Marc Lecomte

Reaction of Metformin with Dicarbonyl Compounds. Possible Implication in the Inhibition of Advanced Glycation End Product Formation

Dicarbonyl compounds such as methylglyoxal and glyoxal are extremely reactive glycating agents involved in the formation of advanced glycation end products (AGEs), which in turn are associated with diabetic vascular complications. Guanidino compounds such as aminoguanidine appear to inhibit AGE formation by reacting with alpha-dicarbonyl compounds. The aim of this work was to study whether the antihyperglycemic agent metformin (a guanidine-like compound) might react with reactive alpha-dicarbonyls. Metformin was incubated at pH 7.4 and 37 degrees in the presence of either methylglyoxal or glyoxal and reaction products analysed by HPLC coupled to mass tandem spectrometry. AGE formation on albumin by methylglyoxal and glyoxal in the presence or absence of metformin was also studied by measuring the fluorescence at 370/440 nm after albumin-AGE isolation by ultrafiltration. As a standard for mass spectra analysis, a metformin-methylglyoxal adduct was chemically synthesised and characterised as a triazepinone (2-amino-4-(dimethyl-amino)-7-methyl-5,7-dihydro-6H-[1,3,5]triazepin+ ++-6-one). The results obtained showed that metformin strongly reacted with methylglyoxal and glyoxal, forming original guanidine-dicarbonyl adducts. Reaction kinetic studies as well as mass fragmentation spectra of the reaction products were compatible with the presence of triazepinone derivatives. In the presence of metformin, AGE-related fluorescence after albumin incubation with either glyoxal or methylglyoxal was decreased by 37% and 45%, respectively. These results suggest that besides its known antihyperglycemic effect, metformin could also decrease AGE formation by reacting with alpha-dicarbonyl compounds. This is relevant to a potential clinical use of metformin in the prevention of diabetic complications by inhibition of carbonyl stress.

Advanced glycation end-products induce apoptosis of bovine retinal pericytes in culture: involvement of diacylglycerol/ceramide production and oxidative stress induction

One of the earliest changes observed in retinal microvessels in diabetic retinopathy is the selective loss of intramural pericytes. We tested the hypothesis that AGE might be involved in the disappearance of retinal pericytes by apoptosis and further investigated the signaling pathway leading to cell death. Chronic exposure of pericytes to methylglyoxal-modified bovine serum albumin (AGE-BSA) (3 microM) leads to a 3-fold increase of apoptosis (8.9 +/- 1.1%), associated with an increase in cellular ceramide (185 +/- 12%) and diacylglycerol (194 +/- 9%) levels. Ceramide formation was almost inhibited (95%) by an acidic sphingomyelinase inhibitor, desipramine (0.3 microM). Dual inhibition of ceramide (95%) and diacylglycerol (80%) production was observed with a phosphatidylcholine-phospholipase C inhibitor, D609 (9.4 microM). Taken together, these results suggest activation of phosphatidylcholine-phospholipase C coupled to acidic sphingomyelinase. However, both inhibitors only partially protected pericytes against apoptosis, suggesting another apoptotic pathway independent of diacylglycerol/ceramide production. Treatments with various antioxidants completely inhibited pericyte apoptosis, suggesting oxidative stress induction during this apoptotic process. Inhibition of diacylglycerol/ceramide production by N-acetyl-L-cysteine suggests that oxidative stress acts upstream of the two metabolic pathways. AGE treated with metal chelators were also able to induce pericyte apoptosis, suggesting a specific effect of AGE on intracellular oxidative stress independent of redox-active metal ions bound to AGE. In conclusion, these results identify new biochemical targets involved in pericyte loss, which can provide new therapeutic perspectives in diabetic retinopathy.

Modification of Enzymatic Antioxidants in Retinal Microvascular Cells by Glucose or Advanced Glycation End Products

Oxidative stress is one possible pathogenic mechanism to explain diabetic microangiopathy. In the present study, we determined the antioxidant enzyme activities in bovine retinal microvessels and cultured retinal microvascular cells: endothelial cells (BREC) and pericytes (BRP). We further investigated the effects of high glucose and advanced glycation end products (AGE) on these enzyme activities in BREC and BRP. Antioxidant enzyme activities in native retinal microvessels and BREC were quite similar but differed markedly from the BRP ones. High glucose decreased Se-GPx activity (about 20%) in BREC compared to mannitol. High concentrations of mannitol or NaCl increased Se-GPx activity (up to 40%) compared to control medium, suggesting that hyperosmolarity could regulate Se-GPx in BREC. No changes in antioxidant enzyme activities were observed when BRP were cultured with glucose or mannitol at high concentrations. AGE-BSA had no effect on enzyme activities in BREC, whereas 20 microM AGE-BSA increased catalase (40%) and superoxide dismutase (60%) activities in BRP. Differences in antioxidant enzyme activities observed between BREC and BRP, cultured with high concentrations of glucose or AGE, might help to explain their different behavior during the pathogenesis of diabetic retinopathy, i.e., early pericyte drop-out and late endothelial cell proliferation.

