Disruption of GABA or glutamate release from POMC neurons in the adult mouse does not affect metabolic end points

Abstract

Proopiomelanocortin (POMC) neurons contribute to the regulation of many physiological processes; the majority of which have been attributed to the release of peptides produced from the POMC prohormone such as α-MSH, which plays key roles in food intake and metabolism. However, it is now clear that POMC neurons also release amino acid transmitters that likely contribute to the overall function of POMC cells. Recent work indicates that constitutive deletion of these transmitters can affect metabolic phenotypes, but also that the expression of GABAergic or glutamatergic markers changes throughout development. The goal of the present study was to determine whether the release of glutamate or GABA from POMC neurons in the adult mouse contributes notably to energy balance regulation. Disturbed release of glutamate or GABA specifically from POMC neurons in adult mice was achieved using a tamoxifen-inducible Cre construct (Pomc-CreER^T2) expressed in mice also carrying floxed versions of Slc17a6 (vGlut2) or Gad1 and Gad2, encoding the vesicular glutamate transporter type 2 and GAD67 and GAD65 proteins, respectively. All mice in the experiments received tamoxifen injections, but control mice lacked the tamoxifen-inducible Cre sequence. Body weight was unchanged in Gad1- and Gad2- or vGlut2-deleted female and male mice. Additionally, no significant differences in glucose tolerance or refeeding after an overnight fast were observed. These data collectively suggest that the release of GABA or glutamate from POMC neurons in adult mice does not significantly contribute to the metabolic parameters tested here. In light of prior work, the data also suggest that amino acid transmitter release from POMC cells may contribute to separate functions in the adult versus the developing mouse.

Keywords: GAD; VGLUT2; energy balance; food intake; proopiomelanocortin.

Fig. 1.

Conditional deletion of vesicular glutamate transporter (vGlut2) mRNA from proopiomelanocortin (Pomc) cells. A: fluorescent in situ hybridization images demonstrating the expression of vGlut2 mRNA in Pomc cells in the arcuate nucleus following tamoxifen administration in mice lacking the Pomc-CreER^T2 transgene. Arrows indicate examples of colocalized cells. B: fluorescent in situ hybridization images demonstrating that arcuate nucleus Pomc cells no longer express vGlut2 mRNA following tamoxifen administration in mice expressing the Pomc-CreER^T2 transgene. 3V, third ventricle. Scale bars = 25 μm.

Fig. 2.

Pomc-vGlut2^−/− mice show normal body weight on standard chow and high-fat diet. A: conditional deletion of vGlut2 from POMC cells does not significantly affect body weight in male mice fed standard chow [Pomc-CreER^T2(−), n = 8 mice; Pomc-CreER^T2(+), n = 8 mice; F(1,14) = 1.90; P = 0.19; two-way repeated-measures ANOVA]. B: conditional deletion of vGlut2 from POMC cells does not significantly affect body weight in female mice fed standard chow [Pomc-CreER^T2(−), n = 11 mice; Pomc-CreER^T2(+), n = 13 mice; F(1,22) = 0.05; P = 0.83; two-way repeated-measures ANOVA]. C: conditional deletion of vGlut2 from POMC cells does not significantly affect body weight in male mice fed a high-fat diet [Pomc-CreER^T2(−), n = 7 mice; Pomc-CreER^T2(+), n = 8 mice; F(1,13) = 0.01; P = 0.91; two-way repeated-measures ANOVA]. D: conditional deletion of vGlut2 from POMC cells does not significantly affect body weight in female mice fed a high-fat diet [Pomc-CreER^T2(−), n = 17 mice; Pomc-CreER^T2(+), n = 7 mice; F(1,22) = 0.38; P = 0.54; two-way repeated-measures ANOVA]. Conditional deletion of vGlut2 was achieved by tamoxifen injection at 8 wk of age, indicated by the top arrow. High-fat diet (HFD) was introduced at 10 wk of age and indicated by the lower arrow.

Fig. 3.

Pomc-vGlut2^−/− mice refed normally after a fast. A: conditional deletion of vGlut2 does not affect food intake in a 2-h refeeding after an overnight fast in male mice [Pomc-CreER^T2(−), n = 8 mice; Pomc-CreER^T2(+), n = 5 mice; t = 0.88; P = 0.55; unpaired t test]. B: conditional deletion of vGlut2 does not affect food intake in a 2-h refeeding after an overnight fast in female mice [Pomc-CreER^T2(−), n = 5 mice; Pomc-CreER^T2(+), n = 7 mice; t = 0.51; P = 0.62; unpaired t test]. POMC, proopiomelanocortin.

Fig. 4.

