SK3 in POMC neurons plays a sexually dimorphic role in energy and glucose homeostasis

Abstract

Background: Pro-opiomelanocortin (POMC) neurons play a sexually dimorphic role in body weight and glucose balance. However, the mechanisms for the sex differences in POMC neuron functions are not fully understood.

Results: We detected small conductance calcium-activated potassium (SK) current in POMC neurons. Secondary analysis of published single-cell RNA-Seq data showed that POMC neurons abundantly express SK3, one SK channel subunit. To test whether SK3 in POMC neurons regulates POMC neuron functions on energy and glucose homeostasis, we used a Cre-loxP strategy to delete SK3 specifically from mature POMC neurons. POMC-specific deletion of SK3 did not affect body weight in either male or female mice. Interestingly, male mutant mice showed not only decreased food intake but also decreased physical activity, resulting in unchanged body weight. Further, POMC-specific SK3 deficiency impaired glucose balance specifically in female mice but not in male mice. Finally, no sex differences were detected in the expression of SK3 and SK current in total POMC neurons. However, we found higher SK current but lower SK3 positive neuron population in male POMC neurons co-expressing estrogen receptor α (ERα) compared to that in females.

Conclusion: These results revealed a sexually dimorphic role of SK3 in POMC neurons in both energy and glucose homeostasis independent of body weight control, which was associated with the sex difference of SK current in a subpopulation of POMC + ERα + neurons.

Keywords: Energy and glucose homeostasis; POMC neurons; SK current; Sexually dimorphism.

Fig. 1

The expression of SKs in ARH neurons. A Percentage of neurons that express SK1, SK2, SK3 and SK4 in ARH. B Percentage of SK + POMC + neurons in POMC neurons. C–E Percentage of POMC + SK+, AgRP + SK + and POMC + AgRP + SK + neurons populations in SK neurons

Fig. 2

POMC specific deletion of SK3 didn’t affect pituitary function. A PCR detection of SK3^flox/flox allele and Cre-induced recombination in the ARH, NTS and pituitary from control or the pomc-SK3 KO mice. B Serum corticosterone level at basal and stressed conditions of male control and pomc-SK3 KO mice. Data are presented as mean ± SEM with individual data points. N = 3–5 mice per group

Fig. 3

POMC specific deletion of SK3 decreased not only feeding but also physical activity in male mice, resulting in unchanged body weight. A-C Body weight (A), body mass (B) and food intake (C) of male control mice and pomc-SK3 KO mice. D-H O₂ consumption (D), CO₂ production (E) and heat production (F) of male control mice and pomc-SK3 KO mice. Left panel: temporal levels of each parameter measured by the TSE PhenoMaster during the dark cycle and light cycle within 24 h. Middle panel: ANCOVA analysis using body weight as a covariate (p value as indicated). Right panel: predicted O₂ consumption, CO₂ production or heat production using CalR analysis . Although two-way ANOVA did not reveal a significant difference, two-tailed unpaired t-tests detected several significantly different time points. G, H Physical activity (X + Y axis) (G) and physical activity (Z axis) (H) of male control mice and pomc-SK3 KO mice. Data are presented as mean ± SEM with individual data points. N = 6–14 mice per group. #P < 0.05 in two-way ANOVA analysis. *P < 0.05 in two-tailed unpaired t-test

Fig. 4

POMC specific deletion of SK3 did not regulate food intake, energy expenditure or body weight in female mice. A Body weight, B body mass, C food intake, D O₂ consumption, E CO₂ production, F heat production, G physical activity (X + Y axis) and H physical activity (Z axis) of female control mice and pomc-SK3 KO mice, the same reading as Fig. 3. Data are presented as mean ± SEM with individual data points. N = 6–14 mice per group

Fig. 5

POMC specific deletion of SK3 impaired glucose and insulin tolerance in female mice but not in male mice. A, C GTT of male (A) and female (C) control and pomc-SK3 KO mice. B, D ITT of male (B) and female (D) control and pomc-SK3 KO mice. Inserted column figures are AUC analysis of GTT and ITT data, y axis is the arbitrary units for AUC. Data are presented as mean ± SEM with individual data points N = 6–14 mice per group. *, P < 0.05 in two-tailed unpaired t-test

Fig. 6

SK current and the expression of SKs in POMC neurons. A The expression of SK3 in male and female POMC neurons. B Fluorescence for tdTomato (left) and bright-field illumination (right) of a recorded POMC neuron in a brain slice. C Representative traces of SK current in male and female POMC neurons. D Quantification of SK current in POMC neurons from fed male and female mice. E Fluorescence for ZsGreen (left) and tdTomato (middle) and bright-field illumination (right) of a recorded POMC + ERα + neuron in a brain slice. F Representative traces of SK current in male and female POMC + ERα + neurons. G Quantification of SK current in POMC + ERα + neurons from male and female mice. H RNAScope using probes targeting POMC, SK3 and ERα in the ARH. White arrow indicated the neurons that co-express genes that encode POMC, SK3 and ERα. I Quantification of the percentage of POMC + SK3 + ERα + neurons out of total POMC neurons in the ARH. Data are presented as mean ± SEM with individual data points. N = 22–32 neurons per group. *, ****, P < 0.5 or 0.0001 in two-tailed unpaired t-test