Role of Amino Acids on Prevention of Lens Proteins Nonenzymatic Glycation In Vitro, in Senile, and Diabetic Cataract

15 Role of Amino Acids on Prevention of Lens Proteins Nonenzymatic Glycation In Vitro, in Senile, and Diabetic Cataract S. Zahra Bathaie*, Fereshteh Bahmani†, Asghar Farajzadeh* * DE PART MENT OF C LINI CAL BI OCHE MIST RY, FACULT Y OF ME DI CAL SC IENCE S, TARBI AT MODARES UNIVERSITY ( TMU), TEHRAN, IRAN † DEPARTMENT OF BIOCHEMISTRY, FACULTY OF MEDICINE, KASHAN UNIVERSITY OF MEDIC AL SCI ENCES , KASHAN, IRAN

CHAPTER OUTLINE Introduction .................................................................................................................................. 246 History and Overview of the Advanced Glycation End-Products ............................................. 246 Glycating Agents .......................................................................................................................... 249 Different Advanced Glycation End-Products .............................................................................. 249 Advanced Glycation End-Products Formation in the Lens ........................................................ 250 Effects of Advanced Glycation End-Products on Proteins Function ......................................... 251 Direct Effect ..............................................................................................................................251 Indirect Effect ...........................................................................................................................255 Biological Detoxification of Advanced Glycation End-Products ............................................... 255 Prevention/Inhibition of Advanced Glycation End-Products Formation .................................. 256 Inhibitory Effects of Amino Acids on Formation of Advanced Glycation End-Products and Cataract .......................................................................................................... 259 In Vitro Studies .........................................................................................................................260 In Vivo Studies ..........................................................................................................................262 Summary Points ........................................................................................................................... 265 References .................................................................................................................................... 266

List of Abbreviation 3-DG AFGP AG AGEs Ala ALI

3-deoxyglucosone alkyl formyl glycosyl pyrrole aminoguanidine advanced glycation end-products alanine arginine-lysine imidazole

Handbook of Nutrition, Diet, and the Eye. https://doi.org/10.1016/B978-0-12-815245-4.00015-6 © 2019 Elsevier Inc. All rights reserved.

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AR Arg Asp CAT CEL CML FL FRAP Fru Glc Glu Gly GLYT1 GLYT2 GO GOLD GSH Lys MGO MOLD RAGEs ROS SDH SOD STZ α-KG α-LA

aldose reductase arginine aspartic acid catalase carboxyethyl-lysine N-carboxymethyl-lysine N-fructosyl-lysine ferric reducing antioxidant power fructose glucose glutamic acid glycine Gly transporters 1 Gly transporters 2 glyoxal glyoxal lysine dimmer glutathione lysine methylglyoxal methylglyoxal lysine dimmer receptor for advanced glycation end-products reactive oxygen species sorbitol dehydrogenase superoxide dismutase streptozotocin alfa-ketoglutarate alfa-lipoic acid

Introduction Application of a group of therapeutic disciplines together with the conventional medicine has been known as a complementary therapy, which can be used for many diseases including diabetes. Complementary therapy can increase the quality of life of patients with chronic diseases, prevent the development or recurrent of the disease, decrease the side effects of the conventional treatments, and/or help the patients to cope better with symptoms caused by a chronic disease. Among these treatments, amino acid therapy has attracted more attention because amino acids familiar to the body and metabolize, easily. This chapter focuses on the use of amino acids in the inhibition of nonenzymatic glycation as the main cause of diabetic complications, and their application to prevent the diabetic cataract development.

History and Overview of the Advanced Glycation End-Products Louis-Camille Maillard (February 4, 1878 to May 12, 1936), the French scientist, undertook studies of the reaction between amino acids and sugars, and his first paper was published in 1912 (Compt. Rend. 1912, 154, 66. Cited in: http://cen.acs.org/articles/90/i40/MaillardReaction-Turns-100.html). He explained the principles of browning phenomena in meat

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that has been exposed to the air for a long-term. This cascade of reactions has been named “the Maillard reaction.” Further studies on this pathway showed the Maillard reaction as an important phenomenon not only in in food and beverage, but also in paper, textile, biopharmaceutical, and even in soil. After 1970s, substantial attention has been given to the Maillard reaction in the in vivo condition.1,2 Then extensive studies on its chemistry under physiological conditions were carried out. The Maillard reaction or nonenzymatic glycation of amino acids and proteins (Fig. 1) is initiated by a nucleophilic addition reaction. In this reaction, a free amine group of an amino acid (such as Arg/Lys in a protein structure or a free amino acid in the medium) acts as a nucleophile, attacking carbonyl group of a reducing sugar, and forms a Schiff

FIG. 1 Glycation of a protein by a sugar and subsequently, AGEs formation. The initial interaction between a reducing sugar (such as glucose) and free amino groups of the proteins results in the formation of a Schiff base. This reaction is reversible. However, in the presence of excess glucose, fructose, etc., the products rearrange to a stable product named Amadori product or ketoamine. With time, these Amadori products directly or via dicarbonyl intermediates (such as 3-DG) interact with other proteins in the medium to form AGEs/ more complex products.

