Current Enzyme Inhibition, 2007, 3, ???-???
1
Aldose Reductase in The Retina
Mahmoud Ahmed Mansour*
Department of Biochemistry- Faculty of Pharmacy, Al-Azhar University, Nasr City, Cairo, Egypt
Abstract: Aldose reductase (E.C. 1.1.1.21), an intracellular enzyme of polyol pathway, catalyzes NADPHdependent reduction of glucose to sorbitol. Under normoglycemia, most of the cellular glucose is
phosphorylated into glucose-6-phosphate by hexokinase. A minor part of non-phosphorylated glucose enters
the polyol pathway, the alternate route of glucose metabolism. However, under hyperglycemia, because of
saturation of hexokinase with ambient glucose, aldose reductase is activated, leading to excessive production
of sorbitol. Intracellular accumulation of sorbitol is thought to result in irreversible damage. In the diabetic
eye, the increased sorbitol accumulation in retina has been implicated in the pathogenesis of retinopathy,
characterized by pericyte loss, basal membrane thickening, the major ocular complications of diabetes. Nearly
all diabetic subjects have the same degree of retinopathy after 20 years of diabetes. 50% of patients with insulin
dependent diabetes mellitus have proliferative retinopathy after 15 years. In addition, macular edema
frequently produces central vision loss and blindness most commonly in non-insulin dependent diabetes
mellitus. Therefore, aldose reductase enzyme inhibition is becoming one of the therapeutic strategies that have
been proposed to prevent or ameliorate long-term diabetic complications.
Keywords: Aldose reductase, glucose, retina hyperglycemia, blindness.
INTRODUCTION
Aldose reductase (AR) is one of the most thoroughly
studied of aldo-keto reductase due to its putative
involvement in the pathogenesis of diabetic eye diseases [1].
AR catalyzes NADPH–dependent reduction of glucose to
sorbitol (polyol pathway), the first step of sorbitol pathway
(Fig. 1). A large body of evidence, derived principally from
experimental animal studies, supported the hypothesis that
complications [2]. Chronic hyperglycemia leads to many
changes in target tissues of diabetic patients including
accumulation of excess polyol, alteration in the relative
abundance of oxidized and reduced nicotinamide co-enzyme,
change in the levels of glycolytic intermediates and loss of
glutathione [3].
It is well established that high blood glucose levels
greatly influence the development and progression of
diabetic retinopathy [4]. Most cells of the retina are affected
Fig. (1). The polyol pathway; glucose-6-phosphate [2].
enhanced metabolism of glucose through polyol pathway
results in biochemical imbalance associated with diabetic
*Address correspondence to this author at the Phrmacology Department
Faculty of Pharmacy King Saud University, P.O Box 2457 Riyadh 11451
Kingdom of Saudi Arabia; Tel: 00966-3-5812839; Fax: 00966 –3-5817174;
E-mail: mansour1960us@yahoo.com
1573-4080/07 $50.00+.00
by the metabolic abnormalities of diabetes [5] but the sight–
threatening manifestations of diabetic retinopathy are
ultimately attributable to capillary damage [6]. Abnormal
permeability of barrier capillaries can cause macular edema
and capillary closure leading to ischemia and unregulated
angiogenesis [7]. Retinopathy is the most severe
complication of diabetes mellitus because it can result in
irreversible blindness [8]. Therefore, elucidating the
© 2007 Bentham Science Publishers Ltd.
2 Current Enzyme Inhibition, 2007, Vol. 3, No. 1
mechanism for initiating diabetic retinopathy is important
for prevention or treatment.
Aldose reductase inhibition represents an attractive
strategy for the prevention of diabetic complications. The
beneficial effect of aldose reductase inhibition in preventing
or substantially delaying the onset of diabetic complications
in experimental models provides strong support to this
hypothesis [2]. Moreover, transgenic animal studies have
shown that over expression of aldose reductase confers an
increased susceptibility to diabetic cataract [9] and
morphological changes in retinal vasculature similar to those
observed in human diabetic retinopathy [10].
A. Retina
The retina is composed of the tissues arising from the optic
vesicle and consists of a pigment epithelium layer, which is
derived from the outer layer of the optic cup and a complex
sensory layer, which is derived from the inner layer [11].
1. Retinal Pigment Epithelium
The retinal pigment epithelium (RPE) is a single layer of
hexagonal, deeply pigmented cells with a regular
arrangement that forms a sheet between Bruch's membrane
and the photoreceptor layer of the sensory retina. This
epithelium extends from the margin of the optic disc to the
ora serrata and then it continues with the pigment epithelium
of the ciliary body [12].
In the posterior part of the eye, the RPE cells are fairly
uniform in size and shape and measure approximately 16 µm
in diameter. In the mid periphery and equatorial areas, the
cells are thinner and larger in diameter. In the far periphery,
the cells are variable in size and this variability increases
with age [13].
The cytoplasm of the RPE cells contains oval or rounded
pigment granules that are located predominantly near the
apical side. Other pigment granules, composed of lipofuscin,
are found in the cytoplasm and accumulate in the cells
during the process of aging, particularly in the macular area
[14].
The functions of the RPE are mainly the storage of
vitamin A and its conversion to a form that can be utilized
by the photoreceptors for synthesis of rhodopsin and also
phagocytosis and degradation of shed lamellar discs of the
photoreceptors [15].
2. Sensory Retina
The sensory retina is a delicate, transparent layer that forms
the lining the inner surface of the eye between the vitreous
and the retinal pigment epithelium. It is firmly attached
posteriorly at the optic disc and anteriorly at the ora serrata,
where it terminates. Elsewhere, the attachment to the
underlying RPE is weak and is maintained by the intraocular
pressure. The contacts between the photoreceptor outer
segment and the RPE villi, depend on a mucopolysaccharide-cement substance surrounding the photoreceptors
and probably active transport processes [15, 16].
