Myostatin knockout in mice increases myogenesis and decreases adipogenesis

Growth differentiation factor-8 (GDF-8), or Myostatin, plays an important inhibitory role during muscle development. Since muscle and adipose tissue develop from the same mesenchymal stem cells, we hypothesized that Myostatin gene knockout may cause a switch between myogenesis and adipogenesis. Male and female wild type (WT) and Myostatin knockout (KO) mice were sacrificed at 4, 8, and 12 weeks of age. The gluteus muscle (GM) was larger in KO mice compared to WT mice at 8 (P < 0.01) and 12 (P < 0.001) weeks. At 12 weeks, KO mice had decreased fat depots (P < 0.01). Compared to 12-week-old WT mice, serum leptin concentration in KO mice was lower (P < 0.001) and leptin mRNA expression was decreased (P < 0.01) in inguinal adipose tissue. CCAAT/enhancer binding protein-alpha (C/EBPalpha) and peroxisome proliferator-activated receptor-gamma (PPARgamma) levels in adipose tissue were significantly lower in KO mice compared to WT mice. Thus, increased muscle development in Myostatin knockout mice is associated with reduced adipogenesis and consequently, decreased leptin secretion.     

The effects of myostatin on adipogenic differentiation of human bone marrow-derived mesenchymal stem cells are mediated through cross-communication between Smad3 and Wnt/beta-catenin signaling pathways

The effects of myostatin on adipogenic differentiation are poorly understood, and the underlying mechanisms are unknown. We determined the effects of human recombinant myostatin protein on adipogenesis of bone marrow-derived human mesenchymal stem cells (hMSCs) and adipose tissue-derived preadipocytes. For both progenitor cell types, differentiation in the presence of myostatin caused a dose-dependent reduction of lipid accumulation and diminished incorporation of exogenous fatty acid into cellular lipids. Myostatin significantly down-regulated the expression of adipocyte markers PPARgamma, C/EBPalpha, leptin, and aP2, but not C/EBPbeta. Overexpression of PPARgamma, but not C/EBPbeta, blocked the inhibitory effects of myostatin on adipogenesis. Myostatin induced phosphorylation of Smad3 in hMSCs; knockdown of Smad3 by RNAi or inhibition of its upstream kinase by an Alk5 inhibitor blocked the inhibitory effect of myostatin on adipogenesis in hMSCs, implying an important role of Smad3 activation in this event. Furthermore, myostatin enhanced nuclear translocation of beta-catenin and formation of the Smad3-beta-catenin-TCF4 complex, together with the altered expression of a number of Wnt/beta-catenin pathway genes in hMSCs. The inhibitory effects of myostatin on adipogenesis were blocked by RNAi silencing of beta-catenin and diminished by overexpression of dominant-negative TCF4. The conclusion is that myostatin inhibited adipogenesis in human bone marrow-derived mesenchymal stem cells and preadipocytes. These effects were mediated, in part, by activation of Smad3 and cross-communication of the TGFbeta/Smad signal to Wnt/beta-catenin/TCF4 pathway, leading to down-regulation of PPARgamma.     

Myostatin-deficient mice lose more skeletal muscle mass than wild-type controls during hindlimb suspension

Myostatin inhibits myogenesis. Therefore, we sought to determine if mice lacking the myostatin gene [Mstn(-/-)] would lose less muscle mass than wild-type mice during 7 days of hindlimb suspension (HS). Male Mstn(-/-) and wild-type (C57) mice were subjected to HS or served as ground-based controls (n = 6/group). Wild-type mice lost 8% of body mass and approximately 13% of wet mass from biceps femoris, quadriceps femoris, and soleus, whereas the mass of extensor digitorum longus (EDL) was unchanged after HS. Unexpectedly, Mstn(-/-) mice lost more body (13%, P < 0.05) and quadriceps femoris (17%, P < 0.05) mass than wild-type mice and lost 33% of EDL mass (P < 0.01) after HS. Protein expression of myostatin in biceps femoris and quadriceps femoris was not altered, whereas expression of MyoD, Myf-5, and myogenin increased in wild-type mice and tended to decrease in muscles of Mstn(-/-) mice. These data suggest that HS induced myogenesis in wild-type mice to counter atrophy, whereas myogenesis was not induced in Mstn(-/-) mice, thereby resulting in a greater loss of muscle mass.     

