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. 2013 Jul;62(7):2295-307.
doi: 10.2337/db12-1629. Epub 2013 Mar 25.

Extracellular signal-regulated kinase in the ventromedial hypothalamus mediates leptin-induced glucose uptake in red-type skeletal muscle

Chitoku Toda  1 , Tetsuya Shiuchi, Haruaki Kageyama, Shiki Okamoto, Eulalia A Coutinho, Tatsuya Sato, Yuko Okamatsu-Ogura, Shigefumi Yokota, Kazuyo Takagi, Lijun Tang, Kumiko Saito, Seiji Shioda, Yasuhiko Minokoshi
Affiliations

Extracellular signal-regulated kinase in the ventromedial hypothalamus mediates leptin-induced glucose uptake in red-type skeletal muscle

Chitoku Toda et al. Diabetes. 2013 Jul.

Abstract

Leptin is a key regulator of glucose metabolism in mammals, but the mechanisms of its action have remained elusive. We now show that signaling by extracellular signal-regulated kinase (ERK) and its upstream kinase MEK in the ventromedial hypothalamus (VMH) mediates the leptin-induced increase in glucose utilization as well as its insulin sensitivity in the whole body and in red-type skeletal muscle of mice through activation of the melanocortin receptor (MCR) in the VMH. In contrast, activation of signal transducer and activator of transcription 3 (STAT3), but not the MEK-ERK pathway, in the VMH by leptin enhances the insulin-induced suppression of endogenous glucose production in an MCR-independent manner, with this effect of leptin occurring only in the presence of an increased plasma concentration of insulin. Given that leptin requires 6 h to increase muscle glucose uptake, the transient activation of the MEK-ERK pathway in the VMH by leptin may play a role in the induction of synaptic plasticity in the VMH, resulting in the enhancement of MCR signaling in the nucleus and leading to an increase in insulin sensitivity in red-type muscle.

