hypoxia (1% O 2); G, glucose.

33
High glucose and/or hypoxia increase the mitochondrial ROS production in
HDFs.

Fibroblasts are well recognized as important effectors involved in chronic
complications in diabetic skin and wound healing (Ngo, Hayes et al. 2005) . The
glucose transport across the plasma membrane of human fibroblasts (HDF) is
mediated by at least three genetically distinct facilitative glucose transporters
(GLUT), i.e. GLUT -1, -3 and -4 (Longo, Bell et al. 1990) . Thus, oxidative damages
induced in diabetic environment may target fibroblasts, also. Hyperglycemia and
hypoxia gathered increasing pathogenic significance related to oxidative damage in
diabetes. Therefore, the ROS production was evaluated in HDF cultured in
hyperglycemia and hypoxia (figure 6 A). Carboxy -methyl -H2DCFDA was used as
ROS detector. The probe is an ester compound that loses the aceta te groups when
cleave d by esterases after cell entry. The cleavage leads to intracellular trapping of the
nonfluorescent 2’,7’ -dichlorofluorescein (DCF) that becomes highly fluorescent when
is oxidized. Thus, a wide spectra of ROS can be detected (Wardman 2007) . High
glucose and hypoxia induced each other the ROS production. Though, their
combination only was able to generate the highest level of ROS in HDF (Figure 6 A
upper panel). Because of its relative less specificity, the quantity of the probe oxidized
does not reflect the amount of free radicals generated. Though, the level of H2DCFDA
oxidation may at least represent a qualitative tool to compare the intracellular oxidant
formation between different cellular conditions. Hence, it may suggest that the ROS
levels are highest when the cells are exposed to hyperglycemia and hypoxia in
combination . Moreover, along with its relative lack of specificity in terms of what
ROS are generated, the level of H2DCFDA oxidation does not necessarily reflects
where these species are originating. Therefore, HDFs were cultured in normal and
high glucose concentrations in the presence or absence of carbonyl cyanide m-
chlorophenyl hydrazine (CCCP), an uncoupler of mitochondrial respir ation. At the
end of the incubation period under different experimental cond itions, the cells were
loaded with MitoSOX Red , a mitochondria selective superoxide indicator (Batandier,
Fontaine et al. 2002) . The excessive ROS production was specifically block ed from
cells exposed to CCCP, demonstrating that it originates in mitochondria (figure 6 A
lower panel) . These data suggest a dose -dependent effect of ROS on mtDNA stability.

34 In other words, the highest level of mtDNA damage was observed only in cells
expo sed to the combination of hyperglycemia and hypoxia , where ROS reach the
highest concentrations (Figure 6 A upper panel). Furthermore, the critical influence of
ROS levels for inducing mtDNA damages was confirmed by the cumulative effect of
H2O2 concentrati ons on the mtDNA damage (figure 6 B Inset) . A similar observation
was also reported by others in different context (Yakes and Van Houten 1997) .
Moreover, it has been reported that mtDNA stability is altered either by glucose alone
but only at extreme high c oncentrations (Li, Meininger et al. 2001) or when the time
of exposure is long enough to permit accumulation of sufficiently high levels of ROS
(Palmeira, Rolo et al. 2007) . As the combination of high glucose and hypoxia,
palmitate is another factor potent ially relevant for the complications in diabetes that
has been shown to induce probably ROS mediated mtDNA damages in vitro (Rachek,
Musiyenko et al. 2007) . Though, the present study is the first observation that
pathogenic mechanisms relevant in diabetes (i.e., hyperglycaemia and hypoxia) can
induce mtDNA lesions through mitochondrially produced ROS.

High glucose and/or hypoxia induce mtDNA damage in HDFs.

MtDNA stability against excessive ROS production was evaluated in HDFs cultured
in different gluco se concentrations and oxygen tensions using long DNA targets by
quantitative polymerase chain reaction (qPCR) (Santos, Meyer et al. 2006) . The
method rely on the fact that lesions in long mt DNA fragments will slow down or
block the progression of DNA polym erase on tem plate that results in the decrease in
DNA amplification in the damaged template when compared with the undamaged
DNA. In order to correct for the variations in mitochondrial copy number, a short
mtDNA fragment was amplified by an additional rea ction. After amplification, both
mtDNA fragments were quantified using a high sensitive fluorescent dye. The ratio
between absolute amplification values for the large and small mtDNA fragments
defines the n ormalized amplification. Assuming a random distrib ution of lesions and
using the Poisson equation for non -damaged templates , the average lesion frequency
per strand expressed the actual mtDNA damage (Yakes and Van Houten 1997) . For a
better accuracy, t he number of mitochondria was also evaluated by quanti tative real –
time polymerase chain reaction .

35 As for the ROS overproduction, when compared with normal conditions
(normoglycemia and normoxia) , mtDNA normalized damage was significantly
increased o nly in HDFs cultured in the combination between high gl ucose and
hypoxia (figure 6 B). To validate the sensitivity of the long fragment PCR
amplification method to detect mtDNA lesions , HDFs were exposed to increased
concentrations of H2O2 as exogenous donor of ROS . The treatment with H2O2 results
in a dose dependent increase of the normalized damage in mtDNA (Inset). In order to
verify if the source of increased ROS production observed in hyperglycemia and
hypoxia together is mitochondria, the cells were cultured in these conditions with or
without CCC P and mtDNA damage was compared with normal conditions (figure
6C). The lack of the excessive accumulation of mtDNA lesions in hyperglycemia and
hypoxia if the respiratory chain is blocked with CCCP certify the mitochondrial origin
of increased ROS product ion in these states. Again, these data confirm for the first
time that pathogenic mechanisms relevant in diabetes (hyperglycemia and hypoxia)
can induce mtDNA damage through mit ochondria -produced ROS (figure 6 C).

In spite of increased ROS levels, d b/db m ice accumulate less mtDN A lesions in
heart and kidney .

It is well known that diabetes targets certain tissues. Among them, the heart and
kidney are classically affected. On the other hand, the leptin receptor deficient db/db
mouse is a very consistent validated animal model for the study of type 2 diabetes.
That is because the db/db mouse progressively develops obesity, insulin -resistance
and type 2 diabetes after 12 weeks of age. The animal model used here – db/db mice
at di fferent ages, has though some essential advantages in comparison with other
observations (Liang, Hughes et al. 1997; Garcia -Ramirez, Francisco et al. 2008) .
First, the animals originate from breeding pairs as an inbred strain, therefore having a
homogenous genetic background. Second, they have different ages that allow a clear
control over the time of exposure of the tissues to diabetes. The “young” (10 weeks)
animals were young -enough before diabetes occurs, while the “old” (34 weeks) ones
were old -enough to develop specific diabetes -related chronic complications. In other
words, if the estimated life expectancy for db/db mouse is about 2 years (i.e. 104
weeks), the mean duration of chronic hyperglycemia for “old” db/db mice was
therefore about 20 weeks. Th at is around 1/5th from the estimated lifetime expectancy.

36 When compared to mean life expectancy of 70 years for a human subject with
diabetes, 1/5th means about 14 years of exposure to chronic hyperglycemia.

The normalized damage in long fragments of mtDNA from heart and kidney of both
“young” and “old” diabetic mice and their age -matched controls was therefore
estimated in the present study (figure 7 A lower panel). The incidence of mtDNA
damage was not increased in control “old” non -diabetic animals, while significantly
less mtDNA damage was observed in both heart and kidney from “old” db/db mice
when compared either with young animals or with non -diabetic “old” littermates
(p<0.01). This apparent surprising result needs to rule out some possible
metho dological errors. First, an eventual bias from a selection after mitochondrial
drop-out was excluded, as no difference in mitochondria numbers is found (data not
shown). Second, a n additional validation of the long fragment QPCR method to
evaluate the mtDN A damage in vivo was performed. The animal model used was the
knock -in mice expressing an exonuclease -deficient version of the mtDNA polymerase
γ (PolgD257A) (PolgAmut/PolgAmut) (Trifunovic, Wredenberg et al. 2004) and their
heterozygous knock -in mice ( +/PolgAmut), also at different ages (9 and 24 weeks,
respectively) , in order to control over the time of exposure of the mtDNA to age. The
heart of these animals has been described to accumulate the highest levels of mtDNA
damages with age (Trifunovic, Wreden berg et al. 2004) . Hence, the widespread
damage of mtDNA in hearts of PolgAmut/PolgAmut mice was measured. A significant
increase of the mtDNA damage with age (p=0.025) was observed (figure 7 A upper
panel).

Thus, mtDNA stability in both tissues analyzed exhibited similar behavior. The result
has consistency, as either hearts or kidneys are preferential targets for chronic
complications of diabetes and have different numbers of mitochondria. The present
observation may have therefore more relevancies comp ared to former data obtained in
peripheral blood mononuclear cells (PBMC) (Kamiya and Aoki 2003; Garcia –
Ramirez, Francisco et al. 2008) . Low mutagenicity of ROS in ageing cells was though
previously reported (Shokolenko, Venediktova et al. 2009) and may validate the
present result. In the same line is the fact of increasing evidence attesting an
overestimated susceptibility of mtDNA to the accumulation of oxidative base
substitution (Anson, Hudson et al. 2000) . Furthermore, it seems that there i s no

37 typical age-dependent mtDNA lesions in animals without diabetes (Wang, Wong et
al. 1997; Bender, Krishnan et al. 2006) . The observation is in line with the decrease of
mtDNA damage in “young” db/db mice reported in the present study. On the other
hand , the lack of mtDNA damage in non diabetic mice at the same age was also
recently reported (Lee, Choi et al. 2010) but identified in older animals (18 months) .
Taking together, all these data demonstrate that the method used here is sensitive
enough to det ect the classical age related mtDNA damage.

Further, the levels of ROS were measured in hearts and kidneys from db/db mice and
their age -matched controls. For this purpose the levels of nitrosylation were evaluated
in tissue samples. Peroxynitrite is con sidered the main reactive species that is
responsible for the nitrosylation of the tyrosine residues from proteins in vivo,
generating 3 -nitrotyrosine (3 -NT). Though, there is data suggesting in vivo
peroxynitrite -independent nitrosylation of proteins that occurs especially in
inflammatory processes (Gaut, Byun et al. 2002; Duncan 2003) . Nitric monoxyde
(NO) may react with oxygen generating nitric dioxide (NO 2), a compound with a high
oxidative potential. Though, NO 2 nitrosylation of tyrosine is less probab le to occurs in
vivo (Crow and Ischiropoulos 1996) . Two main reason may explain this fact. One is
that the reaction is very slow: 25 nM of NO 2 needs 0.7 h to be generated from NO.
Second, NO 2 nitrosylation of tyrosine has very less efficiency. Though, it m ay
generate 3 -NT in acidic environment, such is the gastric cavity (van der Vliet,
Eiserich et al. 1996) . That is why the levels of nitrosylation of tyrosine residues may
be considered a direct consequence of increased levels of peroxynitrite production ,
mainly from the reaction between nitric oxide and superoxide (Ceriello, Mercuri et al.
2001) .

Thus, the hearts of the “old” db/db mice were exhibit higher levels of nitrosylation of
the protein tyrosine residues (figure 7 B), in spite of t he lower incidence of mtDNA
damage. High levels of ROS production in the organs analyzed may also be
confirmed by up -regulation of the two major antioxidant enzymes SOD isoenzyme –
SOD 2 and CAT . In this line is the observation that both CAT and SOD 2 activi ty is
increased in c ells chronically exposed to high g lucose concentrations (Weidig,
McMaster et al. 2004) . The increased activity of SOD 2 but not CAT only in hearts but
not in kidneys observe d in the present study (figure 8 ) may reflect a specific tissue

38 modulation of the antioxidant enzymes. Such data were already reported in other
animal models (Esposito, Melov et al. 1999; Kowluru, Kowluru et al. 2006) .
Furthermore, the differences in antioxidant activity of the enzymes were not reflected
in a different tissue accumulation of mtDNA damage, as it was previously reported in
a different scenario (Esposito, Melov et al. 1999) .

The “old” db/db mice exhibit an increased mitochondrial base excision repair
(BER) system.

MtDNA uses t he base excision repair (BER) system for repairing most lesions that are
generated endogenously or by environmental agents (Hegde, Hazra et al. 2008) . This
pathway represents the second line of defense against oxidative stress for mtDNA .
The repairing machinery involves several excision steps that, along with ROS,
generate apurinic/apyrimidinic (AP) sites. AP -endonuclease (APE) repairs AP sites
generated by both ROS and excision steps. That is why APE is a key enzyme in this
process.

APE activity was therefore analyzed in mitochondrial extracts from the hearts of the
“old” db/db mice (Figure 9 A, upper and middle panels) . An increased specific activity
was observed. The APE up -regulation may be a possible explanation the lower
amount of mtDNA lesions accumulated in these mice. It has to be noted that this is
observed in “old” but diabetic animals. The observation deserves to be underlined, as
the decrease in the BER activity with age has been reported both in mice and humans
(Chen, Hsieh et al. 2003; Imam, Karahalil et al. 2006) . It is not clear why this it
happens in diabetes. One possible explanation is that in diabetic environment the
mitochondrial aggression by ROS triggers a retrograde signaling from the
mitochondria to the nucleus resulting in some protective effects. The phenomenon
was previously described for the anti -apoptotic response in transgenic mice that
develop cardiomyopathy due to th e accumulation of mt DNA muta tions specifically in
the heart (Mott, Zhang et al. 2001) . Thus, in these animals the mechanism of
pathogenesis does not involve increased oxidative stress. On the contrary, the mt DNA
mutations induce a cytoprotective response including increases in pro -survival
proteins that specifically inhibit apoptosi s. Thus, a sig naling pathway between the

39 mitochondrion and the nucleus could mediate the pathogenic effect of mitochondrial
DNA mutations.

Furthermore, the APE was not induced after a 48-h exposure to hyperglyc emia and a
48-h additional exposure to hypoxia (Figure 9 A, lower panel). That could mean that
the induction of the APE needs probably a chronic exposure to ROS , at least in
diabetic environment.

