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. 2024 Apr 24;21(1):22.
doi: 10.1186/s12986-024-00790-3.

Spexin ameliorated obesity-related metabolic disorders through promoting white adipose browning mediated by JAK2-STAT3 pathway

Bihe Zeng #  1   2 , Qin Shen #  1 , Bo Wang  1 , Xuan Tang  1 , Jiaqi Jiang  1 , Yiming Zheng  1 , Hongbiao Huang  1 , Wenyu Zhuo  1 , Wang Wang  1 , Yang Gao  1 , Xuan Li  1 , Shuhui Wang  1 , Wenjie Li  1 , Guanghui Qian  1 , Jie Qin  1 , Miao Hou  3 , Haitao Lv  4
Affiliations

Spexin ameliorated obesity-related metabolic disorders through promoting white adipose browning mediated by JAK2-STAT3 pathway

Bihe Zeng et al. Nutr Metab (Lond). .

Abstract

Background: Spexin, a 14 amino acid peptide, has been reported to regulate obesity and its associated complications. However, little is known about the underlying molecular mechanism. Therefore, this study aimed to investigate the effects of spexin on obesity and explore the detailed molecular mechanisms in vivo and in vitro.

Methods: Male C57BL/6J mice were fed a high-fat diet (HFD) for 12 weeks to induce obesity, and mice fed a standard fat diet were used as controls. Then, these mice were treated with SPX or Vehicle by intraperitoneal injection for an additional 12 weeks, respectively. The metabolic profile, fat-browning specific markers and mitochondrial contents were detected. In vitro, 3T3-L1 cells were used to investigate the molecular mechanisms.

Results: After 12 weeks of treatment, SPX significantly decreased body weight, serum lipid levels, and improved insulin sensitivity in HFD-induced obese mice. Moreover, SPX was found to promote oxygen consumption in HFD mice, and it increased mitochondrial content as well as the expression of brown-specific markers in white adipose tissue (WAT) of HFD mice. These results were consistent with the increase in mitochondrial content and the expression of brown-specific markers in 3T3-L1 mature adipocytes. Of note, the spexin-mediated beneficial pro-browning actions were abolished by the JAK2/STAT3 pathway antagonists in mature 3T3-L1 cells.

Conclusions: These data indicate that spexin ameliorates obesity-induced metabolic disorders by improving WAT browning via activation of the JAK2/STAT3 signaling pathway. Therefore, SPX may serve as a new therapeutic candidate for treating obesity.

