+ MODEL

Nutrition, Metabolism & Cardiovascular Diseases (2013) xx, 1e7

Available online at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/nmcd

VIEWPOINT

AGEs, rather than hyperglycemia, are

responsible for microvascular complications
in diabetes: A “glycoxidation-centric” point
of view
N.C. Chilelli, S. Burlina, A. Lapolla*

Department of Medicine, Division of Metabolic Diseases, University of Padova, Via Giustiniani n 2,

35128 Padova, Italy
Received 21 December 2012; received in revised form 18 March 2013; accepted 12 April 2013

KEYWORDS Abstract Aims: Advanced glycation end products (AGE) excess is one of the most important
Diabetes mellitus; mechanisms involved in the pathophysiology of chronic diabetic complications. This review first
Advanced glycation summarizes the role of these compounds in microvascular pathogenesis, particularly in the light
end products; of recently proposed biochemical mechanisms for diabetic retinopathy, nephropathy and neu-
Microvascular ropathy. Then we focus on the relationship between AGE and metabolic memory, trying to
complications; clarify the former’s role in the missing link between micro- and macrovascular complications.
Oxidative stress; Data synthesis: An excessive AGE formation has been demonstrated in the newly disclosed
Metabolic memory biochemical pathways involved in the microvascular pathobiology of type 2 diabetes, confirm-
ing the central role of AGE in the progression of diabetic neuropathy, retinopathy and nephrop-
athy. As shown by recent studies, AGE seem to be not "actors", but "directors" of processes
conducting to these complications, for at least two main reasons: first, AGE have several intra-
and extracellular targets, so they can be seen as a "bridge" between intracellular and extracel-
lular damage; secondly, whatever the level of hyperglycemia, AGE-related intracellular glyca-
tion of the mitochondrial respiratory chain proteins has been found to produce more reactive
oxygen species, triggering a vicious cycle that amplifies AGE formation. This may help to
explain the clinical link between micro- and macrovascular disease in diabetes, contributing
to clarify the mechanisms behind metabolic memory.
Conclusions: The pathophysiological cascades triggered by AGE have a dominant,
hyperglycemia-independent role in the onset of the microvascular complications of diabetes.
An effective approach to prevention and treatment must therefore focus not only on early gly-
cemic control, but also on reducing factors related to oxidative stress, and the dietary intake of
exogenous AGE in particular.
ª 2013 Elsevier B.V. All rights reserved.

* Corresponding author. Tel.: þ39 (0)498216848; fax: þ39 (0)498216838.

E-mail address: annunziata.lapolla@unipd.it (A. Lapolla).

0939-4753/$ - see front matter ª 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.numecd.2013.04.004