Pomc-vGlut2^−/− mice display normal glucose tolerance. A: blood glucose levels following a 2-mg/g ip injection of glucose in male mice before tamoxifen injection. B: area under the curve (AUC) of the data presented in A. Before the tamoxifen injection, male mice from both genotypes do not show differences in glucose clearance [Pomc-CreER^T2(−), n = 17 mice; Pomc-CreER^T2(+), n = 14 mice; t = 0.69; P = 0.49; unpaired t test]. C: blood glucose levels flowing a 2-mg/g ip injection of glucose in male mice 3 wk after tamoxifen injection. D: AUC of the data presented in C. Deletion of vGlut2 from POMC cells does not affect glucose clearance in male mice [Pomc-CreER^T2(−), n = 9 mice; Pomc-CreER^T2(+), n = 6 mice; t = 1.19; P = 0.25; unpaired t test]. E: blood glucose levels following a 2-mg/g ip injection of glucose in female mice before tamoxifen injection. F: AUC of the data presented in E. Before the tamoxifen injection, female mice from both genotypes do not show differences in glucose clearance [Pomc-CreER^T2(−), n = 13 mice; Pomc-CreER^T2(+), n = 17 mice; t = 1.65; P = 0.11; unpaired t test]. G: blood glucose levels following a 2-mg/g ip injection of glucose in female mice following tamoxifen injection. H: AUC of the data presented in G. Deletion of vGlut2 from POMC cells does not affect glucose clearance [Pomc-CreER^T2(−), n = 5 mice; Pomc-CreER^T2(+), n = 12 mice; t = 0.45; P = 0.65; unpaired t test]. POMC, proopiomelanocortin.

Fig. 5.

Conditional deletion of Gad1/2 mRNA from proopiomelanocortin (Pomc) cells. A: fluorescent in situ hybridization images demonstrating the expression of Gad1/2 mRNA (red) in Pomc cells (green) in the arcuate nucleus after tamoxifen administration in mice lacking the the Pomc-CreER^T2 transgene. Arrows indicate examples of colocalized cells. B: fluorescent in situ hybridization images demonstrating that arcuate nucleus Pomc cells (green) no longer express Gad1/2 mRNA (red) after tamoxifen administration in mice expressing the Pomc-CreER^T2 transgene. 3V, third ventricle. Scale bars = 25 μm.

Fig. 6.

Pomc-Gad1/2^−/− mice show normal body weight on standard chow. A: conditional deletion of Gad1/2 from proopiomelanocortin (POMC) cells does not significantly affect body weight in male mice fed standard chow [Pomc-CreER^T2(−), n = 11 mice; Pomc-CreER^T2(+), n = 8 mice; F(1,17) = 0.26; P = 0.61; two-way repeated-measures ANOVA]. B: conditional deletion of Gad1/2 from POMC cells does not significantly affect body weight in female mice fed standard chow [Pomc-CreER^T2(−), n = 7 mice; Pomc-CreER^T2(+), n = 6 mice; F(1,11) = 0.009; P = 0.92; two-way repeated-measures ANOVA]. Conditional deletion of Gad1/2 was achieved by tamoxifen injection at 10 wk of age, indicated by the arrow.

Fig. 7.

Pomc-Gad1/2^−/− mice refeed normally after a fast. A: before tamoxifen induction of Cre expression, male mice from both genotypes refeed similarly [Pomc-CreER^T2(−), n = 10 mice; Pomc-CreER^T2(+), n = 7 mice; t = 0.46; P = 0.65; unpaired t test]. B: conditional deletion of Gad1/2 does not affect food intake in a 2-h refeeding after an overnight fast in male mice [Pomc-CreER^T2(−), n = 10; Pomc-CreER^T2(+), n = 7 mice; t = 0.06; P = 0.96; unpaired t test]. C: before tamoxifen induction of Cre expression, female mice from both genotypes refeed similarly [Pomc-CreER^T2(−), n = 8 mice; Pomc-CreER^T2(+), n = 6 mice; t = 1.25; P = 0.23; unpaired t test]. D: conditional deletion of Gad1/2 does not affect food intake in a 2-h refeeding after an overnight fast in female mice [Pomc-CreER^T2(−), n = 8 mice; Pomc-CreER^T2(+), n = 6 mice; t = 1.12; P = 0.28; unpaired t test]. POMC, proopiomelanocortin.

Fig. 8.

Pomc-Gad1/2^−/− mice display normal glucose tolerance. A: blood glucose levels after a 2-mg/g ip injection of glucose in male mice before tamoxifen injection. B: area under the curve (AUC) of the data presented in A. Before tamoxifen injection, male mice from both genotypes do not show differences in glucose clearance [Pomc-CreER^T2(−), n = 10 mice; Pomc-CreER^T2(+), n = 8 mice; t = 1.24; P = 0.23; unpaired t test]. C: blood glucose levels after a 2-mg/g ip injection of glucose in male mice 3 wk after tamoxifen injection. D: AUC of the data presented in C. Deletion of Gad1/2 from POMC cells does not affect glucose clearance in male mice [Pomc-CreER^T2(−), n = 11 mice; Pomc-CreER^T2(+), n = 8 mice; t = 0.80; P = 0.43; unpaired t test]. E: blood glucose levels after a 2-mg/g ip injection of glucose in female mice before tamoxifen injection. F: AUC of the data presented in E. Before tamoxifen injection, female mice from both genotypes do not show differences in glucose clearance [Pomc-CreER^T2(−), n = 7 mice; Pomc-CreER^T2(+), n = 5 mice; t = 0.59; P = 0.56; unpaired t test]. G: blood glucose levels after a 2-mg/g ip injection of glucose in female following tamoxifen injection. H: AUC of the data presented in G. Deletion of Gad1/2 from proopiomelanocortin (POMC) cells does not affect glucose clearance [Pomc-CreER^T2(−), n = 7; Pomc-CreER^T2(+), n = 6; t = 0.09; P = 0.93; unpaired t test].