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SJ Conroy, I Green, G Dixon, PM Byrne, J Nolan, YH Abdel-Wahab, N McClenaghan, PR Flatt, and P Newsholme

We have previously reported that newly diagnosed Type-1 diabetic patient sera potently suppressed insulin secretion from a clonal rat pancreatic beta-cell line (BRIN BD11) but did not alter cell viability. Here, we report that apoptosis in BRIN BD11 cells incubated in various sera types (fetal calf serum (FCS), normal human serum and Type-1 diabetic patient) was virtually undetectable. Although low levels of necrosis were detected, these were not significantly different between cells incubated in sera from different sources. ATP levels were reduced by approximately 30% while nitrite production increased twofold from BRIN BD11 cells incubated for 24 h in the presence of Type-1 diabetic patient sera compared with normal human sera. Additionally, ATP levels were reduced by approximately 40% and DNA fragmentation increased by more than 20-fold in BRIN BD11 cells incubated in FCS in the presence of a pro-inflammatory cytokine cocktail (interleukin-1beta, tumour necrosis factor-alpha and interferon-gamma), compared with cells incubated in the absence of cytokines. Nitric oxide production from BRIN BD11 cells was markedly increased (up to 10-fold) irrespective of sera type when the cytokine cocktail was included in the incubation medium. Type-1 diabetic patient sera significantly (P<0.001) raised basal levels of intracellular free Ca(2+ )concentration ([Ca(2+)](i)) in BRIN BD11 cells after a 24-h incubation. The alteration in [Ca(2+)](i) concentration was complement dependent, as removal of the early complement components C1q and C3 resulted in a significant reduction (P<0.01) of sera-induced [Ca(2+)](i )changes. We propose that the mechanism of Type-1 diabetic patient sera-induced inhibition of insulin secretion from clonal beta-cells may involve complement-stimulated elevation of [Ca(2+)](i) which attenuates the nutrient-induced insulin secretory process possibly by desensitizing the cell to further changes in Ca(2+).

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Birgitte N Friedrichsen, Nicole Neubauer, Ying C Lee, Vivian K Gram, Niels Blume, Jacob S Petersen, Jens H Nielsen, and Annette Møldrup

The incretin hormones, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP), have been suggested to act as β-cell growth factors and may therefore be of critical importance for the maintenance of a proper β-cell mass. We have investigated the molecular mechanism of incretin-induced β-cell replication in primary monolayer cultures of newborn rat islet cells. GLP-1, GIP and the long-acting GLP-1 derivative, lira-glutide, increased β-cell replication 50–80% at 10–100 nM upon a 24 h stimulus, whereas glucagon at a similar concentration had no significant effect. The stimulatory effect of GLP-1 and GIP was efficiently mimicked by the adenylate cyclase activator, forskolin, at 10 nM (~90% increase) and was additive (~170–250% increase) with the growth response to human growth hormone (hGH), indicating the use of distinct intracellular signalling pathways leading to mitosis by incretins and cytokines, respectively. The response to both GLP-1 and GIP was completely blocked by the protein kinase A (PKA) inhibitor, H89. In addition, the phosphoinositol 3-kinase (PI3K) inhibitor wortmannin and the mitogen-activated protein kinase kinase (MEK) inhibitor PD98059, both inhibited GLP-1- and GIP-stimulated proliferation. The p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580, had no inhibitory effect on either GLP-1 or GIP stimulated proliferation. Cyclin Ds act as molecular switches for the G0/G1-S phase transition in many cell types and we have previously demonstrated hGH-induced cyclin D2 expression in the insulinoma cell line, INS-1. GLP-1 time-dependently induced the cyclin D1 mRNA and protein levels in INS-1E, whereas the cyclin D2 levels were unaffected. However, minor effect of GLP-1 stimulation was observed on the cyclin D3 mRNA levels. Transient transfection of a cyclin D1 promoter-luciferase reporter construct into islet monolayer cells or INS-1 cells revealed approximately a 2–3 fold increase of transcriptional activity in response to GLP-1 and GIP, and a 4–7 fold increase in response to forskolin. However, treatment of either cell type with hGH had no effect on cyclin D1 promoter activity. The stimulation of the cyclin D1 promoter by GLP-1 was inhibited by H89, wortmannin, and PD98059. We conclude that incretin-induced β-cell replication is dependent on cAMP/PKA, p42 MAPK and PI3K activities, which may involve transcriptional induction of cyclin D1. GLP-1, GIP and liraglutide may have the potential to increase β-cell replication in humans which would have significant impact on long-term diabetes treatment.

