Glucagon-like peptide 1 agonists and the development and growth of pancreatic beta-cells

Glucagon-like peptide 1 (GLP-1) is an intestine-derived insulinotropic hormone that stimulates glucose-dependent insulin production and secretion from pancreatic beta-cells. Other recognized actions of GLP-1 are to suppress glucagon secretion and hepatic glucose output, delay gastric emptying, reduce food intake, and promote glucose disposal in peripheral tissues. All of these actions are potentially beneficial for the treatment of type 2 diabetes mellitus. Several GLP-1 agonists are in clinical trials for the treatment of diabetes. More recently, GLP-1 agonists have been shown to stimulate the growth and differentiation of pancreatic beta-cells, as well as to exert cytoprotective, antiapoptotic effects on beta-cells. Recent evidence indicates that GLP-1 agonists act on receptors on pancreas-derived stem/progenitor cells to prompt their differentiation into beta-cells. These new findings suggest an approach to create beta-cells in vitro by expanding stem/progenitor cells and then to convert them into beta-cells by treatment with GLP-1. Thus GLP-1 may be a means by which to create beta-cells ex vivo for transplantation into patients with insulinopenic type 1 diabetes and severe forms of type 2 diabetes.     

Group VIA phospholipase A2 forms a signaling complex with the calcium/calmodulin-dependent protein kinase IIbeta expressed in pancreatic islet beta-cells

Insulin-secreting pancreatic islet beta-cells express a Group VIA Ca(2+)-independent phospholipase A(2) (iPLA(2)beta) that contains a calmodulin binding site and protein interaction domains. We identified Ca(2+)/calmodulin-dependent protein kinase IIbeta (CaMKIIbeta) as a potential iPLA(2)beta-interacting protein by yeast two-hybrid screening of a cDNA library using iPLA(2)beta cDNA as bait. Cloning CaMKIIbeta cDNA from a rat islet library revealed that one dominant CaMKIIbeta isoform mRNA is expressed by adult islets and is not observed in brain or neonatal islets and that there is high conservation of the isoform expressed by rat and human beta-cells. Binary two-hybrid assays using DNA encoding this isoform as bait and iPLA(2)beta DNA as prey confirmed interaction of the enzymes, as did assays with CaMKIIbeta as prey and iPLA(2)beta bait. His-tagged CaMKIIbeta immobilized on metal affinity matrices bound iPLA(2)beta, and this did not require exogenous calmodulin and was not prevented by a calmodulin antagonist or the Ca(2+) chelator EGTA. Activities of both enzymes increased upon their association, and iPLA(2)beta reaction products reduced CaMKIIbeta activity. Both the iPLA(2)beta inhibitor bromoenol lactone and the CaMKIIbeta inhibitor KN93 reduced arachidonate release from INS-1 insulinoma cells, and both inhibit insulin secretion. CaMKIIbeta and iPLA(2)beta can be coimmunoprecipitated from INS-1 cells, and forskolin, which amplifies glucose-induced insulin secretion, increases the abundance of the immunoprecipitatable complex. These findings suggest that iPLA(2)beta and CaMKIIbeta form a signaling complex in beta-cells, consistent with reports that both enzymes participate in insulin secretion and that their expression is coinduced upon differentiation of pancreatic progenitor to endocrine progenitor cells.     

Pancreatic insulin content regulation by the estrogen receptor ER alpha

The function of pancreatic beta-cells is the synthesis and release of insulin, the main hormone involved in blood glucose homeostasis. Estrogen receptors, ER alpha and ER beta, are important molecules involved in glucose metabolism, yet their role in pancreatic beta-cell physiology is still greatly unknown. In this report we show that both ER alpha and ER beta are present in pancreatic beta-cells. Long term exposure to physiological concentrations of 17beta-estradiol (E2) increased beta-cell insulin content, insulin gene expression and insulin release, yet pancreatic beta-cell mass was unaltered. The up-regulation of pancreatic beta-cell insulin content was imitated by environmentally relevant doses of the widespread endocrine disruptor Bisphenol-A (BPA). The use of ER alpha and ER beta agonists as well as ER alphaKO and ER betaKO mice suggests that the estrogen receptor involved is ER alpha. The up-regulation of pancreatic insulin content by ER alpha activation involves ERK1/2. These data may be important to explain the actions of E2 and environmental estrogens in endocrine pancreatic function and blood glucose homeostasis.     