Please cite this article in press as: Chilelli NC, et al., AGEs, rather than hyperglycemia, are responsible for microvascular complications in
diabetes: A “glycoxidation-centric” point of view, Nutrition, Metabolism & Cardiovascular Diseases (2013), http://dx.doi.org/10.1016/
j.numecd.2013.04.004
 + MODEL
2 N.C. Chilelli et al.
Introduction
Diabetes mellitus is characterized by chronic hyperglyce-
mia and an altered cellular homeostasis, which lead to
diffuse vascular damage and multi-organ dysfunction. In
the long term, diabetic patients risk both micro- and
macrovascular complications: the former result from dam-
age to retinal, renal and neural tissues, which is the cause
of blindness, end-stage renal failure and non-traumatic
lower limb amputation, respectively [1].
The findings of the Diabetes Control and Complications
Trial (DCCT) and the UK Prospective Diabetes Study (UKPDS)
have confirmed that hyperglycemic injury is the primary
causal factor behind microvascular damage, though the
biochemical pathways involved have long remained
unclear.
Brownlee’s theory outlines the mechanisms underlying
microvascular damage, identifying the production of
reactive oxygen species (ROS) as the unifying mechanism
behind the four main pathological pathways triggered by
hyperglycemia, one of which leads to the formation of
heterogenous moieties called advanced glycation end
products (AGE) via non-enzymatic glycation and glyco-
xidation processes [2]. It has been demonstrated that
these compounds increase in the cells and tissues of
patients with diabetes and are associated with the
development of microvascular and macrovascular com-
plications [3,4]
Considering the growing evidence of a clinical rela-
tionship between microvascular and macrovascular com-
plications, much attention has focused on identifying a
possible pathogenic link between them [5]. The most
accredited hypothesis is that microvascular damage is a
non-organ-specific mechanism with a systemic pathogen-
esis that ultimately leads to an increased macrovascular
risk [6]. ROS promoted by hyperglycemia have been
implicated in this pathophysiological mechanism, accused
of persistently damaging the microvasculature and pro-
moting an accelerated atherosclerosis through changes in
the vasa vasorum. This cascade could also contribute to
the “metabolic memory”(or “legacy effect”) and may
provide the basis for the link between micro- and mac-
rovascular diseases [7]. The legacy effect has to do with
the well-known observation that a given period of good
metabolic control produces a long-term beneficial effect
on cardiovascular endpoints, relating to ROS-dependent
structural changes in the microcirculation. There is
increasing evidence of AGEs playing a crucial part in these
mechanisms [8].
Among the biochemical pathways described by Brown-
lee, AGE formation and the activation of AGE receptors
(RAGE) seem to be the most important, interconnected
pathogenic mechanisms, involved in all microvascular
complications. This review first summarizes the specific
role of these compounds in microvascular pathogenesis,
particularly in the light of recently proposed biochemical
mechanisms for diabetic retinopathy, nephropathy and
neuropathy; then we focus on the relationship between
AGEs and metabolic memory, trying to clarify the former’s
role in the missing link between micro- and macrovascular
complications.
Please cite this article in press as: Chilelli NC, et al., AGEs, rather than hyperglycemia, are responsible for microvascular complications in
diabetes: A “glycoxidation-centric” point of view, Nutrition, Metabolism & Cardiovascular Diseases (2013), http://dx.doi.org/10.1016/
j.numecd.2013.04.004
AGE accumulation in diabetic patients e many
roads leading to Rome
The most important process responsible for AGE accumu-
lation in diabetic patients is the non-enzymatic glycation
reaction, or Maillard reaction. This can be globally seen as
a three-step process, the final stage of which comprises a
complex series of oxidation, dehydration and cyclization
reactions, which give rise to so-called endogenous AGEs,
i.e. thermodynamically unstable compounds that typically
accumulate on proteins with a long half-life, though they
have recently been shown to form on proteins with a short
half-life too, such as plasma proteins, lipoproteins, and
intracellular proteins [2,9]. One category of molecules
similar to AGEs are the advanced lipoxidation end products
(ALEs) deriving from the metal-catalyzed oxidation of
fatty acids with the production of lipid hydroperoxides,
