Beta-cell Area Research Articles

High fat feeding unmasks variable insulin responses in male C57BL/6 mouse substrains.

Mouse models are widely used for elucidating mechanisms underlying type 2 diabetes. Genetic background profoundly affects metabolic phenotype; therefore, selecting the appropriate model is critical. Although variability in metabolic responses between mouse strains is now well recognized, it also occurs within C57BL/6 mice, of which several substrains exist. This within-strain variability is poorly understood and could emanate from genetic and/or environmental differences. To better define the within-strain variability, we performed the first comprehensive comparison of insulin secretion from C57BL/6 substrains 6J, 6JWehi, 6NJ, 6NHsd, 6NTac and 6NCrl. In vitro, glucose-stimulated insulin secretion correlated with Nnt mutation status, wherein responses were uniformly lower in islets from C57BL/6J vs C57BL/6N mice. In contrast, in vivo insulin responses after 18 weeks of low fat feeding showed no differences among any of the six substrains. When challenged with a high-fat diet for 18 weeks, C57BL/6J substrains responded with a similar increase in insulin release. However, variability was evident among C57BL/6N substrains. Strikingly, 6NJ mice showed no increase in insulin release after high fat feeding, contributing to the ensuing hyperglycemia. The variability in insulin responses among high-fat-fed C57BL/6N mice could not be explained by differences in insulin sensitivity, body weight, food intake or beta-cell area. Rather, as yet unidentified genetic and/or environmental factor(s) are likely contributors. Together, our findings emphasize that caution should be exercised in extrapolating data from in vitro studies to the in vivo situation and inform on selecting the appropriate C57BL/6 substrain for metabolic studies.

An enzymatically stable GIP/xenin hybrid peptide restores GIP sensitivity, enhances beta cell function and improves glucose homeostasis in high-fat-fed mice

Aims/hypothesisGlucose-dependent insulinotropic polypeptide (GIP) and xenin, regulatory gut hormones secreted from enteroendocrine K cells, exert important effects on metabolism. In addition, xenin potentiates the biological actions of GIP. The present study assessed the actions and therapeutic utility of a (DAla2)GIP/xenin-8-Gln hybrid peptide, in comparison with the parent peptides (DAla2)GIP and xenin-8-Gln.MethodsFollowing confirmation of enzymatic stability, insulin secretory activity of (DAla2)GIP/xenin-8-Gln was assessed in BRIN-BD11 beta cells. Acute and persistent glucose-lowering and insulin-releasing effects were then examined in vivo. Finally, the metabolic benefits of twice daily injection of (DAla2)GIP/xenin-8-Gln was determined in high-fat-fed mice.ResultsAll peptides significantly (p < 0.05 to p < 0.001) enhanced in vitro insulin secretion from pancreatic clonal BRIN-BD11 cells, with xenin (and particularly GIP)-related signalling pathways, being important for this action. Administration of (DAla2)GIP or (DAla2)GIP/xenin-8-Gln in combination with glucose significantly (p < 0.05) lowered blood glucose and increased plasma insulin in mice, with a protracted response of up to 4 h. All treatments elicited appetite-suppressive effects (p < 0.05), particularly (DAla2)GIP/xenin-8-Gln and xenin-8-Gln at elevated doses of 250 nmol/kg. Twice-daily administration of (DAla2)GIP/xenin-8-Gln or (DAla2)GIP for 21 days to high-fat-fed mice returned circulating blood glucose to lean control levels. In addition, (DAla2)GIP/xenin-8-Gln treatment significantly (p < 0.05) reduced glycaemic levels during a 24 h glucose profile assessment. Neither of the treatment regimens had an effect on body weight, energy intake or circulating insulin concentrations. However, insulin sensitivity was significantly (p < 0.001) improved by both treatments. Interestingly, GIP-mediated glucose-lowering (p < 0.05) and insulin-releasing (p < 0.05 to p < 0.01) effects were substantially improved by (DAla2)GIP and (DAla2)GIP/xenin-8-Gln treatment. Pancreatic islet and beta cell area (p < 0.001), as well as pancreatic insulin content (p < 0.05), were augmented in (DAla2)GIP/xenin-8-Gln-treated mice, related to enhanced proliferation and decreased apoptosis of beta cells, whereas (DAla2)GIP evoked increases (p < 0.05 to p < 0.01) in islet number.Conclusions/interpretationThese studies highlight the clear potential of GIP/xenin hybrids for the treatment of type 2 diabetes.

Histological changes in endocrine and exocrine pancreatic tissue from patients exposed to incretin-based therapies.

