Abstract
Background. Myostatin (Mstn) plays an important role in adipocyte growth, differentiation and metabolism, leading to the development of obesity.
Objectives. We aimed to explore the effect of Mstn on white fat browning in a mouse model of type 2 diabetes mellitus (T2DM).
Materials and methods. Twelve wild-type (WT), 12 heterozygous (Mstn(+/−)) and 12 homozygous (Mstn(−/−)) male mice were randomly divided into 6 groups: WT, Mstn(+/−), Mstn(−/−), WT+DM, Mstn(+/−)+DM, and Mstn(−/−)+DM. The first 3 groups were fed normal chow, while the last 3 were fed high-fat diet and administered streptozotocin to generate T2DM. Subsequently, body mass, length, and white and brown fat masses were measured, after which Lee’s index, white−brown ratio and fat index were calculated. The serum free fatty acid (FFA) levels were detected using enzyme-linked immunosorbent assay (ELISA). Hematoxylin and eosin (H&E) staining was used to analyze white and brown fat cell morphology. The relative expression levels of peroxisome proliferator-activated receptor-gamma (PPARγ), peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1α), uncoupling protein 1 (UCP1), and cluster of differentiation 137 (CD137) protein were determined with western blotting.
Results. The Mstn(−/−) group displayed higher levels of PPARγ, PGC-1α and CD137 proteins in white and brown fat compared to the WT and Mstn(+/−) groups, while the expression level of UCP1 protein in the Mstn(−/−) group was higher than in the WT group. The expression levels of PPARγ, PGC-1α, UCP1, and CD137 proteins in the WT+DM group were lower than in the WT group. Moreover, PPARγ, PGC-1α, UCP1, and CD137 proteins were more highly expressed in the Mstn(−/−)+DM group compared to the WT+DM and Mstn(+/−)+DM groups.
Conclusions. The Mstn gene inhibition antagonizes obesity phenotypes, such as white fat accumulation and lipid metabolism derangement caused by T2DM, thus promoting white fat browning.
Key words: diabetes mellitus type 2, obesity, mice, myostatin, brown adipose tissue
Background
Skeletal muscle, brown fat and white fat significantly contribute to maintaining the body’s energy balance, consuming energy through muscle fiber contraction, as well as non-shivering thermogenesis and energy storage, respectively. However, white fat can be converted into brown fat under certain conditions to ameliorate obesity. In type 2 diabetes mellitus (T2DM), energy metabolism is unbalanced, skeletal muscle decreases, white fat increases, and sarcopenic obesity arises. Therefore, increasing skeletal muscle and promoting the browning of white fat is beneficial to treating obesity, T2DM and other metabolic diseases. Skeletal muscle can secrete various myokines, which act as cross-talk messengers between skeletal muscle and fat.1 Myostatin (Mstn), which negatively regulates muscle growth and development, is an important myokine secreted by skeletal muscle.2 After Mstn knockout, mice fed high-fat or regular diet showed decreased fat mass and increased muscle mass,3 suggesting that Mstn can regulate lipid metabolism in T2DM. However, whether Mstn can promote the browning of white fat in T2DM remains unclear.
Objectives
We aimed to observe the expression of a gene associated with white fat browning in diabetic Mstn knockout mice and explore the effect of Mstn on white fat browning in T2DM mice.
Materials and methods
Animals
All animal experiments followed the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines and were performed in accordance with the UK Animal (Scientific Procedures) Act 1986, the relevant guidelines, the EU Directive 2010/63/EU for animal testing, or the management and use guidelines of the National Institute of Health (NIH).
Saiye Biotechnology Co. Ltd. (Guangzhou, China) provided the following 6-week-old male C57BL/6N mice: 12 homozygous Mstn knockout (Mstn(−/−)) mice, 12 heterozygous Mstn knockout (Mstn(+/−)) mice and 12 wild-type (WT) mice. Mice were acclimatized for 1 week at an indoor temperature of 22–24°C, 40–60% humidity, and 12-h day and night cycles and were provided with sufficient food and water before the beginning of the study.
Grouping and modeling
The 12 WT, 12 heterozygous (Mstn(+/−)) and 12 homozygous (Mstn(−/−)) mice were randomly divided into 2 large groups (control and T2DM groups), which were then divided into 6 smaller groups (6 animals per group), as follows: WT group, Mstn(+/−) group, Mstn(−/−) group, WT+DM group, Mstn(+/−)+DM group, and Mstn(−/−)+DM group. The first 3 groups were fed normal chow, while the remaining 3 groups were fed high-fat diet and a small dose of streptozotocin to generate T2DM models. The WT+DM, Mstn(+/−)+DM and Mstn(−/−)+DM groups were fed high-fat diet for 6 weeks and injected intraperitoneally with 2% streptozotocin (Sigma-Aldrich, St. Louis, USA) (prepared with sodium citrate buffer) at 50 mg/kg without fasting. Then, 72 h after the streptozotocin injection, and followed by 6-hour fasting, tail vein blood was collected and fasting blood glucose was measured. The criteria for a successful induction of T2DM in the mice were as follows: mice with fasting blood glucose ≥16.7 mmol/L and the occurrence of polydipsia, polyphagia, polyuria, and weight loss. The WT, Mstn(+/−) and Mstn(−/−) groups were injected intraperitoneally with equal doses of citrate buffer as the control.