B. Aldose Reductase
Aldose reductase (AR) is a cytosolic enzyme present in
most of the mammalian cells. The polyol pathway was first
Mahmoud Ahmed Mansour
identified in the seminal vesicle by Hers [17], who
demonstrated the conversion of blood glucose into fructose,
an energy source for sperm cells. Later, the presence of
sorbitol in diabetic rat lens was reported by Van Heyningen
[18]. This work provided the basis for new research
concerning the pathological role of AR and the polyol
pathway in the development of diabetic complications.
Several studies have suggested that increased reduction of
glucose by AR contributes to the development of secondary
diabetic complications [19]. During hyperglycemic event,
the elevated glucose level enhances the activity of AR by
increasing glucose flux through this pathway [20]. In fact,
AR messenger ribonucleic acid (mRNA) is highly expressed
in the rat lens, retina and sciatic nerve, the major target
organs of diabetic complication [21]. The increased activity
of AR results in decreased NADPH/NADP+ ratio, which has
impact an on other NADPH-dependent enzymes, such as
nitric oxide (NO) synthase and glutathione reductase [22].
The retarded activity of the antioxidant enzyme, glutathione
reductase causes oxidative stress under diabetic conditions.
Increased sorbitol flux through the polyol pathway causes
increases in NADH/NAD+ ratio, which blocks the glycolytic
pathway at the triose phosphate levels. Consequently,
protein KinaseC (PKC) is activated and the formation of
advanced glycation end products is increased. Advanced
glycation end products are toxic. they cause pathological
changes by disrupting protein function and interfering with
cellular receptors and have been implicated in the etiology of
diabetic complications [23].
1. Aldose Reductase as Member of Aldo-Keto Reductase
Superfamily
The aldo-keto-reductase (AKR) comprises a large number
of structurally related enzymes that catalyze the pyridine
nucleotide-dependent reduction of carbonyl groups.
Examples include aldehyde reductase (AKR1A), and aldose
reductase (AKR1B), which are responsible for NADPHlinked formation of alcohol products from aldehydic
functional groups of aromatic and aliphatic hydrocarbons and
aldo- and keto- sugars [24,25], hydroxysteriod
dehydrogenase (AKR1C), which catalyze steroid conversion
and detoxification of polycyclic aromatic hydrocarbons [26],
ketosteriod reductase (AKR1D) and prostaglandin synthase
[27].
Aldose reductase may be considered a typical enzyme of
the AKR superfamily. The enzyme is a small monomeric
protein composed of 315 amino acids residues. The primary
structures, were first determined on rat lens AR [28]. They
demonstrated a high similarity to another NADPHdependant oxido-reductase, human liver aldehyde reductase
(EC 1.1.1.2) [29]. The degree of similarity clearly suggests
that these proteins belong to the same family. Graham et al.
[30] reported that aldose reductase gene is located on
chromosome 7q35. Complementary deoxyribonucleic acid
(cDNA) clones of human placenta, retina and muscle AR
were isolated [31, 32]. The identification of amino acids
sequence of AR from different species revealed a relatively
low sequence identity (82-85%) conserved among human, rat
and other animal species. This could account for the species
differences in the sensitivity of AR to some of the inhibitors
[33].
Aldose Reductase in the Retina
Molecular cloning techniques have identified many
amino acid sequences for cellular proteins (39 proteins) as
members of the aldo-keto reductase superfamily [34]. A wide
variety of proteins from various species constitute this
family, including aldehyde and xylose reductases from
plants, yeast, and bacteria [35]. Nonetheless, the majority of
this family is represented by mammalian aldehyde
reductases, aldose reductases and hydroxysteroid
dehydrogenases. These enzymes are now categorized into
functionally and evolutionarily related groups based on their
genetic origins [36, 37]. Thus, the superfamily of AKRs is
organized into a series of families and subfamilies. While
the overall structural features of the AKR family members
are well conserved, subtle differences near the C-terminal
domain are thought to be responsible for the difference in the
substrate specificity among closely related enzymes.
2. Tertiary Structure of AR
X-ray crystallography and site directed mutagenesis
studies have provided important insights into the structures
of AR. Wilson et al. [38] showed the structure of human
placenta AR. The enzyme molecule contains α/β barrels,
which are still the most prevalent motif with a core of eight
parallel β strands connected by peripheral α helices with a
large hydrophobic active site (Fig. 2). The structure of active
site of AR differs significantly from that of aldehyde
reductase [39]. However, it has been shown that the coenzyme binding site is the same for AR and aldehyde
reductase [2]. The NADP+ co-enzyme molecule is bound to
Current Enzyme Inhibition, 2007, Vol. 3, No. 1 3
the carboxyl terminus of the β barrel in an extended
conformation. The nicotinamide ring is centered in the active
site cavity. This highly hydrophobic active site pocket is
formed by aromatic residues (Trp 20, Tyr 48, Trp 79, Trp
111, Phe 121, Phe 122, and Trp 219) apolar residues (Val
47, Pro 218, Leu 300 and Leu 301) and polar residues (Glu
49, Cys 298 and His 110). The pyrophosphate bridge of
NADPH is tied down by residues 214-230 amino acids,
which hold NADPH tightly in place [40]. The interactions
that favor the binding of NADPH over NADH are due to
Lys 263 and Arg 269 salt linked to the 2'-phosphate of
adenosine moiety of NADPH [2].
3. The Catalytic Mechanism of AR
The proposed catalytic mechanism of AR consists of
stereospecific transfer of the 4-pro-R hydrogen from the
exposed C-4 of the nicotinamide of the bound NADPH to
the carbon of the carbonyl group of the substrate, followed
by the protonation of the substrate carbonyl oxygen by a
proton donor group. Three residues within suitable distance
of the C-4 were identified as the potential proton donor,
including Tyr-48, His-110 and Cys-298. However, site
directed mutagenesis studies implicated Tyr-48 as proton
donor and that His 110 is involved in substrate recognition
[41]. A hydrogen bonding interaction between the phenolic
hydroxyl group of Tyr-48 and the ammonium side chain of
Lys-77 is thought to help to facilitate hydrogen transfer [38].
Cachau et al. [42] further explained a new reaction path in
which proton source was Tyr-48 and His-110 acted as a
Fig. (2). Tertiary Barrel structure of AR. There is eight α helices surrounding a core of eight β strand all in parallel orientation [2].
