The PDZ/coiled-coil domain containing protein PIST modulates insulin secretion in MIN6 insulinoma cells by interacting with somatostatin receptor subtype 5

The multi-domain protein PIST (protein interacting specifically with Tc10) interacts with the SSTR5 (somatostatin receptor 5) and is responsible for its intracellular localization. Here, we show that PIST is expressed in pancreatic beta-cells and interacts with SSTR5 in these cells. PIST expression in MIN6 insulinoma cells is reduced by somatostatin (SST). After stimulation with SST, SSTR5 undergoes internalization together with PIST. MIN6 cells over-expressing PIST display enhanced glucose-stimulated insulin secretion and a decreased sensitivity to SST-induced inhibition of insulin secretion. These data suggest that PIST plays an important role in insulin secretion by regulating SSTR5 availability at the plasma membrane.     

The relationship between GPR40 and lipotoxicity of the pancreatic β-cells as well as the effect of pioglitazone

Free fatty acids (FFAs) acutely stimulate insulin secretion from pancreatic β-cells, whereas impair β-cell function following long term exposure. GPR40, a FFAs receptor, has been demonstrated to be activated by both medium and long chain FFAs and played an important role in insulin release. This study was performed to determine the contribution of GPR40 to short- and/or long-term effects of FFAs on glucose-stimulated insulin secretion (GSIS) and the expression of PDX-1 and GLUT2 in pancreatic β-cells, as well as the intervenient effects of pioglitazone on lipotoxicity of β-cells. βTC6 cell line stably expressing GPR40shRNA were established and the intervention of FFAs and pioglitazone on GSIS and expression of PDX-1 and GLUT2 in βTC6 cells was investigated. Results showed that 1-h exposure to FFAs significantly enhanced GSIS and increased expression of PDX-1 and GLUT2 in pSilencer-control transfected cells, but not in cells transfected with GPR40shRNA. While 48-h exposure to FFAs significantly impaired GSIS in pSilencer-control transfected cells as well as cells transfected with GPR40shRNA. Furthermore, pioglitazone enhanced insulin secretion in pSilencer-control transfected cells exposed to FFAs for 48 h, but not in cells transfected with GPR40shRNA. These results indicate that GPR40 mediates the short-term effects of FFAs on GSIS, but does not mediate the chronic lipotoxicity on β-cells. The reverse role of pioglitazone on lipotoxicity of β-cells may be related to GPR40.     

SSTR2 mediates the somatostatin-induced increase in intracellular Ca(2+) concentration and insulin secretion in the presence of arginine vasopressin in clonal beta-cell HIT-T15

The effects of somatostatin (SRIF) are mediated through the seven transmembrane receptor family that signals via Gi/Go. To date, five distinct SRIF receptors have been characterized and designated SSTR1-5. We have characterized the SRIF receptor that mediates the increase in [Ca(2+)](i) and insulin secretion in HIT-T15 cells (Simian virus 40-transformed Syrian hamster islets) using high affinity, subtype selective agonists for SSTR1 (L-797,591), SSTR2 (L-779,976), SSTR3 (L-796,778), SSTR4 (L-803,087), SSTR5 (L-817,818) and PRL-2903, a specific SSTR2 antagonist. In the presence of arginine vasopressin (AVP), SRIF increased [Ca(2+)](i) and insulin secretion. Treatment with the SSTR2 agonist L-779,976 resulted in similar responses to SRIF. In addition, L-779,976 increased both [Ca(2+)](i) and insulin secretion in a dose-dependent manner. Treatment with L-779,976 alone did not alter [Ca(2+)](i) or basal insulin secretion. In the presence of AVP, all other SRIF receptor agonists failed to increase [Ca(2+)](i) and insulin secretion. The effects of SRIF and L-779,976 were abolished by the SSTR2 antagonist PRL-2903. Our results suggest that the mechanism underlying SRIF-induced insulin secretion in HIT-T15 cells be mediated through the SSTR2.     

