A Hypothesis: Fructosamine-3-Kinase-Related-Protein (FN3KRP) Catalyzes Deglycation of Maillard Intermediates Directly Downstream from Fructosamines.

Non-enzymatic glycation (a.k.a. Maillard reaction) is a series of random spontaneous reactions between reducing sugars and amines, resulting in the formation of irreversible advanced glycation endproducts (AGE's). In food chemistry, this process is beneficial by contributing to the flavor, aroma, texture, and appearance of cooked foods. In vivo, however, Maillard reaction is deleterious because uncontrolled modification and crosslinking of biological macromolecules impairs their function. Consequently, chronic hyperglycemia of diabetes mellitus, for instance, leads to increased non-enzymatic glycation and diverse, multi-organ pathologies of diabetic complications. Based on the fact that toxic compounds, such as free radicals, are detoxified in vivo by specific defense mechanisms, one would expect to find mechanisms to control glucose toxicity as well. Thus far, only one such enzyme, fructosamine-3-kinase (FN3K), has been characterized. It operates intracellularly by catalyzing ATP-dependent removal of Maillard adducts, D-fructoselysines, from proteins, thereby reducing the Maillard reaction flux from glucose to AGE's. When FN3K was isolated, a closely related but distinct protein copurified with it. Unlike FN3K, however, this enzyme, fructosamine-3-kinase-related protein (FN3KRP), does not phosphorylate D-fructoselysines but it does phosphorylate several other (non-physiological) substrates. Interestingly, the distribution of FN3KRP in nature appears to be nearly universal whereas that of FN3K is limited to endotherms. In this article, it is suggested that the function of FN3KRP is deglycation of Maillard adducts downstream from fructoselysines. Such a mechanism, if proven correct, would be valuable given reports on apparent correlations between FN3KRP and some chronic conditions and/or diseases, such as a recent publication which proposes that the FN3KRP gene may be a longevity gene.

A Hypothesis: Moderate Consumption of Alcohol Contributes to Lower Prevalence of Type 2 Diabetes Due to the Scavenging of Alpha-Dicarbonyls by Dietary Polyphenols.

The world is experiencing an epidemic of type-2-diabetes mellitus (T2DM). This has led to increased morbidity and mortality, explosive growth in health care budgets, and an even greater adverse, if indirect, impact on societies and economies of affected countries. While genetic susceptibility to T2DM is a major determinant of its prevalence, changes in lifestyles also play a role. One such change has been a transition from traditional diets characterized by low caloric and high nutrient density to calorie-rich but nutrient-poor Western diets. Given this, one solution to the epidemic of T2DM would be to abandon Western diets and revert to traditional eating patterns. However, traditional diets cannot provide enough calories for the increasing global population, so transition from traditional to Western foodstuffs appears to be irreversible. Consequently, the only practical solution to problems caused by these changes is to modify Western diets, possibly by supplementing them with functional foods containing nutrients that would compensate for these dietary deficits. I present in this study a hypothesis to explain why shifts from traditional to Western diets have been so problematic and to suggest nutrients that may counteract these adverse effects. I postulate that the components of traditional diets that may compensate for deficiencies of Westerns diets are scavengers of reactive α-dicarbonyls produced as unavoidable by-products of glucose and lipid metabolism. Most important among these scavengers are some plant secondary metabolites: polyphenols, phlorotannins, and carotenoids. They are found in alcoholic beverages and are abundant in seasonings, cocoa, coffee, tea, whole grains, pigmented vegetables, fruits, and berries.

Reactions between methylglyoxal and its scavengers in-vivo appear to be catalyzed enzymatically.

Methylglyoxal (MGO) is thought to be an important contributor to the development of diabetic complications. In this paper I propose that MGO, not detoxified by the glyoxalase system, is removed from circulation by MGO-scavengers. Furthermore, since intrinsic rates of reactions between MGO and its scavengers are low, I propose that, in-vivo, these reactions are catalyzed enzymatically.

Potential of birds to serve as pathology-free models of type 2 diabetes, part 2: do high levels of carbonyl-scavenging amino acids (e.g., taurine) and low concentrations of methylglyoxal limit the production of advanced glycation end-products?