4 Current Enzyme Inhibition, 2007, Vol. 3, No. 1
proton shuttle. After donation a of proton from Tyr-48, His110+ intermediates formed, which resulted in the release of
the proton to the substrate along with a hydride transfer from
C-4 of nicotinamide of NADPH. A water bridge and
correlation of movements between His-110 and Lys-66
oriented lysine toward polarized Tyr-48, which explained the
loss of catalytic activity in Lys-77 mutants. The importance
of Asp-43 was evident, as a salt bridge replaced the initial
interaction of Lys with NADP+ after the reaction [42]. The
reaction catalyzed by AR proceeds through an order
sequential kinetic mechanism with NADPH binding first
and NADP+ being released last [43]. The Asp 45- Lys 80Tyr 50 system maintains an overall neutral charge on the
enzymes active site between pH 6-8, allowing a proton to be
transferred to the carbonyl oxygen of the substrate and a
negative charge to develop on the tyrosyl hydroxide.
Elegant kinetic study showed that NADP+ release
represents the rate-limiting step in the reaction of AR [44].
As the rate of co-enzyme release limits the catalytic rate, it
can be seen that perturbations of the interactions that
stabilize co-enzyme binding can have dramatic effects on
Vmax [45].
4. Post-translational Regulation of AR Activity
Several investigators have reported evidence for an
activated form of AR distinguishable from the native
enzyme form by differences in Km for aldehyde substrates
and marked reduction to AR inhibitors [46]. Previous
studies have shown that levels of activated form were found
to be elevated in diabetic tissues. AR isolated from diabetic
or hyperglycemic tissues is less susceptible to inhibition and
is kinetically different from the enzyme purified from normal
or euglycemic human or animal tissues [47]. These changes
in the inhibitor sensitivity and the kinetic properties have
been reported upon thiol oxidation of purified protein in
vitro, suggesting that diabetic changes in AR may be due to
redox modification of its cysteine residues [48]. Due to the
presence of a hyperreactive cysteine residue (Cys-298)
located at the active site of the enzyme; AR is very sensitive
to oxidants [49]. These functional changes are largely
prevented if the enzyme is complexed with NADP(H) prior
to the treatment with modifying agents. Since cysteine side
chain is involved in the stabilization of the co-enzyme in
such a way that the reactive thiol group becomes
inaccessible. Therefore, the enzyme should be susceptible to
modification through Cys-298 only during a catalytic cycle
when nucleotide exchange occurs. Chandra et al. [50]
showed that exposure to nitric oxide (NO) donor results in
rapid and selective modification of AR at cysteine residue
(Cys-298). These observations suggested that NO may be a
physiological regulator of AR activity in vivo and that
changes in NO availability during diabetes could alter AR
catalysis and consequently, the polyol pathway activity.
Chandra et al. [51] showed that NO donors inhibit AR
activity and sorbitol accumulation in erythrocytes. These
observations are consistent with the view that NO is a
physiological regulator of AR. The inhibitory effect of NO
on AR catalysis and the polyol pathway activity was also
evident in diabetes. The erythrocytes of diabetic animals
when incubated with glucose or tissues isolated from
diabetic rats accumulated significantly less sorbitol when
treated with NO donors [51]. This observation provides
Mahmoud Ahmed Mansour
strong evidence supporting a post-translational mechanism.
However, the extent of inhibition of sorbitol formation in
diabetic erythrocytes was much less than that observed in
normal euglycemic cells, indicating that diabetes increased
resistance of AR to the inhibitors. During diabetes, NO
availability is decreased and therefore the net effect is the
stimulation of sorbitol synthesis. However, it has been
reported that NO could also affect the transcription of AR
gene. Stimulation of vascular smooth muscle cells in culture
with NO donors results in an increase in AR mRNA and
inhibiting NO synthesis prevents the increase in AR [52].
Chandra et al. [51] suggested that transcriptional activation
of AR gene in vivo by NO is marginal and that this effect is
overcome by the post-translational inhibition of AR activity.
The AR gene is stimulated by the toicity of high glucose
[49] as well as growth factors, oxidants, mitogens [53] and
cytokines [54]. Most of these stimuli are affected by diabetes
and could change the AR expression during diabetes and
therefore the stimulation of sorbitol synthesis. These
observations explained the high level of AR in diabetic
tissues.
5. General Kinetic Features of AR
Aldose reductase has been isolated and studied from a
variety of species and tissues. Placenta and muscle are
typical human tissue sources for enzyme purification [31,
55], although enzyme levels may be highest in the adrenal
gland [56]. Kinetic and structural properties appear to be
consistent, regardless of tissue source. In relative terms,
glucose is a poor substrate for AR. The apparent Km value
for glucose has been reported by various groups to be in the
range of 50-200 mM, well above normal physiological
levels [55]. The catalytic efficiency of AR is approximately
four orders of magnitude lower for D-glucose than for lipidderived aldehydes, including 4-hydroxy-2-nonenal [57]. The
poor apparent kinetic efficiency of AR with glucose suggests
that the enzyme is maximally active only when intracellular
hexose concentrations rise to abnormally high levels. Major
endogenous substrates linked to metabolic diseases are
glucose and galactose, which are converted to sorbitol and
galactitol, respectively. Reactive sugars and trioses derived
from aldohexoses, including methylglyoxal, glucosone and
deoxyglucosone are excellent substrates for AR [57]. In
diabetic animals, sorbitol production correlates with tissue
levels of AR. Low levels of sorbitol accumulate in the
diabetic mouse lens, which contains low levels of AR [58].
Rat and gerbil lenses, which are endowed with an abundance
of AR, accumulate pathological levels of polyols when
exposed to higher sugar levels [59].
C. Physiological Significance of AR
Current evidence suggests that AR contributes to
metabolic imbalances associated with diabetes and its
complications in the eye and peripheral nerves system [60].
While it is generally accepted that AR-mediated
pathogenesis is dependent on chronically elevated ambient
hexose levels as in diabetes mellitus and galactosemia.
However, the beneficial role(s) of AR in the cells when
hexose levels are normal are still under investigations [61].
