Short-term exercise training early in life restores deficits in pancreatic B-cell mass associated with growth restriction in adult male rats

Fetal growth restriction is associated with reduced pancreatic ß-cell mass, contributing to impaired glucose tolerance and diabetes. Exercise training increases ß-cell mass in animals with diabetes and has long-lasting metabolic benefits in rodents and humans. We studied the effect of exercise training on islet and ß-cell morphology and plasma insulin and glucose, following an intraperitoneal glucose tolerance test (IPGTT) in juvenile and adult male Wistar-Kyoto rats born small. Bilateral uterine vessel ligation performed on day 18 of pregnancy resulted in Restricted offspring born small compared with shamoperated Controls and also sham-operated Reduced litter offspring that had their litter size reduced to five pups at birth. Restricted, Control, and Reduced litter offspring remained sedentary or underwent treadmill running from 5 to 9 or 20 to 24 wk of age. Early life exercise increased relative islet surface area and ß-cell mass across all groups at 9 wk, partially restoring the 60–68% deficit (P = 0.05) in Restricted offspring. Remarkably, despite no further exercise training after 9 wk, ß-cell mass was restored in Restricted at 24 wk, while sedentary littermates retained a 45% deficit (P = 0.05) in relative ß-cell mass. Later exercise training also restored Restricted ß-cell mass to Control levels. In conclusion, early life exercise training in rats born small restored ß-cell mass in adulthood and may have beneficial consequences for later metabolic health and disease.

B-cell loss and B-cell apoptosis in human type 2 diabetes are related to islet amyloid deposition

Amyloid deposition and reduced β-cell mass are pathological hallmarks of the pancreatic islet in type 2 diabetes; however, whether the extent of amyloid deposition is associated with decreased β-cell mass is debated. We investigated the possible relationship and, for the first time, determined whether increased islet amyloid and/or decreased β-cell area quantified on histological sections is correlated with increased β-cell apoptosis. Formalin-fixed, paraffin-embedded human pancreas sections from subjects with (n = 29) and without (n = 39) diabetes were obtained at autopsy (64 ± 2 and 70 ± 4 islets/subject, respectively). Amyloid and β cells were visualized by thioflavin S and insulin immunolabeling. Apoptotic β cells were detected by colabeling for insulin and by TUNEL. Diabetes was associated with increased amyloid deposition, decreased -cell area, and increased β-cell βapoptosis, as expected. There was a strong inverse correlation between β-cell area and amyloid deposition (r=0.42, P < 0.001). β-Cell area was selectively reduced in individual amyloid-containing islets from diabetic subjects, compared with control subjects, but amyloid-free islets had β-cell area equivalent to islets from control subjects. Increased amyloid deposition was associated with β-cell apoptosis (r= 0.56, P < 0.01). Thus, islet amyloid is associated with decreased β-cell area and increased β-cell apoptosis, suggesting that islet myloid deposition contributes to the decreased β-cell mass that characterizes type 2 diabetes.

The influence of genetic background on the induction of oxidative stress and impaired insulin secretion in mouse islets

Aims/hypothesis We determined whether high-glucose-induced beta cell dysfunction is associated with oxidative stress in the DBA/2 mouse, a mouse strain susceptible to islet failure.

Materials and methods Glucose- and non-glucose-mediated insulin secretion from the islets of DBA/2 and control C57BL/6 mice was determined following a 48-h exposure to high glucose. Flux via the hexosamine biosynthesis pathway was assessed by determining O-glycosylated protein levels. Oxidative stress was determined by measuring hydrogen peroxide levels and the expression of anti-oxidant enzymes.

Results Exposure to high glucose levels impaired glucose-stimulated insulin secretion in DBA/2 islets but not C57BL/6 islets, and this was associated with reduced islet insulin content and lower ATP levels than in C57BL/6 islets. Exposure of islets to glucosamine for 48 h mimicked the effects of high glucose on insulin secretion in the DBA/2 islets. High glucose exposure elevated O-glycosylated proteins; however, this occurred in islets from both strains, excluding a role for O-glycosylation in the impairment of DBA/2 insulin secretion. Additionally, both glucosamine and high glucose caused an increase in hydrogen peroxide in DBA/2 islets but not in C57BL/6 islets, an effect prevented by the antioxidant N-acetyl-l-cysteine. Interestingly, while glutathione peroxidase and catalase expression was comparable between the two strains, the antioxidant enzyme manganese superoxide dismutase, which converts superoxide to hydrogen peroxide, was increased in DBA/2 islets, possibly explaining the increase in hydrogen peroxide levels.

