Of mice and MEN1: Insulinomas in a conditional mouse knockout

Patients with multiple endocrine neoplasia type 1 (MEN1) develop multiple endocrine tumors, primarily affecting the parathyroid, pituitary, and endocrine pancreas, due to the inactivation of the MEN1 gene. A conditional mouse model was developed to evaluate the loss of the mouse homolog, Men1, in the pancreatic beta cell. Men1 in these mice contains exons 3 to 8 flanked by loxP sites, such that, when the mice are crossed to transgenic mice expressing cre from the rat insulin promoter (RIP-cre), exons 3 to 8 are deleted in beta cells. By 60 weeks of age, >80% of mice homozygous for the floxed Men1 gene and expressing RIP-cre develop multiple pancreatic islet adenomas. The formation of adenomas results in elevated serum insulin levels and decreased blood glucose levels. The delay in tumor appearance, even with early loss of both copies of Men1, implies that additional somatic events are required for adenoma formation in beta cells. Comparative genomic hybridization of beta cell tumor DNA from these mice reveals duplication of chromosome 11, potentially revealing regions of interest with respect to tumorigenesis.     

Inhibition of Ca2+-independent phospholipase A2 results in insufficient insulin secretion and impaired glucose tolerance

Islet Ca2+-independent phospholipase A2 (iPLA2) is postulated to mediate insulin secretion by releasing arachidonic acid in response to insulin secretagogues. However, the significance of iPLA2 signaling in insulin secretion in vivo remains unexplored. Here we investigated the physiological role of iPLA2 in beta-cell lines, isolated islets, and mice. We showed that small interfering RNA-specific silencing of iPLA2 expression in INS-1 cells significantly reduced insulin-secretory responses of INS-1 cells to glucose. Immunohistochemical analysis revealed that mouse islet cells expressed significantly higher levels of iPLA2 than pancreatic exocrine acinar cells. Bromoenol lactone (BEL), a selective inhibitor of iPLA2, inhibited glucose-stimulated insulin secretion from isolated mouse islets; this inhibition was overcome by exogenous arachidonic acid. We also showed that iv BEL administration to mice resulted in sustained hyperglycemia and reduced insulin levels during glucose tolerance tests. Clamp experiments demonstrated that the impaired glucose tolerance was due to insufficient insulin secretion rather than decreased insulin sensitivity. Short-term administration of BEL to mice had no effect on fasting glucose levels and caused no apparent pathological changes of islets in pancreas sections. These results unambiguously demonstrate that iPLA2 signaling plays an important role in glucose-stimulated insulin secretion under physiological conditions.     

Expression of the human insulin gene in transgenic mice

Transgenic mice which specifically express the human insulin gene in the pancreatic beta cells have been obtained. Expression is demonstrated by the presence in the serum of human insulin (determined by a radioimmunoassay for the human C-peptide), the level of which increases upon glucose stimulus, and by the presence of human insulin mRNA in pancreas, but not in the other tissues tested. This result indicates that nucleotide sequences necessary for tissue-specific expression are within the transferred gene and/or in its vicinity. It also suggests that the regulatory molecules are quite similar if not identical in human and mouse.     

Tg-visfatin×ob/ob小鼠表型特征与内脂素表达

目的研究Tg-visfatin×ob/ob小鼠的表型特征并探讨内脂素的作用。方法将visfatin转基因小鼠与ob（＋/-）小鼠杂交,获取visfatin转基因ob/ob小鼠（Tg-visfatin×ob/ob）,以ob/ob小鼠为对照,测定两种小鼠1～9月龄的体重变化,分别在3、5、9月龄测定其腹腔糖耐受和胰岛素耐受情况,并对其从9月龄～11月龄的死亡率进行统计。结果两种小鼠在1～9月龄的体重差异无显著性。腹腔糖耐受和胰岛素耐受实验显示,与ob/ob小鼠相比,3月龄时,Tg-visfatin×ob/ob小鼠的糖耐受受损及胰岛素耐受情况得以改善;5月龄时,Tg-visfatin×ob/ob小鼠仅糖耐受受损得以改善;9月龄时,Tg-visfatin×ob/ob小鼠具有更严重的糖耐受受损及胰岛素耐受。统计结果显示,从9月龄到11月龄之间,Tg-visfatin×ob/ob小鼠的死亡率比ob/ob小鼠提高了44.4%。结论 visfatin体内的高表达对ob/ob小鼠糖耐受和胰岛素耐受有影响,作用效果随着小鼠的年龄不同而变化,在早期起到代偿性的缓解ob/ob小鼠的糖耐受和胰岛素耐受的作用,到后期表现为失代偿性的加重了ob/ob小鼠的糖耐受和胰岛素耐受,并增加了小鼠的死亡率。     

