Search

SUMOylation reduces oxidative stress and preserves islet mass at the expense of robust insulin secretion. To investigate a role for the deSUMOylating enzyme sentrin-specific protease 1 (SENP1) following metabolic stress, we put pancreas/gut-specific SENP1 knockout (pSENP1-KO) mice on a high-fat diet (HFD). Male pSENP1-KO mice were more glucose intolerant following HFD than littermate controls but only in response to oral glucose. A similar phenotype was observed in females. Plasma glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1) responses were identical in pSENP1-KO and wild-type littermates, including the HFD-induced upregulation of GIP responses. Islet mass was not different, but insulin secretion and β-cell exocytotic responses to the GLP-1 receptor agonist exendin-4 (Ex4) and GIP were impaired in islets lacking SENP1. Glucagon secretion from pSENP1-KO islets was also reduced, so we generated β-cell–specific SENP1 KO mice. These phenocopied the pSENP1-KO mice with selective impairment in oral glucose tolerance following HFD, preserved islet mass expansion, and impaired β-cell exocytosis and insulin secretion to Ex4 and GIP without changes in cAMP or Ca2+ levels. Thus, β-cell SENP1 limits oral glucose intolerance following HFD by ensuring robust insulin secretion at a point downstream of incretin signaling.

Background & Aims Patients with ulcerative colitis have low concentrations of the major membrane lipid phosphatidylcholine (PC) in gastrointestinal mucus, suggesting that defects in colonic PC metabolism might be involved in the development of colitis. To determine the precise role that PC plays in colonic barrier function, we examined mice with intestinal epithelial cell (IEC)-specific deletion of the rate-limiting enzyme in the major pathway for PC synthesis: cytidine triphosphate:phosphocholine cytidylyltransferase-α (CTαIKO mice). Methods Colonic tissue of CTαIKO mice and control mice was analyzed by histology, immunofluorescence, electron microscopy, quantitative polymerase chain reaction, Western blot, and thin-layer chromatography. Histopathologic colitis scores were assigned by a pathologist blinded to the experimental groupings. Intestinal permeability was assessed by fluorescein isothiocyanate–dextran gavage and fecal microbial composition was analyzed by sequencing 16s ribosomal RNA amplicons. Subsets of CTαIKO mice and control mice were treated with dietary PC supplementation, antibiotics, or 4-phenylbutyrate. Results Inducible loss of CTα in the intestinal epithelium reduced colonic PC concentrations and resulted in rapid and spontaneous colitis with 100% penetrance in adult mice. Colitis development in CTαIKO mice was traced to a severe and unresolving endoplasmic reticulum stress response in IECs with altered membrane phospholipid composition. This endoplasmic reticulum stress response was linked to the necroptotic death of IECs, leading to excessive loss of goblet cells, formation of a thin mucus barrier, increased intestinal permeability, and infiltration of the epithelium by microbes. Conclusions Maintaining the PC content of IEC membranes protects against colitis development in mice, showing a crucial role for IEC phospholipid equilibrium in colonic homeostasis. SRA accession number: PRJNA562603., Graphical abstract

SUMOylation reduces oxidative stress and preserves islet mass at the expense of robust insulin secretion. To investigate a role for the deSUMOylating enzyme sentrin-specific protease 1 (SENP1) following metabolic stress, we put pancreas/gut-specific SENP1 knockout mice (pSENP1-KO) on a high fat diet (HFD). Male pSENP1-KO mice were more glucose intolerant following HFD than littermate controls, but only in response to oral glucose. A similar phenotype was observed in females. Plasma glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like-peptide 1 (GLP-1) responses were identical in pSENP1-KO and -WT littermates, including the HFD-induced upregulation of GIP responses. Islet mass was not different, but insulin secretion and β-cell exocytotic responses to the GLP-1 receptor agonist Exendin-4 (Ex4) and GIP were impaired in islets lacking SENP1. Glucagon secretion from pSENP1-KO islets was also reduced, so we generated β-cell-specific SENP1 knockout mice (βSENP1-KO). These phenocopied the pSENP1-KO mice with selective impairment in oral glucose tolerance following HFD, preserved islet mass expansion, and impaired β-cell exocytosis and insulin secretion to Ex4 and GIP without changes in cAMP or Ca2+ levels. Thus, β-cell SENP1 limits oral glucose intolerance following HFD by ensuring robust insulin secretion at a point downstream of incretin signaling.

