Myostatin inhibits IGF-I-induced myotube hypertrophy through Akt

Myostatin is a highly conserved negative regulator of skeletal muscle growth. Loss of functional myostatin in cattle, mice, sheep, dogs, and humans results in increased muscle mass. The molecular mechanisms responsible for this increase in muscle growth are not fully understood. Previously, we have reported that phenylephrine-induced cardiac muscle growth and Akt activation are enhanced in myostatin knockout mice compared with controls. Here we report that skeletal muscle from myostatin knockout mice show increased Akt protein expression and overall activity at baseline secondary to an increase in Akt mRNA. We examined the functional role of myostatin modulation of Akt in C2C12 myotubes, a well-established in vitro model of skeletal muscle hypertrophy. Adenoviral overexpression of myostatin attenuated the insulin-like growth factor-I (IGF-I)-mediated increase in myotube diameter, as well as IGF-I-stimulated Akt phosphorylation. Inhibition of myostatin by overexpression of the NH2-terminal portion of myostatin was sufficient to increase myotube diameter and Akt phosphorylation. Coexpression of myostatin and constitutively active Akt (myr-Akt) restored the increase in myotube diameter. Conversely, expression of dominant negative Akt (dn-Akt) with the inhibitory myostatin propeptide blocked the increase in myotube diameter. Of note, ribosomal protein S6 phosphorylation and atrogin-1/muscle atrophy F box mRNA were increased in skeletal muscle from myostain knockout mice. Together, these data suggest myostatin regulates muscle growth at least in part through regulation of Akt.

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Myostatin inhibits proliferation and insulin-stimulated glucose uptake in mouse liver cells

Biochemistry and Cell Biology ◽

10.1139/bcb-2014-0004 ◽

2014 ◽

Vol 92 (3) ◽

pp. 226-234 ◽

Cited By ~ 16

Author(s):

Rani Watts ◽

Mostafa Ghozlan ◽

Curtis C. Hughey ◽

Virginia L. Johnsen ◽

Jane Shearer ◽

...

Keyword(s):

Skeletal Muscle ◽

Cell Proliferation ◽

Glucose Uptake ◽

Liver Cell ◽

Noncoding Rna ◽

Muscle Growth ◽

Liver Cells ◽

Negative Regulator ◽

Kinase Substrate ◽

Akt Phosphorylation

Although myostatin functions primarily as a negative regulator of skeletal muscle growth and development, accumulating biological and epidemiological evidence indicates an important contributing role in liver disease. In this study, we demonstrate that myostatin suppresses the proliferation of mouse Hepa-1c1c7 murine-derived liver cells (50%; p < 0.001) in part by reducing the expression of the cyclins and cyclin-dependent kinases that elicit G1-S phase transition of the cell cycle (p < 0.001). Furthermore, real-time PCR-based quantification of the long noncoding RNA metastasis associated lung adenocarcinoma transcript 1 (Malat1), recently identified as a myostatin-responsive transcript in skeletal muscle, revealed a significant downregulation (25% and 50%, respectively; p < 0.05) in the livers of myostatin-treated mice and liver cells. The importance of Malat1 in liver cell proliferation was confirmed via arrested liver cell proliferation (p < 0.05) in response to partial Malat1 siRNA-mediated knockdown. Myostatin also significantly blunted insulin-stimulated glucose uptake and Akt phosphorylation in liver cells while increasing the phosphorylation of myristoylated alanine-rich C-kinase substrate (MARCKS), a protein that is essential for cancer cell proliferation and insulin-stimulated glucose transport. Together, these findings reveal a plausible mechanism by which circulating myostatin contributes to the diminished regenerative capacity of the liver and diseases characterized by liver insulin resistance.

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The Type 1 Insulin-Like Growth Factor Receptor (IGF-IR) Pathway Is Mandatory for the Follistatin-Induced Skeletal Muscle Hypertrophy

Endocrinology ◽

10.1210/en.2011-1687 ◽

2012 ◽

Vol 153 (1) ◽

pp. 241-253 ◽

Cited By ~ 33

Author(s):

S. Kalista ◽

O. Schakman ◽

H. Gilson ◽

P. Lause ◽

B. Demeulder ◽

...