Retinoic acid promotes the generation of pancreatic endocrine progenitor cells and their further differentiation into beta-cells

The identification of secreted factors that can selectively stimulate the generation of insulin producing beta-cells from stem and/or progenitor cells represent a significant step in the development of stem cell-based beta-cell replacement therapy. By elucidating the molecular mechanisms that regulate the generation of beta-cells during normal pancreatic development such putative factors may be identified. In the mouse, beta-cells increase markedly in numbers from embryonic day (e) 14.5 and onwards, but the extra-cellular signal(s) that promotes the selective generation of beta-cells at these stages remains to be identified. Here we show that the retinoic acid (RA) synthesizing enzyme Raldh1 is expressed in developing mouse and human pancreas at stages when beta-cells are generated. We also provide evidence that RA induces the generation of Ngn3(+) endocrine progenitor cells and stimulates their further differentiation into beta-cells by activating a program of cell differentiation that recapitulates the normal temporal program of beta-cell differentiation.     

Modulation of the pancreatic islet beta-cell-delayed rectifier potassium channel Kv2.1 by the polyunsaturated fatty acid arachidonate

Glucose stimulates both insulin secretion and hydrolysis of arachidonic acid (AA) esterified in membrane phospholipids of pancreatic islet beta-cells, and these processes are amplified by muscarinic agonists. Here we demonstrate that nonesterified AA regulates the biophysical activity of the pancreatic islet beta-cell-delayed rectifier channel, Kv2.1. Recordings of Kv2.1 currents from INS-1 insulinoma cells incubated with AA (5 mum) and subjected to graded degrees of depolarization exhibit a significantly shorter time-to-peak current interval than do control cells. AA causes a rapid decay and reduced peak conductance of delayed rectifier currents from INS-1 cells and from primary beta-cells isolated from mouse, rat, and human pancreatic islets. Stimulating mouse islets with AA results in a significant increase in the frequency of glucose-induced [Ca(2+)] oscillations, which is an expected effect of Kv2.1 channel blockade. Stimulation with concentrations of glucose and carbachol that accelerate hydrolysis of endogenous AA from islet phosphoplipids also results in accelerated Kv2.1 inactivation and a shorter time-to-peak current interval. Group VIA phospholipase A(2) (iPLA(2)beta) hydrolyzes beta-cell membrane phospholipids to release nonesterified fatty acids, including AA, and inhibiting iPLA(2)beta prevents the muscarinic agonist-induced accelerated Kv2.1 inactivation. Furthermore, glucose and carbachol do not significantly affect Kv2.1 inactivation in beta-cells from iPLA(2)beta(-/-) mice. Stably transfected INS-1 cells that overexpress iPLA(2)beta hydrolyze phospholipids more rapidly than control INS-1 cells and also exhibit an increase in the inactivation rate of the delayed rectifier currents. These results suggest that Kv2.1 currents could be dynamically modulated in the pancreatic islet beta-cell by phospholipase-catalyzed hydrolysis of membrane phospholipids to yield non-esterified fatty acids, such as AA, that facilitate Ca(2+) entry and insulin secretion.     