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Figures

FIG. 1.
FIG. 1.
Effects of leptin injection into the VMH on whole-body and muscle glucose metabolism in mice during a hyperinsulinemic-euglycemic clamp. A: Experimental protocol for the hyperinsulinemic-euglycemic clamp. B: Phosphorylation of STAT3 (on Tyr705), ERK (Thr202/Tyr204), and Akt (Ser473) in the VMH at 30 min after the unilateral injection of leptin (5 ng in 0.1 µL) or saline (0.1 µL) into the VMH. The data were evaluated with the ratio of phosphorylated form to the total protein and expressed as percent increase of the ratio to that of the saline-injected group. Representative immunoblots with antibodies to the phosphorylated (p) or total (t) forms of each protein are shown above densitometric quantitation of the relative phosphorylated/total protein ratio. *P < 0.05 vs. corresponding value for saline-injected group. C: Blood glucose levels during the basal and clamp periods. The clamp period begins at time 0. D: GIR required to maintain euglycemia during the clamp period. *P < 0.05 vs. corresponding value for saline-injected group. E: Rate of glucose disappearance (Rd) during the basal and clamp periods in mice injected with leptin or saline into the VMH unilaterally. F: Rate of glucose appearance (Ra) during the basal and clamp periods as well as the percentage suppression of Ra induced by insulin infusion. Ra reflects EGP. G: Rates of whole-body glycolysis and glycogen synthesis during the clamp period. H: 2DG uptake in muscle during the basal and clamp periods. I: Rates of glycolysis and glycogen synthesis in muscle during the clamp period. *P < 0.05 vs. basal, saline in VMH. †P < 0.05 vs. basal, leptin in VMH. #P < 0.05 vs. clamp, saline in VMH (D–I). All quantitative data are means ± SEM (n = 6 or 7 mice).
FIG. 2.
FIG. 2.
Leptin injection into the VMH increases whole-body insulin sensitivity via MEK-ERK and STAT3 signaling in the VMH. A: Phosphorylation of ERK (Thr202/Tyr204), STAT3 (Tyr705), and Akt (Ser473) in the VMH at 30 min after unilateral injection of leptin or saline into the VMH. The MEK inhibitor U0126 or PI3K inhibitor LY294002 was injected unilaterally into the VMH at 1 h before leptin injection, whereas the STAT3 inhibitor was injected into the VMH at both 1 h and 5 min before leptin injection. DMSO at 0.1% was injected as a control for the inhibitors. The data were evaluated with the ratio of phosphorylated form to the total protein and expressed as percent increase of the ratio to that of the saline-injected group. Representative immunoblots with antibodies to the phosphorylated (p) or total (t) forms of the proteins are shown above the quantitative data, which are means ± SEM (n = 6 or 7 mice). *P < 0.05 vs. corresponding saline-injected group. †P < 0.05 vs. corresponding value for leptin + DMSO in VMH. B–E: Effects of leptin and leptin signaling inhibitors on the time course of the increase in GIR (B), on Rd during the clamp period (C), on Ra during the clamp period and on the percentage suppression of Ra induced by insulin infusion (D), and on the rate of whole-body glycolysis or glycogen synthesis during the clamp period (E) for mice subjected to the hyperinsulinemic-euglycemic clamp protocol. The mean of the GIR values from 60 to 105 min is shown in the bar graphs (B). All data are means ± SEM (n = 6 mice). *P < 0.05 vs. corresponding value for saline-injected group. †P < 0.05 vs. corresponding value for leptin + DMSO in VMH. ‡P < 0.05 for leptin + MEK inhibitor in VMH vs. leptin + STAT3 inhibitor in VMH.
FIG. 2.
FIG. 2.
Leptin injection into the VMH increases whole-body insulin sensitivity via MEK-ERK and STAT3 signaling in the VMH. A: Phosphorylation of ERK (Thr202/Tyr204), STAT3 (Tyr705), and Akt (Ser473) in the VMH at 30 min after unilateral injection of leptin or saline into the VMH. The MEK inhibitor U0126 or PI3K inhibitor LY294002 was injected unilaterally into the VMH at 1 h before leptin injection, whereas the STAT3 inhibitor was injected into the VMH at both 1 h and 5 min before leptin injection. DMSO at 0.1% was injected as a control for the inhibitors. The data were evaluated with the ratio of phosphorylated form to the total protein and expressed as percent increase of the ratio to that of the saline-injected group. Representative immunoblots with antibodies to the phosphorylated (p) or total (t) forms of the proteins are shown above the quantitative data, which are means ± SEM (n = 6 or 7 mice). *P < 0.05 vs. corresponding saline-injected group. †P < 0.05 vs. corresponding value for leptin + DMSO in VMH. B–E: Effects of leptin and leptin signaling inhibitors on the time course of the increase in GIR (B), on Rd during the clamp period (C), on Ra during the clamp period and on the percentage suppression of Ra induced by insulin infusion (D), and on the rate of whole-body glycolysis or glycogen synthesis during the clamp period (E) for mice subjected to the hyperinsulinemic-euglycemic clamp protocol. The mean of the GIR values from 60 to 105 min is shown in the bar graphs (B). All data are means ± SEM (n = 6 mice). *P < 0.05 vs. corresponding value for saline-injected group. †P < 0.05 vs. corresponding value for leptin + DMSO in VMH. ‡P < 0.05 for leptin + MEK inhibitor in VMH vs. leptin + STAT3 inhibitor in VMH.
FIG. 3.
FIG. 3.
