Nobuo Sekine

Tyrosine phosphorylation of the EGF receptor by the kinase Jak2 is induced by growth hormone

When growth hormone binds to its receptor, which belongs to the cytokine receptor superfamily, it activates the Janus kinase Jak2 which has tyrosine-kinase activity and initiates an activation of several key intracellular proteins (for example, mitogen-activated protein (MAP) kinases) that eventually execute the biological actions induced by growth hormone, including the expression of particular genes. In contrast to receptors that themselves have tyrosine kinase activity, the signalling pathways leading to MAP kinase activation, that are triggered by growth hormone are poorly understood, but appear to be mediated by the proteins Grb2 and Shc. We now show that growth hormone stimulates tyrosine phosphorylation of the receptor for epidermal growth factor (EGFR) and its association with Grb2 and at the same time stimulates MAP kinase activity in liver, an important target tissue of growth hormone. Expression of EGFR and its mutants revealed that growth-hormone-induced activation of MAP kinase and expression of the transcription factor c-fos requires phosphorylation of tyrosines on EGFR, but not its own intrinsic tyrosine-kinase activity. Moreover, tyrosine at residue 1,068 of the EGFR is proposed to be one of the principal phosphorylation sites and Grb2-binding sites stimulated by growth hormone via Jak2. Our results indicate that the role of EGFR in signalling by growth hormone is to be phosphorylated by Jak2, thereby providing docking sites for Grb2 and activating MAP kinases and gene expression, independently of the intrinsic tyrosine kinase activity of EGFR. This may represent a novel cross-talk pathway between the cytokine receptor superfamily and growth factor receptor.

Free radical modulation of insulin release in INS-1 cells exposed to alloxan.

Generation of free radicals is thought to mediate the cytotoxic action of alloxan on the pancreatic beta-cell. In this investigation, the early effects of alloxan on cell function were studied. When INS-1D insulinoma cells were exposed to alloxan (1 mM) for 45 min followed by a 3-hr recovery period, the drug increased basal insulin release while abolishing the effect of glucose in static incubations. This was associated with impaired stimulation of cellular metabolism by glucose and reduced viability, both monitored colorimetrically with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). These alterations were largely counteracted by the antioxidant butylated hydroxyanisol (BHA). Similar changes occurred when glucose was added directly after 5 min of alloxan treatment, whereas KCl-induced secretion was only partially inhibited. In perifusion, alloxan caused transient insulin secretion to 50% of the rates obtained with glucose 30 min later. Under these conditions, epinephrine abolished the stimulation due to both agents. Membrane potential and cytosolic calcium concentrations ([Ca2+]i) were recorded to clarify the action of alloxan. Alloxan-induced insulin release correlated with depolarization of INS-1D cells and a rise in [Ca2+]i. Alloxan did not augment [Ca2+]i in the presence of BHA or the absence of extracellular calcium. Nickel chloride blocked the effect of alloxan on [Ca2+]i, whereas verapamil was ineffective. This suggests that alloxan promotes Ca2+ influx through channels distinct from L-type channels, perhaps through non-selective cation channels. Thus, alloxan causes changes in INS-1D cells prevented by antioxidant treatment, suggesting that free radicals may modulate the ionic permeability leading to functional activation.

Synergistic activation of NF-?b and inducible isoform of nitric oxide synthase induction by interferon-? and tumor necrosis factor-? in INS-1 cells

Interferon‐γ (IFN‐γ) is known to exert deleterious effects on pancreatic β‐cells and is implicated in the development of type 1 (autoimmune) diabetes mellitus. In this study, we investigated signaling mechanisms mediating the effects of IFN‐γ in pancreatic β‐cells using a differentiated rat insulin‐secreting cell line, INS‐1, with special reference to the activation of transcription factors STAT (signal transducers and activators of transcription)1 and NF‐κB. Exposure of INS‐1 cells to 100 IU/ml IFN‐γ for 24 h resulted in significant inhibition of nutrient‐induced insulin secretion associated with impaired metabolism. In combination with tumor necrosis factor‐α (TNF‐α) (50 ng/ml), IFN‐γ elicited severe cytotoxicity and induced the expression of the inducible isoform of nitric oxide synthase (iNOS) mRNA. IFN‐γ promoted tyrosine phosphorylation and DNA‐binding of STAT1 through Janus kinase (JAK)1 activation without apparent phosphorylation of JAK2. TNF‐α did not affect STAT1 activation, but stimulated DNA‐binding and transcriptional activity of NF‐κB, both of which were further increased by IFN‐γ. These effects of IFN‐γ and TNF‐α seem physiologically relevant, because either inhibition of STAT1 by the tyrosine kinase inhibitor herbimycin A or that of NF‐κB by sulfasalazine resulted in the reduction of iNOS mRNA expression. In conclusion, IFN‐γ activates STAT1 and potentiates TNF‐α–induced NF‐κB activation in INS‐1 cells, thereby inducing iNOS and cell destruction. J. Cell. Physiol. 184:46–57, 2000.