Keywords: Adipose tissue; Browning; JAK2/STAT3 pathway; Obesity; Spexin.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
SPX ameliorates high-fat-diet-induced obesity in mice. (A) Schematic description of animal experiments. 3-week-old C57BL/6J male mice after one-week adaptive feeding were divided randomly into a high-fat diet group (HFD) and a normal diet (ND) group. After 12 weeks, HFD or ND mice were half received SPX injection (30 µg/kg/day; HFD + SPX or ND + SPX) or vehicle injection (HFD + Veh or ND + Veh). (B) The body weight during the protocol. (C) Obesity model successfully established after 12 weeks. (D, E) Body weight of mice after SPX or Veh treatment at the end of the study. (F) H&E staining in WAT (epididymal WAT) (scale bar: 100 μm). (G) Adipocyte size in WAT of the mice. (H) The food intake after SPX or Veh treatment among each group. All values are represented as means with error bars representing S.D. *, P < 0.05; **, P < 0.01. n = 6 for each group
Fig. 2
Fig. 2
SPX promotes mice energy expenditure. (A) The oxygen consumption (VO2) of the mice during a 12 h light/12 h dark cycle. (D) the average values of VO2 for 12 h dark periods against body weight, (G) ANCOVA predicted VO2 at a given body weight of 40 g. (B) The carbon dioxide production (VCO2) of the mice during a 12 h light/12 h dark cycle. (E) the average values of VCO2 for 12 h dark periods against body weight, (H) ANCOVA predicted VCO2 at a given body weight of 40g. (C) the average values of the respiratory exchange ratio (RER) of the mice during a 12 h light/12 h dark cycle. (F) the average values of RER for 12-h light/12-h dark periods. (I) the average values of energy expenditure (EE) for 12 h dark periods against body weight. (J) ANCOVA predicted EE at a given body weight of 40 g. All values are presented as means with error bars representing S.D. In the bar graph, *, P < 0.05; **, P < 0.01. n = 6 for each group
Fig. 3
Fig. 3
SPX improves lipid metabolism and glucose tolerance in HFD feeding-induced obesity. (A ~ D) Serum TG, TCHO, HDL and LDL levels of the mice. (E) Glucose levels during intraperitoneal glucose tolerance test (IPGTT). (F) Glucose area of the curve (AOC) during IPGTT. All values are represented as means with error bars representing S.D. *, P < 0.05; **, P < 0.01. n = 6 for each group
Fig. 4
Fig. 4
SPX induces thermogenic gene expression and browning in WAT of HFD mice. (A) Immunoblotting for indicated proteins in WAT of the mice. The protein (B) and mRNA (D) expression levels of UCP-1, TBX1 and CIDEA in WAT. (C) Immunohistochemical staining of UCP-1 in WAT (scale bar: 100 μm). All values are represented as means with error bars representing S.D. *, P < 0.05; **, P < 0.01. n = 6 for each group
Fig. 5
Fig. 5
SPX increases mitochondrial biogenesis in WAT of HFD mice. (A) The expression levels of mitochondrial biogenesis proteins CYT-B/actin in the mice. (B) Representative transmission electron microscopic images (Red arrows indicate the mitochondria). Original magnification, ×15,000. All values are represented as means with error bars representing S.D. *, P < 0.05; **, P < 0.01. n = 6 for each group
Fig. 6
Fig. 6
SPX increases beige-fat specific markers in 3T3-L1 white adipocytes. (A ~ D) SPX increases the expressions of UCP1, TBX1 and CIDEA in 3T3-L1 adipocytes in a time-dependent and concentration-dependent manner. (E) The mRNA expression levels of UCP1, TBX1 and CIDEA in 100 nM SPX-treated differentiated 3T3-L1 adipocytes after 3 h. (F) Immunofluorescence staining shows increased expression of UCP1 upon 100 nM SPX treatment 3 h (×600 magnification; scale bar = 50 μm). All values are represented as means with error bars representing S.D. *, P < 0.05; **, P < 0.01. n = 6 for each group
Fig. 7
Fig. 7
SPX increases mitochondrial biogenesis in 3T3-L1 white adipocytes. (A) The levels CYT-B/actinin 100 nM SPX-treated differentiated 3T3-L1 adipocytes after 3 h. (B) Representative transmission electronic microscopic images from 100 nM SPX-treated differentiated 3T3-L1 adipocytes. Original magnification, ×15,000 (Red arrows indicate the mitochondria). (C) MitoTracker Green staining of mitochondria in 3T3-L1 cells (Original magnification, 600×, scale bar = 50 μm). All values are represented as means with error bars representing S.D. In the bar graph, *, P < 0.05; **, P < 0.01. n = 6 for each group
Fig. 8
Fig. 8
SPX-induced adipose browning is mediated through JAK2 and STAT3 signaling. (A, B) RNA sequence of the possible involved signaling pathways. (C, D) The expression of P-JAK2/JAK2 and P-STAT3/STAT3 in 3T3-L1 mature adipocyte. (E, F) The expression of P-JAK2/JAK2 and P-STAT3/STAT3 in WAT of mice. All values are represented as means with error bars representing S.D. In the bar graph, *, P < 0.05; **, P < 0.01. n = 6 for each group
Fig. 9
Fig. 9
JAK2-STAT3 signaling pathway inhibitors eliminate the adipose browning induced by SPX. The adipocytes were treated with an inhibitor of JAK2 (AZD1480) or an inhibitor of STAT3 (stattic). (A ~ D) The protein expression of UCP1, TBX1 and CIDEA treated with AZD1480 and stattic. (E) The expression levels of mitochondrial biogenesis proteins CYT-B. (F) The mRNA expression of UCP1, TBX1 and CIDEA. All values are represented as means with error bars representing S.D. In the bar graph, *, P < 0.05; **, P < 0.01. n = 6 for each group
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