Generation of insulin-expressing cells from mouse embryonic stem cells

The therapeutic potential of transplantation of insulin-secreting pancreatic beta-cells has stimulated interest in using pluripotent embryonic stem (ES) cells as a starting material from which to generate insulin secreting cells in vitro. Mature beta-cells are endodermal in origin so most reported differentiation protocols rely on the identification of endoderm-specific markers. However, endoderm development is an early event in embryogenesis that produces cells destined for the gut and associated organs in the embryo, and for the development of extra-embryonic structures such as the yolk sac. We have demonstrated that mouse ES cells readily differentiate into extra-embryonic endoderm in vitro, and that these cell populations express the insulin gene and other functional elements associated with beta-cells. We suggest that the insulin-expressing cells generated in this and other studies are not authentic pancreatic beta-cells, but may be of extra-embryonic endodermal origin.     

Downregulation of the voltage-dependent calcium channel (VDCC) beta-subunit mRNAs in pancreatic islets of type 2 diabetic rats

The present study was undertaken to determine whether altered expression of the VDCC beta-subunits in pancreatic beta-cells could play a role in the changes in beta-cell sensitivity to glucose that occur with diabetes. Application of competitive RT-PCR procedure revealed that in normal Wistar rats, LETO and prediabetic OLETF rats, the beta(2)-subunit mRNA levels were 60-200-fold greater than the levels for the beta(3)-subunit. These findings suggest that the beta(2)-subunit as well as the beta-cell type VDCC1 alpha(1)-subunit may be the predominant form of the VDCC expressed in pancreatic beta-cells. The levels of mRNA encoding the beta-subunits and the beta-cell type alpha(1)-subunit as well as insulin were significantly reduced in diabetic rats. Perfusion experiments revealed that diabetic rats showed the higher basal insulin secretion and profoundly impaired insulin secretory responses to glucose compared with non-diabetic rats. Alternatively, impaired insulin secretory responses to glucose in high dose glucose-infused rats were recovered partly with the elevation of mRNA levels of the VDCC beta(2)- and beta(3)-subunits as well as the alpha(1)-subunit by the treatment with diazoxide. Thus, considering the possibility that the most striking effect of the VDCC alpha(1) beta-subunit coexpression in pancreatic beta-cells might occur on activation kinetics like the skeletal muscle, the impairment of further activation of the VDCCs to acute glucose challenge caused by the reduced expressions of the alpha(1) beta-subunits mRNAs in type 2 diabetic animals might be at least partly associated with the alterations in beta-cell sensitivity to glucose.     

Overexpression of the aldose reductase gene induces apoptosis in pancreatic beta-cells by causing a redox imbalance.

To determine the role of the polyol metabolizing pathway under hyperglycemic conditions, the effects of aldose reductase (AR) on the cellular functions of pancreatic beta-cells were examined. Stable transfectants of rat AR cDNA were obtained with a pancreatic beta-cell line, HIT, in which a negligible amount of AR was originally expressed. Overproduction of AR triggered DNA fragmentation, as judged with the TUNEL method and agarose gel electrophoresis. Morphological analysis by electron microscopy also clearly showed apoptosis of the AR-overexpressing HIT cells. Induction by interleukin-1beta of gene expression such as those of an inducible form of nitric oxide synthase (NOS-II) and Mn-superoxide dismutase (Mn-SOD), was much lower in the transfectants than in the control cells, while the expression of constitutively expressed genes such as those for Cu,Zn-superoxide dismutase and insulin was not changed. The susceptibility to interleukin-1beta stimulation of the expression of the NOS II and Mn-SOD genes was due to suppressed NF-kappaB activity, which is essential for the expression of these genes. In addition, the intracellular NADPH/NADP+ ratio was considerably lower in the AR-transfected cells than in control cells. Thus, the overexpression of AR in pancreatic beta-cells induced apoptosis that may be caused by a redox imbalance.     

