Insulin-like growth factor (IGF-I) induces myotube hypertrophy associated with an increase in anaerobic glycolysis in a clonal skeletal-muscle cell model

In an attempt to address the complex and clinically challenging question of the causes of muscle wasting in patients with cachexia, we have developed a primary adult human skeletal muscle cell model. The cultured cells were characterised by immunocytochemistry using antibodies to the myofibrillar protein constituents desmin and titin. Myotube formation was confirmed biochemically by a fourfold increase in the activity of the muscle-specific enzyme creatinine kinase, and myoblast withdrawal from the cell cycle, which is essential for terminal differentiation, was associated with progressive retinoblastoma protein dephosphorylation. Having successfully confirmed the phenotype of these adult human muscle cells, we assessed their interaction with the insulin-like growth factor (IGF) system. IGF-I is known to stimulate myoblast survival, proliferation and differentiation in cell lines, and, like insulin, is a potent anabolic agent in the regulation of protein metabolism. We have shown that IGF-I stimulated both replication and differentiation of myoblasts, whilst fibroblast growth factor-2 stimulated replication but inhibited differentiation. Examining the IGF system during the process of terminal differentiation, we found that both myoblasts and myotubes expressed insulin, IGF-I and insulin-IGF-I hybrid receptors, with the levels of all three receptor types increasing on differentiation. The cells also produced a wide range of IGF binding proteins (IGFBPs) including IGFBP-2, IGFBP-4 and abundant IGFBP-3, which has not been shown to be produced by any other skeletal muscle cell line examined to date. Both insulin and IGF-I had anabolic effects on myotube protein metabolism at physiological concentrations. Insulin was more potent than IGF-I: use of the IGF analogue long R(3)IGF-I demonstrated that the effects of exogenous IGF-I on protein metabolism were not affected by the high levels of endogenous IGFBP production. In summary, we have developed and characterised a clinically relevant in vitro model with which to address the aetiology of muscle wasting associated with chronic catabolic conditions, and we anticipate that future work will enable the development of novel, effective therapeutic interventions.

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Growth hormone promotes skeletal muscle cell fusion independent of insulin-like growth factor 1 up-regulation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0510033103 ◽

2006 ◽

Vol 103 (19) ◽

pp. 7315-7320 ◽

Cited By ~ 90

Author(s):

A. Sotiropoulos ◽

M. Ohanna ◽

C. Kedzia ◽

R. K. Menon ◽

J. J. Kopchick ◽

...

Keyword(s):

Skeletal Muscle ◽

Growth Hormone ◽

Growth Factor ◽

Cell Fusion ◽

Muscle Cell ◽

Skeletal Muscle Cell ◽

Insulin Like Growth Factor

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Insulin-like growth factor (IGF-I) induces myotube hypertrophy associated with an increase in anaerobic glycolysis in a clonal skeletal-muscle cell model

Biochemical Journal ◽

10.1042/bj3390443 ◽

1999 ◽

Vol 339 (2) ◽

pp. 443-451 ◽

Cited By ~ 43

Author(s):

Christopher SEMSARIAN ◽

Pramod SUTRAVE ◽

David R. RICHMOND ◽

Robert M. GRAHAM

Keyword(s):

