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Differentiation-specific alterations to glutathione synthesis in and hormonally stimulated release from human skeletal muscle cells¹

IAN A. COTGREAVE^*2, LINA GOLDSCHMIDT^*, MICHAIL TONKONOGI^*, and MICHAEL SVENSSON^*,

^* Division of Biochemical Toxicology, Institute of Environmental Medicine, Karolinska Institute, S-17177 Stockholm, Sweden; and
Department of Physiology and Pharmacology, Karolinska Institute, S-11486, Stockholm, Sweden

²Correspondence: Division of Biochemical Toxicology, Institute of Environmental Medicine, Karolinska Institute, Box 210, S-17177 Stockholm, Sweden. E-mail: Ian.Cotgreave{at}I mm.Ki.Se

SPECIFIC AIMS

Human skeletal muscle atrophy or cachexia are debilitating conditions associated with a variety of serious clinical conditions, including postoperative shock, trauma associated with intensive care, local skeletomuscular injury, and various wasting diseases. Many studies have implicated the induction of oxidative stress in muscle in these largely catabolic events, and serious depletions of the major tissue antioxidant glutathione have been implicated. Nevertheless, the muscle-specific biochemistry of glutathione is poorly defined. We compared the sulfur amino acid precursor specificity of synthesis of glutathione in normal human skeletal muscle myoblasts (SKMmbs) and culture and cells stimulated to undergo differentiation to a myotube-like phenotype (SKMmts). We also contrasted the abilities of the cells to extrude glutathione in response to systemic mediators released during trauma and injury in the body.

PRINCIPAL FINDINGS

1. Differentiation alters the sulfur precursor specificity of synthesis of glutathione in human skeletal muscle cells
Using a medium devoid of any low molecular weight sulfur-containing components to deplete intracellular GSH and allow resynthesis in response to specific precursors, we demonstrated that SKMmbs are absolutely unable to use methionine (1 mM) to support assimilation of intracellular GSH. However, differentiation into SKMmts induced this unusual ability, which has been demonstrated for very few normal human cell types, e.g., hepatocytes. Even though SKMmbs are able to use extracellular GSH (100 µM) itself, this ability is completely lost in the differentiated cells (Fig. 1 ). The buthionine sulfoximine sensitivity of this event revealed a dependence on the activity of glutathione synthetase, but anthglutin, a potent inhibitor of -glutamyl transpeptidase, was without inhibitory effect. The ability to use extracellular cystine (25 µM) in SKMmbs is considerably impaired in favor or the use of cysteine (50 µM) in the differentiated cells. Finally, SKMmbs demonstrate a considerable readiness to deacetylate the thiol-containing drug N-acetyl-L-cysteine (100 µM) and use the resultant cysteine for GSH synthesis, a property unaffected by differentiation.

View larger version (17K): [in this window] [in a new window]   Figure 1. Methionine, N-acetyl-L-cysteine, and glutathione-stimulated reassimilation of cellular glutathione in undifferentiated and differentiated muscle cells in culture. Confluent human SKMmbs (A) or SKMmts (B) were initially depleted of their glutathione by an 18 h incubation in M199- medium. The cells were then exposed to either control growth medium or M199- medium supplemented with either methionine (1 mM), N-acetyl-L-cysteine (NAC, 100 µM), or reduced glutathione (GSH, 100 µM). The cells were analyzed at different times for intracellular content of GSH by in situ derivatization with the thiol-reactive florigenic label monobromobimane and resultant HPLC analysis of the adducts. All data were obtained from the same batch of cells and are expressed as mean ± SD, n = 3.

2. Human skeletal muscle cells secrete glutathione, which is greatly enhanced in the presence of stress mediators only in the differentiated state
We demonstrate that SKMmbs and SKMmts continually release reduced GSH into the growth medium, suggesting that this cell type may constitute a major extrahepatic source of systemic GSH in the human body. More important, however, although SKMmbs were insensitive to hormones such as vasopressin (100 nM), glucagon (100 nM), and phenylephrine (10 µM), the differentiated SKMmt cells responded to these stimuli by increased export of the reduced tripeptide (Fig. 2 ).

View larger version (19K): [in this window] [in a new window]   Figure 2. Differentiation facilitates stress hormone-stimulated release of reduced glutathione from human skeletal muscle cells in culture. Accumulation of reduced glutathione from undifferentiated (A) and differentiated (B) skeletal muscle cells was monitored in the absence or presence of glucagon (100 nM), phenylephrine (10 µM), or vasopressin (100 nM). Data are expressed as mean ± SD, n = 3. No significant differences were noted in the data in myoblasts (A), but all data points in treated differentiated cells (B) were significantly different from their respective controls (P<0.001 except for vasopressin: 6 h, P<0.01). Thin dashed line denotes lower level of detection.

