Hiroyo Ota

Attenuation of glucose-induced insulin secretion by intermittent hypoxia via down-regulation of CD38

AIMS Sleep apnea syndrome (SAS) is characterized by recurrent episodes of oxygen desaturation during sleep, the development of daytime sleepiness, and deterioration in the quality of life. Accumulating evidence suggests the association of intermittent hypoxia (IH), a hallmark of SAS, and type 2 diabetes independently on body mass index and waist circumference. In addition to insulin resistance, the progression to type 2 diabetes is dependent on the impairment of glucose-induced insulin secretion (GIS) from pancreatic β-cells. However, the direct effects of IH on GIS are elusive. MAIN METHODS HIT-T15 hamster β-cells and isolated rat islets were exposed to 64 cycles/24 h of IH (5 min hypoxia/10 min normoxia) or normoxia for 24 h. Changes of GIS and gene expression in IH-treated β-cells were analyzed by ELISA and real-time RT-PCR, respectively. KEY FINDINGS After IH treatment, GIS both from IH-treated HIT-T15 cells and isolated rat islets were significantly attenuated. The level of insulin mRNA was unchanged by IH. The mRNA levels of glucose transporter 2 (Glut2), glucokinase (GK), sulfonylurea receptor1 (SUR1), and L-type Ca2+channel1.2 (Cav1.2) in IH-treated-islets were similar to those in normoxia-treated islets. In contrast, the mRNA level of CD38 in IH-treated islets was significantly lower than that in normoxia-treated islets. The reporter gene assay revealed that the transcription of CD38 was attenuated by IH, and the transfection of CD38 expression vector recovered the attenuation of GIS by IH. SIGNIFICANCE These results indicate that IH stress directly attenuates GIS from β-cells via the down-regulation of CD38.

A novel ryanodine receptor expressed in pancreatic islets by alternative splicing from type 2 ryanodine receptor gene

Cyclic ADP-ribose (cADPR), a potent Ca(2+) mobilizing intracellular messenger synthesized by CD38, regulates the opening of ryanodine receptors (RyRs). Increases in intracellular Ca(2+) concentrations in pancreatic islets, resulting from Ca(2+) mobilization from RyRs as well as Ca(2+) influx from extracellular sources, are important in insulin secretion by glucose. In the present study, by screening a rat islet cDNA library, we isolated a novel RyR cDNA (the islet-type RyR), which is generated from the RyR2 gene by alternative splicing of exons 4 and 75. When the expression vectors for the islet-type and the authentic RyRs were transfected into HEK293 cells, the islet-type RyR2 as well as the authentic one showed high affinity [(3)H]ryanodine binding. Intracellular Ca(2+) release in the islet-type RyR2-transfected cells was enhanced in the presence of cADPR but not in the authentic RyR2-transfected cells. The islet-type RyR2 mRNA was expressed in a variety of tissues such as in pancreatic islets, cerebrum, and cerebellum, whereas the authentic RyR2 mRNA was predominantly expressed in heart and aorta. These results suggest that the islet-type RyR2 may be an intracellular target for cADPR signaling.

Pancreatic β cell proliferation by intermittent hypoxia via up-regulation of Reg family genes and HGF gene

