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

ABSTRACT: 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 ? 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.

SUBMITTER: Chhabra Y

PROVIDER: S-EPMC6463913 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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

Chhabra Yash Y Nelson Caroline N CN Plescher Monika M Barclay Johanna L JL Smith Aaron G AG Andrikopoulos Sof S Mangiafico Salvatore S Waxman David J DJ Brooks Andrew J AJ Waters Michael J MJ

FASEB journal : official publication of the Federation of American Societies for Experimental Biology 20190219 5

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 disapp ...[more]

PMID: 30779881

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Project description:The transcription factor signal transducer and activator of transcription (STAT)-5 mediates GH stimulation of IGF-I gene expression in the liver. Previous studies suggested that STAT5 might exert this effect by binding to an IGF-I intron 2 region and a distal 5'-flanking region each containing two STAT5 binding sites. Here we report the identification of three additional chromosomal regions containing a total of five putative STAT5 binding sites that may mediate GH-induced STAT5 activation of IGF-I gene expression in the mouse liver. By comparing an 170-kb mouse genomic DNA containing the IGF-I gene with the corresponding human sequence, we identified 19 putative STAT5 binding sites that bear the consensus sequence of STAT5 binding site and are conserved across the two species. Chromatin immunoprecipitation assays indicated that five chromosomal regions containing a total of nine of the 19 putative STAT5 binding sites were bound by STAT5 in the mouse liver in response to GH administration and that these bindings preceded or coincided with GH-increased IGF-I gene transcription. Two of the five chromosomal regions correspond to those previously identified in other species, and the three new chromosomal regions that contain a total of five putative STAT5 binding sites are IGF-I intron 3 regions located at least 26 kb from the transcription start site. Gel-shift assays confirmed the binding of the five new putative STAT5 binding sites as well as three of the four previously identified STAT5 binding sites to GH-activated STAT5 from the mouse liver. Cotransfection analyses indicated that, although each of the five chromosomal regions was able to mediate STAT5 activation of reporter gene expression, together they mediated greater STAT5 activation of reporter gene expression in response to GH. Overall, these results suggest that GH-induced STAT5 activation of IGF-I gene expression in the mouse liver might be collectively mediated by at least eight STAT5 binding sites located in distal intronic and 5'-flanking regions of the IGF-I gene.

Project description:STAT (signal transducer and activator of transcription) proteins play a critical role in cellular response to a wide variety of cytokines and growth factors by regulating specific nuclear genes. STAT-dependent gene transcription can be finely tuned through the association with co-factors in the nucleus. We showed previously that STAT5 (including 5a and 5b) specifically interacts with a mitochondrial enzyme PDC-E2 (E2 subunit of pyruvate dehydrogenase complex) in both leukemic T cells and cytokine-stimulated cells. However, the functional significance of this novel association remains largely unknown. Here we report that PDC-E2 may function as a co-activator in STAT5-dependent nuclear gene expression. Subcellular fractionation analysis revealed that a substantial amount of PDC-E2 was constitutively present in the nucleus of BaF3, an interleukin-3 (IL-3)-dependent cell line. IL-3-induced tyrosine-phosphorylated STAT5 associated with nuclear PDC-E2 in co-immunoprecipitation analysis. These findings were confirmed by confocal immunofluorescence microscopy showing constant nuclear localization of PDC-E2 and its co-localization with STAT5 after IL-3 stimulation. Similar to mitochondrial PDC-E2, nuclear PDC-E2 was lipoylated and associated with PDC-E1. Overexpression of PDC-E2 in BaF3 cells augmented IL-3-induced STAT5 activity as measured by reporter assay with consensus STAT5-binding sites. Consistent with the reporter data, PDC-E2 overexpression in BaF3 cells led to elevated mRNA levels of endogenous SOCS3 (suppressor of cytokine signaling 3) gene, a known STAT5 target. We further identified two functional STAT5-binding sites in the SOCS3 gene promoter important for its IL-3-inducibility. The observation that both cis-acting elements were essential to detect the stimulatory effect by PDC-E2 strongly supports the role of PDC-E2 in up-regulating the transactivating ability of STAT5. All together, our results reveal a novel function of PDC-E2 in the nucleus. It also raises the possibility of nuclear-mitochondrial crosstalk through the interaction between STAT5 and PDC-E2.

Project description:Mitogenic signal-transduction pathways have not been well defined in pancreatic beta-cells. In the glucose-sensitive rat beta-cell line, INS-1, glucose (6-18 mM) increased INS-1 cell proliferation (>20-fold at 15 mM glucose). Rat growth hormone (rGH) also induced INS-1 cell proliferation, but this was glucose-dependent in the physiologically relevant concentration range (6-18 mM glucose). The combination of rGH (10 nM) and glucose (15 mM) was synergistic, maximally increasing INS-1 cell proliferation by >50-fold. Moreover, glucose-dependent rGH-induced INS-1 cell proliferation was increased further by addition of insulin-like growth factor 1 (IGF-1; 10 nM) to >90-fold at 12 mM glucose. Glucose metabolism and phosphatidylinositol-3'-kinase (PI3'K) activation were necessary for both glucose- and rGH-stimulated INS-1 cell proliferation. Glucose (>3 mM) independently increased tyrosine-phosphorylation-mediated recruitment of growth-factor-bound protein 2 (Grb2)/murine sons of sevenless-1 protein (mSOS) and PI3'K to insulin receptor substrate (IRS)-1 and IRS-2, as well as SH2-containing protein (Shc) association with Grb2/mSOS and downstream activation of mitogen-activated protein kinase and 70 kDa S6 kinase. Glucose-induced IRS- and Shc-mediated signal transduction was enhanced further by the addition of IGF-1, but not rGH. In contrast, rGH was able to activate Janus kinase 2 (JAK2)/signal transducer and activator of transcription 5 (STAT5) signal transduction at glucose concentrations above 3 mM, but neither glucose independently, nor glucose with added IGF-1, were able to activate the JAK2/STAT5 signalling pathway. Thus rGH-mediated proliferation of beta-cells is directly via the JAK2/STAT5 pathway without engaging the Shc or IRS signal-transduction pathways, although activation of PI3'K may play an important permissive role in the glucose-dependent aspect of rGH-induced beta-cell mitogensis. The additive effect of rGH and IGF-1 on glucose-dependent beta-cell proliferation is therefore reflective of rGH and IGF-1 activating distinctly different mitogenic signalling pathways in beta-cells with minimal crosstalk between them.

Dataset Information

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

Publications

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

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