Impact of fetal and neonatal environment on beta cell function and development of diabetes.

The global epidemic of diabetes is a serious threat against health and healthcare expenses. Although genetics is important it does not explain the dramatic increase in incidence, which must involve environmental factors. Two decades ago the concept of the thrifty phenotype was introduced, stating that the intrauterine environment during pregnancy has an impact on the gene expression that may persist until adulthood and cause metabolic diseases like obesity and type 2 diabetes. As the pancreatic beta cells are crucial in the regulation of metabolism this article will describe the influence of normal pregnancy on the beta cells in both the mother and the fetus and how various conditions like diabetes, obesity, overnutrition and undernutrition during and after pregnancy may influence the ability of the offspring to adapt to changes in insulin demand later in life. The influence of environmental factors including nutrients and gut microbiota on appetite regulation, mitochondrial activity and the immune system that may affect beta cell growth and function directly and indirectly is discussed. The possible role of epigenetic changes in the transgenerational transmission of the adverse programming may be the most threatening aspect with regard to the global diabetes epidemics. Finally, some suggestions for intervention are presented.

The L-α-amino acid receptor GPRC6A is expressed in the islets of Langerhans but is not involved in L-arginine-induced insulin release.

GPRC6A is a seven-transmembrane receptor activated by a wide range of L-α-amino acids, most potently by L-arginine and other basic amino acids. The receptor is broadly expressed, but its exact physiological role remains to be elucidated. It is well established that L-arginine stimulates insulin secretion; therefore, the receptor has been hypothesized to have a role in regulating glucose metabolism. In this study, we demonstrate that GPRC6A is expressed in islets of Langerhans, but activation of the receptor by L-arginine did not stimulate insulin secretion. We also investigated central metabolic parameters in GPRC6A knockout mice compared with wildtype littermates and found no difference in glucose metabolism or body fat percentage when mice were administered a standard chow diet. In conclusion, our data do not support a role for GPRC6A in L-arginine-induced insulin release and glucose metabolism under normal physiological conditions.

Transgenic rescue of adipocyte glucose-dependent insulinotropic polypeptide receptor expression restores high fat diet-induced body weight gain.

The glucose-dependent insulinotropic polypeptide receptor (GIPr) has been implicated in high fat diet-induced obesity and is proposed as an anti-obesity target despite an uncertainty regarding the mechanism of action. To independently investigate the contribution of the insulinotropic effects and the direct effects on adipose tissue, we generated transgenic mice with targeted expression of the human GIPr to white adipose tissue or beta-cells, respectively. These mice were then cross-bred with the GIPr knock-out strain. The central findings of the study are that mice with GIPr expression targeted to adipose tissue have a similar high fat diet -induced body weight gain as control mice, significantly greater than the weight gain in mice with a general ablation of the receptor. Surprisingly, this difference was due to an increase in total lean body mass rather than a gain in total fat mass that was similar between the groups. In contrast, glucose-dependent insulinotropic polypeptide-mediated insulin secretion does not seem to be important for regulation of body weight after high fat feeding. The study supports a role of the adipocyte GIPr in nutrient-dependent regulation of body weight and lean mass, but it does not support a direct and independent role for the adipocyte or beta-cell GIPr in promoting adipogenesis.

Suppression of FAT/CD36 mRNA by human growth hormone in pancreatic ß-cells.

Fatty acid-induced damage in pancreatic ß-cells is assumed to play an important role in the development of type 2 diabetes. Lactogens (prolactin, placental lactogen and growth hormone) improve ß-cell survival via STAT5 activation but the molecular targets are incompletely characterized. The aim of this study was to examine the effect of human growth hormone (hGH) on mRNAs of fatty acid transport and binding proteins expressed in pancreatic ß-cells, and to examine this in relation to ß-cell survival after exposure to fatty acids. hGH decreased mRNA levels of FAT/CD36, whereas mRNAs of GPR40, FASN, FABP2, FATP1 and FATP4 were unchanged. RNAi against FAT/CD36 decreased fatty acid-induced apoptosis. Over-expression of constitutively active STAT5 was able to mimic hGH's suppression of FAT/CD36 expression, whereas dominant negative STAT5 was unable to block the effect of hGH indicating that STAT5 did not bind directly to the FAT/CD36 promoter. The hGH-mediated suppression of FAT/CD36 mRNA was associated with a decrease in palmitate uptake and fatty acid-induced basal hyper-secretion of insulin resulting in improved glucose-stimulated insulin secretion. This study suggests that hGH can protect ß-cells against fatty acid-induced damages.

