Suppression of body fat accumulation in myostatin-deficient mice (original) (raw)
Decreased fat accumulation in Mstn–/– mice. We previously reported that Mstn knockout mice have a widespread increase in skeletal muscle mass, leading to a 25–30% increase in overall body weight at 3–6 months of age (3). A comparison of Mstn+/+ and Mstn–/– male mice at older ages, however, revealed that, unlike Mstn+/+ mice, Mstn–/– mice did not continue to gain weight beyond 6 months of age, so that by 9–10 months of age, the total body weights of Mstn+/+ mice were comparable to those of Mstn–/– mice (data not shown). In order to determine whether the normalization of body weights between the two genotypes resulted from a normalization of muscle weights, we compared muscle weights of Mstn+/+ and Mstn–/– mice in the C57BL/6J background at various ages. As shown in Figure 1a for the triceps muscle (similar results were obtained for the pectoralis, quadriceps, gastrocnemius/plantaris, and tibialis anterior), differences in muscle weights were evident even at the youngest age examined (2 months) and were maintained in older animals. In addition, at all ages examined, mice heterozygous for the Mstn mutation had muscle weights that were intermediate between those of Mstn+/+ and Mstn–/– mice, suggesting that the effect of myostatin on muscle mass is dose-dependent.
Mstn deletion suppresses fat accumulation. (a) Increased mass of triceps muscle in Mstn–/– mice at different ages (n = 4–13). Black, Mstn+/+; blue, Mstn+/–; pink, Mstn–/–. Data are expressed as mean ± SEM. (b–f) Mass of male epididymal (b), retroperitoneal (c), and inguinal (d), and female parametrial (e) and retroperitoneal (f) fat pads at different ages (n = 4–13). Orange bars designate the mean, and symbols designate individual animals colored as in a, with diamonds for males and circles for females. #P = 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t test. (g) Serum concentration of leptin to mass of total body fat in individual 8-month-old male mice. (h) Fat cell histology in epididymal fat pads. Scale bar = 50 μm.
Because normalization of body weights occurred even though differences in skeletal muscle weights continued to be maintained throughout the life of the animals, we investigated the possibility that the increase in body weight in older Mstn+/+ mice was due to a higher rate of fat accumulation. As shown in Figure 1, b–f, an analysis of individual fat pad weights revealed no differences between Mstn+/+ and Mstn–/– mice at 2 months of age. By 5–6 months, however, there was a significant difference in males, with individual fat pads of Mstn+/+ mice weighing on average 2.4–4.4 times those of Mstn–/– mice. By 9–10 months, fat pad weights in both male and female Mstn+/+ mice were spread over a large range, with fat pad weights in some animals increasing up to ninefold compared with those at 2 months of age. In contrast, every Mstn–/– mouse examined at 9–10 months remained lean with relatively little fat pad weight gain over the 7- to 8-month interval. In order to rule out the possibility that fat stores were simply redistributed in Mstn–/– mice, we measured total body fat. As shown in Figure 1g, the mean total body fat mass was reduced by 70% in Mstn–/– mice compared with Mstn+/+ mice. Serum leptin levels correlated with the amount of total body fat in individual animals and were therefore significantly lower in Mstn–/– mice (13.0 ± 1.6 ng/ml, Mstn+/+; 2.6 ± 0.2 ng/ml, Mstn–/–; P < 0.001) (Figure 1g). Hence, the differences in total body weight gain over time appeared to result from differences in fat accumulation.
In order to determine whether the differences in fat pad weights reflected differences in fat cell number or cell size, we carried out a more detailed analysis of the gonadal fat pads. Mstn–/– mice had approximately 25% fewer gonadal fat pad cells than Mstn+/+ mice (Table 1). Cell size, however, was also affected, with the average weight of cells in the genital fat pad of Mstn–/– mice being approximately 40% that of Mstn+/+ mice (Table 1; Figure 1h). In addition to lower fat pad weights, adult male Mstn–/– mice also had significantly lower serum triglyceride and cholesterol levels than Mstn+/+ mice (Table 1). Blood glucose control seemed to be unaffected, however, as Mstn+/+ and Mstn–/– mice had similar insulin, fed glucose, and fasting glucose levels (Table 1). They also showed similar responses in glucose tolerance tests, with each reaching a maximum serum glucose value of approximately 220 mg/dl after 15 minutes and returning to base line 2 hours after glucose administration (data not shown).
