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. 2024 Sep 28;16(19):3295.
doi: 10.3390/nu16193295.

Effect of Isoflavone on Muscle Atrophy in Ovariectomized Mice

Sayaka Kawai  1 , Takuro Okamura  1 , Chihiro Munekawa  1 , Yuka Hasegawa  1 , Ayaka Kobayashi  1 , Hanako Nakajima  1 , Saori Majima  1 , Naoko Nakanishi  1 , Ryoichi Sasano  2 , Masahide Hamaguchi  1 , Michiaki Fukui  1
Affiliations

Effect of Isoflavone on Muscle Atrophy in Ovariectomized Mice

Sayaka Kawai et al. Nutrients. .

Abstract

Background: Sarcopenia, characterized by muscle mass decline due to aging or other causes, is exacerbated by decreased estrogen levels after menopause in women. Isoflavones, a class of flavonoids acting on estrogen receptors, may have beneficial effects on metabolic disorders. We examined these effects in ovariectomized mice fed a high-fat, high-sucrose diet (HFHSD).

Methods: At 7 weeks old, female C57BL6/J mice (18-20 g, n = 12) underwent bilateral ovariectomy (OVX), and were then fed a high-fat, high-sucrose diet starting at 8 weeks of age. Half of the mice received isoflavone water (0.1%). Metabolic analyses, including glucose and insulin tolerance tests, were conducted. Muscle analysis involved grip strength assays, next-generation sequencing, quantitative RT-PCR, and western blotting of skeletal muscle after euthanizing the mice at 14 weeks old. Additionally, 16S rRNA gene sequence analysis of the gut microbiota was performed.

Results: The results demonstrated that isoflavone administration did not affect body weight, glucose tolerance, or lipid metabolism. In contrast, isoflavone-treated mice had higher grip strength. Gene expression analysis of the soleus muscle revealed decreased Trim63 expression, and western blotting showed inactivation of muscle-specific RING finger protein 1 in isoflavone-treated mice. Gut microbiota analysis indicated higher Bacteroidetes and lower Firmicutes abundance in the isoflavone group, along with increased microbiota diversity. Gene sets related to TNF-α signaling via NF-κB and unfolded protein response were negatively associated with isoflavones.

Conclusions: Isoflavone intake alters gut microbiota and increases muscle strength, suggesting a potential role in improving sarcopenia in menopausal women.

Keywords: estrogen; gut microbiota; isoflavone; menopause; sarcopenia.

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Conflict of interest statement