Incretin-based therapies have been associated with an increased risk of pancreatitis. Recently, various histological abnormalities have been reported in human pancreatic tissue from brain-dead organ donors who had been exposed to incretin-based drugs. In the present study we examined pancreatic tissue collected at surgery. Human pancreatic tissue from 7 type 2-diabetic patients treated with incretin-based drugs (type 2-I), 6 diabetic patients without incretin treatment (type 2-NI), 11 patients without diabetes (no diabetes group) and 9 brain-dead organ donors (BDOD group) was examined. Fractional beta-cell area was reduced in the type 2-NI group compared to the group without diabetes (P < .05), but there was no difference compared to the type 2-I patients. Alpha-cell area (P = .30), beta-cell replication (P = .17) and alpha-cell replication (P = .91) were not different. There were also no differences in acinar cell (P = .13) and duct cell replication (P = .099). Insulin-positive duct cells were more frequent in the type 2-I and the BDOD groups (P = .034). No co-expression of insulin and glucagon was detected. Pancreatic intraepithelial neoplasia (PanIN) lesions were very rare, all low-grade (PanIN 1a and 1b) and tended to occur more frequently in the type 2-I group (P = .084). The present results did not reveal marked histological abnormalities in the pancreas of incretin-treated patients with type 2 diabetes. Low numbers of specimens available and a large inter-individual variability of the findings warrant caution regarding the interpretation of histological data concerning drug effects on the human pancreas.

The S20G substitution in hIAPP is more amyloidogenic and cytotoxic than wild-type hIAPP in mouse islets.

The S20G human islet amyloid polypeptide (hIAPP) substitution is associated with an earlier onset of type 2 diabetes in humans. Studies of synthetic S20G hIAPP in cell-free systems and immortalised beta cells have suggested that this may be due to increased hIAPP amyloidogenicity and cytotoxicity. Thus, using primary islets from mice with endogenous S20G hIAPP expression, we sought to determine whether the S20G gene mutation leads to increased amyloid-induced toxicity, beta cell loss and reduced beta cell function. Islets from mice in which mouse Iapp was replaced with human wild-type or S20G hIAPP were isolated and cultured in vitro under amyloid-forming conditions. Levels of insulin and hIAPP mRNA and protein, amyloid deposition and beta cell apoptosis and area, as well as glucose-stimulated insulin and hIAPP secretion, were quantified. Islets expressing S20G hIAPP cultured in 16.7mmol/l glucose demonstrated increased amyloid deposition and beta cell apoptosis, reduced beta cell area, decreased insulin content and diminished glucose-stimulated insulin secretion, compared with islets expressing wild-type hIAPP. Amyloid deposition and beta cell apoptosis were also increased when S20G islets were cultured in 11.1mmol/l glucose (the concentration that is thought to be physiological for mouse islets). S20G hIAPP reduces beta cell number and function, thereby possibly explaining the earlier onset of type 2 diabetes in individuals carrying this gene mutation.

Impact of proton pump inhibitor treatment on pancreatic beta-cell area and beta-cell proliferation in humans.

Gastrin has been shown to promote beta-cell proliferation in rodents, but its effects in adult humans are largely unclear. Proton pump inhibitors (PPIs) lead to endogenous hypergastrinaemia, and improved glucose control during PPI therapy has been reported in patients with diabetes. Therefore, we addressed whether PPI treatment is associated with improved glucose homoeostasis, islet cell hyperplasia or increased new beta-cell formation in humans. Pancreatic tissue specimens from 60 patients with and 33 patients without previous PPI therapy were examined. The group was subdivided into patients without diabetes (n = 27), pre-diabetic patients (n = 31) and patients with diabetes (n = 35). Fasting glucose and HbA1c levels were not different between patients with and without PPI therapy (P = 0.34 and P = 0.30 respectively). Beta-cell area was higher in patients without diabetes than in patients with pre-diabetes or diabetes (1.33 ± 0.12%, 1.05 ± 0.09% and 0.66 ± 0.07% respectively; P < 0.0001). There was no difference in beta-cell area between patients with and without PPI treatment (1.05 ± 0.08% vs 0.87 ± 0.08%, respectively; P = 0.16). Beta-cell replication was rare and not different between patients with and without PPI therapy (P = 0.20). PPI treatment was not associated with increased duct-cell replication (P = 0.18), insulin expression in ducts (P = 0.28) or beta-cell size (P = 0.63). These results suggest that in adult humans, chronic PPI treatment does not enhance beta-cell mass or beta-cell function to a relevant extent.

