Hypothalamic extended synaptotagmin-3 contributes to the development of dietary obesity and related metabolic disorders

Abstract

The C₂ domain containing protein extended synaptotagmin (E-Syt) plays important roles in both lipid homeostasis and the intracellular signaling; however, its role in physiology remains largely unknown. Here, we show that hypothalamic E-Syt3 plays a critical role in diet-induced obesity (DIO). E-Syt3 is characteristically expressed in the hypothalamic nuclei. Whole-body or proopiomelanocortin (POMC) neuron-specific ablation of E-Syt3 ameliorated DIO and related comorbidities, including glucose intolerance and dyslipidemia. Conversely, overexpression of E-Syt3 in the arcuate nucleus moderately promoted food intake and impaired energy expenditure, leading to increased weight gain. Mechanistically, E-Syt3 ablation led to increased processing of POMC to α-melanocyte-stimulating hormone (α-MSH), increased activities of protein kinase C and activator protein-1, and enhanced expression of prohormone convertases. These findings reveal a previously unappreciated role for hypothalamic E-Syt3 in DIO and related metabolic disorders.

Keywords: POMC neuron; extended synaptotagmin 3; glucose intolerance; hypothalamus; obesity.

Fig. 1.

Expression of E-Syt3 in mouse hypothalamus. (A and B) The whole-mount X-Gal staining of the adult E-Syt3 heterozygous mouse brain. Ventral (A) and dorsal (B) views are shown. HT, hypothalamus. (Scale bars, 1 mm.) (C–E) Representative images showing X-Gal staining of cerebral cortex (C), thalamus (TH, D) and hippocampus (Hippo, E). D3V, dorsal third ventricle. (Scale bars, 50 µm.) (F) A representative image showing X-Gal staining product appears in the Arc, VMH, and DMH nuclei of the hypothalamus. Dashed line in green indicates the border of the nucleus. 3V, ventral third ventricle. (Scale bar, 50 µm.) (G and H) X-Gal staining of the lateral hypothalamic area (LH, G) and the paraventricular nucleus of hypothalamus (PVH, H). (Scale bars, 50 µm.) (I) Combined X-Gal staining for E-Syt3 and immunohistochemical staining for NeuN and glial fibrillary acidic protein (GFAP) of the hypothalamus of E-Syt3 heterozygous mice. X-Gal staining product appears blue, and immunohistochemical staining product appears red to brown. Arrows indicate double positive cells. (Scale bars, 20 µm.) For each staining, similar data were obtained from 4 or more mice.

Fig. 2.

Ablation of E-Syt3 ameliorates DIO and related comorbidities. (A) Western blot data showing that E-Syt3 protein is depleted in the hypothalamus of E-Syt3 KO mice. β-Actin was used as a loading control. (B) Body-weight gain in male E-Syt3 WT and KO mice fed a chow or HFD from 6 wk of age. n = 9 (WT, Chow), 8 (KO, Chow), 6 (WT, HFD), 7 (KO, HFD). (C and D) Fat mass (C) and lean mass (D) at 36 wk old. n = 7 (WT, Chow), 8 (KO, Chow), or 6 (HFD) per group. (E) Hematoxylin & eosin (H & E) staining of the eWAT of HFD-fed mice. (Scale bars, 50 μm.) (F) Plasma leptin levels. n = 8 (Chow) or 7 (HFD) per group. (G and H) GTT (G) and the AUC of GTT (H) of 32-wk-old mice. n = 8 (Chow), 6 (WT, HFD), or 7 (KO, HFD) per group. (I) Plasma insulin levels. n = 8 (WT, Chow), 7 (KO, Chow), 5 (WT, HFD), or 6 (KO, HFD). (J) Liver TG levels. n = 9 (KO, HFD) or 10 (all other groups) per group. (K) Plasma TG levels. n = 13 (WT, Chow), 15 (KO, Chow), or 8 (HFD) per group. (L) Plasma TC levels. n = 11 (Chow) or 13 (HFD) per group. (M) Food intake assessed during the first 7 wk of diet treatment. n = 10 (WT, Chow), 7 (KO, Chow), 6 (WT, HFD), or 7 (KO, HFD). (N and O) Representative infrared images of HFD-fed E-Syt3 WT and KO mice (N), and the mean temperature in the interscapular (boxed) area (O). n = 8 per group. (P and Q) O₂ consumption (VO₂, P) and energy expenditure (EE, Q) of the mice fed an HFD for 6 wk. Light: light cycle; Dark: dark cycle. lbm, lean body mass. n = 4 per group. Data are presented as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed Student’s t test (O); one-way ANOVA with Newman–Keuls posthoc test (C, F, H–M, P, and Q); two-way ANOVA with Bonferroni’s posthoc test, comparison between E-Syt3 WT and KO mice fed an HFD (B and G).

Fig. 3.

Overexpression of E-Syt3 in Arc neurons leads to obesity-like phenotype. (A) Representative fluorescent images showing the expression of EGFP after the injection of control lentivirus (Ctrl-Lenti) into the Arc nucleus (outlined by white dotted line). Cell nuclei were counterstained with DAPI (blue). V, third ventricle. (Scale bar, 50 µm.) (B) Male E-Syt3 WT mice were injected Ctrl-Lenti or E-Syt3–Lenti viruses into Arc nucleus. Western blot for E-Syt3 in the Arc nucleus and the quantification data are shown. β-Actin was used as a loading control. n = 3 per group. (C–E) Body weight (C), fat mass (D), and lean mass (E) of mice after lentivirus injection. n = 7 per group. (F) Representative H&E staining images of eWAT. (Scale bars, 50 µm.) (G and H) Plasma levels of TG (G) and TC (H). n = 7 (G) or 8 (H) per group. (I and J) GTT (I) and the AUC of GTT (J). n = 7 per group. (K) Food intake of mice assessed during the first 6 wk after surgeries. n = 8 (Ctrl-Lenti) or 7 (E-Syt3–Lenti). (L and M) Representative infrared images (L), and the mean temperature in the interscapular (boxed) area (M). n = 8 per group. (N–P) O₂ consumption (VO₂, N), CO₂ production (VCO₂, O), and energy expenditure (EE, P) of the indicated mice. Light: light cycle; Dark: dark cycle. n = 4 per group. Data are presented as means ± SEM. *P < 0.05, **P < 0.01, two-tailed Student’s t test (B, G, H, J, K and M); one-way ANOVA with Newman–Keuls posthoc test (N–P); two-way ANOVA with Bonferroni’s posthoc test (C and D).

