Insulin resistance is a characteristic feature of Type II diabetes, of uncontrolled Type I diabetes, and is also associated with a number of heterogenous conditions. The major objective of this study is to elucidate biochemical mechanisms which may contribute to insulin resistance in insulinopenic (Type I) diabetes, using rats with streptozotin (STZ)- induced diabetes as a model. l.) Several lines of evidence suggest that processing (N-glycosylation) of the insulin receptor is altered in rats with severe STZ-diabetes, suggesting that processing of other proteins may also be affected. Possible post-translational modification of glucose transporters (GLUT) from liver, muscle and adipose tissue of STZ-diabetic and control rats will be examined by 2-D gel electrophoresis (2-D SDS- PAGE) followed by immunoblotting with site-specific antibodies; with or without antecedent glycosidase treatment. Since 2-D SDS-PAGE reveals several processing isoforms of specific GLUT, cell fractionation studies will test the hypothesis that certain processing isoforms of GLUT4 are preferentially translocated to the plasma membrane in response to insulin. N-linked glycosylation and Golgi processing of a model peptide 125-I-N- octanoyl-Asp-Thyr-Thr-N (OTP) will be studied in hepatocytes of control and diabetic rats, to assess a defect in ER or Golgi processing and/or vesicular transport associated with diabetes. 2.) Increased flux through the hexosamine synthetic pathway has been proposed as a mechanisms of glucose-induced, glucose transport insulin resistance. Products of this pathway.may alter the glycosylation/processing of glycoproteins. The rate limiting enzyme for glucose entry into this pathway is glutamine: fructose-6-P amido transferase (GFAT). The regulation of GFAT, products of the pathway and the biochemical mechanisms by which these products may cause insulin resistance will be studied in tissue culture systems and in isolated muscles. The methods developed in these studies will then be applied in vivo, to evaluate the possible role of this pathway in glucose- induced insulin resistance in muscle and adipose tissue of intact animals, i.e. control rats, controls infused with glucose and diabetic rats. In a pilot study, possible alterations in this pathway will be examined in a murine model of Type II diabetes.
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