Neonatal antibiotics administered to human infants initiate gut microbiota dysbiosis that may have long-term effects on body weight and metabolism. We examined antibiotic-induced adaptations in pancreatic islets of the piglet, a well-accepted model of human infant microbiota and pancreas development. Neonatal piglets randomized to amoxicillin [30 mg/kg body wt/day; n = 7, antibiotic (ANTI)] or placebo [vehicle control; n = 7, control (CON)] from postnatal day ( PND) 0– 13 were euthanized at PND7, 14, and 49. The metabolic phenotype along with functional, immunohistological, and transcriptional phenotypes of the pancreatic islets were studied. The gut microbiome was characterized by 16S rRNA gene sequencing, and microbial metabolites and microbiome-sensitive host molecules were measured. Compared with CON, ANTI PND7 piglets had elevated transcripts of genes involved in glucagon-like peptide 1 ((GLP-1) synthesis or signaling in islets ( P < 0.05) coinciding with higher plasma GLP-1 ( P = 0.11), along with increased tumor necrosis factor α ( Tnf) ( P < 0.05) and protegrin 1 ( Npg1) ( P < 0.05). Antibiotic-induced relative increases in Escherichia, Coprococcus, Ruminococcus, Dehalobacterium, and Oscillospira of the ileal microbiome at PND7 normalized after antibiotic withdrawal. In ANTI islets at PND14, the expression of key regulators pancreatic and duodenal homeobox 1 ( Pdx1), insulin-like growth factor-2 ( Igf2), and transcription factor 7-like 2 ( Tcf7l2) was downregulated, preceding a 40% reduction of β-cell area ( P < 0.01) and islet insulin content at PND49 ( P < 0.05). At PND49, a twofold elevated plasma insulin concentration ( P = 0.07) was observed in ANTI compared with CON. We conclude that antibiotic treatment of neonatal piglets elicited gut microbial changes accompanied by phasic alterations in key regulatory genes in pancreatic islets at PND7 and 14. By PND49, reduced β-cell area and islet insulin content were accompanied by elevated nonfasted insulin despite normoglycemia, indicative of islet stress.

SUMOylation reduces oxidative stress and preserves islet mass; but this happens at the expense of robust insulin secretion. To investigate a role for the deSUMOylating enzyme sentrin-specific protease 1 (SENP1) in glycemia following metabolic stress, we put pancreas/gut-specific SENP1 knockout mice (pSENP1-KO) on an 8-10-week high fat diet (HFD). Male pSENP1-KO mice were more glucose intolerant following HFD than littermate controls, but this was only obvious in response to oral glucose, and a similar but milder phenotype was observed in females. Plasma incretin responses were identical, and glucose-dependent insulinotropic polypeptide (GIP) was equally upregulated after HFD, in pSENP1-KO and - WT littermates. Islet mass was not different, but insulin secretion and β-cell exocytotic responses to Exendin4 (Ex4) and GIP were impaired in islets lacking SENP1. Glucagon secretion from pSENP1-KO islets was also reduced, consistent with the expected SENP1 knockout in all islet cells, so we generated β-cell–specific SENP1 knockout mice (βSENP1-KO). These phenocopied the pSENP1-KO mice with selective impairment in oral glucose tolerance following HFD, preserved islet mass expansion, and impaired β-cell exocytosis and insulin secretion to Ex4 and GIP. Thus, β-cell SENP1 limits glucose intolerance following HFD by ensuring a robust facilitation of insulin secretion by incretins such as GIP.

Cytidine triphosphate:phosphocholine cytidylyltransferase-α (CTα) is the rate limiting enzyme in the major pathway for de novo phosphatidylcholine (PC) synthesis. When CTα is deleted specifically in intestinal epithelial cells of adult mice (CTα

Hyperinsulinemic hypoglycemia syndrome (HIHG) is a rare complication of roux-en-Y gastric bypass surgery. The pathology is associated with an excessive function of pancreatic beta-cells, and requires pancreas resection in patients that are recalcitrant to nutritional and pharmacological interventions. The exact prevalence is not clearly understood and the underlying mechanisms not yet fully characterized. We herein sought to perform histological and molecular examination of pancreatic sections obtained from a patient who developed HIHG as a complication of gastric bypass compared to 3 weight-matched controls. We studied markers of cellular replication and beta-cell differentiation by immunohistochemistry and immunofluorescence. HIHG after gastric bypass was characterized by a profound increase in beta-cell mass. Cellular proliferation was increased in islets and ducts compared to controls, suggesting unrestrained proliferation in HIHG. We also detected beta-cell differentiation markers in duct cells and occasional duct cells displaying both insulin and glucagon immunoreactivity. These histological observations suggest that beta-cell differentiation from ductal progenitor cells could also underly beta-cell mass expansion in HIHG. Altogether, our results can be construed to demonstrate that HIHG after gastric bypass is characterized by abnormal beta-cell mass expansion, resulting from both unrestrained beta-cell replication and neogenesis.

De novo phosphatidylcholine (PC) synthesis via CTP:phosphocholine cytidylyltransferase-alpha (CT alpha) is required for VLDL secretion. To determine the precise role of de novo PC synthesis in intestinal lipid metabolism, we deleted CT alpha exclusively in the intestinal epithelium of mice (CT alpha(IKO) mice). When fed a chow diet, CT alpha(IKO) mice showed normal fat absorption despite a similar to 30% decrease in intestinal PC concentrations relative to control mice, suggesting that biliary PC can fully support chylomicron secretion under these conditions. However, when fed a high-fat diet, CT alpha(IKO) mice showed impaired passage of FAs and cholesterol from the intestinal lumen into enterocytes. Impaired intestinal lipid uptake in CT alpha(IKO) mice was associated with lower plasma triglyceride concentrations, higher plasma glucagon-like peptide 1 and peptide YY, and disruption of intestinal membrane lipid transporters after a high-fat meal relative to control mice. Unexpectedly, biliary bile acid and PC secretion was enhanced in CT alpha(IKO) mice due to a shift in expression of bile-acid transporters to the proximal intestine, indicative of accelerated enterohepatic cycling. These data show that intestinal de novo PC synthesis is required for dietary lipid absorption during high-fat feeding and that the reacylation of biliary lyso-PC cannot compensate for loss of CT alpha under these conditions.

