Vitamin D3 cannot revert desensitization of growth hormone (GH)-induced STAT5-signaling in GH-overexpressing mice non-calcemic tissues.

Growth hormone (GH) binding to a membrane receptor dimer triggers multiple intracellular signaling pathways. Signal transducers and activators of transcription are the most relevant of these pathways for GH action. GH also activates several inhibitory mechanisms, particularly suppressors of cytokine signaling (SOCS/CIS) proteins. GH-overexpressing mice exhibit hepatic desensitization of the JAK2/STAT5 GH-signaling pathway, associated with an increased abundance of CIS. Vitamin D3 has been shown to inhibit GH-induced expression of CIS and SOCS-3 and therefore prolong GH signaling in osteoblast-like cells. The purpose of the present study is to determine if vitamin D3 could attenuate CIS expression in GH-overexpressing mice, and consequently allow GH JAK2/STAT5 signaling in GH-responsive tissues in these animals. The abundance of CIS, SOCS-2, SOCS-3, STAT5b and GHR, as well as STAT5b tyrosine phosphorylation after a GH stimulus, were measured in liver and muscle of GHRH-transgenic mice treated with 1alpha,25-dihydroxyvitamin D3 for 7 days. This treatment did not diminish CIS expression in GH-overexpressing mice tissues, nor did the content of SOCS-2 and SOCS-3 significantly vary. GH-induced STAT5b phosphorylation levels were similar to basal values in transgenic mice liver treated with or without vitamin D; the refractoriness to GH was also present in muscle. Therefore, treatment with vitamin D was not sufficient to revert STAT5 GH signaling desensitization in non-calcemic tissues in GH-overexpressing mice.

Differential regulation of membrane associated-growth hormone binding protein (MA-GHBP) and growth hormone receptor (GHR) expression by growth hormone (GH) in mouse liver.

Growth hormone (GH) binding to GH receptor (GHR) is the initial step that leads to the physiological functions of the hormone. Proteolytical cleavage of the GHR in humans and rabbits and alternative processing of the GHR transcript in rodents generates circulating growth hormone binding protein (GHBP). Moreover, other GHR truncated forms that result from alternative processing of the GHR mRNA transcript have been described. These GHR short forms are inserted in the plasma membrane but they are unable to transduce the signal. In rodents, membrane associated-GHBP (MA-GHBP), which accounts for a significant proportion of liver GH binding capacity, represents the main GHR short form found in membranes, and may therefore function as a negative form of the receptor. In the present study, GHR and MA-GHBP content in liver were analyzed using mutant and transgenic mice expressing different concentrations of growth hormone to evaluate the correlation between GH levels, body weight (BW), GHR and MA-GHBP expression. It was found that GH deficiency was associated with diminished BW, GHR and MA-GHBP expression, while increased GH concentration led to increased BW, GHR and MA-GHBP expression, but MA-GHBP upregulation was more pronounced than the observed increase in GHR expression. Since GHR and MA-GHBP both contribute to liver GH binding capacity, GH-induced enrichment of the dominant negative form would represent a compensatory mechanism triggered by high levels of the hormone. This attempt to attenuate the effects of supraphysiological concentrations of GH may be critical to reduce or prevent their plausible damaging effects on the organism.

Chronic blockade of the AT2 receptor with PD123319 impairs insulin signaling in C57BL/6 mice.

The renin-angiotensin system modulates insulin action. Angiotensin type 1 receptor exerts a deleterious effects while the angiotensin type 2 receptor (AT2R) appears to have beneficial effects providing protection against insulin resistance and type 2 diabetes. Although recent reports indicate that agonism of AT2R ameliorates diabetes and insulin resistance, the phenotype of AT2R-knockout mice seems to be controversial relating this aspect. Thus, in this study we have explored the role of AT2R in the control of insulin action. To that end, C57Bl/6 mice were administered the synthetic AT2R antagonist PD123319 for 21days (10mg/kg/day ip); vehicle treated animals were used as control. Glucose tolerance, metabolic parameters, in vivo insulin signaling in main insulin-target tissues as well as levels of adiponectin, TNF-α, MCP-1 and IL-6 in adipose tissue were assessed. AT2R blockade with PD123319 induced a marginal effect on glucose homeostasis but an important reduction in the insulin-induced phosphorylation of the insulin receptor and Akt in both liver and adipose tissue. Insulin signaling in skeletal muscle remained unaltered after treatment with PD123319, which could explain the minimal effect on glucose homeostasis induced by PD123319. Our current results reinforce the notion that the AT2R has a physiological role in the conservation of insulin action.

Increased SH2-Bbeta content and membrane association in transgenic mice overexpressing GH.

Transgenic mice overexpressing GH present a marked GH signaling desensitization, reflected by low basal phosphorylation levels of the tyrosine kinase JAK2, and signal transducer and activator of transcription-5 (STAT5) and a lack of response of these proteins to a high GH dose. To evaluate the mechanisms involved in the regulation of JAK2 activity by high GH levels in vivo, the content and subcellular distribution of SH2-Bbeta were studied in GH-overexpressing transgenic mice. SH2-B is a member of a conserved family of adapter proteins characterized by the presence of a C-terminal SH2 domain, a central pleckstrin homology (PH) domain, and an N-terminal proline rich region. The isoform SH2-Bbeta modulates JAK2 activity by binding to the phosphorylated enzyme, further increasing its activity. However, it may also interact with non-phosphorylated inactive JAK2 via lower affinity binding sites, preventing abnormal activation of the kinase. SH2-Bbeta may also function as an adapter protein, acting as a GH signaling mediator. We now report that, in an animal model of GH excess in which JAK2 is not phosphorylated, although it is increased in the membrane-fraction, both the level of SH2-Bbeta, and especially its association to membranes, are augmented (67% and 13-fold vs normal mice values respectively), suggesting SH2-Bbeta could modulate JAK2 activity in vivo.