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base. Although this reaction is fast, it is reversible and its rate depends on the concentration of available sugar. Therefore, by lowering the sugar concentration, possibility of degradation of the unstable product is increased. A Schiff base can undergo further rearrangement, through an irreversible process, to form a more stable Amadori product, a ketoamine. In the suitable condition such as high sugar concentration, Amadori products accumulate over time and can undergo additional complex rearrangements giving rise to different types of advanced glycation end-products (AGEs) that are shown in Fig. 2.

FIG. 2 Chemical structure of different types of AGEs. (Up) Fluorescent crosslinking AGEs such as vesperlysine, pentosidine, and crossline. (Middle) Nonfluorescent crosslinking AGEs such as IDL: imadazolium dilysine crosslinks; AFGP: alkyl formyl glycosyl pyrroles; and ALI: arginine-lysine imidazole crosslinks. (Down) Noncrosslinking AGEs such as N-fructosyl-lysine; CEL: N-carboxyethyl-lysine; and CML: N-carboxymethyllysine.

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Glycating Agents In reality, all reducing sugars such as glucose (Glc), fructose (Fru), and certain sugar derivatives, such as ascorbic acid, can initiate the Maillard reaction in vivo. However, because of the slow rate of the reaction of Glc with proteins and its high extracellular concentration in the diabetic patients, it has been thought that AGEs only form at long-lived and/or extracellular proteins. Nevertheless, further studies showed that the short-lived proteins, intracellular, and even the nuclear proteins also can be a target of glycation.3,4 Type of sugar, in addition to the sugar concentration and half-life of biomacromolecules, influences the process of glycation. The rate of glycation is also directly proportional to the percentage of sugar in the open-chain form.5 It has been shown that Glc in the solution is 0.002% in the open-chain form at 20°C, but glyceraldehyde-3-phosphate (an intermediate in the glycolysis) is 100% in the open-chain form.6 Therefore, the latter produces over 200-fold more glycated hemoglobin (HbA1b) than the first, after 72 h incubation.7 In addition, the rate of human serum albumin glycation by Fru has been ten-fold more than Glc, in vitro.8 Thus, precursors other than Glc, such as Glc-6-phosphate, Fru, glyceraldehyde, dihydroxy-acetone-phosphate, glyceraldehyde-3-phosphate, and the dicarbonyl compounds like glyoxal (GO), methylglyoxal (MGO), and 3-deoxyglucosone (3-DG), are of great importance to intracellular Maillard reaction. Because the accumulation of these reactive glycolytic intermediates increases in some metabolic situation in the cell, they have an important role in the in vivo AGEs formation.7–9 In some organs such as ocular lens, Fru is produced through oxidation of sorbitol in an enzymatic reaction catalyzed by sorbitol dehydrogenase (SDH). This pathway, polyol pathway, increases the concentrations of Fru in the same order of magnitude to Glc. Thus, the probability of in vivo glycation by Fru is increased.9,10 In addition, if the dietary intake of Fru is increased, its level is markedly elevated in the lenses. Therefore, up to 23-fold increase in Fru concentration has been reported in the lenses of diabetic patients, which is twice of Glc concentration. Previous in vivo studies on the endogenous glycation reaction by Fru have shown that 10 to 20% of the sugar moieties binds to human ocular lens proteins via carbon C-2.10 It has also been reported that AGEs production can be facilitated by some exogenous compounds derived from foods, tobacco, etc.11 Therefore, AGEs formation progressively increases with age, even in the absence of diseases like diabetes.

Different Advanced Glycation End-Products As mentioned, AGEs are complex, heterogeneous molecules that are produced by crosslinking of proteins and result in the misfolding/malfolding of proteins. This process has been known as browning. Various types of AGEs are identified (Fig. 2), and based on their chemical structure and ability to emit fluorescence can be divided into three main categories. These are as follows: (1) The crosslinking AGEs with fluorescence emission, such as pentosidine, crossline, vesper lysine A, glyoxal lysine dimmer (GOLD), methylglyoxal lysine dimmer (MOLD), and glucosepane.

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(2) The nonfluorescent crosslinking AGEs, such as imidazolium dilysine (IDL), alkyl formyl glycosyl pyrrole (AFGP), and arginine lysine imidazole (ALI) crosslinks. (3) Noncrosslinking and nontoxic AGEs such as pyrraline, N-carboxymethyllysine (CML), N-carboxyethyl-lysine (CEL), and N-fructosyl-lysine (FL).12 AGEs derived from glycolaldehyde or glyceraldehyde are toxic.13 Thus, AGES can also be divided into toxic and nontoxic compounds. As Fig. 2 depicts, the majority of AGEs (e.g., pentosidine, glucosepane, MOLD, GOLD, and crossline) are formed on the free amines of Lys and Arg residues of proteins; however, the N-terminal of proteins is also a candidate for glycation. Such event is happening in the N-terminal valine of hemoglobin β-chain and result in the formation of HbA1c.14 Cysteine is also a candidate for glycation in some proteins.15 The role of different types of AGEs in the pathogenesis of diabetic complications has been extensively studied. The presence of some inconsistencies and contradictions in the reported results increase the complexity of the mechanisms. For example, the serum levels of CML have increased in diabetic patients with retinopathy, but it has not changed in nephropathy. On the other hand, the level of pentosidine has increased in both groups.16 Therefore, to clarify the mechanism(s) involved in production of different glycation products at different situations, there is still something that needs to be discovered. In addition to aging and diabetes, AGEs have been introduced as the markers and causative factors for pathogenesis of neurodegenerative diseases, such as Alzheimer’s and Parkinson’s diseases.12,17,18 It has been shown that AGEs formation under physiological condition is extremely related to the half-life of proteins. So, AGEs accumulate prominently in the long-lived proteins such as lens crystallins.19