Conclusions/interpretation Chronic high glucose culture caused an impairment in glucose-stimulated insulin secretion in DBA/2 islets, which have a genetic predisposition to failure, and this may be the result of oxidative stress.

Amyloid formation results in recurrence of hyperglycaemia following transplantation of human IAPP transgenic mouse islets

Aims/hypothesis Islet transplantation is a potential cure for diabetes; however, rates of graft failure remain high. The aim of the present study was to determine whether amyloid deposition is associated with reduced beta cell volume in islet grafts and the recurrence of hyperglycaemia following islet transplantation.

Methods We transplanted a streptozotocin-induced mouse model of diabetes with 100 islets from human IAPP (which encodes islet amyloid polypeptide) transgenic mice that have the propensity to form islet amyloid (n = 8–12) or from non-transgenic mice that do not develop amyloid (n = 6–10) in sets of studies that lasted 1 or 6 weeks.

Results Plasma glucose levels before and for 1 week after transplantation were similar in mice that received transgenic or non-transgenic islets, and at that time amyloid was detected in all transgenic grafts and, as expected, in none of the non-transgenic grafts. However, over the 6 weeks following transplantation, plasma glucose levels increased in transgenic but remained stable in non-transgenic islet graft recipients (p < 0.05). At 6 weeks, amyloid was present in 92% of the transgenic grafts and in none of the non-transgenic grafts. Beta cell volume was reduced by 30% (p < 0.05), beta cell apoptosis was twofold higher (p < 0.05), and beta cell replication was reduced by 50% (p < 0.001) in transgenic vs non-transgenic grafts. In summary, amyloid deposition in islet grafts occurs prior to the recurrence of hyperglycaemia and its accumulation over time is associated with beta cell loss.

Conclusions/interpretation Islet amyloid formation may explain, in part, the non-immune loss of beta cells and recurrence of hyperglycaemia following clinical islet transplantation.

Amyloid formation in human IAPP transgenic mouse islets and pancreas, and human pancreas, is not associated with endoplasmic reticulum stress

Aims/hypothesis Supraphysiological levels of the amyloidogenic peptide human islet amyloid polypeptide have been associated with beta cell endoplasmic reticulum (ER) stress. However, in human type 2 diabetes, levels of human IAPP are equivalent or decreased relative to matched controls. Thus, we sought to investigate whether ER stress is induced during amyloidogenesis at physiological levels of human IAPP.

Methods Islets from human IAPP transgenic mice that develop amyloid, and non-transgenic mice that do not, were cultured for up to 7 days in 11.1, 16.7 and 33.3 mmol/l glucose. Pancreases from human IAPP transgenic and non-transgenic mice and humans with or without type 2 diabetes were also evaluated. Amyloid formation was determined histologically. ER stress was determined in islets by quantifying mRNA levels of Bip, Atf4 and Chop (also known as Ddit3) and alternate splicing of Xbp1 mRNA, or in pancreases by immunostaining for immunoglobulin heavy chain-binding protein (BIP), C/EBP homologous protein (CHOP) and X-box binding protein 1 (XBP1).

Results Amyloid formation in human IAPP transgenic islets was associated with reduced beta cell area in a glucose- and time-dependent manner. However, amyloid formation was not associated with significant increases in expression of ER stress markers under any culture condition. Thapsigargin treatment, a positive control, did result in significant ER stress. Amyloid formation in vivo in pancreas samples from human IAPP transgenic mice or humans was not associated with upregulation of ER stress markers.

Conclusions/interpretation Our data suggest that ER stress is not an obligatory pathway mediating the toxic effects of amyloid formation at physiological levels of human IAPP.

Exendin-4 increases islet amyloid deposition but offsets the resultant beta cell toxicity in human islet amyloid polypeptide transgenic mouse islets

Aims/hypothesis In type 2 diabetes, aggregation of islet amyloid polypeptide (IAPP) into amyloid is associated with beta cell loss. As IAPP is co-secreted with insulin, we hypothesised that IAPP secretion is necessary for amyloid formation and that treatments that increase insulin (and IAPP) secretion would thereby increase amyloid formation and toxicity. We also hypothesised that the unique properties of the glucagon-like peptide-1 (GLP-1) receptor agonist exendin-4 to maintain or increase beta cell mass would offset the amyloid-induced toxicity.

