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Altered expression and subcellular localization of diacylglycerol-sensitive protein kinase C isoforms in diabetic rat
T Babazono1, J Kapor-Drezgic, J A Dlugosz
1Juvenile Diabetes Foundation/Medical Research Council of Canada Group in Diabetic Nephropathy, Department of Medicine, University of Toronto, Ontario.
Abstract:
Protein kinase C (PKC) is implicated in the pathogenesis of diabetic nephropathy. This study was designed to identify the expression of diacylglycerol (DAG)-sensitive PKC-alpha, -betaII, -delta, and -epsilon isoforms in normal and diabetic rat glomerular cells and to determine the effects of high glucose and insulin on PKC isoform cellular compartmentalization and PKC activity. Diabetic rats treated with or without insulin and normal rats were examined 2 and 4 weeks after streptozotocin/vehicle injection. Renal cortical tissue immunogold-labeled with anti-PKC-alpha, -betaII, -delta, or -epsilon antibody was visualized by electron microscopy. From isolated glomeruli, total cell lysate and cytosol and membrane fractions were immunoblotted with the same anti-PKC isoform antibodies. PKC activity in isolated glomeruli was measured by 32P-phosphorylation of the epidermal growth factor (EGF)-receptor substrate. Immunogold labeling revealed expression of the four PKC isoforms by glomerular visceral epithelial, endothelial, and mesangial cells of both normal and diabetic rats. Immunoblot analysis of the diabetic rat glomeruli at 2 weeks demonstrated a significant increase in membrane-associated PKC-alpha, -delta, and -epsilon and a significant decrease in membrane PKC-betaII content compared with normal, which were similar at 4 weeks. Insulin treatment normalized membrane PKC isoform contents and caused a significant decrease in the cytosol content of PKC-alpha, -betaII, and -delta and total cellular PKC-alpha compared with normal. Although PKC activity in the cells of diabetic rat glomeruli was increased by 20% compared with normal, the difference did not reach statistical significance. In insulin-treated diabetic rat glomeruli, PKC activity was significantly decreased compared with non-insulin-treated diabetic rat glomeruli. In conclusion, DAG-sensitive PKC-alpha, -betaII, -delta, and -epsilon isoforms are all found in the three major glomerular cell types in rats, and the expression, compartmentalization, and activity are modulated independently by high glucose and insulin.
Insights
High glucose alters protein kinase C (PKC) isoform expression and localization in diabetic nephropathy. Insulin therapy helps normalize these changes, impacting PKC activity in kidney cells.
Area of Science:
- Nephrology
- Cell Biology
- Biochemistry
Background:
- Diabetic nephropathy is a major complication of diabetes.
- Protein kinase C (PKC) is implicated in its pathogenesis.
- Understanding PKC isoform regulation is crucial for therapeutic development.
Purpose of the Study:
- To investigate the expression and cellular localization of diacylglycerol (DAG)-sensitive PKC-alpha, -betaII, -delta, and -epsilon isoforms in normal and diabetic rat glomerular cells.
- To determine the effects of high glucose and insulin on PKC isoform compartmentalization and activity.
Main Methods:
- Streptozotocin-induced diabetic rat model.
- Immunogold labeling and electron microscopy for cellular expression.
- Western blotting of glomerular fractions for protein compartmentalization.
- Measurement of PKC activity via substrate phosphorylation.
Main Results:
- All four PKC isoforms are expressed in glomerular cells of normal and diabetic rats.
- Diabetes increased membrane-associated PKC-alpha, -delta, and -epsilon, and decreased membrane PKC-betaII.
- Insulin treatment normalized membrane PKC levels and reduced cytosol content.
- PKC activity was not significantly increased in diabetic glomeruli but was reduced by insulin.
Conclusions:
- DAG-sensitive PKC isoforms are present in rat glomerular cells.
- High glucose and insulin independently modulate PKC expression, compartmentalization, and activity.
- These findings offer insights into the role of PKC in diabetic nephropathy and potential therapeutic targets.