Endogenous GIP ameliorates impairment of insulin secretion in proglucagon-deficient mice under moderate beta cell damage induced by streptozotocin.

Aims/hypothesisThe action of incretin hormones including glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) is potentiated in animal models defective in glucagon action. It has been reported that such animal models maintain normoglycaemia under streptozotocin (STZ)-induced beta cell damage. However, the role of GIP in regulation of glucose metabolism under a combination of glucagon deficiency and STZ-induced beta cell damage has not been fully explored.MethodsIn this study, we investigated glucose metabolism in mice deficient in proglucagon-derived peptides (PGDPs)—namely glucagon gene knockout (GcgKO) mice—administered with STZ. Single high-dose STZ (200 mg/kg, hSTZ) or moderate-dose STZ for five consecutive days (50 mg/kg × 5, mSTZ) was administered to GcgKO mice. The contribution of GIP to glucose metabolism in GcgKO mice was also investigated by experiments employing dipeptidyl peptidase IV (DPP4) inhibitor (DPP4i) or Gcg–Gipr double knockout (DKO) mice.ResultsGcgKO mice developed severe diabetes by hSTZ administration despite the absence of glucagon. Administration of mSTZ decreased pancreatic insulin content to 18.8 ± 3.4 (%) in GcgKO mice, but ad libitum-fed blood glucose levels did not significantly increase. Glucose-induced insulin secretion was marginally impaired in mSTZ-treated GcgKO mice but was abolished in mSTZ-treated DKO mice. Although GcgKO mice lack GLP-1, treatment with DPP4i potentiated glucose-induced insulin secretion and ameliorated glucose intolerance in mSTZ-treated GcgKO mice, but did not increase beta cell area or significantly reduce apoptotic cells in islets.Conclusions/interpretationThese results indicate that GIP has the potential to ameliorate glucose intolerance even under STZ-induced beta cell damage by increasing insulin secretion rather than by promoting beta cell survival.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-016-3935-2) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

A novel chemically modified analogue of xenin-25 exhibits improved glucose-lowering and insulin-releasing properties

Background: Xenin-25 is a K-cell derived gut peptide with insulin-releasing activity which is rapidly degraded following release into the circulation. We hypothesized that substitution of all naturally-occurring Lys and Arg residues with Gln would lead to prolonged enzyme resistance and enhanced biological efficacy.Methods: Peptide stability was assessed using murine plasma, in vitro insulin-releasing actions evaluated in BRIN-BD11 cells and acute glucose-lowering and insulin-releasing actions examined in high fat fed mice. For sub-chronic studies, a range of metabolic parameters and pancreatic histology were assessed in high fat fed mice which had received saline vehicle or xenin-25(gln) twice-daily for 21days.Results: In contrast to native xenin-25, xenin-25(gln) was resistant to plasma-mediated degradation and significantly stimulated insulin secretion in BRIN-BD11 cells. Acute administration of xenin-25(gln) in high fat fed mice significantly reduced blood glucose and increased plasma insulin concentrations. Twice-daily administration of xenin-25(gln) in high fat fed mice did not affect food intake, body weight or circulating insulin concentrations but significantly decreased blood glucose from day 9 onwards. Furthermore, glucose tolerance, glucose-mediated insulin secretion, insulin sensitivity and GIP-stimulated insulin-release were significantly enhanced in xenin-25(gln)-treated mice. Pancreatic immunohistochemistry revealed decreased alpha cell area with increased beta cell area and beta-to-alpha cell ratio in xenin-25(gln)-treated mice. In addition, xenin-25(gln) exerted similar beneficial actions in ob/ob mice as demonstrated by reduced blood glucose, superior glycaemic response and glucose-mediated insulin release.Conclusions: Xenin-25(gln) is resistant to plasma-mediated degradation and exerts sustained and beneficial metabolic actions in high fat fed and ob/ob mice.General significance: Glutamine (gln)-modified analogues of xenin may represent an attractive therapeutic approach for type 2 diabetes.