Advanced Glycation End-Products Formation in the Lens The pathophysiology behind senile cataracts is complex and depends on several factors. Normal aging is accompanied by a progressive increase of AGEs in lens proteins. Advanced glycation occurs during normal aging but to a greater degree in diabetes. Thus, diabetes is considered as a major risk factor for the development of cataract, not only for the nonenzymatic glycation of lens proteins, but also for oxidative stress, and activated polyol pathway.20 The extent of protein glycation in lens fiber cells has been estimated to be up to 10-fold higher than other tissues with usual protein turnover.21 Some of the most important reasons are: (1) Glc uptake in the lens does not depend on insulin, thus is constantly exposed to high concentrations of this sugar, especially in diabetes. (2) Lens crystallins are structural proteins that have little or no turnover; therefore, are particularly vulnerable to glycation.22 (3) Those substantial modifications of lens proteins may stimulate further glycation, oxidation, and consequently formation of water insoluble and high-molecular-weight aggregates.

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Nowadays, the process of lens opacification is completely known and it has been found that coloration of the human lens in certain types of cataracts is also related to the formation of AGEs and accumulation of various Amadori products on lens proteins.22–24 Various structurally characterized AGEs in the lenses are shown in Fig. 3. It has been shown that cataractous lenses contain significantly higher levels of GOLD, MOLD, and Methylglyoxal hydroimidazolones compared with the normal lenses from age-matched subjects. The increase in the serum pentosidine, argpyrimidine, GOLD, and MOLD concentrations has been also reported in the diabetic patients.25

Effects of Advanced Glycation End-Products on Proteins Function Crosslinked species accumulate in different tissues such as the cataractous lens, cornea, skin, smooth muscle, and vascular collagen both as a consequence of diabetes and normal aging, thus affecting the whole function of the organ. Alterations in the organ function are induced both directly, due to the changes in the structure and function of proteins; or indirectly, due to the binding of AGEs to AGE receptors (RAGEs) on the cell surface.

Direct Effect As mentioned, increased nonenzymatic glycation of proteins in the presence of reducing sugars and building up the AGEs alter the proteins structure and folding, which result in the changes in their function. Proteins have different functions, including enzymatic activity, ligand binding, transport of other proteins or ligands, DNA binding activity, and so on. Any changes in the protein structure may affect their activities (Fig. 4A), modify protein half-life, and alter immunogenicity. The results of our recent studies have indicated the named changes in the structure and function of various proteins from different locations. For example, serum proteins like albumin26,27 and HSP70,28 plasma proteins like fibrinogen,29 extracellular proteins like lysozyme,30 cytosolic proteins like crystallins,31–35 and even nuclear proteins like histone H14 are the substrates for glycation reaction and AGE formation. As a result of glycation, many proteins lose their activity,4,26,28,36 but activity of a few proteins like fibrinogen is increased.29 The increased activity of fibrinogen also results in an increase in clot formation, which is the reason for higher chance of atherosclerosis in diabetic patients.29 Loss of activity of the molecular chaperones (e.g., HSP7028 and α-crystallin31) results in the misfolding and alteration in the function of other proteins, which in turn affect many other proteins and systems in the body. Another example of the effect of glycation on decreasing the protein activity is Na+-K+ ATPase, which results in alteration of the intracellular ion concentration and subsequent water movement via osmosis, after in vitro glycation.37 Such an in vivo effect may contribute toward cataract formation in diabetes. The impaired esterase activity of MGO-modified serum albumin compared to unmodified albumin is another example. Moreover, cysteine proteases like

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FIG. 3 Advanced glycation end-products in diabetic and ageing lenses. Various AGEs found in lenses due to aging and diabetes. For details see the text. MG-H1, MG-H2, and MG-H3: hydroimidazolones isomers 1, 2, and 3, respectively; K2P: 1-(5-amino-5-carboxypentyl)-4-(5-amino-5-carboxypentyl-amino)-3-hydroxy-2, 3-dihydropyridinium; OP-Lys: 2-ammonio-6-(3-oxidopyridinium-1-yl)hexanoate. Other abbreviations are defined in the Abbreviation list.