Klotho lacks a vitamin D independent physiological role in glucose homeostasis, bone turnover, and steady-state PTH secretion in vivo

Apart from its function as co-receptor for fibroblast growth factor-23 (FGF23), Klotho is thought to regulate insulin signaling, intracellular oxidative stress, and parathyroid hormone (PTH) secretion in an FGF23 independent fashion. Here, we crossed Klotho deficient (Kl^−/−) mice with vitamin D receptor (VDR) mutant mice to examine further vitamin D independent functions of Klotho. All mice were fed a rescue diet enriched with calcium, phosphorus, and lactose to prevent hyperparathyroidism in VDR mutants, and were killed at 4 weeks of age after double fluorochrome labeling. Kl^−/− mice displayed hypercalcemia, hyperphosphatemia, dwarfism, organ atrophy, azotemia, pulmonary emphysema, and osteomalacia. In addition, glucose and insulin tolerance tests revealed hypoglycemia and profoundly increased peripheral insulin sensitivity in Kl^−/− mice. Compound mutants were normocalcemic and normophosphatemic, did not show premature aging or organ atrophy, and were phenocopies of VDR mutant mice in terms of body weight, bone mineral density, bone metabolism, serum calcium, serum phosphate, serum PTH, gene expression in parathyroid glands, as well as urinary calcium and phosphate excretion. Furthermore, ablation of vitamin D signaling in double mutants completely normalized glucose and insulin tolerance, indicating that Klotho has no vitamin D independent effects on insulin signaling. Histomorphometry of pancreas islets showed similar beta cell volume per body weight in all groups of animals. In conclusion, our findings cast doubt on a physiologically relevant vitamin D and Fgf23 independent function of Klotho in the regulation of glucose metabolism, bone turnover, and steady-state PTH secretion in vivo.     

Hepatocyte growth factor preserves beta cell mass and mitigates hyperglycemia in streptozotocin-induced diabetic mice

Type I diabetes is an autoimmune disease that results in destructive depletion of the insulin-producing beta cells in the islets of Langerhans in pancreas. With the knowledge that hepatocyte growth factor (HGF) is a potent survival factor for a wide variety of cells, we hypothesized that supplementation of HGF may provide a novel strategy for protecting pancreatic beta cells from destructive death and for preserving insulin production. In this study, we demonstrate that expression of the exogenous HGF gene preserved insulin excretion and mitigated hyperglycemia of diabetic mice induced by streptozotocin. Blood glucose levels were significantly reduced in mice receiving a single intravenous injection of naked HGF gene at various time points after streptozotocin administration. Consistently, HGF concomitantly increased serum insulin levels in diabetic mice. Immunohistochemical staining revealed a marked preservation of insulin-producing beta cells by HGF in the pancreatic islets of the diabetic mice. This beneficial effect of HGF was apparently mediated by both protection of beta cells from death and promotion of their proliferation. Delivery of HGF gene in vivo induced pro-survival Akt kinase activation and Bcl-xL expression in the pancreatic islets of diabetic mice. These findings suggest that supplementation of HGF to prevent beta cells from destructive depletion and to promote their proliferation might be an effective strategy for ameliorating type I diabetes.     

Neuro-endocrine basis for altered plasma glucose homeostasis in the Fragile X mouse

Background

The fragile X mouse model shows an increase in seizure susceptibility, indicating an involvement of the GABAergic system via an alteration in cellular excitability. In the brain, we have previously described a reduction in GABA_A receptor expression as a likely basis for this susceptibility. In the brains of fragile X mice, this reduction in receptor expression culminates with a concomitant increase in the expression of glutamic acid decarboxylase (GAD), the enzyme responsible for GABA synthesis. Further, voltage-sensitive calcium channel expression is reduced in the pancreas of the fragile X mouse. Since there are considerable similarities in the GABAergic system in the brain and pancreas, we evaluated the protective role of taurine in pancreatic islet development in both wild type (WT) and fragile X mice (KO).

Methods

One-month-old FVB/NJ males or age-matched fmr1-knockout (KO) mice were supplemented with taurine in drinking water (0.05% w/v) for four weeks. Age-matched controls were fed water only for the same duration. At four weeks, mice were sacrificed and pancreases processed for histology and immunohistochemical studies on changes of insulin, glucagon and somatostatin expression. Additional mice were subjected to a glucose tolerance test.