生长抑素受体在人神经内分泌癌组织中的表达及受体激活对细胞生长的抑制

生长抑素（somatostatin,SST）通过与细胞膜上的G蛋白偶联的生长抑素受体（somatostatin receptors,SSTRs）结合而发挥其抑制细胞增殖的作用,因而生长抑素类似物（somatostatin analogue, SSA）常被用于肿瘤辅助治疗。然而,治疗效果存在相当大的个体差异,推测生长抑素类似物治疗效果不佳,与内源性生长抑素受体表达缺失或者表达量和亚型组合有关。为此,检测各亚型SSTR在几例罕见的神经内分泌肿瘤中的表达,并检测过表达SSTR2和SSTR5以及受体激活对细胞增殖的抑制效果,分析受体激活的可能机制,有助于临床筛选适合SSA肿瘤辅助治疗的病例,预估SSA的治疗效果。免疫组化检测肿瘤组织SSTR1-5的表达。在培养的293T细胞中过表达SSTR2和SSTR5,免疫共沉淀检测受体相互作用,免疫荧光和共聚焦显微镜检测受体细胞内定位。用MTT法检测受体过表达及激活对培养的人肺癌细胞NCI-H460细胞增殖的影响,用流式细胞技术检测细胞周期分布。SSTR1-5在10例神经内分泌肿瘤组织中均有不同程度的表达,表达亚型及表达量与肿瘤类型和年龄无关,SSTR5在所有肿瘤组织中均表达。SSTR2与SSTR5可形成受体相互作用。SSTR2与SSTR5活化后相互作用增加并定位于细胞质。共表达SSTR2和SSTR5显著抑制细胞增殖,并与受体激活剂呈现剂量相关性。SSTR2/SSTR5的共表达及激活显著减少S期的细胞而滞留于G₁期。     

Somatostatin-28 regulates GLP-1 secretion via somatostatin receptor subtype 5 in rat intestinal cultures

Five somatostatin receptors (SSTRs) bind somatostatin-14 (S-14) and somatostatin-28 (S-28), but SSTR5 has the highest affinity for S-28. To determine whether S-28 acting through SSTR5 mediates inhibition of glucagon-like peptide-1 (GLP-1), fetal rat intestinal cell cultures were treated with somatostatin analogs with relatively high specificity for SSTRs 2-5. S-28 dose-dependently inhibited GLP-1 secretion stimulated by gastrin-releasing peptide more potently than S-14 (EC(50) 0.01 vs. 5.8 nM). GLP-1 secretion was inhibited by an SSTR5 analog, BIM-23268, more potently than S-14 and nearly as effectively as S-28. The SSTR5 analog L-372,588 also suppressed GLP-1 secretion equivalent to S-28, but a structurally similar peptide, L-362,855 (Tyr to Phe at position 7), was ineffective. An SSTR2-selective analog was less effective than S-28, and an SSTR3 analog was inactive. Separate treatment with GLP-1-(7-36)-NH(2) increased S-28 and S-14 secretion by three- and fivefold; BIM-23268 abolished S-28 without altering S-14, whereas the SSTR2 analog was inactive. The results indicate that somatostatin regulation of GLP-1 secretion occurs via S-28 through activation of SSTR5. GLP-1-stimulated S-28 secretion is also autoregulated by SSTR5 activation, suggesting a feedback loop between GLP-1 and S-28 modulated by SSTR5.     

AMP-activated protein kinase and pancreatic/duodenal homeobox-1 involved in insulin secretion under high leucine exposure in rat insulinoma β-cells

The effect of leucine on glucose-stimulated insulin secretion (GSIS) in pancreatic β-cells is quite controversial, and mechanism involved in the effect has not been elucidated yet. Consequently, we aimed to investigate effect of leucine on GSIS and its mechanism focusing on contribution of AMP-activated protein kinase (AMPK) and pancreatic/duodenal homeobox-1 (PDX-1). Rat insulinoma β-cells (INS-1, RIN m5F, DN-PDX-1#28 and PDX-1#6) were cultured with or without leucine, AICAR (AMPK agonist) or compound C (AMPK antagonist) for 48 hrs. In contrast to control, AICAR treatment decreased GSIS at high glucose and insulin content, also impaired protein and mRNA expression of PDX-1 and its downstream targets, glucokinase (GCK) and glucose transporter 2 (GLUT2). Compound C treatment had the opposite effects. We observed that neither AICAR nor compound C could affect expression of GCK and GLUT2 when PDX-1 expression was absent. Chronic leucine exposure inhibited GSIS at high glucose and insulin content in a dose-dependent manner, concomitant with an increase in AMPK and a decrease in PDX-1, GCK and GLUT2. The inhibitory effects of leucine was potentiated by AICAR treatment and rescued by compound C treatment. Finally, the inhibition of PDX-1 could potentiate the impaired effects induced by leucine whereas overexpression of PDX-1 could protect the cell from impairment induced by leucine. The study indicated that chronic leucine might result in an increase in AMPK and then a decrease in PDX-l, in turn to depress GCK and GLUT2 resulting in decreased GSIS at high glucose and insulin content.     