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FIG. 4 Overview of cellular AGE interactions and functions. (A) Changing in protein function due to the glycation modification. For example, enzyme-substrate interaction in normal condition (up) is disrupted after glycation of the enzyme (down). (B) AGE formation due to the crosslinking of glycated proteins leads to accumulation of AGEs and tissue rigidity. (C) Interaction of AGE with its membrane bound receptor (RAGE) results in the activation of some signaling pathways and induction of the expression of some genes to produce proteins like TNF-α (Tumor necrosis factor alfa) and IL-6 (Interleukin-6), as the inflammatory mediators. Continued

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FIG. 4, CONT’D (D) Inducing the formation of free radicals; Production of reducing equivalents in the mitochondrial matrix during normal catabolism of Glc that followed by the appearance of superoxide anion (O2 °) and its subsequent elimination by the action of superoxide dismutase (SOD) and catalase (CAT) as the antioxidant defense system. In diabetic condition, due to the glycation and inactivation of the named enzymes and alteration in the activity of complex III of the respiratory chain, free radicals are accumulated and induce the cell damage.

cathepsins are inhibited by MGO modification at the cysteines of the active site. The glycation of low-density lipoprotein (LDL) reduces their uptake by their normal receptors and glycation of superoxide dismutase increases reactive oxygen species generation and amplifies the oxidative stress.12 Glycated histone H1 that was extracted and purified from the liver of diabetic rats has also showed a lower binding affinity to DNA, compared with the histone H1 separated from normal rat liver.4 This alteration can in order lead to the changes in the expression of some genes. Glycoxidatively modified proteins, like fibronectin, can form large aggregates in the extracellular space (Fig. 4B). Crosslinks formed between extracellular matrix components are known to affect cell-matrix interactions, impair matrix assembly, reduce protein turnover (possibly due to a decreased proteolytic digestibility of glycated proteins or altered proteolytic enzyme activity), and increase arterial and myocardial stiffness.38,39 The reduced activity of glycated lysozyme in this compartment can also reduce its function as the innate immune system and result in the inflammation in the extracellular fluid.30 As mentioned, the long-lived structural proteins like lens crystallins are the most important target for nonenzymatic glycation associated with aging and complications of diabetes. Crystallins largely determine the transparency of the eye lens. The extent of glycation of a variety of proteins, including lens crystallins and lens capsule, is approximately two times more in diabetes and aging process. It has been reported that the extent of glycation, AGE formation, and appearance of yellow color aggregates increase with the severity of diabetic complications.40 α-Crystallin, a member of the small heat shock

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protein family, is a major structural element in the protein matrix of the vertebrate eye lens. The chaperoning ability of α-crystallin is believed to be essential for the maintenance of transparency of the lens, thus preventing the formation of cataract.41 The in vitro glycation of α-crystallin disrupted its structural stability, resulting in decreased chaperone activity, similar to that seen in the in vivo studies.36,42

Indirect Effect AGE-RAGE interaction (Fig. 4C) initiates the cascade of activation reactions and signal transduction pathways. Other major features of AGEs-RAGE complex formations relate to their endocytosis, degradation, pro-oxidant, and pro-inflammatory events. Recent studies suggest that interaction of AGEs with their receptors alters all the named processes, including intracellular signaling, expression of some genes, release of free radicals, and proinflammatory molecules that contribute toward the pathology of diabetic complications.43–47 RAGEs are members of the immunoglobulin receptor family and bind several ligands in addition to AGEs, such as HMG-1, S-100 proteins, or β-amyloid peptide. Binding of ligands to RAGEs results in activation of NADPH-oxidase that lead to an increased production of reactive oxygen species (ROS). Furthermore, activation of signaling pathways including ERK, p38/MAPK, JAK/STAT-pathway, rho-GTPases, and phosphoinositol 3-kinase (PI3K) is linked to RAGE activation. One major downstream target of RAGE is the proinflammatory NF-κB pathway, which in turn leads to elevated RAGE expression and perpetuation of the cellular inflammatory state. In addition to RAGEs, other binding proteins have also been described, including oligosaccharyl transferase (OST48, AGE-R1), 80K-H phosphoprotein (AGE-R2), galectin-3 (AGE-R3), CD36, and scavenger receptors II-a and II-b. Taken together, several AGE-binding molecules are involved in binding, signaling, and degradation of AGE. The AGE-mediated effects will therefore depend on the occurrence of these receptors on the individual cell type.43–49 Moreover, exogenous dietary AGEs that were formed from cooked food can enter blood circulation; however, their contribution to pathophysiological processes is under discussion.50 Production of free radicals and oxidative stress is another consequence of AGEs formation (Fig. 4D). Glycation-derived free radicals can cause protein fragmentation and oxidation of nucleic acids and lipids,12 which in order cause some complications including cell apoptosis.

Biological Detoxification of Advanced Glycation End-Products Available data suggest that AGEs are eliminated from the blood mainly by scavenger receptor-mediated uptake in Kupffer cells and liver sinusoidal endothelial cells.51 The liver enzymes like α-ketogluteraldehyde dehydrogenase are capable of inactivating 3-DG and

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preventing AGE formation. The important role of kidneys to eliminate AGEs has also been suggested.51 Macrophages possess receptors enabling them to recognize and remove harmful AGE-proteins through endocytosis, which is associated with the role of lysozyme in the extracellular fluid. A variety of plasma amines, like spermine, may also react with sugar and Amadori carbonyl groups to reduce AGEs.52 Antioxidants can also protect against glycation-derived free radicals.53 The “detoxifying” enzymes such as glutathione-dependent glyoxalase complex (formed from glyoxalase I and glyoxalase II components) act as an effective detoxification system for GO and MGO.54,55 The enzyme complex catalyzes conversion of MGO to s-D-lactoyl-glutathione, then it is subsequently converted to D-lactate by glyoxalase II. In oxidative situations associated with low glutathione concentrations, the antiglycation defense mediated by the glyoxalase system is insufficient; this establishes link between oxidative stress and glycation. Cells that overexpress this enzyme show less accumulation of MGO-derived AGEs.27,55 Glyoxalase-I enzymatic activity becomes progressively impaired with the aging process; this contributes to an accelerated AGE accumulation with aging.56