AR gene expression is widespread, as evidenced by the
presence of gene transcripts in a large number of human
Aldose Reductase in the Retina
tissues. It is suggested that the enzyme might function
physiologically as a general housekeeping enzyme under
normal conditions. In addition, new evidence points to a
potential role for AR in cytokine-mediating signaling
processes.
1. Osmoregulatory Role
The kidney is one of the richest tissue sources of AR,
with most of the enzyme localized in the medullary portion
[62] from which quantities of the enzyme are isolated for
biochemical studies [63]. Sorbitol is one of the organic
osmolytes that balance the osmotic pressure of extracellular
Nacl, fluctuating in accord once with urine osmolality [64].
These findings therefore suggest the osmoregulatory role of
AR in the renal homeostasis. The functional consequence of
AR expression in kidney medulla was made clear when it
was discovered that exposure of a line of renal papillary
epithelial cells to increased extracellular osmolarity
stimulated an increase in intracellular sorbitol [65]. In
addition, ablation of AR gene in mice results in defect in
kidney function. The mice become unable to concentrate
their urine to normal level [62]. However, it is not clear why
the absence of AR leads to a defect in water resorption, as
sorbitol constitutes only 2% of the total osmolality of
mouse kidney [66]. No such phenotype has been reported
when animal models from other species (rat) are treated with
AR inhibitors. This suggests that either AR inhibitors are
not sufficiently effective to reduce sorbitol levels enough to
cause a defect in urinary concentration, or that other
mechanisms, not present in the mouse kidney are able to
functionally compensate for the loss of AR activity.
The increased expression of AR under hyper-osmotic
stress was subsequently reported in a variety of cells of nonrenal origin, such as Chinese hamster ovary cells [67],
cultured human retinal pigment epithelial cells [68], and
human embryonic epithelial cells [69]. Transient transfection
studies with chloramphenicol acetyltransferase reporter
constructs, containing various 5'-flanking regions of aldose
reductase gene, identified the osmotic response element
mediating this hyperosmotic stress-induced increase in the
transcription of AR gene [70-72]. Studies on the factors that
interact with these response elements and augment the
transcription of AR gene may provide insight into the
regulatory mechanisms of the gene expression.
2. Detoxification
While AR is thought to play a major role in the
synthesis of sorbitol as an osmolyte in the kidney medulla,
its distribution among tissues unaffected by extracellular
osmotic stress suggests an alternate metabolic role. In
addition, the marked hydrophobic nature of the active site is
unusual for an enzyme thought to be involved in the
metabolism of aldo- sugars. It has been demonstrated that
hydrophobic substrate is the catalytic preference of AR.
Srivastava et al. [73] indicated that AR reduced lipid
peroxidation-derived aldehydes as well as their glutathione
conjugates. The role of AR in metabolizing products of lipid
peroxidation or their metabolites is further supported by the
observations that the expression of AR is enhanced under
conditions of oxidative stress both in cell culture system and
in vivo [53] and that the inhibition of AR increases the
accumulation of lipid peroxidation products during
Current Enzyme Inhibition, 2007, Vol. 3, No. 1 5
inflammation and ischemia [74]. Additionally, it has been
shown that cells resistant to oxidative stress express higher
levels of AR than the corresponding sensitive cells and that
the inhibition of AR increases the sensitivity of these cells
to cytotoxic aldehydes [75]. This view is further reinforced
by the observation that several structurally different
phospholipids aldehydes are efficiently reduced by the
enzyme AR, suggesting that AR may be an important
component of mechanisms that remove and detoxify these
aldehydes when they are generated in oxidized lipids [76]. It
could be concluded that AR fulfills a role as an oxidative
defense protein.
In a series of aldehyde substrates for human AR
investigated isocorticosteroids [77] and isocaproaldehyde
[78] both with Km values of approximately 1 µM or less,
they are the best physiological substrates known to date. The
next preferred substrates for AR may be aldehydes derived
from biogenic amines [79] and methylglyoxal, a toxic
aldehyde produced nonenzymatically from triose phosphate
and enzymatically from acetone/acetol metabolism [80].
17α-hydroxyprogesterone [81] and 4-hydroxynonenal [82], a
reactive aldehyde produced by oxidative damage to
unsaturated fatty acids, are also excellent substrates for the
enzyme with Km values of 20–30 µM. Another line of study
demonstrated that 3-deoxy-glucosone, one of the crosslinking agents formed as intermediates in non-enzymatic
glycation, is a good substrate for AR [83].
Aldose reductase also catalyzes the reduction of acrolein,
a highly reactive and mutagenic molecule generated during
lipid peroxidation and as a metabolic by-product of
cyclophosphamide [84]. Both 3-deoxyglucosone and acrolein
exhibited a similar range of Km values (40-80 µM) in the
kinetic analysis [85]. Whereas glucose is one of the
endogenous substrates for AR, comparison with other
endogenous aldehydes indicates that glucose is a rather poor
substrate with a Km value of 70 mM [86]. The interpretation
of these findings is that AR in the adrenal gland and
reproductive organs may normally participate in the
synthesis and catabolism of steroids hormones, whereas it is
involved in the metabolism of biogenic amines in the central
nervous system. The enzyme may also act as an extrahepatic
detoxification enzyme in various tissues. Thus, the
significance of AR in the polyol pathway may be quite
limited under non-diabetic conditions: it provides an
osomolyte sorbitol in the renal medulla and supplies
fructose as an energy source of sperm in the seminal vesicle.