Keyword(s):

Skeletal Muscle ◽

Knockout Mice ◽

Muscle Hypertrophy ◽

Critical Role ◽

Muscle Growth ◽

Dominant Negative ◽

Wild Type ◽

Myostatin Inhibition

Myostatin inhibition by follistatin (FS) offers a new approach for muscle mass enhancement. The aim of the present study was to characterize the mediators responsible for the FS hypertrophic action on skeletal muscle in male mice. Because IGF-I and IGF-II, two crucial skeletal muscle growth factors, are induced by myostatin inhibition, we assessed their role in FS action. First, we tested whether type 1 IGF receptor (IGF-IR) is required for FS-induced hypertrophy. By using mice expressing a dominant-negative IGF-IR in skeletal muscle, we showed that IGF-IR inhibition blunted by 63% fiber hypertrophy caused by FS. Second, we showed that FS caused the same degree of fiber hypertrophy in wild-type and IGF-II knockout mice. We then tested the role of the signaling molecules stimulated by IGF-IR, in particular the Akt/mammalian target of rapamycin (mTOR)/70-kDa ribosomal protein S6 kinase (S6K) pathway. We investigated whether Akt phosphorylation is required for the FS action. By cotransfecting a dominant-negative form of Akt together with FS, we showed that Akt inhibition reduced by 65% fiber hypertrophy caused by FS. Second, we evaluated the role of mTOR in FS action. Fiber hypertrophy induced by FS was reduced by 36% in rapamycin-treated mice. Finally, because the activity of S6K is increased by FS, we tested its role in FS action. FS caused the same degree of fiber hypertrophy in wild-type and S6K1/2 knockout mice. In conclusion, the IGF-IR/Akt/mTOR pathway plays a critical role in FS-induced muscle hypertrophy. In contrast, induction of IGF-II expression and S6K activity by FS are not required for the hypertrophic action of FS.

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Skeletal muscle cell hypertrophy induced by inhibitors of metalloproteases; myostatin as a potential mediator

AJP Cell Physiology ◽

10.1152/ajpcell.2001.281.5.c1624 ◽

2001 ◽

Vol 281 (5) ◽

pp. C1624-C1634 ◽

Cited By ~ 30

Author(s):

Clotilde Huet ◽

Zhi-Fang Li ◽

Hai-Zhen Liu ◽

Roy A. Black ◽

Marie-Florence Galliano ◽

...

Keyword(s):

Skeletal Muscle ◽

Growth Factor ◽

Muscle Growth ◽

Matrix Metalloproteases ◽

Proteolytic Processing ◽

Negative Regulator ◽

Paracrine Factors ◽

Skeletal Muscle Growth ◽

Myotube Hypertrophy

Cell growth and differentiation are controlled in many tissues by paracrine factors, which often require proteolytic processing for activation. Metalloproteases of the metzincin family, such as matrix metalloproteases and ADAMs, recently have been shown to be involved in the shedding of growth factors, cytokines, and receptors. In the present study, we show that hydroxamate-based inhibitors of metalloproteases (HIMPs), such as TAPI and BB-3103, increase the fusion of C2C12 myoblasts and provoke myotube hypertrophy. HIMPs did not seem to effect hypertrophy via proteins that have previously been shown to regulate muscle growth in vitro, such as insulin-like growth factor-I, calcineurin, and tumor necrosis factor-α. Instead, the proteolytic maturation of myostatin (growth differentiation factor-8) seemed to be reduced in C2C12 cells treated with HIMPs, as suggested by the presence of nonprocessed myostatin precursor only in hypertrophic myotubes. Myostatin is a known negative regulator of skeletal muscle growth, belonging to the transforming growth factor-β/bone morphogenetic protein superfamily. These results indicate that metalloproteases are involved in the regulation of skeletal muscle growth and differentiation, that the proteolytic maturation of myostatin in C2C12 cells may be directly or indirectly linked to the activity of some unidentified HIMP-sensitive metalloproteases, and that the lack of myostatin processing on HIMP treatment may be a mediator of myotube hypertrophy in this in vitro model.