CONCLUSIONS

Intracellular balance between the production of oxidants and the availability of tissue antioxidants is an important determinant in the development of oxidative stress. The induction of cellular and tissue oxidative stress has been associated with a variety of pathophysiological changes ranging from oxidation of cellular macromolecules to the induction of cell death by apoptosis or necrosis and the loss of tissue structure and function. Glutathione is a major low molecular weight antioxidant present in eukaryotic cells, and losses of muscle GSH have been demonstrated in association with extreme physical activity, skeletomuscular injury, and muscle atrophy associated with postoperative trauma and intensive care. Thus, understanding the basic biochemical and physiological regulation of muscle GSH and its application the development of possible therapeutic regimes for the manipulation of muscle GSH levels is relevant to clinical medicine.

We demonstrate that the state of differentiation clearly affects the sulfur source specificity of synthesis of GSH in human muscle cells in culture as well as the ability to respond to systemic stress-mediating molecules. Differentiation of human skeletal muscle myoblasts induces considerable alterations in the ability to use common low molecular weight sources of organic sulfur. This is reflected by a loss of preference for cystine and acquisition of ability to use extracellular methionine with differentiation. The unusual capacity of SKMmbs to effectively use extracellular glutathione was completely lost upon differentiation. The molecular mechanisms underlying these adaptations are obscure, but it could be speculated to be due primarily to altered expression of carrier uptake mechanisms in cystine and glutathione and in the induction of one or more enzymes in the trans-sulfuration pathway governing conversion of methionine to cysteine (Fig. 3 ). Irrespective of the mechanism underlying these adaptations during differentiation, the changes may reflect localized alterations in the availability of low molecular weight sulfur-containing precursors in the intact tissue. Thus, any attempt to modulate intramuscular glutathione content during trauma, cachexia, or injury must take into account both the precursor specificity of resynthesis and the optimal manner by which to deliver these components to the tissue, i.e., by parenteral nutrition or systemic infusion. However, our data suggest that intramuscular injection/perfusion with glutathione, N-acetyl-L-cysteine, or methionine or combinations of these may provide an attractive alternative for rapid elevation of glutathione, particularly in large locomotory muscles (Fig. 3) .

View larger version (15K): [in this window] [in a new window]   Figure 3. Schematic representation of adaptations to glutathione homeostasis induced by differentiation in vitro. During differentiation, adaptive alterations to carrier-mediated uptake and/or amino acid metabolism lead to alterations in the sulfur amino acid precursor specificity of glutathione synthesis in normal human skeletal muscle cells. Differentiation also sensitizes the cells to release larger amounts of glutathione in response to hormonal stress mediators, perhaps underlying the drainage of glutathione seen in vivo during local and systemic trauma, extremes of physical activity, and musculoskeletal injury. SKMmbs are skeletal muscle myoblasts; SKMmts are skeletal muscle myotube-like cells derived from SKMmbs by growth factor restriction.

Another major finding of this work is the observation that SKMmbs and SKMmts both release glutathione and that this release is under hormonal regulation only in the differentiated cells. The data thus suggest that human skeletal muscle cells may serve as a continuous extrahepatic source of glutathione and that this role may be particularly important during localized or systemic trauma, where the release of stress hormones may greatly enhance the release of the antioxidant (Fig. 3) . The molecular mechanisms underlying induced sensitivity to stress-mediating molecules are unclear. However, vasopressin, glucagon, and phenylephrine have all been shown to induce the release of glutathione from hepatocytes via the activity of a canalicular extrusion protein. The most effective agent was glucagon in SKMmt cells, suggesting some degree of coordinated release of glucose and glutathione from the muscle during extreme physiological responses. Thus, the findings not only help explain the dramatic losses of muscle glutathione during trauma and injury, but also illustrate the importance of this bulky human tissue as a systemic reservoir of glutathione that is able to assist the liver in systemic supply of the tripeptide when required.

In summary, these data offer some of the first evidence that differentiation can alter the sulfur source preference for GSH in human cells and offer a sound biochemical rationale for attempts to clinically modulate muscle GSH levels during the development of muscle atrophy. The findings may also provide a plausible mechanism for the acute muscle GSH losses seen in situations of localized and systemic trauma in association with muscle atrophy.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0685fje; to cite this article, use FASEB J. (January 30, 2002) 10.1096/fj.01-0685fje

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This Article Full Text (PDF) All Versions of this Article: 16/3/435 01-0685fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by COTGREAVE, I. A. Articles by SVENSSON, M. Search for Related Content PubMed PubMed Citation Articles by COTGREAVE, I. A. Articles by SVENSSON, M.