Results

Taurine treatment resulted in a significant increase in the number and size of islets. WT taurine-fed mice, slightly hypoglycemic prior to glucose injection, showed significantly reduced plasma glucose at 30 min post-injection when compared to control mice. KO mice had normal baseline plasma glucose concentration; however, following glucose injection they had higher plasma glucose levels at 30 min when compared to controls. Supplementation of taurine to KO mice resulted in reduced baseline levels of plasma glucose. After glucose injection, the taurine-fed KO mice had reduced plasma glucose at 30 min compared to KO. Concomitant with the increased islets size and glucose tolerance observed in taurine-fed mice there was an increase in insulin, glucagon and somatostatin immunoreactivity in the islets of WT mice. In the KO mice however, insulin levels were not affected whereas glucagon and somatostatin levels were reduced. Exocytosis of these hormones is calcium-dependent, therefore any exacerbation of calcium homeostasis could affect hormone release. We found the expression of the voltage sensitive calcium channels (VSCC) is drastically reduced in the pancreas of fragile X mice.

Conclusions

During early development, the VSCC play an important role in calcium-dependent gene expression. Since these channels are also involved in depolarization and calcium-mediated vesicular release of neurotransmitters and pancreatic hormones, alterations in the expression of VSCC not only will affect calcium-mediated gene expression but also hormonal and neurotransmitter release creating therefore a neuroendocrine perturbation in the fragile X that may potentially affect other organ systems. We find that in the fragile X mouse, taurine treatment may partially restore functionality of the neuro-endocrine pancreas.

     

Brain somatostatin receptors are up-regulated in somatostatin-deficient mice

The peptide somatostatin (SST) is widely synthesized in the brain and periphery and acts through a family of five receptors (SSTR1-5) to exert numerous effects. A gene product related to SST, cortistatin (CST), also interacts with SSTR1-5. Here we have investigated the regulation of SSTR1-5 and of CST in SST knockout (SSTKO) mice. The five SSTRs were quantitated individually by subtype-selective binding analysis, by immunocytochemistry, and by mRNA measurement and showed, in the brain of SSTKO mice, up-regulation of subtypes 1, 2, 4, and 5, and down-regulation of SSTR3. Peripheral tissues displayed both subtype- and tissue-specific changes in SSTR1-5 mRNA levels of expression. Lack of SST did not up-regulate normal CST expression in brain nor did it induce its expression in the periphery. SST-like immunoreactivity, however, was induced in the proximal midgut in SSTKO animals, suggesting intestinal expression of a novel SST-like gene.     

Glucose homeostasis, insulin secretion, and islet phospholipids in mice that overexpress iPLA2beta in pancreatic beta-cells and in iPLA2beta-null mice

Studies with genetically modified insulinoma cells suggest that group VIA phospholipase A(2) (iPLA(2)beta) participates in amplifying glucose-induced insulin secretion. INS-1 insulinoma cells that overexpress iPLA(2)beta, for example, exhibit amplified insulin-secretory responses to glucose and cAMP-elevating agents. To determine whether similar effects occur in whole animals, we prepared transgenic (TG) mice in which the rat insulin 1 promoter (RIP) drives iPLA(2)beta overexpression, and two characterized TG mouse lines exhibit similar phenotypes. Their pancreatic islet iPLA(2)beta expression is increased severalfold, as reflected by quantitative PCR of iPLA(2)beta mRNA, immunoblotting of iPLA(2)beta protein, and iPLA(2)beta enzymatic activity. Immunofluorescence microscopic studies of pancreatic sections confirm iPLA(2)beta overexpression in RIP-iPLA(2)beta-TG islet beta-cells without obviously perturbed islet morphology. Male RIP-iPLA(2)beta-TG mice exhibit lower blood glucose and higher plasma insulin concentrations than wild-type (WT) mice when fasting and develop lower blood glucose levels in glucose tolerance tests, but WT and TG blood glucose levels do not differ in insulin tolerance tests. Islets from male RIP-iPLA(2)beta-TG mice exhibit greater amplification of glucose-induced insulin secretion by a cAMP-elevating agent than WT islets. In contrast, islets from male iPLA(2)beta-null mice exhibit blunted insulin secretion, and those mice have impaired glucose tolerance. Arachidonate incorporation into and the phospholipid composition of RIP-iPLA(2)beta-TG islets are normal, but they exhibit reduced Kv2.1 delayed rectifier current and prolonged glucose-induced action potentials and elevations of cytosolic Ca(2+) concentration that suggest a molecular mechanism for the physiological role of iPLA(2)beta to amplify insulin secretion.     