In our previous publication, we reported on the advantages of using birds as a pathology-free model of type 2 diabetes mellitus (T2DM). Using this new perspective, we observed that birds are missing the RAGE gene, considered an important factor in the development of diabetic complications. In this article, we identify two additional Maillard reaction-related characteristics of birds that have the potential to account, in part, for avian ability to cope successfully with chronic hyperglycemia. First, compared to mammals, blood plasma of birds has significantly higher concentrations of taurine and other free amino acids that act as scavengers of reactive carbonyls. Second, there are also indications that avian blood plasma contains lower concentrations of methylglyoxal (MG) due, in part, to its decreased production by avian erythrocytes. Our deductions are based on relatively meager experimental data and are therefore speculative. One certain outcome of our study, however, is the idea that birds can be a useful model for the study of Maillard reactions and etiology of diabetic complications. We anticipate and hope that results of future studies will support the hypothesis identifying MG as a key intermediate in the etiology of diabetic complications. If this is indeed the case, then prevention and control of diabetic complications may become transformed into a more circumscribed, defined, and tractable problem whose goals will be to minimize the production of MG and to maximize its elimination by detoxification or scavenging.

Potential of birds to serve as a pathology-free model of type 2 diabetes, Part 1: Is the apparent absence of the rage gene a factor in the resistance of avian organisms to chronic hyperglycemia?

Diabetes mellitus is a global pandemic that accounts for ever-increasing rates of morbidity and mortality and consumes a growing share of national health care budgets. In spite of concerted efforts, a solution to this problem has not yet been found. One reason for this situation is lack of good animal models. Such models have been used successfully in many areas of biomedical research, but they have proven less than satisfactory in studies on diabetic complications. In this article, we propose to supplement traditional animal models of diabetes that use longitudinal, prospective studies of sick animals (mammals) with retrospective/comparative investigations of healthy animals (birds). Avians are promising models for such studies because they live healthy lives with chronic hyperglycemia that would be fatal to humans. We outline the advantages of the new perspective and show how, by implementing this approach, we observed that birds appear to be missing an important gene linked to diabetic complications. The protein encoded by this gene is a receptor for advanced glycation end products (RAGEs). Although the absence of RAGEs from birds has yet to be confirmed at the protein level, other differences between humans and birds may also be important in accounting for the ability of birds to live with chronic hyperglycemia. Two such additional such characteristics are currently being explored, and it is probable that more will emerge in time. We believe that the proposed perspective may improve the understanding of diabetes mellitus and may help in developing new means for controlling and preventing diabetic complications.

Maillard reactions in hyperthermophilic archaea: implications for better understanding of non-enzymatic glycation in biology.

Maillard reactions are an unavoidable feature of life that appear to be damaging to cell and organisms. Consequently, all living systems must have ways to protect themselves against this process. As of 2012, several such defense mechanisms have been identified. They are all enzymatic and were found in mesophilic organisms. To date, no systematic study of Maillard reactions and the relevant defense mechanisms has been conducted in thermophiles (50°C-80°C) or hyperthermophiles (80°C-120°C). This is surprisingly because Maillard reactions become significantly faster and potent with increasing temperatures. This review examines this neglected issue in two well-defined sets of hyperthermophiles. My analysis suggests that hyperthermophiles cope with glycation stress by several mechanisms: • Absence of glycation-prone head groups (such as ethanoalamine) from hyperthermophilic phospholipids • Protection of reactive carbohydrates and labile metabolic intermediates by substrate channeling. • Conversion of excess reactive sugars such as glucose to non-reactive compounds including trehalose, di-myo-inositol-phosphate and mannosylglycerate. • Detoxification of methylglyoxal and other ketoaldehydes by conversion to inert products through a variety of reductases and dehydrogenases. • Scavenging of the remaining carbonyls by nucleophilic amines, including a variety of novel polyamines. Disruption of the Maillard process at its early stages, rather than repair of damage caused by it at later stages, appears to be the preferred strategy in the organisms examined. The most unique among these mechanisms appears to be a polyamine-based scavenging system. Undertaking research of the Maillard process in hyperthermophiles is important in its own right and is also likely to provide new insights for the control of these reactions in humans, especially in diseases such as diabetes mellitus.