In conclusion, the present study shows for the first time that hyperglyc emia and
hypoxia, together, are able to induce mtDNA damage through induction of
mitochondrial ROS production. The pathogenic mechanism was successfully
compensated for in diabetic animals, possibly by the repair mechanisms and therefore
may not represent a major factor for the development of chronic complications.

40

Figure 6 . High glucose and/or hypoxia increase the mitochondrial reactive
oxygen species production and induce mitochondrial DNA (mtDNA) damage in
human dermal fibroblasts. Human dermal fibroblasts were exposed to different
glucose con centrations and oxygen tensions (< 1% for hypoxia) in the presence or
absence of an uncoupler of mitochondrial respiration – carbonyl cyanide m –
chlorophenyl hydrazone. (A) Reactive oxygen species production as evaluated by
CM-H2DCFDA (upper panel) or MitoS OX Red fluorescent probe (lower panel) is
expressed in fluorescence units as percentage of control. Values are mean  standard
error of mean (SEM) from 9 to 21 observations. *p<0.05, Bonferoni test versus
control; #p<0.05 versus normoxia and high glucose. (B) mtDNA integrity is measured
as normalized damage in long fragments. Upper panel: Values are mean  SEM from
nine observations. Analysis of variance one way (Bonferoni test versus control).
*p<0.05. Inset: A dose -dependent increase of normalized damage frequency in
mtDNA is induced by treatment with H2O2. Cells grown in normal glucose
concentration and normoxia were exposed for 30 min to different concentrations of
H2O2. Values are mean  SEM from six observations. Lower panel: In the presence
of carbony l cyanide m -chlorophenyl hydrazone, mDNA integrity is preserved. Values
are mean  SEM from four to five observations. *p<0.01.

41

Figure 7 . Db/db mice accumulate less mitochondrial DNA (mtDNA) lesions in
the heart and kidneys despite increase in reactive oxygen species. (A) Normalized
damage in the long fragment (10 kB) of mtDNA from heart (lower left panel) and the
kidney (lower right panel) in “old” and “young” db/db mice compared with their age –
matched controls (heterozygotes). Values are the m ean  standard error of the mean
from eight observations. Analysis of variance one way (Tukey test) between groups:
*p<0.001 for heart and p<0.003 for kidney versus control. Note instead a significant
(p=0.025) increase of damage in mtDNA fragments from mt DNA mutator mice hearts
(upper panel). Values are mean  standard error of the mean from four observations
(t-test versus control). (B) Whole tissue homogenate from the hearts of “old” db/db
mice and their age -matched littermates were prepared and analyzed by competitive
chemiluminescence enzyme -linked immunosorbent assay for nitrosylated proteins.
Graph shows the nitro -bovine serum albumin equivalents ( g/ml) from seven
different samples. *p<0.001 (t -test).

42

Figure 8 . Specific activity of serum superoxide dismutase 2 and catalase is
different in the tissues of db/db mice. Enzymatic activity for superoxide dismutase 2
(A) and catalase (B) in the heart and kidney from “old” and “young” db/db mice
compared with their age -matched controls. Values are mean  standard error of the
mean from 8 to 12 observations. *p<0.05 (analysis of variance one way).

43

Figure 9 . The mitochondrial base excision repair system is increased in the “old”
db/db mice and is not modulated in mitochondrial fractions isolated from human
dermal fibroblasts exposed to hyperglycemia and hypoxia. (A). Mitochondrial
extracts were isolated from “old” db/db mice heart and their age -matched littermates
and from human dermal fibroblasts cultured 48 h in hyperglycemia and 48 h
additional in hypoxia. Equal amounts of mouse (upper panel) and human (lower
panel) mitochondrial protein extracts were incubated with labeled tetrahydrofuran
substrate for 1 h at 37 C. Formamide (80%) w as added to the mix to stop reactions,
and the reaction products were analyzed by electrophoresis in 25% polyacrylamide, 8
M urea gels. Repaired substrate (arrow) is quantified as percent versus control (A1,
lower panel). Values represent means of OD  standard error of the mean for three
observations. Human apurinic/apyrimidinic endonuclease or mitochondrial protein
extract was used as negative control. *p=0.045 (t -test). (B) and (C). Purity of the
mouse (upper panels) and human (lower panels) mitochondria l extracts was verified
by immunoblotting against c -AMP response element binding protein (B) and by
lactate dehydrogenase activity assay (C).

44

Section I – Original Contributions

I.2. Evaluation of the antioxidant status and oxidative stress in the serum and
erythrocytes of patients with different stages of diabetes mellitus, especially at
the first medical onset

45

I.2. Evaluation of the antioxidant status and oxidative st ress in the serum and
erythrocytes of patients with different stages of diabetes mellitus, especially at
the first medical onset – the state of the art

I.2.1. Antioxidant status of the blood

An antioxidant system exerts its specific effect by stopping a radical reaction or
stabilizing a free radical (Gutteridge and Halliwell 2007) . The antioxidants are most
convenient divided into two categories, i.e. enzymatic and non -enzymatic. The latter
are further classified as hydrophylic and lypophylic (Gutteridge and Halliwell 2007) .

I.2.1.1. Enzymatic antioxidants

The enzymes act as antioxi dants have intra -cellular distribution. The antioxidant
enzymes essentially involved in the metabolism of superoxide anion and peroxide
consist of: superoxid dismutase (Lipcsey, Soderberg et al.) , catalase (CAT),
glutathion peroxidase (GPx) and peroxyredo xins (Prdx).

Superoxid dismutase (Lipcsey, Soderberg et al.) catalyzes the dismutation of
superoxide anion to anion peroxide, as follows: O 2 + O 2 + 2H+  H2O2 + O 2.
Three SOD iso -enzymes has been described in humans: the cytosolic Cu,Zn -SOD
(SOD 1), the mitosolic Mn -SOD (SOD 2), and the extracellular SOD (SOD 3). In the
catalytic center of the enzyme, the copper is essential, while zinc only stabilizes the
protein (Fridovich 1995) . It seems that SOD 2 is essential for survival, as the knock -out
mice die few days after born by renal failure (Lebovitz, Zhang et al. 1996) . The
resulting hydrogen peroxide (H 2O2) is furthermore essentially neutralized by
combinant action of CAT and Prdx.

Catalase (CAT) is essential for the neutralization of the H2O2. Several mutations
have described in certain diseases, such as diabetes mellitus, hypertension or vitiligo
(Goth, Rass et al. 2004) .

46

Peroxidases also neutralizes the H 2O2, using different substrates as reducti ve agents,
as follows: H 2O2 + DH 2  D + 2H 2O. When the reduced gluthathione (GSH) is used,
the enzymes are called gluthathione peroxidases (GPx).

Gluthathione peroxidases ( GPx ) are selenium dependent antioxidant enzymes that
might neutralize various peroxides, and include at least four different families. They
generate the oxidized gluthathione (GSSG) that has to be further reduced by
gluthathion e reductase (GRed) – a NADPH depe ndent antioxidant enzyme. As the
NADPH mainly originates from the pentoses path, the intra -cellular level of GSH is
essentially dependent by this metabolic shunt. The first GPx iso -enzyme (GPx 1) is
considered the most important member of the family, probab ly due to its
mitochondria expression and the presumed protection against free -radicals damage
(Legault, Carrier et al. 2000) .

Gluthathione transferase (GST) is essential for the neutralization of different
organic peroxides by reduced gluthathione (GSH) (Vos and Van Bladeren 1990) . The
GSH regeneration is also mediated by gluthathione reductase that is a process
dependent of intra -cellular concentration of NADPH derived from pentose pathway.

Peroxyredoxines (Prdx) consist of six types of cysteine dependent antioxidant
enzymes that are organized in three different categories, in accordance with their
common function – that also is the peroxides -neutralization (Rhee, Chae et al. 2005) .
Their ubiquous cellular distrib ution highly suggests a paracrine -type of peroxide –
neutralization (Wood, Schroder et al. 2003) . The cysteine along with thioredoxin and
NADPH are essential cofactors for the Prdx enzymatic activity (Rhee, Chae et al.
2005) .

The erythrocytes (RBCs) are pe rmanently exposed to significant levels of
superoxide anion essentially derived from hemog lobin (Hb) (Sadrzadeh, Graf et al.
1984) . From this perspective the enzymes neutralizing this particular free radical are
essential for the RBCs function and stabilit y. Hence, the erythrocytes contain CuZn
SOD (EC 1.15.1.1) – SOD 1, catalase (EC 1.11.1.6), – CAT, seleno -dependent

47 gluthathion peroxidase (EC 1.11.1.9) – GPx, and g luthathion transferase (EC
2.5.1.18) – GST. The pentose pathway is therefore essential in the se cells as NADPH
is essential for gluthathione regeneration.

I.2.1.2 . Non-enzymatic antioxidants in plasma are essentially divided in two main
groups, i.e. water – soluble (mainly albumin, uric acid, bilirubin and thiols), and lipid
– soluble (especially tocopherols, ubiquinone, and carotenoids). The efficacy of the
antioxidant response is a difficult issue. On one side, there is always a dynamic
interaction between the specific internal deposits, dietary source and intensity of the
free radic al aggression. On the other hand, the cooperative interactions between
different antioxidants at a certain state explain the synergistic effect that provide
better protection against free radical aggression than any single antioxidant alone
(Packer, Slater et al. 1979) . There is a general agreement that albumin, uric acid and
bilirubin are the most important components of the antioxidant defence in plasma.

Bilirubin has controversial antioxidant effects. In vitro studies (Stocker 2004) suggest
either the s cavanger efficiency against peroxyl radicals of unconjugated bilirubin or
the ineficiency of conjugated bilirubin as scavanger against superoxide anion and
hydrogen peroxyde. In vivo studies has been shown that, when in normal range for
healthy adults, the conjugated bilirubin efficiently scavanges peroxyl radicals and
protects fatty acids against oxidation (Stocker, Glazer et al. 1987) . Furthermore,
several epidemiological studies (Breimer, Wannamethee et al. 1995; Mayer 2000)
suggest a decrease in cardiovascular risk with lower plasma bilirubin concentration.

Uric acid (UA) is responsible for about 1/3rd of plasma total antioxidant capacity
(Waring, Maxwell et al. 2002) . Depending of its concentrations and the oxidative
statu s of plasma, UA might act either as antioxidant. The hydroxyl and peroxynitrite
radicals neutralization is mainly responsible for the antioxidant effect of UA (Scott
and Hooper 2001) . Especially when excessive the UA is pro -oxidant by several
mechanisms. Along with reduced copper accelerates LDL oxidation (Bagnati,
Perugini et al. 1999) ; with peroxynitrite further generates aminocarbonyl radical, thus
accelerating initial tissue damages (Santos, Anjos et al. 1999) ; activates platelets and
increases the ris k of thrombosis in patients with coronary heart disease (Johnson,
Kivlighn et al. 1999) . Recent studies have suggested that hyperuricemia is a risk

48 factor for cardiovascular disease (CVD) in the general population (Meisinger, Koenig
et al. 2008) and may pl ay a causal role in the development of metabolic syndrome
(Nakagawa, Hu et al. 2006) . Hypouricemia may be encountered in patients with either
type 1 (Shichiri, Iwamoto et al. 1987) or type 2 diabetes (Shichiri, Iwamoto et al.
1990) . The condition reflects an increased of uric acid renal clearance and occurs only
in patients with normal GFR levels (Shichiri, Iwamoto et al. 1987) .

Albumin encounters for more than 70% of serum antioxidant activity (Bourdon and
Blache 2001) . The main mechanism behind the antioxidant proprieties of albumin
consists in its ability to bind various molecules (Peters 1996) . Essentially, the albumin
is always an antioxidant; it never has pro -oxidant activity. Currently, it is accepted
that three e ssential factors contributes to antioxidant abilities of albumin (Halliwell
and Gutteridge 1990; Roche, Rondeau et al. 2008) , i.e. its capacity to bind transitional
metals; its propriety to scavenge free radicals that is mediated by several active sites
located in specific amino acidic residues; and its structural integrity. It has to be noted
that the structural integrity of the albumin molecule has only recently accepted as
essential for its antioxidant proprieties. Hence (Kagan, Tyurin et al. 2003) , a ch ange
in the protein conformation that is induced by either excessive binding of free fatty
acids (i.e. diabetes or pre -eclampsia) or hyperglycemia mediated glycosylation (i.e.
diabetes) might induce protein bind copper discharge. Thus, the plasma free copp er
concentration increases that arguments its availability for redox reactions therefore
consuming plasma antioxidants.

I.2.2. Antioxidant status of the blood in type 2 diabetes

The chronic hyperglycemia, as the main metabolic characteristic of type 2 d iabetes,
generates excessive reactive oxygen species (ROS) that virtually affects all the tissues
and organs, including the pancreatic β-cells (Newsholme, Haber et al. 2007) .
Excessive oxidative damage specifically deteriorates pancreatic β -cell, therefore
essentially contributing to the pathogenesis of diabetes (Kaneto, Katakami et al.
2007) . Type 2 diabetes angiopathy, both microvascular or macrovascular, is
essentially associated with o xidative stress (Giugliano, Ceriello et al. 1996) . Though,
it is gene rally accepted that in diabetic environment the chronic hyperglycemia is
associated with increased oxidative stress (Wright, Scism -Bacon et al. 2006) . It also a

49 general agreement about the fact that the a ntioxidant systems are involved in redox
regulation of the cell acting as the major tools to neutralize the oxidative stress and
therefore protect ing the cells from oxidative stress -induced damages. Furthermore, the
contribution of the imbalanced redox mechanism to the diabetes pathogenesis or its
specific complications has been already raised (Ceriello 2000; Opara 2002) . However,
the antioxidant status of plasma in type 2 diabetic patients remains controversial
(Lodovici, Bigagli et al. 2009; El Boghdady and Badr 2012) . The controversy persists
also as essential issue in patients with type 2 diabetes, in early stages – i.e. at the first
medical onset – meaning those of no previous medical history of type 2 diabetes – or
wihtout apparent evidence of specific chronic complications (Guldiken, Demir et al.
2009; Lodovici, Bigagli et al. 2009; El Boghdady and Badr 2012) .
The nature of this controversy is though difficult to be explained. On one side it might
be the fact that in diabetes different types of (plasma) antioxidants may be generated
in response to specific types of ROS produced in a certain cellular or tissular state
(Halliwell and Gutteridge 1995) . On the other side the differences may reside in the
type of method used to measure a specific antioxidant (Feillet -Coudray, Rock et al.
1999) . Hence, su ch type of differences were previously described in experimental
diabetes and were correlated with the plasma levels of vitamin E (Feillet -Coudray,
Rock et al. 1999) .