Neogenesis and proliferation of beta-cells induced by human betacellulin gene transduction via retrograde pancreatic duct injection of an adenovirus vector

Betacellulin (BTC) has been shown to have a role in the differentiation and proliferation of beta-cells both in vitro and in vivo. We administered a human betacellulin (hBTC) adenovirus vector to male ICR mice via retrograde pancreatic duct injection. As a control, we administered a beta-galactosidase adenovirus vector. In the mice, hBTC protein was mainly overexpressed by pancreatic duct cells. On immunohistochemical analysis, we observed features of beta-cell neogenesis as newly formed insulin-positive cells in the duct cell lining or islet-like cell clusters (ICCs) closely associated with the ducts. The BrdU labeling index of beta-cells was also increased by the betacellulin vector compared with that of control mice. These results indicate that hBTC gene transduction into adult pancreatic duct cells promoted beta-cell differentiation (mainly from duct cells) and proliferation of pre-existing beta-cells, resulting in an increase of the beta-cell mass that improved glucose tolerance in diabetic mice.     

The expression and function of a group VIA calcium-independent phospholipase A2 (iPLA2beta) in beta-cells

Many cells express a Group VIA phospholipase A2, designated iPLA2beta, that does not require calcium for activation, is stimulated by ATP, and is sensitive to inhibition by a bromoenol lactone suicide substrate (BEL). Studies in various cell systems have led to the suggestion that iPLA2beta has a role in phospholipid remodeling, signal transduction, cell proliferation, and apoptosis. We have found that pancreatic islets, beta-cells, and glucose-responsive insulinoma cells express an iPLA2beta that participates in glucose-stimulated insulin secretion but is not involved in membrane phospholipid remodeling. Additionally, recent studies reveal that iPLA2beta is involved in pathways that contribute to beta-cell proliferation and apoptosis, and that various phospholipid-derived mediators are involved in these processes. Detailed characterization of the enzyme suggests that the beta-cells express multiple isoforms of iPLA2beta, and we hypothesize that these participate in different cellular functions.     

Fatty acids potentiate interleukin-1beta toxicity in the beta-cell line INS-1E

Evidence for "lipotoxicity," i.e., negative effects of fatty acids (FA) on pancreatic beta-cells is incomplete. Here, we tested whether non-toxic concentrations of FA potentiate toxic effects of interleukin-1beta (IL-1beta). Culture of INS-1E clonal beta-cells for 2-6 days with 70 microM docosahexaenoic acid (DHA), eicosapentaenoic acid, arachidonic acid, 0.1mM linoleic acid, or 0.1-0.2mM oleic acid exerted no or minor negative effects on cell viability (MTT assay). Viability was reduced by 0.5 ng/ml IL-1beta. All tested FA significantly aggravated this effect after 6 days of culture. IL-1beta also exerted negative effects on cellular insulin content and DHA and oleic acid aggravated these effects. L-NAME, an inhibitor of constitutive nitric oxide (NO) synthase, reduced the negative effects of IL-1beta per se but did not abolish the potentiating effects of FA. CONCLUSION: FA potentiate toxic effects of IL-1beta on beta-cells by mechanisms that include NO-independent ones.     

Hyperinsulinism in infancy: understanding the pathophysiology

Hyperinsulinism in infancy (HI) is the commonest cause of persistent and recurrent hypoglycaemia in the infancy and childhood period. HI is a heterogeneous disorder with respect to clinical presentation, histology, molecular biology and genetics. Recent advances have provided unique insights into the pathophysiology of this intriguing disease as well as providing an understanding of the normal physiological and biochemical mechanisms regulating insulin secretion from pancreatic beta-cells. The histological differentiation of focal and diffuse forms of HI has radically changed the surgical management to this disease. So far mutations in five different genes have been described which lead to dysregulated insulin secretion from beta-cells. Despite these advances the genetic defect is still unknown in about 60% of cases.     

