Growth arrest specific protein (GAS) 6: a role in the regulation of proliferation and functional capacity of the perinatal rat beta cell.

AIMS/HYPOTHESIS: Maternal low-protein (LP) diet during gestation results in a reduced beta cell mass in the offspring at birth and this may hamper the ability to adapt to high-energy food and sedentary lifestyle later in life. To investigate the biology behind the LP-offspring phenotype, this study aimed to identify differentially expressed genes in the pancreas and their potential role in the fetal programming. METHODS: Wistar rats were given either an LP diet or normal-chow (NC) diet during gestation and differentially expressed genes in the offspring around the time of birth were identified using RNA microarray and quantitative PCR. The role of a differentially expressed gene, growth arrest specific protein 6 (GAS6), was evaluated in vitro using neonatal rat islets. RESULTS: The mRNA level of Gas6, known to be mitogenic in other tissues, was reduced in LP offspring. The mRNA content of Mafa was increased in LP offspring suggesting an early maturation of beta cells. When applied in vitro, GAS6 increased proliferation of neonatal pancreatic beta cells, while reducing glucose-stimulated insulin secretion without changing the total insulin content of the islets. In addition, GAS6 decreased the mRNA content of Mafa. CONCLUSIONS/INTERPRETATION: We propose a role for GAS6 in the regulation of pancreatic beta cells in the critical period around the time of birth. Our results support the hypothesis that the reduced beta cell mass seen in LP offspring is caused by a change in the intra-uterine environment that favours premature maturation of the beta cells.

Multifactorial treatment increases endothelial progenitor cells in patients with type 2 diabetes.

AIMS/HYPOTHESIS: Endothelial progenitor cells (EPC) augment vascular repair and neovascularisation. Patients with type 2 diabetes have reduced EPC and increased risk of cardiovascular disease (CVD), which is reduced by multifactorial intervention. Our aim, therefore, was to evaluate in type 2 diabetic patients whether the numbers of EPC derived from peripheral blood mononuclear cells is influenced by a multifactorial treatment strategy. METHODS: We enrolled 28 patients newly referred for initiation of multifactorial treatment, which consisted of improving glycaemic, lipid and blood pressure control, as well as antithrombotic therapy and lifestyle modification. EPC count was assessed by in vitro cultures at baseline and after 90 days of treatment. After 7 days in culture, we identified EPC by fluorescent staining of attached cells. Patients were treated with metformin, aspirin, statins and angiotensin II receptor blockers, and divided accordingly into groups of mono-, dual-, triple- or quadruple therapy. RESULTS: After 90 days of treatment, glycaemic control improved and total cholesterol decreased. Multifactorial intervention for 90 days significantly increased EPC count in cultures by 35% (from 105 [SE 8] to 140 [11] cells per field [p = 0.002]). The change in EPC among patients with quadruple therapy was higher (63%) than in untreated patients (-32%, p = 0.043). CONCLUSIONS/INTERPRETATION: Numbers of EPC derived from peripheral blood mononuclear cells increased significantly after multifactorial intervention in type 2 diabetic patients. It remains to be shown whether these changes contribute to the beneficial effects of multifactorial intervention on diabetic micro- and macroangiopathy.

Lysine deacetylases are produced in pancreatic beta cells and are differentially regulated by proinflammatory cytokines.