Sirt1 regulates insulin secretion by repressing UCP2 in pancreatic beta cells

Sir2 and insulin/IGF-1 are the major pathways that impinge upon aging in lower organisms. In Caenorhabditis elegans a possible genetic link between Sir2 and the insulin/IGF-1 pathway has been reported. Here we investigate such a link in mammals. We show that Sirt1 positively regulates insulin secretion in pancreatic beta cells. Sirt1 represses the uncoupling protein (UCP) gene UCP2 by binding directly to the UCP2 promoter. In beta cell lines in which Sirt1 is reduced by SiRNA, UCP2 levels are elevated and insulin secretion is blunted. The up-regulation of UCP2 is associated with a failure of cells to increase ATP levels after glucose stimulation. Knockdown of UCP2 restores the ability to secrete insulin in cells with reduced Sirt1, showing that UCP2 causes the defect in glucose-stimulated insulin secretion. Food deprivation induces UCP2 in mouse pancreas, which may occur via a reduction in NAD (a derivative of niacin) levels in the pancreas and down-regulation of Sirt1. Sirt1 knockout mice display constitutively high UCP2 expression. Our findings show that Sirt1 regulates UCP2 in beta cells to affect insulin secretion.     

Factors influencing insulin and glucagon secretion in lean and genetically obese mice.

The control of insulin and glucagon secretion from isolated pancreatic islets of lean and genetically obese mice has been compared. The enlarged islets of obese mouse pancreas and islets of obese mouse pancreas and islets of obese mice maintained on a restricted diet manifested a greater response to glucose stimulation of insulin secretion than the lean mice islets. The glucagon content of the islets, the secretion of glucagon in a medium containing 150 mg% glucose and the stimulation of glucagon secretion by arginine did not differ significantly in the two groups. Adrenaline stimulated glucagon secretion in vitro from obese mice but not from lean mice. Antinsulin serum injections into obese mice increased the plasma glucagon levels about twofold and had no effect on glucagon levels in lean mice, although the level of hyperglycaemia was the same in both groups. It is suggested that the suppression of glucagon release by glucose requires a higher concentration of insulin in the obese mouse pancreas than in lean mice.     

Autophagy is important in islet homeostasis and compensatory increase of beta cell mass in response to high-fat diet

Autophagy is an evolutionarily conserved machinery for bulk degradation of cytoplasmic components. Here, we report upregulation of autophagosome formation in pancreatic beta cells in diabetic db/db and in nondiabetic high-fat-fed C57BL/6 mice. Free fatty acids (FFAs), which can cause peripheral insulin resistance associated with diabetes, induced autophagy in beta cells. Genetic ablation of atg7 in beta cells resulted in degeneration of islets and impaired glucose tolerance with reduced insulin secretion. While high-fat diet stimulated beta cell autophagy in control mice, it induced profound deterioration of glucose tolerance in autophagy-deficient mutants, partly because of the lack of compensatory increase in beta cell mass. These findings suggest that basal autophagy is important for maintenance of normal islet architecture and function. The results also identified a unique role for inductive autophagy as an adaptive response of beta cells in the presence of insulin resistance induced by high-fat diet.     

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.     

Somatostatin inhibits insulin release via SSTR2 in hamster clonal beta-cells and pancreatic islets

Somatostatin (SST) inhibits pancreatic endocrine secretion. It is generally accepted that SSTR2 and SSTR5 mediate the inhibition of glucagon and insulin release, respectively. The present study was performed to test the hypothesis that SSTR2, but not SSTR5, mediates SST-induced inhibition of insulin release in hamster beta-cells. Both hamster clonal beta-cells HIT-T15 and pancreatic islets were used to test this hypothesis. Both SST and a nonpeptide SSTR2 agonist L-779,976 (1-100 nM) inhibited insulin release from HIT-T15 and islets in a concentration-dependent manner. In contrast, nonpeptide agonists for SSTR1, 3, 4 and 5 at the highest concentration studied (1 microM) failed to inhibit insulin release. PRL-2903, a peptide SSTR2 antagonist (0.1-1 muicroM), antagonized SST-induced inhibition of insulin release in a concentration-dependent manner. Taken together, we conclude that, in hamster beta-cells, SST inhibits insulin release via SSTR2 but not SSTR5.     

