Background Adiponectin (Ad) is a fat-derived protein that plays an important role in insulin sensitivity. Low circulating Ad levels are associated with insulin resistance and hyperinsulinemia and may be responsible for an increased risk of atherosclerosis. A reciprocal relationship has been observed between insulin resistance and Ad levels; however, the factors leading to low Ad levels in insulin resistant states have not yet been identified.
Methods We studied the effects of insulin on Ad production and release by treating cultured 3T3-L1 adipocytes with increasing concentrations of insulin. Pharmacological inhibitors of mitogen-activated protein (MAP) kinase (PD98059) and of phosphatidylinositol (PI) 3-kinase (wortmannin) were used to identify the signaling pathway through which insulin affects Ad. Intracellular Ad content and secreted Ad in cell culture media were measured by Western blot, whereas Ad mRNA levels were determined by real-time RT-PCR.
Results Insulin treatment resulted in a dose-and time-dependent decrease in intracellular Ad, with an associated increase in secreted Ad. The maximal effect was observed with 100nM insulin after 24 hours incubation, resulting in a 24% decrease in intracellular Ad (p≤0.05), and a 53% increase in secreted Ad (p≤0.05). The half-maximal effect was observed at a physiologic concentration of insulin (0.7nM) at 5 hours. Insulin in concentrations of 1nM, 10nM, and 100nM had no effect on Ad mRNA. Pre-treatment of 3T3-L1 adipocytes with wortmannin but not PD98059 significantly inhibited insulin-stimulated Ad secretion (p≤0.05). Inhibition of either the PI 3-kinase- or MAP-kinase-dependent pathway diminished basal production of Ad. Both wortmannin and PD98059 inhibited adiponectin mRNA levels by approximately 50% (p≤0.01).
Conclusions Our results demonstrate a stimulatory effect of insulin on Ad secretion that is dependent on the PI 3-kinase signaling pathway. We also find that both the PI 3-kinase- and MAP kinase-dependent pathways are necessary for basal Ad production. These results suggest an explanation for the observed hypoadiponectinemia in patients with insulin resistance, when defects in the PI 3-kinase-dependent signaling pathway lead to decreased Ad production, inadequate Ad secretion, and therefore low circulating Ad levels.
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