Free access

A Shirakami, T Toyonaga, K Tsuruzoe, T Shirotani, K Matsumoto, K Yoshizato, J Kawashima, Y Hirashima, N Miyamura, CR Kahn, and E Araki

Insulin receptor substrate 1 (IRS-1) gene polymorphisms have been identified in type 2 diabetic patients; however, it is unclear how such polymorphisms contribute to the development of diabetes. Here we introduced obesity in heterozygous IRS-1 knockout (IRS-1(+/-)) mice by gold-thioglucose (GTG) injection and studied the impact of reduced IRS-1 expression on obesity-linked insulin resistance. GTG injection resulted in approximately 30% weight gain in IRS-1(+/-) and wild type (WT) mice, compared with saline-injected controls. There was no difference in insulin sensitivity between lean IRS-1(+/-) and lean WT. Elevated fasting insulin levels but no change in fasting glucose were noted in obese IRS-1(+/-) and WT compared with the respective lean controls. Importantly, fasting insulin in obese IRS-1(+/-) was 1.5-fold higher (P<0.05) than in obese WT, and an insulin tolerance test showed a profound insulin resistance in obese IRS-1(+/-) compared with obese WT. The islets of obese IRS-1(+/-) were 1.4-fold larger than those of obese WT. The expression of insulin receptor and IRS-1 and IRS-2 was decreased in obese IRS-1(+/-), which could in part explain the profound insulin resistance in these mice. Our results suggest that IRS-1 is the suspected gene for type 2 diabetes and its polymorphisms could worsen insulin resistance in the presence of other additional factors, such as obesity.

Free access

Ronald Gonzalez, Benjamin K Reingold, Xiaodong Gao, Mandeep P Gaidhu, Robert G Tsushima, and Suraj Unniappan

Nesfatin-1 is a recently discovered multifunctional metabolic hormone abundantly expressed in the pancreatic islets. The main objective of this study is to characterize the direct effects of nesfatin-1 on insulin secretion in vitro using MIN6 cells and islets isolated from C57BL/6 mice. We also examined the expression of the nesfatin-1 precursor protein, nucleobindin 2 (NUCB2) mRNA, and nesfatin-1 immunoreactivity (ir) in the islets of normal mice and in the islets from mice with streptozotocin-induced type 1 diabetes and diet-induced obese (DIO) mice with type 2 diabetes. Nesfatin-1 stimulated glucose-induced insulin release in vitro from mouse islets and MIN6 cells in a dose-dependent manner. No such stimulation in insulin secretion was found when MIN6 cells/islets were incubated with nesfatin-1 in low glucose. In addition, a fourfold increase in nesfatin-1 release from MIN6 cells was observed following incubation in high glucose (16.7 mM) compared to low glucose (2 mM). Furthermore, we observed a significant reduction in both NUCB2 mRNA expression and nesfatin-1-ir in the pancreatic islets of mice with type 1 diabetes, while a significant increase was observed in the islets of DIO mice. Together, our findings indicate that nesfatin-1 is a novel insulinotropic peptide and that the endogenous pancreatic islet NUCB2/nesfatin is altered in diabetes and diet-induced obesity.

Free access

Rhonda D Prisby, Joshua M Swift, Susan A Bloomfield, Harry A Hogan, and Michael D Delp

Osteopenia and an enhanced risk of fracture often accompany type 1 diabetes. However, the association between type 2 diabetes and bone mass has been ambiguous with reports of enhanced, reduced, or similar bone mineral densities (BMDs) when compared with healthy individuals. Recently, studies have also associated type 2 diabetes with increased fracture risk even in the presence of higher BMDs. To determine the temporal relationship between type 2 diabetes and bone remodeling structural and mechanical properties at various bone sites were analyzed during pre-diabetes (7 weeks), short-term (13 weeks), and long-term (20 weeks) type 2 diabetes. BMDs and bone strength were measured in the femora and tibiae of Zucker diabetic fatty rats, a model of human type 2 diabetes. Increased BMDs (9–10%) were observed in the distal femora, proximal tibiae, and tibial mid- shafts in the pre-diabetic condition that corresponded with higher plasma insulin levels. During short- and long-term type 2 diabetes, various parameters of bone strength and BMDs were lower (9–26%) in the femoral neck, distal femora, proximal tibiae, and femoral and tibial mid-shafts. Correspondingly, blood glucose levels increased by 125% and 153% during short- and long-term diabetes respectively. These data indicate that alterations in BMDs and bone mechanical properties are closely associated with the onset of hyperinsulinemia and hyperglycemia, which may have direct adverse effects on skeletal tissue. Consequently, disparities in the human literature regarding the effects of type 2 diabetes on skeletal properties may be associated with the bone sites studied and the severity or duration of the disease in the patient population studied.