Pancreatic glucose-dependent insulinotropic polypeptide (GIP) (1–30) expression is upregulated in diabetes and PEGylated GIP(1–30) can suppress the progression of low-dose-STZ-induced hyperglycaemia in mice

Glucose-dependent insulinotropic polypeptide (GIP) is a peptide hormone released from gut K cells. While the predominant form is GIP(1-42), a shorter form, GIP(1-30), is produced by pancreatic alpha cells and promotes insulin secretion in a paracrine manner. Here, we elucidated whether GIP(1-30) expression is modulated in mouse models of diabetes. We then investigated whether PEGylated GIP(1-30) can improve islet function and morphology as well as suppress the progression to hyperglycaemia in mice treated with low-dose streptozotocin (LD-STZ). We examined pancreatic GIP immunoreactivity in rodent diabetic models. We synthesised [D-Ala(2)]GIP(1-30) and modified the C-terminus with polyethylene glycol (PEG) to produce a dipeptidyl peptidase-4 (DPP-4)-resistant long-acting GIP analogue, [D-Ala(2)]GIP(1-30)-PEG. We performed i.p.GTT and immunohistochemical analysis in non-diabetic and LD-STZ diabetic mice, with or without administration of [D-Ala(2)]GIP(1-30)-PEG. Pancreatic GIP expression was concomitantly enhanced with alpha cell expansion in rodent models of diabetes. Treatment with DPP-4 inhibitor decreased both the GIP- and glucagon-positive areas and preserved the insulin-positive area in LD-STZ diabetic mice. Body weight was not affected by [D-Ala(2)]GIP(1-30)-PEG in LD-STZ or non-diabetic mice. Treatment with GIP significantly ameliorated chronic hyperglycaemia and improved glucose excursions in LD-STZ mice. Treatment with GIP also reduced alpha cell expansion in the islets and suppressed plasma glucagon levels compared with non-treated LD-STZ mice. Additionally, [D-Ala(2)]GIP(1-30)-PEG preserved beta cell area via inhibition of apoptosis in LD-STZ mice. Our data suggest that GIP(1-30) expression is upregulated in diabetes, and PEGylated GIP(1-30) can suppress the progression to STZ-induced hyperglycaemia by inhibiting beta cell apoptosis and alpha cell expansion.

Beneficial metabolic actions of a stable GIP agonist following pre-treatment with a SGLT2 inhibitor in high fat fed diabetic mice

The purpose of the present study was to examine if a stable glucose-dependent insulinotropic polypeptide (GIP) agonist could exert beneficial metabolic control in diabetic mice which had been pre-treated with sodium-glucose-cotransporter-2 (SGLT2) inhibitor dapagliflozin (DAPA). High fat fed mice administered low dose streptozotocin (STZ) received vehicle, DAPA once-daily over 28 days, or DAPA once-daily for 14 days followed by (dAla2)GIP once-daily for 14 days. Energy intake, body weight, glucose and insulin concentrations were measured at regular intervals. Glucose tolerance, insulin tolerance test, dual-energy X-ray absorptiometry (DEXA) and pancreatic histology were examined. Once-daily administration of (dAla2)GIP for 14 days in high fat fed diabetic mice pre-treated with DAPA demonstrated significant decrease in body weight, blood glucose and increased insulin concentrations which were independent of changes in energy intake. Similarly, glucose tolerance, glucose-stimulated insulin secretion, insulin sensitivity and HOMA-β were significantly enhanced in (dAla2)GIP-treated mice. DEXA analysis revealed sustained percentage body fat loss with no changes in lean mass, bone mineral content and density. Pancreatic immunohistochemical analysis revealed decreased islet number and increases in islet area, beta cell area and pancreatic insulin content. The DAPA-induced increase in alpha cell area was also reversed. Additional acute in vitro and in vivo experiments confirmed that the impaired action of (dAla2)GIP under hyperglycaemic-induced conditions was significantly reversed by DAPA treatment. These data demonstrate that (dAla2)GIP can exert beneficial metabolic control in high fat fed diabetic mice pre-treated with DAPA. The results highlight possibility of a targeted and personalized approach using a GIP agonist and SGLT2 inhibitor for the treatment of type 2 diabetes.

Hepatic role in an early glucose-lowering effect by a novel dipeptidyl peptidase 4 inhibitor, evogliptin, in a rodent model of type 2 diabetes