Heat shock factor protein 1 (HSF1) is a transcription factor that regulates the expression of key molecular chaperones, thereby orchestrating the cellular response to stress. This system was recently implicated in the control of insulin sensitivity and is therefore being scrutinised as a novel therapeutic avenue for type 2 diabetes. However, the regulation and biological actions of HSF1 in beta cells remain elusive. Herein, we sought to investigate the regulation of HSF1 in pancreatic beta cells and to study its potential role in cell survival. We exposed human islets and beta cell lines to glucolipotoxicity and thapsigargin. HSF1 activity was evaluated by gel shift assay. HSF1 acetylation and interaction with the protein acetylase cAMP response element binding protein (CBP) were investigated by western blot. We measured the expression of HSF1 and its canonical targets in islets from Goto–Kakizaki (GK) rat models of diabetes and delineated the effects of HSF1 acetylation using mutants mimicking constitutive acetylation and deacetylation of the protein. Glucolipotoxicity promoted HSF1 acetylation and interaction with CBP. Glucolipotoxicity-induced HSF1 acetylation inhibited HSF1 DNA binding activity and decreased the expression of its target genes. Restoration of HSF1 activity in beta cells prevented glucolipotoxicity-induced endoplasmic reticulum stress and apoptosis. However, overexpression of a mutant protein (K80Q) mimicking constitutive acetylation of HSF1 failed to confer protection against glucolipotoxicity. Finally, we showed that expression of HSF1 and its target genes were altered in islets from diabetic GK rats, suggesting that this pathway could participate in the pathophysiology of diabetes and constitutes a potential site for therapeutic intervention. Our results unravel a new mechanism by which HSF1 inhibition is required for glucolipotoxicity-induced beta cell apoptosis. Restoring HSF1 activity may represent a novel strategy for the maintenance of a functional beta cell mass. Our study supports the therapeutic potential of HSF1/heat shock protein-targeting agents in diabetes treatment.

The Nr4a subfamily of nuclear receptor comprises three members in mammalian cells: Nur77/Nr4a1, Nurr1/Nr4a2, and Nor1/Nr4a3. Nr4a proteins play key roles in the regulation of glucose homeostasis in peripheral metabolic tissues. However, their biological functions in β-cells remain relatively uncharacterized. Here we sought to investigate the potential role of Nor1 in the regulation of β-cell mass and, in particular, β-cell survival/apoptosis. We used histological analysis to examine the consequences of genetic deletion of either Nur77 and Nor1 on β-cell mass, investigated the expression patterns of Nr4as in human islets and INS cells and performed gain- and loss-of-function experiments to further characterize the role of Nor1 in β-cell apoptosis. Surprisingly, Nor1 knockout mice displayed increased β-cell mass, whereas mice with genetic deletion of Nur77 did not exhibit any significant differences compared with their WT littermates. The increase in β-cell mass in Nor1 knockout mice was accompanied by improved glucose tolerance. A gene expression study performed in both human islets and INS cells revealed that Nor1 expression is significantly increased by pro-inflammatory cytokines and, to a lesser extent, by elevated concentrations of glucose. Nor1 overexpression in both INS and human islet cells caused apoptosis, whereas siRNA-mediated Nor1 knockdown prevented cytokine-induced β-cell death. Finally, Nor1 expression was up-regulated in islets of individuals with type 2 diabetes. Altogether, our results uncover that Nor1 negatively regulates β-cell mass. Nor1 represents a promising molecular target in diabetes treatment to prevent β-cell destruction.

Nor1, the third member of the Nr4a subfamily of nuclear receptor, is garnering increased interest in view of its role in the regulation of glucose homeostasis. Our previous study highlighted a proapoptotic role of Nor1 in pancreatic beta cells and showed that Nor1 expression was increased in islets isolated from type 2 diabetic individuals, suggesting that Nor1 could mediate the deterioration of islet function in type 2 diabetes. However, the mechanism remains incompletely understood. We herein investigated the subcellular localization of Nor1 in INS832/13 cells and dispersed human beta cells. We also examined the consequences of Nor1 overexpression on mitochondrial function and morphology. Our results show that, surprisingly, Nor1 is mostly cytoplasmic in beta cells and undergoes mitochondrial translocation upon activation by proinflammatory cytokines. Mitochondrial localization of Nor1 reduced glucose oxidation, lowered ATP production rates, and inhibited glucose-stimulated insulin secretion. Western blot and microscopy images revealed that Nor1 could provoke mitochondrial fragmentation via mitophagy. Our study unveils a new mode of action for Nor1, which affects beta-cell viability and function by disrupting mitochondrial networks.

Type 2 diabetes mellitus is a disorder characterized by insulin resistance and a relative deficit in insulin secretion, both of which result in elevated blood glucose. Understanding the molecular mechanisms underlying the pathophysiology of diabetes could lead to the development of new therapeutic approaches. An ever-growing body of evidence suggests that members of the NR4A family of nuclear receptors could play a pivotal role in glucose homeostasis. This review aims to present and discuss advances so far in the evaluation of the potential role of NR4A in the regulation of glucose homeostasis and the development of type 2 diabetes.