Leptin injection into the VMH increases glucose utilization in red-type muscle via MEK-ERK signaling in the VMH, whereas it enhances insulin-induced suppression of glycogen phosphorylase a activity in the liver via STAT3 in the VMH. Effects of leptin and the MEK inhibitor U0126 on 2DG uptake (A and B) as well as on glycolysis and glycogen synthesis (C) in soleus, Gastro-R, Gastro-W, or epiWAT during the clamp period of the hyperinsulinemic-euglycemic clamp protocol. Effects of leptin, the STAT3 inhibitor, and the PI3K inhibitor LY294002 on 2DG uptake in soleus (D), Gastro-W (E), or epiWAT (F) during the clamp period. G: Effects of leptin and leptin signaling inhibitors on glycogen phosphorylase a activity in liver during the clamp period. All tissue samples were obtained from the mice studied in Fig. 2B–E. All data are means ± SEM (n = 6 mice). *P < 0.05 vs. corresponding value for saline-injected group. †P < 0.05 vs. corresponding value for leptin + DMSO in VMH.
FIG. 4.
FIG. 4.
Systemic injection of leptin increases whole-body glucose utilization and glucose uptake in soleus muscle via MEK-ERK signaling in the VMH. A: Phosphorylation of STAT3 (on Tyr705), ERK (Thr202/Tyr204), and Akt (Ser473) in the VMH at 1 h after intraperitoneal injection of leptin (5 mg/kg) or saline (leptin i.p. or saline i.p.). The MEK inhibitor U0126 or DMSO (0.01%) was injected into the VMH bilaterally 1 h before injection of leptin or saline. The data were evaluated with the ratio of phosphorylated form to the total protein and expressed as percent increase of the ratio to that of saline i.p. + DMSO group. Representative immunoblots with antibodies to the phosphorylated (p) or total (t) forms of the proteins are shown above the quantitative data (n = 6 or 7 mice). *P < 0.05 vs. corresponding value for saline i.p. + DMSO in VMH. §P < 0.05 vs. corresponding value for saline i.p. + MEK inhibitor in VMH. †P < 0.05 vs. leptin i.p. + DMSO in VMH. B: Immunohistofluorescence analysis of phosphorylated forms of STAT3 (Tyr705) and ERK (Thr202/Tyr204) in the VMH at 1 h after intraperitoneal injection of saline (c, e, and g) or leptin (a, d, f, and h). Panels d, f, and h are digital zoom images corresponding to the boxed area in panel a, whereas panels c, e, and g represent the equivalent area in the VMH. Immunofluorescence of pSTAT3 (c and d), pERK (e and f), and both phosphorylated proteins (a, g, and h) is shown. The dashed trace in panels a and b represents the VMH. Panel b shows a Nissl-stained section of the medial hypothalamus from a C57BL/6 mouse and is modified with permission from Hof et al. (40). 3V, third ventricle. Scale bars, 20 µm. C–F: Effects of injection of the MEK inhibitor U0126 into the VMH on changes in glucose metabolism induced by intraperitoneal injection of leptin in mice subjected to the hyperinsulinemic-euglycemic clamp protocol. U0126 or DMSO was injected into the VMH bilaterally 1 h prior to leptin or saline injection (n = 5 or 6 mice). C: Glucose infusion rate during the clamp period. *P < 0.05 vs. corresponding value for saline i.p. + DMSO in VMH. †P < 0.05 vs. corresponding value for leptin i.p. + DMSO in VMH. D: Rd in basal and clamp periods. E: 2DG uptake in soleus, Gastro-W, and epiWAT during the basal and clamp periods. F: Ra during the basal and clamp periods as well as the percentage suppression of Ra induced by insulin infusion. The Ra in the basal period is equal to Rd in the basal period. *P < 0.05 vs. corresponding value for saline i.p. + DMSO in VMH in the basal period. #P < 0.05 vs. corresponding value for saline i.p. + DMSO in VMH in the clamp period. §P < 0.05 vs. corresponding value for saline i.p. + MEK inhibitor in VMH in the clamp period. ‡P < 0.05 vs. corresponding value for the basal period. †P < 0.05 vs. corresponding value for leptin i.p. + DMSO in VMH (D–F). All quantitative data are means ± SEM.
FIG. 4.
FIG. 4.
Systemic injection of leptin increases whole-body glucose utilization and glucose uptake in soleus muscle via MEK-ERK signaling in the VMH. A: Phosphorylation of STAT3 (on Tyr705), ERK (Thr202/Tyr204), and Akt (Ser473) in the VMH at 1 h after intraperitoneal injection of leptin (5 mg/kg) or saline (leptin i.p. or saline i.p.). The MEK inhibitor U0126 or DMSO (0.01%) was injected into the VMH bilaterally 1 h before injection of leptin or saline. The data were evaluated with the ratio of phosphorylated form to the total protein and expressed as percent increase of the ratio to that of saline i.p. + DMSO group. Representative immunoblots with antibodies to the phosphorylated (p) or total (t) forms of the proteins are shown above the quantitative data (n = 6 or 7 mice). *P < 0.05 vs. corresponding value for saline i.p. + DMSO in VMH. §P < 0.05 vs. corresponding value for saline i.p. + MEK inhibitor in VMH. †P < 0.05 vs. leptin i.p. + DMSO in VMH. B: Immunohistofluorescence analysis of phosphorylated forms of STAT3 (Tyr705) and ERK (Thr202/Tyr204) in the VMH at 1 h after intraperitoneal injection of saline (c, e, and g) or leptin (a, d, f, and h). Panels d, f, and h are digital zoom images corresponding to the boxed area in panel a, whereas panels c, e, and g represent the equivalent area in the VMH. Immunofluorescence of pSTAT3 (c and d), pERK (e and f), and both phosphorylated proteins (a, g, and h) is shown. The dashed trace in panels a and b represents the VMH. Panel b shows a Nissl-stained section of the medial hypothalamus from a C57BL/6 mouse and is modified with permission from Hof et al. (40). 