AIMS Although accumulating evidence suggests the associations between sleep apnea syndrome (SAS) and type 2 diabetes, the direct effect of intermittent hypoxia (IH) on pancreatic β cell proliferation remains a missing piece of the puzzle. MAIN METHODS Rat RINm5F β cells, hamster HIT-T15 β cells, and human 1.1B4 β cells were exposed to normoxia (21% O2, 5% CO2, and balance N2), to sustained hypoxia (SH: 1% O2, 5% CO2, and balance N2), or to intermittent hypoxia (IH: 64 cycles of 5 min SH and 10 min normoxia) for 24 h. After the treatment, cellular proliferation and apoptosis were measured by WST-8 assay and TUNEL method, respectively. The expression of regenerating gene (Reg) family, interleukin (IL)-6, and hepatocyte growth factor (HGF) was determined by real-time RT-PCR. KEY FINDINGS The cellular proliferation of HIT-T15, RINm5F and 1.1B4 cells by IH was significantly increased, whereas apoptosis of these cells was unchanged. Real-time RT-PCR revealed that the mRNA levels of Reg family genes, IL-6, a typical Reg family gene inducer, and HGF, an inhibitor of high-concentration of Reg protein-induced apoptosis, were increased in IH-treated cells. In addition, siRNAs against rat Reg family genes except for PAP I/Reg 2 attenuated IH-induced β cell proliferation. SIGNIFICANCE IH stress stimulates pancreatic β cell to induce IL-6 gene expression. By the IL-6 stimulation, β cells over-express Reg family genes as well as HGF gene. Reg family proteins stimulate β cell proliferation and HGF inhibits apoptosis of β cells. As a result, β cell numbers are increased by IH.

Synergistic activations of REG I α and REG I β promoters by IL-6 and Glucocorticoids through JAK/STAT pathway in human pancreatic β cells.

Reg (Regenerating gene) gene was originally isolated from rat regenerating islets and its encoding protein was revealed as an autocrine/paracrine growth factor for β cells. Rat Reg gene is activated in inflammatory conditions for β cell regeneration. In human, although five functional REG family genes (REG Iα, REG Iβ, REG III, HIP/PAP, and REG IV) were isolated, their expressions in β cells under inflammatory conditions remained unclear. In this study, we found that combined addition of IL-6 and dexamethasone (Dx) induced REG Iα and REG Iβ expression in human 1.1B4 β cells. Promoter assay revealed that a signal transducer and activator of transcription- (STAT-) binding site in each promoter of REG Iα (TGCCGGGAA) and REG Iβ (TGCCAGGAA) was essential for the IL-6+Dx-induced promoter activation. A Janus kinase 2 (JAK2) inhibitor significantly inhibited the IL-6+Dx-induced REG Iα and REG Iβ transcription. Electrophoretic mobility shift assay and chromatin immunoprecipitation revealed that IL-6+Dx stimulation increased STAT3 binding to the REG Iα promoter. Furthermore, small interfering RNA-mediated targeting of STAT3 blocked the IL-6+Dx-induced expression of REG Iα and REG Iβ. These results indicate that the expression of REG Iα and REG Iβ should be upregulated in human β cells under inflammatory conditions through the JAK/STAT pathway.

Intermittent hypoxia induces the proliferation of rat vascular smooth muscle cell with the increases in epidermal growth factor family and erbB2 receptor

Obstructive sleep apnea is characterized by intermittent hypoxia (IH), and associated with cardiovascular diseases, such as stroke and heart failure. These cardiovascular diseases have a relation to atherosclerosis marked by the proliferation of vascular smooth muscle cells (VSMCs). In this study, we investigated the influence of IH on cultured rat aortic smooth muscle cell (RASMC). The proliferation of RASMC was significantly increased by IH without changing the level of apoptosis. In order to see what induces RASMC proliferation, we investigated the influence of normoxia (N)-, IH- and sustained hypoxia (SH)-treated cell conditioned media on RASMC proliferation. IH-treated cell conditioned medium significantly increased RASMC proliferation compared with N-treated cell conditioned medium, but SH-treated cell conditioned medium did not. We next investigated the epidermal growth factor (EGF) family as autocrine growth factors. Among the EGF family, we found significant increases in mRNAs for epiregulin (ER), amphiregulin (AR) and neuregulin-1 (NRG1) in IH-treated cells and mature ER in IH-treated cell conditioned medium. We next investigated the changes in erbB family receptors that are receptors for ER, AR and NRG1, and found that erbB2 receptor mRNA and protein expressions were increased by IH, but not by SH. Phosphorylation of erbB2 receptor at Tyr-1248 that mediates intracellular signaling for several physiological effects including cell proliferation was increased by IH, but not by SH. In addition, inhibitor for erbB2 receptor suppressed IH-induced cell proliferation. These results provide the first demonstration that IH induces VSMC proliferation, and suggest that EGF family, such as ER, AR and NRG1, and erbB2 receptor could be involved in the IH-induced VSMC proliferation.