A novel mouse model of Niemann-Pick type C disease carrying a D1005G-Npc1 mutation comparable to commonly observed human mutations.

We have identified a point mutation in Npc1 that creates a novel mouse model (Npc1(nmf164)) of Niemann-Pick type C1 (NPC) disease: a single nucleotide change (A to G at cDNA bp 3163) that results in an aspartate to glycine change at position 1005 (D1005G). This change is in the cysteine-rich luminal loop of the NPC1 protein and is highly similar to commonly occurring human mutations. Genetic and molecular biological analyses, including sequencing the Npc1(spm) allele and identifying a truncating mutation, confirm that the mutation in Npc1(nmf164) mice is distinct from those in other existing mouse models of NPC disease (Npc1(nih), Npc1(spm)). Analyses of lifespan, body and spleen weight, gait and other motor activities, as well as acoustic startle responses all reveal a more slowly developing phenotype in Npc1(nmf164) mutant mice than in mice with the null mutations (Npc1(nih), Npc1(spm)). Although Npc1 mRNA levels appear relatively normal, Npc1(nmf164) brain and liver display dramatic reductions in Npc1 protein, as well as abnormal cholesterol metabolism and altered glycolipid expression. Furthermore, histological analyses of liver, spleen, hippocampus, cortex and cerebellum reveal abnormal cholesterol accumulation, glial activation and Purkinje cell loss at a slower rate than in the Npc1(nih) mouse model. Magnetic resonance imaging studies also reveal significantly less demyelination/dysmyelination than in the null alleles. Thus, although prior mouse models may correspond to the severe infantile onset forms of NPC disease, Npc1(nmf164) mice offer many advantages as a model for the late-onset, more slowly progressing forms of NPC disease that comprise the large majority of human cases.

The physiological substrates of fructosamine-3-kinase-related-protein (FN3KRP) are intermediates of nonenzymatic reactions between biological amines and ketose sugars (fructation products).

The physiological function of fructosamine-3-kinase (FN3K) is relatively well understood. As shown in several studies, most conclusively by data on the FN3K-KO mouse, this enzyme breaks down compounds produced by the non-enzymatic glycation of proteins by D-glucose. In contrast with FN3K, very little is known about the function of the fructosamine-3-kinase-related-protein (FN3KRP) even though it has a 65% amino-acid sequence identity with FN3K. We do know that this enzyme is a kinase as evidenced by its ability to phosphorylate non-physiological compounds such a psicosamines, ribulosamines, erythrulosamines, and glucitolamines. However, FN3KRP does not phosphorylate any of the numerous Amadori products that are the physiological substrates of FN3K. The fact that FN3KRP is highly conserved in all vertebrates and present throughout nature suggests that it plays an important role in cellular metabolism and makes identification of its physiological substrates an important objective. In this paper, we propose that FN3KRP phosphorylates products resulting from a non-enzymatic glycation of amines by ketoses (fructation) that involves a 2,3-enolization and produces the stable Amadori intermediate, 2-amino-2-deoxy-D-ribo-hex-3-ulose (ADRH). This ketosamine is then phosphorylated to 2-amino-2-deoxy-D-ribo-hex-3-ulose-4-phosphate (ADRH-4-P). Since phosphates are much better leaving groups than hydroxyls, this destabilizes the C-2 amine bond and results in a spontaneous ß-elimination of the phosphate to regenerate an unmodified amine with the concomitant production of 4-deoxy-2,3-diulose. Consequently, we postulate that the principal physiological function of FN3KRP is the breakdown of nonenzymatic fructation products. If confirmed in future studies, this hypothesis opens up new perspectives for an improved understanding of biological Maillard reactions and mechanisms for their control and/or reversal.

Identification of two gene clusters and a transcriptional regulator required for Pseudomonas aeruginosa glycine betaine catabolism.