Skeletal Muscle ◽

Muscle Hypertrophy ◽

Cell Model ◽

Muscle Growth ◽

C2c12 Cells ◽

Anaerobic Glycolysis ◽

Skeletal Muscle Hypertrophy ◽

Igf I ◽

Control Myotubes ◽

Myotube Hypertrophy

Insulin-like growth factor-I (IGF-I) is an important autocrine/paracrine mediator of skeletal-muscle growth and development. To develop a definitive cultured cell model of skeletal-muscle hypertrophy, C2C12 cells were stably transfected with IGF-I and clonal lines developed and evaluated. Quantitative morphometric analysis showed that IGF-I-transfected myotubes had a larger area (2381±60 µm2 versus 1429±39 µm2; P< 0.0001) and a greater maximum width (21.4±0.6 µm versus 13.9±0.3 µm; P< 0.0001) than control C2C12 myotubes, independent of the number of cell nuclei per myotube. IGF-I-transfected myotubes had higher levels of protein synthesis but no difference in DNA synthesis when compared with control myotubes, indicating the development of hypertrophy rather than hyperplasia. Both lactate dehydrogenase and alanine aminotransferase activities were increased (3- and 5-fold respectively), and total lactate levels were higher (2.3-fold) in IGF-I-transfected compared with control myotubes, indicating an increase in anaerobic glycolysis in the hypertrophied myotubes. However, expression of genes involved in skeletal-muscle growth or hypertrophy in vivo, e.g. myocyte nuclear factor and myostatin, was not altered in the IGF-I myotubes. Finally, myotube hypertrophy could also be induced by treatment of C2C12 cells with recombinant IGF-I or by growing C2C12 cells in conditioned media from IGF-I-transfected cells. This quantitative model should be uniquely useful for elucidating the molecular mechanisms of skeletal-muscle hypertrophy.

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Nerve growth factor stimulation of ERK1/2 phosphorylation requires both p75NTR and α9β1 integrin and confers myoprotection towards ischemia in C2C12 skeletal muscle cell model

Cellular Signalling ◽

10.1016/j.cellsig.2012.08.008 ◽

2012 ◽

Vol 24 (12) ◽

pp. 2378-2388 ◽

Cited By ~ 13

Author(s):

Keren Ettinger ◽

Shimon Lecht ◽

Hadar Arien-Zakay ◽

Gadi Cohen ◽

Shlomit Aga-Mizrachi ◽

...

Keyword(s):

Skeletal Muscle ◽

Nerve Growth Factor ◽

Growth Factor ◽

Muscle Cell ◽

Cell Model ◽

Skeletal Muscle Cell ◽

Nerve Growth ◽

Stimulation Of

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Activation of insulin-like growth factor gene expression during work-induced skeletal muscle growth

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1990.259.1.e89 ◽

1990 ◽

Vol 259 (1) ◽

pp. E89-E95 ◽

Cited By ~ 53

Author(s):

D. L. DeVol ◽

P. Rotwein ◽

J. L. Sadow ◽

J. Novakofski ◽

P. J. Bechtel

Keyword(s):

Gene Expression ◽

Skeletal Muscle ◽

Growth Factor ◽

Muscle Growth ◽

Experimental Period ◽

Mrna Levels ◽

Insulin Like Growth Factor ◽

Muscle Weight ◽

Skeletal Muscle Growth ◽

Igf I

We have investigated the hypothesis that there is local regulation of insulin-like growth factor (IGF) gene expression during skeletal muscle growth. Compensatory hypertrophy was induced in the soleus, a predominantly slow-twitch muscle, and plantaris, a fast-twitch muscle, in 11- to 12-wk-old female Wistar rats by unilateral cutting of the distal gastrocnemius tendon. Animals were killed 2, 4, or 8 days later, and muscles of the nonoperated leg served as controls. Muscle weight increased throughout the experimental period, reaching 127% (soleus) or 122% (plantaris) of control values by day 8. In both growing muscles, IGF-I mRNA, quantitated by a solution-hybridization nuclease-protection assay, rose by nearly threefold on day 2 and remained elevated throughout the experimental period. IGF-II mRNA levels also increased over controls. A more dramatic response was seen in hypophysectomized rats, where IGF-I mRNA levels rose by 8- to 13-fold, IGF-II values by 3- to 7-fold, and muscle mass increased on day 8 to 149% (soleus) or 133% (plantaris) of the control contralateral limb. These results indicate that signals propagated during muscle hypertrophy enhance the expression of both IGF genes, that modulation of IGF-I mRNA levels can occur in the absence of growth hormone, and that locally produced IGF-I and IGF-II may play a role in skeletal muscle growth.

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Intravenous infusion of insulin-like growth factor I in fetal sheep reduces hepatic IGF-I and IGF-II mRNAs

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.1996.271.6.r1632 ◽

1996 ◽

Vol 271 (6) ◽

pp. R1632-R1637 ◽

Cited By ~ 3

Author(s):

K. L. Kind ◽

J. A. Owens ◽

F. Lok ◽

J. S. Robinson ◽

K. J. Quinn ◽

...