3. Unique Tissue Distribution Pattern of AR
Recent investigations disclosed the unexpected
distribution pattern of AR not only in different species but
in tissues other than “target” organs of diabetic
complications. In human tissues, it is quite abundant in the
epithelial cells lining the collecting tubules in the renal
medulla. Additionally, AR is also found in many other
tissues such as seminal vesicles, retina, lens and muscle
[87]. In mouse, AR mRNA was most abundantly expressed
in the testis, whereas a very low level of the transcript was
detected in the sciatic nerve and lens [88]. These results
suggest that mouse AR may possess a significant role in the
testicular metabolism. On the other hand, the low expression
of the enzyme in the nerve and lens was in marked contrast
with the findings in rat, which indicated the localization of
6 Current Enzyme Inhibition, 2007, Vol. 3, No. 1
the enzyme transcript in these “target” organs of diabetic
complications [21]. Consistent with these findings is the
absence of cataract formation during the course of
hyperglycemia in mouse [58], in contrast with the finding in
rat, the first experimental model of sugar cataract formation
[1]. Immunoblot and immunohistochemical analyses in rat
tissues further showed high levels of AR protein in the
adrenal gland and various reproductive organs, including the
granulosa cells of rat ovary [89]. Of particular interest is the
fact that cyclic changes in the expression and localization of
AR were observed in rat ovary during the estrous cycle [90].
These changes in the enzyme expression were indicated to be
under hormonal control, and the study suggests another
functional role of AR in the female reproductive organ,
which can be deranged under diabetic conditions.
D. Aldose Reductase in Glucose Toxicity
Aldose reductase is the first, and rate-limiting enzyme of
the polyol pathway [19]. Under euglycemic conditions, AR
plays a minor role in glucose metabolism, however, during
diabetes, its contribution is significantly enhanced [1].
Increased AR activity by hyperglycemia has been proposed
to greatly influence the development and progression of
secondary diabetic complications (retinopathy, neuropathy
and nephropathy) [51]. Retinal capillary pericytes contain the
enzyme AR and the accumulation of excess sugar alcohol
catalyzed by AR in pericytes, has been linked to their
degradation [4].
1. Effect of Accelerated Polyol Pathway
Increased activity of AR and the polyol pathway has been
suggested to be underlying biochemical cause of secondary
diabetic complications [19]. The induction of osmotic stress
from the excess intracellular accumulation of sugar alcohol
(polyol) can lead to altered membrane permeability and
subsequently to biochemical changes that result in the
initiation of cellular lesion (polyol osmotic theory) [8]. In
addition, it has been shown that transgenic over expression
of AR gene in mice causes sorbitol accumulation in target
tissues following diabetes induction [91]. Moreover, the role
of AR in the pathogenesis of diabetic complications is
further supported by the evidence showing that inhibition of
the enzyme prevents and/or delays the development of
diabetic cataracts, neuropathy, nephropathy and retinopathy
[1]. These observations suggested a major role for polyol
pathway in glucose toxicity proven to be responsible for
secondary diabetic complications. However, in cell culture
studies, hyperglycemia induces progressive resistantce of the
enzyme to inhibition [92]. AR isolated from diabetic
humans or animals is more resistant to pharmacological
inhibition than that from normal euglycemic tissues [46].
In the retina, the localization of AR in retinal
microvessels was demonstrated in various animal species
including human [8, 93, 94]. In hyperglycemia, glucose may
accumulate to supernormal intra-cellular level as a result of
an increased uptake and decreased efflux [95]. The glucose
accumulation in the retina in diabetic patients is greater than
that in normal retina [96]. Enhanced AR activity is a
mechanism for cardinal manifestation of diabetic retinal
microangiopathy common to date in human [6].
Additionally, it has been reported that increased prevalence
Mahmoud Ahmed Mansour
of retinopathy correlates with increased levels of erythrocytes
AR activity [97]. Treatment with AR inhibitors was shown
to prevent capillary basement membrane thickening, the
early structural lesion observed in the retina [98-100].
Recently, it has been showed that human retina expressed
sorbitol dehydrogenase (SDH) activity as indicated by
increased fructose level after 24 and 48 hrs incubation of
retinal cells in high glucose (unpublished observation).
Therefore, fructose is the end product of polyol pathway in
the retina. Because fructose and its metabolites fructose-3phosphate and 3-deoxyglucosone are more potent nonenzymatic glycating agent than glucose, therefore, flux of
glucose through the polyol pathway would increase advanced
glycation end product (AGE) formation [101]. In the ocular
lens, the accumulation of polyol induces hyperosmotic
swelling and deranges the cell membrane, resulting in the
leakage of amino acids and glutathione to provoke cataract
formation [102]. In other “target” organs of diabetic
complications, however, such osmotic stress is currently
considered to play a minor role in the tissue damage.
Instead, depletion of cofactors used in the pathway by
accelerated flux of glucose is postulated to elicit various
metabolic disturbances in those tissues. Under
normoglycemic conditions, polyol pathway accounts for
approximately 3% of glucose utilization [103], whereas more
than 30% of glucose is metabolized through this pathway
under hyperglycemia [104]. The increased flux of glucose
through this pathway and consequent expenditure of
cofactors for AR (NADPH) and sorbitol dehydrogenase
(NAD + ) leads to a redox state change and a cascade of
interrelated metabolic imbalances. Glutathione reductase and
nitric oxide (NO) synthase are substantially affected because
of the depletion of the cofactor NADPH. As glutathione
reductase is an antioxidative enzyme that maintains the level
of tissue glutathione, the overall effect would be the
increased susceptibility to oxidative stress under diabetic
conditions [101]. Indeed, increased susceptibility to H2O 2
along with a reduced level of glutathione was reported in the
endothelial cells cultured in high glucose medium [105].
Similarly, the production of NO from L-arginine by NO
synthase is suppressed resulting from the depletion of
NADPH, thereby reducing the release of NO to elicit
microvascular derangement and the slowing of nerve
conduction [106,107].
2. Aldose Reductase and Other Factors in Glucose
Toxicity
Along with the increased flux of glucose through the
polyol pathway, there are other putative mechanisms that
may take part in the toxic effects of hyperglycemia. Among
the well-documented factors are activation of protein kinase
C [108, 109], enhanced nonenzymatic glycation [110], and
augmentation of oxidative stress [101]. Some of these are
postulated to be correlated with each other. Both increased
AR activity and oxidative stress have been implicated in the
pathogenesis of diabetic complications [111]. The important
role of two mechanisms in diabetic retinopathy is supported
biochemical [112], functional [113] and biological [114]
abnormalities in the retina. Both AR inhibitors and
antioxidants prevent formation of retinal pericyte ghosts and
acellular capillaries [112], increased vascular permeability
[115] and decreased blood flow. Various mechanisms are
postulated to account for augmented oxidative stress in
Aldose Reductase in the Retina
diabetes. A generation of oxygen free radicals was enhanced
because of auto-oxidation of glucose. The protection against
oxidative stress is attenuated because of reduced glutathione
availability and inactivation of superoxide dismutase.