Although multiple dipeptidyl peptidase 4 (DPP4) inhibitors have shown glucose-lowering effects by preserving pancreatic cells in high-fat diet (HFD)/streptozotocin (STZ)-induced diabetic mice, the hepatic role in regulation of glucose homeostasis by DPP4 inhibitors in HFD/STZ mice remains elusive. In herein study, parallel comparison of effects on the liver (expression of gluconeogenic genes and the linked signaling molecules) and pancreas (islet morphology and relative area of alpha or beta cells) in combination with glucose-lowering effects were made at the end of 2- and 10-week of evogliptin treatment in HFD/STZ mice. Significant control of hyperglycemia was observed from the second week and persisted during 10-week treatment of 0.3% evogliptin in HFD/STZ mice. This effect was accompanied by increased level of plasma glucagon-like peptide-1 and preserved pancreas islet structure. Furthermore, the hepatic increases in gluconeogenic gene expression in HFD/STZ mice was significantly reduced by evogliptin treatment, which was accompanied by the suppression of cAMP response element-binding protein (CREB) phosphorylation and expression of transducer of regulated CREB protein 2. This hepatic effect of evogliptin treatment was reproduced in 2-week study, however, pancreatic beta-cell area was not altered yet although the expression of pancreatic and duodenal homeobox protein 1 was increased. We conclude that the suppression of hepatic gluconeogenesis by evogliptin is followed by preservation of pancreatic islet, leading to remarkable and persistent glucose-lowering effect in HFD/STZ mice. Our findings provide further insight for the hepatic role in DPP4 inhibitor-mediated glucose control in diabetes.

Prenatal Testosterone Exposure Leads to Gonadal Hormone-Dependent Hyperinsulinemia and Gonadal Hormone-Independent Glucose Intolerance in Adult Male Rat Offspring.

Elevated testosterone levels during prenatal life lead to hyperandrogenism and insulin resistance in adult females. This study evaluated whether prenatal testosterone exposure leads to the development of insulin resistance in adult male rats in order to assess the influence of gonadal hormones on glucose homeostasis in these animals. Male offspring of pregnant rats treated with testosterone propionate or its vehicle (control) were examined. A subset of male offspring was orchiectomized at 7 wk of age and reared to adulthood. At 24 wk of age, fat weights, plasma testosterone, glucose homeostasis, pancreas morphology, and gastrocnemius insulin receptor (IR) beta levels were examined. The pups born to testosterone-treated mothers were smaller at birth and remained smaller through adult life, with levels of fat deposition relatively similar to those in controls. Testosterone exposure during prenatal life induced hyperinsulinemia paralleled by an increased HOMA-IR index in a fasting state and glucose intolerance and exaggerated insulin responses following a glucose tolerance test. Prenatal androgen-exposed males had more circulating testosterone during adult life. Gonadectomy prevented hyperandrogenism, reversed hyperinsulinemia, and attenuated glucose-induced insulin responses but did not alter glucose intolerance in these rats. Prenatal androgen-exposed males had decreased pancreatic islet numbers, size, and beta-cell area along with decreased expression of IR in gastrocnemius muscles. Gonadectomy restored pancreatic islet numbers, size, and beta-cell area but did not normalize IRbeta expression. This study shows that prenatal testosterone exposure leads to a defective pancreas and skeletal muscle function in male offspring. Hyperinsulinemia during adult life is gonad-dependent, but glucose intolerance appears to be independent of postnatal testosterone levels.

I10W]tigerinin-1R enhances both insulin sensitivity and pancreatic beta cell function and decreases adiposity and plasma triglycerides in high-fat mice.

We have previously described the insulinotropic activities of [I10W]tigerinin-1R (RVCSAIPLPWCH.NH2) in vitro. In this study, we investigated the effects of the peptide on nutrient homoeostasis in mice with diet-induced obesity and insulin resistance. Male NIH Swiss mice were maintained on a high-fat diet for 12 weeks prior to the study. Twice-daily intraperitoneal injections of [I10W]tigerinin-1R (75nmol/kg body weight) were administered for 28days. Body weight, energy intake, body fat content, and plasma concentrations of triglyceride, cholesterol, non-fasting glucose and insulin were monitored. Effects of the peptide on glycaemic control were measured by glucose tolerance and insulin sensitivity tests. Pancreatic hormone content and insulin secretory responses of islets isolated from treated and untreated mice were examined. Immunohistochemical analysis was performed to study possible changes in islet morphology. Administration of [I10W]tigerinin-1R to high-fat-fed mice produced significant (P<0.05) decreases in plasma glucose, glucagon and triglyceride concentrations and an increase in plasma insulin compared to high-fat-fed controls. No changes in body weight or energy intake were observed with peptide treatment, but glycaemic control was significantly improved in response to oral or intraperitoneal glucose. Insulin sensitivity and secretory responses of islets to established insulin secretagogues were also significantly improved in peptide-treated mice. Total body fat, pancreatic insulin and glucagon contents, islet, beta and alpha cell areas were all significantly decreased in treated mice. This study shows that [I10W]tigerinin-1R improves insulin sensitivity, islet function and glycaemic control in high-fat-fed mice and has potential as a template for development of novel anti-diabetic agents.