Docosahexaenoic Acid Is a Major n-3 Polyunsaturated Fatty Acid in Bovine Retinal Microvessels

Abstract: The aim of this study was to purify microvessels from bovine retina and also to cultivate bovine retinal endothelial cells (BRECs) or intramural pericytes, to determine their fatty acid composition. Microvessels were obtained after Dounce homogenization of the retina followed by centrifugation on albumin cushion and finally microvessels in the pellet were trapped on a 100‐µm nylon filter. Contamination of microvessel preparations by neuronal tissue, assessed after both microscopic examination and western blotting with a monoclonal antibody raised against rhodopsin, was minor. In the entire bovine retina, docosahexaenoic acid (DHA) represented 23.3% of the total fatty acids and there was about three times less arachidonic acid (AA) (8.2%) than DHA. In contrast, DHA and AA levels were almost equivalent in the retinal microvessels with ∼10% of total fatty acids. When compared with intact microvessels, the DHA proportion of confluent monolayers of both BRECs or pericytes in primary cultures dropped to ∼2% of the total fatty acids, whereas AA was unchanged. Culture medium supplementation with unesterified DHA (10 µM) restored the DHA proportion of BRECs close to the microvascular value at the expense of linoleic acid without affecting AA very much. In contrast, DHA supplementation in pericytes increased the DHA proportion of these cells at the expense of AA. In conclusion, DHA of intact microvessels represented 10% of the total fatty acids, which was close to the AA proportion. Mild DHA supplementation of BRECs or pericytes in primary cultures restored their DHA proportion to the original microvessel value. This high percentage of polyunsaturated fatty acids in retinal microvessels should allow us to test the hypothesis that oxidation products derived from these fatty acids may be involved in the pathogenic process leading to diabetic retinopathy.

IN VITRO AND IN VIVO ALTERATIONS OF ENZYMATIC GLYCOSYLATION IN DIABETES

Carbohydrate composition changes of glycoconjugates constituting the glycocalix of microvascular cells could be involved in the alterations of cell-cell interactions observed in diabetic retinopathy. In this field, we have recently reported that advanced glycation end products (AGEs) modify galactose, fucose and sialic acid contents of specific cellular glycoproteins. To better understand the mechanisms involved in glycoprotein modifications in diabetes, we now investigate whether glucose and AGEs could affect the activities of enzymes involved in galactose, fucose and sialic acid metabolism : glycosyltransferases (synthesis) and glycosidases (catabolism). For this, bovine retinal endothelial cells (BREC) and pericytes (BRP) were cultured in the presence of high glucose concentration or AGEs, and cell glycosidase and glycosyltransferase activities were measured. The same enzymatic activities were studied in the whole retina from streptozotocin-treated rats. The results show that high glucose concentration did not affect glycosidases and glycosyltransferases neither in BRP nor in BREC except for galactosyltransferase activities in BREC. Concerning BRP, only galactosyltransferase activities were altered by AGEs. In contrast, in BREC, AGEs increased beta-D galactosidase, alpha-L fucosidase and neuraminidase activities (+37%, +56%, 36% respectively) whereas galactosyltransferase, fucosyltransferase and sialyltransferase activities were decreased (-11%, -24% and -23% respectively). In the retina from diabetic rats, beta-D galactosidase, alpha-L fucosidase and neuraminidase activities increased (+70%, +57%, +78% respectively) whereas fucosyl and sialyltransferase decreased (-7% and -15% respectively). The possible consequence of these enzymatic activity changes could be a defect in the carbohydrate content of some glycoproteins that might participate in the endothelial cell dysfunctions in diabetic microangiopathy.