AIMS/HYPOTHESIS: Cytokine-induced beta cell toxicity is abrogated by non-selective inhibitors of lysine deacetylases (KDACs). The KDAC family consists of 11 members, namely histone deacetylases HDAC1 to HDAC11, but it is not known which KDAC members play a role in cytokine-mediated beta cell death. The aim of the present study was to examine the KDAC gene expression profile of the beta cell and to investigate whether KDAC expression is regulated by cytokines. In addition, the protective effect of the non-selective KDAC inhibitor ITF2357 and interdependent regulation of four selected KDACs were investigated. METHODS: The beta cell line INS-1 and intact rat and human islets were exposed to cytokines with or without ITF2357. Expression of mRNA was assessed by real-time PCR and selected targets validated at the protein level by immunoblotting. Effects on cytokine-induced toxicity were investigated by in vitro assays. RESULTS: Hdac1 to Hdac11 were expressed and differentially regulated by cytokines in INS-1 cells and rat islets. HDAC1, -2, -6 and -11 were found to be expressed and regulated by cytokines in human islets. ITF2357 protected against cytokine-induced beta cell apoptosis and counteracted cytokine-induced attenuation of basal insulin secretion. In addition, cytokine-induced regulation of Hdac2 and Hdac6, but not Hdac1 and Hdac11, was reduced by KDAC inhibition. CONCLUSIONS/INTERPRETATION: All classical KDAC genes are expressed by beta cells and differentially regulated by cytokines. Based on the relative expression levels and degree of regulation by cytokines, we propose that HDAC1, -2, -6 and -11 are of particular importance for beta cell function. These observations may help in the design of specific KDAC inhibitors to prevent beta cell destruction in situ and in islet grafts.

IL-1beta-induced chemokine and Fas expression are inhibited by suppressor of cytokine signalling-3 in insulin-producing cells.

AIMS/HYPOTHESIS: Chemokines recruit activated immune cells to sites of inflammation and are important mediators of insulitis. Activation of the pro-apoptotic receptor Fas leads to apoptosis-mediated death of the Fas-expressing cell. The pro-inflammatory cytokines IL-1beta and IFN-gamma regulate the transcription of genes encoding the Fas receptor and several chemokines. We have previously shown that suppressor of cytokine signalling (SOCS)-3 inhibits IL-1beta- and IFN-gamma-induced nitric oxide production in a beta cell line. The aim of this study was to investigate whether SOCS-3 can influence cytokine-induced Fas and chemokine expression in beta cells. METHODS: Using a beta cell line with inducible Socs3 expression or primary neonatal rat islet cells transduced with a Socs3-encoding adenovirus, we employed real-time RT-PCR analysis to investigate whether SOCS-3 affects cytokine-induced chemokine and Fas mRNA expression. The ability of SOCS-3 to influence the activity of cytokine-responsive Fas and Mcp-1 (also known as Ccl2) promoters was measured by reporter analysis. RESULTS: IL-1beta induced a time-dependent increase in Mcp-1 and Mip-2 (also known as Cxcl2) mRNA expression after 6 h of stimulation in insulinoma (INS)-1 and neonatal rat islet cells. This induction was inhibited when Socs3 was expressed in the cells. In INS-1 cells, IL-1beta + IFN-gamma induced a tenfold and eightfold increase of Fas mRNA expression after 6 and 24 h, respectively. This induction was inhibited at both time-points when expression of Socs3 was induced. In promoter studies SOCS-3 significantly inhibited the cytokine-induced activity of Mcp-1 and Fas promoter constructs. CONCLUSIONS/INTERPRETATION: SOCS-3 inhibits the expression of cytokine-induced chemokine and death-receptor Fas mRNA.

Functional SOCS1 polymorphisms are associated with variation in obesity in whites.

AIMS/HYPOTHESIS: The suppressor of cytokine signalling 1 (SOCS1) is a natural inhibitor of cytokine and insulin signalling pathways and may also play a role in obesity. In addition, SOCS1 is considered a candidate gene in the pathogenesis of both type 1 diabetes (T1D) and type 2 diabetes (T2D). The objective was to perform mutation analysis of SOCS1 and to test the identified variations for association to T2D-related quantitative traits, T2D or T1D. METHODS: Mutation scanning was performed by direct sequencing in 27 white Danish subjects. Genotyping was carried out by TaqMan allelic discrimination. A total of more than 8100 individuals were genotyped. RESULTS: Eight variations were identified in the 5' untranslated region (UTR) region. Two of these had allele frequencies below 1% and were not further examined. The six other variants were analysed in groups of T1D families (n = 1461 subjects) and T2D patients (n = 1430), glucose tolerant first-degree relatives of T2D patients (n = 212) and normal glucose tolerant (NGT) subjects. The rs33977706 polymorphism (-820G > T) was associated with a lower body mass index (BMI) (p = 0.004). In a second study (n = 4625 NGT subjects), significant associations of both the rs33977706 and the rs243330 (-1656G > A) variants to obesity were found (p = 0.047 and p = 0.015) respectively. The rs33977706 affected both binding of a nuclear protein to and the transcriptional activity of the SOCS1 promoter, indicating a relationship between this polymorphism and gene regulation. CONCLUSIONS/INTERPRETATION: This study demonstrates that functional variations in the SOCS1 promoter may associate with alterations in BMI in the general white population.