Beta-cell dysfunction induced by non-cytotoxic concentrations of Interleukin-1ß is associated with changes in expression of beta-cell maturity genes and associated histone modifications.

Decreased insulin secretory capacity in Type 2 diabetes mellitus is associated with beta-cell dedifferentiation and inflammation. We hypothesize that prolonged exposure of beta-cells to low concentrations of IL-1ß induce beta-cell dedifferentiation characterized by impaired glucose-stimulated insulin secretion, reduced expression of key beta-cell genes and changes in histone modifications at gene loci known to affect beta-cell function. Ten days exposure to IL-1ß at non-cytotoxic concentrations reduced insulin secretion and beta-cell proliferation and decreased expression of key beta-cell identity genes, including MafA and Ucn3 and decreased H3K27ac at the gene loci, suggesting that inflammatory cytokines directly affects the epigenome. Following removal of IL-1ß, beta-cell function was normalized and mRNA expression of beta-cell identity genes, such as insulin and Ucn3 returned to pre-stimulation levels. Our findings indicate that prolonged exposure to low concentrations of IL-1ß induces epigenetic changes associated with loss of beta-cell identity as observed in Type 2 diabetes.

The aromatic amino acid sensor GPR142 controls metabolism through balanced regulation of pancreatic and gut hormones.

OBJECTIVES: GPR142, which is highly expressed in pancreatic islets, has recently been deorphanized as a receptor for aromatic amino acids; however, its physiological role and pharmacological potential is unclear. METHODS AND RESULTS: We find that GPR142 is expressed not only in ß- but also in α-cells of the islets as well as in enteroendocrine cells, and we confirm that GPR142 is a highly selective sensor of essential aromatic amino acids, in particular Trp and oligopeptides with N-terminal Trp. GPR142 knock-out mice displayed a very limited metabolic phenotype but demonstrated that L-Trp induced secretion of pancreatic and gut hormones is mediated through GPR142 but that the receptor is not required for protein-induced hormone secretion. A synthetic GPR142 agonist stimulated insulin and glucagon as well as GIP, CCK, and GLP-1 secretion. In particular, GIP secretion was sensitive to oral administration of the GPR142 agonist an effect which in contrast to the other hormones was blocked by protein load. Oral administration of the GPR142 agonist increased [3H]-2-deoxyglucose uptake in muscle and fat depots mediated through insulin action while it lowered liver glycogen conceivably mediated through glucagon, and, consequently, it did not lower total blood glucose. Nevertheless, acute administration of the GPR142 agonist strongly improved oral glucose tolerance in both lean and obese mice as well as Zucker fatty rat. Six weeks in-feed chronic treatment with the GPR142 agonist did not affect body weight in DIO mice, but increased energy expenditure and carbohydrate utilization, lowered basal glucose, and improved insulin sensitivity. CONCLUSIONS: GPR142 functions as a sensor of aromatic amino acids, controlling GIP but also CCK and GLP-1 as well as insulin and glucagon in the pancreas. GPR142 agonists could have novel interesting potential in modifying metabolism through a balanced action of gut hormones as well as both insulin and glucagon.

STAT5 activity in pancreatic beta-cells influences the severity of diabetes in animal models of type 1 and 2 diabetes.

Pancreatic beta-cell growth and survival and insulin production are stimulated by growth hormone and prolactin through activation of the transcription factor signal transducer and activator of transcription (STAT)5. To assess the role of STAT5 activity in beta-cells in vivo, we generated transgenic mice that expressed a dominant-negative mutant of STAT5a (DNSTAT5) or constitutive active mutant of STAT5b (CASTAT5) under control of the rat insulin 1 promoter (RIP). When subjected to a high-fat diet, RIP-DNSTAT5 mice showed higher body weight, increased plasma glucose levels, and impairment of glucose tolerance, whereas RIP-CASTAT5 mice were more glucose tolerant and less hyperleptinemic than wild-type mice. Although the pancreatic insulin content and relative beta-cell area were increased in high-fat diet-fed RIP-DNSTAT5 mice compared with wild-type or RIP-CASTAT5 mice, RIP-DNSTAT5 mice showed reduced beta-cell proliferation at 6 months of age. The inhibitory effect of high-fat diet or leptin on insulin secretion was diminished in isolated islets from RIP-DNSTAT5 mice compared with wild-type islets. Upon multiple low-dose streptozotocin treatment, RIP-DNSTAT5 mice exhibited higher plasma glucose levels, lower plasma insulin levels, and lower pancreatic insulin content than wild-type mice, whereas RIP-CASTAT5 mice maintained higher levels of plasma insulin. In conclusion, our results indicate that STAT5 activity in beta-cells influences the susceptibility to experimentally induced type 1 and type 2 diabetes.

