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-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.