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Different impacts of saturated and unsaturated free fatty acids on COX-2 expression in C2C12 myotubes

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00293.2009 ◽

2009 ◽

Vol 297 (6) ◽

pp. E1291-E1303 ◽

Cited By ~ 38

Author(s):

Akito Kadotani ◽

Yo Tsuchiya ◽

Hiroyasu Hatakeyama ◽

Hideki Katagiri ◽

Makoto Kanzaki

Keyword(s):

Skeletal Muscle ◽

Fatty Acids ◽

Free Fatty Acids ◽

Molecular Mechanisms ◽

C2c12 Myotubes ◽

Prostaglandin E ◽

Akt Phosphorylation ◽

Intracellular Signals ◽

Cox 2 ◽

Oxidative Function

In skeletal muscle, saturated free fatty acids (FFAs) act as proinflammatory stimuli, and cyclooxygenase-2 (COX-2) is a pro/anti-inflammatory enzyme induced at sites of inflammation, which contributes to prostaglandin production. However, little is known about the regulation of COX-2 expression and its responses to FFAs in skeletal muscle. Herein, we examined the effects of saturated and unsaturated FFAs, including a recently identified lipokine (lipid hormone derived from adipocytes), palmitoleate, on COX-2 expression in C2C12 myotubes as a skeletal muscle model. Exposure of myotubes to saturated FFAs [palmitate (16:0) and stearate (18:0)], but not to unsaturated FFAs [palmitoleate (16:1), oleate (18:1), and linoleate (18:2)], led to a slow-onset induction of COX-2 expression and subsequent prostaglandin E2 production via mechanisms involving the p38 MAPK and NF-κB but not the PKCθ signaling cascades. Pharmacological modulation of mitochondrial oxidative function failed to interfere with COX-2 expression, suggesting the mitochondrial overload/excessive β-oxidation contribution to this event to be minimal. On the contrary, unsaturated FFAs appeared to effectively antagonize palmitate-induced COX-2 expression with markedly different potencies (linoleate > oleate > palmitoleate), being highly associated with the suppressive profile of each unsaturated FFA toward palmitate-evoked intracellular signals, including p38, JNK, ERK1/2 MAPKs, and PKCθ, as well as IκB degradation. In addition, our data suggest little involvement of PPAR in the protective actions of unsaturated FFAs against palmitate-induced COX-2 expression. No direct contribution of the increased COX-2 activity in generating palmitate-induced insulin resistance was detected, at least in terms of insulin-responsive Akt phosphorylation and GLUT4 translocation. Taken together, our data provide a novel insight into the molecular mechanisms responsible for the FFA-induced COX-2 expression in skeletal muscle and raise the possibility that, in skeletal myocytes, COX-2 and its product prostaglandins may play an important role in the complex inflammation responses caused by elevated FFAs, for example, in the diabetic state.

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Anabolic Activity of a Soy Extract and Three Major Isoflavones in C2C12 Myotubes

Planta Medica ◽

10.1055/a-0598-4812 ◽

2018 ◽

Vol 84 (14) ◽

pp. 1022-1029 ◽

Cited By ~ 4

Author(s):

Wenya Zheng ◽

Marie Hemker ◽

Mingyong Xie ◽

Sebastian Soukup ◽

Patrick Diel

Keyword(s):

Molecular Mechanisms ◽

Control Cell ◽

Muscle Growth ◽

In Vitro Study ◽

C2c12 Cells ◽

C2c12 Myotubes ◽

Gene Expressions ◽

Insulin Growth Factor ◽

Anabolic Activity

AbstractIsoflavones have been reported to stimulate muscle growth. The aim of this in vitro study was to examine anabolic activity and associated molecular mechanisms of a soy extract (SoyEx), isoflavone aglycones, and a mixture simulating the composition of SoyEx in C2C12 myotubes. C2C12 cells were differentiated into myotubes. The effects of SoyEx, genistein, daidzein, glycitein, and the mixture of genistein-daidzein-glycitein (Mix) on myotube diameter and number were determined. In addition, the expression of genes and proteins associated with anabolic activity was analyzed. Treatment with SoyEx, genistein, and Mix led to a significant increase of myotube diameter and an increase of the number of myotubes per area compared to the control cell. The increase of diameter by SoyEx was antagonized by an antiestrogen, not by an antiandrogen. Furthermore, gene expressions of insulin growth factor (IGF)-1 and its receptor (IGF-1R), as well as protein expression of myosin heavy chain (MHC), were significantly increased by SoyEx, genistein, and Mix. The effects induced by genistein and Mix were comparable to SoyEx. In conclusion, SoyEx displays an anabolic activity in C2C12 myotubes by binding to ER and modulating IGF-1 and MHC expression. Our studies with isoflavone aglycones and Mix indicate that the isoflavone aglycone with the highest anabolic bioactivity in SoyEx is genistein.