It has been previously shown in type 2 diabetes that oxidative damage is different in a
certain cell or organ and is more expressed in tissues with high metabolic demans,
such as blood cells (Sailaja, Baskar et al. 2003) . From this perspective the red blood
cells (RBCs) are essential effectors of the defense system against oxidative stress i n
blood (Prior and Cao 1999) . Recent data raised showing that in patients with late type
2 diabetes, the RBCs are exposed to higher oxidative damage and expressed a
decreased life span (Calderon -Salinas, Munoz -Reyes et al. 2011; Maellaro, Leoncini
et al. 2 011). It is well established that the erythrocytes possess enzymatic and non –
enzymatic antioxidants that protect the cell from the damages induced by ROS
overproduction . If the importance of superoxide anion for the chronic complications
in diabetes is con sidered, than the study at least three enzymes – the superoxide
dismutase (Lipcsey, Soderberg et al.) that converts superoxide into hydrogen
peroxide, together with the glutathione peroxidase (GPX) and catalase (CAT) that
convert hydrogen peroxide to water – might be significant . Hence, all these

50 antioxidant enzymes in blood have been reported as markers of vascular injury in type
2 diabetes (King 2008) . Thought, data about their activities are still conflicting
(Sundaram, Bhaskar et al. 1996; Peuchant, Del mas-Beauvieux et al. 1997; Kesavulu,
Giri et al. 2000; Seghrouchni, Drai et al. 2002) . Furthermore, when data about the
non-enzymatic antioxidant capacity in RBCs are analyzed, very few reports are
available (Calderon -Salinas, Munoz -Reyes et al. 2011; Mael laro, Leoncini et al.
2011) that refers to patients with late type 2 diabetes .

I.2. Evaluation of the antioxidant status and oxidative st ress in the serum and
erythrocytes of patients with different stages of diabetes mellitus, especially at
the first me dical onset – original contribution beyond the state of the art

Another important domain of my research activity is the evaluation of the antioxidant
status and markers of oxidative st ress (i.e., total antioxidant capacity – TAC, different
antiox idant enz ymes ) in the serum and erhytrocytes (red blood cells – RBCs) of
patients with different stages of diabetes mellitus, especially at the first medical onset
– meaning those of no previous medical history of type 2 diabetes (T2DM).

I have shown that TAC increases in the serum and RBCs of the patients with early
T2DM (at first clinical onset or with no evidence of chronic complications) when
compared with the standard antioxidant power of a water soluble equivalent of
vitamin D (TROLO X). I therefore here made the first observation on the TAC
alteration in human RBCs in diabetes. Furthermore, I was able to show that specific
antioxidant capacity (i.e., the activity of SOD and CAT) increases in the erythrocytes
of the patient with T2DM a t first clinical onset, and occur even in RBCs with a
decreased life span (i.e., increased caspase -3 activity).

Oxidative stress and total antiox idant status of plasma increased in patients with
early type 2 diabetes.

The levels of ROS were evaluated in blood samples of all subjects included in the
analysis (i.e. subjects without diabetes – as controls, and patients with type 2 diabetes
either at first clinical onset or without evidence of diabetes -related chronic
compl ications – as target group) by measuring the concentration of lipid peroxides.

51 The plasma instead of serum was used to estimate oxidative stress parameters having
in mind the fact that the platelet activation is followed by free radicals release (Leo,
Pratico et al. 1997) . There is sufficient data showing that lipid peroxides are essential
effectors of the ROS -mediated oxidative damage. Furthermore, the measurement of
malondialdehyde (MDA) -TBA adduct is a wide -accepted and validated method used
for the quan tification of lipid peroxidation (Kedziora -Kornatowska, Luciak et al.
1998) , and recently have been shown that it positively correlates with type 2 diabetes
duration, irrespective of age, gender, HbA1c and plasma SOD (Nakhjavani,
Esteghamati et al. 2010) . ROS overproduction was documented in plasma samples of
all patients included in target group, either diabetic patients at first clinical onset
(figures 1 0 B) or without evidence of chronic complications (figures 1 1 C).

As already mentioned, the effect of antioxidants in human plasma is synergic
(Valkonen and Kuusi 1997) . Hence, the total antioxidant capacity (TAC) of a sample
better estimates the free radical neutralizing efficacy when compared to any specific
activity of a certain antioxidant present in that sample. Though, the plasma also
contains some traceable compou nds that might influence its oxidative status, such is
ascorbic acid, vitamin E, and flavonoids. Their specific activity may be better
expressed by the antioxidant gap (GAP) or the residual antioxidant activity. That is
calculated by subtracting the antiox idant activity of albumin and uric acid (the
principal antioxidants in human plasma) from the total antioxidant capacity.

The total and residual antioxidant capacity was therefore estimated in plasma of all
subjects included in the analysis. A significan t increase of TAC and GAP was
observed in patients with diabetes (figure 10 A and figures 11 A and 1 1 B). These
results are in line with previous data published in plasma of late type 2 diabetic
patients, where a similar increase was observed irrespective of chronic complications
(Guldiken, Demir et al. 2009; El Boghdady and Badr 2012) . Thus, TEAC was
increased in plasma of patients with type 2 diabetes and peripheric neuropathy when
compared to subjects wi thout diabetes (El Boghdady and Badr 2012) and also in
plasma of patients with type 2 diabetes and stroke when compared to subjects (with or
without type 2 diabetes ) without cerebrovascular events (Guldiken, Demir et al.
2009) . The method used was similar with that in the present study. That might be of
importance if the heterogeneity of the reports about antioxidant status of plasma in

52 diabetes is considered. Thus, it is well known that plasma antioxidant status can vary
depending of which antioxidant is predominantly detected by the method used
(Feillet -Coudray, Rock et al. 1999) . In experimental diabetes such differences were
observed and correlated with plasma levels of vitamin E (Feillet -Coudray, Rock et al.
1999) .

Figur e 10. Oxidative stress and total antioxidant status of plasma increase s in
early type 2 diabetes patients. Plasma from type 2 diabetes patients at first clinical
onset and from non -diabetes age -matched subjects was evaluated for Trolox
equivalent antioxidant capacity (TEAC) (A) and malondialdehyde -thiobarbituric acid
(MDA -TBA) adducts (B). Values are mean  SE. *P<0.01, #P<0.001 vs. controls
(analysis of variance Kruskal -Wallis test).

53

Figure 11 . Total and residual plasma antioxidant activities and plasma
concentrations of peroxide, albumin, and uric acid in pati ents with
uncomplicated type 2 diabetes and healthy control subjects. (A) Total antioxidant
activity; (B) residual antioxidant activity; (C) lipid peroxides; (D) albumin and uric
acid. ABTS, 2,2’ -azino -bis(3 -ethylbenzothiazoline -6-sulphonic acid); MDA,
malondialdehyde. Data presented as mean  SE. The entire study cohort is not
included in all graphs due to sample hemolysis or exclusion of outliers (mean  2
SD).

54

Oxidative stress and total antioxidant status of RBCs is increased in type 2
diabetes patients at first clinical onset.

Recent evidence suggest that the erythrocytes (RBCs) are mainly targeted by
oxidative damage in patients with type 2 diabetes (Jay, Hitomi et al. 2006) .
Furthermore, it has been shown that, as probably the most proeminent cellular
components of the blood tissue, the RBCs are essential effectors of the defense
system against oxidative stress in blood stream (Ahmed, Naqvi et al. 2006) . As
previously mentio ned, the erythrocytes are permanently exposed to significant levels
of superoxide anion essentially derived from hemog lobin (Sadrzadeh, Graf et al.
1984) . Moreover, the present data have shown that lipid peroxidation is increased in
plasma of patients with diabetes (figures 10 B and figures 11 C), suggesting that the
blood cells, and therefore the RBCs, are exposed to an environment with excessive
free radicals production. On the other hand, it has been reported that the erythrocytes’
membrane is permeable to free radicals, i.e. superoxide anion and hydrogen peroxide
(Lynch and Fridovich 1978; Mathai and Sitaramam 1994) .

Thus, the magnitude of lipid peroxidation and non -enzymatic antioxidant activity in
RBCs of subjects with type 2 diabetes at first clinic al onset was investigated . The
malondialdehyde (MDA) -TBA adduct and t he Trolox equivalent a ntioxidant capacity
(TEAC) respectively, were therefore measured. A significant increase of
malondialdehy de (MDA) -TBA adduct (figure 7 B ) and TAC (figure 12 A) in RB Cs of
patients with diabetes when compared with non -diabetic subjects was observed .

As probably expected, the increased lipid peroxides shown here in RBCs exposed to
diabetic environment are in line with previous studies that do cumented significantly
higher levels of various markers of ROS in type 2 diabetic subjects (Nourooz -Zadeh,
Tajaddini -Sarmadi et al. 1995; Aguirre, Martin et al. 1998; Pasaoglu, Sancak et al.
2004) . Interesting though , the total antioxidant capacity was increased in erythrocytes
of patients with early type 2 diabetes (figure 12 A). It is important to mention that
TEAC assay is validated as standard method to measure the total antioxidant capacity
of a biological sample (Prior and Cao 1999) . To date, the present study is the first

55 report that uses TEAC assay to evaluate the non -enzymatic antioxidant defense in
RBCs.

Figure 12 . Oxidative stress and total antioxidant status of RBCs is increased in
type 2 diabetes patients at first clinical onset. Red blood cells (RBCs) from early
type 2 diabetes patients and from non -diabetes age -matched subjects were evaluated
for TEAC (A) and MDA -TBA adducts (B). Values are mean  SE. *P<0.01.
#P<0.001 vs. controls (analysis of variance Kruskal -Wallis test). Hg, hemoglobin.

Antioxidant enzymatic activity is early up -regulated in erythrocytes from
patients with type 2 diabetes .

It has been previously outlined the fact that SOD, CAT and GPx are the main
enzymatic antioxidants in the RBCs and also the major enzymes neutralizing
superoxide anion and hydrogen peroxide radical.

Hence, the enzymatic antioxidants in RBCs of all subjects included in the present
study were analyzed by measuring the SOD, CAT and GPx ’s activities (figure 13 ). A
significant increase for SOD and GPx, but not for CAT activity was therefore
observed in patients with type 2 diabetes . The significant increase of SOD and GPx
activities only shown here might have some possible explanations. First, the

56 difference in activity could be a consequence of a tissue -specific modulation of the
antioxidant enzymes (Wassmann, Wassmann et al. 2004) . Second, it has been shown
that different antioxidant enzymes have various substrate affinities. That is the case
for increased affinity for hydrogen peroxide of GPx when compared to CAT (Jones,
Eklow et al. 1981; Michiels, Raes et al. 1994) . Third, it has been reported that under
some circumstances an increased SOD activity induces increased GPx and reduced
CAT activities as compensation (Ceba llos, Delabar et al. 1988; Kelner and Bagnell
1990) .

Figure 13 Antioxidant enzymatic activity is up -regulated early in erythrocytes
from type 2 diabetes patients. Superoxide dismutase (Lipcsey, Soderberg et al.) (A),
catalase (CAT) (B), and glutathione peroxidase (GPX) (C) activity was measured in
erythrocytes from type 2 diabetes patients at first clinical onset and from non -diabetes
age-matched individuals. Values are mean  SE. *P<0.05, #P<0.01 vs. controls
(analysis of variance, one -way Tukey test).

57

Up to this point the present study has show n that since early diabetes, RBCs are
exposed to ROS overproduction originating either from inside or outside of the cell,
and exhibit an increased both nonezymatic and enzymatic antioxidant defense. Early
activation of the antioxidant defense observed here could be an argument for the
controversial effects of antioxidant supplements in patients with diabetes (Neri,
Calvagno et al. 2010; Golbidi, Ebadi et al. 2011; Bjela kovic, Nikolova et al. 2012) .
Though, t he compensatory efficiency of the response remains to be elucidated,
especially when oxidative stress mediated apoptosis of RBCs in diabetes is taken into
account (Maellaro, Leoncini et al. 2011) .

Apoptosis is activa ted in RBCs of type 2 diabetes patients since first clinical
onset.

When senescent, experience various injuries or clinical disorders the erythrocytes
should be removed from circulation. Under such conditions, a progressive process of
coordinated, program med cell death that eventually leads to disposal of affected cells
and precedes hemolysis (i.e. does not involve rupture of the cell membrane and
release of intracellular material ) has been recently described as erythrocyte apoptosis
or eryptosis (Lang, Gulbins et al. 2010) . In spite of controversies, the phenomenon is
nowadays accepted as particular for the RBCs since it shares but also lacks several
hallmarks of apoptosis (Lang, Gulbins et al. 2010) . Thus, eryptosys exhibits essential
steps as apoptosis , such is cell shrinkage, cell memb rane blebbing, and cell membrane
scrambling leading to phosphatidylserine (PS) exposure at the cell surface (Lang,
Gulbins et al. 2010) , and the dysfunctional erythrocytes are recognized and destroyed
by macrophages (Lang , Gulbins et al. 2010) . Instead, as do not have nuclei and
mitochondria, the RBCs undergoing apoptosis do not exhibit mitochondrial
depolarization and condensation of nuclei (Lang, Gulbins et al. 2010) .

An enhanced eryptosis has been described in several diseases characterized by high
levels of oxidative stress, such is diabetes and chronic kidney disease (Calderon –
Salinas, Munoz -Reyes et al. 2011; Maellaro, Leoncini et al. 2011) . Hence, Calderon –

58 Salinas and colab. ( 197) have studied the externalization of PS in erytrhrocytes of
patients with type 2 diabetes with or without chronic kidney disease (CKD). They
found that that erythrocytes from diabetic patients have a rise in erythrocyte PS
externalization, that was furthermore increased by t he duration of the diabetic
condition and the presence of CKD . Maellaro and colab. ( 198) investigate in RBCs
from patients with late type 2 diabetes whet her the activation of caspase -3 is
operative . The authors concluded t hat in diabetic erythrocytes caspase -3 activa tion
occurs and is correlated to the deg ree of hyperglycaemia and plasma (rather then
intracellular) level of oxidative stress, suggesting tha t extracellular oxidative stress,
likely induced by hyperglycaemia, could play a causative role.