Coexpression of human somatostatin receptor-2 (SSTR2) and SSTR3 modulates antiproliferative signaling and apoptosis

Background

Somatostatin (SST) via five G_i coupled receptors namely SSTR1-5 is known to inhibit cell proliferation by cytostatic and cytotoxic mechanisms. Heterodimerization plays a crucial role in modulating the signal transduction pathways of SSTR subtypes. In the present study, we investigated human SSTR2/SSTR3 heterodimerization, internalization, MAPK signaling, cell proliferation and apoptosis in HEK-293 cells in response to SST and specific agonists for SSTR2 and SSTR3.

Results

Although in basal conditions, SSTR2 and SSTR3 colocalize at the plasma membrane and exhibit heterodimerization, the cell surface distribution of both receptors decreased upon agonist activation and was accompanied by a parallel increase in intracellular colocalization. Receptors activation by SST and specific agonists significantly decreased cAMP levels in cotransfected cells in comparison to control. Agonist-mediated modulation of pERK1/2 was time and concentration-dependent, and pronounced in serum-deprived conditions. pERK1/2 was inhibited in response to SST; conversely receptor-specific agonist treatment caused inhibition at lower concentration and activation at higher concentration. Strikingly, ERK1/2 phosphorylation was sustained upon prolonged treatment with SST but not with receptor-specific agonists. On the other hand, SST and receptor-specific agonists modulated p38 phosphorylation time-dependently. The receptor activation in cotransfected cells exhibits G_i-dependent inhibition of cell proliferation attributed to increased PARP-1 expression and TUNEL staining, whereas induction of p21 and p27^Kip1 suggests a cytostatic effect.

Conclusion

Our study provides new insights in SSTR2/SSTR3 mediated signaling which might help in better understanding of the molecular interactions involving SSTRs in tumor biology.     

Increased DNA methylation and decreased expression of PDX-1 in pancreatic islets from patients with type 2 diabetes

Mutations in pancreatic duodenal homeobox 1 (PDX-1) can cause a monogenic form of diabetes (maturity onset diabetes of the young 4) in humans, and silencing Pdx-1 in pancreatic β-cells of mice causes diabetes. However, it is not established whether epigenetic alterations of PDX-1 influence type 2 diabetes (T2D) in humans. Here we analyzed mRNA expression and DNA methylation of PDX-1 in human pancreatic islets from 55 nondiabetic donors and nine patients with T2D. We further studied epigenetic regulation of PDX-1 in clonal β-cells. PDX-1 expression was decreased in pancreatic islets from patients with T2D compared with nondiabetic donors (P = 0.0002) and correlated positively with insulin expression (rho = 0.59, P = 0.000001) and glucose-stimulated insulin secretion (rho = 0.41, P = 0.005) in the human islets. Ten CpG sites in the distal PDX-1 promoter and enhancer regions exhibited significantly increased DNA methylation in islets from patients with T2D compared with nondiabetic donors. DNA methylation of PDX-1 correlated negatively with its gene expression in the human islets (rho = -0.64, P = 0.0000029). Moreover, methylation of the human PDX-1 promoter and enhancer regions suppressed reporter gene expression in clonal β-cells (P = 0.04). Our data further indicate that hyperglycemia decreases gene expression and increases DNA methylation of PDX-1 because glycosylated hemoglobin (HbA1c) correlates negatively with mRNA expression (rho = -0.50, P = 0.0004) and positively with DNA methylation (rho = 0.54, P = 0.00024) of PDX-1 in the human islets. Furthermore, while Pdx-1 expression decreased, Pdx-1 methylation and Dnmt1 expression increased in clonal β-cells exposed to high glucose. Overall, epigenetic modifications of PDX-1 may play a role in the development of T2D, given that pancreatic islets from patients with T2D and β-cells exposed to hyperglycemia exhibited increased DNA methylation and decreased expression of PDX-1. The expression levels of PDX-1 were further associated with insulin secretion in the human islets.     

Complete clinical remission and disappearance of liver metastases after treatment with somatostatin analogue in a 40-year-old woman with a malignant insulinoma positive for somatostatin receptors type 2