Association between interleukin-6 polymorphism and age-at-onset of type 1 diabetes. Epistatic influences of the tumor necrosis factor-alpha and interleukin-1beta polymorphisms

Multiple immune mediators have been mentioned as playing a role in the pathomechanism of type1 DM. Interleukin (IL)-1beta, and tumor necrosis factor (TNF)-alpha play a central role in the autoimmune destruction of pancreatic beta-cells, whereas IL-6 inhibits TNF-alpha secretion, and may have some protecting effects. In our study, we aimed to investigate the association between these three cytokines' single nucleotide polymorphisms (IL-6 gene G(-174)C, TNF-alpha gene G(-308)A and IL-1beta gene C(3954)T polymorphisms) and age-at-onset of type 1 diabetes mellitus (T1DM) in 165 diabetic children (median age: 17 years). Polymorphisms were determined using the PCR-RFLP method. We found that the age-at-onset of T1DM was significantly different in patients with a different IL-6 genotype (median age-at-onset of T1DM was: 8, 6 and 4.5 years in children with the (-174)GG, GC and CC genotypes, respectively; p < 0.01). Adjusted for TNF-alpha and IL-1beta polymorphisms, patients with a IL-6 (-174)CC genotype have a 3.0-fold (95% CI: 1.2-7.1) increased risk of developing diabetes before the age of 6 years than (-174)G allele carrier patients. However, we found this association to be present only in patients who carried the TNF-alpha (-308)A or IL-1beta (3954)T allele, i.e. in patients with high TNF-alpha and high IL-1beta producer genotypes. We suppose that in the case of high TNF-alpha and IL-1beta producer genotypes, elevated proinflammatory cytokine levels result in a higher production of IL-6 in (-174)G allele carrier patients. This elevated IL-6 level may have a protective effect against the development of T1DM and may delay the destruction of pancreatic beta-cells.     

Diabetes and tumor formation in transgenic mice expressing Reg I

To examine the effect of overexpressed regenerating gene (Reg) I on pancreatic beta-cells, we generated transgenic mice expressing Reg I in islets (Reg-Tg mice). Three lines of Reg-Tg mice were established. In line-1 Reg-Tg mice, the expression level of Reg I mRNA in islets was 7 times higher than those in lines 2 and 3 of Reg-Tg mice, and line 1 mice developed diabetes by apoptosis of beta-cells, as well as various malignant tumors. In addition to the decrease in beta-cells, compensatory islet regeneration and proliferation of ductal epithelial cells were observed in line-1 Reg-Tg mice. Because Reg I protein was secreted primarily into pancreatic ducts from acinar cells, it may primarily stimulate the proliferation of ductal epithelial cells, and not beta-cells, and their differentiation into islets. Moreover, the tumor-promoting activity of Reg I protein should be considered for its possible clinical applications.     

Interleukin-1 beta inhibits proinsulin conversion in rat beta-cells via a nitric oxide-dependent pathway.

Exposure of pancreatic beta-cells to interleukin-1 beta (IL-1 beta) alters their protein expression and phenotype. Previous work has shown that IL-1 beta inhibited proinsulin conversion in rat islets, but the mechanism of this inhibition remained unknown. To investigate this phenomenon further, we examined purified rat beta-cells for IL-1 beta-induced inhibition of proinsulin conversion and nitric oxide (NO)-dependency of this inhibitory process. Rat beta-cells were cultured for 24 h with or without IL-1 beta and the inducible-nitric-oxide-synthase (iNOS) inhibitor N(G)-methyl-L-arginine (NMA). Exposure to IL-1 beta suppressed proinsulin-1 and proinsulin-2 synthesis by more than 50 %. Conversion of both proinsulin isoforms was also delayed. The suppressive effects of IL-1 beta on proinsulin synthesis and conversion were prevented by addition of NMA. Exposure to IL-1 beta also decreased the expression of the proinsulin convertase (PC) PC2. This decrease in PC2 expression was NO-dependent. In conclusion, IL-1 beta inhibition of proinsulin conversion in rat beta-cells occurs via an NO-mediated pathway.