OBJECTIVE The glucoincretin hormone glucagon-like peptide 1 (GLP-1) enhances glucose-stimulated insulin secretion and stimulates pancreatic β-cell mass expansion. We have previously shown that the forkhead transcription factor FoxO1 is a prominent transcriptional effector of GLP-1 signaling in the β-cell. FoxO1 activity is subject to a complex regulation by Akt-dependent phosphorylation and SirT1-mediated deacetylation. In this study, we aimed at investigating the potential role of SirT1 in GLP-1 action. RESEARCH DESIGN AND METHODS FoxO1 acetylation levels and binding to SirT1 were studied by Western immunoblot analysis in INS832/13 cells. SirT1 activity was evaluated using an in vitro deacetylation assay and correlated with the NAD+-to-NADH ratio. The implication of SirT1 in GLP-1–induced proliferation was investigated by BrdU incorporation assay. Furthermore, we determined β-cell replication and mass in wild-type and transgenic mice with SirT1 gain of function after daily administration of exendin-4 for 1 week. RESULTS Our data show that GLP-1 increases FoxO1 acetylation, decreases the binding of SirT1 to FoxO1, and stunts SirT1 activity in β-INS832/13 cells. GLP-1 decreases both the NAD+-to-NADH ratio and SirT1 expression in INS cells and isolated islets, thereby providing possible mechanisms by which GLP-1 could modulate SirT1 activity. Finally, the action of GLP-1 on β-cell mass expansion is abolished in both transgenic mice and cultured β-cells with increased dosage of SirT1. CONCLUSIONS Our study shows for the first time that the glucoincretin hormone GLP-1 modulates SirT1 activity and FoxO1 acetylation in β-cells. We also identify SirT1 as a negative regulator of β-cell proliferation.

Insulin resistance and relative insulin deficiency contribute to the pathogenesis of type 2 diabetes. Defective insulin secretion from pancreatic β-cells results from the progressive deterioration of pancreatic β-cell mass and function. Glucagon-like peptide 1 (GLP-1), an incretin hormone secreted by intestinal L cells in response to a meal, improves glycemic control in patients with type 2 diabetes by addressing both the insulin secretion defect as well as the decline in β-cell mass. These observations fostered the development of new therapeutic agents targeting GLP-1 signaling. This review gives an overview our current knowledge of the molecular mechanisms by which GLP-1 enhances β-cell mass and function.

Gain-of-function mutations in NOTCH1 are common in T-cell lymphoblastic leukemias and lymphomas (T-ALL), making this receptor a promising target for drugs such as γ-secretase inhibitors, which block a proteolytic cleavage required for NOTCH1 activation. However, the enthusiasm for these therapies has been tempered by tumor resistance and the paucity of information on the oncogenic programs regulated by oncogenic NOTCH1. Here we show that NOTCH1 regulates the expression of PTEN (encoding phosphatase and tensin homolog) and the activity of the phosphoinositol-3 kinase (PI3K)-AKT signaling pathway in normal and leukemic T cells. Notch signaling and the PI3K-AKT pathway synergize in vivo in a Drosophila melanogaster model of Notch-induced tumorigenesis, and mutational loss of PTEN is associated with human T-ALL resistance to pharmacological inhibition of NOTCH1. Overall, these findings identify transcriptional control of PTEN and regulation of the PI3K-AKT pathway as key elements of the leukemogenic program activated by NOTCH1 and provide the basis for the design of new therapeutic strategies for T-ALL., This work was supported by the Fondazione Città Della Speranza (G. Basso), the Spanish Ministerio de Educacion y Ciencia and Asociación Española Contra el Cancer (M.D.), NIH grant CA120196, the WOLF Foundation, the Charlotte Geyer Foundation, the Golfers Against Cancer Foundation and the Leukemia and Lymphoma Society (grant 1287-08) (A.A.F.). Adolfo Ferrando is a Leukemia & Lymphoma Society Scholar.

Activating transcription factor 3 (ATF3) is a stress-inducible gene and encodes a member of the ATF/CREB family of transcription factors. However, the physiological significance of ATF3 induction by stress signals is not clear. In this report, we describe several lines of evidence supporting a role of ATF3 in stress-induced beta-cell apoptosis. First, ATF3 is induced in beta cells by signals relevant to beta-cell destruction: proinflammatory cytokines, nitric oxide, and high concentrations of glucose and palmitate. Second, induction of ATF3 is mediated in part by the NF-kappaB and Jun N-terminal kinase/stress-activated protein kinase signaling pathways, two stress-induced pathways implicated in both type 1 and type 2 diabetes. Third, transgenic mice expressing ATF3 in beta cells develop abnormal islets and defects secondary to beta-cell deficiency. Fourth, ATF3 knockout islets are partially protected from cytokine- or nitric oxide-induced apoptosis. Fifth, ATF3 is expressed in the islets of patients with type 1 or type 2 diabetes, and in the islets of nonobese diabetic mice that have developed insulitis or diabetes. Taken together, our results suggest ATF3 to be a novel regulator of stress-induced beta-cell apoptosis.

