Abstract
Insulin resistance in diabetes mellitus has been associated with aberrant expression of glucose transporter proteins. We examined the effect of gestational diabetes on skeletal muscle Glut-4 and liver Glut-2 protein expression in the macrosomic offspring of the hyperglycemic rat. Five time-dated pregnant Sprague-Dawley rats were made mildly hyperglycemic (plasma glucose 100-300mg/dl) by intraperitoneal injection of 37 mg/kg streptozotocin on day five of gestation. Two rats were injected with vehicle alone to serve as controls. Macrosomia was selectively defined as birth weight >2SD of mean control pup birth weight (Control pups' mean birth weight: 6.29g±0.49, Macrosomia pups' mean birth weight: 7.40g±0.16). Animals were sacrificed at 10-12 weeks of age. Skeletal muscle and liver samples were quickly removed and frozen in liquid nitrogen. 100 mg of muscle or liver homogenate was solubulized in Laemmli buffer. Protein content was determined by Bio-Rad assay with bovine serum albumin as the standard. Samples were separated by SDS-PAGE and transferred to nitrocellulose. Membranes were incubated with primary antibody (rabbit anti-rat Glut-4 for muscle and rabbit anti-rat Glut-2 for liver) followed by anti-rabbit Ig, horseradish peroxidase linked secondary antibody. Western blot analysis was performed using Amersham ECL reagent. Densitometric quantitation data (OD units) are as follows: Control muscle Glut-4 9.18±0.14, Macrosomic muscle Glut-4 3.51±0.07 p<0.01; Control liver Glut-2 2.38 ±0.54, Macrosomic liver Glut-2 3.49 ±0.19 p <0.05. In the macrosomic offspring of the diabetic dam, muscle GLUT-4 protein expression was decreased by 62% and liver Glut-2 protein expression was increased by 46% compared to controls. Decreased Glut-4 protein expression in muscle peripherally, in contrast to increased Glut-2 protein expression in liver centrally, may partially account for the presence of insulin resistance known to occur in the offspring of the gestational diabetic.
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