Trefoil factors are expressed in human and rat endocrine pancreas: differential regulation by growth hormone.

Trefoil factors (TFFs) 1, 2, and 3 are expressed in mucosal epithelia. TFFs are particular abundant in the intestine in which they play a crucial role in maintenance and restitution of the epithelium. Because pancreas developmentally arises from the primitive foregut, we explored the expression of TFFs in the pancreas in man and rat. Immunocytochemical staining of adult human pancreas showed abundant TFF3 immunoreactivity in pancreatic islets and some duct cells, whereas weak TFF1 and no TFF2 staining were detected. In the islets TFF3 localized to most insulin and some glucagon and pancreatic polypeptide-producing cells. TFF3 immunoreactivity was colocalized with insulin and glucagon in distinct cell clusters in human fetal pancreas at wk 14 and in the newborn rat pancreas. In isolated human and rat islets, TFF3 and TFF1 mRNA was identified by RT-PCR, and TFF3 protein was detected in human pancreas and islets by ELISA. Exposure of neonatal rat islets or insulinoma cells to GH, a known beta-cell growth factor, resulted in markedly increased TFF3 but decreased TFF1 mRNA levels. The effect of GH on TFF3 expression was confirmed by Western blot. Culture of neonatal rat islets in the presence of TFF3 resulted in attachment and migration of the islet cells, but no effects on proliferation, insulin secretion or cytokine-induced apoptosis were seen. These data demonstrate expression of TFFs in the endocrine pancreas, but their possible functions remain unknown.

Glucose triggers protein kinase A-dependent insulin secretion in mouse pancreatic islets through activation of the K+ATP channel-dependent pathway.

OBJECTIVE: To assess the significance of protein kinase A (PKA) in glucose triggering of ATP-sensitive K(+) (K(+)(ATP)) channel-dependent insulin secretion and in glucose amplification of K(+)(ATP) channel-independent insulin secretion. METHODS: Insulin release from cultured perifused mouse pancreatic islets was determined by radioimmunoassay. RESULTS: In islets cultured at 5.5 mmol/l glucose, and then perifused in physiological Krebs-Ringer medium, the PKA inhibitors, H89 (10 micromol/l) and PKI 6-22 amide (30 micromol/l) did not inhibit glucose (16.7 mmol/l)-induced insulin secretion, but inhibited stimulation by the adenylyl cyclase activator, forskolin (10 micromol/l). In the presence of 60 mmol/l K(+) and 250 micromol/l diazoxide, which stimulates maximum Ca(2+) influx independently of K(+)(ATP) channels, H89 (10 micromol/l) inhibited Ca(2+)-evoked insulin secretion, but failed to prevent glucose amplification of K(+)(ATP) channel-independent insulin secretion. In the presence of 1 mmol/l ouabain and 250 micromol/l diazoxide, which cause modest Ca(2+) influx, glucose amplification of K(+)(ATP) channel-independent insulin secretion was observed without concomitant Ca(2+) stimulation of PKA activity. In islets cultured at 16.7 mmol/l glucose, glucose (16.7 mmol/l)-induced insulin secretion in physiological Krebs-Ringer medium was augmented and now inhibited by H89 (10 micromol/l), implicating that culture at 16.7 mmol/l glucose may increase Ca(2+)-sensitive adenylyl cyclase activity and hence PKA activity. In accordance, Ca(2+)-evoked insulin secretion at 60 mmol/l K(+) and 250 micromol/l diazoxide was improved, whereas glucose amplification of K(+)(ATP) channel-independent insulin secretion was unaffected. CONCLUSIONS: Glucose may activate PKA through triggering of the K(+)(ATP) channel-dependent pathway. Glucose amplification of K(+)(ATP) channel-independent insulin secretion, on the other hand, occurs by PKA-independent mechanisms.

A critical role for CK2 in cytokine-induced activation of NFκB in pancreatic ß cell death.