Nakajima, H. received individual compensation from Kowa Pharmaceutical Co., Ltd., Kyowa Hakko Kirin Co., Ltd., and Nippon Boehringer Ingelheim Co., Ltd. Nakanishi, N. received individual compensation from Kowa Pharmaceutical Co., Ltd., Novo Nordisk Pharma Ltd., Nippon Boehringer Ingelheim Co., Ltd., and TERUMO CORPORATION. Hamaguchi, M. received grants from AstraZeneca K.K., Ono Pharma Co., Ltd., and Kowa Pharmaceutical Co., Ltd.; and also received individual compensation from AstraZeneca K.K., Ono Pharma Co., Ltd., Eli Lilly Japan K.K., Sumitomo Dainippon Pharma Co., Ltd., Daiichi Sankyo Co., Ltd., Mitsubishi Tanabe Pharma Corp., Sanofi K.K., and Kowa Pharmaceutical Co., Ltd. outside of the submitted work. Fukui, M. received grants from Ono Pharma Co., Ltd., Oishi Kenko Inc., Yamada Bee Farm, Nippon Boehringer Ingelheim Co., Ltd., Kissei Pharmaceutical Co., Ltd., Mitsubishi Tanabe Pharma Corp., Daiichi Sankyo Co., Ltd., Sanofi K.K., Takeda Pharmaceutical Co., Ltd., Astellas Pharma Inc., MSD K.K., Kyowa Kirin Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd., Kowa Pharmaceutical Co., Ltd., Novo Nordisk Pharma Ltd., Sanwa Kagaku Kenkyusho Co., Ltd., Eli Lilly Japan K.K., Taisho Pharmaceutical Co., Ltd., Terumo Corp., Teijin Pharma Ltd., Nippon Chemiphar Co., Ltd., Abbott Japan Co., Ltd., and Johnson & Johnson K.K. Medical Co., TERUMO CORPORATION, and also received individual compensation from Nippon Boehringer Ingelheim Co., Ltd., Kissei Pharmaceutical Co., Ltd., Mitsubishi Tanabe Pharma Corp., Daiichi Sankyo Co., Ltd., Sanofi K.K., Takeda Pharmaceutical Co., Ltd., Astellas Pharma Inc., MSD K.K., Kyowa Kirin Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd., Kowa Pharmaceutical Co., Ltd., Novo Nordisk Pharma Ltd., Ono Pharma Co., Ltd., Sanwa Kagaku Kenkyusho Co., Ltd., Eli Lilly Japan K.K., Taisho Pharmaceutical Co., Ltd., Bayer Yakuhin, Ltd., AstraZeneca K.K., Mochida Pharmaceutical Co., Ltd., Abbott Japan Co., Ltd., Teijin Pharma Ltd., Arkray Inc., Medtronic Japan Co., Ltd., and Nipro Corp., TERUMO CORPORATION, outside of the submitted work. Sasano, R. was employed by the company AiSTI SCIENCE Co., Ltd. The remaining 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
Administration of isoflavone did not change body weight, glucose tolerance, and lipid metabolism in OVX mice. (a) Body weight (n = 6); (b) change in dietary and isoflavone water intake (n = 6); (c) results from the intraperitoneal glucose tolerance test (iPGTT; 1 g/kg body weight) along with area under the curve (AUC) analysis (n = 6); (d) findings from the insulin tolerance test (ITT; 0.5 U/kg body weight), including AUC analysis (n = 6); (e) serum levels of albumin (Alb), alanine aminotransferase (ALT), triglycerides (TG), total cholesterol (T-Chol), and nonesterified fatty acids (NEFA) (n = 6); (f) serum estradiol levels (n = 6). Data are presented as mean ± SD values and were analyzed using a paired t-test. OVX: ovariectomy.
Figure 2
Figure 2
Isoflavone administration enhanced grip strength in mice and decreased the expression levels of muscle atrophy-related genes in OVX mice. (a) Shown are representative images of hematoxylin and eosin-stained sections of the plantaris muscle, collected from 14-week-old mice, with a scale bar of 100 μm; (b) the cross-sectional area and diameter of the plantaris muscle were measured (n = 6). (c) Both absolute and relative grip strength were recorded (n = 6); (d) the absolute and relative weights of the plantaris and soleus muscles were measured in 14-week-old mice (n = 6 in each group); (e) gene set enrichment analysis (GSEA) showed enrichment plots of three glycolysis-related gene sets, comparing isoflavone-treated mice with controls. A heatmap of 100 core genes is also provided for these comparisons; (f) western blot analysis detected MuRF1 levels in the gastrocnemius muscle, and the relative intensity of MuRF-1 was quantified (n = 6). Data are presented as mean ± SD values. Data were analyzed using a paired t-test. *** p < 0.001 and **** p < 0.0001.
Figure 3
Figure 3
Administration of isoflavone changed the gut microbiota in OVX mice. (a) Constituents of the gut microbiota. Relative abundance of gut microbiota at the phylum levels (n = 3); (b) operational taxonomic units (OTUs) (n = 3), Shannon index, Chao1 (n = 3), and Gini–Simpson index (n = 3); (c) Firmicutes/Bacteroidetes ratio; (d) linear discriminant analysis (LDA) scores of gut microbiota of the control mice (green) and the isoflavone mice (red); (e) the structure of co-occurrence OTU networks is delineated. Nodes symbolize OTUs, while edges depict statistically significant positive correlations between each pair of OTUs. The dimensions of nodes reflect the relative abundance of OTUs within the dataset; (f) relative abundance of co-abundance gene groups (CAGs). Data are presented as mean ± SD values. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.
Figure 4
Figure 4
Correlation of serum isoflavones with intestinal microbiota and small intestinal gene cluster. (a) Fold change of concentration in serum of equol, genistein, and daidzein; (b) chemical structure of daidzein, genistein and equol; (c) circos plot showing the gene expression in relation to 12 differential CAGs and three isoflavone metabolites, and gene set related TNF-α signaling via NF-κB and unfolded protein response. Only pairs with an absolute value of correlation coefficient greater than 0.3 are connected at the edges. Red links stand for positive correlations and blue links stand for negative correlations. Data are presented as mean ± SD values. * p < 0.05. ns: not significant.
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