Regulation of beta-cell mass by hormones and growth factors.

Substantial new information has accumulated on molecular mechanisms of pancreas development, regulation of beta-cell gene expression, and the role of growth factors in the differentiation, growth, and regeneration of beta-cells. The present review focuses on some recent studies on the mechanism of action of cytokines such as growth hormone (GH) and prolactin (PRL) in beta-cell proliferation and gene expression-in particular, the role of signal transducers and activators of transcription (STAT) proteins. The implication of the discovery of suppressors of cytokine signaling (SOCS) proteins for the interaction between stimulatory and inhibitory cytokines, including GH, PRL, leptin, and the proinflammatory cytokines interleukin-1 and interferon-gamma, in beta-cell survival is not yet clear. Recent studies indicate a role of cell adhesion molecules and the delta-like protein preadipocyte factor 1/fetal antigen 1 (Pref-1/FA-1) in cytokine-induced beta-cell growth and development. Surprisingly, glucagon-like peptide-1 (GLP-1) was recently found to stimulate not only insulin secretion but also beta-cell replication and differentiation, which may present a new perspective in treatment of type 2 diabetes. Together with the intriguing reports on positive effects of insulin on both beta-cell growth and function, a picture is emerging of an integrated network of signaling events acting in concert to control beta-cell mass adaptation to insulin demand.

Mechanism of protein tyrosine phosphatase 1B-mediated inhibition of leptin signalling.

Upon leptin binding, the leptin receptor is activated, leading to stimulation of the JAK/STAT signal transduction cascade. The transient character of the tyrosine phosphorylation of JAK2 and STAT3 suggests the involvement of protein tyrosine phosphatases (PTPs) as negative regulators of this signalling pathway. Specifically, recent evidence has suggested that PTP1B might be a key regulator of leptin signalling, based on the resistance to diet-induced obesity and increased leptin signalling observed in PTP1B-deficient mice. The present study was undertaken to investigate the mechanism by which PTP1B mediates the cessation of the leptin signal transduction. Leptin-induced activation of a STAT3 responsive reporter was dose-dependently inhibited by co-transfection with PTP1B. No inhibition was observed when a catalytically inactive mutant of PTP1B was used or when other PTPs were co-transfected. PTP1B was able to dephosphorylate activated JAK2 and STAT3 in vitro, whereas either no or a minimal effect was observed with cluster of differentiation 45 (CD45), PTPalpha and leukocyte antigen-related (LAR). By utilisation of a selective PTP1B inhibitor, the leptin-induced STAT3 activation was enhanced in cells. In conclusion, these results suggested that the negative regulatory role of PTP1B on leptin signalling is mediated through a direct and selective dephosphorylation of the two signalling molecules, JAK2 and STAT3.

Regulation of pancreatic beta-cell mass and proliferation by SOCS-3.