Prevention/Inhibition of Advanced Glycation End-Products Formation A major goal in the diabetes treatment is controlling the blood glucose level. Although diet is very important, it is not easy to control for a diabetic patient. Thus, drug interventions are also taking place.53,57,58 In addition, other treatments that prevent/ inhibit AGEs formation are also considered. Complement therapy using natural products, phytochemicals,35,59 hot tub therapy28,60,61 , and so on are some examples of such treatments, which have been used from ancient times. The main reason of diabetic complications is glycation of biomacromolecules, especially proteins. Therefore, protection of proteins against glycation either by prevention or by dealing with the consequences of glycation has been considered. Since glycation and AGE formation is a spontaneous process and does not require an enzyme (nonenzymatic), the suggested inhibitors with a long half-life, no toxicity, no immune reaction, and preferably familiar with the physiological condition are the best. However, compounds with all of these characteristics are not abundant. Fig. 5 shows various stages of protein glycation reaction and AGE formation, emphasizing the inhibitor(s) used at each stage. Additionally, this figure indicates the mechanism of action of these compounds that are considered as therapeutic agent/complement in diabetes. Here, we summarize the effects of these compounds. (1) Inhibitors of glucose biosynthesis. Metformin (or N,N-dimethylimidodicarbonimidic diamide, a biguanine compound) activates AMP-activated protein kinase (AMPK), which is an enzyme that plays an important role in insulin signaling, whole body

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FIG. 5 Schematic representation of the sites of action of antidiabetic compounds to inhibit protein glycation and AGE formation. Each of the named compounds can affect more than one step in the process. Inhibition of endogenous reducing sugars production is an important strategy for diabetes treatment and prevention. While some compounds potentiate glucose entry to the cells or increase its metabolism, breaking the AGEs or interfere with its interaction with receptor is another strategy. For more information, please see the text. “a” indicates the direct production of an Amadori product from a Schiff base in a reversible reaction, while “b” represents formation of a dicarbonyl intermediate from a Schiff base in an irreversible reaction. Dotted arrows represent the multiple steps in the mentioned reaction.

energy balance, and metabolism of glucose and fats; therefore inhibits the biosynthesis of glucose, gluconeogenesis, by liver cells.62 (2) Blockers of free amino groups on protein structure, in both side chain and/or N-terminal region, prevent the nucleophilic addition reaction. They can prevent the glycation reaction through covering the active amine groups. One of the famous examples of this group of materials is aspirin (acetyl salicylic acid) that can acetylate the free amine groups especially epsilon amine in Lys residue of proteins. Both in vivo

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and in vitro results have shown the inhibition of protein (like α-crystallin and albumin) glycation by aspirin.53,63–65 This mechanism is distinct from the antiinflammatory effect of other nonsteroid antiinflammatory drugs (NSAIDS). (3) Blockers of the carbonyl groups on reducing sugars, Amadori products and dicarbonyl intermediates (3-DG, MGO, etc.) effectively reduce glycation and AGE formation. Compounds such as aminoguanidine or pimagedine reduce level of AGEs through interacting with 3-DG and thus make it unavailable to protein amino groups. Aminoguanidine slows down the progression of lens opacification in moderately diabetic rats.66 This drug is capable of protecting skin proteins (elastin and collagen), nerves, eye lens, and kidney proteins from crosslinking; however, its toxic effect on higher doses should also be considered. Carnosine (beta-alanyl-L-histidine) that can react with sugars and prevent their interaction with proteins26,58 can also be included in this group of antiglycating compounds. Free amino acids (like Lys and Gly) and other amino containing compounds (like polyamines) also fall in this category of materials that will be discussed in detail in the following section. (4) Chemical chaperones from polyols family (like glycerol and inositol), which stabilize protein structure and prevent their modifications and conformational changes, are also considered as another group of antiglycating compounds. (5) Antioxidants can protect cells and proteins against free radicals derived via autoxidative glycation, glycoxidation, and AGE compounds. Amino acids like Lys,26 dipeptides like carnosine or N-acetyl carnosine,67 and some drugs like aspirin53 can also act through this mechanism. Carnosine has attracted much attention as a natural antioxidant and transition-metal ion sequestering agent. In addition, carnosine can nonenzymatically react with detrimental hexoses, pentoses, and trioses and protect proteins, such as α-crystallin, against glycation. Therefore, it can decrease crosslinking induced by sugars, diminish the modification of α-crystallin, and disaggregate glycated α-crystallin.68 Some studies have indicated alfa-lipoic acid (α-LA), a naturally occurring dithiol compound, is an antioxidant that acts through direct radical scavenging and metal chelating, interacting with other antioxidants, and increasing the reduced intracellular glutathione and vitamin C levels. Studies have showed that α-LA facilitates nonoxidative and oxidative Glc metabolism and increases Glc uptake leading to improved Glc utilization both in vitro and in vivo. Other reports have also indicated that α-LA may prevent protein glycation in cultured lens under hyperglycemic conditions.69 The ability of α-LA to prevent cataractogenesis has also been demonstrated in vitro in rat lens cell cultures exposed to high concentrations of Glc, and also in vivo in an oxidative stress model of cataract, as well as in nutritionally induced type 2 diabetic rats.70 Its biosynthesis decreases as people age and is reduced in people with compromised health. These powerful hypoglycemic and antioxidant effects led to the use of α-LA supplementation in treatment of diabetic cataract and neuropathy.71