This study aimed to assess the role of constitutive protein kinase CK2 in cytokine-induced activation of NFκB in pancreatic ß cell death. The CK2 inhibitors DRB (5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole) (50 µM) and DMAT (2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole) (5 µM), which decreased CK2 activity by approx. 65 %, rescued INS-1E ß cells and mouse islets from cytokine (IL-1ß, TNF-α plus IFN-γ)-induced ß cell death without affecting H2O2- or palmitate-induced ß cell death. Western blot analysis revealed that while DRB or DMAT did not influence cytokine-induced IκBα degradation, they inhibited NFκB-dependent IκBα resynthesis, demonstrating that cytokine-induced NFκB activity is dependent on CK2. Both DRB and DMAT inhibited the constitutive phosphorylation of NFκB p65 at serine 529, while leaving cytokine-induced phosphorylations of NFκB p65 at serines 276 and 536 unaltered. In comparison, putative phosphorylation sites for CK2 on HDACs 1, 2, and 3 at serines 421/423, 394, and 424, respectively, which may stimulate NFκB transcriptional activity, were unchanged by cytokines and CK2 inhibitors. Whereas IL-1ß and TNF-α stimulate IκBα degradation and NFκB activation, IFN-γ potentiates cytokine-induced ß cell death through activation of STAT1. DRB and DMAT inhibited IFN-γ-stimulated phosphorylation of STAT1 at serine 727, while leaving IFN-γ-induced phosphorylation of STAT1 at tyrosine 701 unaffected. Inhibition of cytokine-induced ß cell death by CK2 inhibitors was, however, not dependent on IFN-γ, and IFN-γ did not affect CK2-dependent IκBα turnover. In conclusion, it is suggested that cytokine-induced activation of NFκB in ß cells is dependent on CK2 activity, which phosphorylates NFκB p65 at serine 529.

AKAP 18 alpha and gamma have opposing effects on insulin release in INS-1E cells.

A-kinase anchoring proteins (AKAPs) are known to compartmentalise protein kinase(s) to discrete cellular locations. Here we show that silencing of AKAP 18 alpha or gamma expression results in decreased or increased glucose-stimulated insulin secretion in INS-1E cells. Glucose stimulates AKAP 18 alpha and inhibits AKAP 18 gamma mRNA expressions while palmitate markedly reduces AKAP 18 alpha expression. Human growth hormone (GH) stimulates AKAP 18 alpha expression and attenuates palmitate-induced suppression of AKAP 18 alpha mRNA level. The roles of AKAP 18 alpha and gamma in mediating insulin release are consistent with their respective regulations by glucose.

Serum albumin protects from cytokine-induced pancreatic beta cell death by a phosphoinositide 3-kinase-dependent mechanism.

The present study was undertaken to investigate the biological activity of serum albumin when pancreatic beta cells were challenged by cytokines and pro-apoptotic reactive oxygen species like H(2)O(2). Culture of mouse islets or INS-1E beta cells for 24 h in the presence of H(2)O(2) (25 micromol/l) increased cell death. This demise was prevented by serum albumin, dependent on its free sulfhydryl group, emphasizing that albumin may scavenge H(2)O(2) due to its antioxidant properties. Culture for 48 h with a cytokine mixture of IL-1beta (160 pg/ml), IFN-gamma (200 ng/ml), and TNF-alpha (2 ng/ml) revealed that albumin, also protected against cytokine-induced death of both mouse islets and INS-1E beta cells. This protective effect against cytokine-induced beta cell death was, however, not dependent on albumins free sulfhydryl group, but was inhibited by the phosphoinositide 3-kinase (PI3K) inhibitors LY294002 (25 micromol/l) and wortmannin (1 micromol/l), suggesting that albumin may rescue beta cells from cytokine-induced cell death by activation of PI3K. In accordance, albumin stimulated phosphorylation of Akt, a down-stream target for PI3K. In conclusion, it is suggested that albumin may be a survival factor for pancreatic beta cells through scavenging of reactive oxygen species and by PI3K-dependent activation of Akt.

G protein-coupled receptor 39 deficiency is associated with pancreatic islet dysfunction.

G protein-coupled receptor (GPR)-39 is a seven-transmembrane receptor expressed mainly in endocrine and metabolic tissues that acts as a Zn(++) sensor signaling mainly through the G(q) and G(12/13) pathways. The expression of GPR39 is regulated by hepatocyte nuclear factor (HNF)-1alpha and HNF-4alpha, and in the present study, we addressed the importance of GPR39 for glucose homeostasis and pancreatic islets function. The expression and localization of GPR39 were characterized in the endocrine pancreas and pancreatic cell lines. Gpr39(-/-) mice were studied in vivo, especially in respect of glucose tolerance and insulin sensitivity, and in vitro in respect of islet architecture, gene expression, and insulin secretion. Gpr39 was down-regulated on differentiation of the pluripotent pancreatic cell line AR42J cells toward the exocrine phenotype but was along with Pdx-1 strongly up-regulated on differentiation toward the endocrine phenotype. Immunohistochemistry demonstrated that GRP39 is localized selectively in the insulin-storing cells of the pancreatic islets as well as in the duct cells of the exocrine pancreas. Gpr39(-/-) mice displayed normal insulin sensitivity but moderately impaired glucose tolerance both during oral and iv glucose tolerance tests, and Gpr39(-/-) mice had decreased plasma insulin response to oral glucose. Islet architecture was normal in the Gpr39 null mice, but expression of Pdx-1 and Hnf-1alpha was reduced. Isolated, perifused islets from Gpr39 null mice secreted less insulin in response to glucose stimulation than islets from wild-type littermates. It is concluded that GPR39 is involved in the control of endocrine pancreatic function, and it is suggested that this receptor could be a novel potential target for the treatment of diabetes.