The frog skin host-defense peptide CPF-SE1 improves glucose tolerance, insulin sensitivity and islet function and decreases plasma lipids in high-fat fed mice

The frog skin host-defense peptide CPF-SE1 has previously been shown to stimulate the in vitro release of insulin from clonal BRIN-BD11 β-cells. In this study, the in vivo effects of the peptide were investigated in male NIH Swiss mice maintained on a high-fat diet to induce obesity and insulin resistance. Insulin-secretory responses of islets isolated from treated and untreated mice and changes in islet morphology were also examined. Total body fat, plasma glucagon, triglyceride and cholesterol concentrations were measured at the end of the treatment period. Acute intraperitoneal administration of CPF-SE1 (75nmol body weight) to high-fat fed mice together with glucose (18mmol/kg body weight) improved glucose tolerance and insulin responses compared to high-fat fed controls. Long term administration of CPF-SE1 (twice-daily administration of 75nmol/kg body weight for 28 days) did not affect body weight or energy intake but decreased circulating glucose and increased insulin concentrations. Insulin sensitivity and insulin-secretory responses of islets to secretagogues were also significantly improved at 28 days in peptide-treated mice. In addition, significant decreases in plasma glucagon concentrations, pancreatic insulin and glucagon content, islet and beta cell area, body fat and plasma triglyceride levels were observed in CPF-SE1 treated with mice. In conclusion, CPF-SE1 improves beta cell function, insulin sensitivity and glycaemic control whilst reducing total body fat and circulating triglyceride levels. The peptide shows potential for development into an agent for treatment of patients with metabolic syndrome and type 2 diabetes.

High-fat diet feeding significantly attenuates anagliptin-induced regeneration of islets of Langerhans in streptozotocin-induced diabetic mice.

BackgroundDPP-4 inhibitors reportedly exert effects on both alpha and beta cells, and promote the proliferation and survival of beta cells. We investigated the effects of anagliptin on structurally-impaired islets of Langerhans in streptozotocin (STZ)-treated mice, fed either a normal or a high-fat diet. Pdx-1 expression in the pancreas and serum insulin/glucagon concentrations were also examined.FindingsAnagliptin treatment significantly up-regulated pancreatic Pdx-1 expression, with elevated serum glucagon-like peptide-1 concentrations, regardless of whether the diet was normal or high-fat. However, interestingly, the beta cell regeneration, structural normalization of islets of Langerhans including alpha cell: beta cell area ratios, and serum insulin elevation, all observed with anagliptin administration in the animals fed a normal diet, were markedly suppressed in the high-fat fed group.ConclusionsHigh-fat diet feeding clearly weakened the regenerative effects of anagliptin on the islets of Langerhans in STZ-treated mice. Our findings suggest the importance of normalizing lipid metabolism for full manifestation of DPP-4 inhibitor effects on the islets of Langerhans.Electronic supplementary materialThe online version of this article (doi:10.1186/s13098-015-0047-y) contains supplementary material, which is available to authorized users.

Increment of serum C-peptide measured by glucagon test closely correlates with human relative beta-cell area.

Pancreatic beta-cell mass contributes to glucose tolerance. The aim of this study was to evaluate the relationships between human beta-cell mass and various clinical parameters, including insulin secretory capacity. The study included 32 Japanese patients who underwent pancreatectomy and were naive to oral hypoglycemic agents and insulin. They were classified into those with normal glucose tolerance (n=13), impaired glucose tolerance (n=9) and diabetes (n=10), and their insulin secretory capacity and insulin resistance were evaluated. Immunohistochemistry was used to determine relative beta-cell area (%) which represented the proportion of insulin-positive cell area to whole pancreatic section. Increment of C-peptide immunoreactivity level by glucagon test (ΔC-peptide, increment of serum C-peptide [nmol/L] at 6 min after intravenous injection of 1-mg glucagon; r=0.64, p=0.002), homeostasis model assessment of beta-cell function (HOMA-beta, fasting immunoreactive insulin [μIU/mL] x 20 / (fasting plasma glucose [mmol/L] - 3.5); r=0.50, p=0.003), C-peptide index (CPI, fasting C-peptide [nmol/L] / fasting plasma glucose [mmol/L]; r=0.36, p=0.042), and fasting immunoreactive insulin (F-IRI [pmol/L]; r=0.36, p=0.044) correlated significantly and positively with the relative beta-cell area. The area under the curve of plasma glucose level from 0 to 120 min by 75 g-OGTT (AUC0-120) also correlated significantly and inversely with the relative beta-cell area (r=-0.36, p=0.045). Stepwise multiple regression analysis identified ΔC-peptide as the only independent and significant determinant of the relative beta-cell area. We conclude that ΔC-peptide, HOMA-beta, CPI, F-IRI and AUC0-120 correlated closely with the relative beta-cell area, and ΔC-peptide was the most valuable index for the prediction of the area.