Measurement of body mass and length
The body mass and length (from the nasal tip to the anus) for each group were measured before and after diabetes induction. Lee’s index was calculated and used to assess the degree of obesity in mice, as follows (Equation 1):
(1)
Detection of blood lipids
After 6 h of fasting, 0.2 mL of mouse tail vein blood was collected and centrifuged at 3000 rpm for 5 min. The serum was collected to detect the triacylglycerol (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) levels using an automatic biochemical analyzer (AU400; Beckman Coulter, Brea, USA). The serum free fatty acid (FFA) level was detected with the use of an FFA enzyme-linked immunosorbent assay (ELISA) kit (Cloud-Clone, Houston, USA) and a microplate reader (ELx808; BioTeK, Winooski, USA).
Measurement of white and brown fat mass
The mice were sacrificed by cervical dislocation, after which white adipose (groin, epididymis and mesenteric) and brown adipose (scapula) tissues were quickly dissected and weighed. The white–brown ratio and fat index were calculated as follows (Equation 2 and Equation 3):
(2)
(3)
Half of the white adipose tissue in the groin and half of the brown adipose tissue in the scapula were stored in liquid nitrogen, while the other halves were preserved in 4% neutral buffered formalin and embedded in paraffin by a tissue-dehydrating machine (JT-12S; Junjie, Shenzen, China).
Hematoxylin and eosin staining and the analysis of adipose tissue
Instruments and reagents for hematoxylin and eosin (H&E) staining and the analysis of adipose tissue are detailed in the Supplementary Materials. Images of adipose tissue sections were viewed under an optical microscope (E100; Nikon Corp., Tokyo, Japan), and the adipose tissue morphology was analyzed using ImageJ software (National Institutes of Health, Bethesda, USA).
Detection of white fat browning-related gene expression
Western blotting was used to measure the expression levels of the following proteins: peroxisome proliferator-activated receptor gamma (PPARγ), peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1α), uncoupling protein 1 (UCP-1), and cluster of differentiation 137 (CD137). Briefly, 50 mg of adipose tissue was treated with protein lysis solution to completely lyse the tissue, which was then centrifuged at 12,000 rpm for 10 min, and the supernatant was collected (Supplementary Materials).
Detection of Mstn expression in the gastrocnemius muscle and serum
After the mice were sacrificed, the left gastrocnemius muscle was dissected. Western blotting was used to detect the expression level of Mstn in the gastrocnemius using the same procedure as above. The serum Mstn level was detected using Mstn ELISA kit (Cloud-Clone), according to the manufacturer’s instructions.
Statistical analyses
The IBM SPSS v. 22.0 (IBM Corp., Armonk, USA) and GraphPad Prism v. 8.0 (GraphPad Software, San Diego, USA) were utilized to analyze the experimental data and present the results, respectively. A bootstrapped one-way analysis of variance (ANOVA) was used for comparisons among the 6 groups, followed by Tukey’s post hoc test. Representative results are shown as mean ± standard deviation (M ±SD), and p < 0.05 was considered statistically significant.
Results
Indicators of growth and obesity
Lee’s index and the white−brown ratio were significantly lower in the Mstn(−/−) group than in the WT and Mstn(+/−) groups (p < 0.05). There was no significant difference in the fat index between the Mstn(−/−) group and the WT or Mstn(+/−) groups (p > 0.05). Lee’s index was lower in the WT+DM group than in the WT group, while the white−brown ratio and fat index were higher in the WT+DM group than in the WT group (p < 0.05). The Mstn(−/−)+DM group displayed lower Lee’s index and white−brown ratio compared to the WT+DM and Mstn(+/−)+DM groups (p < 0.05). The fat index was lower in the Mstn(−/−)+DM group than in the WT+DM group (p < 0.05), but it was not significantly different compared with that in the Mstn(+/−)+DM group (p > 0.05) (Supplementary Table 1 and Figure 1).
