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. 2020 Feb;8(1):e000951.
doi: 10.1136/bmjdrc-2019-000951.

Loss of Voltage-Gated Proton Channel Hv1 Leads to Diet-Induced Obesity in Mice

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Free PMC article

Loss of Voltage-Gated Proton Channel Hv1 Leads to Diet-Induced Obesity in Mice

Huimin Pang et al. BMJ Open Diabetes Res Care. .
Free PMC article

Abstract

Objective: The voltage-gated proton channel Hv1 has been proposed to mediate NADPH oxidase (NOX) function by regulating intracellular pH during respiratory bursts. In our previous work, we showed that Hv1 is expressed in pancreatic β cells and positively regulates insulin secretion. Here, we investigated the role of Hv1 in adipose tissue differentiation, metabolic homeostasis and insulin sensitivity using Hv1 knockout (KO) mice.

Design: Mice with genetic deletion of Hv1 are treated with high-fat diet (HFD) similar to wild-type (WT) mice. Body weight gain, adiposity, insulin sensitivity and gene expressions in both adipose tissue and liver were analyzed.

Results: Mice with genetic deletion of Hv1 display overt obesity with higher body weight gain and accumulation of adipose tissue compared with similarly HFD-treated WT. Hv1-deficient mice develop more glucose intolerance than WT, but no significant difference in insulin resistance, after fed with HFD. Deficiency of Hv1 results in a remarkable increase in epididymal adipocyte weight and size, while the gene expressions of proinflammatory factors and cytokines are obviously enhanced in the HFD-fed mice. Furthermore, the gene expression of Hv1 is increased in the HFD-fed mice, which is accompanied by the increase of NOX2 and NOX4. In addition, there is more severely diet-induced steatosis and inflammation in liver in KO mice.

Conclusion: Our data demonstrated that lacking of Hv1 results in diet-induced obesity in mice through inflammation and hepatic steatosis. This study suggested that Hv1 acts as a positive regulator of metabolic homeostasis and a potential target for antiobesity drugs in therapy and may serve as an adaptive mechanism in cooperating with NOX to mediate reactive oxygen species for adipogenesis and insulin sensitivity.

Keywords: adipogenesis; hepatic steatosis; inflammation; obesity.

Conflict of interest statement

Competing interests: None declared.