Activin A increases intracellular free calcium concentrations in rat pancreatic islets

Activin A stimulated insulin secretion in rat pancreatic islets, an effect that was attenuated by reduction of extracellular Ca2+ and abolished by either nitrendipine or verapamil. Activin A increased intracellular the free Ca2+ concentration, [Ca2+]i in fura‐2‐loaded islets. Activin A‐mediated elevation of [Ca2+]i was abolished by the reduction of extracellular Ca2+ or the addition of nifedipine. In addition, activin A did not increase [Ca2+]i in the presence of diazoxide, an opener of ATP‐sensitive K+ channels. These results suggest that activin A increases insulin secretion by stimulating Ca2+ entry.

Glucose-Induced Insulin Secretion in INS-1 Cells Depends on Factors Present in Fetal Calf Serum and Rat Islet–Conditioned Medium

To study the regulation of growth and differentiated function of insulin-secreting cells, the rat insulinoma cell line INS-1 was cultured in a defined serum-free medium containing prolactin, IGF-I, and triiodothyronine, which was originally reported to maintain insulin secretion of islet cells. Growth and viability, as well as cellular insulin content of INS-1 cells in the defined medium, were comparable to the control cells cultured in the complete medium containing 10% fetal calf serum. However, after a 3-day culture in this medium, insulin secretion in response to glucose, pyruvate, and leucine was markedly blunted compared with the control cells (−78, −68, and −56%, respectively), whereas the response to 30 mmol/1 K+ was only slightly decreased. In these cells: 1) nutrient metabolism assessed by tetrazolium salt reduction was reduced in response to pyruvate and leucine, which are mainly metabolized in the mitochondria; 2) oxidation of both [3,4-14C]glucose and [1-14C]pyruvate was decreased (−22 and −32%, respectively); 3) glucose failed to depolarize the membrane potential, whereas tolbutamide was fully active; 4) video imaging analysis of cytosolic Ca2+ showed a decrease in the population of glucose-responsive cells, while the response to 30 mmol/1 K+ was preserved; 5) serum replenishment for 3 days restored glucose-induced insulin secretion. Interestingly, conditioned serum-free medium from rat islets maintained the insulin secretory function of INS-1 cells, although glucagon, somatostatin, and some other factors failed to restore the function. In contrast, conditioned media from HepG2, PC12, and human umbilical vein endothelial cells did not substitute for serum. Thus, the impaired insulin secretion of the cells cultured in the defined medium is best explained by defective mito-chondrial metabolism. Islet cells, but not INS-1 cells, produce factors required for normal signal generation by nutrient secretagogues.

GH Inhibits Interferon-γ-Induced Signal Transducer and Activator of Transcription-1 Activation and Expression of the Inducible Isoform of Nitric Oxide Synthase in INS-1 Cells

Interferon-γ and TNFα synergistically induce the inducible isoform of nitric oxide synthase and elicit severe cytotoxicity in pancreatic β-cells. We demonstrate here that GH, the well knownβ -cell mitogen, inhibits nitric oxide production by reducing inducible nitric oxide synthase gene induction by the two cytokines and counteracts their cytotoxic effect in insulin-secreting INS-1 cells. To elucidate the underlying mechanism, we examined activation of the transcription factors implicated in the induction of inducible nitric oxide synthase, signal transducer and activator of transcription-1, and nuclear factor-κB. GH inhibited tyrosine phosphorylation and DNA binding of signal transducer and activator of transcription-1 promoted by interferon-γ, whereas nuclear factor-κB activation by TNFα was not affected by GH. GH was found to induce suppressor of cytokine signaling-1 and -3, both of which are able to inhibit interferon-γ activation of signal transducer and activator of transcription-1, suggesting that ...