Fig. 4.

POMC neuron-specific deletion of E-Syt3 mitigates DIO and related metabolic disorders. (A–C) Male mice were placed on an HFD starting at 6 wk old. Body weight (A), fat mass (B), and lean mass (C) were then assessed. n = 11 (A), 11 (E-Syt3^Loxp/Loxp in B or C) or 10 (other groups in B or C) per group. (D) Representative H&E staining images of eWAT. (Scale bars, 50 µm.) (E and F) Distribution of area (E) and the mean area of eWAT adipocyte (F). E-Syt3^L/L, E-Syt3^Loxp/Loxp; P, E-Syt3^L/L, POMC-Cre, E-Syt3^Loxp/Loxp. n = 3 per group. (G and H) Plasma levels of TG (G) and TC (H). n = 8 (POMC-Cre), 9 (E-Syt3^Loxp/Loxp, or POMC-Cre, E-Syt3^Loxp/Loxp) in G, or 6 (H) per group. (I and J) GTT (I) and the AUC of GTT (J). n = 9 per group. (K) Daily HFD intake. n = 13 (POMC-Cre), 8 (E-Syt3^Loxp/Loxp), or 9 (POMC-Cre, E-Syt3^Loxp/Loxp). (L and M) Representative infrared images of HFD-fed mice (L), and quantification of the mean temperature in the interscapular (boxed) area (M). n = 11 (POMC-Cre), 12 (E-Syt3^Loxp/Loxp), or 13 (POMC-Cre, E-Syt3^Loxp/Loxp). (N and O) O₂ consumption (VO₂, N) and EE (O) of HFD-fed mice. Light: light cycle; Dark: dark cycle. n = 4 per group. Data are presented as means ± SEM. *P < 0.05, **P < 0.01, one-way ANOVA with Bonferroni’s (B, F, J, K, and M–O) or Newman–Keuls (G and H) posthoc test; two-way ANOVA with Bonferroni’s posthoc test (A and I).

Fig. 5.

Depletion of E-Syt3 enhances the processing of POMC to α-MSH. (A and B) Hypothalamic contents of α-MSH (A) and POMC (B) peptides in HFD-fed E-Syt3 WT and KO mice. n = 7 (A) or 8 (B) per group. (C) Molar ratio of α-MSH versus POMC. n = 7 per group. (D) Relative mRNA level of POMC in the hypothalamus of HFD-fed mice. n = 7 (WT) or 6 (KO) per group. (E) HFD-fed mice were briefly fasted, and then i.c.v. administered SHU9119, a potent MC3/4R antagonist, or aCSF as control. Food intake during the following 12 h is shown. n = 6 per group. (F) HFD-fed mice were briefly fasted, and then i.c.v. administered aCSF or SHU9119. Food intake during the following 4 h is shown. P, E-Syt3^L/L, POMC-Cre, E-Syt3^Loxp/Loxp. n = 6 (Ctrl), 8 (POMC-Cre, SHU9119), and 7 (POMC-Cre, E-Syt3^Loxp/Loxp, SHU9119) per group. (G and H) HFD-fed mice were i.c.v. injected aCSF or SHU9119. O₂ consumption (VO₂, G) and EE (H) at 6-h posttreatment are shown. n = 4 per group. (I) Relative hypothalamic mRNA levels of PC1/3 and PC2 in HFD-fed mice. au, arbitrary unit. n = 5 (WT) or 7 (KO) in PC1/3 assay; n = 8 (WT) or 7 (KO) in PC2 assay. (J) Representative Western blots for PC1/3 and PC2 in the hypothalamus of chow-, 2-, and 4-wk HFD-fed mice. β-Actin was used as a loading control. (K) Quantification of the Western blots for PC1/3 and PC2. n = 3 per group. (L) Elevated PKC kinase activity in the Arc of HFD-fed E-Syt3 KO mice. n = 7 per group. (M and N) Overexpression of E-Syt3 (E-Syt3-L) reduced the promoter activities of mouse PC1/3 (M) and PC2 (N) genes in Neuro2a cells. n = 6 (M) or 3 (N) per group. (O and P) Crispri-mediated knockdown of E-Syt3 increased the promoter activities of mouse PC1/3 (O) and PC2 (P) genes in Neuro2a cells, while suppressing the activity of PKC by Calphostin C, or the activity of AP-1 by SR11302, significantly abolished these effects (O and P). In O n = 9 (DMSO, dimethyl sulphoxide), 3 (Calphostin C), 6 (SR11302); in P, n = 9 (DMSO), 6 (Calphostin C), or 3 (SR11302) per group. Data are presented as means ± SEM. *P < 0.05, **P < 0.01, two-tailed Student’s t test (A, C, I, and L–N); one-way ANOVA with Fisher’s LSD (E and F), Newman–Keuls (G and H) or Bonferroni’s (K, O, and P) posthoc test.