Pancreatic islet insulin secretion and metabolism in adult rats malnourished during neonatal life.

Pancreatic islets were isolated from rats that had been nursed by dams fed with a control or an 8.7% protein diet during the first 12 d of the lactation period. Glucose-induced insulin secretion from islets in the 8.7% protein group was reduced 50%. The islet insulin and DNA content were similar, whereas the pancreatic insulin content was reduced by 30 % in the rats fed 8.7 % protein. In order to elucidate the mechanism responsible for the attenuation of insulin secretion, measurements were performed of the activity of several islet enzymes that had previously been supposed to be involved in the coupling of glucose stimulation to insulin secretion. Islet glucose oxidation was unaffected, but glucose-stimulated hydrolysis of phosphatidylinositol was reduced by one-third in the islets of rats fed 8.7% protein. The activity of mitochondrial glycerophosphate dehydrogenase was similar in islets of rats fed the 8.7% protein diet and those fed the control diet. The activity of Ca-independent phospholipase A2 was increased fourfold in the islets of rats fed 8.7% protein. It is concluded that impairment of glucose-induced insulin secretion in rats fed a low-protein diet may be caused by attenuation of islet phosphatidylinositol hydrolysis, and it is tentatively suggested that the increased activity of Ca-independent phospholipase A2 in islets of rats fed a low-protein diet may participate in the stimulation of apoptosis.

Imidazoline NNC77-0074 stimulates Ca2+-evoked exocytosis in INS-1E cells by a phospholipase A2-dependent mechanism.

We have previously demonstrated that the novel imidazoline compound (+)-2-(2-(4,5-dihydro-1H-imidazol-2-yl)-thiopene-2-yl-ethyl)-pyridine (NNC77-0074) increases insulin secretion from pancreatic beta-cells by stimulation of Ca(2+)-dependent exocytosis. Using capacitance measurements, we now show that NNC77-0074 stimulates exocytosis in clonal INS-1E cells. NNC77-0074-stimulated exocytosis was antagonised by the cytoplasmic phospholipase A(2) (cPLA(2)) inhibitors ACA and AACOCF(3) and in cells treated with antisense oligonucleotide against cPLA(2)alpha. NNC77-0074-evoked insulin secretion was likewise inhibited by ACA, AACOCF(3), and cPLA(2)alpha antisense oligonucleotide treatment. In pancreatic islets NNC77-0074 stimulated PLA(2) activity. We propose that cPLA(2)alpha plays an important role in the regulation of NNC77-0074-evoked exocytosis in insulin secreting beta-cells.

Phosphatidylinositol 4-kinase serves as a metabolic sensor and regulates priming of secretory granules in pancreatic beta cells.

Insulin secretion is controlled by the beta cell's metabolic state, and the ability of the secretory granules to undergo exocytosis increases during glucose stimulation in a membrane potential-independent fashion. Here, we demonstrate that exocytosis of insulin-containing secretory granules depends on phosphatidylinositol 4-kinase (PI 4-kinase) activity and that inhibition of this enzyme suppresses glucose-stimulated insulin secretion. Intracellular application of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] stimulated exocytosis by promoting the priming of secretory granules for release and increasing the number of granules residing in a readily releasable pool. Reducing the cytoplasmic ADP concentration in a way mimicking the effects of glucose stimulation activated PI 4-kinase and increased exocytosis whereas changes of the ATP concentration in the physiological range had little effect. The PI(4,5)P(2)-binding protein Ca(2+)-dependent activator protein for secretion (CAPS) is present in beta cells, and neutralization of the protein abolished both Ca(2+)- and PI(4,5)P(2)-induced exocytosis. We conclude that ADP-induced changes in PI 4-kinase activity, via generation of PI(4,5)P(2), represents a metabolic sensor in the beta cell by virtue of its capacity to regulate the release competence of the secretory granules.