Central resistin induces hepatic insulin resistance via neuropeptide Y

Abstract

Sensing of peripheral hormones and nutrients by the hypothalamus plays an important role in maintaining peripheral glucose homeostasis. The hormone resistin impairs the response to insulin in liver and other peripheral tissues. Here we demonstrate that in normal mice resistin delivered in the lateral cerebral ventricle increased endogenous glucose production during hyperinsulinemic-euglycemic clamp, consistent with induction of hepatic insulin resistance. In agreement, central resistin inhibited Akt phosphorylation and increased the expression of glucose-6-phosphatase, the enzyme regulating glucose output in the liver. Central resistin induced expression of proinflammatory cytokines as well as suppressor of cytokine signaling-3, a negative regulator of insulin action in liver. Central infusion of resistin was associated with neuronal activation in the arcuate, paraventricular and dorsomedial nuclei, and increased neuropeptide Y (NPY) expression in the hypothalamus. The effects of central resistin on glucose production as well as hepatic expression of proinflammatory cytokines were abrogated in mice lacking NPY. Pretreatment of wild-type mice with antagonists of the NPY Y1 receptor, but not the Y5 receptor, also prevented the effects of central resistin. Together, these results suggest that resistin action on NPY neurons is an important regulator of hepatic insulin sensitivity.

Figure 1.

Central resistin induces hepatic insulin resistance. A, D, Hyperinsulinemic-euglycemic clamp in wild-type (A) or retn^−/− (D) mice treated with intracerebroventricular resistin (white bars) or vehicle (black bars). Several markers of hepatic insulin sensitivity are altered by central resistin treatment before clamp. B, E, Ratio of phosphorylated to total Akt, SOCS3 to GAPDH, and phosphorylated STAT3 to total STAT3 protein. C, F, G6Pase, TNFα, and IL6 mRNA levels. Data are means ± SEM, n = 4–6/group. *p < 0.05; **p < 0.01 versus vehicle.

Figure 2.

Central resistin stimulates Fos immunoreactivity (ir) specifically in hypothalamic regions. A, Photomicrographs of coronal brain sections showing Fos immunostaining in the arcuate (Arc), paraventricular (PVN), and dorsomedial nucleus (DMN), after intracerebroventricular resistin (white bars) or vehicle (black bars) treatment. B, C, Number of Fos-ir cells per section in hypothalamic (B) and extrahypothalamic (C) regions. Data are mean Fos-ir cells/hemisphere ± SEM, n = 4. *p < 0.05; **p < 0.001 versus vehicle. 3v, Third ventricle; me, median eminence; SCN, suprachiasmatic nucleus, MPO, medial preoptic nucleus, LHA, lateral hypothalamic area; VMH, ventromedial hypothalamus; AHA, anterior hypothalamic area; PeV, periventricular nucleus; Ctx, cortex (primary motor); BLA, basolateral amygdala; BSTL, lateral bed nucleus of the stria terminalis; DMX, dorsal vagal nucleus; LC, locus ceruleus; NTS, nucleus tractus solitarius; PB, parabrachial nucleus; Rt, reticular thalamus. Scale bar, 250 μm.

Figure 3.

Central resistin increases NPY expression in the hypothalamus. A, Effect of intracerebroventricular resistin (white bar) versus vehicle (black bar) on neuropeptide mRNA levels in the hypothalamus. Data are mean ± SEM, n = 6–8. *p < 0.05. B, Darkfield photomicrographs of coronal sections of the hypothalamus showing NPY mRNA expression in the Arc and dorsomedial nucleus (DMN). Scale bars: Arc, 200 μm; DMN, 100 μm. C, Intensity of signal was quantified using NIH Image J. Data are mean ± SEM, n = 4/group. *p < 0.05; **p < 0.01 versus vehicle. D, Brightfield photomicrographs showing colocalization of For-ir (brown nuclear stain) and silver grains corresponding to NPY mRNA in Arc and DMN. Scale bars, 50 μm. Red arrow depicts a Fos-ir neuron, black arrow depicts a double-labeled neuron, and green arrow depicts a neuron expressing only NPY mRNA.

Figure 4.

NPY mediates induction of hepatic insulin resistance by resistin. A, Hyperinsulinemic-euglycemic clamp in npy−/− mice treated with intracerebroventricular resistin (white bars) or vehicle (black bars). B, Effects of intracerebroventricular resistin on protein. C, mRNA expression of mediators of insulin signaling in the liver during hyperinsulinemic-euglycemic clamp. Data are means ± SEM, n = 7–8/group.

Figure 5.

Pharmacological blockade of NPY Y1 receptors, but not of Y5 receptors, prevents the induction of hepatic insulin resistance by resistin. A,B, Hyperinsulinemic-euglycemic clamp and hepatic protein levels in wild-type mice treated with intracerebroventricular NPY Y1 receptor antagonist, BIBP3226, or vehicle followed by intracerebroventricular resistin (white bars) or vehicle (hatched bars), n = 6/group. C,D, Hyperinsulinemic-euglycemic clamp and hepatic protein levels in wild-type mice treated with intracerebroventricular NPY Y5 receptor antagonist, CGP71683 or vehicle followed by intracerebroventricular resistin (white bars) or vehicle (hatched bars), n = 4/group. Data are means ± SEM *p < 0.05 versus vehicle/vehicle (black bars); **p < 0.01 versus vehicle/vehicle; ^δp < 0.05 versus antagonist/vehicle.