Figure 5.

Chronic insulin exposure attenuates leptin-induced phosphorylation of key signaling molecules. rHypoE-19 and mHypoA-2/10 neurons were pretreated with PBS vehicle or 100 nM insulin for 24 hours. The cells were then washed and allowed to recover in fresh medium for 1 hour before being rechallenged with 10 nM leptin or PBS vehicle. Protein was isolated at 15 minutes after treatment and analyzed using Western blot analysis with phospho-specific antibodies against AKT (a), ERK1/2 (b), or STAT3 (c and d), and protein levels were normalized to total AKT, total ERK1/2, or total STAT3 levels, respectively. Data are shown with SEMs (n = 3–5 independent experiments). *, P < .05; **, P < .001; as per t tests.