Free access

K Fosgerau, P Galle, T Hansen, A Albrechtsen, C de Lemos Rieper, B Klarlund Pedersen, L Kongskov Larsen, A Randrup Thomsen, O Pedersen, M Bagge Hansen, and A Steensberg

Abstract

Interleukin-6 (IL6) is critically involved in inflammation and metabolism. About 1% of people produce IL6 autoantibodies (aAb-IL6) that impair IL6 signaling in vivo. We tested the hypothesis that the prevalence of such aAb-IL6 is increased in type 2 diabetic patients and that aAb-IL6 plays a direct role in causing hyperglycemia. In humans, the prevalence of circulating high-affinity neutralizing aAb-IL6 was 2.5% in the type 2 diabetic patients and 1% in the controls (odds ratio 2.5, 95% confidence interval 1.2–4.9, P=0.01). To test for the role of aAb-IL6 in causing hyperglycemia, such aAb-IL6 were induced in mice by a validated vaccination procedure. Mice with plasma levels of aAb-IL6 similar to the 2.5% type 2 diabetic patients developed obesity and impaired glucose tolerance (area under the curve (AUC) glucose, 2056±62 vs 1793±62, P=0.05) as compared with sham-vaccinated mice, when challenged with a high-fat diet. Mice with very high plasma levels of aAb-IL6 developed elevated fasting plasma glucose (mM, 4.8±0.4 vs 3.3±0.1, P<0.001) and impaired glucose tolerance (AUC glucose, 1340±38 vs 916±25, P<0.001) as compared with sham-control mice on normal chow. In conclusion, the prevalence of plasma aAb-IL6 at levels known to impair IL6 signaling in vivo is increased 2.5-fold in people with type 2 diabetes. In mice, matching levels of aAb-IL6 cause obesity and hyperglycemia. These data suggest that a small subset of type 2 diabetes may in part evolve from an autoimmune attack against IL6.

Free access

N M Whalley, L E Pritchard, D M Smith, and A White

Proglucagon is cleaved to glucagon by prohormone convertase 2 (PC2) in pancreatic α-cells, but is cleaved to glucagon-like peptide-1 (GLP-1) by PC1 in intestinal L-cells. The aim of this study was to identify mechanisms which switch processing of proglucagon to generate GLP-1 in the pancreas, given that GLP-1 can increase insulin secretion and β-cell mass. The α-cell line, αTC1-6, expressed PC1 at low levels and GLP-1 was detected in cells and in culture media. GLP-1 was also found in isolated human islets and in rat islets cultured for 7 days. High glucose concentrations increased Pc1 gene expression and PC1 protein in rat islets. High glucose (25 mM) also increased GLP-1 but decreased glucagon secretion from αTC1-6 cells suggesting a switch in processing to favour GLP-1. Three G protein-coupled receptors, GPR120, TGR5 and GPR119, implicated in the release of GLP-1 from L-cells are expressed in αTC1-6 cells. Incubation of these cells with an agonist of TGR5 increased PC1 promoter activity and GLP-1 secretion suggesting that this is a mechanism for switching processing to GLP-1 in the pancreas. Treatment of isolated rat islets with streptozotocin caused β-cell toxicity as evidenced by decreased glucose-stimulated insulin secretion. This increased GLP-1 but not glucagon in the islets. In summary, proglucagon can be processed to GLP-1 in pancreatic cells. This process is upregulated by elevated glucose, activation of TGR5 and β-cell destruction. Understanding this phenomenon may lead to advances in therapies to protect β-cell mass, and thereby slow progression from insulin resistance to type 2 diabetes.