Islet amyloid formation is an important determinant for inducing islet inflammation in high-fat-fed human IAPP transgenic mice.

Amyloid deposition and inflammation are characteristic of islet pathology in type 2 diabetes. The aim of this study was to determine whether islet amyloid formation is required for the development of islet inflammation in vivo. Human islet amyloid polypeptide transgenic mice and non-transgenic littermates (the latter incapable of forming islet amyloid) were fed a low-fat (10%) or high-fat (60%) diet for 12 months; high-fat feeding induces islet amyloid formation in transgenic mice. At the conclusion of the study, glycaemia, beta cell function, islet amyloid deposition, markers of islet inflammation and islet macrophage infiltration were measured. Fasting plasma glucose levels did not differ by diet or genotype. Insulin release in response to i.v. glucose was significantly greater in both high vs low fat groups, and significantly lower in both transgenic compared with non-transgenic groups. Only high-fat-fed transgenic mice developed islet amyloid and showed a trend towards reduced beta cell area. Compared with islets from low-fat-fed transgenic or high-fat-fed non-transgenic mice, islets of high-fat-fed transgenic mice displayed a significant increase in the expression of genes encoding chemokines (Ccl2, Cxcl1), macrophage/dendritic cell markers (Emr1, Itgax), NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome components (Nlrp3, Pycard, Casp1) and proinflammatory cytokines (Il1b, Tnf, Il6), as well as increased F4/80 staining, consistent with increased islet inflammation and macrophage infiltration. Our results indicate that islet amyloid formation is required for the induction of islet inflammation in this long-term high-fat-diet model, and thus could promote beta cell dysfunction in type 2 diabetes via islet inflammation.

A novel DPP IV-resistant C-terminally extended glucagon analogue exhibits weight-lowering and diabetes-protective effects in high-fat-fed mice mediated through glucagon and GLP-1 receptor activation

Modification of the structure of glucagon could provide useful compounds for the potential treatment of obesity-related diabetes. This study evaluated N-acetyl-glucagon, (D-Ser(2))glucagon and an analogue of (D-Ser(2))glucagon with the addition of nine amino acids from the C-terminal of exendin(1-39), namely (D-Ser(2))glucagon-exe. All analogues were resistant to dipeptidyl peptidase IV degradation. N-Acetyl-glucagon lacked acute insulinotropic effects in BRIN BD11 cells, whereas (D-Ser(2))glucagon and (D-Ser(2))glucagon-exe evoked significant (p < 0.001) insulin release. (D-Ser(2))glucagon-exe stimulated cAMP production (p < 0.001) in glucagon- and GLP-1-receptor (GLP-1R)-transfected cells but not in glucose-dependent insulinotropic polypeptide-receptor-transfected cells. In normal mice, N-acetyl-glucagon and (D-Ser(2))glucagon retained glucagon-like effects of increasing (p < 0.001) plasma glucose and insulin levels. (D-Ser(2))glucagon-exe was devoid of hyperglycaemic actions but substantially (p < 0.001) increased plasma insulin levels. (D-Ser(2))glucagon-exe reduced the glycaemic excursion (p < 0.01) and increased the insulin secretory (p < 0.01) response following a glucose challenge 12 h after administration. Studies in GLP-1R knockout mice confirmed involvement of the GLP-1R pathway in the biological actions of (D-Ser(2))glucagon-exe. Twice-daily administration of (D-Ser(2))glucagon-exe to high-fat-fed mice for 28 days significantly (p < 0.05 to p < 0.001) reduced body weight, energy intake and non-fasting glucose levels, as well as increasing insulin concentrations. Glucose tolerance and insulin sensitivity were significantly (p < 0.01) improved and energy expenditure, O2 consumption and locomotor activity were (p < 0.05 to p < 0.001) augmented. The metabolic benefits were accompanied by increases in pancreatic islet number (p < 0.001) and area (p < 0.05), as well as beta cell area (p < 0.05). Beneficial effects were largely retained for 14 days following cessation of treatment. This study emphasises the potential of (D-Ser(2))glucagon-exe for the treatment of obesity-related diabetes.

Incretin receptor null mice reveal key role of GLP-1 but not GIP in pancreatic beta cell adaptation to pregnancy.

