GABA type B receptor signaling in proopiomelanocortin neurons protects against obesity, insulin resistance, and hypothalamic inflammation in male mice on a high-fat diet

Abstract

There is evidence suggesting that the GABA system in the arcuate nucleus, where orexigenic neuropeptide Y and agouti-related peptide as well as anorexigenic proopiomelanocortin (POMC) are expressed, plays an important role in energy balance. In this study, we generated POMC-specific GABAB receptor-deficient [knock-out (KO)] mice. Male KO mice on a high-fat diet (HFD) showed mild increases in body weight (BW) at the age of 9 weeks compared to wild-type (WT) mice, and the differences remained significant until 16 weeks old. However, there was no difference in BW in females between genotypes. While food intake was similar between genotypes, oxygen consumption was significantly decreased in the male KO mice. The insulin tolerance test revealed that the male KO mice were less insulin sensitive compared to WT mice at the age of 8 weeks, when there was no significant difference in BW between genotypes. Despite increased BW, POMC mRNA expression in the arcuate nucleus was significantly decreased in the KO mice compared to WT mice at the age of 16 weeks. Furthermore, the expression of TNFα as well as IL-6, proinflammatory markers in the hypothalamus, was significantly increased in the KO mice on a HFD compared to WT mice. This demonstrates that the deletion of GABAB receptors in POMC neurons in the male mice on a HFD results in obesity, insulin resistance, and hypothalamic inflammation. Furthermore, the decreased POMC expression in the obese KO mice suggests that the regulation of POMC expression through GABAB receptors is essential for proper energy balance.

Figure 1.

Generation of POMC-specific GABA_BR-deficient mice. A, Deletion efficiency of POMC-Cre as assessed by immunohistochemistry for EGFP. GABA_B1R^+/+; Z/EG control hypothalamus (left) compared with POMC-GABA_B1R^−/[minus]; Z/EG hypothalamus (right). ARC, Arcuate nucleus; 3V, third ventricle. B, Detection of deletion of GABA_B1R alleles (Δ) in POMC-GABA_B1R^−/− (KO) mice compared with GABA_B1R^+/+ (WT) mice. DNA was extracted from different tissues, and deletion of the floxed allele was detected by PCR. P, Pituitary; H, hypothalamus; HB, hindbrain; C, cerebral cortex; L, liver; M, skeletal muscle; F, fat. Recombination was detected only in pituitary, hypothalamus, and hindbrain of KO mice. A PCR reaction with GAPDH was used as an internal control. C, GABA_BR protein levels in WT mice compared with KO mice as determined by immunoblotting. β-actin protein levels are shown as a loading control. Hypo, Hypothalamus; brain, cerebral cortex. D, POMC cells detected with immunohistochemistry in hypothalamus in WT mice and KO mice at the age of 16 weeks on the CHD.

Figure 2.

Body weight and composition. A, B, BW of male (A) and female (B) POMC-GABA_B1R^−/− (KO) mice and GABA_BR^+/+ (WT) mice on a HFD. C, D, BW of male (C) and female (D) KO and WT mice on the CHD. E–H, Epididymal fat pad weight (E), serum leptin levels (F), brown adipose tissue weight (G), and nose–rump length (H) of male KO and WT mice at the age of 16 weeks on a HFD. All values are mean ± SE. n = 8 per genotype. *p < 0.05 versus WT mice.

Figure 3.

Analysis of energy metabolism. A, Cumulative food intake for 3 d. B, POMC-GABA_B1R^−/− (KO) mice have increased feed efficiency compared with GABA_B1R^+/+ (WT) mice. C, KO mice have decreased energy expenditure as indicated by decreased resting V_O2 during the dark cycle compared with WT mice. D, KO mice have increased RQ values during the dark cycle compared with WT mice. E, F, Locomotor activity (E) and body temperature (F) were not different between genotypes. Data were collected from male mice on a HFD at the age of 8 weeks (A, B) and 16 weeks (C–F). All values are mean ± SE. n = 7 per genotype. *p < 0.05 versus WT mice.

Figure 4.

ITT and GTT. Insulin sensitivity and glucose homeostasis are impaired in POMC-GABA_B1R^−/− (KO) mice. A, B, ITT (A) and GTT (B) in male KO mice and GABA_B1R^+/+ (WT) mice at the age of 8 weeks on a HFD. All values are mean ± SE. n = 7 per genotype. *p < 0.05 versus WT mice.

Figure 5.

POMC, NPY, and AgRP mRNA expression in hypothalamus analyzed by RT-PCR. A–C, Expression of POMC mRNA (A), NPY mRNA (B), and AgRP mRNA (C) in GABA_B1R^+/+ (WT) and POMC-GABA_B1R^−/− (KO) mice at ages 2 and 16 weeks on the CHD or HFD. All values are mean ± SE. n = 6–8 per genotype. *p < 0.05 versus WT mice.

Figure 6.

POMC mRNA expression analyzed by in situ hybridization. A, Representative photographs showing POMC mRNA expression in the arcuate nucleus of male GABA_B1R^+/+ (WT) and POMC-GABA_B1R^−/− (KO) mice at the age of 16 weeks on the HFD. B, POMC mRNA expression was significantly decreased in KO mice compared to WT mice. All values are mean ± SE. n = 8 per genotype. *p < 0.05 versus WT mice.

Figure 7.

Changes in GABAergic and glutamatergic POMC neurons. A, Representative photographs showing the staining of POMC (magenta) and GAD67 (green) in the arcuate nucleus in male WT and KO mice at the age of 16 weeks on the HFD. B, GABAergic POMC neurons were significantly decreased in WT mice but not in KO mice on the HFD. C, Representative photographs showing the staining of POMC (magenta) and vGLUT2 (green) in the arcuate nucleus in male WT and KO mice at the age of 16 weeks on the HFD. D, There were no differences in numbers of glutamatergic POMC neurons between WT mice and KO mice on the HFD or CHD. E, Expression of Synapsin 1 in the region of POMC neurons. POMC neurons (magenta) and Synapsin 1 expression (green) in the arcuate nucleus in WT and KO mice at the age of 16 weeks on the HFD are shown. POMC neurons colocalized with GAD 67 or vGLUT2 were shown by arrows, whereas those not thus colocalized were shown by arrowheads. All values are mean ± SE. n = 6 per genotype. *p < 0.05 versus WT mice.

Figure 8.

Hypothalamic inflammatory signaling. Hypothalamic expression of inflammation signals (IL-6, TNFα) were increased in POMC-GABA_B1R^−/− (KO) mice on the HFD at the age of 16 weeks compared to KO mice on the CHD, or GABA_B1R^+/+ (WT) mice on the CHD or HFD. Expression levels of microglia-specific (Gfap) and astrocyte-specific (CD68) markers were not different between groups. All values are mean ± SE. n = 5–8 per genotype. *p < 0.05 versus KO mice on the CHD, WT mice on the CHD, or WT mice on the HFD.

Figure 9.

Possible mechanisms by which POMC expression was affected in POMC-specific GABA_BR KO mice. A, GABA_BRs are located presynaptically and postsynaptically in POMC neurons, which release GABA and glutamate. B, GABAergic POMC neurons are increased in GABA_BR KO mice compared to WT mice on a HFD. Deletion of GABA_BRs in GABAergic terminals leads to increases in GABA release, whereas that in glutamatergic terminals results in increases in glutamate. Increased GABA release could decrease POMC expression through action on GABA_ARs.