The Stretch-Activated Channel Blocker Gd3+ Reduces Palytoxin Toxicity in Primary Cultures of Skeletal Muscle Cells

In this study, primary cultures of trout skeletal muscle cells were used to investigate the main signal transduction pathways of insulin and IGF-I receptors in rainbow trout muscle. At different stages of in vitro development (myoblasts on day 1, myocytes on day 4, and fully developed myotubes on day 11), we detected in these cells the presence of immunoreactivity against ERK 1/2 MAPK and Akt/PKB proteins, components of the MAPK and the phosphatidylinositol 3-kinase-Akt pathways, respectively, two of the main intracellular transduction pathways for insulin and IGF-I receptors. Both insulin and IGF-I activated both pathways, although the latter provoked higher immunoreactivity of phosphorylated MAPKs and Akt proteins. At every stage, increases in total MAPK immunoreactivity levels were observed when cells were stimulated with IGF-I or insulin, while total Akt immunoreactivity levels changed little under stimulation of peptides. Total Akt and total MAPK levels increased as skeletal muscle cells differentiated in culture. Moreover, when cells were incubated with IGF-I or insulin, MAPK-P immunoreactivity levels showed greater increases over the basal levels on days 1 and 4, with no effect observed on day 11. Although Akt-P immunoreactivity displayed improved responses on days 1 and 4 as well, a stimulatory effect was still observed on day 11. In addition, the present study demonstrates that purified trout insulin receptors possess higher phosphorylative activity per unit of receptor than IGF-I receptors. In conclusion, these results indicate that trout skeletal muscle culture is a suitable model to study the insulin and IGF-I signal transduction molecules and that there is a different regulation of MAPK and Akt pathways depending on the developmental stage of the muscle cells.

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Glucose uptake and metabolism by cultured human skeletal muscle cells: rate-limiting steps

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.2001.281.1.e72 ◽

2001 ◽

Vol 281 (1) ◽

pp. E72-E80 ◽

Cited By ~ 28

Author(s):

Laureta M. Perriott ◽

Tetsuro Kono ◽

Richard R. Whitesell ◽

Susan M. Knobel ◽

David W. Piston ◽

...

Keyword(s):

Skeletal Muscle ◽

Glucose Metabolism ◽

Glucose Transport ◽

Human Skeletal Muscle ◽

Primary Cultures ◽

Insulin Response ◽

Muscle Cells ◽

Skeletal Muscle Cells ◽

Insulin Effect ◽

Human Skeletal Muscle Cells

To use primary cultures of human skeletal muscle cells to establish defects in glucose metabolism that underlie clinical insulin resistance, it is necessary to define the rate-determining steps in glucose metabolism and to improve the insulin response attained in previous studies. We modified experimental conditions to achieve an insulin effect on 3- O-methylglucose transport that was more than twofold over basal. Glucose phosphorylation by hexokinase limits glucose metabolism in these cells, because the apparent Michaelis-Menten constant of coupled glucose transport and phosphorylation is intermediate between that of transport and that of the hexokinase and because rates of 2-deoxyglucose uptake and phosphorylation are less than those of glucose. The latter reflects a preference of hexokinase for glucose over 2-deoxyglucose. Cellular NAD(P)H autofluorescence, measured using two-photon excitation microscopy, is both sensitive to insulin and indicative of additional distal control steps in glucose metabolism. Whereas the predominant effect of insulin in human skeletal muscle cells is to enhance glucose transport, phosphorylation, and steps beyond, it also determines the overall rate of glucose metabolism.

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Mechanical stimulation increases expression of acetylcholinesterase in cultured myotubes

AJP Cell Physiology ◽

10.1152/ajpcell.1997.273.6.c2002 ◽

1997 ◽

Vol 273 (6) ◽

pp. C2002-C2009 ◽

Cited By ~ 24

Author(s):

Douglas A. Hubatsch ◽

Bernard J. Jasmin

Keyword(s):

Skeletal Muscle ◽

Mechanical Stimulation ◽

Primary Cultures ◽

Early Time ◽

Muscle Cells ◽

Skeletal Muscle Cells ◽

Trophic Factors ◽

Molecular Forms ◽

Neural Activation ◽

Ache Expression

We tested the hypothesis that acetylcholinesterase (AChE) expression in skeletal muscle cells is increased by passive mechanical stimulation. To this end, primary cultures of myotubes were subjected to repeated cycles of stretch-relaxation for 5 min, 30 min, 3 h, and 24 h, using the Flexercell FX-2000 strain unit. Although mechanical stimulation did not affect AChE expression at early time points, it led to a significant increase (42%; P < 0.05) in total AChE activity at 24 h. This increase reflected a general elevation in the activity of all AChE molecular forms as opposed to a preferential increase in a specific form. Tetrodotoxin (TTX) treatment did not prevent the increase in AChE expression, whereas nifedipine partially blocked it. These changes in enzyme expression were accompanied by increases in the levels of AChE mRNA, suggesting the involvement of pretranslational regulatory mechanisms. Together, these results illustrate that, in addition to neural activation and trophic factors, passive mechanical forces modulate expression of AChE in skeletal muscle cells. Because TTX did not prevent the increase in AChE expression, it appears that the effects of mechanical stimulation are independent of electrical activity, which further indicates the use of an alternate signaling pathway.

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