Islet adaptations to pregnancy were explored in C57BL6/J mice lacking functional receptors for glucagon-like peptide 1 (GLP-1) and gastric inhibitory polypeptide (GIP). Pregnant wild type mice and GIPRKO mice exhibited marked increases in islet and beta cell area, numbers of medium/large sized islets, with positive effects on Ki67/Tunel ratio favouring beta cell growth and enhanced pancreatic insulin content. Alpha cell area and glucagon content were unchanged but prohormone convertases PC2 and PC1/3 together with significant amounts of GLP-1 and GIP were detected in alpha cells. Knockout of GLP-1R abolished these islet adaptations and paradoxically decreased pancreatic insulin, GLP-1 and GIP. This was associated with abolition of normal pregnancy-induced increases in plasma GIP, L-cell numbers, and intestinal GIP and GLP-1 stores. These data indicate that GLP-1 but not GIP is a key mediator of beta cell mass expansion and related adaptations in pregnancy, triggered in part by generation of intra-islet GLP-1.

Toll-like receptor 4 agonistic monoclonal antibody reverses new onset type 1 diabetes (BA12P.106)

Abstract Viral, parasitic or bacterial infections can prevent T1D in NOD mice. The “hygiene hypothesis” proposes that the incidence of autoimmunity is increasing due to decreased immune stimulation in childhood, offering a rationale for stimulation of innate immunity as a treatment for T1D. We demonstrate that a TLR4/MD-2 specific agonistic monoclonal antibody, UT18, can not only prevent T1D, but also reverse new onset T1D (diagnosed by polyuria, weight loss, and elevated blood glucose). UT15, a control TLR4/MD-2 non-agonistic monoclonal antibody, neither prevented nor reversed T1D. In the prevention studies, UT18 treatment resulted in decreased insulitis vs. UT15 treatment. UT18 treatment permanently reversed new onset T1D in 9/13 mice, while UT15 treated mice rapidly progressed to end-stage diabetes (&amp;gt;600 mg/dl). Mice successfully treated with UT18 had less severe islet infiltration and increased insulin positive beta-cell area compared to UT15 controls. Transfer of CD4+ and CD8+ T-cells (from mice successfully treated with UT18) did not cause diabetes for up to 75 days post transfer, whereas cells transferred from UT15 and non-diabetic NOD mice rapidly caused disease. The lack of T cell mediated diabetes transfer suggests that T cell tolerance was restored in new onset diabetic mice treated with UT18. Restoration of T cell tolerance via TLR-4/MD-2 mediated innate immune stimulation is a promising new pathway for treatment of T1D and other T cell mediated autoimmune diseases.

The role of caspase-8 in amyloid-induced beta cell death in human and mouse islets

Reduced beta cell mass due to increased beta cell apoptosis is a key defect in type 2 diabetes. Islet amyloid, formed by the aggregation of human islet amyloid polypeptide (hIAPP), contributes to beta cell death in type 2 diabetes and in islet grafts in patients with type 1 diabetes. In this study, we used human islets and hIAPP-expressing mouse islets with beta cell Casp8 deletion to (1) investigate the role of caspase-8 in amyloid-induced beta cell apoptosis and (2) test whether caspase-8 inhibition protects beta cells from amyloid toxicity. Human islet cells were cultured with hIAPP alone, or with caspase-8, Fas or amyloid inhibitors. Human islets and wild-type or hIAPP-expressing mouse islets with or without caspase-8 expression (generated using a Cre/loxP system) were cultured to form amyloid. Caspase-8 and -3 activation, Fas and FLICE inhibitory protein (FLIP) expression, islet beta cell and amyloid area, IL-1β levels, and the beta:alpha cell ratio were assessed. hIAPP treatment induced activation of caspase-8 and -3 in islet beta cells (via Fas upregulation), resulting in apoptosis, which was markedly reduced by blocking caspase-8, Fas or amyloid. Amyloid formation in cultured human and hIAPP-expressing mouse islets induced caspase-8 activation, which was associated with Fas upregulation and elevated islet IL-1β levels. hIAPP-expressing mouse islets with Casp8 deletion had comparable amyloid, IL-1β and Fas levels with those expressing hIAPP and Casp8, but markedly lower beta cell apoptosis, higher beta:alpha cell ratio, greater beta cell area, and enhanced beta cell function. Beta cell Fas upregulation by endogenously produced and exogenously applied hIAPP aggregates promotes caspase-8 activation, resulting in beta cell apoptosis. The prevention of amyloid-induced caspase-8 activation enhances beta cell survival and function in islets.