Glycine betaine (GB), which occurs freely in the environment and is an intermediate in the catabolism of choline and carnitine, can serve as a sole source of carbon or nitrogen in Pseudomonas aeruginosa. Twelve mutants defective in growth on GB as the sole carbon source were identified through a genetic screen of a nonredundant PA14 transposon mutant library. Further growth experiments showed that strains with mutations in two genes, gbcA (PA5410) and gbcB (PA5411), were capable of growth on dimethylglycine (DMG), a catabolic product of GB, but not on GB itself. Subsequent nuclear magnetic resonance (NMR) experiments with 1,2-(13)C-labeled choline indicated that these genes are necessary for conversion of GB to DMG. Similar experiments showed that strains with mutations in the dgcAB (PA5398-PA5399) genes, which exhibit homology to genes that encode other enzymes with demethylase activity, are required for the conversion of DMG to sarcosine. Mutant analyses and (13)C NMR studies also confirmed that the soxBDAG genes, predicted to encode a sarcosine oxidase, are required for sarcosine catabolism. Our screen also identified a predicted AraC family transcriptional regulator, encoded by gbdR (PA5380), that is required for growth on GB and DMG and for the induction of gbcA, gbcB, and dgcAB in response to GB or DMG. Mutants defective in the previously described gbt gene (PA3082) grew on GB with kinetics similar to those of the wild type in both the PAO1 and PA14 strain backgrounds. These studies provided important insight into both the mechanism and the regulation of the catabolism of GB in P. aeruginosa.

Fructosamine-3-kinase-related-protein phosphorylates glucitolamines on the C-4 hydroxyl: novel substrate specificity of an enigmatic enzyme.

Fructosamine-3-kinase (FN3K) phosphorylates fructosamines to fructosamine-3-phosphates. Recent data from FN3K-knockout mouse indicate that this phosphorylation results in deglycation of proteins modified by non-enzymatic glycation process. A homolog of FN3K, the FN3K-related-protein (FN3KRP) displays 65% amino acid sequence identity with FN3K and is highly conserved in evolution. However, FN3KRP does not phosphorylate substrates of FN3K such as fructoselysine and its physiological function remains unknown. We observed that human erythrocytes that contain both enzymes phosphorylate N-methylglucamine (meglumine) to two products. One of these is meglumine-3-phosphate (Meg3P), an activity consistent with the known substrate specificity of FN3K. Here, we identify the second product as meglumine-4-phosphate (Meg4P) and show that it is produced specifically by FN3KRP. While it is unlikely that meglumine is the physiological target of FN3KRP, this novel specificity, along with FN3KRPs known phosphorylation of some ketosamines on the C-3 hydroxyl may prove useful in identifying the physiological substrates of this kinase.

Fructosamine-6-phosphates are deglycated by phosphorylation to fructosamine-3,6-bisphosphates catalyzed by fructosamine-3-kinase (FN3K) and/or fructosamine-3-kinase-related-protein (FN3KRP).

Nonenzymatic glycation of proteins and some phospholipids by glucose and other reducing sugars (a.k.a Maillard reaction) is an unavoidable result of the coexistence of these sugars and the affected macromolecules in living systems. The consequences of this process are deleterious both in the intracellular and extracellular environments as evidenced by the close association between increased nonenzymatic glycation and complications of diabetes. Because of these considerations, we have proposed that the intrinsic toxicity of glucose and other sugars is counteracted in vivo by active deglycation mechanisms including transglycation of Schiff's bases and FN3K-dependent breakdown of fructosamines. While this modified hypothesis is receiving increasing experimental support, several issues regarding glycation/deglycation remain unresolved. Two such important questions are In this paper we propose a resolution of both these quandaries by proposing that fructosamine-6-phosphates are deglycated by phosphorylation to fructosamine-3,6-bisphosphates catalyzed by FN3KRP and/or possibly FN3K. We provide some preliminary evidence in support of this hypothesis and outline experimental approaches for definitive tests of this hypothesis. The potential medical implications of this finding are not clear yet but, if correct, this observation is likely to have a major impact on our understanding of the very basic and hitherto unexplored aspect of glucose metabolism and chemistry in vivo. One can imagine that, at some point in the future, measurement of FN3K/FN3KRP activity may be of diagnostic value in assessing an individual's susceptibility to diabetic complications. Further down the road, one can also envision a gene therapeutic intervention to bolster FN3K/FN3KRP-based antiglycation defenses.