We have provided evidence that glucagon-like peptide-1, a potential therapeutic agent in the treatment of diabetes, activates phosphatidylinositol-3 kinase/protein kinase B signalling in the pancreatic beta cell. Since this pathway promotes cell survival in a variety of systems, we tested whether glucagon-like peptide-1 protects beta cells against cell death induced by elevated glucose and/or non-esterified fatty acids.Human islets and INS832/13 cells were cultured at glucose concentrations of 5 or 25 mmol/l in the presence or absence of palmitate. Apoptosis was evaluated by monitoring DNA fragmentation and chromatin condensation. Wild-type and protein kinase B mutants were overexpressed in INS832/13 cells using adenoviruses. Nuclear factor-kappa B DNA binding was assayed by electrophoretic mobility shift assay.In human pancreatic beta cells and INS832/13 cells, glucagon-like peptide-1 prevented beta cell apoptosis induced by elevated concentrations of (i) glucose (glucotoxicity), (ii) palmitate (lipotoxicity) and (iii) both glucose and palmitate (glucolipotoxicity). Overexpression of a dominant-negative protein kinase B suppressed the anti-apoptotic action of glucagon-like peptide-1 in INS832/13 cells, whereas a constitutively active protein kinase B prevented beta cell apoptosis induced by elevated glucose and palmitate. Glucagon-like peptide-1 enhanced nuclear factor-kappa B DNA binding activity and stimulated the expression of inhibitor of apoptosis protein-2 and Bcl-2, two anti-apoptotic genes under the control of nuclear factor-kappa B. Inhibition of nuclear factor-kappa B by BAY 11-7082 abolished the prevention of glucolipotoxicity by glucagon-like peptide-1.The results demonstrate a potent protective effect of glucagon-like peptide-1 on beta cell gluco-, lipo- and glucolipotoxicity. This effect is mediated via protein kinase B activation and possibly its downstream target nuclear factor-kappa B.

We previously provided evidence that glucagon-like peptide 1 (GLP-1) induces pancreatic beta-cell growth nonadditively with glucose in a phosphatidylinositol (PI) 3-kinase- and protein kinase C zeta-dependent manner. However, the exact mechanism by which the GLP-1 receptor (GLP-1R), a member of the G protein-coupled receptor (GPCR) superfamily, activates the PI 3-kinase signaling pathway to promote beta-cell growth remains unknown. We hypothesized that the GLP-1R could activate PI 3-kinase and promote beta-cell proliferation through transactivation of the epidermal growth factor (EGF) receptor (EGFR), an event possibly linked to GPCRs via activation of c-Src and the production of putative endogenous EGF-like ligands. Both the c-Src inhibitor PP1 and the EGFR-specific inhibitor AG1478 blocked GLP-1-induced [(3)H]thymidine incorporation in INS(832/13) cells as well as in isolated rat islets, while only AG1478 inhibited the proliferative action of betacellulin (BTC), an EGFR agonist. Both compounds also suppressed GLP-1-induced PI 3-kinase activation. A time-dependent increase in tyrosine phosphorylation of the EGFR in response to GLP-1 was observed in INS(832/13) cells. This transactivation of the EGFR was sensitive to both the pharmacological agents PP1 and AG1478. The action of GLP-1 and BTC on INS cell proliferation was found to be not additive. Overexpression of a dominant-negative EGFR in INS cells with a retroviral expression vector curtailed GLP-1-induced beta-cell proliferation. GLP-1 treatment of INS cells caused a decrease in cell surface-associated BTC, as shown by FACS analysis. Also, the metalloproteinase inhibitor GM6001 and an anti-BTC neutralizing antibody suppressed the GLP-1 proliferative effect. Finally, coculturing the prostatic cancer cell line LNCaP that lacks GLP-1 responsiveness with INS cells increased LNCaP cell proliferation in the presence of GLP-1, thus revealing that INS cells secrete a growth factor in response to GLP-1. GM6001 and an anti-BTC neutralizing antibody suppressed increased LNCaP cell proliferation in the presence of GLP-1 in the coculture experiments. The results are consistent with a model in which GLP-1 increases PI 3-kinase activity and enhances beta-cell proliferation via transactivation of the EGFR that would require the proteolytic processing of membrane-anchored BTC or other EGF-like ligands.

The leucine-to-glutamate (Leu→Glu) pathway, which metabolizes the carbon atoms of l-leucine to form l-glutamate, was studied by incubation of rat tissue segments with l-[U-14C]leucine and estimation of the [14C]glutamate formed. Metabolism of the leucine carbon chain occurs in most rat tissues, but maximal activity of the Leu→Glu pathway for glutamate formation is limited to the thoracic aorta and pancreas. In rat aorta, the Leu→Glu pathway functions to relax the underlying smooth muscle; its functions in the pancreas are unknown. This report characterizes the Leu→Glu pathway of rat pancreas and develops methods to examine its functions. Pancreatic segments effect net formation of glutamate on incubation with l-leucine, l-glutamine, or a mix of 18 other plasma amino acids at their concentrations in normal rat plasma. Glutamate formed from leucine remains mainly in the tissue, whereas that from glutamine enters the medium. The pancreatic Leu→Glu pathway uses the leucine carbons for net glutamate formation; the α-amino group is not used; the stoichiometry is as follows: 1 mol of leucine yields 2 mol of glutamate (2 leucine carbons per glutamate) plus 2 mol of CO2. Comparison of the Leu→Glu pathway in preparations of whole pancreatic segments, isolated acini, and islets of Langerhans localizes it in the acini; relatively high activity is found in cultures of the AR42J cell line and very little in the INS-1 832/13 cell line. Pancreatic tissue glutamate concentration is homeostatically regulated in the range of ∼1–3 μmol/g wet wt. l-Valine and leucine ethyl, benzyl, and tert-butyl esters inhibit the Leu→Glu pathway without decreasing tissue total glutamate.