Methods Islets from amyloid-forming human IAPP transgenic and control non-transgenic mice were cultured for 48 h in 16.7 mmol/l glucose alone (control) or with exendin-4, potassium chloride (KCl), diazoxide or somatostatin. Human IAPP and insulin release, amyloid deposition, beta cell area/islet area, apoptosis and AKT phosphorylation levels were determined.

Results In control human IAPP transgenic islets, amyloid formation was associated with increased beta cell apoptosis and beta cell loss. Increasing human IAPP release with exendin-4 or KCl increased amyloid deposition. However, while KCl further increased beta cell apoptosis and beta cell loss, exendin-4 did not. Conversely, decreasing human IAPP release with diazoxide or somatostatin limited amyloid formation and its toxic effects. Treatment with exendin-4 was associated with an increase in AKT phosphorylation compared with control and KCl-treated islets.

Conclusions/interpretation IAPP release is necessary for islet amyloid formation and its toxic effects. Thus, use of insulin secretagogues to treat type 2 diabetes may result in increased islet amyloidogenesis and beta cell death. However, the AKT-associated anti-apoptotic effects of GLP-1 receptor agonists such as exendin-4 may limit the toxic effects of increased islet amyloid.

Investigational agents that protect pancreatic islet β-cells from failure

Type 2 diabetes is associated with insulin resistance and reduced insulin secretion, which results in hyperglycaemia. This can then lead to diabetic complications such as retinopathy, neuropathy, nephropathy and cardiovascular disease. Although insulin resistance may be present earlier in the progression of the disease, it is now generally accepted that it is the deterioration in insulin-secretory function that leads to hyperglycaemia. This reduction in insulin secretion in Type 2 diabetes is due to both islet β-cell dysfunction and death. Therefore, interventions that maintain the normal function and protect the pancreatic islet β-cells from death are crucial in the treatment of Type 2 diabetes so that plasma glucose levels may be maintained within the normal range. Recently, a number of compounds have been shown to protect β-cells from failure. This review examines the evidence that the existing therapies for Type 2 diabetes that were developed to lower plasma glucose (metformin) or improve insulin sensitivity (thiazolidinediones) may also have islet-protective function. Newer emerging therapeutic agents that are designed to increase the levels of glucagon-like peptide-1 not only stimulate insulin secretion but also appear to increase islet β-cell mass. Evidence will also be presented that the future of drug therapy designed to prevent β-cell failure should target the formation of advanced glycation end products and alleviate oxidative and endoplasmic reticulum stress.

High glucose-induced impairment in insulin secretion is associated with reduction in islet glucokinase in a mouse model of susceptibility to islet dysfunction

Type 2 diabetes is characterized by islet dysfunction resulting in hyperglycemia, which can then lead to further deterioration in islet function. A possible mechanism for hyperglycemia-induced islet dysfunction is the accumulation of advanced glycation end products (AGE). The DBA/2 mouse develops pancreatic islet dysfunction when exposed to a high glucose environment and/or obesity-induced insulin resistance. To determine the biochemical cause of dysfunction, DBA/2 and C57BL/6 control islets were incubated in 11.1 mM or 40 mM glucose in the absence or presence of the AGE inhibitor aminoguanidine (AG) for 10 days. Basal (2.8 mM glucose) insulin release was increased in both DBA/2 and C57BL/6 islets incubated with 40 mM vs 11.1 mM glucose for 10 days. Chronic exposure to hyperglycemia decreased glucose (20 mM)-stimulated insulin secretion in DBA/2 but not in C57BL/6 islets. AG significantly increased fold-induced insulin release in high glucose cultured DBA/2 mouse islets, but did not affect C57BL/6 islet function. DBA/2 islet glucokinase was significantly reduced following 40 mM glucose culture, compared with 11.1 mM glucose cultured DBA/2 islets and 40 mM glucose cultured C57BL/6 islets. Incubation of islets with AG resulted in a normalization of DBA/2 islet glucokinase levels. In conclusion, chronic high glucose-induced increases in AGE can result in islet dysfunction and this is associated with reduced glucokinase levels in a mouse model with susceptibility to islet failure.