Figure 14 . Caspase -3 is activated in RBCs of type 2 diabetes patients from first
clinical onset. Caspsase -3 activity was measured in RBCs from early type 2 diabetes
patients and from non -diabetes age -matched subjects. Values are mean  SE. #P<0.05
vs. controls (analysis of variance Kruskal -Wallis test).

59

We there fore investigated the ca spase -3 activity in all our subjects . Caspase -3
activation was found to o ccur early in diabetes (figure 14 ). Interestingly, we found a
negative correlation between caspase -3 activity and MDA -TBA adducts values in
diabetic erythrocytes that reach the significance in RBCs from patients with type 2
diabetes and hypertension only (Spearman r= -0.662, p=0,0219). Moreover, caspase -3
activity positively correlated with the levels of TAC in RBCs from patients with
diabetes , especially in hypertensive subgroup (Spearman r=0 .724 vs 0.8172, and
p=0.004 9 vs 0.0019, respectively). The result suggests that lipid peroxides exhibits a
strong inhibitory effect on caspase activity. A possible explanation might be that the
caspases are a family of cysteine proteases containing a thiol group as active site
necessary for their activity (Thornberry and Lazebnik 1998) . This thiol group renders
them particularly susceptible to redox modification by S -nitrosylation or oxidation.
Such modifications result in the inhibition of their catalytic activities (Curtin,
Donovan et al. 2002) .

In conclusion, our study shows that the pattern of defense against free radicals
aggression is early activated in blood stream of patients with type 2 diabetes , and
occurs even in RBCs with an increased apoptotic activity. By showing that
antioxidant defense is up regulated since first clinical onset in type 2 diabetes, we
question the beneficial effects of antioxidant supplements for clinical practice.
Nevertheless, the evaluation of antioxidant status has to be c onsidered for clinical
applicability despite the presence of any specific diabetes chronic complication.

60
Secti on I – Original contributions

I.3. Involvement of the erythrocytes in L -arginine dependent nitric oxide
metabolism in diabetes

61
I.3. Involvement of the erythrocytes in L -arginine dependent nitric oxide
metabolism in diabetes – the state of the art

Nowadays, it is well known that L-Arginine (L -Arg) is an important source to
generate nitric oxide (NO) in many human cells and that L -Arg exerts its important
cardiovascular role mainly via nitric oxide (NO) -dependent processes (Wu and Morris
1998) . Besides, L -Arg has other NO -independent roles in various physiological
processes (Wu and Morris 1998) . Thus, L -Arg is involved in post -translatio nal
modifications of some proteins that allow to be targeted for degradation by the
ubiquitin -dependent proteolytic pathway . L-Arg is also regulator of its own
metabolism, by modulating an essential cofactor of carbamoyl -phosphate synthase I, a
key enzyme for arginine dependent urea synthesis. L -Arg is also an essential
stimulator for hormone secretion, i.e. insulin or growth hormone.

L-Arg is considered as a semi -essential amino -acid, meaning that in certain conditions
(i.e. children or during recovery a fter infection and inflammation) the endogenous
synthesis may not fulfill the physiological requirements (Wu and Morris 1998) . The
sources of L -Arg that is found in adult plasma in post -prandial state are exogenous
(i.e. diet) and endogenous (i.e. protein degradation and citrulline) (Wu and Morris
1998) . Hence, citrulline is the main de novo source for L -Arg in adults, and is
essentially derived from glutamate that originates in the mitochondria of enterocytes
(Wu and Morris 1998) . Following release from th e small intestine, the kidneys take
up primarily the citrulline for L -Arg production . Thus, the circulation of the citrulline
from the enterocytes to the proximal tubules of the kidney is also know n as the
intestinal -renal axis of arginine synthesis. The l iver is not essentially involved in
extracting either citrulline or L -Arg from the blood stream. Furthermore, this organ
contains an exceedingly arginase activity to hydrolyze Arg into urea plus ornithine
thus explaining why t he hepatic urea cycle does not lead to a net synthesis of L -Arg
(Wu and Morris 1998) . The citrulline is up -taken into the cells by a selective transport
system (i.e. N -system) that is specific for amino -acids having amide group as side –
chain (Wu and Brosnan 1992) .

62
L-Arg degradation produces NO, citrulline, ornithine, urea, polyamines, proline,
creatine, glutamate, and/or agmatine and involves several pathways (Wu and Morris
1998) . These pathways are initiated by arginases, three isoforms of NOS, L –
Arg:glycine amidinotransferase, and L-Arg decarboxylase.
The arginase pathway is quantitatively most important for L -Arg catabolism in human
generating urea and ornithine (Wu and Morris 1998) . Two arginase i soforms are
expressed . While type II arginase is synthesized in all mitochondria -containing
extrahepatic cells (Li, Meininger et al. 2001) , type I arginase is produced mainly in
hepatocytes (Morris 2007) , and, to a limited extent, in extrahepatic cells (Li,
Meininger et al. 2002) , including red blood cells (RBCs) (Kim, Iyer et al. 2002) .
In the cells expressing NOS, L -Arg degradation might generate citrulline along with
NO. Though, in some extent, the citrulline can be furthermore recycled to L -Arg by
the combined action of two specific enzymes (i.e. argininosuccinate synthetase and
argin inosuccinate lyase ) through a pathway called the citrulline -NO cycle (Hecker,
Sessa et al. 1990) . The significance of this metabolic alternative it might be the
potential support of citrulline instead of L -Arg in sustaining NO synthesis. This
hypothesis is mainly accepted in the light of in vitro studies, showing higher levels of
induced NO in vascular smooth cells over -expressing a rgininosuccinate synthetase
(Xie and Gross 1997) . Though, the contribution of the a rginine recycling pathway to
NO synthesis in vivo is completely unknown (Wu and Morris 1998) .

The critical role that n itric oxide (NO) plays in vascular homeostasis in well known
(Ignarro 1989) . The synthesis of NO generally follows two main routes. On one side,
NO can be enzymatically synthesized in a reaction catalyzed by nitric oxide synthase
(NOS) (Alderton, Cooper et al. 2001) . Nowadays, there are three isoforms of NOS
already described: neuronal NOS (nNOS or NOS1), inducible NOS (iNOS or NOS2),
and endothelial NOS (eNOS or NOS3). Currently, th e existence of mitochondrial
NOS isofo rm (mtNOS) is still debated. In spite of controversy (Chen and Popel
2009) , recent data (Kleinbongard, Schulz et al. 2006) have shown that NOS3 is
expressed in RBCs and is potentially used as vascular source of NO. On the other
side, when NO production from NOS is impaired the non -enzymatic NO synthesis is
activated (Li, Cui et al. 2008) . This alternative route of nitrite -derived NO production

63 is particularly active under ischemia conditions (Zweier, Wang et al. 1995 ) in either
different tissues (Li, Cui et al. 2008) , and erythrocytes (Huang, Shiva et al. 2005) .

Decreased plasma levels of L -Arg characterize the endothelial dysfunction in patients
with diabetes (Wu and Meininger 2000; Kashyap, Lara et al. 2008) . Howe ver, data
based on clinical studies using L -arginine supplementation in subjects with diabetes
are still conflicting (Wu and Meininger 2000; Kashyap, Lara et al. 2008) .

Thus, oral supplementation of L-arg at a dosage of 7 g/day for 4 weeks in patients
with type 1 diabetes mellitus did not improve endothelial function (Mullen, Wright et
al. 2000) . Under contrary, long -term oral L -Arg administration (9 g/day for 3 months)
has been shown to improve but not completely normalize peripheral and hepatic
insulin sensitivity in patients with type 2 diabetes (Piatti, Monti et al. 2001) . Similar
results were obtained by Lucotti P et al. (Lucotti, Setola et al. 2006) where 8.3 g/day
L-Arg continuously administ ered for 21 days resulted in an additive effect compared
with a diet and exercise training program alone on glucose metabolism and insulin
sensitivity. Furthermore, it improved endothelial function, oxidative stress, and
adipokine release in obese type 2 diabetic patients with insulin resistance.
When L -Arg L-arginine (1 g/min) was intravenous infused during the last 30 min of
the glucose clamp ( at 18 mmol/l for 2 h ) to newly diagnosed complication -free diet –
treated type 2 diabetic patients , has reduced blood pressure and improved
hemodynamic function (Marfella, Nappo et al. 2000) . Though, the acute intravenous
L-arginine perfusion (625 mg/min x 10 minutes) in normotensive nonsmoking
patients with type 2 diabetes and angiographically normal coronary arteries and
normal plasma cholesterol was followed by no improve ment of coronary artery
responses to physiological stimuli (Nitenberg, Paycha et al. 1998) . On the contrary, an
intravenous bolus of L -Arg up to 5 g administered to patients with type 1 diabetes
reduced mean blood pressure and decreased platelet aggregatio n (Giugliano, Marfella
et al. 1997) .

Up to date, the implication of the RBCs in NO -dependent L -Arg metabolic pathways
has been studied in several reports (Chen and Mehta 1998) . Though, the present study
investigates for the first time the involvement of RBCs in Arg metabolic pathways in
type 2 diabetes at first clinical onset.

64
I.3. Involvement of the erythrocytes in L -arginine dependent nitric oxide
metabolism in diabetes – original contribution beyond the state of the art

As another distinctive part o f my research activity, I have also studied the
involvement of the RBCs in L -arginine (L -Arg) dependent nitric oxide (NO)
metabolism in diabetes. Thus, L-Arg content , arginase activity and nitrate/nitrite
concentrations were assayed in the RBCs and plasma collected from patients with
type 2 diabe tes at first clinical onset and age-matched non-diabetes subjects as
controls.

I found that L-Arg content remained unchanged in RBCs while decreased in the
plasma of patients with type 2 diabetes . The RBCs expressed lower arginase activity
while plasma exhibit an increased enzymatic activity in patients with diabetes. Both
nitrite and nitrate concentrations where higher in RBCs of patients with type 2
diabetes, while remained similar in the plasma of all subjects analyzed .

In this original contribution I was able to show for the first time that in patients with
type 2 diabetes at first clinical onset the L -Arg catabolism is driven mainly towards
NO synthesis, suggesting that the RBC pool represent s a potentially compensatory
intravascular compartment for the endothelial dysfunction in diabetes.

L-Arginine pool is unchanged in RBCs and decreased in the plasma of patients
with diabetes.

The L -arginine (L -Arg) po ol in RBCs and the plasma of all subjects included was
measured by capillary electrophoresis (CE). The method is well recognized as a
sensitive approach for quantification of ionic charged trace analytes (Chien and Burgi
1991) . Thus, it permits both a rapid and efficient separation of charged compounds
present in small sample volumes. When adequate conditions are chosen, the
separation can often be achieved directly in aqueous media, without sample
pretreatment, giving simple, fast, reli able, a nd easily automated methods (Barbas,
Adeva et al. 1998) . CE also has a good detection limit 5 -50 mg/l, wide linearity range

65 20-1000 mg/l, good reproducibility, short time of analysis (more than 30 compounds
can be analyzed in 30 -40 min) (Soga and Im aizumi 2001) .

As show in Figure 15 A and 15 B, the L-Arg content remained similar in RBCs
(Figure 15 A) while decreased in the plasma (Figure 1 5 B) of all the patients with
type 2 diabetes, even when hypertension was documented at first clinical onset.

Figure 1 5. L-Argin ine pool is unchanged in RBCs (A) and decreased in plasma
(B) of patients with diabetes. Erythrocytes (RBCs) and plasma from patients with
type 2 diabetes at first clinical onset and from non -diabetes age -matched subjects
were assayed for L -Arginine content. Values are mean  SEM. P=0.045 vs. controls,
ANOVA Kruskal -Wallis test.

66
This original contribution represent the first report to measure the L -Arg content in
RBCs from patients with no previous medical history of diabetes . In the RBCs of
subjects with type 2 diabetes at different stages of the disease, Ramirez -Zamora et al.
(Ramirez -Zamora, Mendez -Rodriguez et al. 2013) , have found lower L -Arg when
compared with healthy individuals. The authors suggested that L -Arg pool is capable
of regulating serum levels of L -Arg through buffering its concentrations. In other
words, the L -Arg is released from erythrocytes in order to maintain within normal
range the serum level of L -Arg in patients with diabetes.

The present data show that the lack of difference has maintained even when
hype rtension was documented in subjects with diabetes (Figure 1 5 A).

It seems that documented systemic hypertension has by itself a repressive effect on
the rate of L -Arg cellular transport. It has been shown that t he transport of L -Arg in
RBCs is mediated via the cationic amino acid transport (CAT) systems y+ and y+L
(Deves, Chavez et al. 1992) . Thus, Moss et al. (Moss, Brunini et al. 2004) has
previously show that the bi oavailability of L -Arg from the red blood cells is decreased
in patients with known medical history of systemic arterial hypertension. The authors
concluded that the diminished rate of L -Arg transport in human erythrocytes in
hypertension is mediated selec tively by a down regulation of the specific transport
system y+L activity (Moss, Brunini et al. 2004) . Hence, the mechanisms by which
system y +L is impaired in hypertension and also its relevance for further insights into
the pathophysiolo gy and treatment of this particular condition are not yet clearly
revealed (Moss, Brunini et al. 2004) .

Under contrary , in other chronic medical conditions that are characterized by systemic
high blood pressure, i.e. uremia and heart failure, an adaptive increase in the activity
of the cationic amino acid transport system y+, elevating the transport of L -Arg in red
blood cells is reported (Hanssen, Brunini et al. 1998; Mendes Ribeiro, Brunini et al.
1999) . The occurrence of the same phenomenon, i.e. increase in transport capacity for
L-Arg, in various clinical syndromes characterized by very different causes suggests a
possible common trigger. Accumulating data has been shown that some circulating
cytokines that are increased in both conditions (i.e. IL -1, IL -6, TNF -alpha) might up –

67 regulate the expression of the cationic amino acid transport system and iNOS in
platelets and RBCs precursors. Hence, the pre -thrombotic state and potential
thrombotic events that characterize either heart or renal failure might be further
minimi zed. On the other hand, the iNOS -mediated increases in NO synthesis is a
failing contra -regulatory mechanism, as the wide -spread vasoconstriction encountered
in heart failure and progressive systemic hypertension documented in renal failure
cannot be rever sed.