Garcinia cambogia extract ameliorates visceral adiposity in C57BL/6J mice fed on a high-fat diet

The aim of present study is to evaluate the effects of Garcinia cambogia on the mRNA levels of the various genes involved in adipogenesis, as well as on body weight gain, visceral fat accumulation, and other biochemical markers of obesity in obesity-prone C57BL/6J mice. Consumption of the Garcinia cambogia extract effectively lowered the body weight gain, visceral fat accumulation, blood and hepatic lipid concentrations, and plasma insulin and leptin levels in a high-fat diet (HFD)-induced obesity mouse model. The Garcinia cambogia extract reversed the HFD-induced changes in the expression pattern of such epididymal adipose tissue genes as adipocyte protein aP2 (aP2), sterol regulatory element-binding factor 1c (SREBP1c), peroxisome proliferator-activated receptor gamma2 (PPARgamma2), and CCAT/enhancer-binding protein alpha (C/EBPalpha). These findings suggest that the Garcinia cambogia extract ameliorated HFD-induced obesity, probably by modulating multiple genes associated with adipogenesis, such as aP2, SREBP1c, PPARgamma2, and C/EBPalpha in the visceral fat tissue of mice.     

Calcineurin-independent inhibition of 3T3-L1 adipogenesis by Pasteurella multocida toxin: suppression of Notch1, stabilization of beta-catenin and pre-adipocyte factor 1

Pasteurella multocida toxin (PMT) is a potent mitogen and a specific activator of Gq-dependent signalling pathways. PMT impairs osteoblast differentiation and causes bone loss and fat reduction in vivo. We examined the effect of PMT on cell signalling pathways involved in 3T3-L1 adipocyte differentiation. We demonstrate that PMT treatment before or together with differentiation induction factors inhibits adipogenesis and prevents upregulation of important adipocyte markers - peroxisome-proliferator-activated receptor gamma (PPARgamma) and CAATT enhancer-binding protein alpha (C/EBPalpha). Moreover, PMT completely downregulates PPARgamma and C/EBPalpha expression in mature adipocytes. Differentiation of pre-adipocytes into adipocytes requires the suppression of pre-adipocyte factor 1 (Pref1) and Wnt signalling, along with the degradation of beta-catenin. PMT prevents downregulation of Pref1 and beta-catenin under differentiation-inducing conditions. In addition, PMT treatment downregulates expression of Notch1, a protein responsible for cell fate decision and implicated in regulation of adipogenesis in 3T3-L1 cells. PMT action on adipogenesis was not reversed by cyclosporin A, an inhibitor of Galphaq-PLC-calcium-dependent calcineurin activation. Our results reveal new pathways involved in PMT action on cellular physiology and differentiation. Our study further demonstrates that the effect of PMT on Pref1/PPARgamma/C/EBPalpha expression and adipogenesis does not occur just through activation of the Galphaq-calcium-calcineurin pathway, but involves Wnt/beta-catenin and Notch1 signalling pathways, two signalling pathways strongly linked to cancer predisposition, neurological and immunological dysfunctions, and fat and bone development.     

Defective adipocyte differentiation in mice lacking the C/EBPbeta and/or C/EBPdelta gene.

To investigate the role of C/EBP family members during adipocyte differentiation in vivo, we have generated mice lacking the C/EBPbeta and/or C/EBPdelta by gene targeting. Approximately 85% of C/EBPbeta(-/-).delta(-/-) mice died at the early neonatal stage. By 20 h after birth, brown adipose tissue of the interscapular region in wild-type mice contained many lipid droplets, whereas C/EBPbeta(-/-).delta(-/-) mice did not accumulate droplets. In addition, the epidydimal fat pad weight of surviving adult C/EBPbeta(-/-).delta(-/-) mice was significantly reduced compared with wild-type mice. However, these adipose tissues in C/EBPbeta(-/-).delta(-/-) mice exhibit normal expression of C/EBPalpha and PPARgamma, despite impaired adipogenesis. These results demonstrated that C/EBPbeta and C/EBPdelta have a synergistic role in terminal adipocyte differentiation in vivo. The induction of C/EBPalpha and PPARgamma does not always require C/EBPbeta and C/EBPdelta, but co-expression of C/EBPalpha and PPARgamma is not sufficient for complete adipocyte differentiation in the absence of C/EBPbeta and C/EBPdelta.     