Lipid metabolism indicators
The serum TG and TC levels were both lower in the Mstn(−/−) group than in the WT and Mstn(+/−) groups (both p < 0.05). Moreover, the LDL-C, HDL-C and FFA levels in the Mstn(−/−) group were not significantly different compared with those in the WT and Mstn(+/−) groups (p > 0.05). The TG, TC, LDL-C, and FFA levels were higher in the WT+DM group than in the WT group, while the HDL-C level was lower in the WT+DM group than in the WT group (p < 0.05). The TC level was lower in the Mstn(−/−)+DM group than in the WT+DM and Mstn(+/−)+DM groups, while the HDL-C level was higher in the Mstn(−/−)+DM group compared to the WT+DM and Mstn(+/−)+DM groups (p < 0.05). The serum TG, LDL-C and FFA levels were lower in the Mstn(−/−)+DM group than in the WT+DM group (p < 0.05) and showed no significant difference compared with those in the Mstn(+/−)+DM group (p > 0.05) (Supplementary Table 2 and Figure 2).
Morphology of white adipocytes
White adipocytes in the WT group displayed a full shape and uniform size. Interestingly, when compared with the WT group, the number of white adipocytes in the Mstn(−/−) group was significantly increased, while the volume was significantly reduced, and they showed an irregular shape. The performance of the Mstn(+/−) group was between that of the WT and Mstn(−/−) groups. Compared with the WT group, the volume of white adipocytes in the WT+DM group was significantly greater, with variable sizes and fused cells. The number of white adipocytes in the Mstn(−/−)+DM group was significantly increased, whereas the size of their white adipocytes was significantly reduced compared to the WT+DM group. As expected, the results for the Mstn(+/−)+DM group were between that of the WT+DM and Mstn(−/−)+DM groups (Figure 3A).
Morphology of brown adipocytes
The brown adipocytes in the WT, Mstn(+/−) and Mstn(−/−) groups all showed multilocular morphology, and no single-vesicle adipocytes were observed. When compared to the WT and Mstn(+/−) groups, the Mstn(−/−) group showed a more dense distribution of brown adipocytes. In the WT+DM group, many single-vesicle adipocytes were present among the multilocular brown adipocytes, and there was a circular lipid droplet in the center of each cell. In the Mstn(−/−)+DM group, multilocular brown adipocytes were distributed among the single-vesicle adipocytes, the number of which was significantly reduced, and the volume of lipid droplets was significantly decreased compared with the WT+DM group. As expected, the results for the Mstn(+/−)+DM group were between those of the WT+DM and Mstn(−/−)+DM groups (Figure 3B).
Expression levels of PPARγ, PGC-1α, UCP1, and CD137 protein in white and brown fat
The expression levels of PPARγ, PGC-1α and CD137 proteins in white and brown fat in the Mstn(−/−) group were higher than those in the WT and Mstn(+/−) groups (p < 0.05). The UCP1 protein in white and brown fat demonstrated higher expression in the Mstn(−/−) group compared to the WT group (p < 0.05). The PPARγ, PGC-1α, UCP1, and CD137 protein expression levels in white and brown fat were reduced in the WT+DM group compared to the WT group (p < 0.05). Moreover, the expression levels of PPARγ, PGC-1α, UCP1, and CD137 proteins in white and brown fat were higher in the Mstn(−/−)+DM group than in the WT+DM and Mstn(+/−)+DM groups (p < 0.05) (Supplementary Table 3,4 and Figure 4, Figure 5).
Expression levels of Mstn in the gastrocnemius muscle and serum
The expression levels of Mstn in the gastrocnemius muscle and serum of WT heterozygous and homozygous mice gradually decreased with the decrease of gene abundance. The Mstn was not detected in the Mstn(−/−) and Mstn(−/−)+DM groups, suggesting that protein expression was completely inhibited after Mstn knockout. The Mstn expression level was correlated with gene abundance. Compared with the same-genotype non-diabetic group, the expression level of Mstn protein in the gastrocnemius of the WT+DM and Mstn(+/−)+DM groups significantly increased (p < 0.05), suggesting that skeletal muscle secretion of Mstn increases in T2DM (Supplementary Table 5 and Figure 6).
Discussion
In this study, we examined the effect of Mstn on white fat browning in T2DM mice. Obesity is a global public health problem and a significant cause of T2DM and other metabolic disorders. Recently, the browning of white fat has been a topic of great interest in the treatment of obesity. Exploring the sensing and regulatory mechanisms of the conversion of white fat to brown fat promoted by an external stimulus will help develop new drugs for the treatment of obesity.
