Figure 10. Effects of insulin on glucose uptake, lipogenesis, and activation of PKB in adipocytes from 3- to 3.

-month-old WT and Pde3b-KO mice. (A and B) Adipocytes (0.2 ml 15% cells [vol/vol] in A; 1 ml 2.0% cells [vol/vol] in B) were incubated with the indicated concentrations of insulin for 10 minutes (A; n = 3) and 3 hours (B; n = 5). Uptake of 2-[1-³H]-deoxyglucose (A) and incorporation of D-[³H]-glucose into lipids (B) were measured as described in Methods and expressed as fold increase relative to nonstimulated cells. In KO adipocytes, basal lipogenesis was 56% ± 0.08% (mean ± SEM; P = 0.0001) that of WT adipocytes. Data (mean ± SEM) are from 5 independent experiments, each of which used adipocytes pooled from 2–3 mice. *P < 0.05; **P < 0.01. (C) Western blot analysis of FAS from WT and KO mice (20 μg protein/lane; n = 3) using anti-FAS antibody. (D) Adipocytes (each batch consisted of adipocytes from 2 of a total 6 WT and 6 KO mice) were incubated for 10 minutes with or without 1 nM insulin. Adipocyte fractions, prepared as described in Methods, were subjected to Western blotting with antibody recognizing PKB phosphorylated on serine 473 and, after stripping, with anti-PKB antibody. One Western blot representative of 3 is shown. PKB/phosphorylated PKB bands from 6 WT and 6 KO mice were quantified.