Growth hormone and prolactin are important growth factors for pancreatic beta-cells. The effects exerted by these hormones on proliferation and on insulin synthesis and secretion in beta-cells are largely mediated through the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway. Suppressors of cytokine signaling (SOCS) proteins are specific inhibitors of the JAK/STAT pathway acting through a negative-feedback loop. To investigate in vivo effects of SOCS-3 in growth hormone (GH)/prolactin signaling in beta-cells we generated transgenic mice with beta-cell-specific overexpression of SOCS-3. The relative beta-cell proliferation and volume in the mice were measured by morphometry. Beta-cell volume of transgenic female mice was reduced by over 30% compared with beta-cell volume in wild-type female mice. Stimulation of transgenic islets in vitro with GH showed a reduced tyrosine phosphorylation of STAT-5 when compared with wild-type islets. Transduction of primary islet cultures with adenoviruses expressing various SOCS proteins followed by stimulation with GH or glucagon-like peptide-1 (GLP-1) revealed that SOCS-3 inhibited GH- but not GLP-1-mediated islet cell proliferation, indicating that the decreased beta-cell volume observed in female transgenic mice could be caused by an inhibition of GH-induced beta-cell proliferation by SOCS-3. In spite of the reduced beta-cell volume the transgenic female mice exhibited enhanced glucose tolerance compared with wild-type littermates following an oral glucose-tolerance test. Together these data suggest that SOCS-3 modulates cytokine signaling in pancreatic beta-cells and therefore potentially could be a candidate target for development of new treatment strategies for diabetes.

Beta cell proliferation and growth factors.

Formation of new beta cells can take place by two pathways: replication of already differentiated beta cells or neogenesis from putative islet stem cells. Under physiological conditions both processes are most pronounced during the fetal and neonatal development of the pancreas. In adulthood little increase in the beta cell number seems to occur. In pregnancy, however, a marked hyperplasia of the beta cells is observed both in rodents and man. Increased mitotic activity has been seen both in vivo and in vitro in islets exposed to placental lactogen (PL), prolactin (PRL) and growth hormone (GH). Receptors for both GH and PRL are expressed in islet cells and are upregulated during pregnancy. By mutational analysis we have identified different functional domains of the cytoplasmic part of the GH receptor. Thus the mitotic signaling only requires the membrane proximal part of the receptor and activation of the tyrosine kinase JAK2 and the transcription factors STAT1 and 3. The activation of the insulin gene however also requires the distal part of the receptor and activation of calcium uptake and STAT5. In order to identify putative autocrine growth factors or targets for growth factors we have cloned a novel GH/PRL stimulated rat islet gene product, Pref-1 (preadipocyte factor-1). This protein contains six EGF-like motifs and may play a role both in embryonic pancreas differentiation and in beta cell growth and function. In summary, the increasing knowledge about the mechanisms involved in beta cell differentiation and proliferation may lead to new ways of forming beta cells for treatment of diabetes in man.

Mutation of the SHP-2 binding site in growth hormone (GH) receptor prolongs GH-promoted tyrosyl phosphorylation of GH receptor, JAK2, and STAT5B.

Binding of GH to GH receptor (GHR) rapidly and transiently activates multiple signal transduction pathways that contribute to the growth-promoting and metabolic effects of GH. While the events that initiate GH signal transduction, such as activation of the Janus tyrosine kinase JAK2, are beginning to be understood, the signaling events that terminate GH signaling, such as dephosphorylation of tyrosyl-phosphorylated signaling molecules, are poorly understood. In this report, we examine the role of the SH2 (Src homology-2) domain-containing protein tyrosine phosphatase SHP-2 in GH signaling. We demonstrate that the SH2 domains of SHP-2 bind directly to tyrosyl phosphorylated GHR from GH-treated cells. Tyrosine-to-phenylalanine mutation of tyrosine 595 of rat GHR greatly diminishes association of the SH2 domains of SHP-2 with GHR, and tyrosine-to-phenylalanine mutation of tyrosine 487 partially reduces association of the SH2 domains of SHP-2 with GHR. Mutation of tyrosine 595 dramatically prolongs the duration of tyrosyl phosphorylation of the signal transducer and activator of transcription STAT5B in response to GH, while mutation of tyrosine 487 moderately prolongs the duration of STAT5B tyrosyl phosphorylation. Consistent with the effects on STAT5B phosphorylation, tyrosine-to-phenylalanine mutation of tyrosine 595 prolongs the duration of tyrosyl phosphorylation of GHR and JAK2. These data suggest that tyrosine 595 is a major site of interaction of GHR with SHP-2, and that GHR-bound SHP-2 negatively regulates GHR/JAK2 and STAT5B signaling.

Growth hormone-releasing factor induces c-fos expression in cultured primary pituitary cells.