Vascular dysfunction and the resulting derangement in tissue
perfusion under diabetic conditions induce ischemia and
reperfusion process, which further generate oxygen free
radicals [116]. On the other hand, it has been generally
accepted that advanced glycation end product participates in
the production of oxygen free radicals [117]. Inactivation of
superoxide dismutase in diabetes was demonstrated to result
from glycation of the two lysine residues on the enzyme
protein [118]. The polyol pathway may act upon this
enhanced glycation process, supplying a reactive glycation
agent fructose. Reduced glutathione availability under
hyperglycemia is attributed to the accelerated polyol pathway
flux, depleting the cofactor NADPH for glutathione
reductase In this context, most of the putative mechanisms
implicated in the toxic effects of hyperglycemia can be
interrelated to each other and linked to enhanced polyol
pathway activity. Obrosova et al., [119] indicated a major
contribution of AR to high glucose-induced oxidative stress
in retinal epithelial cells and showed that fidarestat (AR
inhibitor) can arrest hyperglycemia-induced reactive oxygen.
Diabetes associated phenomena such as increased retinal
AR activity, oxidative stress and increased vascular
permeability are interrelated. AR triggers the whole cascade
by causing oxidative stress, which in turn leads to the
overexpression of vascular endothelial growth factor (VEGF)
[120] responsible for increased vascular permeability [121].
Indeed, all three components of this cascade have been found
preventable by an AR inhibitors treatment [119].
Activation of protein kinaseC was reported in vascular
smooth muscle and endothelial cells after the exposure to
hyperglycemia. The rise in NADH/NAD+ ratio via enhanced
polyol pathway may also facilitate the diacylglycerol (DAG)
synthesis by increasing the availability of dihydroxyacetone
phosphate as well as favoring its reduction to glycerol 3phosphate, the intermediates of DAG synthesis [122]. The
increased protein kinase C activity attenuates contractile
responses of aortic vascular smooth muscle cells to such
pressor hormones as angiotensin II and arginine vasopressin.
The activation of protein kinase C increases sodium-proton
pump activity that regulates intracellular pH, cell growth,
and differentiation and also augments expression of various
matrix proteins such as fibronectin, and type IV collagen
[109]. All these biochemical changes could be relevant to
diabetes-induced vascular dysfunction. This phenomenon
was demonstrated in several tissues including retina, aorta,
and renal glomeruli. A specific inhibitor for the β isoform of
protein kinase C was shown to ameliorate vascular
dysfunctions in diabetic rat [123].
3. Aldose Reductase and Thickening of Basement
Membrane
Diabetic retinal complications result from retinal
capillaries functional and morphological alterations:
increased permeability to albumin and macromolecules,
vascular dysfunction, loss of pericytes and basement
membrane thickening.
Current Enzyme Inhibition, 2007, Vol. 3, No. 1 7
Capillary basement membrane thickening and
hypertrophy of extravascular matrix are common features of
diabetic microvascular complications. The link between high
plasma glucose levels and tissues damage is due in part to
the formation and accumulation of advanced glycation
endproducts (AGEs) in tissues [124]. AGEs accumulate in
extracellular matrix proteins as a physiological process
during aging [125]. However, this accumulation happens
earlier, and with an accelerated rate in diabetes mellitus than
in non-diabetic individuals [126]. Increased serum and tissue
levels of AGEs due to reduced removal by kidney have been
evidenced in end stage renal failure [127] and are more
important in diabetic than non-diabetic patients [128]. A
highly significant correlation has been shown between the
importance of AGEs deposits and the severity of diabetic
complications [129]. In vitro and In vivo studies have
indicated that AGEs induce irreversible cross-links in longliving matrix structural proteins such as collagen [129].
AGEs are implicated in the basement membrane thickening
through these alterations, via a reduction in susceptibility of
matrix proteins to proteolytic degradation [130]. These
architectural changes also alter the functional properties of
the basement membrane including permeability. Advanced
glycation of proteoglycans induces a decrease in the
electronegative charges [131] and therefore modifies selective
filtration properties of the basement membrane. These
alterations lead to macular edema secondary to the leakage of
macromolecules. Capillary closures are responsible for nonperfused areas (ischemic retinopathy), which induce the
secretion of vascular endothelial growth factor (VEGF) and
the development of neo-vessels (proliferative retinopathy).
Evidence of this role relies on the results of studies
indicating that the deleterious effects of AGEs on retinal
capillary pericytes and endothelial cells are inhibited by
AGEs receptors antibodies.
E. A Potential Target for the Prevention of Diabetic
Complication
Aldose reductase has been implicated in the etiology of
diabetic complications. A variety of compounds have been
observed to inhibit AR. Orally active inhibitors of the
enzyme have been investigated for many years. Although
several of these compounds have progressed to the clinical
level, only one such drug is currently on the market [132].
Due to the limited number of a valuable drug for the
treatment of diabetic complication, a number of rational
approaches for the discovery of AR inhibitors have under
been taken since the determination of the 3-dimensional
structure of the enzyme. There are a variety of structurally
diverse AR inhibitors.
1. Clinical Trials of AR Inhibitors
El-Kabbani et al. [2] showed that the incidence of
retinopathy was significantly influenced by mean blood
glucose levels and study participants prasticing intensive as
compared to conventional glycemic control experienced a
marked reduction in the incidence and progression of
diabetic retinopathy. The protective effect was less noticeable
if intensive control was instituted after some progression
occurred. Similar results were found in the study of
galactosemic dogs, which showed that correction of
8 Current Enzyme Inhibition, 2007, Vol. 3, No. 1
hyperglycemia through removal of dietary galactose did not
stop the progressive appearance of retinal abnormalities
associated with diabetic retinopathy [133]. Taken together,
these studies emphasize that retinopathy followed a long
term pathogenesis and that once initiated, pharmacological
efforts to interfere are far less likely to be successful than
prophylactic treatment prior to onset of early and perhaps,
irreversible tissue changes. Therefore, even if the therapeutic
basis for AR inhibition is valid, efficacy for a given drug
might be impossible to establish if the clinical study design
does not take into consideration known risk factors for
retinopathy such as duration of diabetes, rigor of glycemic
control and existence of early changes in retinal vasculature.