The tumor suppressor gene ST18 was originally characterized as the third member of the neural zinc finger transcription factor family. However, little is known about its biological functions. Herein, we demonstrate that, in the pancreas, ST18 expression is restricted to endocrine cells. The detection of ST18 expression in pancreatic β-cells prompted us to investigate its regulation and its role in β-cell mass and function. We show that ST18 expression and activity are increased by cytotoxic concentrations of fatty acids and cytokines in INS832/13 cells. Furthermore, ST18 is also increased in islets of diet-induced obese animals. Overexpression and RNA interference knockdown studies demonstrate that ST18 induces β-cell apoptosis and curtails β-cell replication. Finally, our data suggest that ST18 impairs insulin secretion. Taken together, our findings indicate that ST18 could represent a novel transcriptional mediator of lipotoxicity and cytokine-induced β-cell death. We suggest that genetic or pharmacologic manipulations of ST18 could help maintain a functional β-cell mass.

To better understand the link between fatty acid signaling and the pleiotropic effects of fatty acids in the pancreatic beta-cell, we investigated whether fatty acids regulate immediate-early response genes (IEGs) coding for transcription factors implicated in cell proliferation, differentiation, and apoptosis. Palmitate and oleate, but not long-chain polyunsaturated fatty acids, caused a pronounced accumulation of c-fos and nur-77 mRNAs in beta-cells (INS cells) to an extent similar to that produced by the protein kinase C (PKC) activator phorbol myristate acetate (PMA). The effect was dose dependent and occurred at concentrations between 0.1 and 0.5 mmol/l in the presence of 0.5% albumin. The action of the fatty acid occurred at the transcriptional level, and the mRNA accumulation displayed a bell-shaped kinetics with a maximal effect at 1 h. 2-Bromopalmitate was ineffective, indicating that fatty acids must be metabolized to cause their effect. Neither fatty acid was able to induce c-fos and nur-77 in PKC-downregulated cells or cells incubated in the presence of the Ca2+ channel blocker nifedipine or the Ca2+ chelator EGTA, suggesting involvement of the PKC and Ca2+ signaling pathways. Palmitate and oleate also increased c-fos protein expression and DNA binding activity of the transcription factor AP-1. Oleate, but not palmitate, increased [3H]thymidine incorporation in INS cells. Finally, both palmitate and oleate caused c-fos and nur-77 mRNA accumulation in isolated rat islets. It is suggested that IEG induction by the most abundant circulating fatty acids plays a role in the adaptive process of the beta-cell to hyperlipidemia. These results have implications for our understanding of obesity-associated diabetes and the link between fatty acids and tumorigenesis.

Type 2 diabetes is characterized by both insulin resistance and progressive deterioration of β-cell function. The forkhead transcription factor FoxO1 is a prominent mediator of insulin signaling in β-cells. We reasoned that identification of FoxO1 target genes in β-cells could reveal mechanisms linking β-cell dysfunction to insulin resistance. In this study, we report the characterization of Nov/Ccn3 as a novel transcriptional target of FoxO1 in pancreatic β-cells. FoxO1 binds to an evolutionarily conserved response element in the Ccn3 promoter to regulate its expression. Accordingly, CCN3 levels are elevated in pancreatic islets of mice with overexpression of a constitutively active form of FoxO1 or insulin resistance. Our functional studies reveal that CCN3 impairs β-cell proliferation concomitantly with a reduction in cAMP levels. Moreover, CCN3 decreases glucose oxidation, which translates into inhibition of glucose-stimulated Ca(2+) entry and insulin secretion. Our results identify CCN3, a novel transcriptional target of FoxO1 in pancreatic β-cells, as a potential target for therapeutic intervention in the treatment of diabetes.

Glucagon-like peptide-1 (GLP-1), a glucoincretin hormone secreted by intestinal L cells, is a potent growth factor for the pancreatic β-cell. The development of GLP-1 mimetics and enhancers as a novel class of anti-diabetes medications underpins the importance of elucidating the molecular basis of GLP-1 signaling. In the present study, we sought to test the hypothesis that β-arrestin-mediated recruitment of c-Src underlies the proliferative action of GLP-1 in β-cells. Our results show that GLP-1 increased c-Src phosphorylation in INS832/13 cells, an effect inhibited by siRNA-mediated β-arrestin1 knockdown. Pharmacological inhibition of c-Src and overexpression of a dominant-negative c-Src mutant protein curtailed GLP-1-induced β-cell proliferation. Co-immunoprecipitation experiments showed a physical association between c-Src and both β-arrestin1 and GLP-1R upon GLP-1 treatment. Moreover, expression of β-arrestin1 mutants that lack the ability to bind c-Src blunted GLP-1-induced proliferation. Conversely, expression of a β-arrestin1 mutant that fails to target G protein-coupled receptors to clathrin-coated pits for sequestration/degradation maximally increased β-cell proliferation. We propose that the formation of a signaling complex comprising the agonist-stimulated GLP-1R, β-arrestin1 and c-Src is required for the action of GLP-1 on β-cell mass.

