Insulin inhibits neuropeptide Y gene expression in the arcuate nucleus through GABAergic systems

Abstract

Neuropeptide Y (NPY) in the arcuate nucleus is an orexigenic hormone of which levels are regulated by humoral as well as neural signals. In this study, we examined the regulation of NPY gene expression in the arcuate nucleus in hypothalamic organotypic cultures. Dexamethasone (DEX) (10(-9) to 10(-7) M) significantly increased NPY mRNA expression, and the effects were not influenced by coincubation with the sodium channel blocker tetrodotoxin (TTX), indicating that the action of DEX is independent of action potentials. Conversely, insulin (10(-11) to 10(-9) M) significantly inhibited NPY expression stimulated by DEX, and the inhibitory action of insulin was abolished in the presence of TTX. Because GABA and its receptors are expressed in the arcuate nucleus in vivo, we examined whether GABAergic systems were involved in the insulin action. The GABAB agonist baclofen significantly inhibited NPY expression stimulated by DEX, and the inhibitory action of insulin was completely abolished in the presence of either the GABAA antagonist bicuculline or the GABAB antagonist CGP35348 (p-3-aminopropyl-p-diethoxymethyl phosphoric acid). Furthermore, increases in the GABA-synthesizing enzyme glutamic acid decarboxylase 65 (GAD65) mRNA expression preceded decreases in NPY mRNA expression in the arcuate nucleus in the cultures. Experiments in vivo also demonstrated that increases in GAD65 mRNA expression in the arcuate nucleus preceded decreases in the NPY mRNA expression in a fasting-refeeding paradigm and that intracerebroventricular injection of insulin increased GAD65 mRNA expression in the arcuate nucleus in fasted rats. These data suggest that insulin inhibits NPY gene expression in the arcuate nucleus through GABAergic systems.

Figure 1.

Effects of DEX and insulin on NPY mRNA expression in the arcuate nucleus in the organotypic cultures. a, NPY expression was significantly increased with DEX (10^–7 m) at 4 h and continued to increase until 24 h. b, Incubation with DEX (10^–9 to 10^–7 m) for 24 h increased NPY mRNA expression dose dependently. c, Incubation with insulin (10^–11 to 10^–9 m) for 24 h did not affect NPY mRNA expression significantly in the absence of DEX. d, Incubation with insulin (10^–11 to 10^–9 m) for 24 h inhibited NPY mRNA expression stimulated by DEX (10^–8 m) dose dependently. e, Incubation with insulin (10^–9 m) significantly inhibited NPY mRNA expression stimulated by DEX (10^–8 m) at 12 and 24 h. f, Whereas incubation with DEX (10^–8 m) for 24 h increased NPY mRNA expression significantly even in the presence of 10^–6 m TTX, incubation with insulin (10^–9 m) for 24 h did not inhibit NPY mRNA expression stimulated by DEX (10^–8 m) in the presence of TTX. g, Representative autoradiographs showing NPY mRNA expression in the arcuate nucleus in the organotypic cultures are shown. The mean NPY expression levels in the absence of DEX (control) are expressed as 100 and are shown as dashed lines (d, e). The results are expressed as means ± SE (n = 10). *p < 0.05 versus control (a, b) or values stimulated by DEX (d, e). n.s., Not significant.

Figure 2.

Effects of GABA agonists and antagonists and glutamate antagonists on NPY mRNA expression in the arcuate nucleus in the organotypic cultures. a, Incubation with GABA_B agonist baclofen (10^–6 to 10^–4 m) did not affect NPY mRNA expression in the absence of DEX. b, Incubation with baclofen (10^–6 to 10^–4 m) significantly inhibited NPY mRNA expression stimulated by DEX (10^–8 m). c, Incubation with GABA agonist muscimol (10^–6 to 10^–4 a m) for 24 h did not significantly affect NPY mRNA expression stimulated by DEX (10^–8 m). d, Incubation with NMDA glutamate receptor antagonist AP-5 (100μm) or non-NMDA glutamate receptor antagonist CNQX (20 μm) did not significantly affect NPY mRNA expression stimulated by DEX. e, GABA antagonist bicuculline (10^–4 a m) significantly increased NPY mRNA expression stimulated by DEX, and insulin did not inhibit NPY expression significantly in the presence of bicuculline. f, Whereas incubation with GABA_B antagonist CGP35348 (1 mm) by itself did not affect NPY mRNA expression significantly, the inhibitory action of insulinon NPY expression was abolished in the presence of CGP35348. The mean NPY expression levels in the absence of DEX are expressed as 100 and are shown as dashed lines. The results are expressed as means ± SE (n = 10). *p < 0.05 versus values stimulated by DEX. n.s., Not significant.

Figure 3.

Expression of mRNA for receptors of GABA_A (GABA_AR) and GABA_B (GABA_BR) in the hypothalamic organotypic cultures. The mRNA for GABA_A and GABA_B receptors were expressed throughout the hypothalamic slices (a, b), whereas no visible signals were detected with sense probes (c, d). Expressions for GABA_A (e) and GABA_B (f) receptor mRNAs were compared with NPY mRNA expression (g, h) in the arcuate nucleus in adjacent slices. Scale bars, 50 μm.

Figure 4.

Effects of insulin on GAD65 mRNA expression in the arcuate nucleus in the organotypic cultures. a, GAD mRNA (shown by silver grains) is expressed in digoxigenin-stained NPY neurons. b, Insulin treatment significantly increased the GAD65 mRNA expression at 6 h, and the levels remained increased at 24 h. c, NPY and GAD65 mRNA expression in adjacent slices incubated with vehicle (control) or 10^–9m insulin for 24 h is shown. The results are expressed as means ± SE (n = 10). *p < 0.05 versus control. Scale bar, 20 μm.

Figure 5.

Changes in NPY (a) and GAD65 (b) mRNA in the arcuate nucleus, serum insulin levels (c), blood glucose levels (d), and cumulative food intake (e) in rats that had been deprived of food for 48 h and refed with normal rat chow for the duration indicated. The mean levels of NPY and GAD mRNA in rats that had access to food ad libitum were expressed as 100 and are shown as dashed lines. f, NPY and GAD65 mRNA expression in adjacent slices before and 24 h after refeeding is shown. *p < 0.05 versus values in rats fasted for 48 h (shown as time 0 in each panel).