3V, third ventricle. Scale bars, 20 µm. C–F: Effects of injection of the MEK inhibitor U0126 into the VMH on changes in glucose metabolism induced by intraperitoneal injection of leptin in mice subjected to the hyperinsulinemic-euglycemic clamp protocol. U0126 or DMSO was injected into the VMH bilaterally 1 h prior to leptin or saline injection (n = 5 or 6 mice). C: Glucose infusion rate during the clamp period. *P < 0.05 vs. corresponding value for saline i.p. + DMSO in VMH. †P < 0.05 vs. corresponding value for leptin i.p. + DMSO in VMH. D: Rd in basal and clamp periods. E: 2DG uptake in soleus, Gastro-W, and epiWAT during the basal and clamp periods. F: Ra during the basal and clamp periods as well as the percentage suppression of Ra induced by insulin infusion. The Ra in the basal period is equal to Rd in the basal period. *P < 0.05 vs. corresponding value for saline i.p. + DMSO in VMH in the basal period. #P < 0.05 vs. corresponding value for saline i.p. + DMSO in VMH in the clamp period. §P < 0.05 vs. corresponding value for saline i.p. + MEK inhibitor in VMH in the clamp period. ‡P < 0.05 vs. corresponding value for the basal period. †P < 0.05 vs. corresponding value for leptin i.p. + DMSO in VMH (D–F). All quantitative data are means ± SEM.
FIG. 5.
FIG. 5.
MCR in the VMH mediates leptin-induced whole-body glucose utilization and glucose uptake in soleus muscle, but not leptin-induced enhancement of the suppressive effect of insulin on EGP. The effects of the MCR antagonist SHU9119 injected into the VMH on changes in glucose metabolism induced by unilateral injection of leptin into the VMH were evaluated with the hyperinsulinemic-euglycemic clamp. SHU9119 or saline was injected into the VMH 1 h prior to leptin or saline injection. A: GIR during the clamp period. *P < 0.05 vs. corresponding value for saline + saline in VMH. †P < 0.05 vs. corresponding value for leptin + saline in VMH. B: Rd during basal and clamp periods. C: 2DG uptake in soleus, Gastro-W, and epiWAT during basal and clamp periods. D: Ra during basal and clamp periods as well as the percentage suppression of Ra by insulin infusion. Ra in the basal period is equal to Rd in the basal period. *P < 0.05 vs. corresponding value for saline + saline in VMH in the basal period. #P < 0.05 vs. corresponding value for saline + saline in VMH in the clamp period. §P < 0.05 vs. corresponding value for saline + SHU9119 in VMH in the clamp period. ‡P < 0.05 vs. corresponding value for the basal period. †P < 0.05 vs. corresponding value for leptin + saline in VMH (B–D). All data are means ± SEM (n = 6 or 7 mice).
FIG. 6.
FIG. 6.
An MCR agonist in the VMH increases whole-body glucose utilization and glucose uptake in soleus muscle but does not enhance the suppressive effect of insulin on EGP. The effects of injection of the MCR agonist MT-II into the VMH on glucose metabolism were evaluated with the hyperinsulinemic-euglycemic clamp. The MEK inhibitor U0126 or DMSO (0.01%) was injected into the VMH unilaterally at 1 h before injection of MT-II or saline. A: GIR during the clamp period. *P < 0.05 vs. corresponding value for saline + DMSO in VMH. B: Rd during the basal and clamp periods. C: 2DG uptake in soleus, Gastro-W, and epiWAT during the clamp period. D: Ra during the basal and clamp periods as well as the percentage suppression of Ra induced by insulin infusion. Ra in the basal period is equal to Rd in the basal period. *P < 0.05 vs. corresponding value for saline + DMSO in VMH in the basal period. #P < 0.05 vs. corresponding value for saline + DMSO in VMH in the clamp period. §P < 0.05 vs. corresponding value for saline + MEK inhibitor in VMH in the clamp period. ‡P < 0.05 vs. corresponding value for the basal period (B and D). E: Phosphorylation of synapsin in the VMH after injection of leptin and the MEK inhibitor U0126. The MEK inhibitor U0126 or DMSO (0.01%) was injected into the VMH unilaterally at 1 h before injection of leptin or saline without the hyperinsulinemic-euglycemic clamp. The VMH was collected at 30 min after leptin injection. The data were evaluated with the ratio of phosphorylated form (p) to the total (t) protein and expressed as percent increase of the ratio to that of saline + DMSO in the VMH group. *P < 0.05 vs. corresponding value for saline + DMSO in VMH. †P < 0.05 vs. corresponding value for leptin + DMSO in VMH. ns, not significant. All data are means ± SEM (n = 6 or 7 mice).
FIG. 7.
FIG. 7.
Model for the mechanism of regulation of glucose metabolism in muscle and liver by leptin in the VMH. Ob-Rb in the VMH plays a key role in the regulation of glucose metabolism and insulin sensitivity in muscle and liver by leptin. Leptin-activated MEK-ERK signaling in the VMH increases insulin sensitivity and glucose utilization in red muscle through activation of MCR in the VMH. Ob-Rb–expressing VMH neurons likely activate POMC neurons either in the ARC itself (14) or at their synaptic connections with VMH neurons through the MEK-ERK pathway. The MEK-ERK pathway then stimulates synaptic plasticity for POMC neurons and MCR-expressing neurons in the VMH. Whereas other brain sites may contribute to the leptin-induced enhancement of the suppressive effect of insulin on hepatic glucose production, leptin-activated STAT3 signaling in the VMH mediates this enhancement by inhibiting glycogen phosphorylase a activity in liver. 3V, third ventricle.
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