It is encouraging that new inhibitors are becoming available
[134, 119]; Some with novel structures free from the
hydantoin nucleus found in inhibitors such as sorbinil,
better tissues penetration and activity than the carboxylic
acid inhibitors and higher selectivity against AR [135]. A
similar rationale applies for AR inhibitors studies to
establish efficacy toward diabetic neuropathy. In addition to
these potential design flaws in prior clinical trials, failure to
demonstrate inhibitor efficacy may be related to poor
pharmacokinetic profiles of the investigated compounds. For
example, inadequate nerve penetration almost certainly
contributed to the failure of ponalrestat in clinical trials for
diabetic neuropathy. In addition, unexpected toxicity was a
factor leading to the termination of clinical trials of sorbinil
and tolrestat [136]. However, a promising effect of aldose
reductase inhibitor on nerve conduction velocity was
reported. When diabetic patients without any symptomatic
neuropathy were treated with the aldose reductase inhibitor
sorbinil, significant improvement in the conduction velocity
was observed in all three nerves tested: the peroneal motor
nerve, the median motor nerve, and the median sensory
nerve [137]. Subsequently, numerous clinical studies were
carried out to evaluate the efficacy of sorbinil. However, the
major adverse reaction of sorbinil was a hypersensitivity
reaction in the early weeks of therapy, which is similar to
that seen with other hydantoins. The efficacy of another class
of inhibitor, tolrestat, was modest in diabetic patients
already symptomatic of neuropathy [138], although the
progress of mild diabetic autonomic and peripheral
neuropathy could be halted [139]. The only adverse reaction
reported on tolrestat was an increase in serum levels of
alanine aminotransferase or aspartate aminotransferase,
although some patients in the placebo group also exhibited
similar clinical findings during the study [140].
2. Classes of AR Inhibitor
A list of the most c o m m o n l y available inhibitors
contained either a cyclicimide groups, such as
spirohydantoin group or spirosuccinimide group, an acetic
acid moiety. The best known of the spirohydantoincontaining compounds, sorbinil, has been thoroughly
analyzed by structural analysis of the AR and NADPH, and
several clinical studies. Other examples of these groups
include fidarestat and its stereoisomers. There are numerous
compounds that incorporate the acetic acid moiety, and they
include tolrestat, ponalrestat (statil) and zopolrestat. It is
noted that the cyclicimide or acetic acid moieties bind to an
essentially hydrophilic area of the active side of AR, which
contains the Tyr-48, His-110 and Trp-111 residues.
Mahmoud Ahmed Mansour
Another common feature among the various inhibitors is
the presence of one or more aromatic groups, which may
include phthalazinyl group (ponalrestat), a naphthyl group
(tolrestat), a benzothiazole group (zopolrestat), a 2' -thioxo1,3-thiazolan-4-one group (epalrestat) and a halogenated
benzylgroup (ponalrestat). These aromatic groups bind in the
hydrophobic pocket of AR, Trp-111, Phe-122 and Leu-300
residues, either through hydrogen bonding or hydrophobic
contact.
Inhibitors containing the cyclicimide or carboxylic
groups exhibit similar in vitro but different in vivo activities
[141]. The carboxylic acid-containing inhibitors have lower
in vivo activity, which has been attributed to relatively lower
pKa values, thus causing ionization at physiological pH and
an inability to traverse cell membranes. Cyclicimides have
higher pKa and are only partially ionized at physiological
pH, thus being able to pass through cell membranes and
have better pharmacokinetic properties. Sorbinil possessed
all these attributes, but its development as a therapeutic
agent was halted due to hypersensitive reaction [142].
The flavonoids (2-phenyl-4H-1-benzopyran-4-one) are
also good inhibitors of AR [143] but do not contain either
the carboxylic acid or cyclicimides moieties. This class of
inhibitors, both naturally occurring and synthetic, has higher
pKa values than the carboxylic acid [144] and also has
antioxidant properties [145], which prevent 4-hydroxy-2,3trans-nonenal (HNE)-induced cataract formation.
A newer class of AR inhibitors is the phenylsulfonylnitromethanes [146], which exhibited potent activity against
AR and some of which also showed irreversible inhibition.
The departure of the NADP+ from the enzyme is believed
to be the rate-determining step and this is when the current
AR inhibitors inhibit enzyme action. It was shown that the
inhibitors that bind to hydrophobic pocket were better AR
inhibitors. Such knowledge has been utilized in the design
of potent and specific inhibitors of AR. However, modeling
studies have shown that inhibitors specific to AR should
interact with the C-terminal residues by binding to
specificity pocket [41]. The hydrogen-bonding interactions
between the inhibitors and active site residues (Tyr-48, His110, Trp-111) oriented the inhibitor in the active site.
3. Variable Levels of Aldose Reductase in Diabetic
Patients
Substantial variations in the levels of AR expression in
various tissues exist among individuals with or without
diabetes. Marked variability in AR activity was reported for
enzyme preparations isolated from human placentas [86]. AR
purified from erythrocytes exhibited a nearly three-fold
variation in activity among diabetic patients [147]. Such
differences in the activity of aldose reductase may influence
the susceptibility of patients to glucose toxicity via
acceleration of polyol pathway when these individuals are
maintained under equivalent glycemic control. To test this
hypothesis, it is necessary to determine the levels of AR in
numerous diabetic subjects. In the previous studies,
investigators examined variations in AR by isolating the
enzyme from placenta or erythrocytes and assaying its
activity [86,147]. The isolation of the enzyme was necessary
because of the presence of other structurally related members
of aldo-keto reductase family, particularly aldehyde
Aldose Reductase in the Retina
reductase, in crude tissue preparations. These enzymes share
overlapping substrate specificity with AR. A newly
developed immunoassay method using a specific antibody
directed against AR could circumvent such difficulties [148].