Thus, the result reported here in the present paper suggested that L -Arg pool from red
blood cells is not yet depleted in early stages of diabetes, even when systemic
hypertension was documented.

Arginase activity decreased in RBCs and increased in the plasma of patients with
diabetes.

As previously mentioned, the arginase pathway is quantitatively most important for
L-Arg catabolism (Wu and Morris 1998) . That is why the erythrocyte and plasma
arginase activity in all subjects recruited was further analyzed . The arginase activity
was lower in RBCs (Figure 16 A) while increased in the plasma (Figure 16 B) of all
patients with early type 2 diabetes when compared with subjects without diabetes .

Previous reports analyzing protein disregulation in red blood cells membranes of the
patients with type 2 diabetes (Jiang, Jia et al. 2003) show increased arg inase activity
in RBC membranes. In spite of both cytoplasm and membrane activity, up to date
there is no direct comparison of the enzymatic activities of arginase in both the
membrane and cytoplasm of RBCs in diabetes (Jiang, Ding et al. 2006) . This may be
of interest as long as indirect evidences (Jiang, Ding et al. 2006) suggest different
activities bet ween the two cellular compartments even that are presumed to be the
consequence of the different ratio of amount of arginase in membrane and cytoplasm .
It could be speculated that these differences are generated by arginase -flotilin
interactions in RBC mem brane (Jiang, Ding et al. 2006) . Human f lotillins are highly
conserved hydrophobic proteins isolated from caveolae/lipid raft domains of cellular
membranes consist ing of two family members: flotillin -1 (reggie -2) and flotillin -2
(reggie -1) (Malaga -Trillo, Laessing et al. 2002) . Flotillin -1 and flotillin -2 are the most

68 abundant membr ane proteins expressed in red blood cells lipid rafts (Baumann, Ribon
et al. 2000) . They have been implicated in numerous cellular processes including
signal transduction and membrane trafficking (Baumann, Ribon et al. 2000) . Flotillin –
1 plays an important role in the second signaling pathway required for insulin –
stimulated glucose transport, i.e. insulin -stimulated translocation of glucose
transporter 4 in adipose and muscle c ells (Bryant, Govers et al. 2002; Ducluzeau,
Fletcher et al. 2002) .

Figure 16 . Arginase activity decreased in RBCs and increased in plasma of
patients with diabetes. RBCs and plasma from patients with type 2 diabetes at first
clinical onset and from non -diabetic age -matched individuals were evaluated for
arginase activity. Values are mean  SEM. P=0.001 vs. controls (A and B), ANOVA
Kruskal -Wallis test.

RBCs Arginase activity
(μmol of urea/g Hb/min)
Plasma Arginase activity (μM/min)

69
It is notable that decreased arginase activity was maintained in RBCs of patients with
diabetes and documented high blood pressure (Figure 16 A). This observation could
represent an adap tive response of RBCs against endothelial dysfunction. That is,
arginase inhibits the NO production through several mechanisms, including
competition with NOS for the substrate L -Arg (Durante, Johnson et al. 2007) .
Therefore, upregulation of arginase in endothelial cells inhibits eNOS -derived NO
production, and may con tribute to endothelial damage in different conditions such as
hypertension and diabetes (Durante, Johnson et al. 2007) . Furthermore, it has been
shown that intraplatelet arginase activity in patients with systemic arterial
hypertension is not affected (Mos s, Siqueira et al. 2010) , suggesting that other blood
cells may exhibit similar adap tive behavior. Hence, t he arginase -mediated up-
regulation of NO synthesis in blood cells would not further deplete intravascular
concentration of NO.

When plasma samples were assayed in all subjects included in the present study L –
Arg content was decreased (Figure 15 B) while argina se activity increased (Figure 16
B) in patients with early diabetes. The same tendency was maintained when systemic
hypertension was documented in subjects with diabetes (Figure 15 B and Figure 16
B).

These results are in line with previous data (Kashyap, Lara et al. 2008) . Kashyap et al.
were measured plasma arginase activity, arginine metabolites, and skeletal muscle
NO synthase (NOS) activit y in patients with uncomplicated type 2 diabetes and age –
/BMI -matched subjects without diabetes and examined the effect of physiologic
hyperinsulinemia on plasma arginase activity before and following a 4 -h euglycemic –
hyperinsulinemic clamp . Percutaneous m uscle biopsies were collected from the vastus
lateralis muscle 60 minutes before the start of the clamp (Kashyap, Lara et al. 2008) .
The main results of the study were that plasma arginase activity is increased and NOS
activity is impaired in subjects with type 2 diabetes and is markedly reduced by short –
term (4 h) physiological hyperinsulinemia. These data sugges t an increased arginine
catabolism in the plasma of patients with type 2 diabetes.

70 As for the L -Arg content of the red blood cells, the plasma l evels of L -Arg is different
in patients with systemic hypertension alone compared to various clinical syndromes
characterized by hypertension. Hence, the plasma levels of L -Arg increase in patients
with systemic arterial hypertension (Moss, Brunini et al. 2004) and decrease in
medical conditions characterized by hypertension, such as chronic renal or cardiac
failure (Mendes Ribeiro, Brunini et al. 2001) . On the other hand, Kashyap et al.
(Kashyap, Lara et al. 2008) reported instead l ow concentrations of L -Arg in plasma
samples collected from normotensive patients with type 2 diabetes. There is no clear
explanation for these differences. Though, t he most important mechanisms behind
these variations include alterations of L -Arg transport or arginase activity, and
increased plasma concentrations of L -Arg analogues, i.e. ADMA (asymmetric
dimethyl -L-arginine) and SDMA (symmetric dimethyl -L-arginine). All together,
these data suggest a complex modulation of plasma L -Arg in patients with diabetes
and hypertension at first clinical onset.

There is one more important aspect that needs to be discussed here. Hence, it has to
be mentioned that L -Arg may be found either in plasma and RBC (i.e., RBC/plasma
L-arginine ratio). In other words, the above -mentioned ratio might potentially reflect a
relationship between plasma levels and cellular transport of L -Arg in various
conditions. In terms of the present results, o ne therefore could suppose that the
reduced plasma L -arginine (F igure 1 5 B) could compromise its uptake by RBC
(Figure 1 5 A). Although it cannot be excluded, this alternative is questionable since
the “arginine paradox” is taken into consideration.

The concept has been extensively discussed in relative recent reviews (Mann,
Yudilevich et al. 2003; Moss, Brunini et al. 2004; Dioguardi 2011) .

Hence, the “arginine paradox” has been primarily described in endothelial cells,
where acute supplementation with exogenous L -Arg induced a paradoxical increase in
NO production, in spite of the fact that the cytoplasm of these cells exhibits an
intracellular concentration of arginine that is more than sufficient to saturate the
eNOS activity (Kurz and Harrison 1997) . In other words, in spite of high levels of
cellular L -Arg in tis sue culture media (i.e. 400 μM), because the Km of arginase for
L-Arg is high (1 –3 mM) whereas the Km of NOS isoforms for L -Arg is relatively low

71 (3–10 μM) (Buga, Singh et al. 1996) , in cell -free systems (i.e. cultured endothelial
cells) arginase may not n ecessarily limit the availabi lity of intracellular L -Arg for NO
production . Nevertheless, Waddington et al. have been reported that the inhibition of
NO synthesis in renal mesangial cells is not associated as probably expected with
enhanced arginase activi ty (Waddington, Tam et al. 1998) . Moreover, further
experimental data suggest that intracellular transport and metabolism of the L -Arg is
rather independently modulated by plasma arginine concentrations (Dioguardi 2011) .
Thus, overexpression of the specifi c arginine receptors induces both arginine uptake
and nitric oxide production, while their specific blockade (i.e. by competitive amino
acids) reduces arginine uptake but not nitric oxide production. Furthermore, the
presence of citrulline in the cell cult ure media has no influence upon intracellular
concentration of L -Arg, while it stimulates nitric oxide production even in a medium
containing saturating levels of arginine (Dioguardi 2011) .

The “arginine paradox” phenomenon appears to be even more exace rbated by the
observations resulting from in vivo data. Thus, it seems that L -Arg supply is rate
limiting for NO synthesis (i.e. reducing NO production) in patients with
hypercholesterolemia and impaired endothelium -dependent vasodilation (Creager,
Gallagh er et al. 1992) . Moreover, the long -term supplementation with L -Arg does not
improved eNOS activity, as it has been shown by in vitro and in vivo data on healthy
human vessels (Chin -Dusting, Alexander et al. 1996) . Further, when L -Arg has been
chronically supplemented in patients recovering after myocardial infarction, an
increased of cardio -vascular mortality has been reported instead of the expected effect
of promoting vasodilation (Schulman, Becker et al. 2006) . Taken together, all these in
vivo observat ions support the persistence of the “arginine paradox” when L -Arg is
also chronically supplemented in various clinical scenarios.

These data suggest a particular intracellular transport, metabolism and NO regulation
of L-Arg, and a discrepancy in the sens itivity of eNOS to extracellular L -arginine in
cell-free sys tems and studies in vivo irrespective of high intracellular and circulating
levels of L -arginine.

In their review Mann et al. (Mann, Yudilevich et al. 2003) suggest several potential
mechanisms explaining the phenomenon. First, in patients with renal and heart

72 failure, hypercholesterol emia and atherosclerosis the presence of increased plasma
concentrations of ADMA (asymmetric dimethylarginine) could be one explanati on for
the L -Arg paradox (Boger 2003) . Then, the co -localization of eNOS and CAT
(chloramphenicol acetyltransferase) transporters in the caveolae (i.e. invaginations of
the plasma membrane or “lipid rafts”) of the endothelial cells could al so help t o
explain the phenomenon (Solomonson, Flam et al. 2003) . That is in spite of the fact
that NOS is cytosolic in macrophages and other cells . Moreover, Mann et al. have
found no evidence that arginase and eNOS are co -localized in plasmalemmal caveolae
(Mann, Yudilevich et al. 2003) . Finally, another report suggested that, in
macrophages and endothelial cells, iNOS and eNOS respectively, have access to
different intracellular L -Arg pools (Closs, Scheld et al. 2000) .

In his quite recent review, Dioguardi (Diog uardi 2011) proposes as more consistent
explanation for the endothelial arginine paradox the tight coupling of NO production
to the citrulline -NO cycle (Flam, Eichler et al. 2007) . Therefore, NO production
depends mostly on the efficient recycling of argin ine-derived citrulline back to
arginine, and not so much on exogenous arginine supply.

Furthermore, in order to understand the clinical paradox of arginine, that is reflected
in its inefficiency or even damaging effect when supplemented chronically, the same
author (Dioguardi 2011) suggests as possible mechanism the profound repressive
impact on NOS activity exerted by arginase -driven arginine metabolism, that is even
more amplified by ageing (Santhanam, Christianson et al. 2008) or diabetic
environment (Kashyap, Lara et al. 2008) , i.e. degree of hyperglycemia and insulin
deficiency. Exogenous supplementation with L -Arg in such conditions may be
therefore potentially harmful, as it has been demonstrated that induces the expression
of both types of arginases (Grody, Argyle et al. 1989) . In addition, oral arginine
supplementation may inhibit the recycling of citrulline to arginine by urea produced in
excess and also promotes insulin resistance , as arginine is recognized as a potent
stimulator of glucagon synthesis (Dioguardi 2011) .

As concluding remarks, the “arginine paradox” has been primarily described in
endothelial cells and refers to the discrepancy that, even at physiological L -Arg
concentrations, eNOS should be well saturated with substrate, and the addition of

73 exogenous L -Arg should not affect the enzyme’s activity (Boger 2004) . There have
been several explanations postulated, although none of them can fully explain the
phenomenon (Moss, Brunini et al. 2004; Dioguardi 2011) .

The erythrocytes NO production increas ed in patients with diabetes

The measurement of nitrate and nitrite levels in blood stream (i.e. plasma) is
considered a valuable index of estimating NO production (Viinikka 1996) .
Furthermore, accumulating data have been suggested that hemoglobin (Hb) is playing
important roles in NO metabolism in blood stream , being recognized as a powerful
catalyst of NO oxidation (Kim -Shapiro, Schechter et al. 2006) . Therefore, the L –
Arg/NO metabolic pathw ay in the erythrocytes and plasma of all subjects included in
the present study was further investigated . Figure 3 shows significantly higher levels
of NO production in RBCs from all early type 2 diabetes patients when comp ared
with non -diabetic subjects .

Figure 17 . RBC NO production increased in patients with diabetes. Nitrite (A)
and nitrate (Nox ) (B) were measured in RBCs from early type 2 diabetic patients and
from non -diabetic age -matched subjects. Values are mean  SEM. P<0.001 vs.
controls (A and B), ANOVA one -way Tukey test.

74

The present study reported for the first time that NO production in the RBCs of
patients with early type 2 diabetes was significantly increased when compared w ith
non-diabetic subjects. D ata reported here are in line with very recent observation of
Ramirez -Zamora et al. showing increased nitrites concentration in RBCs from
patients with type 2 diabetes at different stages of the disease (Ramirez -Zamora,
Mendez -Rodriguez et al. 2013) . Palmerini et al. (Palmerini, Zucchi et al. 2009)
observed similar results on patients with metabolic syndrome and ere ctile
dysfunction.

The same result was observed in RBCs of patients with diabetes and documented
hypertension in the current study (Figure 17 ). Hence, t his observation suggests that
RBCs could maintain their production of NO in spite of the reduced plasma levels of
L-Arg (Fi gure 1 5 B). A possible explanation of the phenomenon could be ac tivation
of the L -Arg transport. The effect has been already described in uraemic platelets and
mononuclear cells, which therefore may provide a protective mechanism against
increased aggrega tion and accelerated atherosclerosis characteristic of renal failure
(Mendes Ribeiro, Brunini et al. 2001; Brunini, Roberts et al. 2002) . As increased
aggregation and accelerated atherosclerosis characterizes the diabetic environment, a
putative NO -mediate d protective mechanism originating from the erythrocytes might
be also speculated.