GH-releasing factor (GRF) and somatostatin regulates the secretion and biosynthesis of GH as well as the proliferation of GH-producing cells. In order to further characterize the mitogenic effect of GRF, we studied the expression of the proto-oncogene c-fos in primary pituitary cells. Maximal induction of c-fos mRNA was observed 20-60 min after stimulation with 5 nM GRF, returning to basal levels after 2 h. Somatostatin-14 (5 nM) partially inhibited the GRF induced c-fos expression. Forskolin and phorbol 12, 13 dibutyrate induced c-fos gene in cultured primary pituitary cells with similar kinetics. Transcription of the fos gene was accompanied by biosynthesis of the fos protein. Indirect immunofluorescence using a fos specific antibody, showed exclusive nuclear localization of the fos protein. These data demonstrate that GRF and somatostatin, in addition to regulating GH secretion and somatotroph proliferation, can also regulate the expression of c-fos proto-oncogene in primary somatotrophs.

Inhibition of somatotroph growth and growth hormone biosynthesis by activin in vitro.

Activin-A, a homodimeric protein composed of two inhibin beta A-subunits, was first isolated from gonadal fluids based upon its ability to stimulate FSH secretion and biosynthesis, but was also observed to suppress GH secretion. The present report describes the effects of activin on the biosynthesis of GH and the proliferation of pituitary somatotrophs. In pituitary cells cultured in the presence of 0.7 nM activin for 3 days, GH secretion was decreased by 50% compared to the control value. Inhibition of GH biosynthesis, measured by quantitative immunoprecipitation of [35S]methionine-labeled cells, could be observed after 24 h of activin treatment, and maximal (70%) inhibition of GH biosynthesis was observed after 3 days. Activin inhibited basal as well as GH-releasing factor (GRF)-, glucocorticoid-, and thyroid hormone-stimulated GH biosynthesis. Inhibin, which is known to reverse the effect of activin on FSH secretion, did not reverse the effect of activin on GH biosynthesis. Treatment of somatotrophs with activin for 3 days completely inhibited the growth-promoting effect of GRF on somatotrophs. However, no effect of activin on GRF-stimulated expression of the c-fos protooncogene was observed. These data demonstrate that activin, in addition to its stimulatory effect on FSH secretion, is able to inhibit both expression of GH and growth of somatotropic cells.

Identification of a growth hormone-responsive STAT5-binding element in the rat insulin 1 gene.

GH and PRL stimulate both proliferation and insulin production in pancreatic beta-cells as well as in the rat insulinoma cell line RIN-5AH, We report here that human GH increases insulin mRNA levels in RIN-5AH cells via both somatogenic and lactogenic receptors. GH stimulated the rat insulin 1 promoter activity 2-fold, and this stimulation was abolished by introduction of a block mutation in a gamma-interferon-activated sequence (GAS)-like element (GLE) with the sequence 5'-TTCTGGGAA-3' located in the rat insulin 1 enhancer at position -330 to -322. This element, termed Ins-GLE, was able to confer GH responsiveness to a heterologous promoter. GH induced the binding of two protein complexes to the Ins-GLE. An antibody directed against the transcription factor STAT5 (signal transducer and activator of transcription) supershifted the GH-induced complexes. Furthermore, in COS7 cells transiently transfected with STAT5 and GH receptor cDNAs, it was found that expression of STAT5 was necessary for GH induction of these two DNA-binding complexes. These results suggest that GH stimulates insulin 1 promoter activity by inducing the binding of STAT5 to Ins-GLE.

The role of the growth hormone (GH) receptor and JAK1 and JAK2 kinases in the activation of Stats 1, 3, and 5 by GH.

