Loss of growth hormone-mediated signal transducer and activator of transcription 5 (STAT5) signaling in mice results in insulin sensitivity with obesity.

Growth hormone (GH) has an important function as an insulin antagonist with elevated insulin sensitivity evident in humans and mice lacking a functional GH receptor (GHR). We sought the molecular basis for this sensitivity by utilizing a panel of mice possessing specific deletions of GHR signaling pathways. Metabolic clamps and glucose homeostasis tests were undertaken in these obese adult C57BL/6 male mice, which indicated impaired hepatic gluconeogenesis. Insulin sensitivity and glucose disappearance rate were enhanced in muscle and adipose of mice lacking the ability to activate the signal transducer and activator of transcription (STAT)5 via the GHR (Ghr-391-/-) as for GHR-null (GHR-/-) mice. These changes were associated with a striking inhibition of hepatic glucose output associated with altered glycogen metabolism and elevated hepatic glycogen content during unfed state. The enhanced hepatic insulin sensitivity was associated with increased insulin receptor ß and insulin receptor substrate 1 activation along with activated downstream protein kinase B signaling cascades. Although phosphoenolpyruvate carboxykinase (Pck)-1 expression was unchanged, its inhibitory acetylation was elevated because of decreased sirtuin-2 expression, thereby promoting loss of PCK1. Loss of STAT5 signaling to defined chromatin immunoprecipitation targets would further increase lipogenesis, supporting hepatosteatosis while lowering glucose output. Finally, up-regulation of IL-15 expression in muscle, with increased secretion of adiponectin and fibroblast growth factor 1 from adipose tissue, is expected to promote insulin sensitivity.-Chhabra, Y., Nelson, C. N., Plescher, M., Barclay, J. L., Smith, A. G., Andrikopoulos, S., Mangiafico, S., Waxman, D. J., Brooks, A. J., Waters, M. J. Loss of growth hormone-mediated signal transducer and activator of transcription 5 (STAT5) signaling in mice results in insulin sensitivity with obesity.

Growth hormone activated STAT5 is required for induction of beige fat in vivo.

OBJECTIVE: The anti-obesity actions of growth hormone (GH) led us to investigate if GH signaling is able to regulate beige/brite fat development of white adipose tissue (WAT). METHODS: We studied WAT in GHR-391 mice engineered to be unable to activate STAT5 in response to GH, in mice with adipose specific deletion of GHR, in GHR-/- mice and in bGH transgenic mice. QPCR, immunoblots and immunohistochemistry were used to characterize WAT. The in vivo effects of ß-3 adrenergic activation with CL-316,243 and that of FGF21 infusion were also studied. RESULTS: GHR-391 mice had lower surface temperature than WT, with deficiency in ß-oxidation and beiging transcripts including Ucp1. Oxidative phosphorylation complex subunit proteins were decreased dramatically in GHR-391 inguinal white adipose tissue (iWAT), but increased in bGH iWAT, as were proteins for beige/brown markers. In accord with its lack of ß-3 adrenergic receptors, iWAT of GHR-391 mice did not beige in response to administration of the ß-3 specific agonist CL-316,243 in contrast to WT mice. GHR-391 mice are deficient in FGF21, but unlike WT, infusion of the purified protein was without effect on extent of beiging. Finally, fat-specific deletion of the GHR replicated the loss of beiging associated transcripts. CONCLUSION: In addition to promoting lipolysis, our study suggests that GH is able to promote formation of beige adipose tissue through activation of STAT5 and induction of Adrb3. This sensitizes WAT to adrenergic input, and may contribute to the anti-obesity actions of GH.

Inflammation regulates TMPRSS6 expression via STAT5.

