Induction of aldose reductase in cultured human microvascular endothelial cells by advanced glycation end products.

Accelerated formation and accumulation of advanced glycation end products, as well as increased flux of glucose through polyol pathway, have been implicated in the pathogenesis of diabetic vascular complications. We investigated effects of advanced glycation end products on the levels of aldose reductase mRNA, protein, and activity in human microvascular endothelial cells. When endothelial cells were cultured with highly glycated bovine serum albumin, aldose reductase mRNA in endothelial cells demonstrated concentration-dependent elevation. The increase in aldose reductase mRNA was accompanied by elevated protein expression and enzyme activity. Significant increase in the enzyme expression was also observed when endothelial cells were cultured with serum obtained from diabetic patients with end-stage renal disease. Pretreatment of the endothelial cells with probucol or vitamin E prevented the advanced glycation end products-induced increases in aldose reductase mRNA and protein. Electrophoretic mobility shift assays using the nuclear extracts of the endothelial cells treated with advanced glycation end products showed enhancement of specific DNA binding activity for AP-1 consensus sequence. These results indicate that accelerated formation of advanced glycation end products in vivo may elicit activation of the polyol pathway, possibly via augmented oxidative stress, and amplify endothelial cell damage leading to diabetic microvascular dysfunction.

Increased levels of aldose reductase in peripheral mononuclear cells from type 2 diabetic patients with microangiopathy.

Aldose reductase (AR) protein was measured in peripheral mononuclear cells (PMCs) from 55 patients with type 2 diabetes by a two-site ELISA using anti-human AR monoclonal antibody. AR levels did not correlate with age, duration of diabetes, and HbAlc. Furthermore, no significant differences were found in AR levels between the patients and healthy subjects. Thirty seven patients had at least one of diabetic microangiopathy; retinopathy, neuropathy, or nephropathy. AR levels were significantly higher in the patients with microangiopathy than in those without it (52.3 +/- 15.7 vs. 43.0 +/- 15.2 ng/10(6) cells, P < 0.05). The patients with neuropathy had significantly higher AR levels than those without neuropathy (53.7 +/- 15.8 vs. 42.7 +/- 14.3 ng/l0(6) cells, P < 0.05). The same result applied to the patients with retinopathy (54.5 + 15.4 vs. 44.6 +/- 15.3 ng/10(6) cells, P < 0.05). The AR levels in the patients with nephropathy tended to give a higher value than those without it. However, there were no significant differences between the two (53.9 +/- 3.6 vs. 46.4 +/- 2.6 ng/10(6) cells, NS). These results indicate that AR levels in PMCs from type 2 diabetic patients are associated with the presence of microangiopathy. The measurement of AR proteins in PMCs with this ELISA system is a useful tool for the clinical study of diabetic complications, and would increase our understanding of the pathogenesis of the disease.

Reversal by glucose of pancreatic amylase insufficiency in chronic alcoholic rats.

Carbohydrate consumption regulates pancreatic amylase synthesis in rats. The Lieber-DeCarli 36% alcohol diet employed in chronic alcohol studies and the isocaloric control diet contain 11 and 47% of total calories from carbohydrates, respectively. Young rats fed ad libitum the 36% ethanol diet for 2 weeks obtained 1.2 g/day of carbohydrate, whereas those pair-fed with control diet received 5.8 g/day. Rats fed the 36% ethanol diet and given an intramuscular injection of a solution of 1.5 g of glucose daily for 2 weeks received twofold greater amounts of carbohydrate than saline-injected controls (2.7 versus 1.2 g). These changes in carbohydrate intake produced proportionate changes in pancreatic amylase levels. The secretory responses to cholecystokinin-octapeptide (CCK8) of acini from control and glucose-injected rats were significantly higher compared with those in the saline-injected or noninjected alcohol groups. The blood alcohol levels in glucose-injected rats were markedly reduced compared with other alcohol groups (71.7 versus 274.9 mg/dl) despite similar amounts of ethanol ingestion daily (2.4 g) in the three groups. In vitro experiments with acini from rats fed a nutritionally optimal diet revealed that high pharmacologic concentrations of ethanol, while inducing basal secretion, inhibited CCK8-stimulated amylase secretion. These results indicate that: (a) the amount of alcohol consumption does not correlate with either the levels of blood alcohol or of pancreatic amylase; (b) the carbohydrate availability in rats regulates pancreatic amylase levels despite significant levels of alcohol in blood; (c) blood alcohol levels observed in vivo may not affect synthetic and secretory processes of amylase in pancreatic acini.

Regulation of pancreatic amylase by dietary carbohydrate in chronic alcoholic rats.

The nutritional adequacy of dietary ingredients is essential for optimal food consumption and growth of animals. Dietary carbohydrate levels regulate pancreatic amylase synthesis. Ethanol diets with 36% of total calories from ethanol and 11% from carbohydrate are nutritionally inadequate, whereas a 26% ethanol diet made isocaloric to the 36% alcohol diet by the addition of maltose-dextrins provides all nutrients in amounts recommended for normal growth. Young rats fed the modified ethanol diet for 3 months consume 101.4 ml of diet daily compared to 66.5 ml by those on the 36% ethanol diet. Increased food consumption results in (a) similar amounts of alcohol consumption (3.6 vs. 3.3 g/day), (b) a threefold enhancement in carbohydrate intake (5.1 vs. 1.7 g/day), and (c) a normal growth rate (6.7 vs. 3.1 g/day). Both the acinar content of amylase (20.2 +/- 0.3 micrograms/mg of protein) and the acinar response to cholecystokinin-octapeptide in 36% ethanol diet-fed rats are significantly reduced compared to those of 26% ethanol diet-fed rats (34.1 +/- 5.6 micrograms/mg of protein). These results confirm (a) the nutritional adequacy of the 26% ethanol diet compared to the 36% ethanol diet, and (b) that carbohydrate inadequacy, and not ethanol consumption per se, is the primary cause of pancreatic amylase insufficiency in chronic alcoholic rats.