GH has been shown to activate the GH receptor (GHR)-associated tyrosine kinase JAK2 and the Src homology 2 domain-containing transcription factors Stats (signal transducers and activators of transcription) 1, 3, and 5. The present work investigates the role of GHR and JAK2 in the activation of Stats 1, 3, and 5 by GH. The ability of GH to stimulate the tyrosyl phosphorylation of these Stats was assessed in Chinese hamster ovary (CHO) cells expressing truncated and mutated GHR. GH was observed to stimulate tyrosyl phosphorylation of Stats 1, 3, and 5 in CHO cells expressing GHRs that bind JAK2 [GHR1-638 (full-length) and GHR1-454 (lacks approximately half of the cytoplasmic domain)] but not in CHO cells expressing GHR that do not bind JAK2 (GHR1-318 or GHR1-294). GH-dependent tyrosyl phosphorylation of Stat5, but not Stats 1 or 3, was reduced in CHO cells expressing GHR1-454. GH-dependent tyrosyl phosphorylation of Stats 3 and 5 was severely reduced and undetectable for Stat1 in cells expressing GHR1-454 in which tyrosines 333 and 338 (the only tyrosines phosphorylated within 1-454) are mutated to phenylalanine (GHR1-454Y333, 338F). However, GH-dependent phosphorylation of Stats 1, 3, and 5 was observed in cells expressing full-length GHR in which tyrosines 333 and 338 are mutated to phenylalanine (GHR1-638Y333, 338F) GH, whose receptor lacks previously defined Stat1- or Stat3-binding sites, was found in 3T3-F442A fibroblasts and 2fTGH-GHR cells to stimulate tyrosyl phosphorylation of JAK2 to a substantially greater extent than, and JAK1 to a similar extent as, leukemia inhibitory factor (LIF) and/or interferon gamma (IFN gamma), ligands whose receptors contains Stat3- and Stat1-binding sites and activate Stat3 and Stat1, respectively, better than GH. These findings suggest that: 1) JAK2 is required for GH-dependent phosphorylation of Stats 1, 3, and 5; 2) tyrosines 333 and/or 338 are required for maximal tyrosyl phosphorylation of Stats 1, 3, and 5; 3) Stat5 binds to a phosphorylated tyrosine(s) within amino acids 454-638 in addition to tyrosines 333 and/or 338; 4) GH stimulates tyrosyl phosphorylation of JAK1 in addition to JAK2 with JAK2 having a much greater response; 5) some Stat3 and Stat5 (and possibly Stat1) may bind to nonphosphorylated amino acids in GHR or to phosphorylated tyrosines in proteins that bind to GHR (e.g. JAK22) to be maximally activated; and 6) if JAK2, which contains Stat3-binding motifs, does serve as a docking site for some Stat proteins, Stat-JAK2 binding is likely to be more important for GH than LIF or IFN gamma in 3T3-F442A cells since GH induces 15 times more tyrosyl-phosphorylated JAK2 than LIF or IFN gamma.

Characterization of the inhibitory effect of growth hormone on primary preadipocyte differentiation.

GH exerts adipogenic activity in several preadipocyte cell lines, whereas in primary rat preadipocytes, GH has an antiadipogenic activity. To better understand the molecular mechanism involved in adipocyte differentiation, the expression of adipocyte-specific genes was analyzed in differentiating preadipocytes in response to GH. We found that the expression of both adipocyte determination and differentiation factor 1 (ADD1) and peroxisome proliferator activated receptor gamma(PPARgamma) was induced in preadipocytes during differentiation. In the presence of GH, which markedly inhibited triglyceride accumulation, no reduction in the expression level of ADD1 was observed in response to GH, whereas there was a 50% reduction in the expression of PPARgamma. The DNA binding activity of the PPARgamma/retinoid X receptor-alpha(RXRalpha) to the ARE7 element from the aP2 gene was also reduced by approximately 50% in response to GH. GH inhibited the expression of late markers of adipocyte differentiation, fatty acid synthase, aP2, and hormone-sensitive lipase by 70-80%. The antiadipogenic effect of GH was not affected by the mitogen-activated protein (MAP) kinase/ extracellular-regulated protein (ERK) kinase inhibitor PD 98059, indicating that the mitogen-activated protein kinase pathway was not involved in GH inhibition of preadipocyte differentiation. The expression of preadipocyte factor-1/fetal antigen 1 was decreased during differentiation, and GH treatment prevented this down-regulation of Pref1/FA1. A possible role for Pref-1/FA1 in mediating the antiadipogenic effect of GH was indicated by the observation that FA1 inhibited differentiation as effectively as GH. These data suggest that GH exerts its inhibitory activity in adipocyte differentiation at a step after the induction of ADD1 but before the induction of genes required for terminal differentiation.