Elevated glucose and saturated fatty acids synergize in inducing apoptosis in INS832/13 cells and in human islet cells. In order to gain insight into the molecular mechanism(s) of glucolipotoxicity (Gltox), gene profiling and metabolic analyses were performed in INS832/13 cells cultured at 5 or 20 mm glucose in the absence or presence of palmitate. Expression changes were observed for transcripts involved in mitochondrial, lipid, and glucose metabolism. At 24 h after Gltox, increased expression of lipid partitioning genes suggested a promotion of fatty acid esterification and reduced lipid oxidation/detoxification, whereas changes in the expression of energy metabolism genes suggested mitochondrial dysfunction. These changes were associated with decreased glucose-induced insulin secretion, total insulin content, ATP levels, AMP-kinase activity, mitochondrial membrane potential and fat oxidation, unchanged de novo fatty acid synthesis, and increased reactive oxygen species, cholesterol, ceramide, and triglyceride levels. However, the synergy between elevated glucose and palmitate to cause ß-cell toxicity in term of apoptosis and reduced glucose-induced insulin secretion only correlated with triglyceride and ceramide depositions. Overexpression of endoplasmic reticulum glycerol-3-phosphate acyl transferase to enhance lipid esterification amplified Gltox at intermediate glucose (11 mm), whereas reducing acetyl-coenzyme A carboxylase 1 expression by small interfering RNA to shift lipid partitioning to fat oxidation reduced Gltox. The results suggest that Gltox entails alterations in lipid partitioning, sterol and ceramide accumulation, mitochondrial dysfunction, and reactive oxygen species production, all contributing to altering ß-cell function. The data also suggest that the early promotion of lipid esterification processes is instrumental in the Gltox process.

Glucagon-like peptide-1 (GLP-1) is a glucoincretin hormone most intensively studied for its actions on insulin secreting beta-cells. GLP-1 and its receptor are also found in brain and accumulating evidence indicates that GLP-1 has neuroprotective actions. Here, we investigated whether GLP-1 protects neuronal cells from death evoked by nerve growth factor (NGF) withdrawal. Compromised trophic factor signaling may underlie neurodegenerative diseases ranging from Alzheimer disease to diabetic neuropathies. We report that GLP-1 provides sustained protection of cultured neuronal PC12 cells and sympathetic neurons from degeneration and death caused by NGF deprivation. Past work shows that NGF deprivation induces the pro-apoptotic protein Bim which contributes to neuron death. Here, we find that GLP-1 suppresses Bim induction promoted by NGF deprivation. Thus, GLP-1 may protect neurons, at least in part, by suppressing Bim induction. Our findings support the idea that drugs that mimic or elevate GLP-1 represent potential therapeutics for neurodegenerative diseases.

Diabetes is associated with decreased pancreatic beta-cell function and mass. It is unclear whether diabetes treatment should aim at restoring beta-cell performance/mass or at inducing "beta-cell rest" to prevent further deterioration. The transcription factor Foxo1 protects beta-cells against oxidative stress induced by hyperglycemia and prevents beta-cell replication in insulin-resistant states. Here we show that these combined effects are associated with a concerted repression of genes involved in glycolysis, nitric-oxide synthesis, G protein-coupled receptor signaling, and ion transport. Conversely, Foxo1 increases expression of several neurotransmitter receptors and fails to regulate target genes predicted from Caenorhabditis elegans and Drosophila studies. Functional analyses show decreased glucose utilization and insulin secretion in beta-cells overexpressing Foxo1. We propose the definition of "metabolic diapause" for the changes induced by Foxo1 to protect beta-cells against oxidative stress. The data provide genetic underpinning for the concept of beta-cell rest as a treatment goal in diabetes.

The glucoincretin hormone glucagon-like peptide-1 (GLP-1) increases pancreatic beta-cell proliferation and survival through sequential activation of the epidermal growth factor receptor (EGFR), phosphatidylinositol-3 kinase (PI 3-kinase), and Akt. We investigated the role of transcription factor FoxO1 in the proliferative and antiapoptotic actions of GLP-1 in beta-cells. GLP-1 inhibited FoxO1 through phosphorylation-dependent nuclear exclusion in pancreatic beta (INS832/13) cells. The effect of GLP-1 was suppressed by inhibitors of EGFR (AG1478) and PI 3-kinase (LY294002). In contrast, LY294002 but not AG1478 suppressed insulin-induced FoxO1 phosphorylation. Expression of constitutively nuclear FoxO1 in beta-cells prevented the proliferative and antiapoptotic actions of GLP-1 in cultured beta-cells and the increase in pancreatic beta-cell mass in response to Exendin4 in transgenic mice. Gene expression and chromatin immunoprecipitation assays demonstrated that GLP-1 increases pancreatic and duodenal homeobox gene-1 and Foxa2 expression and inhibits FoxO1 binding to both promoters. We propose that FoxO1 mediates the pleiotropic effects of the glucoincretin hormone on cell proliferation and survival.

