Absence of the Inhibitory Effect of Guanine Nucleotides on Adenylate Cyclase Activity in White Adipocyte Membranes of the Ob/ob Mouse. Effect of the Ob Gene

In mice homozygous for the ob gene (ob/ob), the response of adipose tissue adenylate cyclase to stimulation by lipolytic hormones is abnormally low in comparison to that in lean mice (+/+). Studies on the kinetics of adenylate cyclase activation in white adipocyte membranes under a variety of conditions show the following differences between +/+ and ob/ob mice. 1) The inhibitory effects of GTP and guanyl-5'-yl imidodiphosphate, which were clearly seen in +/+ membranes, were absent in the ob/ob membranes. 2) Half-maximal activation by GTP (in the presence of isoproterenol) required at least 10 times more GTP in ob/ob than in +/+ membranes. 3) Increasing the magnesium concentration (up to 10 mM) of the assay medium facilitated the activation of cyclase by modulatory ligands proportionately more in ob/ob than in +/+ membranes; in the +/+ membranes, 10 mM Mg2+ abolished the inhibitory effects of GTP. 4) Treatment with pertussis toxin attenuated the inhibitory effects of guanine nucleotides in +/+ membranes; no effect of the treatment was seen in the ob/ob membranes. 5) Pretreatment of membranes with cholera toxin facilitated cyclase activation proportionately more in ob/ob than in +/+ membranes; in addition, this treatment led to a shift to the left of the GTP dose-response curve in the ob/ob membranes. Cholera and pertussis toxins catalyzed the incorporation of ADP-ribose into their respective substrates in both the +/+ and the ob/ob membranes, showing that the alpha subunits of the stimulatory and inhibitory proteins of the regulatory component Ns and Ni, respectively are present in both types of membranes. Taken together, the results are consistent with the hypothesis that an excess of beta subunit (either primary or secondary to an altered interaction between beta and Ni alpha or Ns alpha) is responsible for the altered sensitivity to activating ligands of the adipocyte adenylate cyclase of the ob/ob mouse. In addition to these findings, we report an effect of the ob gene on the expression of adenylate cyclase activity, since adipose tissue cyclase from heterozygous lean mice (+/ob) showed characteristics which were intermediate between those of +/+ and ob/ob membranes.