which are subsequently processed to epoxyhydroxy,
ketohydroxy, and cyclic derivatives that may in turn break
down to form aldehydes, ketones or alcohols, or become
condensed to form polymers. The products of lipid per-
oxidation form ALEs through reaction with proteins,
generating stable or labile adducts or cross-links in pro-
teins, some of which are colored or fluorescent [10]. Other
substances, such as methylglyoxal (MGO), are formed in
the context of metabolic pathways with enzyme-catalyzed
steps, i.e. via glycolysis or the hexose-monophosphate
route [11].
The second way in which these compounds accumulate
in diabetic patients is through a defective renal excretion
of AGEs typical of diabetic nephropathy because renal
clearance is inversely related to plasma levels of AGEs
[12,13]. This gradually creates a vicious circle as the
greater circulating pool of plasma AGEs in turn changes the
shape and structure of many proteins at glomerulus level
(particularly type IV collagen and the mesangium), as well
as interacting with specific receptors. These interactions
are associated with a worsening chronic kidney disease and
further impairment of glycoxidation product clearance
[14,15].
Much attention has recently been paid to the so-called
exogenous AGEs, harmful products of "browning" (or the
Maillard reaction) in various foods. Together with endoge-
nous AGEs, these compounds form the majority of
glycation-free adducts, the greater proportion of circu-
lating AGEs in diabetic and non-diabetic subjects [16].
Among the various food processing methods, heating,
sterilizing, and microwaves contribute to the generation of
exogenous AGEs, all of which tend to accelerate the non-
enzymatic addition of non-reducing sugars to free NH2
groups of proteins and lipids [16].
It has been well documented that exogenous (dietary)
AGEs are related directly to plasma levels of AGEs in
healthy, diabetic and nephropathic individuals, contrib-
uting to the promotion of oxidative, inflammatory and
degenerative processes that underlie the pathogenesis
of many chronic diseases [17]. Numerous studies
have also shown that restricting diet-derived AGEs not
only stops the progression of atherosclerosis and renal
injury, but even improves insulin resistance in animal
models [18].
 + MODEL
AGEs e responsible for microvascular complications in diabetes
AGEs in the pathobiology of microvascular
complications e a special “piece in the
puzzle”
Hyperglycemia promotes microvascular damage through at
least five main mechanisms: an increased flux of glucose
and other sugars through the polyol pathway; an increased
intracellular AGE formation; interaction between AGEs and
their receptors (termed RAGEs) leading to intracellular
signaling, which disrupts cell function [19]; a persistent
activation of protein kinase C (PKC) isoforms [20]; and an
increased hexosamine pathway activity [21]. Selectively
inhibiting each of these biochemical pathways has pro-
duced disappointing results, however [2], prompting the
suggestion that the pathogenesis of microvascular compli-
cations is due to a single hyperglycemia-induced process
overwhelming all others. According to Brownlee, this single
unifying process involves the overproduction of superoxide
by the mitochondrial electron transport chain [2]; although
the relationship between hyperglycemia and oxidative
stress had been suggested more than 40 years ago, these
authors were the first to clarify the biochemical link be-
tween them.
Assuming the role of ROS production as the lowest
common denominator in the pathobiology of diabetic
complications, AGE formation and the actions they trigger
seem to play an important part in oxidative stress pro-
cesses, also in terms of the therapeutic opportunities
deriving from the development of products with anti-
glycation properties [22,23].
The main mechanisms behind the tissue damage caused
by AGEs are intracellular glycation, cross-link formation,
and interaction with RAGEs. The intracellular accumulation
of AGEs alters cytoplasmic and nuclear factors, including
the proteins involved in regulating gene transcription [10].
Another important mechanism is the formation of stable
abnormal cross-links on collagen, which causes chemical
and physical changes in the collagen’s structure and
consequent functional changes typical of chronic diabetic
complications, such as basement membrane thickening
with a resistance to proteolytic digestion [24]. Finally, the
AGEeRAGE interaction leads to cellular signaling, including
nuclear factor-kB (NF-kB) activation, increased cytokine