Prevention of Reg I-induced β-cell apoptosis by IL-6/dexamethasone through activation of HGF gene regulation.

Reg (regenerating gene) product, Reg protein, is induced in pancreatic β-cells and acts as autocrine/paracrine growth factor for regeneration via the cell surface Reg receptor. However, high concentrations of Reg I protein induced β-cell apoptosis. In the present study, we found that hepatocyte growth factor (HGF) attenuated the β-cell apoptosis induced by the high concentrations of Reg I protein and that the combined stimulation of interleukin-6 (IL-6) and dexamethasone (Dx) induced the accumulation of HGF mRNA as well as Reg I mRNA in β-cells. The accumulation of the HGF mRNA was caused by the activation of the HGF promoter. Deletion analysis revealed that the region of -96 to -92 of the HGF gene was responsible for the promoter activation by IL-6+Dx. The promoters contain a consensus transcription factor binding sequence for signal transducer and activator of transcription (STAT). Site-directed mutations of STAT-binding motif in the region markedly attenuated the HGF promoter activity. Chromatin immunoprecipitation assay showed that STAT3 is located at the active HGF promoter in response to IL-6+Dx stimulation. These results strongly suggest that the combined stimulation of IL-6 and glucocorticoids induces the activation of both Reg and HGF genes and that the anti-apoptotic effects of HGF against the Reg I-induced apoptosis may help β-cell regeneration by Reg I protein.

Human retinal pigment epithelial cell proliferation by the combined stimulation of hydroquinone and advanced glycation end-products via up-regulation of VEGF gene

Although recent research showed that advanced glycation endproduct (AGE) and hydroquinone (HQ) are related to the pathogenesis of age-related macular degeneration (AMD), the mechanism how AGE and HQ induce or accelerate AMD remains elusive. In the present study, we examined the effects of AGE and HQ on changes of human retinal pigment epithelial (RPE) cell numbers and found that the viable cell numbers were markedly reduced by HQ by apoptosis and that AGE prevented the decreases of HQ-treated cell numbers by increased replicative DNA synthesis of RPE cells without changing apoptosis. Real-time RT-PCR revealed that vascular endothelial growth factor (VEGF)-A mRNA was increased by HQ treatment and the addition of HQ+AGE resulted in a further increment. The increase of VEGF secretion was confirmed by ELISA, and inhibition of VEGF signaling by chemical inhibitors and small interfering RNA decreased the HQ+AGE-induced increases in RPE cell numbers. The deletion analysis demonstrated that −102 to −43 region was essential for the VEGF-A promoter activation. Site-directed mutaions of specificity protein 1 (SP1) binding sequences in the VEGF-A promoter and RNA interference of SP1 revealed that SP1 is an essential transcription factor for VEGF-A expression. These results indicate that HQ induces RPE cell apoptosis, leading to dry AMD, and suggest that AGE stimulation in addition to HQ enhances VEGF-A transcription via the AGE-receptor for AGE pathway in HQ-damaged cells. As a result, the secreted VEGF acts as an autocrine/paracrine growth factor for RPE and/or adjacent vascular cells, causing wet AMD.