Elements of diabetic nephropathy in a patient with GLUT 2 deficiency.

The Fanconi-Bickel syndrome is caused by homozygosity or compound heterozygosity for mutations of the facilitated glucose transporter 2 gene (GLUT2). Glycogen accumulates in renal tubular cells and they fail to reabsorb multiple filtered solutes because of impairment in GLUT2-mediated efflux of glucose. We describe a 10-year-old male child with GLUT2 deficiency who produced massive amounts of 3-deoxyfructose (3-DF) in the kidneys. Since 3-DF is a detoxification product of a potent glycating agent, 3-deoxyglucosone, a precursor of advanced glycation end-products, this suggests a massive accumulation of glucose within tubular cells probably as a consequence of GLUT2 deficiency. The level of 3-DF in the urine of this atypical patient, who also manifested renal glomerular hyperfiltration, microalbuminuria, and glomerular mesangial expansion, was higher than in any patient examined with diabetes mellitus. Elevated levels of glucose and/or its metabolites in renal tubular cells may be necessary but not sufficient for the development of both the renal tubulopathy and diabetic-like glomerular disease in GLUT2 deficiency.

Susceptibility to diabetic nephropathy is related to dicarbonyl and oxidative stress.

Dicarbonyl and oxidative stress may play important roles in the development of diabetes complications, and their response to hyperglycemia could determine individual susceptibility to diabetic nephropathy. This study examines the relationship of methylglyoxal, 3-deoxyglucosone (3DG), and oxidative stress levels to diabetic nephropathy risk in three populations with diabetes. All subjects in the Overt Nephropathy Progressor/Nonprogressor (ONPN) cohort (n = 14), the Natural History of Diabetic Nephropathy study (NHS) cohort (n = 110), and the Pima Indian cohort (n = 45) were evaluated for clinical nephropathy, while renal structural measures of fractional mesangial volume [Vv(Mes/glom)] and glomerular basement membrane (GBM) width were determined by electron microscopy morphometry in the NHS and Pima Indian cohorts. Methylglyoxal and 3DG levels reflected dicarbonyl stress, while reduced glutathione (GSH) and urine 8-isoprostane (8-IP) measured oxidative stress. Cross-sectional measures of methylglyoxal production by red blood cells incubated in 30 mmol/l glucose were increased in nephropathy progressors relative to nonprogressors in the ONPN (P = 0.027) and also reflected 5-year GBM thickening in the NHS cohort (P = 0.04). As nephropathy progressed in the NHS cohort, in vivo levels of methylglyoxal (P = 0.036), 3DG (P = 0.004), and oxidative stress (8-IP, P = 0.007 and GSH, P = 0.005) were seen, while increased methylglyoxal levels occurred as nephropathy progressed (P = 0.0016) in the type 2 Pima Indian cohort. Decreased glyceraldehyde-3-phosphate dehydrogenase activity also correlated with increased methylglyoxal levels (P = 0.003) in the NHS cohort. In conclusion, progression of diabetic nephropathy is significantly related to elevated dicarbonyl stress and possibly related to oxidative stress in three separate populations, suggesting that these factors play a role in determining individual susceptibility.

Carnosine and anserine act as effective transglycating agents in decomposition of aldose-derived Schiff bases.

There are numerous publications describing the positive effects of carnosine (beta-alanyl-histidine) and anserine (beta-alanyl-1-N-methyl-histidine) on cell and organ function. Of special interest to us is the fact that these dipeptides act to retard and (in one instance) reverse non-enzymatic glycation. To date, the primary explanation for these anti-glycating effects has been the fact that carnosine and anserine can serve as alternative and competitive glycation targets, thereby protecting proteins from this deleterious process. In this paper, we document another mechanism by which these two peptides can retard or reverse glycation. The process involves decomposition of the very first intermediates of the non-enzymatic glycation cascade (aldosamines a.k.a. Schiff bases) by nucleophilic attack of carnosine and/or anserine on the preformed aldosamine such as glucosyl-lysine. If future research shows this reaction is to be physiologically important, this mechanism could explain some of the beneficial effects of carnosine and anserine as anti-glycating agents.