and adhesion molecule expression, induction of oxidative
stress, and an increase in cytosolic ROS [25]. It is worth
noting at this point that, in addition to the RAGEs, a number
of RAGE ligands have been identified in diabetic patients,
including members of the S100 calgranulin family and high-
mobility group box 1 (HMGB1). These ligands’ interaction
with RAGEs can trigger a subsequent interaction with an
innate immune system signaling molecule, the toll-like re-
ceptor 4 (TLR-4) [25].
As for the extracellular actions, a new class of molecules
e the soluble RAGE (sRAGE) e has recently added its
contribution to this complex scenario. sRAGEs reflect the
total pool of soluble RAGEs in plasma and are implicated in
diffuse micro- and macrovascular damage, even in non-
diabetic subjects [26]. There are several variants: in
particular, the endogenous soluble RAGEs (esRAGEs) are
circulating truncated variants of the RAGE isoform capable
of neutralizing AGE-mediated damage by competing with
Please cite this article in press as: Chilelli NC, et al., AGEs, rather than hyperglycemia, are responsible for microvascular complications in
diabetes: A “glycoxidation-centric” point of view, Nutrition, Metabolism & Cardiovascular Diseases (2013), http://dx.doi.org/10.1016/
j.numecd.2013.04.004
3
cell-surface RAGEs for ligand binding, and they are there-
fore an expression of antioxidant status with a demon-
strated role in protecting against early extracoronary
macro-angiopathy [25]. We recently suggested that esR-
AGE upregulation might be part of the cell’s defenses
against carotid plaque formation induced by oxidative
stress in patients with type 2 diabetes [27].
Among all the biochemical mechanisms involved, the
AGE pathway seems to be the most important in the
pathogenesis and progression of microvascular complica-
tions. For a start, as mentioned earlier, AGEs have several
intra- and extracellular targets, so they can be seen as a
“bridge” between intracellular and extracellular damage.
In addition, whatever the level of hyperglycemia, AGE-
related intracellular glycation of the mitochondrial respi-
ratory chain proteins has been found to produce more ROS
[28], which promote AGE formation. An excessive AGE
formation has also been implicated in the newly disclosed
biochemical pathways involved in the microvascular
pathobiology of type 2 diabetes, confirming its central role
in the progression of diabetic neuropathy, retinopathy and
nephropathy.
Role of AGEs in diabetic neuropathy
Autonomic and peripheral nerves are both affected in dia-
betic neuropathy: autonomic nerve involvement could be
one of the causes of sudden death, while the peripheral
nerve involvement associated with atherosclerosis is a
major determinant of lower limb morbidity (ulceration,
amputation, etc). Non-enzymatic glycation and AGE for-
mation have an important role in the onset of these com-
plications. There are reports of carboxymethyllysine (CML),
MGO-derived hydroimidazolone, frucosyl-lysine and 3-DG
accumulating in the peripheral nerves of streptozocin-
induced diabetic rats [29], and human diabetic patients
[30]. In human models, CML was identified in vascular
endothelial cells, pericytes, axons, Schwann cells and
basement membrane, the structural proteins of which were
modified (e.g. tubulin, neurofilament, axonal Na/K ATPase,
and myelin proteins in Schwann cells) [31]. Extracellular
matrix (ECM) proteins were found modified by AGEs too,
and this may severely affect cell function: ECM protein
glycation modifies functionally important arginine residues,
causing loss of charge and structural distortions associated
with a lower integrin binding affinity and cell detachment,
leading to basement membrane thickening [32].
These AGE-induced protein modifications cause struc-
tural and functional changes in the peripheral nerve.
Neuronal cells and Schwann cells incubated with AGEs
in vitro undergo apoptosis [33]. The interrupted axonal
transport caused by neurofilament and tubulin modification
may contribute to the development of atrophy and nerve
fiber degeneration [31]. Changes in the myelin protein in
Schwann cells may cause nerve fiber demyelination [33].
Jimenez et al. also demonstrated that glycation of laminin
and fibronectin (ECM proteins) prompts a reduced
neurotrophin-stimulated neurite outgrowth and precondi-
tioned neurite outgrowth in streptozocin-induced diabetes,
indicating a mechanism for the failure of axonal regener-
ation [32].
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4 N.C. Chilelli et al.