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NF-κB-Inducing Kinase (NIK) Mediates Skeletal Muscle Insulin Resistance: Blockade by Adiponectin

Endocrinology ◽

10.1210/en.2011-1343 ◽

2011 ◽

Vol 152 (10) ◽

pp. 3622-3627 ◽

Cited By ~ 26

Author(s):

Sanjeev Choudhary ◽

Sandeep Sinha ◽

Yanhua Zhao ◽

Srijita Banerjee ◽

Padma Sathyanarayana ◽

...

Keyword(s):

Insulin Resistance ◽

Skeletal Muscle ◽

Glucose Uptake ◽

Kinase Activity ◽

Pi3 Kinase ◽

Dominant Negative ◽

Muscle Insulin Resistance ◽

Akt Phosphorylation ◽

Skeletal Muscle Insulin Resistance ◽

Obese Subjects

Enhanced levels of nuclear factor (NF)-κB-inducing kinase (NIK), an upstream kinase in the NF-κB pathway, have been implicated in the pathogenesis of chronic inflammation in diabetes. We investigated whether increased levels of NIK could induce skeletal muscle insulin resistance. Six obese subjects with metabolic syndrome underwent skeletal muscle biopsies before and six months after gastric bypass surgery to quantitate NIK protein levels. L6 skeletal myotubes, transfected with NIK wild-type or NIK kinase-dead dominant negative plasmids, were treated with insulin alone or with adiponectin and insulin. Effects of NIK overexpression on insulin-stimulated glucose uptake were estimated using tritiated 2-deoxyglucose uptake. NF-κB activation (EMSA), phosphatidylinositol 3 (PI3) kinase activity, and phosphorylation of inhibitor κB kinase β and serine-threonine kinase (Akt) were measured. After weight loss, skeletal muscle NIK protein was significantly reduced in association with increased plasma adiponectin and enhanced AMP kinase phosphorylation and insulin sensitivity in obese subjects. Enhanced NIK expression in cultured L6 myotubes induced a dose-dependent decrease in insulin-stimulated glucose uptake. The decrease in insulin-stimulated glucose uptake was associated with a significant decrease in PI3 kinase activity and protein kinase B/Akt phosphorylation. Overexpression of NIK kinase-dead dominant negative did not affect insulin-stimulated glucose uptake. Adiponectin treatment inhibited NIK-induced NF-κB activation and restored insulin sensitivity by restoring PI3 kinase activation and subsequent Akt phosphorylation. These results indicate that NIK induces insulin resistance and further indicate that adiponectin exerts its insulin-sensitizing effect by suppressing NIK-induced skeletal muscle inflammation. These observations suggest that NIK could be an important therapeutic target for the treatment of insulin resistance associated with inflammation in obesity and type 2 diabetes.

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Signal Transducer and Activator of Transcription 3 (STAT3) Suppresses STAT1/Interferon Signaling Pathway and Inflammation in Senescent Preadipocytes

Antioxidants ◽

10.3390/antiox10020334 ◽

2021 ◽

Vol 10 (2) ◽

pp. 334

Author(s):

Aisha Y. Madani ◽

Yasser Majeed ◽

Houari B. Abdesselem ◽

Maha V. Agha ◽

Muneera Vakayil ◽

...