Mechanism of inhibition of growth hormone receptor signaling by suppressor of cytokine signaling proteins.

In this study we have investigated the role of suppressor of cytokine signaling (SOCS) proteins in GH receptor-mediated signaling. GH-induced transcription was inhibited by SOCS-1 and SOCS-3, while SOCS-2 and cytokine inducible SH2-containing protein (CIS) had no effect By using chimeric SOCS proteins it was found that the ability of SOCS proteins to inhibit GH-mediated transcription was located in the amino-terminal 40-80 amino acids. In SOCS-3, 46 amino acids C-terminal to the SH2 domain were required for the inhibitory activity, while a truncated SOCS-1 having only 2 amino acids C-terminal to the SH2 domain was able to inhibit GH-mediated transcription. Both SOCS-1 and SOCS-3 were able to inhibit GH-induced STAT5 (signal transducer and activator of transcription) activation. SOCS-1 inhibited the tyrosine kinase activity of Janus kinase 2 (JAK2) directly, while SOCS-3 only inhibited JAK2 when stimulated by the GH receptor. All four SOCS proteins were able to bind to a tyrosine-phosphorylated glutathione-S-transferase-GH receptor fusion protein, and SOCS-3 required the same 46 C-terminal amino acids for GH receptor binding as it did for inhibition of GH-mediated transcription and STAT5 activation. These data suggest that SOCS-1 and -3 can suppress GH-induced transcriptional activity, presumably by inhibiting the kinase activity of JAK2 either directly in the case of SOCS-1 or via binding to the tyrosine-phosphorylated GH receptor in the case of SOCS-3.

Multiple growth hormone-binding proteins are expressed on insulin-producing cells.

The insulin-producing rat islet tumor cell line, RIN-5AH, expresses somatogen binding sites and responds to GH by increased proliferation and insulin production. Affinity cross-linking shows that RIN-5AH cells contain two major GH-binding subunits of Mr 100-130K (110K), which appear to exist as disulfide-linked multimers of Mr 270-350K (300K). In addition, a minor Mr 180K GH-binding protein is identified which does not appear to be associated with other proteins by disulfide bridges. A plasma membrane-enriched fraction accounts for 86% of the RIN-cell GH-binding activity while cytosol and intracellular organelles are low in GH-binding activity. The plasma membrane-bound activity is soluble in Triton X-100 with intact hormone binding characteristics. The apparent KD in detergent solution is estimated to 18 ng/ml (8 x 10(-10) M). 125I-hGH-affinity cross-linking to intact and detergent-solubilized membranes as well as hGH-affinity purified protein reveals labeled proteins of Mr 180K and Mr 285-350K. In contrast to the cross-linked Mr 300K complexes of intact cells those of disintegrated cellular material are resistant to reduction with dithiothreitol, and it is speculated that this is due to intersubunit cross-linking of the disulfide-linked Mr 110K GH-binding subunits. The GH-binding proteins are purified approximately 100-fold by one cycle of hGH-affinity chromatography and five major proteins of Mr 180K, 94K, 86K, 64K, and 54K are identified by silver staining in the purified fraction. It is concluded that the RIN-5AH cells have multiple GH-binding proteins which may mediate signals for either proliferation and/or insulin production.

Growth hormone is a growth factor for the differentiated pancreatic beta-cell.