Finally, Chan et al. recently discovered extra-pancreatic
proinsulin-producing bone-marrow-derived cells (PI-BMDC)
in the sciatic nerve and neurons of the dorsal root ganglion
(DRG) of mice and rats with diabetic neuropathy [34]. The
authors showed that hyperglycemia e through poly(ADP-
ribose) polymerase-1 (PARP-1) overexpression and the
subsequent overproduction of AGEs and free radicals e
induces fusogenic PI-BMDCs that travel to the peripheral
nervous system, where they fuse with Schwann cells and
DRG neurons, causing neuron dysfunction and death, a sine
qua non condition for diabetic neuropathy.
[37]. Muller macroglia have a fundamental role in retinal
physiology and are severely dysfunctional during hypergly-
cemia, due largely to AGE accumulation at this level, where
they increase glial fibrillary acidic protein (GFAP) expres-
sion, NO production and glutamate synthesis (as a function
of glutamate transporter disruption) by the diabetic retina.
All these changes may contribute to retinal neuron exci-
totoxicity [38].
Role of AGEs in diabetic nephropathy

A progressive loss of kidney function has been clearly

Role of AGEs in diabetic retinopathy
Diabetic retinopathy involves structural and functional
changes in the retinal vasculature, with abnormal vessel
development leading to hemorrhage, ischemia and infarc-
tion. AGEs have been shown to contribute to this condition.
Through the above-mentioned ROS actions, AGE accumu-
lation leads to vessel thickening, hypertension, endothelial
dysfunction and loss of pericytes. AGEs also reduce platelet
survival and increase platelet aggregation, promoting a
procoagulant state and leading to ischemia and the induc-
tion of growth factors, with angiogenesis and neo-
vascularization [35].
This traditional view of diabetic retinopathy focusing on
vascular damage seems to be incomplete, however,
because neuronal damage might also contribute to its
pathogenesis and progression. Experimental studies have
shown that diabetes adversely affects the whole neuro-
sensory retina, accelerating neuronal apoptosis and acti-
vating or altering neuroretinal supporting cell metabolism
[36], suggesting that diabetic retinopathy could be a sen-
sory neuropathy, like peripheral diabetic neuropathy. AGEs
play a part in these recently hypothesized mechanisms
demonstrated in diabetic patients, correlating with
increasing circulating AGE levels [38]. Patients with diabetic
nephropathy have a dual form of damage, i.e. an increased
formation of serum AGEs and their reduced clearance [39].
Given the kidney’s complex structure, numerous harmful
mechanisms e including the matrix protein cross-linking
properties of AGEs and their activation of downstream
signaling e are responsible for disrupting the renal archi-
tecture and interfering with kidney function [40].
Glycation of type IV collagen and laminin alters these
proteins’ function, causing an increase in the vascular
permeability of albumin. On the other hand, AGE-driven
structural changes in other extracellular matrix proteins
impair their degradation by metalloproteinases, leading to
basement membrane thickening, a well-known histological
feature of diabetic nephropathy [14].
Another target of non-enzymatic glycation in the kidney
is the mesangium, where changes induced by AGEs
(increased pericyte apoptosis and vascular endothelial
growth factor expression) cause glomerular hyperfiltration,
an early renal dysfunction in diabetes [41].
Finally, AGEeRAGE interactions have been shown to play
a pivotal part in the pathogenesis of diabetic nephropathy.

Figure 1 Schematic overview of the different etiological contributions of hyperglycemia, AGEs and oxidative stress in different
phases of diabetes mellitus. In particular, a "micro" metabolic memory (at mitochondrial level) presumably frees the AGE-mediated
action from chronic hyperglycemia, while a "macro" metabolic memory, resulting from the chronic interaction between oxidative
stress and glycation at tissue/vessel level, forms the bridge between macro- and microvascular damage. AGEs, advanced glycation
end products; mtDNA, mitochondrial DNA; OS, oxidative stress; PKC, protein kinase C; RCPs, respiratory chain proteins.

Please cite this article in press as: Chilelli NC, et al., AGEs, rather than hyperglycemia, are responsible for microvascular complications in
diabetes: A “glycoxidation-centric” point of view, Nutrition, Metabolism & Cardiovascular Diseases (2013), http://dx.doi.org/10.1016/
j.numecd.2013.04.004
 + MODEL
AGEs e responsible for microvascular complications in diabetes 5

