Regulation of Glucocorticoid Receptor α and β Isoforms and Type I 11β-Hydroxysteroid Dehydrogenase Expression in Human Skeletal Muscle Cells: A Key Role in the Pathogenesis of Insulin Resistance?

0021-972X/01/$03.00/0 The Journal of Clinical Endocrinology & Metabolism Copyright © 2001 by The Endocrine Society Vol. 86, No. 5 Printed in U.S.A. Regulation of Glucocorticoid Receptor ␣ and ␤ Isoforms and Type I 11␤-Hydroxysteroid Dehydrogenase Expression in Human Skeletal Muscle Cells: A Key Role in the Pathogenesis of Insulin Resistance? Endocrinology and Metabolism Unit, Southampton General Hospital School of Medicine (C.B.W., S.J.D.), and Department of Chemical Pathology (P.J.W.) and Medical Research Council Environmental Epidemiology Unit (D.I.W.P.), Southampton General Hospital, Southampton, United Kingdom SO16 6YD; and Division of Biomedical Sciences, University of Portsmouth (S.J.D.), Portsmouth, United Kingdom P01 2UP ABSTRACT Glucocorticoid excess frequently results in obesity, insulin resistance, glucose intolerance, and hypertension and may be the product of altered glucocorticoid hormone action. Tissue sensitivity to glucocorticoid is regulated by the expression of glucocorticoid receptor isoforms (GR␣ and GR␤) and 11␤-hydroxysteroid dehydrogenase type I (11␤HSD1)-mediated intracellular synthesis of active cortisol from inactive cortisone. We have analyzed the expression of GR␣, GR␤, and 11␤HSD1 and their hormonal regulation in skeletal myoblasts from men (n ⫽ 14) with contrasting levels of adiposity and insulin resistance. Immunohistochemical, Northern blot, and Western blot analysis indicated abundant expression of GR␣ and 11␤HSD1 under basal conditions. The apparent Km and maximum velocity for the conversion of cortisone to cortisol were 440 ⫾ 14 nmol/L and 75 ⫾ 7 pmol/mg protein䡠h and 437 ⫾ 16 nmol/L and 33 ⫾ 6 pmol/mg protein䡠h (mean ⫾ SEM; n ⫽ 4) in the presence and absence of 20% serum. Incubation of myoblasts with increasing concentrations of glucocorticoid (50 –1000 nmol/L) resulted in a dose-dependent decline in GR␣ expression and a dose-dependent increase in GR␤ expression. 11␤HSD1 activity was sensitively up-regulated by increasing concentrations of glucocorti- C USHING’S SYNDROME and glucocorticoid excess due to exposure to supraphysiological doses of glucocorticoid frequently result in a spectrum of clinical features distinguished by obesity, insulin resistance, glucose intolerance, and hypertension (1). These characteristics bear marked similarities to key features of the metabolic syndrome and type II diabetes (2, 3) and may be the product of increased glucocorticoid antagonism of insulin action. Indeed, glucocorticoids promote gluconeogenesis and glycogen synthesis (4, 5), inhibit glycogenolysis, and reduce the disposal of glucose to the intracellular compartment as a consequence of inhibition of the translocation of the glucose transporter, GLUT-4, to the cell membrane (6 – 8). Moreover, glucocorticoids promote the differentiation of preadipocytes into mature fat cells (9), diminish glucose uptake, and stimulate lipoprotein lipase activity in ad- Received October 30, 2000. Revision received February 5, 2001. Accepted February 7, 2001. Address all correspondence and requests for reprints to: Dr Christopher B. Whorwood, Endocrinology and Metabolism Unit, Level D, South Block, Southampton General Hospital School of Medicine, Tremona Road, Southampton, United Kingdom SO16 6YD. E-mail: . coid (50 –1000 nmol/L: P ⬍ 0.05). Abolition of these effects by the GR antagonist, RU38486, indicates that regulation of GR␣, GR␤, and 11␤HSD1 expression is mediated exclusively by the GR␣ ligandbinding variant. In contrast, 11␤HSD1 was down-regulated by insulin (20 –100 mU/mL: P ⬍ 0.01) in the presence of 20% serum, whereas incubation with insulin under serum-free conditions resulted in a dose-dependent increase in 11␤HSD1 activity (P ⬍ 0.05). Incubation with insulin-like growth factor I resulted in a similar pattern of 11␤HSD1 activity. Although neither testosterone nor androstenedione (5–200 nmol/L) affected 11␤HSD1 activity, incubation of myoblasts with dehydroepiandrosterone (500 nmol/L) resulted in a decline in 11␤HSD1 activity (P ⬍ 0.05). These data suggest that glucocorticoid hormone action in skeletal muscle is determined principally by autoregulation of GR␣, GR␤, and 11␤HSD1 expression by the ligand-binding GR␣ isoform. Additionally, insulin and insulinlike growth factor I regulation of 11␤HSD1 may represent a novel mechanism that maintains insulin sensitivity in skeletal muscle tissue by diminishing glucocorticoid antagonism of insulin action. (J Clin Endocrinol Metab 86: 2296 –2308, 2001) ipose tissue, which result in an increase in lipid mobilization and triglyceride sequestration in visceral fat depots (7, 8). Additionally, glucocorticoids inhibit the activity of lipoprotein lipase in skeletal muscle and diminish the uptake of circulating triglyceride, which contribute to the clinical and atherogenic features of dislipidemia that frequently accompany glucocorticoid excess and insulin resistance (10). Importantly, the close parallel between the clinical features of the metabolic syndrome and glucocorticoid excess suggest that abnormalities of glucocorticoid hormone action may contribute to the pathogenesis of key features of the metabolic syndrome (11) and the development of premature atherosclerosis and cardiovascular disease (12–14). Cross-sectional studies have revealed strong positive associations among circulating levels of cortisol, blood pressure, glucose intolerance, and hypertriglyceridemia and have led to suggestions that chronic activation of the hypothalamic-pituitary-adrenal (HPA) axis may underlie this relationship (15, 16). However, in most obese, insulin-resistant subjects, circulating levels of cortisol are normal or may even be slightly decreased (17). Furthermore, there is evidence to 2296 C. B. WHORWOOD, S. J. DONOVAN, P. J. WOOD, AND D. I. W. PHILLIPS GC HORMONE ACTION IN HUMAN SKELETAL MUSCLE sues may be considered to play a key role in the regulation of tissue sensitivity to glucocorticoid through two interdependent mechanisms: the direct regulation of intracellular levels of cortisol, and the corollary of increased intracellular levels of cortisol upon the regulation of GR␣. The tissue-specific regulation of isoforms of 11␤HSD is poorly understood. However, several studies have produced evidence in support of the hypothesis that the regulation of 11␤HSD may be mediated through the actions of a number of hormones and growth factors. However, much of this research is contradictory and either predates the discovery of 11␤HSD2 or fails to characterize the species- and tissuespecific mechanisms that underlie the regulation of 11␤HSD in an isoform-specific manner. Nevertheless, these data have served to highlight key elements underlying the hormonal regulation of 11␤HSD, including glucocorticoids (30, 32, 33), insulin (34, 35), GH (35), thyroid hormones (36), and the sex steroids (37, 38) and support the hypothesis that dysregulation of enzyme activity may underlie the etiology of a spectrum of diseases, includin (...truncated)