Plasma NO production remains unmodified in patients with dia betes

Once formed, NO diffuses out of the cells and enters the extravascu lar and
intraluminal spaces . In the va scular wall, NO diffuse into the vascular smooth muscle
and reacts with soluble guanylate cyclase (sGC), which catalyzes the formation of
3’,5’ -cyclic guanosine monophosphate (cGMP) to induce vasodilation (Lancaster
1994) . When extravascular , NO inhibit s mitochondrial respiration by binding to the
heme group of cytochrome c oxidase, thus regulating oxygen delivery to the tissues
(Cooper and Brown 2008) . Inside the lumen, an important part of NO enters the
erythrocyte where is mainly scavenged in normoxia by reacting with oxygenated
hemoglobin (Eich, Li et al. 1996) . When intravascular, NO may also regulate certain

75 rheologic proprieties of the blood by interaction with the heme group of guanylate
cyclase expressed on erythrocytes and platelets thus modulati ng erythrocyte
deformability and platelet -platelet adhesion (Cerwinka, Cooper et al. 2002; Bor –
Kucukatay, Wenby et al. 2003) . Moreover, it can react with plasma constituents to
form various active compounds, such is: nitrosyl ated species ( i.e. nitrosothiol s),
nitrated lipids, and nitrite . Thus, through these species NO is able of transducing its
bioactivity fa r from where is generated (Cosby, Partovi et al. 2003) .

Figure 18 . Plasma NO production remains unmodified in patients with diabetes.
Nitrite (A) and nitrite plus nitrate (Nox) (B) were assayed in plasma from patients
with type 2 diabetes at first clinical onset and from age -matched non -diabetic
subjects.

Conflicting data are referring to NO bioavailability in the serum of patients with
diabetes. Thus, NO metabolites are either increased (Zahedi Asl, Ghasemi et al. 2008;
Ramirez -Zamora, Mendez -Rodriguez et al. 2013) , decreased (Huszka, Kaplar et al.
1997; Vanizor, Orem et al. 2001) or unchanged (Ferlito and Gallina 1999) in patients
with early type 2 diabetes mellitus. The same conflicting results are also reported in
the presence of chronic complications of diabetes (Maejima, Nakano et al. 2001) . In
the present study no differences were reported in NO metabolites in the plasma of al l

76 patients with type 2 diabetes included when compared with non-diabetic controls
(Figure 18 ).

The presented data here suggest a putative increase of RBC NO availability. It is well
known that RBCs are the major intravascular storage sites of nitrite in human blood
(Dejam, Hunter et al. 2005) and also that they have important roles in NO
bioavailability (Kim -Shapiro, Schechter et al. 2006; Chen and Popel 2009) .
Production of intracellular nitrite in RBCs involves several pathways (Dejam, Hunter
et al. 2005) . However, rec ent data (Sibmooh, Piknova et al. 2008) provide evidences
for the transport of NO via hemoglobin within RBCs. Nitrite has the potential to be a
major intravascular NO storage molecule in humans that is capable of transducing NO
bioactivity distal to its si te of formation (Cosby, Partovi et al. 2003) .

In other words, the erythrocytes seem to play a dual rol e in regulating NO availability.

On one side they scavenge and transform NO according to intracellular oxygen status,
as follows: under aerobic conditions oxyhemoglobin (HbO 2) converts NO to inactive
nitrate (NO 3-) and methemoglobin (MetHb, where heme irons are ferric) (Eich, Li et
al. 1996) while under (relatively) anaerobiosis NO is converted by deoxyhemoglobin
(Hb, deoxygenated hemoglobin) to iron-nitrosyl -hemoglobin (HbNO ) that was
classically considered a stable and inert end product (Cassoly and Gibson 1975) .

On the other side, they can serve as a site for NO-related reac tions involving iron –
nitrosyl -hemoglobin (HbNO ) – recently reconsidered as an important source of
bioactive NO (Nagababu, Ramasamy et al. 2003) , S-nitroso hemoglobin ( SNOHb ),
nitrite reduction, and enzymatic cleavage by NOS 3 expressed on erythrocytes (Chen
and Popel 2009) .

The reaction of NO with HbO 2 is predicted to be the major way by which the
erythrocytes inactivate intravascular NO. Though, there are several mechanisms that
might limit the intensity of the (natural) NO scavenging capacity of the RBCs (Kim –
Shapiro, Schechter et al. 2006) . First, blood flow velocity gradients are generated
along the close proximity of the endothelial wall. Thus, in the center of the blood
vessels the pressure is higher than near the vessel wall. Therefore, a cell -free a nd a

77 hemoglobin -free zone are created along the walls that allow NO to escape the
scavenging effect of RBCs in a higher extent. Second, an intrinsically slower rate of
NO uptake by RBCs when compared with cell -free Hb has been described, although it
is exp ected that NO to have similar properties in traversing membranes as oxygen.
Third, the e xtravasation of cell-free Hb into the subendothelial space allows Hb to
come closer to the source of NO that could increase NO consumption.

The S -NOHb contributes to the preservation of NO bioactivity in the RBCs (Chen and
Popel 2009) . The proposed mechanism assumes that first NO binds to deoxygenated
(ferrous) hemoglobin to form Hb(Fe2+)NO. Then, during Hb oxigenation the NO
group is transferred to the cysteine residue at position 93 in the β chain (ß -93
cysteine) of the hemo globin molecule, thus forming the SNOHb . Further, under
hypoxic conditions, SNOHb transfer s of the NO group to the thiols of the anion
exchange protein present on the membrane. The NO is exported from the erythrocytes
and might regulate blood flow . In spite current data supporting that the
intraerythrocytic SNOHb could be an important NO source in the intraluminal region,
the role of SNOHb in hypoxic vasodi lation is still debated.

The erythrocyte’s NOS 3 expression is another mechanism that potentially preserves
RBCs NO bioactivity (Chen and Popel 2009) . In spite of controversies it is thought
that NO synthesized by NOS 3 located in erythrocytes can escape scavenging by
hemoglobin and it could serve as another important source regulating vascular tone
and hemorheological properties of erythrocytes and platelets . Though, it is not
established how much NO from this source is available to the intraluminal region or
the vascular wall.

Nitrite is actually considered the major vascular storage pool of NO (Cosby, Partovi
et al. 2003) . Here, the e rythrocytes contain the majority of intravascular nitrite in the
whole blood (Cosby, Partovi et al. 2003) . This assumption was validated also in v ivo
on healthy individuals where has been shown that two thirds of intravascular nitrite is
located in erythrocytes, the majority of nitrite in erythrocytes is located in the cytosol,
and its concentration reach to 300 nM (Dejam, Hunter et al. 2005) . The e rythrocytic
NO is a relatively stable because its reaction rate with heme proteins is really low
compared to that of authentic NO. Recent experimental e vidence suggests that nitrite

78 represents in fact a reservoir that preserves NO bioactivity that can be e nzymatically
reduced to NO by hemoglobin under hypoxic conditions (Huang, Shiva et al. 2005) .
This is the reductase activity of hemoglobin (Huang, Shiva et al. 2005) . That specific
enzymatic activity of the hemoglobin is possible due to the equilibrium bet ween the
oxygenated and deoxygenated state of the hemoglobin, which is essential particularly
at the pre -capillary arterioles where the exchanges between erythrocytic hemoglobin
and tissue bed are performed. In other words, in oxygenated state the hemoglob in has
a reduced redox potential for the hemes – a condition that is thermodynamically
favorable for nitrite reduction, while the deoxygenated hemoglobin exposes more
ligand -free hemes as the reaction site with nitrite (Huang, Shiva et al. 2005) . Apart
from the nitrite reduction by hemoglobin, other certain proteins located in the
extracellular region (i.e. xanthine reductase or mitochondri al proteins such is
cytochrome c), might reduce nitrite to NO and therefore regulate tissue oxygenation
(Lundberg an d Weitzberg 2005; Basu, Azarova et al. 2008) .

The significance of present original observation may rely in the fact that erythrocytes
are essential in mediating NO bioavailability under specific conditions characterizing
endothelial dysfunction. Hence, as previously mentioned there are data suggesting
that RBC nitrite reservoir can be enzymatically reduced to NO by hemoglobin under
hypoxic conditions (Cosby, Partovi et al. 2003) . These results suggest that RBC
hemoglobin acts as a nitrite reductase, poten tially contributing to hypoxic vasodilation
in tissues with low oxygen tension. In medical conditions characterized by chronic
hypoxia, such as diabetes mellitus, these mechanisms may represent a potential
adap tive response against endothelial damage.

Taking together, the present study show that L -Arg catabolism is driven mainly
towards NO synthesis in the RBCs of patients with type 2 diabetes, even when
hypertension was documented at first clinical onset. Despite previous data stating that
production of intracellular nitrite in RBCs involves several pathways (Dejam, Hunter
et al. 2005) , the actual observation – showing a decrease in RBCs arginase activity –
could be considered a potential mechanism of increased RBC NO production in early
diabetes. Hence, the RBC pool would represent a potentially compensatory
intravascular compartment for endothelial dysfunction in diabetes.

79

80
Secti on I – Original contributions

I.4. The impact of basal insulin analogues on glucose variability in patients with
type 2 dia betes undergoing renal replacement therapy for end stage renal disease

81
I.4. The impact of basal insulin analogues on glucose variability in patients with
type 2 diabetes undergoing renal replacement therapy for end stage renal disease
– the state of the art

As for the number of subjects with diabetes (Shaw, Sicree et al. 2010) , there is a
continuous increase in patients with diabetes who re ach end stage renal disease
(ESRD) (Collins, Foley et al. 2012) . It is nowadays established the essential
contri bution of chronic hyperglycemia to the development of vascular complications
in diabetes, including in subjects with ESRD (Mehrotra, Kalantar -Zadeh et al. 2011) .
It has been also extensively shown that hypoglycemic events per se have crucial
impact on mort ality and vascular outcomes in patients with type 2 diabetes ( T2DM )
(Patel, MacMahon et al. 2008; Bonds, Miller et al. 2010) . Accumulating data
demonstrate the importance of acute fluctuations of blood glucose reflected as glucose
variability (GV), and mor e recently of HbA1c variability for the progression of
diabetes complications, including subjects with established diabetic nephropathy
(Smith -Palmer, Brandle et al. 2014) . It is now accepted that acute GV is increased in
patients with T2DM under maintenan ce hemodialysis ( HD) for ESRD, with no
evidence for a specific underlying mechanism (Kazempour -Ardebili, Lecamwasam et
al. 2009; Riveline and Hadjadj 2009) . Moreover, the variability in HbA1c might be of
importance in the development of nephropathy in pati ents with T2DM (Smith -Palmer,
Brandle et al. 2014) . In a very recent paper, it has been shown that higher HbA1c
level is correlated with higher variability in chronic glycemic control assessed by
standard deviation (SD) for HbA1c, and use of insulin therap y is associated with
increased risk of hypoglycemia in individuals with diabetes and ESRD (Williams,
Garg et al. 2014) . However, limited amount of information is available about the
HbA1c variability in T2DM patients o n dialysis. It has been shown that patients with
T2DM and ESRD exhibit insulin resistance (IR) as demonstrated by significantly
higher HOMA -IR values when compared to the ESRD patients without diabetes
(Bodlaj, Berg et al. 2006) . Nevertheless, GV is increased in patients with diabetes and
IR (Pitsillides, Anderson et al. 2011; Giordani, Di Flaviani et al. 2014) . The efficacy
of basal insulin therapy with human analogues on GV in patients with diabetes has
been proved in various studies (Smith -Palmer, Brandle et al. 2014) . Though , there is a
lack of information about the influence of different basal insulin regimens on GV in

82 patients on dialysis with T2DM and IR. The present study aims to evaluate the
influence of basal insulin analogues on GV – reflected as either acute blood glucose
fluctuatio ns or HbA1c variability in patients with T2DM on HD as replacement
therapy for ESRD .

I.4. The impact of basal insulin analogues on glucose variability in patients with
type 2 diabetes undergoing renal replacement therapy for end stage renal disease
– original contributions beyond the state of the art

As distinctive part of my research interest, I analyzed the impact of basal insulin
analogues on glucose variability (GV) in patients with type 2 diabetes (DM)
undergoing renal replacement therapy. Thus, vari ous glycemic profiles (Coefficient of
variation – CV of mean glucose) over 5 days period of continuous glucose monitoring
were compared between the day on (HD -on) and the day off (HD -off) dialysis. The
CV of at least 3 values of HbA1c (HPLC) since replacem ent therapy has been applied
was used to assay the long term GV. Endogenous insulin and insulin resistance
(HOMA using fasting glucose and C -peptide levels), fasting lipid profile, quantitative
C-reactive protein (CRP) and ferritin (values adjusted for Hb) were measured in
serum at inclusion.

I found that, while all subjects were insulin resistant, the overnight HD -off and HD -on
short term (CV CGMS) GV, overall long term (CV of HbA1c) GV, CRP and ferritin
were reduced in subjects treated with detemir .

This original contribution show for the first time that i nsulin resistant patients with
type 2 diabetes undergoing hemodialysis for end stage renal disease on insulin
detemir exhibit lower glycemic variability and pro -inflammatory profile than with
insulin glargine .

Insulin detemir was more efficient than insulin glargine in reducing intraday
nocturnal glycemic variability in patients with type 2 on hemodialysis.

83 The subgroup on glargine sequentially included subjects on basal – bolus insulin
regimen, wh ile the subgroup on detemir consisted of part icipants on basal insulin,
only. Therefore, the overnight g lucose variability was only assessed , in order to limit
the bias of short acting analog insulin regimen on glucose profiles . Moreover, we
observed a sig nificant reduction of BMI in subjects treated with insulin detemir than
with insulin glargine (35.1±1.9 vs. 28.4±1.5 kg/m2, p=0.018, T -paired). In spite of
strong clinical evidence of weight reduction effect induced by insulin detemir vs.
glargine (Niswender, Piletic et al. 2014) ; (Swinnen, Simon et al. 2011) , our result
might reflect a possible weight gain effect in patients treated with insulin glargine, as
all the subjects included in this subgroup received basal bolus insulin regimen.