The Mstn is mainly expressed in the skeletal muscle and negatively regulates its growth and development.2 Moreover, previous studies have shown that Mstn may regulate fat metabolism.3 For example, the subcutaneous fat content to body weight ratio and the serum TG levels were significantly lower in the Mstn(−/−) Meishan pigs than in the WT pigs.4 Our research demonstrated several interesting results, namely that Lee’s index and the serum HDL-C levels were lower in the WT+DM group than in the WT group, whereas the fat index and the serum TG, TC, LDL-C, and FFA levels were higher in the WT+DM group when compared to the WT group. Lee’s index, fat index, and serum TG, TC, LDL-C, and FFA levels were lower in the Mstn(−/−)+DM group than in the WT+DM group, while the serum HDL-C level was higher in the Mstn(−/−)+DM group than in the WT+DM group. These results suggest that Mstn knockout significantly antagonizes obesity characteristics, such as fat accumulation and lipid metabolism disorderliness, caused by T2DM. The decrease in fat mass observed in the Mstn(−/−) mice may be associated with Mstn-induced inhibition of the differentiation of pre-adipocytes into mature adipocytes. This decrease in the number of mature adipocytes leads to a reduction in total fat.5
White adipose tissue is mainly distributed in subcutaneous and internal organs, such as mesenteric organs and gonads. White fat cells contain a single large lipid droplet and a high level of TG. The main function of the white adipose tissue is to store excess energy in the body in the form of fat, which then becomes the main factor leading to obesity. Conversely, brown adipose tissue is mainly distributed between the shoulder blades, the back of the neck, the axilla, the mediastinum, and around the kidneys. The body’s brown fat gradually decreases with age. Brown adipocytes contain small multilocular lipid droplets and many mitochondria, which consume TG stored in white fat through uncoupled oxidative phosphorylation, generating heat and regulating the body’s temperature balance.6 Beige fat is an intermediate form of white fat in the process of being converted to brown fat. It is activated by cold and other external stimuli, and exerts a heat-generating effect like brown fat, leading to white fat browning.6 Both brown and beige fat improve body fat metabolism and reduce obesity, and the cells of both fat cells carry highly expressed marker genes. Studies have confirmed that PPARγ, PGC-1α, UCP1, PR domain-containing 16 (PRDM16), and cell death-inducing DNA fragmentation factor α-like effector A (CIDEA) are highly expressed in brown adipocytes, while CD137 and transmembrane protein 26 (TMEM26) are highly expressed in beige adipocytes. Therefore, these genes are markers of white fat browning.7, 8, 9
The Mstn has also been shown to regulate white fat browning. Studies have reported that Mstn(−/−) mice display increased energy utilization and resistance to genetic or diet-induced obesity. Moreover, subcutaneous white fat demonstrates some of the characteristics of brown fat, in which the expression levels of PGC-1α, UCP1, CD137, and Tmem26 were increased.10
This study shows that the expression levels of PPARγ, PGC-1α, UCP1, and CD137 in the white and brown fat were higher in the Mstn(−/−) group than in the WT group, lower in the WT+DM group than in the WT group, and higher in the Mstn(−/−)+DM group compared to the WT+DM group. Combined with the H&E staining of white and brown adipose tissues, these results suggested that T2DM may decrease the expression of brown fat and beige fat marker genes and increase white fat phenotypes and energy storage. Thus, it may be surmised that Mstn knockout significantly antagonizes this phenomenon, promotes white fat browning, increases heat production in the body, and promotes the consumption of white fat, thereby reducing obesity. The Mstn can inhibit the differentiation of brown adipocytes by activating Smad3 phosphorylation.11 We found that after Mstn treatment of brown adipocytes, the expression of the PPARγ, UCP1, PGC-1, and PRDM16 genes was downregulated. The expression of these genes was further supressed after β-catenin activator treatment. Furthermore, there are interactions between the Smad3 and Wnt/β-catenin pathways.11 The Mstn can induce phosphorylation of Smad3, further increasing the stability of β-catenin, and the Wnt/β-catenin pathway can inhibit the differentiation of brown adipocytes.11, 12, 13 However, the specific mechanism involved requires further study.
Limitations
This study was beset by certain limitations, including the small sample size and a lack of an investigation into the underlying molecular mechanisms. The specific mechanism underlying the role of Mstn in white fat browning requires further studies. Moreover, female mice have different fat metabolism characteristics compared to male mice. To exclude the effect of estrogen, male mice were chosen for this experiment. We expect similar results in female mice, in which fat deposition and type are strongly dependent on estrogen and its signaling.
Conclusions
This study suggests that inhibiting Mstn expression may antagonize obesity phenotypes, such as white fat accumulation and lipid metabolism derangement caused by T2DM, upregulate the expression of PPARγ, PGC-1α, UCP1, and CD137, promote white fat browning, and alleviate obesity.
Supplementary data
The Supplementary materials are available at https://doi.org/10.5281/zenodo.8392519. The package contains the following files:
Supplementary Table 1. Growth and obesity indicators.
Supplementary Table 2. Lipid metabolism indicators.
Supplementary Table 3. Expression levels of PPARγ, PGC-1α, UCP1, and CD137 protein in white fat.
Supplementary Table 4. Expression levels of PPARγ, PGC-1α, UCP1, and CD137 protein in brown fat.
Supplementary Table 5. Expression levels of Mstn in the gastrocnemius and serum.
Supplementary File 1. Materials and methods description.