Some clues as to the regulation, expression, function, and distribution of fructosamine-3-kinase and fructosamine-3-kinase-related protein.

Fructosamine-3-kinase (FN3K) and the more recently discovered fructosamine-3-kinase-related protein (FN3KRP) appear to protect proteins from nonenzymatic glycation. To gain a better understanding of these enzymes we performed a series of investigations including (1) in silico comparisons of their promoters; (2) real-time PCR analysis of their expression in human tissues; (3) effects of hyperglycemia, interleukin-1beta (IL-1beta), and nuclear factor kappa-B (NFkappaB) activation on their mRNA levels; (4) effects of small interfering RNA (siRNA) suppression of FN3K expression (knockdown) in cultured cells and (5) search of FN3K and FN3KRP homologs in available genomic and EST (expressed sequence tag) databases. Our results indicate that (1) both FN3K and FN3KRP promoters are TATA-less and CAAT-less and contain several homologous CpG islands and Sp1 binding sites. (2) Both genes are expressed in all human tissue examined, with FN3K showing significantly higher levels in tissues susceptible to nonenzymatic glycation and diabetic complications. (3) Treatment of fibroblasts with high glucose, IL-1beta, and activation of NFkappaB does not affect the expression of either FN3K or FN3KRP. (4) Knockdown of FN3K in cultured cells inhibits or arrests their growth. (5) FN3K-like genes are widely distributed in nature, with the notable exception of insects and yeasts. These data suggest that FN3K and FN3KRP are constitutive "housekeeping" genes and that they play an important role in cell metabolism, possibly as deglycating enzymes.

Transglycation--a potential new mechanism for deglycation of Schiff's bases.

Nonenzymatic glycation is believed to play a major role in the development of diabetic complications. Over the past several years we and others have shown that in cells this nonenzymatic process can be reversed by an ATP-dependent reaction catalyzed by fructosamine-3-kinase (FN3K) and possibly by its isozyme, fructosamine-3-kinase-related protein (FN3KRP). In this study we provide the first evidence that this FN3K-dependent deglycation, acting on the Amadori products, is complemented by another deglycation process operating on the very first product of nonenzymatic glycation, glucosylamines (Schiff's bases). We postulate that the first step in this Schiff's-base deglycation process occurs by transfer of the sugar moiety from macromolecule-bound glucosylamine to one of the low-molecular weight intracellular nucleophiles-in particular, glutathione. We term this reaction transglycation, and in this study we demonstrate that it occurs readily and spontaneously in vitro. We further propose that one of the spontaneously formed glucose-glutathione adduct(s) is subsequently removed from cells by a multidrug-resistance pump (MRP, MDR-protein, ATP-binding-cassette protein), metabolized, and excreted in urine. In support of this latter contention, we show that at least one transglycation product, glucose-cysteine, is found in human urine and that its concentrations are increased in diabetes.

Intrinsic toxicity of glucose, due to non-enzymatic glycation, is controlled in-vivo by deglycation systems including: FN3K-mediated deglycation of fructosamines and transglycation of aldosamines.