Figure 19 Insulin detemir was more efficient than insulin glargine in reducing
intraday nocturnal glycemic variability in patients with type 2 diabetes on
hemodialysis Coefficient of variation (CV) for blood glucose levels over 5 days
period of continuous glucose monitoring. (A) HD -on=day of dialysis; (B) HD -off=the
following inter -dialytic period. P= 0.0001 (A) and P=0.0011 (B), paired t -test.

Though, we here report the first prospective study comparing the influence of basal
insulin analogues on glucose variabi lity in patients with type 2 d iabet es ongoing HD
for ESRD (figure 19 ).

84 Nocturnal hypoglycemic events were reduced either as number and duration
with insulin detemir than with insulin glargine in patients with type 2 diabetes
on hemodialysi s.

Several mech anisms may explain the higher incidence rate of h ypoglycemia in
patients with renal failure , i.e. impaired renal gluconeogenesis, malnutrition, altered
metabolism of insulin and hypoglycemic agents or as a consequence of HD itself
(Haviv, Sharkia et al. 2000; Jackson, Holland et al. 2000) . Insulin resistance is
common in uraemic patients, which may favour the progression of diabetic
nephropathy (Rigalleau and Gin 2005) . Thus, HD patients with type 2 diabetes might
not be only insulin resistant but also pr one to hypoglycaemia, with mild
hypoglycaemic episodes frequently preceding severe or even fatal hypoglycaemia
(Haviv, Sharkia et al. 2000) . Therefore, CGM can be efficiently used to detect acute
and unrecognized blood glucose oscillations and thus to redu ce the high
cardiovascular mortality in these patients (Riveline, Teynie et al. 2009) . We were able
to show that n octurnal hypoglycemia events were lower either as number and duration
in the subgroup of patient s treated with detemir (Figure 20 ). No severe hypoglycemia
was reported.

Figure 20 . Nocturnal hypoglycemic events were reduced either as number and
duration with insulin detemir than with insulin glargine in patients with type 2
diabetes on hemodialysis HD-on=day of dialysis; HD -off=the following inter –
dialytic period.

85
Variability in chronic glycemic control in patients with type 2 diabetes on
hemodialysis on insulin detemir was lower than with insulin glargine .

Very recent data on diabetic hemodialysis patients (Williams, Garg et al. 2014)
demonstrate that higher baseline HbA1c categories were associated with greater mean
SDs of HbA1c levels over 3 -year of analysis and increased odds ratio of
hypoglycemia hospitalization. That is, high variability in ch ronic glycemic control is
associated with high risk of hypoglycemia related hospitalization in these patients.
The lower CV for HbA1c reported in the present study with insulin detemir than with
insulin glargine (figure 21 ) might suggest an immediate benef icial impact of reducing
variability in chronic glycemic control on hypoglycemia events. However, large -scale
studies are required to confirm these findings on hypoglycemia related cardiovascular
risk in HD patients with type 2 diabetes.

Figure 21 Variability in chronic glycemic control in patients with type 2 diabetes
on hemodialysis on insulin detemir was lower than with insulin glargine

86 Coefficient of variation (CV) for HbA1c from at least 3 most recent values. P= 0.036
between groups, paired t -test.

Lower pro -inflammatory profile in patients with type 2 diabetes on hemodialysis
on insulin detemir than glargine.

End-stage renal disease affects HDL metabolism in patients with diabetes (Tan 1986;
Cacciagiu, Gonzalez et al. 2014) . In general, pat ients with this disorder exhibit a
decrease of HDL cholesterol levels (Tan 1986; Cacciagiu, Gonzalez et al. 2014) .
When compared with non -diabetes subjects on HD, the atherogenic effect of lipid
alterations in HD patients with type 2 diabetes is probably e xplained by concomitant
decrease in HDL cholesterol and increase in triglyceride -rich remnant lipoproteins, as
in spite of levels above 150 mg/dl there is no difference in triglyceride values, and the
paradoxical LDL cholesterol decrease is more likely lin ked to malnutrition and
inflammatory state (Cacciagiu, Gonzalez et al. 2014) . Besides, the chronic
inflammation is also an emerging risk factor for accelerated atherosclerosis in diabetic
patients with ESRD (Zoccali, Mallamaci et al. 2004) . Among various m arkers, ferritin
and quantitative C -reactive protein levels might be considered inflammatory proteins
that predict cardiovascular disease in patients with ESRD (Zoccali, Mallamaci et al.
2004) . Though, in the presence of insulin resistance conditions, neit her the pro –
inflammatory profile nor the atherogenic lipoprotein levels, but HDL cholesterol only
has been reduced in HD patients with type 2 diabetes when compared with HD non –
diabetes patients (Cacciagiu, Gonzalez et al. 2014) . Moreover, it has been show n
(Bodlaj, Berg et al. 2006) that low HDL cholesterol level was the only predictor for
higher HOMA -IR in HD patients with type 2 diabetes.

87 We found similar low HDL cholesterol values between two subgroups, while total
cholesterol, HOMA -IR and both marker s of inflammation were reduced in patients on
detemir only (figure 22 ). Thus, our result might suggest an efficient modulation of
pro-inflammatory profile by insulin detemir than insulin glargin in patients with type
2 diabetes and insulin resistance under going renal replacement therapy for end stage
renal disease.

Figure 22 Lower pro -inflammatory profile in patients with type 2 diabetes on
hemodialysis on insulin detemir than glargine. P<0.001 between groups, paired t –
test.

#
#

88

Section I– Original Contributions

I.5. Concluding remarks

89
I.5. Concluding remarks

I.5.1. HIF -1α stabilization is essential for wound healing whereas mitochondrial
ROS mediated mtDNA damages are successfully compensated by intrinsic
repairing mechanisms in diabetes

Specific c hronic complications of diabetes are nowadays considered as a major
medical and economical concern.

In spite of the important progress in understanding the specific pathogenic
mechanisms or in developing differ ent therapeutic strategies, the morbidity and
mortality related with diabetes is still important suggesting a lack of efficacy in
management . Therefore, a better understanding of the specific pathogenic
mechanisms involved in chronic complications of diabetes might contri bute to
progress in therapeutic approaches and future drug development.

Therefore, by focusing on mitochondrial DNA stability and HIF behavior as main tool
for oxygen sensing in chronic hyperglycemia characterizing the diabetic environment,
I have primari ly investigated the significance of these two p athogenic mechanisms for
the chron ic complications of diabetes in order to identify new therapeutic approaches.

Furthermore, I have specifically characterized the mechanisms of glucose -mediated
inference on HIF-1α stability and function , and have also described the efficacy of
HIF induction in diabetic wound healing process . Besides, I have specifically
analysed the combined effect of chronic hyperglycemia and hypoxia together on
mtDNA stability, and I have al so investigated how mitochondrial ROS production
influence the mtDNA integrity in diabetes.

By original data provided here I was able to demonstrate that, in spite of the VHL –
mediated repressive effect of high glucose on its stability and function, HIF-1α is an
essential factor and a valuable potential therapeutic target for improving the defective
wound healing process in diabetes , as its local stabilization activated all the defective
essential steps of this process .

90
Moreover, the original data showed here represent the first observation that
pathogenic mechanisms relevant in diabetes ( i.e. the combined hyperglycemia and
hypoxia) can induce critical levels of mitochondria originating ROS responsible for
mtDNA damage s that were successfully compensated b y the repairing mechanisms in
diabetic animals. The evidence of compensation further suggests that, at least in
diabetic animals, the combined hyperglycemia -hypoxia induced mitochondria ROS
production may therefore not represent a major factor for the dev elopment of chronic
complications.

I.5.2. The antioxidant status is early activated in plasma and erythrocytes of
patients with type 2 diabetes

The blood should be probably one of the first organs specifically targeted in diabetes.

In spite of sufficie nt data attesting that redox imbalance is essentially involved in
diabetes progression and occurrence of specific chronic complications, the antioxidant
status of plasma is controver sial and there is a lack of information related to the
antioxidant defens e of the eryt hrocytes in diabetes. Besides, the controversial effect of
antioxida nts supplementation in diabetes has constantly emerged in spite of
accumulating efforts reflected by numerous clinical studies.

Therefore, I have primarily investigated the antioxidant status and markers of
oxidative stess in the serum and erhytrocytes of patients with early type 2 diabetes ,
focusing on special subgroups – i.e. those with no previous medical history or with no
evidence of specific cronic complications of diabetes.

The original data provided here demonstrate that, in spite of increased oxidative
aggression, both non -enzymatic and enzymatic antioxidant defense is early activated
in the bl ood stream of patients with type 2 diabetes , and occurs even in RBCs w ith a
shortened life span .

The present result could support a possible explanation for the controversial effects of
antioxidants supplementation in diabetes .

91
Furthermore, as perspective, the evaluation of antioxidant status (in blood) has
probably to b e considered for clinical applicab ility since early clinical stages in
patients with type 2 diabetes.

I.5.3. L-Arginine drives to NO synthesis in erythrocytes of early type 2 diabetic
patients

L-Arginine is an important source of nitric oxide in many NOS expressing human
cells and exerts its cardiovascular role essentially via nitric oxide -dependent
processes . Besides, its main metabolic pathway implies the arginase -mediated
conversion to ornithine into urea cycle. The erythrocyte’s pool is generally acce pted
as the most important intravascular nitrate originating NO reservoir.

In spite evidence of the erythrocyte’s implications in NO-dependent L-Arginine
metabolic pathways , clinical studies using L -arginine supplementation in subjects
with diabetes are s till conflicting while there is a lack of information analyzing the
potential involvement of the RBCs in L -Arginine metabolism in early diabetes.

Hence, I have investigated the L -Arginine -nitric oxide metabolic pathway and
arginase activity in RBCs and pl asma of subjects with type 2 diabetes with no
previous medical history of diabetes.

The original results showed here represent the first observation that L -Arginine pool
is driven to NO synthesis when low arginase activity is expressed in erythrocytes of
patients with early type 2 diabetes.

Hence, the L -Arginine erythrocyte’s pool drives to NO synthesis that may constitute
an intravascular compensatory mechanism for endothelial dysfunction in early type 2
diabetes.

I.5.4. I nsulin resistant patients with type 2 diabetes undergoing hemodialysis for
end stage renal disease on insulin detemir exhibit lower glycemic variability and
pro-inflammatory profile than with insulin glargine.

92
Patients with diabetes who reach end stage renal disease (ESRD) are at higher risk of
morbidity and mortality. A ccumulating data suggest that acute fluctuations of blood
glucose reflected as gl ucose variability (GV) and, more recently HbA1c variability
are important for the progression of diabetes complications, also in patie nts with
established diabetic nephropathy .

In spite of significant data demonstrate the efficacy of basal insulin therapy with
human analogues o n glucose variability in patients with diabetes there is a lack of
information about the influence of differen t basal insulin regimens on GV in patients
on dialysis with diabetes, including those with type 2 diabetes (T2DM).

Therefore, I have evaluate d the influence of basal insulin analogues on GV – reflected
as either acute blood glucose fluctuations or HbA1c v ariability in patients with T2DM
on hemodialysis ( HD) as replacement therapy for ESRD.

The original data reported here represent the first proof of a different influence of
various basal insulin regimens on glucose variability in subjects with diabetes, i nsulin
resistance and ESRD.

93

Section II – Perspectives

General aim

Continuous, open, and undoubted professional development, either from medical,
research or academic perspective, that certifies valid recognition and solid and
promoting career.

Specific aims

– To develop a multi -disciplinary research team able to achieve and manage
competitive research projects.
– To obtain research grants able to self -sustain and development.
– To establish and consolidate a competitive research unit able to p erform excellence
activities in the field of diabetes.

Research directions

– Sirtuin, L -arginine mediated NO production and redox status in relation to caloric
restriction in type 2 diabetes.
– Amyloid, s irtuin , and AGE receptors response to diet AGEs in type 1 diabetes.

Background and aims

Sirtuins (SIRT) are enzymes involved in post -translational modifications of proteins,
that possess either mono -ADP -ribosyltransferase or deacylase activity (Michan and
Sinclair 2007) . Three d ifferent classes of de -acylase are described (Michan and
Sinclair 2007) . SIRT are NAD -dependent class III de -acetylases that transfer the
acetyl group of lysine to the ADP -ribose moiety to form O -acetyl -ADP -ribose,
through hydrolysis of NAD, and generating free nicotin -amide. Therefore, SIRT
might be considered a class of epigenetic regulators that modulate the activity of their
targets by removing covalently attached acetyl groups (Dang 2014) . Hence, by
interfering with NAD/NADH intracellular ratio SIRT in fluence the nutritional and

94 redox state of the cell, while by generating O -acetyl -ADP -ribose interfere with DNA
structure, facilitating the heterochromatin formation and silencing (Denu 2005) .

There are seven (1 -7) SIRT in mammals, which are differently localized and involved
in various cellular processes (Dang 2014) .

The most studied is SIRT -1 that is localized in the nucleus, but little is known for
SIRT -2 and SIRT -3, which are localized mostly in cytoplasm and mitochondria,
respectively (Dang 2014) . Accumulating data suggest that SIRT -1 are novel targets
for treating some diseases associated with aging, and perhaps essential for extending
human life span (Michan and Sinclair 2007) . Further, very recent data (Guedes -Dias
and Oliveira 2013) suggests that SIRT -2 is essentially involved in neurodegeneration
and accelerating aging in diabetes. SIRT -related genes have also been associated with
increased longevity under caloric restriction (Guarente 2005) and they appear to be
implicated in the regulation of d ifferent aspects of caloric restriction responses, such
as glucose homeostasis, insulin secretion, fat metabolism, and stress resistance (Chen
and Guarente 2007) .