Keyword(s):

Adipose Tissue ◽

Molecular Mechanisms ◽

Adenosine Monophosphate ◽

Human Adipose Tissue ◽

Premature Aging ◽

Guanosine Monophosphate ◽

Negative Regulator ◽

Signal Transducer ◽

Interferon Signaling

Obesity promotes premature aging and dysfunction of white adipose tissue (WAT) through the accumulation of cellular senescence. The senescent cells burden in WAT has been linked to inflammation, insulin-resistance (IR), and type 2 diabetes (T2D). There is limited knowledge about molecular mechanisms that sustain inflammation in obese states. Here, we describe a robust and physiologically relevant in vitro system to trigger senescence in mouse 3T3-L1 preadipocytes. By employing transcriptomics analyses, we discovered up-regulation of key pro-inflammatory molecules and activation of interferon/signal transducer and activator of transcription (STAT)1/3 signaling in senescent preadipocytes, and expression of downstream targets was induced in epididymal WAT of obese mice, and obese human adipose tissue. To test the relevance of STAT1/3 signaling to preadipocyte senescence, we used Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9 (CRISPR/Cas9) technology to delete STAT1/3 and discovered that STAT1 promoted growth arrest and cooperated with cyclic Guanosine Monophosphate-Adenosine Monophosphate (GMP-AMP) synthase-stimulator of interferon genes (cGAS-STING) to drive the expression of interferon β (IFNβ), C-X-C motif chemokine ligand 10 (CXCL10), and interferon signaling-related genes. In contrast, we discovered that STAT3 was a negative regulator of STAT1/cGAS-STING signaling—it suppressed senescence and inflammation. These data provide insights into how STAT1/STAT3 signaling coordinates senescence and inflammation through functional interactions with the cGAS/STING pathway.

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Mechanical signal transduction in skeletal muscle growth and adaptation

Journal of Applied Physiology ◽

10.1152/japplphysiol.01178.2004 ◽

2005 ◽

Vol 98 (5) ◽

pp. 1900-1908 ◽

Cited By ~ 103

Author(s):

James G. Tidball

Keyword(s):

Skeletal Muscle ◽

Signal Transduction ◽

Mechanical Activation ◽

Signaling Pathways ◽

Mechanical Stimulation ◽

Muscle Growth ◽

Mammalian Target Of Rapamycin ◽

Target Of Rapamycin ◽

Mechanical Signal ◽

Igf I

The adaptability of skeletal muscle to changes in the mechanical environment has been well characterized at the tissue and system levels, but the mechanisms through which mechanical signals are transduced to chemical signals that influence muscle growth and metabolism remain largely unidentified. However, several findings have suggested that mechanical signal transduction in muscle may occur through signaling pathways that are shared with insulin-like growth factor (IGF)-I. The involvement of IGF-I-mediated signaling for mechanical signal transduction in muscle was originally suggested by the observations that muscle releases IGF-I on mechanical stimulation, that IGF-I is a potent agent for promoting muscle growth and affecting phenotype, and that IGF-I can function as an autocrine hormone in muscle. Accumulating evidence shows that at least two signaling pathways downstream of IGF-I binding can influence muscle growth and adaptation. Signaling via the calcineurin/nuclear factor of activated T-cell pathway has been shown to have a powerful influence on promoting the slow/type I phenotype in muscle but can also increase muscle mass. Neural stimulation of muscle can activate this pathway, although whether neural activation of the pathway can occur independent of mechanical activation or independent of IGF-I-mediated signaling remains to be explored. Signaling via the Akt/mammalian target of rapamycin pathway can also increase muscle growth, and recent findings show that activation of this pathway can occur as a response to mechanical stimulation applied directly to muscle cells, independent of signals derived from other cells. In addition, mechanical activation of mammalian target of rapamycin, Akt, and other downstream signals is apparently independent of autocrine factors, which suggests that activation of the mechanical pathway occurs independent of muscle-mediated IGF-I release.

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Abstract 292: TRPA1 Promoting Myofibroblast Differentiation Mediate Pressure Overload-Induced Cardiac Fibrosis

Circulation ◽

10.1161/circ.138.suppl_2.292 ◽

2018 ◽

Vol 138 (Suppl_2) ◽

Author(s):

Shuang Li ◽

Dong Han ◽

Dachun Yang

Keyword(s):