Effects of Mstn mutation on fat cells and serum levels of triglycerides, cholesterol, glucose, and insulin
Mstn–/– mice failed to accumulate fat despite the fact that they did not have a decreased rate of food consumption. Food consumption in 5.5-month-old Mstn–/– mice was actually 16% greater than that in age-matched Mstn+/+ mice, although, when expressed as a percentage of body weight, food consumption was very similar between Mstn–/– and Mstn+/+ mice (Table 2). Body temperatures of Mstn–/– and Mstn+/+ mice either under normal conditions or in response to a 60-minute cold tolerance test were also similar, even though brown fat weights were reduced by approximately 50% in Mstn–/– mice (Table 2). We also examined expression levels of mRNAs encoding the uncoupling proteins, Ucp1, Ucp2, and Ucp3. The UCP proteins are mitochondrial inner membrane proteins that uncouple the proton gradient from ATP synthesis and have been implicated in thermogenesis (17). As shown in Figure 2, expression levels of each of these mRNAs were not increased in Mstn–/– mice as compared with Mstn+/+ mice. Expression levels of Ucp2 and Ucp3 in skeletal muscle were actually slightly decreased in Mstn–/– mice. Finally, we investigated the possibility that the reduced fat pad weights could be caused by an increase in metabolic rate in the knockout mice. As shown in Table 2, analysis of 6-month-old animals in a metabolic chamber revealed that Mstn–/– mice had higher rates of total and resting O2 consumption compared with Mstn+/+ mice (14% and 8% higher, respectively), which is expected given the higher body weights of Mstn–/– mice. If the data are expressed as a function of body weight, however, Mstn–/– mice actually had lower rates of total and resting O2 consumption compared with Mstn+/+ mice (10% and 15% lower, respectively) (Table 2). The respiratory exchange ratio, the ratio of CO2 produced to O2 consumed, in Mstn–/– mice was identical to that of Mstn+/+ mice (data not shown).
Expression of uncoupling proteins. Fifteen micrograms of total RNA isolated from various tissues in Mstn+/+ and Mstn–/– mice was electrophoresed, blotted, and probed with Ucp1, Ucp2, and Ucp3. Each blot was also hybridized with an S26 ribosomal protein probe as a loading control.
Food intake, brown adipose tissue, body temperature, and metabolic rate in Mstn+/+ and _Mstn_–/– male mice
Suppression of obesity and glucose metabolism in Ay, Mstn–/– mice. The lack of fat accumulation in Mstn–/– mice raised the possibility that inhibition of myostatin might be an effective method of suppressing the development of obesity in settings of abnormal fat accumulation. Therefore, we analyzed the effect of the Mstn mutation in mouse genetic models of obesity. Ay is a dominant mutation that causes obesity by increasing food intake and fuel efficiency (18, 19). The Ay mutation causes abnormal expression of agouti protein, which antagonizes melanocortin receptors in the hypothalamus (18, 19). As in a/a animals, individual muscle weights of Ay/a, Mstn–/– mice were approximately twice as high as those of Ay/a, Mstn+/+ mice (data not shown). Despite the increase in muscle mass, total body weights of Ay/a, Mstn–/– mice at 7 months of age were either comparable to (in the case of males) or actually lower than (in the case of females) those of age-matched Ay/a, Mstn+/+ mice (data not shown). As shown in Figure 3a, the appearance of adult female Ay/a, Mstn–/– mice was more similar to that of a/a, Mstn–/– mice than to that of Ay/a, Mstn+/+ mice. The reduced body weights and altered appearance of Ay/a, Mstn–/– mice resulted from a reduction in fat accumulation. Although individual fat pad weights were higher in Ay/a, Mstn–/– mice than in a/a, Mstn–/– mice, fat pads weighed less than half as much in Ay/a, Mstn–/– mice than in Ay/a, Mstn+/+ mice (Figure 3, b and c). Hence, the presence of the Mstn mutation partially suppressed the development of obesity in Ay/a mice.