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(6) Amadoriases has been known as enzymes that deglycate Amadori products or inactivate intermediates such as 3-DG.72 Some amadoriases function as fructosamine oxidases, which convert fructosyllysine to Lys, hydrogen peroxide, and glucosone. Another class of enzymes that destabilize the glycated proteins is fructosamine 3-kinase, which phosphorylates the Amadori compounds at C3, producing a fructosamine 3-phosphate residue on protein, which then decomposes spontaneously to release phosphate and 3-DG.73 (7) AGE-crosslink breakers like alagebrium and N-phenacylthiazolium bromide offer the potential of reversing diabetic complications, especially cardiovascular disease. It was postulated that alagebrium reverses the stiffening of blood vessel walls, thus is effective in reducing blood pressure and providing therapeutic effect for patients with diastolic heart failure.33 (8) RAGE regulators that prevent or inhibit progression of diabetic complications through inhibition of AGE-RAGE interaction.74 RAGE blockers could prevent the consequences of AGEs-RAGE complex formation and suppress the cellular and inflammatory changes associated with the development of diabetic complications. It has also been shown that after cleavage of RAGE from membrane the soluble RAGE (sRAGE) is formed that can bind to AGEs and work as decoy receptors against ligand-RAGE interaction.45 (9) Chelators of transition metals, such as EDTA that reduce the glycation-derived free radicals. But, in fact, their complete removal may be undesirable and cause distortion of the reactions needing trace elements. In addition, application of these compounds in the in vivo condition is under question. (10) Antibodies may be used to block Amadori products or RAGEs. The specificity of this approach is more than other methods that recognize carbonyl groups; however, antibody therapy is accompanied with some limitation that affects its ordinary use. Fig. 6 shows the structure of some of the mentioned compounds in this section.

Inhibitory Effects of Amino Acids on Formation of Advanced Glycation End-Products and Cataract For the first time, in 1961, Reddy et al. had shown that some amino acids can be actively transported from plasma into the posterior aqueous humor and from there into the lens.75 Therefore, amino acids have role in lens and any distortion in the transfer system results in some eye diseases. On the other hand, disorders due to the deficiency in these transfer systems can be compensated by administration of amino acids. By these hypotheses, several studies have been done on the distribution of amino acids in eye and the beneficial effect of their free form on eye diseases like cataract.76–78 Both in vitro and in vivo studies show antiglycating effects of free amino acids on lens proteins. High solubility of some amino acids especially Gly in comparison with other

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FIG. 6 Chemical structures of some of the named antiglycating compounds in Fig. 5.

anticataract agents is a very important aspect of their application.79 Some examples of these studies are presented here.

In Vitro Studies Antiglycating effects of several amino acids including Lys, Gly, Ala, Glu, and Asp on lens proteins have been reported.36,78,80,81 It has been shown that varying concentrations of Lys and Gly can significantly reduce the extent of glycation of lens proteins in Glc-treated homogenates of normal humans and goats lens.82 In addition, Ala, Asp, and Glu, which are present in relatively larger amounts in the lens, have been found to undergo nonenzymatic glycation and significantly reduce the extent of in vitro glycation of lens proteins in the Glc-treated homogenates of normal human lens.80 Incubation of lens homogenate with galactose has also increased glycation of proteins; however, addition of the mentioned amino acids decreased the glycation of lens proteins.78

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700 Cry 600

Cry+Glc Cry+Glc+Lys

500

FI (AU)

Cry+Glc+Gly 400

300

200

100

0 400

410

420

430

440

450

460

470

Wavelength (nm) FIG. 7 Effect of Gly/Lys treatment on the AGE formation of α-crystallin in the in vitro experiment. The fluorescence intensity (FI) in the arbitrary unit (AU) of the α-crystallin solution in the pure form (Cry), in the presence of glucose (Cry + Glc) and in the presence of Glc with either Gly or Lys (Cry + Glc + Gly or Cry + Glc + Lys), at different wavelengths at the end of the in vitro experiment.

Another study has shown reduced nonenzymatic glycation of lens proteins by acidic amino acids. In this study, lens homogenates of the goat, rat, and human cataractous lens proteins were incubated with different concentrations of Glc, and the effects of Asp and Glu were studied individually. The results indicated that these two amino acids can protect lens proteins from glycation.81 Our in vitro studies using α-crystallin, a purified protein from Cow lenses, showed that incubation of this protein with Glc in the presence or absence of either Lys or Gly, prevents its glycation and conformational change.36 Fig. 7 shows an increase in the fluorescence intensity (FI) of AGEs due to the incubation of α-crystallin with high glucose concentration at about 430 nm. But, in the presence of Gly/ Lys, this value is remained close to the normal value. It means that Gly/ Lys prevents the binding of Glc to α-crystallin and fluorescence AGE formation.31,83 We also showed the inhibitory role of these amino acids on glycation of other proteins like humans or bovine serum albumin.26 Another study, using a set of forty urea/thiourea derivatives of some amino acids, including Phe-, Tyr-, Glu-, and Lys-benzisoxazole hybrids, has indicated the antiglycating activity of these compounds, in vitro. The authors showed the best antiglycation activity

