Endotoxin-induced growth hormone resistance in skeletal muscle

Abstract

Inflammation-induced skeletal muscle wasting is a serious clinical problem and arises in part because of resistance to GH-stimulated IGF-I expression. Although it is established that in the liver, resistance develops because of impaired signaling through the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 5 (STAT5) transduction pathway, together with a more distal defect in STAT5 DNA-binding activity, the situation in skeletal muscle is unclear. Accordingly, we set out to characterize the mechanisms behind the skeletal muscle resistance to GH in rats with acute inflammation induced by endotoxin. Endotoxin caused significant declines in GH-stimulated STAT5a/b phosphorylation and IGF-I gene expression, and this occurred despite a lack of change in signaling protein levels or phosphorylation of JAK2. In whole muscle, GH-stimulated phospho-STAT5a/b levels were reduced by half, and in the nucleus, phospho-STAT5b levels were similarly reduced. Furthermore, the binding of phosphorylated STAT5b to DNA was reduced and to a similar extent to the reduction in nuclear phosphorylated STAT5b. Interestingly, GH-induced androgen receptor gene expression was also suppressed. Thus, it appears that skeletal muscle resistance to GH-stimulated IGF-I expression in acute endotoxemia arises from a defect in STAT5b signaling, with a proportionate reduction in STAT5b DNA binding. Finally, it appears that resistance to GH-induced androgen receptor expression also develops and, together with the attenuated GH-induced IGF-I expression, likely plays an important role in the muscle wasting that arises in endotoxin-induced inflammation.

Figure 1

Acute endotoxemia does not affect skeletal muscle GH receptor, JAK2, or STAT5a/b protein levels but inhibits GH-induced STAT5a/b protein tyrosine phosphorylation. A and B, Western immunoblots; A, basal level of GHR (∼110 kDa) in muscle of V (vehicle)-treated control (Con) and LPS rats; B, GH-stimulated tyrosine phosphorylation of JAK2 and STAT5a/b and their protein levels in whole lysates prepared from the gastrocnemius muscle of LPS and control rats obtained 15 min after an iv bolus of GH, 100 μg/kg, or V. The samples from V-treated animals were pooled (V pool). C, Relative phosphorylation of JAK2 and STAT5a/b proteins 15 min after a GH bolus. Phosphoprotein signals were corrected for the specific protein levels, and the ratios were normalized to the control mean, assigned a value of 100. Bars indicate mean ± sem n = 6 rats per group. *, P < 0.001. Phospho-JAK2 (p-JAK2) was detected in immunoblots of JAK2 immunoprecipitates with an anti-phosphotyrosine antibody (clone 4G10). Phospho-STAT5a/b was detected without immunoprecipitation using a phospho-STAT5a/b-specific antibody.

Figure 2

GH-induced nuclear accumulation of phosphorylated STAT5 is attenuated in skeletal muscle of acutely endotoxic rats. Nuclear extracts were prepared from the individual animals depicted in Fig. 1, namely vehicle (V)-treated control (Con) and LPS-treated rats, and subjected to Western immunoblotting in the same order. Phospho-STAT5a/b (p-STAT5a/b) was detected directly in nuclear extracts with a p-STAT5a/b antibody. To detect p-STAT5a and p-STAT5b, nuclear extracts were immunoprecipitated with an antibody that recognizes either STAT5a or STAT5b. Immunoprecipitates were then separated in different gels and the respective phosphorylated STAT detected with an antibody against p-STAT5a/b.

Figure 3

Myocardial GH-induced STAT5a/b protein tyrosine phosphorylation is depressed in endotoxemia. Western immunoblots of STAT5a/b protein and phosphorylated STAT5a/b (pSTAT5a/b) in whole heart lysates from vehicle (V)-treated control (Con) and LPS-treated rats are shown.

Figure 4

Gel mobility shift analysis shows that GH-induced STAT5b binding to DNA is impaired in skeletal and cardiac muscle of endotoxin-treated rats. Nuclear extracts were prepared from gastrocnemius muscle (A) and heart (B) of rats treated with vehicle (V) (Con; control) or LPS 4 h earlier and then killed 15 min after receiving iv GH,100 μg/kg, or V. Nuclear STAT5b DNA-binding activity was assayed with a ³²P-labeled β-casein promoter probe. Distinct STAT5-DNA complexes (lower arrow) are prominent in the GH-treated compared with V-treated rats and are attenuated in the LPS group. An essentially complete supershift (upper arrow) was induced when samples were preincubated with a STAT5b-specific antibody.

Figure 5

GH stimulation of skeletal muscle IGF-I mRNA expression is acutely impaired in endotoxemia. Rats treated with LPS or saline (Con; control) were given either bGH or vehicle (V) by ip injection 15 min and again 180 min after the LPS or saline and killed 1 h later. Gastrocnemius muscle total IGF-I mRNA levels were measured by quantitative real-time PCR and corrected for the internal housekeeping gene 18S rRNA. The results, mean ± sem of 8 rats per group, are expressed relative to the control group assigned a mean value of 100.