Up-regulation of selenoprotein P and HIP/PAP mRNAs in hepatocytes by intermittent hypoxia via down-regulation of miR-203

Sleep apnea syndrome is characterized by recurrent episodes of oxygen desaturation and reoxygenation (intermittent hypoxia [IH]) and is a risk factor for insulin resistance/type 2 diabetes. However, the mechanisms linking IH stress and insulin resistance remain elusive. We exposed human hepatocytes (JHH5, JHH7, and HepG2) to experimental IH or normoxia for 24 h, measured mRNA levels by real-time reverse transcription polymerase chain reaction (RT-PCR), and found that IH significantly increased the mRNA levels of selenoprotein P (SELENOP) — a hepatokine — and hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein (HIP/PAP) — one of REG (Regenerating gene) family. We next investigated promoter activities of both genes and discovered that they were not increased by IH. On the other hand, a target mRNA search of micro RNA (miRNA) revealed that both mRNAs have a potential target sequence for miR-203. The miR-203 level of IH-treated cells was significantly lower than that of normoxia-treated cells. Thus, we introduced miR-203 inhibitor and a non-specific control RNA (miR-203 inhibitor NC) into HepG2 cells and measured the mRNA levels of SELENOP and HIP/PAP. The IH-induced expression of SELENOP and HIP/PAP was abolished by the introduction of miR-203 inhibitor but not by miR-203 inhibitor NC. These results demonstrate that IH stress up-regulates the levels of SELENOP in human hepatocytes to accelerate insulin resistance and up-regulates the levels of HIP/PAP mRNAs to proliferate such hepatocytes, via the miR-203 mediated mechanism.

Expression of Ins1 and Ins2 genes in mouse fetal liver

A possible cure for diabetes is explored by using non-pancreatic cells such as fetal hepatocytes. The expression of insulin and transcription factors for insulin is investigated in mouse fetal liver. We detected mRNAs for insulin I (Ins1) and insulin II (Ins2) and proinsulin- and mature insulin-positive cells in mouse fetal liver by reverse transcription plus the polymerase chain reaction and immunohistochemistry. Glucagon, somatostatin and pancreatic polypeptide were not expressed throughout development. Mouse Ins2 and Ins1 promoters were transiently activated in mouse fetal hepatocytes of embryonic days 13.5 and 16.5, respectively. Pancreatic and duodenal homeobox 1 (Pdx1) mRNA was not expressed during development of the liver. In contrast, mRNAs and proteins of neurogenic differentiation (NeuroD)/β cell E-box transactivator 2 (Beta2) and v-maf musculoaponeurotic fibrosarcoma oncogene homolog (MafA) were almost simultaneously expressed with insulin genes in the liver. Ins2 and Ins1 promoters were activated in hepatoma cells by the transfection of the expression vector for NeuroD/Beta2 alone and by the combination of NeuroD/Beta2 and MafA, respectively. These results indicate that the expression of NeuroD/Beta2 and MafA is linked temporally with the transcription of Ins2 and Ins1 genes in mouse fetal liver and suggest the potential usage of fetal hepatocytes to make insulin-producing β cells by introducing transcription factors.

Intermittent Hypoxia in Pancreatic beta Cells

Sleep apnea syndrome (SAS) a highly prevalent disorder, and characterized by repetitive episodes of intermittent hypoxia (IH), i.e. recurrent episodes of oxygen desaturation during sleep, the development of daytime sleepiness, and deterioration in the quality of life. Multiple epidemiological studies have provided evidence implicating the presence of SAS as a risk factor for insulin resistance and type 2 diabetes and have reported that type 2 diabetes is associated with SAS independently of age, sex, and body habitus. One of the postulated mechanisms for the metabolic alterations associated with SAS is that IH leads to substantial alterations in both pancreatic β cell function and organ glucose homeostasis. On the other hand, hyperglycemia is known to increase the rate of β cell replication, which can provide an increased source of insulin to combat insulin resistance. Although accumulating evidence suggests associations between SAS and type 2 diabetes, the direct effect of IH on pancreatic β cell has been unknown. In this review, we focus on the impact of IH on pancreatic β cells, particularly β cell dysfunction and cell proliferation.