Mstn deletion partially suppresses fat accumulation and glucose intolerance in Ay mice. (a) Effect of Mstn deletion on appearance of a/a and Ay/a female mice. (b and c) Effects of Mstn deletion on fat pad weights in 7-month-old Ay male (b) (n = 6–9) and female (c) (n = 8–20) mice. Orange bars designate the mean, and symbols designate individual animals. Black, Ay/a, Mstn+/+; pink, Ay/a, Mstn–/–. Diamonds, males; circles, females. (d and e) Suppression of abnormality of glucose tolerance tests in male (d) (n = 4) and female (e) (n = 5–13) Mstn_–/–_ mice. Symbols are as in b and c. Some measurements exceeded the upper detection limit of the glucose test (600 mg/dl). *P < 0.05, ***P < 0.001, Student’s t test.
Loss of myostatin also affected glucose metabolism in Ay/a mice. Ay/a mice have been shown to develop insulin resistance and have therefore been used as a model for type 2 diabetes (18, 19). As described previously (18, 20, 21), male Ay/a, Mstn+/+ mice had elevated fed glucose and insulin levels (Table 3) compared with normal mice (Table 1) and had grossly abnormal serum glucose levels in response to a glucose tolerance test (Figure 3d). In contrast, virtually all Ay/a male mice that were also Mstn–/– had normal fed glucose and insulin levels (Table 3) and had dramatically lower glucose levels following an exogenous glucose load than Ay/a, Mstn+/+ mice (Figure 3d). Similar effects were also observed in female mice (Table 3; Figure 3e), although the abnormalities in glucose metabolism are known to be less severe in female Ay/a mice than in male Ay/a mice (20). Although the Mstn mutation did not completely eliminate the effects of the Ay/a mutation on glucose metabolism (glucose tolerance tests of Ay/a, Mstn–/– mice were still abnormal relative to those of a/a, Mstn+/+ and a/a, Mstn–/– mice), these results suggest that the Mstn mutation can have beneficial effects with respect to the development of type 2 diabetes in Ay/a mice.
Effects of the Mstn mutation on blood glucose and serum insulin levels in 7-month-old Ay animals
Suppression of obesity and glucose metabolism in Lepob/ob, Mstn–/– mice. We also investigated the effects of the Mstn mutation in Lepob/ob mice. Loss of leptin signaling in Lepob/ob mice causes severe obesity as a result of improper regulation of food intake and energy expenditure (22, 23). Although some of the effects of leptin are known to be mediated through melanocortin receptor pathways, leptin is also known to have effects that are melanocortin-independent (24–26). The increase in muscle mass due to loss of myostatin was delayed in Lepob/ob mice as compared with Lep+/+ mice. Individual muscles were only 35–72% heavier in Lepob/ob, Mstn–/– mice than in Lepob/ob, Mstn+/+ mice at 8 weeks of age, although they were 100% heavier at 3 months of age (data not shown). Nevertheless, as in Ay/a mice, the Mstn mutation also suppressed fat accumulation in Lepob/ob mice, which was evident upon examination of individual fat pad weights. At 8 weeks of age, Lepob/ob, Mstn–/– mice had a statistically significant reduction in retroperitoneal and parametrial fat pad weights compared with Lepob/ob, Mstn+/+ mice (Figure 4, a and b). The Lepob/ob mutation is also known to cause abnormalities in glucose metabolism, which is most prominent in young mice (22). The Mstn deletion delayed the development of hyperglycemia in Lepob/ob mice of both sexes (Figure 4, c and d). In female Lepob/ob mice, the Mstn mutation completely suppressed the development of hyperglycemia in animals at 6 and 8 weeks of age, whereas the effect in male mice was most prominent at the youngest age examined (6 weeks).
Mstn deletion partially suppresses fat accumulation and delays hyperglycemia in Lepob/ob mice. (a and b) Decrease in fat pad weights in male (a) (n = 5–8) and female (b) (n = 5–6) Lepob/ob, Mstn–/– 8-week-old mice. Orange bars designate the mean, and symbols designate individual animals. Black, Lepob/ob, Mstn+/+; pink, Lepob/ob, Mstn–/–. Diamonds, males; circles, females. (c and d) Suppression of abnormal fed glucose levels in Lepob/ob, Mstn–/– male (c) (n = 6–22) and female (d) (n = 8–26) mice. Symbols are as in a and b. *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t test.