We have proposed the "glucolipotoxicity" hypothesis in which elevated free fatty acids (FFAs) together with hyperglycemia are synergistic in causing islet beta-cell damage because high glucose inhibits fat oxidation and consequently lipid detoxification. The effects of 1-2 d culture of both rat INS 832/13 cells and human islet beta-cells were investigated in medium containing glucose (5, 11, 20 mM) in the presence or absence of various FFAs. A marked synergistic effect of elevated concentrations of glucose and saturated FFA (palmitate and stearate) on inducing beta-cell death by apoptosis was found in both INS 832/13 and human islet beta-cells. In comparison, linoleate (polyunsaturated) synergized only modestly with high glucose, whereas oleate (monounsaturated) was not toxic. Treating cells with the acyl-coenzyme A synthase inhibitor triacsin C, or the AMP kinase activators metformin and 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside that redirect lipid partitioning to oxidation, curtailed glucolipotoxicity. In contrast, the fat oxidation inhibitor etomoxir, like glucose, markedly enhanced palmitate-induced cell death. The data indicate that FFAs must be metabolized to long chain fatty acyl-CoA to exert toxicity, the effect of which can be reduced by activating fatty acid oxidation. The results support the glucolipotoxicity hypothesis of beta-cell failure proposing that elevated FFAs are particularly toxic in the context of hyperglycemia.

Glucagon-like peptide-1 (GLP-1), an insulinotropic and glucoincretin hormone, is a potentially important therapeutic agent in the treatment of diabetes. We previously provided evidence that GLP-1 induces pancreatic beta-cell growth nonadditively with glucose in a phosphatidylinositol-3 kinase (PI-3K)-dependent manner. In the present study, we investigated the downstream effectors of PI-3K to determine the precise signal transduction pathways that mediate the action of GLP-1 on beta-cell proliferation. GLP-1 increased extracellular signal-related kinase 1/2, p38 mitogen-activated protein kinase (MAPK), and protein kinase B activities nonadditively with glucose in pancreatic beta(INS 832/13) cells. GLP-1 also caused nuclear translocation of the atypical protein kinase C (aPKC) zeta isoform in INS as well as in dissociated normal rat beta-cells as shown by immunolocalization and Western immunoblotting analysis. Tritiated thymidine incorporation measurements showed that the p38 MAPK inhibitor SB203580 suppressed GLP-1-induced beta-cell proliferation. Further investigation was performed using isoform-specific pseudosubstrates of classical (alpha, beta, and gamma) or zeta aPKC isoforms. The PKCzeta pseudosubstrate suppressed the proliferative action of GLP-1, whereas the inhibitor of classical PKC isoforms had no effect. Overexpression of a kinase-dead PKCzeta acting as a dominant negative protein suppressed GLP-1-induced proliferation. In addition, ectopic expression of a constitutively active PKCzeta mutant stimulated tritiated thymidine incorporation to the same extent as GLP-1, and the glucoincretin had no growth-promoting action under this condition. The data indicate that GLP-1-induced activation of PKCzeta is implicated in the beta-cell proliferative signal of the insulinotropic hormone. The results are consistent with a model in which GLP-1-induced PI-3K activation results in PKCzeta translocation to the nucleus, which may play a role in the pleiotropic effects (DNA synthesis, metabolic enzymes, and insulin gene expression) of the glucoincretin.

Aims/hypothesis. Glucagon-like peptide-1 is a potent glucoincretin hormone and a potentially important drug in the treatment of Type II (non-insulin-dependent) diabetes mellitus. We have investigated whether it acts as a growth factor in beta (INS-1)-cells and have studied the signalling pathways and transcription factors implicated in this process. Methods. Cell proliferation was assessed by tritiated thymidine incorporation measurements. We have examined the action of glucagon-like peptide-1 on the enzymatic activity of phosphatidylinositol 3-kinase. The DNA binding activity of transcription factors was investigated by electrophoretic mobility shift assay. Measurements of mRNA were done using the northern technique. Results. Glucagon-like peptide-1 caused an increase in tritiated thymidine incorporation in beta (INS-1)-cells and phosphatidylinositol 3-kinase activity in a dose-dependent manner non-additively with glucose. The phosphatidylinositol 3-kinase inhibitors wortmannin and LY294 002 blocked the effects of glucagon-like peptide-1 on DNA synthesis. Transcription factor pancreatic and duodenal homebox gene 1 (PDX-1) DNA binding activity was increased by glucagon-like peptide-1 at 3 or 11 mmol/l glucose and the phosphatidylinositol 3-kinase inhibitor LY294 002 suppressed the action of glucagon-like peptide-1 on PDX-1 DNA binding activity. Glucagon-like peptide-1 and glucose alone did not change activating protein-1 DNA binding activity. They synergised, however, to increase the activity of activating protein-1. Glucagon-like peptide-1 also increased the expression of PDX-1, glucose transporter 2, glucokinase and insulin mRNAs. Finally, glucagon-like peptide-1 increased the incorporation of tritiated thymidine in isolated rat islets. Conclusion/interpretation. The results suggest that glucagon-like peptide-1 may act as a growth factor for the beta cell by a phosphatidylinositol 3-kinase mediated event. Glucagon-like peptide-1 could also regulate the expression of the insulin gene and genes encoding enzymes implicated in glucose transport and metabolism through the phosphatidylinositol 3-kinase/PDX-1 transduction signalling pathway. [Diabetologia (1999) 42: 856–864]