Figure 3. Insulin response requires TRPC channel activation and PI3 kinase.

(A–E) Representative traces of the insulin-induced currents in the presence of the TRPC channel blocker 2-APB (100 μM) (A) or TRPC5 channel enhancers La³⁺ (100 μM) (B) and rosiglitazone (C and D) or PI3K inhibitor wortmannin (100 nM) (E). V_hold = −60 mV. Similar to La³⁺, rosiglitazone (100 μM) potentiated insulin-activated inward currents. (F) Bar graphs summarizing the effects of Wort, 2-APB, and TRPC 4,5 channel enhancers La³⁺ and rosiglitazone (100 μM) on the insulin-induced inward currents. La ³⁺ (100 μM) augmented the current by 5 fold and rosiglitazone (Rg) by about 2 fold. ***p < 0.001; **p < 0.01, *p < 0.05 significantly different from the control group (black bar). Cell numbers are indicated. (G) The I–V relationship for the rosiglitazone (100 μM) -induced current was obtained by digital subtraction. Note the similar double rectifying I/V plots and reversal potentials for insulin and rosiglitazone. (H) Immunoreactive TRPC5 channel protein is expressed in POMC neurons. Representative photomicrographs illustrating low- (a, b, c) and high- power (d, e, f) images of immunoreactive β-endorphin (a and d; POMC product labeled with Cy2, green), Immunoreactive TRPC5 (b and e; labeled with Cy3, red), and the combined images of the same cells (c and f; yellow). TRPC5 was expressed in the majority of β-endorphin neurons. Note, arrows indicate the cells with co-localization of β-endorphin and TRPC5. IR-TRPC5 is also present in unidentified arcuate neurons (red cells without arrow). Scale bars = 45 μm. (I) Based on qPCR, TRPC5 transcipts are expressed many fold higher in POMC versus NPY/AgRP neurons.