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for a methoxy substituent of Tyr, particularly at the para position.84 Cys has also showed the inhibitory effect on AGE formation both in vitro and in vivo.27

In Vivo Studies Antiglycating effects of Gly and Lys were also investigated in both animal models of diabetes and diabetic patients. Besides being a stimulator of insulin secretion, Gly and Lys showed antidiabetic effect by other mechanisms.29,31,79,85 Diabetic rats treated with Gly showed less enlargement of the glomerular basal membrane than controls. These rats showed a diminution in the microaneurysms in the eyes. In addition, the isolated peripheral blood mononuclear cells from Gly-treated diabetic rats showed a better proliferative response to mitogens phytohemogluttinin or concavalin A, compared with those obtained from nontreated diabetic rats. Gly treated rats had less intense corporal weight loss compared with nontreated animals.79 In another study, Gly diminished hyperglycemia, hypercholesterolemia, and glycated hemoglobin concentrations in diabetic rats. It has been suggested that the effect of Gly may be secondary to its higher solubility, which prevents the formation and precipitation of AGE products.86 Lys has also been known as an inhibitor of protein glycation; however, its long-term use in diabetes treatment considering different aspects of diabetic complications is limited in the literature. In our previous study, the effect of Lys, as a chemical chaperone, on both animal model of diabetes and type 2 diabetic patients was investigated. Results indicated an improvement in some metabolic and lipid parameters in rats, after 5 months treatment with 0.1% Lys26 and after 3 months of treatment with coadministration of 3 mg/day of Lys with glibenclamide and metformin in diabetic patients.29 This treatment decreased fasting serum Glc and insulin resistance, and improved antioxidant defense system and lipid profile in both rat and human. We also investigated the inhibitory effect of Gly/Lys, on the progression of eye lens opacification in the STZ-induced diabetic rats. Our results indicated the beneficial effect of both amino acids.36,83 In these studies, we reported the changes in the activity of various enzymes and proteins in the lens of normal and diabetic rats due to Gly/Lys treatment.31,83,87 As it was shown, all three important pathways in the lenses were changed in the diabetic rats (D) compared with the normal group (N), but due to Gly/Lys administration (DG/DL) these parameters returned to the normal values or closed to it. Both Gly and Lys had no effect on the normal group (NG/NL). Fig. 8 compares the eyes of a normal and a diabetic rat. As seen, the diabetic rat eyes are completely opaque after 12 weeks. This figure also shows the score and incidence of cataract in these rat groups. These parameters were biweekly determined by a specialist in this field using a handheld ophthalmoscope that was equipped with a slit lamp. As seen, there are significant differences between the percentage of cataract (from clear to completely opaque or grade 4) in the lenses of diabetic animals with those treated with Gly/Lys in comparison with the untreated group.31,83

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(A) 4

DL NL N D DG NG

3

Cataract Score

(B)

2 1 0 0

2

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100

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75

8

10

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Grade 1 Grade 3 66.6% 55.5%

50

44.4%

44.4%

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25 11.1%

0

(D)

6

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N

D

DG

NG

DL

NL

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FIG. 8 Effect of Gly/Lys treatment on the opacity of the lenses in STZ-diabetic rats. Photographs of a normal rat with a clear eye (A) and a diabetic rat at the end of 12 weeks, with eye opacification (B). (C) Cataract Score in different groups of diabetic rats throughout the experimental period. (D) Maturation of the cataract in diabetic rats after 12 weeks in different groups. The percentages of each grade of opacity in each group are shown in the figure. Different grades are shown by a range of color from white (clear) to black (grade 4) of opacity. D and N, diabetic and normal groups; DG and NG, diabetic and normal groups received Gly; DL and NL, diabetic and normal groups received Lys.

Several mechanisms have been proposed for inhibition of protein glycation by amino acids, especially in the lenses. Some of them are: •

Interaction of free amino acids with sugars. Lys and Gly, like some other amino acids, were found to react with Glc at physiological pH and temperature, and undergo nonenzymatic glycation. Formation of the glycated Lys has been shown by paper/ thinlayer chromatography and HPLC. Confirmation was made by studies on incorporation