Insulinoma is the most common pancreatic endocrine tumor, accounting for 40% of all pancreatic functional neoplasm, and is characterized by hypersecretion of insulin and hypoglycemia. Elective treatment for insulinomas is surgical enucleation. Medical therapy with diazoxide, followed by somatostatin analogues in some cases, may be necessary to treat the hypoglycemic symptoms. We report a case of a patient affected by metastatic insulinoma with severe hypoglycemia. After surgery, histopathology confirmed the presence of a malignant insulinoma with multiple metastases in the liver. Due to the persistence of hypoglycemia, the patient was started on octreotide LAR treatment, which determined a complete clinical remission with regression of the metastatic lesions in the liver after one year. Repeated CT scans 2 and 3 years after surgery confirmed the remission. To our knowledge, the complete regression of the disease in insulinomas treated with long-standing somatostatin analogue therapy has never been reported. Immunohistochemical analysis in tissue specimens showed a strong membrane immunoreactivity for somatostatin receptors type 2 (SSTR2) in both the primary nodule and the metastases. The capacity of somatostatin analogues to negatively regulate cell proliferation through indirect and direct mechanisms has been experimentally demonstrated. Furthermore, SSTR2 activation may exert pro-apoptotic effects in neoplastic cells. Thus, both mechanisms may have been responsible of the remission of the disease in this patient. This case underlies the potential impact of the treatment of pancreatic insulinomas with somatostatin analogues, and, if confirmed, the usefulness of SSTR determination in these neoplastic specimens.     

生长抑素受体的生理功能及动力学特征

生长抑素受体家族(somatostatin receptors,SSTRs)是一类介导生长抑素及其类似物,具有多种生物学效应的G蛋白偶联受体家族,其生理功能和作用机制长期以来倍受关注.研究表明,这些细胞膜上存在的特定膜受体包括SSTR1、SSTR2、SSTR3、SSTR4以及SSTR5,可以通过cAMP、PTP和MAPK信号通路,在调控GH分泌、诱导细胞凋亡、抑制肿瘤细胞增生、抑制胰岛素作用和抑制细胞生长等生物学过程发挥重要的作用,同时表现出与其它G蛋白偶联受体性质相似的动力学特征.本文将SSTRs的结构、分布和生理功能、配体选择性、下游信号通路,以及该受体家族的动力学特征最新研究进展作一综述.     

Upregulated Expression of SSTR1 is Involved in Neuronal Apoptosis and is Coupled to the Reduction of bcl-2 Following Intracerebral Hemorrhage in Adult Rats

Somatostatins are peptide hormones that regulate diverse cellular processes, such as neurotransmission, cell proliferation, apoptosis, and endocrine signaling as well as inhibiting the release of many hormones and other secretory proteins. SSTR1 is a member of the superfamily of somatostatin receptors possessing seven-transmembrane segments. Aberrant expression of SSTR1 has been implicated in several human diseases, including pseudotumor cerebri, and oncogenic osteomalacia. In this study, we investigated a potential role of SSTR1 in the regulation of neuronal apoptosis in the course of intracerebral hemorrhage (ICH). A rat ICH model in the caudate putamen was established and subjected to behavioral tests. Western blot and immunohistochemistry indicated a remarkable up-regulation of SSTR1 expression surrounding the hematoma after ICH. Double-labeled immunofluorescence showed that SSTR1 was mostly co-localized with neurons, and was rarely distributed in activated astrocytes and microglia. Additionally, SSTR1 co-localized with active-caspase-3 and bcl-2 around the hematoma. The expression of active-caspase-3 was parallel with that of SSTR1 in a time-dependent manner. In addition, SSTR1 knockdown specifically resulted in reduced neuronal apoptosis in PC12 cells. All our findings suggested that up-regulated SSTR1 contributed to neuronal apoptosis after ICH, which was accompanied with reduced expression of bcl-2.     

SSTR5 ablation in islet results in alterations in glucose homeostasis in mice

Somatostatin (SST) peptide is a potent inhibitor of insulin secretion and its effect is mediated via somatostatin receptor 5 (SSTR5) in the endocrine pancreas. To investigate the consequences of gene ablation of SSTR5 in the mouse pancreas, we have generated a mouse model in which the SSTR5 gene was specifically knocked down in the pancreatic beta cells (betaSSTR5Kd) using the Cre-lox system. Immunohistochemistry analysis showed that SSTR5 gene expression was absent in beta cells at three months of age. At the time of gene ablation, betaSSTR5Kd mice demonstrated glucose intolerance with lack of insulin response and significantly reduced serum insulin levels. Insulin tolerance test demonstrated a significant increase of insulin clearance in vivo at the same age. In vitro studies demonstrated an absence of response to SST-28 stimulation in the betaSSTR5Kd mouse islet, which was associated with a significantly reduced SST expression level in betaSSTR5Kd mice pancreata. In addition, betaSSTR5Kd mice had significantly reduced serum glucose levels and increased serum insulin levels at 12 months of age. Glucose tolerance test at an older age also indicated a persistently higher insulin level in betaSSTR5Kd mice. Further studies of betaSSTR5Kd mice had revealed elevated serum C-peptide levels at both 3 and 12 months of age, suggesting that these mice are capable of producing and releasing insulin to the periphery. These results support the hypothesis that SSTR5 plays a pivotal role in the regulation of insulin secretion in the mouse pancreas.