Increased sensitivity to GH in liver of Ames dwarf (Prop1df/Prop1df) mice related to diminished CIS abundance.

To investigate the influence of chronic GH deficiency on GH signaling in vivo, we have analyzed Janus kinase (JAK) 2/signal transducers and activators of transcription (STAT) 5 GH signaling pathway, and its regulation by the suppressors of the cytokine signaling SOCS and by the JAK2-interacting protein SH2-Bbeta, in liver of Ames dwarf (Prop1df/Prop1df) mice, which are severely deficient in GH, prolactin and TSH, and of their normal littermates. Prop1df/Prop1df mice displayed unaltered GH receptor, JAK2 and STAT5a/b protein levels. No significant differences in the basal tyrosine-phosphorylation levels of JAK2 and STAT5a/b were found between both groups of animals. After in vivo administration of a high GH dose (5 microg/g body weight (BW)), the tyrosine-phosphorylation levels of JAK2 and STAT5a/b increased significantly, reaching similar values in normal and dwarf mice. However, after stimulation with lower GH doses (50 and 15 ng/g BW) the tyrosine-phosphorylation level of STAT5a/b was higher in dwarf mice. The protein content of CIS, a SOCS protein that inhibits STAT5 signaling, was approximately 80% lower in dwarf mice liver, while SOCS-2 and SOCS-3 levels were unaltered. The content of SH2-Bbeta, a modulator of JAK2 activity, was reduced by approximately 30% in dwarf mice, although this was associated with normal JAK2 response to a high GH dose. In summary, Prop1df/Prop1df mice display increased hepatic sensitivity to GH, an effect that could be related to the lower abundance of CIS in this tissue. Furthermore, the lower CIS content found in this model of GH deficiency suggests that CIS protein levels are regulated by GH in vivo.

Cytokine-inducible SH2 protein up-regulation is associated with desensitization of GH signaling in GHRH-transgenic mice.

The effects of continuous high GH levels on GH signal transduction through the GH receptor (GHR)/Janus kinase 2 (JAK2)/signal transducer and activator of transcription 5 (STAT5) pathway as well as the desensitization of this pathway by suppressors of cytokine signaling (SOCS) were studied in transgenic mice overexpressing GHRH. In transgenic mice, hepatic GHR levels were 4.5-fold higher than in normal animals, whereas the protein contents of JAK2, STAT5a, and STAT5b did not vary. This same pattern was found for basal tyrosine phosphorylation (PY-): PY-GHR was 4.5-fold increased in transgenic mice, whereas there were no differences in PY-JAK2 and PY-STATs between normal and transgenic animals. After GH administration, tyrosine phosphorylation of GHR, JAK2, and STAT5s increased 3- to 7-fold in normal mice, but no significant changes were found in transgenic mice, indicating a decreased GH sensitivity in these animals. The content of cytokine-inducible SH2 protein, a member of the SOCS family, was 18-fold higher in GHRH-transgenic than in normal mice. Conversely, SOCS-3, present in normal mice, was hardly seen in transgenic animals, whereas SOCS-2 levels did not vary. These findings suggest that cytokine-inducible SH2 protein, significantly induced by continuously elevated GH levels, may be the SOCS protein responsible for the GH signaling desensitization in transgenic animals.

Prolonged exposure to GH impairs insulin signaling in the heart.

Acromegaly is associated with cardiac hypertrophy, which is believed to be a direct consequence of chronically elevated GH and IGF1. Given that insulin is important for cardiac growth and function, and considering that GH excess induces hyperinsulinemia, insulin resistance, and cardiac alterations, it is of interest to study insulin sensitivity in this tissue under chronic conditions of elevated GH. Transgenic mice overexpressing GH present cardiomegaly and perivascular and interstitial fibrosis in the heart. Mice received an insulin injection, the heart was removed after 2  min, and immunoblotting assays of tissue extracts were performed to evaluate the activation and abundance of insulin-signaling mediators. Insulin-induced tyrosine phosphorylation of the insulin receptor (IR) was conserved in transgenic mice, but the phosphorylation of IR substrate 1 (IRS1), its association with the regulatory subunit of the phosphatidylinositol 3-kinase (PI3K), and the phosphorylation of AKT were decreased. In addition, total content of the glucose transporter GLUT4 was reduced in transgenic mice. Insulin failed to induce the phosphorylation of the mammalian target of rapamycin (mTOR). However, transgenic mice displayed increased basal activation of the IR/IRS1/PI3K/AKT/mTOR and p38 signaling pathways along with higher serine phosphorylation of IRS1, which is recognized as an inhibitory modification. We conclude that GH-overexpressing mice exhibit basal activation of insulin signaling but decreased sensitivity to acute insulin stimulation at several signaling steps downstream of the IR in the heart. These alterations may be associated with the cardiac pathology observed in these animals.