Roles of CTPL/Sfxn3 and Sfxn family members in pancreatic islet

Pancreatic AR42J cells have the feature of pluripotency of the precursor cells of the gut endoderm. Betacellulin (BTC) and activin A (Act) convert them into insulin‐secreting cells. Using mRNA differential display techniques, we have identified a novel mitochondrial transporter, which is highly expressed during the course of differentiation, and have designated it citrate transporter protein‐like protein (CTPL). Recently sideroflexin 1 (Sfxn1) was shown to be a susceptible gene of flexed‐tail (f/f) mice, and CTPL has turned out to be a rat orthologous protein of Sfxn3, a member of sideroflexin family. CTPL/Sfxn3 was targeted to mitochondrial membrane like Sfxn1. The expression levels of CTPL/Sfxn3, Sfxn2, and Sfxn5 were upregulated in the early phase of differentiation into insulin‐secreting cells but the expression levels of Sfxn1 and Sfxn3 did not change. All Sfxn family members were expressed in rat pancreatic islet. The expression levels of CTPL/Sfxn3, Sfxn2, and Sfxn5 were also upregulated in islets of streptozotocin‐induced diabetic rats compared to normal rats. The downregulation of CTPL/Sfxn3 in a rat insulinoma cell line, INS‐1, with the antisense oligonucleotide did not affect the insulin secretion. Taken together, CTPL/Sfxn3 and some other family members might be important in the differentiation of pancreatic β‐cells as a channel or a carrier molecule and be related to the regeneration of pancreatic endocrine cells.

Bimodal effect of transforming growth factor-β on insulin secretion in MIN6 cells

The effects of transforming growth factor-beta (TGF-beta) on insulin secretion were investigated using a glucose-responsive clonal cell line, MIN6. One hundred pM TGF-beta stimulated insulin release during 0.5-24 h of incubation in the presence of 5.5 mM glucose, but not after 48 h; 1 nM TGF-beta also stimulated insulin release up to 2 h of exposure, but the effect was not seen after 6 h of exposure. When cells were incubated with 25 mM glucose for 24 h, 100 pM TGF-beta significantly inhibited glucose-stimulated insulin release, whereas insulin release was not altered at 0 or 2.8 mM glucose. On the contrary, forskolin- (10 microM) and tolbutamide- (40 microM) induced insulin release were not affected by TGF-beta. TGF-beta affected neither the cell growth nor the cellular insulin content. An addition of 1 microM nitrendipine abolished TGF-beta-induced insulin secretion at 5.5 mM glucose. The presence study shows that TGF-beta exerts a bimodal effect on glucose-induced insulin secretion from MIN6 cells, depending on dose, time of exposure and concentrations of coexisting glucose. These effects might be mediated by the Ca(2+)-dependent mechanism.

Prostaglandin A1 enhances c-fos expression and activating protein-1 activity

Prostaglandin A1 (PGA1) increases heat shock element (HSE)-mediated transcription, thereby enhancing expression of HSE-bearing genes, including heat shock proteins. Because we recently found functional HSEs in the human and rodent c-fos promoters, we hypothesized that PGA1 might increase c-fos expression through the HSE. In this study, we revealed that PGA1 induces c-fos expression at least partly by increasing the binding between heat shock factor-1 and the HSE, and that PGA1 enhances activity of activating protein-1 (AP-1). Interestingly, so far as PGA, is present in the medium, AP-1-mediated transcription enhanced by PGA1 cannot be detected by the standard luciferase reporter gene assay. Instead, it can be detected by either checking luciferase mRNA levels in the presence of PGA1 or measuring luciferase activities just after removal of PGA1. These results showed that protein products of some stress-responsive genes can increase, not during the stressful condition, but immediately after recovery from stress.

Myocardial Injury without Electrocardiographic Changes after a Suicide Attempt by an Overdose of Glimepiride and Zolpidem: A Case Report and Literature Review.

A 40-year-old diabetic man was admitted to our hospital for poor glycemic control. During hospitalization, he took 42 mg glimepiride and 50 mg zolpidem as a suicide attempt. The following day, the creatine kinase-MB fraction and troponin I levels were elevated to 112 IU/L and 8.77 ng/mL, respectively, without any electrocardiographic abnormalities. The patient recovered completely without any complications. Four weeks later, coronary computed tomography angiography and myocardial perfusion scintigraphy revealed moderate one-vessel coronary disease without the evidence of myocardial ischemia or old infarction. Cardiac-specific markers must be considered in sulfonylurea-induced hypoglycemic patients, particularly when the patient is unconscious and does not exhibit any clinical manifestations.