As demonstrated immunohistochemically in kidney sam- may be responsible for a “macro-metabolic memory” that
ples, RAGEs are expressed by glomerular podocytes and involves the extracellular compartment and causes mac-
tubular cells. Interaction with AGEs thus promotes patho- rovascular damage.
logical changes in these cells (mainly by enhancing ROS As suggested previously, micro- and macrovascular
production), contributing to chronic diabetic kidney lesions damage would influence each other, and the combined
[42], which include glomerular sclerosis and tubulointer- effects of "micro" and "macro" metabolic memory may be
stitial fibrosis. An impaired nitric oxide (NO) action appears the link between micro- and macrovascular complications
to be important in the increased expression of hypoxia- in diabetes.
inducible factor-1a (HIF-1 a) and TGF-b in diabetic kid- This overview (Fig. 1) clearly shows that hyperglycemia
neys, leading to tubulointerstitial ischemia and fibrosis has a key, early role in the disease, while oxidative and
[43]. The AGEeRAGE axis may be involved in this scenario glycoxidative stress mainly perpetuates tissue damage and
to some degree, since it has been shown to specifically the progression of complications in the longer term.
reduce endothelial NO activity and NO production through a Figure 2 summarizes the respective triggers for hypergly-
series of complex interactions with multiple enzymes [42]. cemia and AGE formation. While the therapeutic options
for the former are well established, there are currently
only trials underway for the latter. The imbalance between
AGEs and metabolic memory e not just a oxidative stress and antioxidant status in diabetic patients
matter of glycemia is currently under investigation and we have recently
demonstrated that it is an early, hyperglycemia-
Although hyperglycemia remains a hallmark in the patho- independent determinant of diabetic cardiomyopathy
physiology of chronic diabetic complications, it is now clear [48]. The dietary intake of exogenous AGEs, which has
that therapies should also address a number of factors only become increasingly prevalent in recent years, is another
partially related to glycemic control [10,44]. Many of these
factors appear to stem from an imbalance in diabetic pa-
tients between oxidative stress and antioxidant capacity,
which is seen as a bridge between hyperglycemia and the
multiple biochemical cascades that lead to microvascular
complications [45]. One such pathway involves excessive
AGE formation, which links the two most important pro-
cesses behind vascular damage in diabetes, i.e. glycation
and oxidation. The relationship between hyperglycemia
and AGEs is by no means straightforward, however: suffice
it to remember that a hypothetical blood glucose level of
0 mg/dl does not coincide with 0 mmol/mol of HbA1c,
which is one of the most important AGEs [46]. In a previous
analysis, we reported that diabetic patients with and
without complications have a different individual pro-
pensity for oxidation and/or differing oxidation kinetics
relating to behavioral and environmental factors [47].
Returning to this concept, we suggest that the patho-
genic mechanism behind the role of AGEs in the genesis of
microvascular damage may involve two temporally sepa-
rate phases (Fig. 1): in the early years of the disease, we
can imagine a linear relationship between hyperglycemia,
increased oxidative stress and excessive AGE formation,
with all three factors linked by a causal association; later
on, persistent respiratory chain protein glycation and DNA
damage in the mitochondria generate a hyperglycemia-
independent vicious cycle [8], in which oxidative stress is
self-supporting and AGEs "feed" this process. This picture
could represent a sort of “micro-metabolic memory” taking
effect at intracellular level.
The main downstream effects of this physiopathological
scenario include changes in the composition and structure
of the extracellular matrix, mediated by inflammatory
processes induced by receptor binding of AGEs or oxidative
stress [6]. Subsequent fibrosis and the extension of the
extracellular structures interfere with capillary blood flow, Figure 2 Factors triggering hyperglycemia and AGE forma-
reducing capillary density in particular [6]. These structural tion in diabetes: Light grey fields refer to etiological factors
changes reduce the flow reserve and this can directly affect with known therapeutic options, dark grey fields to etiological
upstream arteries, causing endothelial dysfunction (over factors postulated as future therapeutic targets. AGEs,
time) and then atherosclerosis. This proposed mechanism advanced glycation end products.

Please cite this article in press as: Chilelli NC, et al., AGEs, rather than hyperglycemia, are responsible for microvascular complications in
diabetes: A “glycoxidation-centric” point of view, Nutrition, Metabolism & Cardiovascular Diseases (2013), http://dx.doi.org/10.1016/
j.numecd.2013.04.004
 + MODEL
6 N.C. Chilelli et al.
important factor that may contribute significantly to this
mechanism, acting as a “throttle” in both of the above-
described phases [16].
Conclusion
This review investigated the contribution of AGEs to the
microvascular complications of diabetes, emphasizing their
pivotal role in oxidative stress: as shown by recent studies
on the pathophysiology of mitochondria, AGEs seem to be
not “actors”, but “directors” of this process.
What distinguishes the role of AGEs from other
biochemical pathways discussed by Brownlee is that these
compounds derive from both endogenous and exogenous
sources, and take effect at both intracellular and extra-
cellular level. This points to a pathophysiological link be-
tween micro- and macrovascular disease in diabetes e a
relationship suggested by a number of important clinical
trials [5] e and the present hypothesis may help to explain
some of these results. The pathophysiology of these com-
pounds could also contribute considerably to explaining the
mechanisms behind metabolic memory.
All the above considerations support the current
conviction that the proper therapeutic approach to pre-
venting complications of diabetes is to concentrate not only
an early glycemic control, but also on reducing factors
related to oxidative stress, and the dietary intake of
exogenous AGEs in particular.
Finally, although hyperglycemia and AGEs should be
considered in parallel rather than in any causeeeffect
relationship for the time being, there is already a hypoth-
esis that turns this paradigm upside down, seeing AGEs as
etiologic agents in type 2 diabetes [49].
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