Keyword(s):

Gene Expression ◽

Skeletal Muscle ◽

Growth Factor ◽

Intravenous Infusion ◽

Feedback Inhibition ◽

Fetal Liver ◽

Plasma Concentrations ◽

Insulin Like Growth Factor ◽

Fetal Sheep ◽

Igf I

Liver contains the highest concentrations of insulin-like growth factor (IGF) I mRNA in adult rats and sheep and is a major source of circulating IGF-I. In rats, inhibition of hepatic IGF-I production by exogenous IGF-I has been reported. In fetal sheep, skeletal muscle and liver are major sites of IGF-I synthesis and potential sources of circulating IGF-I. To determine whether feedback inhibition of IGF gene expression in fetal liver or muscle by IGF-I occurs, IGF-I and IGF-II mRNAs were measured in these tissues after intravenous infusion of recombinant human IGF-I into fetal sheep. Infusion of IGF-I (26 +/- 4 micrograms.h-1.kg-1; n = 6) or saline (n = 6) commenced on day 120 of pregnancy (term = 150 days) and continued for 10 days. Plasma concentrations of IGF-I were threefold higher in infused fetuses at 130 days of gestation (P < 0.0003), whereas those of IGF-II were unchanged. IGF-I infusion reduced the relative abundance of IGF-I mRNA (P < 0.0002) and IGF-II mRNA (P < 0.01) in fetal liver by approximately 50% but did not alter IGF-I or IGF-II mRNA in skeletal muscle. These results indicate that IGF-I inhibits the expression of both IGF-I and IGF-II genes in fetal liver and that IGF gene expression in fetal liver and muscle is differentially regulated by IGF-I.

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Insulin-like growth factor I stimulates degradation of an mRNA transcript encoding the 14 kDa ubiquitin-conjugating enzyme

Biochemical Journal ◽

10.1042/bj3190455 ◽

1996 ◽

Vol 319 (2) ◽

pp. 455-461 ◽

Cited By ~ 44

Author(s):

Simon S WING ◽

Nathalie BEDARD

Keyword(s):

Skeletal Muscle ◽

Growth Factor ◽

Binding Proteins ◽

Mrna Levels ◽

Insulin Like Growth Factor ◽

Mrna Transcript ◽

Factor I ◽

Igf I ◽

Ubiquitin Conjugating Enzyme ◽

Growth Factor I

Upon fasting, the ubiquitin-dependent proteolytic system is activated in skeletal muscle in parallel with the increases in rates of proteolysis. Levels of mRNA encoding the 14 kDa ubiquitin-conjugating enzyme (E214k), which can catalyse the first irreversible reaction in this pathway, rise and fall in parallel with the rates of proteolysis [Wing and Banville (1994) Am. J. Physiol. 267, E39-E48], indicating that the conjugation of ubiquitin to proteins is a regulated step. To characterize the mechanisms of this regulation, we have examined the effects of insulin, insulin-like growth factor I (IGF-I) and des(1–3) insulin-like growth factor I (DES-IGF-I), which does not bind IGF-binding proteins, on E214k mRNA levels in L6 myotubes. Insulin suppressed levels of E214k mRNA with an IC50 of 4×10-9 M, but had no effects on mRNAs encoding polyubiquitin and proteasome subunits C2 and C8, which, like E214k, also increase in skeletal muscle upon fasting. Reduction of E214k mRNA levels was more sensitive to IGF-I with an IC50 of approx. 5×10-10 M. During the incubation of these cells for 12 h there was significant secretion of IGF-I-binding proteins into the medium. DES-IGF-I, which has markedly reduced affinity for these binding proteins, was found to potently reduce E214k mRNA levels with an IC50 of 3×10-11 M. DES-IGF-I did not alter rates of transcription of the E214k gene, but enhanced the rate of degradation of the 1.2 kb mRNA transcript. The half-life of the 1.2 kb transcript was approximately one-third that of the 1.8 kb transcript and can explain the more marked regulation of this transcript observed previously. This indicates that the additional 3´ non-coding sequence in the 1.8 kb transcript confers stability. These observations suggest that IGF-I is an important regulator of E214k expression and demonstrate, for the first time, stimulation of degradation of a specific mRNA transcript by this hormone, while overall RNA accumulates.

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