Very few data are referring to SIRT -3 implications in diabetes or as regulators of
mitochon drial oxidative stress. Thus, recently Calabrese and colab. have found a
down -regulation in SIRT -1 and -2 activity as indicators of systemic cellular stress
response in patients with type 2 diabetes (Calabrese, Cornelius et al. 2012) , while
Canton et al. (Caton, Richardson et al. 2013) reported that SIRT -3 regulates mouse
pancreatic beta cell function and is suppressed in pancreatic islets isolated from
patients with type 2 diabetes. Further, mitochondrial SIRT -3 seems to be involved in
chronic complication s of diabetes, such is heart disease (Pillai, Sundaresan et al.
2010) or nephropathy (Maeda, Koya et al. 2011) . Nevertheless, calorie restriction
reduces oxidative stress by SIRT -3-mediated SOD 2 activation (Qiu, Brown et al.
2010) .

Intensive research is also evolved to find out the possible therapeutic opportunities of
SIRT modulators in aging diseases (Milne, Lambert et al. 2007; Nogueiras, Habegger
et al. 2012) .

95 Thought, accumulating data in very recent years gathered much controv ersy around
“canonical” SIRT functions. Hence, classical effect of life span extension when SIRT –
1 is overexpressed or SIRT -2 mediated life span extension of calorie restriction are
questioned (Dang 2014) . Therefore, this recent studies rise an intense deb ate around
SIRT functions in aging, calorie restriction, as well as age -related diseases.

In a previous study (Savu, Iosif et al. 2014) I and my colab. were able to show that
erythrocytes (RBCs) pool would represent a potentially compensatory intravascula r
compartment for endothelial dysfunction in diabetes. Our observation – showing a
decrease in RBCs arginase activity – could be considered a potential mechanism of
increased RBC -NO production in early diabetes. Hence, L -Arginine (L -Arg) becomes
an importa nt source for NO synthesis in the RBCs of patients with type 2 diabetes at
first clinical onset. In a different research article (Savu, Bradescu et al. 2013) I and
my research team found that erythrocyte caspase -3 and antioxidant defense (ei ther
non-enzyma tic and enzymatic – i.e. SOD, CAT and GPx activity) is activated in RBCs
and plasma of type 2 diabetes patients at first clinical onset. This result suggests that
the pattern of defense against free radical aggression is activated early in the blood
stream of patients with type 2 diabetes, and occurs even in RBCs with a shortened life
span.

Thought, there is a lack of data about SIRT activity in RBCs relative to oxidative
stress and diabetic environment. Hence, it has been shown that caloric restriction
increases SIRT -1 and -2 gene expression in polymorphonuclear cells (PBMCs)
(Crujeiras, Parra et al. 2008) through different mechanisms (Lamming, Latorre –
Esteves et al. 2005; Wang, Nguyen et al. 2007) and this effect is accompanied by an
improvement in antio xidant status in overweight subjects (Crujeiras, Parra et al.
2008) .

Hence, it would be of interest to evaluate the activity of SIRT -2 and -3 in erythrocytes
and mononuclear cells (as cellular response to stress) in relation to L -arg mediated
NO productio n from erythrocytes (as response against endothelial dysfunction) and
redox status (as specific antioxidant response to oxidative environment) in the blood
of patient s with type 2 diabetes stratified according to weight and duration of the
disease under ca loric restriction.

96
The increased consumption of highly processed foods in last decades (Cordain, Eaton
et al. 2005) increased the exposure to advanced glycation endproducts (AGEs). The
detrimental effect of high ingestion of AGEs and proteins on health an d disease
promoting is well recognized (Poulsen, Hedegaard et al. 2013) . Serum concentration
of AGEs has been positively correlated with the severity and progre ssion of diabetes
complications either microvascular or macrovascular, both in type 1 and type 2
diabetes (Poulsen, Hedegaard et al. 2013) . Accumulating data show that serum AGEs
correlates with dietary AGEs, and the latter also essentially contribute to the
endogenous production of AGEs and associate with inflammation and oxidative stress
in patient s with diabetes (Uribarri, Peppa et al. 2003; Chao, Huang et al. 2010) . In
spite of increased heterogeneity (Poulsen, Hedegaard et al. 2013) , it is considered as
general agreement that carboxy -methyl -lysine (CML) – and in less extent
methylglyoxal (MGO) – is one of the most studied AGE compound, both in patients
with diabetes and in healthy subjects (Van Puyvelde, Mets et al. 2014) . The beneficial
effects of AGE -restricted diet have been recently confirmed (Uribarri, Cai et al. 2011)
in a 4 -month follow -up study on patients with type 2 diabetes without renal disease or
overt cardiovascular disease. The authors reported a significant improve of plasma
insulin, markers of inflammation and oxidative stress. Low dietary AGEs for 6 weeks
have been also reported a s reducing plasma levels of inflammatory molecules in
patients with well -controlled type 1 and type 2 diabetes (Vlassara, Cai et al. 2002) .
Interestingly, no adverse effect of low dietary AGEs has been reported for patients
with diabetes (Kellow and Savige 2013) . Irrespective of its nature, i t has to be noted
that the effect cannot be finally attributed to the dietary AGE contents per se. From
this point of view, there is a lot of debate if either the deleterious impact of high
content or the protective con sequence of reduced composition it can be attributed to
the effects of heated foods or diet AGEs itself (Poulsen, Hedegaard et al. 2013) . In
spite of these controversies, when the effect of low dietary AGEs is analyzed the meal
plan is prepared based mainl y on a food questionnaire as recommended by Goldberg
et al (Goldberg, Cai et al. 2004) or following instructions on how to modify cooking
time and temperature but not the quantity or nutrient composition of food (Uribarri,
Cai et al. 2011) . In addition, as deleterious effect of high protein intake in relation to
dietary AGEs is well documented (Uribarri and Tuttle 2006) protein intake may be
used as estimate for dietary AGEs (Meek, LeBoeuf et al. 2013) .

97
The potential toxic effects of dieta ry AGEs are essentially modulated by the
interactions with specific AGEs receptors (Poulsen, Hedegaard et al. 2013) . Among
the AGE binding proteins, a receptor for AGEs (Hartter, Svoboda et al.) and
oligosaccharyl transferase complex protein 48 (OST -48 or AGER1 ) seem to be the
most important . Five of 6 isoforms of RAGE lack the transmembrane domain and are
probably secreted from cells. Generally these isoforms are referred to as sRAGE
(soluble RAGE) or esRAGE (endogenous secretory RAGE) (Poulsen, Hedegaard et
al. 2013) . RAGE is widely expressed in tissues, and its activation induces vascular
cell dysfunction mainly via activation of oxidative stress and inflammation. The
importance of these receptors for diabetes occurrence resides in the animal studies,
where it has been shown that RAGE -deficient mice developed protection against
diabetic complications (Poulsen, Hedegaard et al. 2013) . Though, the interaction
between AGEs and RAGE is much debated in vivo, where other specific ligands are
described, including beta-amyloid (Thornalley 1998) . AGER1 is expressed on many
cells, mainly macrophages, both on membrane and subcellular compartments, and
mediates the AGEs uptake and degradation (Poulsen, Hedegaard et al. 2013) . The
mechanisms behind these actions include suppression of NF -kB activity and MAPK
phosphorylation (Lu, He et al. 2004; Cai, Ramdas et al. 2012) and also the activation
of NAD -dependent SIRT -1 (Uribarri, Cai et al. 2011) . The protective synergism
between AGER1 and SIRT -1 is evidenced in conditions characterized by high levels
of AGEs and oxidative stress, such is type 2 diabetes (Uribarri, Cai et al. 2011) . Thus,
on one side, the AGE -induced hyper -acetylation is followed by AGER1 inactivation
and reflects SIRT -1 inefficiency; on the other side, AGER 1 overexpression blocks
AGE -induced suppression of SIRT -1 and prevents AGE -induced impaired insulin
signaling, at least in adipocytes (Uribarri, Cai et al. 2011) . Furthermore, the presence
of diabetic complications in patients with type 1 diabetes correlat es with low
expression of AGER1 in mononuclear cells and elevated serum AGEs (He,
Koschinsky et al. 2001) .

Insulin amyloid polypeptide (IAPP) is a 37 amino acid peptide that results from the
conversion of a pre -pro-protein of 89 amino acid lengths in endoplasmic reticulum
and secretory granules of beta cells (Westermark, Andersson et al. 2011) . Non –
significant amounts are also expressed in humans from the pyloric antrum

98 (Toshimori, Narita et al. 1991) . IAPP is co -secreted with C -peptide and insulin as
response to glucose in an echi -molecular ratio (Kahn, D'Alessio et al. 1990) , and it
seems that exerts at least hormonal paracrine effects (Martinez, Kapas et al. 2000) .
IAPP aggregation as amyloid has been recently hypothesized to initiate intra -cellular
in the secretory granules of the beta cells (Paulsson, Andersson et al. 2006) and is
described in almost all pat ients with type 2 diabetes or in some patients after islet
transplantation (Westermark, Andersson et al. 2011) . Islet amyloid is extensively
linked to type 2 diabetes (Westermark, Andersson et al. 2011) , though has been also
described in subjects without d iabetes (Bell 1959) . It is to note that high levels of
IAPP exerts toxic effects on cells, mainly when insulin is absent (Paulsson and
Westermark 2005) . In a very recent paper, Paulsson et al. (Paulsson, Ludvigsson et al.
2014) found high plasma levels of IAPP in 25 of 244 children with newly diagnosed
type 1 diabetes that were not correlated with C -peptide nor with autoantibody against
IAA.

Up to date, there is no data available about the importance of IAPP for the
progression of type 1 diabetes.

The s ignificance of exogenous AGEs might be therefore of great interest in relation to
amyloid for the development of type 1 diabetes. The assumption is furthermore
emphasized if the importance of cumulative exposure to AGEs on type 1 diabetes is
considered fro m animal studies. Hence, it has been shown that only 30% of non -obese
diabetic (NOD) mice fed with AGE -restricted diet developed type 1 diabetes at first
generation. Further, the maintenance of t he same restricted diet in both dams and
offspring decreased the incidence of type 1 diabetes to less than 15% in the next two
generations. Finally, the disease recurred when offspring were returned to the
standard diet (Peppa, He et al. 2003) .

Hence, it would be of interest to evaluate the amyloid, sirtuin and AGE receptors
response to diet AGEs in blood from patients with type 1 diabetes at different stages
of disease.

Team establishment

99 The material resources gained once the funds have been achieved allow the
establishment of a permanent research team t hat will be composed from clinicians,
biochemists and biologists. The team will also include students in their final years of
study and young PhD students, who will therefore benefit from support aimed to
fulfill their scientific activity from bench to bed side.

Funding resources

Diabetes mellitus is nowadays a serious health problem, that is spreading almost
epidemic, either worldwide or in our country. Thus, while in Romanian population it
already affects more than 1.5 million adults, the global prevalen ce is expected to
reach 366 million people by 2030.
Hence, diabetes becomes a major health, social and economical concern that
generates a tremendous interest from both scientific community and governmental
authorities aimed to reduce the impact on medic al resources. As consequence, the
number of specific financed research programs has been constantly increased.
The funding resources those are potentially available may include as follows:
1. Direct financial support from European Union research platforms, such is Horizon
2020, Fit for Health and IMI programs.
2. Research programs financed from Romanian Government, such is:
a. Funding resources targeting internal research, i.e. “Programul Parteneriate” or
“Programul Operational Sectorial pentru Cresterea C ompetitivitatii Economice”
(POSCCE).
b. Funding resources supporting young researchers, i.e. small internal grants
supported by universities.
c. Bilateral cooperative programs with specific research teams from countries inside
or outside of European Union .
3. Research funds or fellowships supported by international organizations for study of
diabetes, i.e. EASD or IDF.

Risk analysis

1. Human resources drop out. In actual social -economic climate this estimated risk
might have several possible explanation s. Probably one of the first reasons is low self –

100 motivation for research activities as a direct consequence of insufficient specific
salary for a generally highly time -consuming work. The problem is moreover
exacerbated due to financial instability affecti ng general economy.
2. Delayed or cessation of funds procurement. The main reason behind this possible
risk is the financial instability affecting general economy, also. Exchange variability
and funds insufficiency might be other major pitfalls, too.
3. Economic crisis.

Expected impact

1. Positive impact on my professional development.
– Increase ability in obtaining scientific funds and grants.
– Increase ability in managing scientific funds and grants.
2. Positive impact on human resources.
– Increase their performance for good research practice, laboratory skills, and
scientific writing.
– Increase their ability to understand scientific information.
– Increase their capacities to be enrolled and/or to obtain scientific projects, funds and
grants for the future activity as independent researchers/scientists.
3. Positive impact on my professional network.
– Establish and extend new professional contacts.
– Establish and extend future research directions, scientific projects and funding
sources.
4. Benefit of the patients suffering from diabetes, who live especially in Romania.
– Underline at least that relative simple modification in their life -style (i.e. calorie
restriction and ingestion of healthy prepared food) might have important
consequences on the occurrence and progression of their disease (i.e. diabetes).
5. Benefit of the pharmaceutical companies.
– The output of the research will be a substantial body of data that might form the
basis for further study and development work dedicated to sp ecific drug discovery.
Specifically, potential use for sirtuin treatment along with insul in therapy in type 1
diabetes will be of interest.
6. Development of a center of excellence dedicated for the study of diabetes.

101 SWOT analysis

Strengths

– Medical expertise in diabetes, nutrition and metabolic disease as clinician working
in the “N Paulescu” National Institute for Diabetes, Nutrition and Metabolic Diseases.
– Experience in clinical trials.
– Work -experience in two prestigious clinics from abroad, i. e. Udine, Italy and
Karolinska Institutet, Sweden.
– Licentiate degree in medical sciences at Karolinska Institutet, Sweden.
– Very good skills in elaborating and conducting scientific work, from lab bench,
through the final original scientific paper able to be published in international
databases of medical journals.
– Good skills for team working.

Weaknesses

– Limited experience in obtaining scientific funds and grants.
– Limited experience in managing scientific funds and grants.

Opportunities

– Effectively working in the “N Paulescu” National Institute for Diabetes, Nutrition
and Metabolic Diseases.
– Working as collaborator assistant professor in Univ. Med. Ph. “Carol Davila”.
– Permissive access to professional -targeted training programs.
– Permissive access to national and international research framework.
– Access to medical literature and databases through faculty library.

Threats

– Increasing competition to access research funds.
– Increasing costs for publication, as a consequence of e xtended number of open –
access ISI journals.

102 – Economic crisis.

103
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