Molecular mechanism of 1,25-dihydroxyvitamin D3 inhibition of adipogenesis in 3T3-L1 cells

We have investigated the molecular mechanism whereby 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] inhibits adipogenesis in vitro. 1,25(OH)2D3 blocks 3T3-L1 cell differentiation into adipocytes in a dose-dependent manner; however, the inhibition is ineffective 24-48 h after the differentiation is initiated, suggesting that 1,25(OH)2D3 inhibits only the early events of the adipogenic program. Treatment of 3T3-L1 cells with 1,25(OH)2D3 does not block the mitotic clonal expansion or C/EBPbeta induction; rather, 1,25(OH)2D3 blocks the expression of C/EBPalpha, peroxisome proliferator-activated receptor-gamma (PPARgamma), sterol regulatory element-binding protein-1, and other downstream adipocyte markers. The inhibition by 1,25(OH)2D3 is reversible, since removal of 1,25(OH)2D3 from the medium restores the adipogenic process with only a temporal delay. Interestingly, although the vitamin D receptor (VDR) protein is barely detectable in 3T3-L1 preadipocytes, its levels are dramatically increased during the early phase of adipogenesis, peaking at 4-8 h and subsiding afterward throughout the rest of the differentiation program; 1,25(OH)2D3 treatment appears to stabilize the VDR protein levels. Consistently, adenovirus-mediated overexpression of human (h) VDR in 3T3-L1 cells completely blocks the adipogenic program, confirming that VDR is inhibitory. Inhibition of adipocyte differentiation by 1,25(OH)2D3 is ameliorated by troglitazone, a specific PPARgamma antagonist; conversely, hVDR partially suppresses the transacting activity of PPARgamma but not of C/EBPbeta or C/EBPalpha. Moreover, 1,25(OH)2D3 markedly suppresses C/EBPalpha and PPARgamma mRNA levels in mouse epididymal fat tissue culture. Taken together, these data indicate that the blockade of 3T3-L1 cell differentiation by 1,25(OH)2D3 occurs at the postclonal expansion stages and involves direct suppression of C/EBPalpha and PPARgamma upregulation, antagonization of PPARgamma activity, and stabilization of the inhibitory VDR protein.     

Role of Wnt10b and C/EBPalpha in spontaneous adipogenesis of 243 cells

This report examines the balance of positive and negative adipogenic factors in a line of immortalized 243 embryonic fibroblasts that undergo spontaneous preadipocyte differentiation. Control of adipogenesis reflects the interplay of factors that promote or inhibit expression of C/EBPalpha and PPARgamma. The 243 cells express C/EBPalpha early and at elevated levels compared to 3T3-F442A preadipocytes or adipocytes. Cell clones were derived from the heterogeneous 243 population for ability or inability to differentiate into adipocytes. Wnt10b, a secreted protein that inhibits adipogenesis, is expressed at high levels in cells with low adipogenic potential and is undetectable in preadipocytes that spontaneously differentiate. In contrast, C/EBPalpha is expressed at reduced levels in cells with low adipogenic potential, and is expressed at high levels in preadipocytes that spontaneously differentiate. These data are consistent with a model in which decreased Wnt10b, coupled with increased C/EBPalpha, results in induction of PPARgamma and spontaneous adipogenesis of 243 cells.     

Differences in muscle protein synthesis and anabolic signaling in the postabsorptive state and in response to food in 65-80 year old men and women

Women have less muscle than men but lose it more slowly during aging. To discover potential underlying mechanism(s) for this we evaluated the muscle protein synthesis process in postabsorptive conditions and during feeding in twenty-nine 65-80 year old men (n = 13) and women (n = 16). We discovered that the basal concentration of phosphorylated eEF2(Thr56) was approximately 40% less (P<0.05) and the basal rate of MPS was approximately 30% greater (P = 0.02) in women than in men; the basal concentrations of muscle phosphorylated Akt(Thr308), p70s6k(Thr389), eIF4E(Ser209), and eIF4E-BP1(Thr37/46) were not different between the sexes. Feeding increased (P<0.05) Akt(Thr308) and p70s6k(Thr389) phosphorylation to the same extent in men and women but increased (P<0.05) the phosphorylation of eIF4E(Ser209) and eIF4E-BP1(Thr37/46) in men only. Accordingly, feeding increased MPS in men (P<0.01) but not in women. The postabsorptive muscle mRNA concentrations for myoD and myostatin were not different between sexes; feeding doubled myoD mRNA (P<0.05) and halved that of myostatin (P<0.05) in both sexes. Thus, there is sexual dimorphism in MPS and its control in older adults; a greater basal rate of MPS, operating over most of the day may partially explain the slower loss of muscle in older women.     