The amount of the enzyme determined by the immunoassay
highly correlates with the activity of AR isolated from the
erythrocytes of the same individuals [149]. By using this
assay method, the association between the AR level in the
erythrocyte, and various clinical parameters determined in
patients with non-insulin-dependent diabetes mellitus
(NIDDM) were investigated. Several fold difference in the
erythrocyte enzyme level was depicted among diabetic
patients, whereas no significant difference in the mean
enzyme level was demonstrated between the healthy and
diabetic individuals. The enzyme level did not correlate with
age, duration of diabetes, fasting blood glucose, or
glycosylated hemoglobin (HbA1c) levels, which represent
glycemic control of the patient. However, data obtained from
two different groups of diabetic subjects suggest that a high
level of erythrocyte AR may affect the susceptibility and
prognosis of diabetic retinopathy [8, 150]. In another study
group, 95 NIDDM patients were classified according to the
results of seven nerve function tests, and the association
between the enzyme level and the clinical findings was
investigated [151]. The erythrocyte AR level was
significantly higher in those patients showing overt
neuropathy compared with those without demonstrable
neuropathy. A higher level of AR is one of the independent
risk factors for overt neuropathy. Accordingly, these results
support the hypothesis that a difference in the level of AR is
responsible for the susceptibility of diabetic patients to toxic
effects of glucose. The activity of AR fractionated from the
erythrocytes was reported to be significantly higher in IDDM
patients with complications compared with those showing
no signs of complications [152]. Increased levels of AR
protein were also demonstrated by immunoblot analysis in
the mononuclear cells isolated from IDDM patients with
apparent diabetic complications [153]. The level of AR
expressed in the erythrocyte seems to be stable, as no
apparent alteration in the enzyme level was observed during
the follow-up period of 12 months in the studied patients
[151]. In this study, the enzyme level remained unchanged
irrespective of improved or stably high HbA1c levels during
the follow-up period. These findings indicate that the
expression of the erythrocyte enzyme is unaffected by the
glycemic control of the patients. It can, therefore, be
speculated that different levels of AR observed in diabetic
patients may be genetically determined. To explore this
possibility, two regions on the aldose AR relevant to the
enzyme expression were examined: the promoter region
containing a TATA box [154], and the region containing
identified osmotic response sequences [72]. However, in the
DNA sampled from 700 NIDDM patients with different
enzyme levels in the erythrocyte, there was no change in
either of these regions associated with differences in the
expression of AR levels [155]. Thus, the reason for the
variable expression of AR in human subjects has yet to be
elucidated. The understanding of the mechanisms defining
the expression levels in the targeted tissues may lead to new
avenues of preventive therapy for diabetic complications. A
hypothesis as to whether the high enzyme level predisposes
the patients to the development of complications has to be
further tested by the prospective study carried out through
Current Enzyme Inhibition, 2007, Vol. 3, No. 1 9
the prolonged time course of diabetes. Also to be considered
is the relevance of the AR level in the erythrocyte in
predicting the enzyme level in the “target” tissues of diabetic
complications. Whether a high level of enzyme expression in
the erythrocyte reflects the level in the different cell lineage
has to be determined. It will take some time before all the
data become available; nonetheless, a high level of AR in
the erythrocyte was demonstrated to be a risk factor for
vascular and neural derangement observed in diabetic
patients. Identification of a subset of patients who have a
high level of AR expression, and thereby are more
susceptible to toxic effects of glucose, may enable us to
target these patients for clinical intervention trial by use of
new AR inhibitors. The data on the enzyme levels may also
aid in the optimization of administration of the inhibitors to
match the extent of enzyme suppression when exploring
their efficacy in diabetic individuals.
CONCLUSION
Diabetes mellitus is recognized as a leading cause of new
cases of blindness and is associated with increased risk for
painful neuropathy, heart diseases and kidney failure. Many
theories have been advanced to explain mechanisms leading
to diabetic complications including accelerated protein
glycation, altered signaling involving PKC, excessive
oxidative stress and stimulation of glucose metabolism by
the polyol pathway. It has been demonstrated that polyol
pathway is the major source of diabetes induced oxidative
stress, as AR activity depletes its co-factors NADPH, which
is required for glutathione reductase to regenerate GSH. In
addition to the formation of fructose and its metabolites,
which are potent non-enzymatic glycating agents, AR
increases AGE. Therefore, designing and screening specific
inhibitors for AR is becoming the therapeutic strategies that
have been proposed to delay or prevent diabetic
complications.
The tertiary structure of aldose reductase, including the
active site and the interaction with inhibitors of diverse
chemical structures has been resolved. However, much still
remains to be elucidated regarding the pathophysiological
significance of the enzyme and the regulatory mechanisms of
AR expression in various human tissues. In diabetic animal
models, promising effects of AR inhibitors were
demonstrated. However, most of the clinical trials carried
out so far, produced rather modest or disappointing effects of
the inhibitors on the functional and morphological
improvements in diabetic retinopathy. There could be several
reasons that account for the disparity in the inhibitor effects
observed between animal and clinical studies. Possible
explanations include the chronic nature of diabetes in human
subjects and the ensuing loss of ability to reconstitute the
structural derangement once triggered under hyperglycemia.
Secondly, the relative abundance of the aldo-keto reductase
family, such as aldehyde reductase, which is colocalized in
human tissues and later may interfere with the action of
inhibitors to suppress AR. Lastly, the efficacy of AR
inhibitors may depend on the enzyme level expressed in
diabetic individuals. The variable levels of the enzyme
expressed in the “target” tissues may affect the extent of
involvement of the polyol pathway in the pathogenic
mechanisms of diabetic complications. If multiple
10 Current Enzyme Inhibition, 2007, Vol. 3, No. 1
mechanisms are involved in the pathogenesis of diabetic
complications, the extent of the effectiveness of AR
inhibitors is likely to be determined by the extent of the
polyol pathway involvement in the toxic effects of
hyperglycemia. This extent is likely to be variable among
individuals having different levels of AR in “target” tissues.
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