Free access

Sachiko Kitanaka, Utako Sato, and Takashi Igarashi

Mutations in hepatocyte nuclear factor-1β (HNF-1β) lead to type 5 maturity-onset diabetes of the young (MODY5). Moreover, mutations in the HNF-1β gene might cause multiorgan abnormalities including renal diseases, genital malformations, and abnormal liver function. The objective of this study was to investigate the molecular mechanism of diabetes mellitus, intrauterine growth retardation, and cholestasis observed in MODY5 patients. We analyzed the transactivity of wild-type and three mutant HNF-1β on native human insulin, IGF-I, and multidrug resistance protein 2 (MRP2) promoters in combination with HNF-1α, using a reporter-assay system in transiently transfected mammalian cells. In the human insulin gene promoter, we found that the cooperation of HNF-1α and HNF-1β is prominent. Absence of this cooperation was observed in all of the HNF-1β mutants. In the human IGF-I and MRP2 promoters, we found that the HNF-1β His153Asn (H153N) mutant had a mutant-specific repressive effect on both HNF-1α and wild-type HNF-1β transactivity. Absence of the cooperation of HNF-1β mutants with HNF-1α in the human insulin gene promoter might be one cause of defective insulin secretion. The H153N mutant-specific repression of HNF-1α and HNF-1β transactivity in human IGF-I and MRP2 promoters might explain the case-specific clinical features of growth retardation and cholestasis observed only in early infancy. We found differential property of HNF-1α/HNF-1β activity and the effect of HNF-1β mutants by the promoters. We consider that analyses of HNF-1β mutants on the intended human native promoters in combination with HNF-1α may be useful in investigating the molecular mechanisms of the various features in MODY5.

Free access

Benjamin J Lamont and Sofianos Andrikopoulos

Incretin-based therapies appear to offer many advantages over other approaches for treating type 2 diabetes. Some preclinical studies have suggested that chronic activation of glucagon-like peptide 1 receptor (GLP1R) signalling in the pancreas may result in the proliferation of islet β-cells and an increase in β-cell mass. This provided hope that enhancing GLP1 action could potentially alter the natural progression of type 2 diabetes. However, to date, there has been no evidence from clinical trials suggesting that GLP1R agonists or dipeptidyl peptidase-4 (DPP4) inhibitors can increase β-cell mass. Nevertheless, while the proliferative capacity of these agents remains controversial, some studies have raised concerns that they could potentially contribute to the development of pancreatitis and hence increase the risk of pancreatic cancer. Currently, there are very limited clinical data to directly assess these potential benefits and risks of incretin-based therapies. However, a review of the preclinical studies indicates that incretin-based therapies probably have only a limited capacity to regenerate pancreatic β-cells, but may be useful for preserving any remaining β-cells in type 2 diabetes. In addition, the majority of preclinical evidence does not support the notion that GLP1R agonists or DPP4 inhibitors cause pancreatitis.

Free access

Elisabet Estil.les, Noèlia Téllez, Joan Soler, and Eduard Montanya

Interleukin-1β (IL1B) is an important contributor to the autoimmune destruction of β-cells in type 1 diabetes, and it has been recently related to the development of type 2 diabetes. IGF2 stimulates β-cell proliferation and survival. We have determined the effect of IL1B on β-cell replication, and the potential modulation by IGF2 and glucose. Control-uninfected and adenovirus encoding for IGF2 (Ad-IGF2)-infected rat islets were cultured at 5.5 or 22.2 mmol/l glucose with or without 1, 10, 30, and 50 U/ml of IL1B. β-Cell replication was markedly reduced by 10 U/ml of IL1B and was almost nullified with 30 or 50 U/ml of IL1B. Higher concentrations of IL1B were required to increase β-cell apoptosis. Although IGF2 overexpression had a strong mitogenic effect on β-cells, IGF2 could preserve β-cell proliferation only in islets cultured with 10 U/ml IL1B, and had no effect with 30 and 50 U/ml of IL1B. In contrast, IGF2 overexpression induced a clear protection against IL1B-induced apoptosis, and higher concentrations of the cytokine were needed to increase β-cell apoptosis in Ad-IGF2-infected islets. These results indicate that β-cell replication is highly sensitive to the deleterious effects of the IL1B as shown by the inhibition of replication by relatively low IL1B concentrations, and the almost complete suppression of β-cell replication with high IL1B concentrations. Likewise, the inhibitory effects of IL-β on β-cell replication were not modified by glucose, and were only modestly prevented by IGF2 overexpression, in contrast with the higher protection against IL1B-induced apoptosis afforded by glucose and by IGF2 overexpression.