In vivo effect of 8-epi-PGF2α on retinal circulation in diabetic and non-diabetic rats

Retinal hemodynamic responses to a F2-isoprostane, 8-epi-PGF2alpha, were quantitated in vivo in non-diabetic and diabetic rats using a video fluorescein angiography system. Vascular diameters and retinal mean circulation time were determined before and after 5 microl intra-vitreous injection of 8-epi-PGF2alpha (10(-5) to 10(-3) M), 10(-4) M 8-epi-PGF2alpha, + 10(-3) M SQ29,548 or 10(-3) M LCB2853 (two inhibitors of TXA2 receptor), 10(4) M 9beta-PGF2alpha, or the carrier in non-diabetic animals. Diabetic rats received either 8-epi-PGF2alpha 10(-4) M, or the carrier. Compared to control animals, diabetic rats presented in the basal state a venous vasodilation (P<0.01), without modification of retinal mean circulation time or blood flow. After intravitreous injection of 8-epi-PGF2alpha, a significant arterial vasoconstriction was observed in control but not in diabetic animals. This vasoconstriction was concomitant with increased retinal mean circulation time in control but not in diabetic rats, inducing an impaired reduction of blood flow. No vasoconstriction was observed after injection of either the carrier, 9beta-PGF2alpha or the isoprostane associated to the inhibitors of TXA2 receptors. This is the first direct observation that the isoprostane 8-iso-PGF2alpha is a potent vasoconstricting agent in the retina. It occurs at the arterial but not venous level, and is likely mediated through a TXA2-like receptor. Differences observed between control and diabetic animals suggest altered adaptative mechanisms toward vasoconstrictor substances (such as isoprostanes) in diabetic rats.

Albumin antioxidant capacity is modified by methylglyoxal

OBJECTIVE Oxidative stress seems to play a major role in diabetic vascular complication development. Plasma albumin, via its thiol groups, is the main extracellular antioxidant molecule. Methylglyoxal (MG) is a very reactive dicarbonyl compound increased in diabetes which strongly modifies proteins by non-enzymatic glycosylation. The aim of this work was to study if MG could modify albumin antioxidant capacity. METHODS Bovine serum albumin was incubated with 1 mM MG at 37 degrees C for 7 days (MG-BSA). Albumin physico-chemical changes were evaluated by tryptophan autofluorescence measurement in the presence or in the absence of a quencher (acrylamide). Albumin antioxidant capacity was determined by thiol measurement using Ellmans reagent as well as in a cellular system (HeLa cells stressed by H2O2). RESULTS MG-BSA exhibited important modifications as shown by conformational changes, decreased tryptophan autofluorescence (30%) and significant thiol loss (40%). MG-BSA led to important modifications resulting in oxidation and loss of albumin antioxidant capacity. MG-BSA modifications were close to the one observed in albumin isolated from diabetic patients. CONCLUSION Our results suggest that deleterious effects induced by carbonyl stress in diabetes could also originate from a loss of albumin antioxidant capacity by dicarbonyl compound attack. The biological consequences of these findings have now to be investigated.

Effects of advanced glycation end products on glycosphingolipids of retinal pericytes

Diabetic retinopathy is a chronic diabetic complication characterized by alterations of cell-cell and cell-matrix interactions in retinal microvessels. Advanced glycation end products (AGES) accumulate in microvessels during diabetes and they affect vascular cell biology. Glycosphingolipids are components of the cell plasma membrane and they have an important role in cell-cell interactions and cell growth regulation. The aim of this study was to investigate the effect of AGES on glycosphingolipid metabolism in retinal microvascular cells such as pericytes. Bovine retinal pericytes were cultured with either albumin or AGES. AGES were prepared by incubation of albumin with methylglyoxal at 37°C during 50h. Glycosphingolipids were metabolically radiolabelled with 14C-galactose, extracted and analysed by HPTLC and autoradiography. GM3 fatty acids of pericytes were analysed by GC-MS after derivatization with PFB and BSTFA. The results obtained show that AGES affect the glycosphingolipid composition of retinal pericytes in culture. AGES increased the three major neutral glycosphingolipidsdetected in these cells: globoside (+30%), CTH (+20%) and CMH (+80%). GM3 and GD3 were the main gangliosides detected. GM3 was increased by AGES (+30%) whereas GD3 was decreased (-35%), suggesting an alteration of the sialyltransferase II activity which is responsible for GD3 synthesis. GM3 fatty acid analysis suggests that AGES induced only quantitative but not qualitative changes on retinal pericyte glycosphingolipids. These results show a new metabolic effect of AGES modifying the composition of cell glycosphingolipids which play a main role in cell-cell interactions and cell growth. Further investigations will be needed to determine whether these modifications could be involved in the microvascular alterations observed in diabetes.