The regulation of the growth of the pancreatic beta-cell is poorly understood. There are previous indications of a role of GH in the growth and insulin production of the pancreatic islets. In the present study we present evidence for a direct long-term effect of GH on proliferation and insulin biosynthesis of pancreatic beta-cells in monolayer culture. In culture medium RPMI 1640 supplemented with 2% normal human serum islets or dissociated islet cells from newborn rats maintained their insulin-producing capacity. When supplemented with 1-1000 ng/ml pituitary or recombinant human GH the islet cells attached, spread out, and proliferated into monolayers mainly consisting of insulin-containing cells. The number of beta-cells in S-phase was increased from 0.9-6.5% as determined by immunochemical staining of bromodeoxyuridine incorporated into insulin-positive cells. The increase in cell number was accompanied with a continuous increase in insulin release to the culture medium reaching a 10- 20-fold increase after 2-3 months with a half-maximal effect at about 10 ng/ml human GH. The biosynthesis of (pro)insulin was markedly increased with a normal rate of conversion of proinsulin to insulin. It is concluded that GH is a potent growth factor for the differentiated pancreatic beta-cell.

Growth hormone binding to specific receptors stimulates growth and function of cloned insulin-producing rat insulinoma RIN-5AH cells.

Binding of 125I-labeled human GH (hGH) to a cloned rat insulin-producing cell line RIN-5AH in monolayer culture was studied along with some physiological effects of the hormone on these cells. Binding was time and temperature dependent, and steady state binding was observed in 60 min at 37 C with [125I]hGH at 4.2 pM, whereas at 24 C, binding had not reached a steady state after 120 min. The binding was largely reversible, since 80% of initially bound [125I]hGH dissociated from the cells upon incubation in hGH-free buffer for 120 min. Half-maximal binding was obtained when cells were incubated in the presence of 3.0 X 10(-10) M unlabeled hGH. Rat GH as well as human placental lactogen were able to compete for binding sites, but with less affinity. Other non-GH peptides at 6.7 micrograms/ml did not affect [125I]hGH binding. Scatchard analysis revealed curvilinear plots, and approximately 2700 high affinity binding sites were calculated. Culture of RIN-5AH in the presence of 1 microgram/ml hGH for 4 days resulted in an 80% increase in insulin content as well as an 18% increase in cell number and DNA and protein content compared to those in cells cultured in the absence of hGH. The dose dependence of the insulinotropic effect showed that half-maximal and maximal stimulation were observed in cells cultured in the presence of 10 and 100 ng/ml, respectively. Insulin release to the medium during the 4-day culture period was not affected by hGH. These data suggest that GH, through binding to specific receptors in the cell membrane, directly stimulates proliferation and function of pancreatic beta-cells.

The stimulatory effect of growth hormone, prolactin, and placental lactogen on beta-cell proliferation is not mediated by insulin-like growth factor-I.

The effects of GH, PRL, and placental lactogen (PL) on the proliferation of pancreatic beta-cells in vitro were studied as well as the possible effect of insulin-like growth factor-I (IGF-I) in mediating this effect. Proliferating beta-cells were identified by staining with a monoclonal antibody to bromodeoxyuridine (BrdU) after cells were incubated for 1 h in the presence of 10 microM BrdU. By double staining with insulin antibodies it was found that 6.3% of the beta-cells had incorporated BrdU when cultured for 7 days in the presence of 1 microgram/ml human GH (hGH) compared to 0.6% when cultured in the absence of hGH. Similar results were obtained using rat GH. The half-maximal effect of hGH on beta-cell proliferation was observed at 10 ng/ml, and the maximal effect at 100 ng/ml. Islet cells cultured in the presence of PRL or PL caused a dose-dependent increase in beta-cell proliferation similar to that caused by hGH. GH, PRL, and PL had no effect on the proliferation of glucagon- or somatostatin-producing cells. The addition of 100 ng/ml IGF-I to either control or GH-stimulated islet cells did not affect the labeling index. When GH-stimulated proliferation of beta-cells was measured in the presence of neutralizing concentrations of a rabbit IGF-I antiserum, the percentage of beta-cells incorporating BrdU was unaffected. Using Northern blot analysis, no IGF-I transcripts could be detected in RNA from GH-stimulated islets, whereas IGF-I transcripts were readily detected in RNA isolated from rat liver tissue. These data suggest that the stimulatory effect of GH, PRL, and PL on beta-cell proliferation is not mediated by IGF-I, but, rather, is a direct mitogenic effect on the beta-cell.