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. 2022 Aug 31:13:889122.
doi: 10.3389/fendo.2022.889122. eCollection 2022.

Gabra5 plays a sexually dimorphic role in POMC neuron activity and glucose balance

Zhou Pei  1   2 , Yang He  2 , Jonathan C Bean  2 , Yongjie Yang  2 , Hailan Liu  2 , Meng Yu  2 , Kaifan Yu  2 , Ilirjana Hyseni  2 , Xing Cai  2 , Hesong Liu  2 , Na Qu  2 , Longlong Tu  2 , Kristine M Conde  2 , Mengjie Wang  2 , Yongxiang Li  2 , Na Yin  2 , Nan Zhang  2 , Junying Han  2 , Camille Hs Potts  2 , Nikolas A Scarcelli  2 , Zili Yan  2 , Pingwen Xu  3 , Qi Wu  2 , Yanlin He  4 , Yong Xu  2   5 , Chunmei Wang  2
Affiliations

Gabra5 plays a sexually dimorphic role in POMC neuron activity and glucose balance

Zhou Pei et al. Front Endocrinol (Lausanne). .

Abstract

Pro-opiomelanocortin (POMC) neurons are important for the regulation of body weight and glucose balance. The inhibitory tone to POMC neurons is mediated primarily by the GABA receptors. However, the detailed mechanisms and functions of GABA receptors are not well understood. The α5 subunit of GABAA receptor, Gabra5, is reported to regulate feeding, and we found that Gabra5 is highly expressed in POMC neurons. To explore the function of Gabra5 in POMC neurons, we knocked down Gabra5 specifically from mature hypothalamic POMC neurons using the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 strategy. This POMC-specific knock-down of Gabra5 did not affect body weight or food intake in either male or female mice. Interestingly, the loss of Gabra5 caused significant increases in the firing frequency and resting membrane potential, and a decrease in the amplitude of the miniature inhibitory postsynaptic current (mIPSC) in male POMC neurons. However, the loss of Gabra5 only modestly decreased the frequency of mIPSC in female POMC neurons. Consistently, POMC-specific knock-down of Gabra5 significantly improved glucose tolerance in male mice but not in female mice. These results revealed a sexually dimorphic role of Gabra5 in POMC neuron activity and glucose balance, independent of body weight control.

Keywords: GABAA receptor; GABAergic input; POMC neurons; glucose tolerance; sex differences.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
The expression of Gabra subunits of GABAA receptors in ARH POMC neurons. (A) Gabra1, (B) Gabra2, (C) Gabra3, (D) Gabra4, (E) Gabra5. On the first row are pie graphs showing the percentage of three neuron populations: POMC+/GABA receptor subunit+ or both, POMC+/GABA receptor subunit- and POMC-/GABA receptor subunit+, in all the neurons that express either POMC or each stated GABA receptor subunit or both. The bar graphs showed the expression of each GABA receptor subunit in POMC neurons between male vs female mice fed on chow diet (the second row), among fed vs fasting vs refeeding conditions from male mice fed on chow diet (the third row), and from male mice fed on chow vs HFD (the fourth row). ns, not significant. *, **, **** P < 0.05, 0.01 or 0.0001 in unpaired t-tests.
Figure 2
Figure 2
The expression of Gabrb subunits of GABAA receptors in ARH POMC neurons. (A) Gabrd1, (B) Gabrd2, (C) Gabrd3. Pie graphs and bar graphs were made the same way as described in Figure 1 . ns, not significant. *, **, **** P < 0.05, 0.01 or 0.0001 in unpaired t-tests.
Figure 3
Figure 3
The expression of Gabrg subunits of GABAA receptors in ARH POMC neurons. (A) Gabrg1, (B) Gabrg2, (C) Gabrg3. Pie graphs and bar graphs were made the same way as described in Figure 1 . ns, not significant. *, ****P < 0.05 or 0.0001 in unpaired t-tests.
Figure 4
Figure 4
The expression of Gabre and Gabrq subunits GABAA receptors in ARH POMC neurons. (A) Gabre and (B) Gabrq. Pie graphs and bar graphs were made the same way as described in Figure 1 . ns, not significant. *, **, **** P < 0.05, 0.01 or 0.0001 in unpaired t-tests.
Figure 5
Figure 5
Gabra5 deficiency increased POMC neuron activities in a sexually dimorphic manner. (A) Generation of pomc-Gabra5 KD mice and controls by stereotaxic injection of AAV-Gabra5sgRNA-tdTomato virus with or without AAV/DJ-CMV7-DIO-saCas9 virus into the ARH of POMC-CreERT2 mice followed by tamoxifen induction. (B) Bright-field illumination (upper) and fluorescence for tdTomato (lower) of the recorded POMC neuron in a brain slice. (C) Representative current clamp traces in POMC neurons from chow-fed pomc-Gabra5 KD and control mice 4 weeks after virus injection. (D–G) Average firing frequency (D), resting membrane potential (E), frequency (F) and amplitude (G) of mIPSC of POMC neurons from chow-fed pomc-Gabra5 KD and control mice 4 weeks after virus injection. Data are presented as mean ± SEM with individual data points. N = 14–44 per group. #P < 0.05 in t-tests. **P < 0.01 or ****P < 0.0001 in one-way ANOVA followed by Turkey’s tests. ns, not significant.
Figure 6
Figure 6
pomc-Gabra5 KD male mice developed glucose intolerance. (A) Generation of pomc-Gabra5 KD and controls by stereotaxic injection of AAV-Gabra5sgRNA-tdTomato virus and AAV/DJ-CMV7-DIO-saCas9 virus together into the ARH of POMC-Cre mice or their wild type littermate mice. (B–E) Weekly body weight (B, D) and food intake (C, E) in male (B, C) and female (D, E) pomc-Gabra5 KD and control mice. (F–I) GTT in male (F, G) and female (H, I) pomc-Gabra5 KD and control mice fed with chow diet for 4 weeks after injection (F, H) or HFD for 4 weeks (G, I). Data are presented as mean ± SEM with individual data points. N=15–27 per group. Inserted column figures are AUC analysis of GTT data, y axis is the artificial units for AUC. #, P < 0.05 in unpaired t-tests. *, P < 0.05 in two-way ANOVA followed by post hoc Sidak’s tests.
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