L-4F treatment reduces adiposity, increases adiponectin levels, and improves insulin sensitivity in obese mice

We hypothesized that the apolipoprotein mimetic peptide L-4F, which induces arterial anti-oxidative enzymes and is vasoprotective in a rat model of diabetes, would ameliorate insulin resistance and diabetes in obese mice. L-4F (2 mg/kg/d) administered to ob/ob mice for 6 weeks limited weight gain without altering food intake, decreased visceral (P < 0.02) and subcutaneous (P < 0.045) fat content, decreased plasma IL-1beta and IL-6 levels (P < 0.05) and increased insulin sensitivity, resulting in decreased glucose (P < 0.001) and insulin (P < 0.036) levels. In addition, L-4F treatment increased aortic and bone marrow heme oxygenase (HO) activity and decreased aortic and bone marrow superoxide production (P < 0.001). L-4F treatment increased serum adiponectin levels (P < 0.037) and decreased adipogenesis in mouse bone marrow (P < 0.039) and in cultures of human bone marrow-derived mesenchymal stem cells (P < 0.022). This was manifested by reduced adiposity, improved insulin sensitivity, improved glucose tolerance, increased plasma adiponectin levels, and reduced IL-1beta and IL-6 levels in obese mice. This study highlights the existence of a temporal relationship between HO-1 and adiponectin that is positively affected by L-4F in the ob/ob mouse model of diabetes, resulting in the amelioration of the deleterious effects of diabetes.     

Glucose-dependent insulinotropic polypeptide receptor null mice exhibit compensatory changes in the enteroinsular axis

The incretins glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are gut hormones that act via the enteroinsular axis to potentiate insulin secretion from the pancreas in a glucose-dependent manner. Both GLP-1 receptor and GIP receptor knockout mice (GLP-1R(-/-) and GIPR(-/-), respectively) have been generated to investigate the physiological importance of this axis. Although reduced GIP action is a component of type 2 diabetes, GIPR-deficient mice exhibit only moderately impaired glucose tolerance. The present study was directed at investigating possible compensatory mechanisms that take place within the enteroinsular axis in the absence of GIP action. Although serum total GLP-1 levels in GIPR knockout mice were unaltered, insulin responses to GLP-1 from pancreas perfusions and static islet incubations were significantly greater (40-60%) in GIPR(-/-) than in wild-type (GIPR(+/+)) mice. Furthermore, GLP-1-induced cAMP production was also elevated twofold in the islets of the knockout animals. Pancreatic insulin content and gene expression were reduced in GIPR(-/-) mice compared with GIPR(+/+) mice. Paradoxically, immunocytochemical studies showed a significant increase in beta-cell area in the GIPR-null mice but with less intense staining for insulin. In conclusion, GIPR(-/-) mice exhibit altered islet structure and topography and increased islet sensitivity to GLP-1 despite a decrease in pancreatic insulin content and gene expression.     

沉默长链非编码RNA Meg3损伤小鼠胰岛细胞的胰岛素分泌功能

MEG3是一种长链非编码RNA。已有研究证明,鼠源Meg3参与小鼠诱导多能干细胞、神经元和视网膜的分化过程。最新报道,MEG3在人胰岛β细胞中高表达,但其对维持成年胰岛β细胞的功能尚不清楚。本研究旨在探讨Meg3在小鼠胰岛细胞胰岛素分泌功能中的作用。实时定量PCR揭示,与Balb/c小鼠心、肝、脾、肺、肌、肾等组织/器官比较,Meg3在胰腺组织中高表达。在非糖尿病小鼠发生自发性糖尿病的第8、12周,Meg3在胰岛中的表达水平分别下调24%±8%和29%±9% (P<0.01);而当血糖升高20 mmol/L,小鼠胰岛中Meg3表达下调72%±16%(P<0.01)。在MIN6细胞中采用RNA干扰敲减Meg3的表达,在高糖浓度（20 mmol/L）刺激条件下,胰岛素分泌显著减少。小鼠静脉注射siRNA,结合血糖测定或葡萄糖耐受试验（IPGTT）显示,si-Meg3小鼠血清胰岛素水平显著下降。注射葡萄糖前血糖升高,注射葡萄糖后耐受能力降低;免疫组化分析显示,si-Meg3小鼠胰岛素阳性细胞的面积减少。实验结果提示,Meg3通过参与胰岛素的合成和分泌维持成年小鼠胰岛功能。Meg3表达失调可能参与I型糖尿病（T1DM）发病过程。