Increased energy expenditure and leptin sensitivity account for low fat mass in myostatin-deficient mice

Myostatin deficiency causes dramatically increased skeletal muscle mass and reduced fat mass. Previously, myostatin-deficient mice were reported to have unexpectedly low total energy expenditure (EE) after normalizing to body mass, and thus, a metabolic cause for low fat mass was discounted. To clarify how myostatin deficiency affects the control of body fat mass and energy balance, we compared rates of oxygen consumption, body composition, and food intake in young myostatin-deficient mice relative to wild-type (WT) and heterozygous (HET) controls. We report that after adjusting for total body mass using regression analysis, young myostatin-deficient mice display significantly increased EE relative to both WT (+0.81 ± 0.28 kcal/day, P = 0.004) and HET controls (+0.92 ± 0.31 kcal/day, P = 0.005). Since food intake was not different between groups, increased EE likely accounts for the reduced body fat mass (KO: 8.8 ± 1.1% vs. WT: 14.5 ± 1.3%, P = 0.003) and circulating leptin levels (KO: 0.7 ± 0.2 ng/ml vs. WT: 1.9 ± 0.3 ng/ml, P = 0.008). Interestingly, the observed increase in adjusted EE in myostatin-deficient mice occurred despite dramatically reduced ambulatory activity levels (-50% vs. WT, P < 0.05). The absence of hyperphagia together with increased EE in myostatin-deficient mice suggests that increased leptin sensitivity may contribute to their lean phenotype. Indeed, leptin-induced anorexia (KO: -17 ± 1.2% vs. WT: -5 ± 0.3%) and weight loss (KO: -2.2 ± 0.2 g vs. WT: -1.6 ± 0.1, P < 0.05) were increased in myostatin-deficient mice compared with WT controls. We conclude that increased EE, together with increased leptin sensitivity, contributes to low fat mass in mice lacking myostatin.     

Xanthine oxidoreductase is a regulator of adipogenesis and PPARgamma activity

In an effort to identify novel candidate regulators of adipogenesis, gene profiling of differentiating 3T3-L1 preadipocytes was analyzed using a novel algorithm. We report here the characterization of xanthine oxidoreductase (XOR) as a novel regulator of adipogenesis. XOR lies downstream of C/EBPbeta and upstream of PPARgamma, in the cascade of factors that control adipogenesis, and it regulates PPARgamma activity. In vitro, knockdown of XOR inhibits adipogenesis and PPARgamma activity while constitutive overexpression increases activity of the PPARgamma receptor in both adipocytes and preadipocytes. In vivo, XOR -/- mice demonstrate 50% reduction in adipose mass versus wild-type littermates while obese ob/ob mice exhibit increased concentrations of XOR mRNA and urate in the adipose tissue. We propose that XOR is a novel regulator of adipogenesis and of PPARgamma activity and essential for the regulation of fat accretion. Our results identify XOR as a potential therapeutic target for metabolic abnormalities beyond hyperuricemia.     

Postnatal expression of myostatin propeptide cDNA maintained high muscle growth and normal adipose tissue mass in transgenic mice fed a high-fat diet

Myostatin plays a robust, negative role in controlling muscle mass. A disruption of myostatin function by transgenic expression of its propeptide (the 5'region, 866 nucleotides) results in significant muscle growth (Yang et al., 2001. Mol Rep Dev 60:351-361). Studies from myostatin and the propeptide transgene mRNA indicated that myostatin mRNA was detected at day 10.5 postcoitum in fetal mice. Its level remained low, but increased by 180% during the postnatal fast-growth period (day 0-10). An early, high-level postnatal expression of the transgene was identified as being responsible for a highly muscled phenotype. High-fat diet induces adiposity in rodents. To study the effects of dietary fat on muscle growth and adipose tissue fat deposition in the transgenic mice, we challenged the mice with a high-fat diet (45% kcal fat) for 21 weeks. Transgenic mice showed 24%-50% further enhancement of growth on the high-fat diet compared to the normal-fat diet (P = 0.004) from 17 to 25 weeks of age. The total mass of the main muscles of transgenic mice showed a 27% increase on the high-fat diet compared to the normal-fat diet (P = 0.004), while the white adipose tissue mass of the transgenic mice was not significantly different from that of wild-type mice fed a normal-fat diet (P = 0.434). The high-fat diet induced wild-type mice developed 190% greater mass of white adipose tissues compared to the normal-fat diet (P = 0.008), which primarily resulted from enlarged adipocytes. These results demonstrate that disruption of myostatin function by its propeptide shifted dietary fat utilization toward muscle tissues with minimal effects on adiposity. These results suggest that enhancing muscle growth by myostatin propeptide or other means during the early developmental stage may serve as an effective means for obesity prevention.