Sundeep Lal

Fructose-3-phosphate production and polyol pathway metabolism in diabetic rat hearts

Previous studies have suggested that polyol-pathway and nonenzymatic glycation may be involved in the development of cardiac myopathy, a well-known manifestation of diabetes. Although the exact etiology of this complication is not fully understood, it is likely to be multifactorial. In this study, we investigated the metabolic consequences of diabetes and the effect of aldose reductase inhibitor (ARI) treatment on cardiac tissues of Sprague-Dawley rats. Perchloric acid (PCA) extracts of hearts from the animals were examined using 31P-nuclear magnetic resonance (NMR), gas chromatography/mass spectrometry (GC/MS), and high-performance liquid chromatography (HPLC). In 31P-NMR spectra of diabetic animals, a peak resonating at the chemical shift of 5.8 ppm with a coupling constant of 10 Hz was identified as fructose-3-phosphate (F3P). Undetectable in controls (< approximately 20 nmol/g), this metabolite was present at a concentration of 81.3 +/- 16.3 nmol/g wet weight (n = 4) in diabetic rat hearts. GC/MS analysis of these extracts from diabetics also identified a decomposition product of F3P, 3-deoxyglucosone (3DG), at a concentration of 9.4 +/- 3.5 nmol/g (n = 3), compared with 0.98 +/- 0.43 nmol/g (n = 3) in controls. No evidence was found for the expected detoxification products of 3-DG, 3-deoxyfructose and 2-keto 3-deoxygluconate. Concomitant with the elevation of F3P and 3DG, fructose and sorbitol levels were also elevated in diabetic animals. Surprisingly, ARI treatment was found to have no effect on the levels of these metabolites. These data suggest that either the heart may be unique in its production of fructose or it may not readily transport the ARI sorbinil. Production of the potent glycating agents F3P and 3DG in diabetics suggests that these compounds may be contributing factors in the glycation of cardiac proteins in the diabetic rat heart.

31P–Nuclear Magnetic Resonance Evidence of an Activated Hexose-Monophosphate Shunt in Hyperglycemic Rat Lenses In Vivo

Using 31P–nuclear magnetic resonance spectroscopy, we have identified elevated concentrations of sedoheptulose-7-phosphate (S-7-P) in lenses from three animal models of hyperglycemia: streptozotocin-induced diabetic rats, galactose-fed rats, and xylose-fed rats. This observation provides a unique and independent confirmation of the activation of the hexose monophosphate shunt (HMPS) pathway in the hyperglycemic lens in vivo. While the elevation in concentration of S-7-P was very dramatic, the other HMPS metabolites in these tissues were below the threshold of detection, as expected for the HMPS pathway near equilibrium. In terms of nonenzymatic glycation, these results suggest that the only HMPS metabolite of importance in the hyperglycemic rat lens is S-7-P. Although in the diabetic lens its role appears to be relatively minor, in the galactosemic lens this compound may be an important contributor to the increased production of advanced glycosylation end products.

The Role of 3-Deoxyglucosone and the Activity of its Degradative Pathways in the Etiology of Diabetic Microvascular Disease

Summary To address the role of 3-deoxyglucosone (3DG) and the activity of its major degradative pathways in diabetic nephropathy and retinopathy we have measured 3DG and its degradation products [3-deoxyfructose (3DF) and 3-deoxy-2-keto-gluconic acid (DGA)] in plasma and RBCs. In addition to these compounds, we have quantified HbA lc , renal dysfunction [glomerular filtration rate (GFR) and urinary albumin excretion(UAE)] and diabetic retinal sequelae over 2-3 years in 25 IDDM subjects with minimal complications during the earliest stages of diabetic microangiopathy. They were also measured in 58 subjects with NIDDM and 30 non-diabetic subjects. Plasma 3DG, 3DF and erythrocyte DGA were significantly elevated in diabetic subjects relative to controls. We also found a highly significant association between plasma 3DG and HbA lc concentrations, indicating that glycemic control is an important determinant of 3DG levels. An increased flux of 3DG to DGA and 3DF was associated with greater degrees of hyperglycemia, since both degradation products correlated with HbA lc values. Increasing rates of GFR also correlated with plasma 3DG levels and 3DG levels increased with increasing urinary albumin excretion (UAE). By contrast, there was an inverse correlation between plasma 3DF and UAE. Subjects showing progression of retinopathy also showed reduced plasma levels of the 3DG product 3DF relative to nonprogressors. As retinal perfusion decreased, we also observed increased 3DG levels.

Production and Metabolism of 3-Deoxyglucosone in Humans

3-deoxyglucosone (3DG), a reactive dicarbonyl sugar involved in nonenzymatic glycation of proteins is found in elevated concentrations in diabetic patients and has been implicated in the pathogenesis of diabetic complications. In this study, we provide ex-vivo and in-vivo evidence for production of 3DG via the fructose-3-phosphokinase pathway. Evidence of further metabolism of this carbohydrate by oxidative and reductive pathways to 2-keto-3-deoxygluconate (DGA) and 3-deoxyfructose (3DF), respectively, is presented. Levels of these three sugars, 3DG, DGA and 3DF, in blood components and fluids from normoglycemic and diabetic humans are summarized. A comparison of the relative levels of 3DG and 3DF in urine from normoglycemics and diabetics suggests a possible impairment of the detoxification of 3DG to 3DF in diabetics.

Effect of pH on the bioenergetics of perfused porcine lenses

Extracellular and intracellular pH has been shown to be an important physiological variable in many biological systems. However, studies on the effect of pH on the metabolic status of the mammalian lens have been few. It has been shown previously that a change in perfusate pH has a significant effect on the lens weight, ion transport, opacification and membrane potential. In this study we show that the bioenergetic status of the perfused pig lens, as assessed by 31P-NMR spectroscopy, is critically dependent on the pH of the bathing medium. At pHs of 7.6 and above, these perfused lenses maintained their nucleotide triphosphates concentrations steady for up to 4 days. In contrast, lenses perfused with identical media at pH 7.0, rapidly lost their nucleotide triphosphates. Our findings suggest that the pH of the extracellular pH may be an important parameter in maintaining the functional competence of the lens.