Knockout Mice ◽

Molecular Mechanisms ◽

Cardiac Fibrosis ◽

Transient Receptor Potential ◽

Transforming Growth Factor ◽

Ventricular Remodeling ◽

Cardiac Fibroblasts ◽

Gene Transfection ◽

Pressure Overload

Background: Hypertensive ventricular remodeling is a common cause of heart failure. Activation and accumulation of cardiac fibroblasts is the key contributors to this progression. Our previous studies indicate that transient receptor potential ankyrin 1 (TRPA1), a Ca 2+ channel necessary and sufficient, play a prominent role in ventricular remodeling. However, the molecular mechanisms regulating remain poorly understood. Methods: We used TRPA1 agonists cinnamaldehyde (CA) pretreatment and TRPA1 knockout mice to understand the role of TRPA1 in ventricular remodeling of hypertensive heart. We also examine the mechanisms through gene transfection and in vitro experiments. Results: TRPA1 overexpression fully activated myofibroblast transformation, while fibroblasts lacking TRPA1 were refractory to transforming growth factor β (TGF-β) -induced transdifferentiation. TRPA1 knockout mice showed hypertensive ventricular remodeling reversal following pressure overload. We found that the TGF-β induced TRPA1 expression through calcineurin-NFAT-Dyrk1A signaling pathway via the TRPA1 promoter. Once induced, TRPA1 activates the Ca 2+ -responsive protein phosphatase calcineurin, which itself induced myofibroblast transdifferentiation. Moreover, inhibition of calcineurin prevented TRPA1-dependent transdifferentiation. Conclusion: Our study provides the first evidence that TRPA1 regulation in cardiac fibroblasts transformation in response to hypertensive stimulation. The results suggesting a comprehensive pathway for myofibroblast formation in conjunction with TGF-β, Calcineurin, NFAT and Dyrk1A. Furthermore, these data indicate that negative modulation of cardiac fibroblast TRPA1 may represent a therapeutic strategy against hypertensive cardiac remodeling.

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Interaction of CREB and PGC-1α Induces Fibronectin Type III Domain-Containing Protein 5 Expression in C2C12 Myotubes

Cellular Physiology and Biochemistry ◽

10.1159/000494655 ◽

2018 ◽

Vol 50 (4) ◽

pp. 1574-1584 ◽

Cited By ~ 6

Author(s):

Xiu-ying Yang ◽

Margaret C.L. Tse ◽

Xiang Hu ◽

Wei-hua Jia ◽

Guan-hua Du ◽

...

Keyword(s):

Skeletal Muscle ◽

Molecular Mechanisms ◽

Metabolic Effects ◽

Hek293 Cells ◽

Metabolic Phenotype ◽

C2c12 Myotubes ◽

Type Iii ◽

Fibronectin Type Iii ◽

Fibronectin Type Iii Domain ◽

Fibronectin Type

Background/Aims: Fibronectin type III domain-containing protein 5 (FNDC5), also known as irisin, is a myokine secreted from muscle in response to exercise. However, the molecular mechanisms that regulate FNDC5 expression and the functional significance of irisn in skeletal muscle remain unknown. In this study, we explored the potential pathways that induce FNDC5 expression and delineated the metabolic effects of irisin on skeletal muscle. Methods: C2C12 myotubes were treated with drugs at various concentrations and durations. The expression and activation of genes were measured by real-time polymerase chain reaction (qRT-PCR) and Western blotting. Oxidative phosphorylation was quantified by measuring the oxygen consumption rate (OCR). Results: We found that the exercise-mimicking treatment (cAMP, forskolin and isoproterenol) increased Fndc5 expression in C2C12 myotubes. CREB over-expressed C2C12 myotubes displayed higher Fndc5 expression. CREB over-expression also promoted peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α) expression. PGC-1α-induced Fndc5 expression was blocked when the dominant negative form of CREB (S133A) was present. PGC-1α mutation (S570A) also decreased Fndc5 expression. Immunoprecipitation showed that overexpressed PGC-1α complexed with CREB in HEK293 cells. C2C12 myotubes treated with forskolin also increased endogenous CREB and PGC-1α binding. Functionally, irisin treatment increased mitochondrial respiration, enhanced ATP production, promoted fatty acid oxidation but decreased glycolysis in myotubes. Conclusion: Our observation indicates that cAMP-mediated PGC-1α/CREB interaction triggers Fndc5 expression, which acts as an autocrine/paracrine to shape the metabolic phenotype of myotubes.

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