Adipocyte beta-adrenergic sensitivity is compromised in animal models of obesity and type 2 diabetes. Although changes in the membrane concentrations of G-protein alpha subunits (Galpha) have been implicated, it remains to be determined how these changes are affected by insulin resistance in the different animal models. Because previous studies used young animals, we measured the concentrations of Galpha and Gbeta subunits in epididymal fat from aged (48 weeks old) db/db mice and from their lean littermates to more closely reproduce the model of type 2 diabetes mellitus. Levels of immunoreactive Galphas, Galphai(1/2), Galphao and Galphaq/11 were all significantly greater in adipocyte membranes from the db/db mice than in membranes from their lean non-diabetic littermate controls. Levels of Galphai(1) and Galphai(2) were also individually determined and although they appeared to be slightly higher in db/db membranes, these differences were not significant. Although the levels of both Galphas isoforms were elevated, levels of the 42 and 46 kDa proteins rose by approximately 42% and 20% respectively, indicating differential protein processing of Galphas. By contrast, levels of Galphai3 were similar in the two groups. The levels of common Gbeta and Gbeta2 were also elevated in db/db mice, whereas Gbeta1 and Gbeta4 levels were not different. To determine whether these changes were due to insulin resistance per se or to elevated glucocorticoid production, G-protein subunit levels were quantified in whole cell lysates from 3T3-L1 adipocytes that were stimulated with different concentrations of either insulin or corticosterone. Although none of the subunit levels was affected by insulin, the levels of both Galphas isoforms were increased equally by corticosterone in a concentration-dependent manner. Since glucocorticoids are known regulators of Galphas gene expression in many cell types and in adipocytes from diabetic rodents, the results presented herein appear to more accurately reflect diabetic pathophysiology than do those of previous studies which report a decrease in Galphas levels. Taken together, these results indicate that most of the selective changes in G-protein subunit production in adipocytes from this animal model of type 2 diabetes may not be due to diminished insulin sensitivity, but may be due to other endocrine or metabolic abnormalities associated with the diabetic phenotype.