benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Diabetes-Mellitus

Apoptosis of retinal capillary cells begins early in diabetes and likely contributes to the capillary obliteration that is an important feature of diabetic retinopathy. Caspases are proteolytic enzymes that are closely involved in the induction and execution phases of apoptosis, but their role in the development of diabetic retinopathy has not been studied previously. Our study focused on the measurement of activities of multiple caspases in retinas of mice at different durations of diabetes. Several caspases (including caspases-1, -2, -6, -8, and -9) were activated as early as 2 months of diabetes. The caspases activity pattern changed with increasing duration of disease, suggesting a slowly developing caspases cascade. Activities of executioner caspases (e.g., cas-6 and -3) became elevated after longer duration of diabetes, and the induction of cas-3 activity was associated with the duration of diabetes at which capillary cells begin to show evidence of undergoing apoptosis. Retinas from patients with type 2 diabetes likewise showed a significant increase in activities of cas-1, -3, -4, and -6. For comparison, retinal caspases were also measured in experimental galactosemia, another model that develops a diabetic-like retinopathy. The pattern of caspases activation differed between diabetes and galactosemia, but cas-1 activity became elevated soon after elevation of blood hexose concentration in both. Caspases offer new therapeutic targets to test the role of apoptosis in the development of diabetic retinopathy.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Biomarkers; Caspase 1; Caspase 3; Caspases; Cattle; Cells, Cultured; Cysteine Proteinase Inhibitors; Diabetes Mellitus; Diabetes Mellitus, Experimental; Endothelium, Vascular; Enzyme Activation; Galactosemias; Humans; Male; Mice; Mice, Inbred C57BL; Reference Values; Retina; Retinal Vessels

Ceramide has been implicated as a mediator of insulin resistance induced by tumor necrosis factor-alpha (TNF) in adipocytes. Adipocytes contain numerous caveolae, sphingolipid and cholesterol-enriched lipid microdomains, that are also enriched in insulin receptor (IR). Since caveolae may be important sites for crosstalk between tyrosine kinase and sphingolipid signaling pathways, we examined the role of increased caveolar pools of ceramide in regulating tyrosine phosphorylation of the IR and its main substrate, insulin receptor substrate-1 (IRS-1). Neither exogenous short-chain ceramide analogs nor pharmacologic increases in endogenous caveolar pools of ceramide inhibited insulin-induced tyrosine phosphorylation of the IR and IRS-1. However, inhibition of TNF-induced caveolar ceramide production reversed the decrease in IR tyrosine phosphorylation in response to TNF. These results suggest that TNF-independent increases in caveolar pools of ceramide are not sufficient to inhibit insulin signaling but that in conjunction with other TNF-dependent signals, caveolar pools of ceramide are a critical component for insulin resistance by TNF.

Topics: 3T3 Cells; Adipocytes; Amino Acid Chloromethyl Ketones; Animals; Caveolae; Ceramides; Cysteine Proteinase Inhibitors; Diabetes Mellitus; Glucosyltransferases; Insulin; Insulin Resistance; Mice; Morpholines; Obesity; Receptor Cross-Talk; Receptor, Insulin; Sphingolipids; Tumor Necrosis Factor-alpha