Insulin excites anorexigenic proopiomelanocortin neurons via activation of canonical transient receptor potential channels

Abstract

Proopiomelanocortin (POMC) neurons within the hypothalamic arcuate nucleus are vital anorexigenic neurons. Although both the leptin and insulin receptors are coupled to the activation of phosphatidylinositide 3 kinase (PI3K) in POMC neurons, they are thought to have disparate actions on POMC excitability. Using whole-cell recording and selective pharmacological tools, we have found that, similar to leptin, purified insulin depolarized POMC and adjacent kisspeptin neurons via activation of TRPC5 channels, which are highly expressed in these neurons. In contrast, insulin hyperpolarized and inhibited NPY/AgRP neurons via activation of KATP channels. Moreover, Zn(2+), which is found in insulin formulations at nanomolar concentrations, inhibited POMC neurons via activation of KATP channels. Finally, as predicted, insulin given intracerebroventrically robustly inhibited food intake and activated c-fos expression in arcuate POMC neurons. Our results show that purified insulin excites POMC neurons in the arcuate nucleus, which we propose is a major mechanism by which insulin regulates energy homeostasis.

Figure 1. Insulin excites guinea pig POMC and kisspeptin neurons and inhibits NPY /AgRP neurons

(A) Identification of arcuate neurons following whole-cell recording with scRT-PCR. Representative gels illustrating the mRNA expression of POMC, Kiss1 and NPY in ten arcuate neurons that were harvested after whole-cell recording. The expected sizes for the PCR products are as follows: for POMC, 206 bp; for Kiss1, 67 bp; for NPY, 126 bp; and for GAPDH, 212 bp. Cells that were negative for POMC, Kiss1 and NPY were tested for GAPDH to confirm the presence of mRNA. Hypothalamic tissue RNA was also included as positive control (+, with RT) and negative control (−, without RT). MM, molecular markers. (B) GP insulin (20 nM) depolarized POMC neurons and induced firing. (C) In voltage clamp, insulin induced an inward current. (D) The insulin-activated inward current and its I–V relationship in POMC neurons. a, Rapid application of 20 nM insulin induced a robust inward current with an internal solution containing 130 mM Cs⁺ gluconate and Ca²⁺ and K⁺ channel blockers in the extracellular CSF (see the Experimental Procedures), V_hold = −40 mV. b, Voltage ramps from 0 to 100 mV were applied (over 2 s) before and during the treatment with insulin. The I–V relationship for the insulin-induced current was obtained by digital subtraction of the control I–V from the I–V in the presence of insulin (20 nM). The reversal potential of the nonselective cation current was 10 mV. (E) Similar to POMC neurons, insulin induced an inward current in kisspeptin neurons that reversed at −10 mV (I/V not shown). (F and G) GP insulin induced an outward current in NPY/AgRP neurons (F), and the I–V plot of the cell shows that the reversal potential is close to E_K+ (−90 mV; G).

Figure 2. Insulin depolarizes and excites mouse POMC neurons and hyperpolarizes and inhibits NPY/AgRP neurons

(A) GP insulin (20 nM) depolarized and induced firing in a mouse POMC neuron. (B) The I–V relationship for the insulin-induced current was obtained by digital subtraction of the control I–V from the I–V in the presence of insulin (20 nM) with a Cs⁺-based internal solution and K⁺ channel blockers in the extracellular CSF (see the Experimental Procedures). The reversal potential of the nonselective cation current was −10 mV (n=3). The I–V relationship showed a typical doubly rectifying shape. (C) Summary of the effects of bovine insulin (150 nM, Sigma-Aldrich I1882), and human recombinant insulin (150 nM, Sigma-Aldrich I9278) to depolarize mouse POMC neurons in comparison to purified guinea pig insulin (20 nM, Nationl Institutes of Health). (D) GP insulin (20 nM) inhibited the firing and hyperpolarized (−10.7 mV) a NPY/AgRP neuron, and the effects of insulin were antagonized by the K_ATP channel blocker tolbutamide (200 μM). (E) I/V plot revealed that the insulin-induced outward current had a reversal potential close to E_K⁺. The voltage protocol consisted of 1s steps every 10 mV from −50 to −110 mV (Data not shown). V_hold = −60 mV. (F) Change in the membrane potential of arcuate NPY/AgRP neurons with application of 20 nM guinea pig insulin and after the addition of 200 μM tolbutamide (n=6). Data points represent the mean ± SEM. *p < 0.05.

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.

Figure 4. Human insulin formulation and Zn²⁺ inhibit POMC neurons

(A) Human insulin preparation (Humulin R) hyperpolarized and inhibited the firing of mouse POMC neurons, which was reversed by tolbutamide (200 μM). (B) The effect was mimicked by Zn²⁺ (0.1–10 μM). (C) In the presence of TTX to block action potential firing, Zn²⁺ hyperpolarized POMC neurons. (D) A representative I–V plot illustrating that the Zn²⁺-activated currents, measured in voltage clamp and in the presence of TTX, reversed at −90 mV (E_K⁺), indicative of the activation of a K⁺ current. (E) A logistics fit of the concentration-response curve for the Zn²⁺-induced outward current yielded an EC₅₀ = 75 nM, which is similar to the Zn²⁺ concentration in 150 nM human insulin preparations. The maximum outward current was obtained at 1 μM Zn²⁺ with higher concentrations showing no further increase in outward current.

Figure 5. Guinea pig insulin given intracerebroventrically robustly inhibited food intake and increased energy expenditure and expression of c-fos in the arcuate nucleus

(A–C) Insulin decreased food intake (A) concomitant with decreases in meal size (B) and frequency (C). (D–F) Insulin increased O₂ consumption (D) as well as CO₂ (E) and metabolic heat production (F). Bars represent means ± SEM (of the incremental hourly intake, meal size, and meal frequency or of the O₂ consumption, CO₂ production and metabolic heat production observed, respectively) in animals fed a standard, grain-based diet and treated with either insulin (4 mU; ICV), or its 0.9% saline vehicle (2 μl; ICV). The asterisk shows values from insulin-treated animals that are significantly different (repeated measures, multifactorial ANOVA/ least significant difference; p< 0.05) than those from vehicle-treated controls (n = 5 in each group). (G and H) Insulin stimulated c-fos expression in the arcuate nucleus. c-fos immunofluorescent micrographs of the arcuate nucleus from a saline (G–a)- and insulin (G–b)- treated guinea pig. The scale bar represents 100 μm. (H) The bar graph represents mean (± SEM) of c-fos positive neurons per section. (I) The bar graph depicts the mean percentage (± SEM) of POMC neurons that express c-fos following ICV insulin versus vehicle in anesthetized mice. Inset displays coexpression (yellow) of a POMC neuron and c-fos. Insulin increased c-fos expression by 2-fold (p<0.001, Student’s t-test).

Figure 6. A cellular model of insulin and leptin signaling via TRPC channel activation in the POMC neurons

Based on the current findings and other published data, we propose convergence of leptin and insulin signaling via IRS-PI3K to activate TRPC1 and TRPC5 channels in POMC neurons, which generates an inward cationic current to depolarize POMC neurons and increase their excitability. PI3 kinase (p85/p110) will also accelerate the insertion of TPRC channels into the membrane (Bezzerides et al., 2004).