Along with oxygen, glucose is an essential macronutrient for most cells, a source of carbons for biosynthesis and energy. However, alongside this indispensable role for cell survival and growth, glucose is intrinsically toxic by reacting with primary amines such as lysine in proteins in a non-enzymatic glycation process (a.k.a. Maillard reaction) especially important in long-lived, homeothermic organisms where temperatures of 37-44 degrees C accelerate its rate. Products of Maillard reactions are known to have adverse effects on protein function and have been implicated in the development of diabetic complications and possibly in neurodegenerative diseases. Because of the unavoidable nature of non-enzymatic glycation and its deleterious effects, we propose that glucose-utilizing organisms, especially the homeothermic ones, possess mechanisms to control this process at its earliest stages. In the intracellular milieu two such mechanisms are apparent at present; a fructosamine-3-kinase(FN3K)-dependent process which is ubiquitous in all warm-blooded animals and a FN3K-independent deglycation pathway present in all animals, including ones which do not have FN3K, such as insects. We propose that of the two pathways, the FN3K-independent mechanism is more important due to the fact that it breaks down the very first intermediate of the Maillard reaction, the Schiff base (a.k.a aldosamine). We postulate that this, FN3K-independent, deglycation occurs by transglycation, in which carbohydrate moieties of glycated amines, such as glucoselysines on proteins, are removed by intracellular nucleophiles including free amino acids and peptides such as glutathione, carnosine and anserine. Furthermore, we hypothesize that one or more of these nucleophile-aldose adducts, formed as by-products of transglycation, are actively removed from cells by one or more of the multi-drug-resistance [MDR] proteins or similar pumps. In the extracellular space, non-enzymatic glycation and deglycation occur as well. We also postulate that, in that setting, transglycation products are removed from the system by the kidneys or similar excretory organs. Our hypothesis leads to several testable predictions including: The deglycation hypothesis offers new paradigm for thinking about non-enzymatic glycation and diabetic complications and offers possible strategies for intervention in this and possibly other degenerative conditions.

Identification of diadenosine-triphosphate in mature bovine lenses.

Mature bovine lenses contain 75-100 microM of a previously unidentified nucleoside polyphosphate. Using (31)P NMR spectroscopy we have identified this compound as diadenosine-5',5'''-triphosphate. The accumulation of this compound in the lens may be a consequence of the high levels of activities of t-RNA synthetases during lens differentiation and growth. The function, if any, of this compound in the bovine lenses is presently unknown.

The expression of the genes for fructosamine-3-kinase and fructosamine-3-kinase-related protein appears to be constitutive and unaffected by environmental signals.

Fructosamine-3-kinase (FN3K) and the more recently discovered fructosamine-3-kinase related protein (FN3KRP) appear to protect proteins from nonenzymatic glycation. To elucidate the patterns of transcriptional regulation of these two genes, we performed in silico comparisons of their promoters along with real-time PCR assays of their expression in a variety of human tissues. Both promoters were TATA-less and CAAT-less, and contained several homologous CpG islands and Sp1 binding sites. The genes were expressed in all human tissues examined, with FN3K showing significantly higher levels in organs susceptible to nonenzymatic glycation and diabetic complications. Cultured fibroblasts treated with conditions mimicking the hormonal and biochemical profile of the diabetic state showed no changes in FN3K and FN3KRP expression relative to untreated cells. These data suggest that FN3K and FN3KRP act as protein repair enzymes and are expressed constitutively in human cells independently of some of the variables altered in the diabetic state.

Glyceraldehyde-3-phosphate dehydrogenase activity as an independent modifier of methylglyoxal levels in diabetes.

Methylglyoxal (MG) may be an important cause of diabetic complications. Its primary source is dihydroxyacetone phosphate (DHAP) whose levels are partially controlled by glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Using a human red blood cell (RBC) culture, we examined the effect of modifying GAPDH activity on MG production. With the inhibitor koningic acid (KA), we showed a linear, concentration-dependent GAPDH inhibition, with 5 microM KA leading to a 79% reduction of GAPDH activity and a sixfold increase in MG. Changes in redox state produced by elevated pH also resulted in a 2.4-fold increase in MG production at pH 7.5 and a 13.4-fold increase at pH 7.8. We found substantial inter-individual variation in DHAP and MG levels and an inverse relationship between GAPDH activity and MG production (R=0.57, P=0.005) in type 2 diabetes. A similar relationship between GAPDH activity and MG was observed in vivo in type 1 diabetes (R=0.29, P=0.0018). Widely varying rates of progression of diabetic complications are seen among individuals. We postulate that modification of GAPDH by environmental factors or genetic dysregulation and the resultant differences in MG production could at least partially account for this observation.