TMPRSS6 is a regulated gene, with a crucial role in the regulation of iron homeostasis by inhibiting hepcidin expression. The main regulator of iron homeostasis, the antimicrobial peptide hepcidin, which also has a role in immunity, is directly upregulated by inflammation. In this study, we analyzed whether inflammation is also a modulator of TMPRSS6 expression in vitro and in vivo and we determined the mechanism of this regulation A Human Hepatoma cell line was treated with interleukin-6 and mice were injected with lipopolysaccharide and TMPRSS6 expression and the regulatory mechanism were addressed. In this study, we demonstrate that inflammation downregulates TMPRSS6 expression in vitro and in vivo. The downregulation of Tmprss6 by inflammation in mice is not dependent on the Bmp-Smad pathway but occurs through a decrease in Stat5 phosphorylation. Moreover, Stat5 positively regulates Tmprss6 expression directly by binding to a Stat5 element located on the Tmprss6 promoter. Importantly, our results highlight the functional role of inflammatory modulation of TMPRSS6 expression in the regulation of hepcidin. TMPRSS6 inhibition via decreased STAT5 phosphorylation may be an additional mechanism by which inflammation stimulates hepcidin expression to regulate iron homeostasis and immunity.

GH-dependent STAT5 signaling plays an important role in hepatic lipid metabolism.

GH deficiency is known to be clinically associated with a high incidence of nonalcoholic fatty liver disease, and this can be reversed by GH administration. Here we investigated the mechanistic basis for this phenomenon using engineered male mice lacking different signaling elements of the GH receptor, hepatic stat5a/b(-/-) mice and a mouse hepatoma line. We found deficient GH-dependent signal transducer and activator of transcription (STAT)-5 signaling correlates with steatosis, and through microarray analysis, quantitative PCR, and chromatin immunoprecipitation, identified putative targets of STAT5 signaling responsible for the steatosis seen on a normal diet. These targets were verified with liver-specific stat5a/b deletion in vivo, and in vitro we show that dominant-negative (DN) STAT5 increases lipid uptake in a mouse hepatoma line. Because loss of STAT5 signaling results in elevated STAT1 and STAT3 activity and intracellular lipid accumulation, we have used DN-STAT5a/b, DN-STAT1, constitutively active (CA)-STAT3, or addition of oleate/palmitate in the hepatoma line to assign which of these apply to individual targets in STAT5 signaling deficiency. These findings and published mouse models of steatosis enable us to propose elevated cd36, pparγ, and pgc1α/ß expression as primary instigators of the steatosis along with elevated fatty acid synthase, lipoprotein lipase, and very low-density lipoprotein receptor expression. Decreased fgf21 and insig2 expression may also contribute. In conclusion, despite normal plasma free fatty acids and minimal obesity, absent GH activation leads to steatosis because activated STAT5 prevents hepatic steatosis. These results raise the possibility of low-dose GH treatment for nonalcoholic fatty liver disease.

In vivo targeting of the growth hormone receptor (GHR) Box1 sequence demonstrates that the GHR does not signal exclusively through JAK2.

GH is generally believed to signal exclusively through Janus tyrosine kinases (JAK), particularly JAK2, leading to activation of signal transducers and activators of transcription (STAT), ERK and phosphatidylinositol 3-kinase pathways, resulting in transcriptional regulation of target genes. Here we report the creation of targeted knock-in mice wherein the Box1 motif required for JAK2 activation by the GH receptor (GHR) has been disabled by four Pro/Ala mutations. These mice are unable to activate hepatic JAK2, STAT3, STAT5, or Akt in response to GH injection but can activate Src and ERK1/2. Their phenotype is identical to that of the GHR(-/-) mouse, emphasizing the key role of JAK2 in postnatal growth and the minimization of obesity in older males. In particular, they show dysregulation of the IGF-I/IGF-binding protein axis at transcript and protein levels and decreased bone length. Because no gross phenotypic differences were evident between GHR(-/-) and Box1 mutants, we undertook transcript profiling in liver from 4-month-old males. We compared their transcript profiles with our 391-GHR truncated mice, which activate JAK2, ERK1/2, and STAT3 in response to GH but not STAT5a/b. This has allowed us for the first time to identify in vivo Src/ERK-regulated transcripts, JAK2-regulated transcripts, and those regulated by the distal part of the GHR, particularly by STAT5.