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of U-[14C] Glc into Lys and Gly. These amino acids also formed adducts after incubation with galactose at physiological pH and temperature. Studies on Lys interaction showed that the extent of glycation of the free amino acid increased with time.78 Direct interaction of amino acids with sugars results in decreased free sugar concentration in the medium; thus, availability of these toxic agents to form protein adduct is reduced. Preventing protein loss in the diabetic lenses. It has been shown that the total and soluble protein content of the cataractous lenses is significantly reduced. Formation of high-molecular-weight aggregates of proteins may be the reason for the observed decrease in protein concentration. Administration of the mentioned amino acids compensates it and makes both soluble and total protein content of lenses close to the normal value.31,88,89 Potentiation of the antioxidant defense system. Oxidative damage to the constituents of the eye lens has been considered as another important mechanism in the development of cataract. The decreased glutathione (GSH) and the altered activities of the antioxidant enzymes, including catalase (CAT) and superoxide dismutase (SOD), are due to the increased oxidative stress in diabetic conditions. Such alteration was also seen in various types of cataract.90 Lys and Gly significantly increased the antioxidant capacity in diabetic rat lenses.36 Suppression of polyol pathway. The dicarbonyl intermediates of the Maillard reaction, such as GO, MGO, and the amount of AGEs, increase during hyperglycemia. The dicarbonyl intermediates are the substrates of aldose reductase (AR) and the AGEs are also its inducers. Thus, when these compounds increase due to hyperglycemia, the activity of the polyol pathway is also increased. As our results indicated, the activity of the AR was significantly increased in diabetic rats and the treatment of these rats with Lys or Gly caused a significant decrease in the activity of this enzyme. However, SDH activity was not significantly altered in the diabetic group.36 Specific transporters for some amino acids in the lens cortex and nucleus.91,92 In rat lens, Gly uptake is mediated by a family of Na/Cl-dependent neurotransmitter transporter proteins. These transporters were named as Gly transporters I and 2 (GLYT1 and GLYT2). Although GLYT1 and GLYT2 are likely to mediate Gly uptake in cortical fiber cells, GLYT2 alone appears to be responsible for the accumulation of Gly in the lens center. The lens core lacks the capacity to synthesize GSH and the hemichannels formed by connexin 4693 and connexin 5094 are responsible for GSH entry into the fiber cells and protecting lenses against oxidants such as H2O2. Therefore, the role of Gly in this region remained to be clear. However, increasing Gly delivery into the core via this transporter may represent a pathway for protecting the lens proteins against some modifications associated with cataract and ageing.91 Hypoglycemic effect by metabolic control. The serum Glc level of diabetic rats was reduced significantly after treatment with Lys and Gly, not only through direct interaction with sugars, but also because of their metabolic effect. For example, catabolism of L-Lys in the liver was down by using 2 mole of α-ketoglutarate (α-KG), as acceptor and cosubstrate. Thus, catabolism of more Lys caused entrance of more Glc to

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•

•

the liver (insulin independent entry) to produce more α-KG to catabolize excess L-Lys, which in turn caused a decrease in blood Glc.26 In humans, it has been found that Gly administration increases the response to insulin.95 Oral Gly stimulates the secretion of a gut hormone that potentiates the insulin effect on Glc removal from the circulation.96 Enhancing both secretion of insulin and activity of insulin receptor. It has been reported that Lys administration (1g/day), accompanied with the antidiabetic tablets (glyciphage or chlorformine), can enhance insulin receptor tyrosine kinase activity in monocytes of type2 diabetes.97 Our results have also indicated the increase in insulin secretion in both diabetic human and rats after Lys administration26,29 The nature of amino acids, which are water-soluble molecules, physiologically available, and nontoxic for body tissues, is an important aspect for their application as complementary therapy. In several studies including ours, many parameters were also measured in normal rats and human treated with Lys/Gly. The results indicated there are no differences between the determined parameters in the normal subjects who were treated with these amino acids when compared with those not treated. Therefore, these amino acids are nontoxic and have no harmful effect on healthy subjects. In addition, their administration even decreased the mortality of diabetic animals.26,29,36,79

In conclusion, a variety of antiglycating agents, natural or synthetic, have been applied to reduce the complication/harms of diabetes and aging. According to our obtained results in more than a decade, oral administration of amino acids, especially Lys and Gly, can significantly delay the onset and progression of diabetic cataract in model animals and human. Amino acids, being highly soluble in water, are rapidly absorbed and transported by mechanisms that do not require energy. These naturally occurring metabolites are less toxic than the synthetic products and may prove to be suitable for preventing nonenzymatic glycation and hence senile and diabetic cataract. Scavenging intracellular sugar and thereby protecting all proteins, including the lens proteins from excessive glycation, appears to be the most important mechanism by which amino acids, especially Lys and Gly, show their beneficial effect on cataract. These studies are continuing to find other involved mechanisms of Gly/Lys antidiabetic function.

Summary Points 1 Glycation is a nonenzymatic and spontaneous reaction between sugar and biomacromolecules such as proteins. 2 Glycation plays an important role in several complications related with aging and diabetes; for example, in senile and diabetic cataract. 3 The antiglycating effect of some amino acids has been shown in the in vivo and in vitro studies. 4 Some amino acids inhibit binding of sugar with proteins, which is the first step in the glycation pathway.

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5 Free amino acids mitigate the glycation of lens proteins and delay cataract. Thus, they can be used as effective complement therapy to treat or prevent cataract. 6 Some amino acids with free-radical scavenging/antioxidant activities are able to preserve cells in the direct or indirect process, through regenerating other antioxidants such as glutathione, vitamins C and E. 7 Some amino acids improve Glc utilization, through improvement of cellular uptake and/or interfere with Glc metabolic pathway. 8 Some amino acids can block the inflammatory processes (the root cause of diabetic complications, cardiovascular disease, arthritis, and some cancers) at the cellular level. 9 Some amino acids can chelate metals that may be implicated in disease promotion through the creation of particularly aggressive free radicals.

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