11beta-hydroxysteroid dehydrogenase type 1 regulates glucocorticoid-induced insulin resistance in skeletal muscle

Abstract

Objective: Glucocorticoid excess is characterized by increased adiposity, skeletal myopathy, and insulin resistance, but the precise molecular mechanisms are unknown. Within skeletal muscle, 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) converts cortisone (11-dehydrocorticosterone in rodents) to active cortisol (corticosterone in rodents). We aimed to determine the mechanisms underpinning glucocorticoid-induced insulin resistance in skeletal muscle and indentify how 11beta-HSD1 inhibitors improve insulin sensitivity.

Research design and methods: Rodent and human cell cultures, whole-tissue explants, and animal models were used to determine the impact of glucocorticoids and selective 11beta-HSD1 inhibition upon insulin signaling and action.

Results: Dexamethasone decreased insulin-stimulated glucose uptake, decreased IRS1 mRNA and protein expression, and increased inactivating pSer(307) insulin receptor substrate (IRS)-1. 11beta-HSD1 activity and expression were observed in human and rodent myotubes and muscle explants. Activity was predominantly oxo-reductase, generating active glucocorticoid. A1 (selective 11beta-HSD1 inhibitor) abolished enzyme activity and blocked the increase in pSer(307) IRS1 and reduction in total IRS1 protein after treatment with 11DHC but not corticosterone. In C57Bl6/J mice, the selective 11beta-HSD1 inhibitor, A2, decreased fasting blood glucose levels and improved insulin sensitivity. In KK mice treated with A2, skeletal muscle pSer(307) IRS1 decreased and pThr(308) Akt/PKB increased. In addition, A2 decreased both lipogenic and lipolytic gene expression.

Conclusions: Prereceptor facilitation of glucocorticoid action via 11beta-HSD1 increases pSer(307) IRS1 and may be crucial in mediating insulin resistance in skeletal muscle. Selective 11beta-HSD1 inhibition decreases pSer(307) IRS1, increases pThr(308) Akt/PKB, and decreases lipogenic and lipolytic gene expression that may represent an important mechanism underpinning their insulin-sensitizing action.

FIG. 1.

Dex treatment (1 μmol/l, 24 h) in C2C12 rodent skeletal myocytes decreases IRS1 total protein expression, increases pSer³⁰⁷ IRS1, but does not change pTyr⁶⁰⁸ IRS1. These observations are reversed by coincubation with the glucocorticoid antagonist RU38486. IRS2 expression increased after Dex treatment. Although Akt/PKB expression does not change, activating pSer⁴⁷³ Akt/PKB decreases but is not recovered by coincubation with RU38486. Total AS160 protein expression increased after Dex pretreatment and was reversed with RU38486. Representative Western blots are shown in panel 1A with quantitation relative to β-actin as internal loading control shown in subsequent panels (IRS1 [total IRS1: black bars, pSer³⁰⁷ IRS1: white bars, pTyr⁶⁰⁸ IRS1: gray bars] [B], IRS2 [C], akt/PKB [total Akt/PKB: black bars, pSer⁴⁷³ Akt/PKB: white bars] [D], and AS160 [E]) (*P < 0.05 vs. control, †P < 0.05, ††P < 0.01 vs. Dex). In C2C12 cells stably overexpressing IRS1, Dex increases Ser³⁰⁷ but does not induce Ser²⁴ phosphorylation (F). Ctrl, control. PMA, phorbol myristate acetate.

FIG. 2.

The endogenous rodent glucocorticoid, CORT, induces a dose- (A) and time- (B) dependent decrease in total IRS1 protein expression (black bars) and increase in pSer³⁰⁷ IRS1 (white bars). Data presented are the means of n = 4–6 experiments with representative Western blots inserted above (*P < 0.05, **P < 0.01 vs. control).

FIG. 3.

Functional 11β-HSD1 enzyme oxo-reductase (A) and dehydrogenase (B) activity is present in explants of mouse quadriceps muscle with levels comparable with that seen in adipose tissue and liver. 11β-HSD1 activity is also observed in differentiated C2C12 rodent skeletal myocytes (data presented as picomole per milligram of protein per hour for C2C12 cells). Although dehydrogenase activity is present, the predominant activity is oxo-reductase generating active glucocorticoid. Coincubation of skeletal muscle explants with the nonselective 11β-HSD inhibitor, glycyrrhetinic acid, significantly decreases activity (data shown are the means ± SE of n = 3–6 experiments, *P < 0.05). In addition, the selective 11β-HSD1 inhibitor, A1 (1 μmol/l, 24 h), decreases oxo-reductase activity in rodent whole-tissue quadriceps explants, differentiated C2C12 skeletal myocytes, and primary cultures of human skeletal myocytes (C) (data shown are the means ± SE of n = 3–6 experiments, *P < 0.05, §P < 0.005) (control = black bars, A1 = white bars). GE, glycyrrhetinic acid.

FIG. 4.

Both CORT and 11DHC decrease IRS1 expression (black bars) and increase pSer³⁰⁷ IRS1 (white bars). The activity of 11DHC is dependent upon its activation to CORT by 11β-HSD1. Inhibition of 11β-HSD1 using glycyrrhetinic acid (A) or the selective 11β-HSD1 inhibitor, A1 (B), reverses the effect of 11DHC upon IRS1 expression and phosphorylation. Similarly, in primary cultures of human myotubes, A1 blocks the cortisone-induced decrease in pThr³⁰⁸ Akt/PKB (white bars) after insulin stimulation without changing total Akt/PKB expression (black bars) (C). Data presented are the mean of n = 4 experiments with representative Western blots inserted (*P < 0.05, **P < 0.01 vs. control, †P < 0.05 vs. 11DHC or cortisone). Ctrl, control.

FIG. 5.

In cortisone pellet–implanted KK mice, the selective 11β-HSD1 inhibitor, A2, increases IRS1 total protein expression, decreases inactivating pSer³⁰⁷ IRS1, and further downstream enhances insulin-stimulated pThr³⁰⁸ Akt/PKB in quadriceps muscle explants. Representative Western blots are shown (A2 vs. vehicle) (A). Real-time PCR analysis endorsing observation from the Genecard analysis presented in Table 3. Gene expression from whole-tissue quadriceps explants obtained from cortisone pellet–implanted KK mice treated for 4 days with the selective 11β-HSD1 inhibitor, A2 (white bars), or vehicle (black bars) (*P < 0.05, **P < 0.01) (n = 3 per group) (B).