Figures

Figure 1
Figure 1
Deletion of Hv1 results in diet-induced obesity. (A) Experimental design. (B and C) Representative images of body size (B) and intra-abdominal fat (C) following 16 weeks of HFD feeding. (D) Body weight of WT and KO mice under 21 weeks of NCD (WT: n=15; KO: n=18) and under 5 weeks of NCD following with 16 weeks of HFD (WT: n=16; KO: n=12). (E) Time course of body weight for WT (n=16) and KO (n=12) littermates following 16 weeks of HFD feeding. (F) Body weight gain in WT and KO mice over the course of 16 weeks being fed high fat. (G) Food intake after introduction of 16 weeks HFD (WT: n=6; KO: n=6). (H) Energy efficiency in WT (n=6) and KO (n=7) mice being fed HFD. It expresses the ratio of the total weight gain to the total calorie intake during the 16 weeks experimental period on HFD feeding. (I) Representative images of separate epididymal white adipose depots (a), livers (b) and kidney+renal white adipose depots (c) from WT and KO mice following 16 weeks HFD feeding. Scale bars: 1 cm. (J) weight of epididymal white adipose tissue and livers, weighed immediately after dissection from WT (n=5) and KO mice (n=6) fed the HFD for 16 weeks. (K) Representative images of H&E staining of histological sections for epididymal white adipose tissue (a) and livers (b) from WT (NCD: n=5; HFD: n=5) and KO mice (NCD: n=6; HFD: n=7) fed the diets for 16 weeks after 5 weeks of normal chow feeding. Scale bars: 100 µm. (L) The area (a) and Feret’s diameter (b) of adipocytes were determined with H&E staining of histological sections. Results represent means±SEM. *P<0.05; **p<0.01; ***p<0.001 versus corresponding WT. ###P<0.001 versus WT mice fed with HFD. HFD, high-fat diet; NCD, normal chow diet; WT, wild type.
Figure 2
Figure 2
Glucose and insulin homeostasis in WT and KO mice fed with HFD. (A) Glucose tolerance test (GTT; 2 g/kg glucose, intraperitoneally (ip)) for WT (n=6) and KO (n=7) mice following 16-week HFD feeding. (B) Area-under-curve of GTT. (C) Time-dependent serum insulin content of WT (n=6) and KO (n=7) mice being fed with HFD after ip glucose (2 g/kg glucose). (D–F) Fasting serum glucose concentration (D), fasting insulin content (E) and insulin resistance index (HOMA-IR) (F) for WT and KO mice under 21 weeks of NCD (WT: n=9; KO: n=11) and under 5 weeks of NCD following with 16 weeks of HFD (WT: n=10; KO: n=10). HOMA-IR was calculated as follows: fasting glucose (mmol/L)×fasting insulin (mU/L)/22.5. (G) Insulin tolerance test (ITT; 0.75 U/kg, ip) for WT (n=5) and KO (n=5) mice following 16-week HFD feeding. (H and I) Serum total cholesterol (H) and triglyceride (I) from WT (NCD: n=5; HFD: n=5) and KO mice (NCD: n=4; HFD: n=6). Data are presented as means±SEM. *P<0.05; **p<0.01; ***p<0.001 versus WT. ##P<0.01 versus WT mice fed with HFD. HFD, high-fat diet; NCD, normal chow diet; WT, wild type.
Figure 3
Figure 3
HFD-induced adipose and liver tissue inflammation and fat accumulation in Hv1-deficient mice. (A) Representative images of immunohistochemistry staining of epididymal adipose sections using CD68 antibody. Scale bars: 100 µm. (B–E) Quantitative RT-PCR analyses of macrophage markers CD68 (B) and CD11b (C), and cytokines interleukin-6 (D) and TNF-α (E) for epididymal adipose tissues from WT and KO mice under 21 weeks of NCD (WT: n=5; KO: n=5,) and under 5 weeks of NCD following with 16 weeks of HFD (WT: n=5; KO: n=5), normalized to WT mice fed with NCD. (F and G) Representative images of liver sections stained by ORO (F) and oil red area calculated as a percentage of the total area (G), from WT (NCD: n=3; HFD: n=3) and KO mice (NCD: n=3; HFD: n=3) under 21 weeks of NCD and under 5 weeks of NCD following with 16 weeks of HFD. Scale bars: 50 µm. (H and I) Representative images of liver immunohistochemistry stained by CD68 antibody (H) and the stained percentage from WT (NCD: n=6; HFD: n=6) and KO mice (NCD: n=5; HFD: n=5) fed with HFD for 16 weeks after 5 weeks of NCD feeding, and under 21 weeks of NCD only. Scale bars: 100 µm. Data are shown as means±SEM. *P<0.05; **p<0.01; ***p<0.001 versus WT mice fed with NCD. #P<0.05; ##p<0.01 versus WT mice fed with HFD. HFD, high-fat diet; NCD, normal chow diet; WT, wild type.
Figure 4
Figure 4
Loss of Hv1 results in HFD-induced upregulations of leptin, NOX2 and NOX4, and possible mechanisms of Hv1 in regulation of cell metabolism. (A–E) Quantitative RT-PCR was used to determine levels of leptin (A), adiponectin (B), and Hv1 (C), NOX4 (D) and NOX2 (E) mRNA in epididymal adipose tissue from NCD-fed and HFD-fed mice (WT: n=5, WT; KO: n=5, KO), normalized to NCD-fed WT mice. Data are presented as means±SEM. *P<0.05 versus NCD-fed WT mice. #P<0.05 versus HFD-fed KO mice. (F) The possible mechanism that Hv1 regulates adipocyte metabolism. Excess glucose activates the pentose phosphate pathway (PPP) by insulin signal in adipocytes, which is a major source of cellular NADPH and leads to NOX activation. NOXs transfer electrons from NADPH to oxygen with the cooperation of Hv1 to extrude proton, generating superoxide and H2O2. Hv1 exerts a protective effect against the onset of diet-induced insulin resistance and adipogenesis in adipocytes. (G) The possible mechanism that loss of Hv1 leads to HFD-induced obesity. Deficiency of Hv1 contributes to HFD-induced obesity and insulin resistance, which results in increased adipocyte differentiation and hypertrophy and gives rise to WAT accumulation, macrophage infiltration in vivo. These alterations are latent but can be brought into light by nutritional challenge resulting in overt obesity, insulin resistance and hepatic steatosis. HFD, high-fat diet; NCD, normal chow diet; WT, wild type.

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