Wholegrain fermentation affects gut microbiota composition, phenolic acid metabolism and pancreatic beta cell function in a rodent model of type 2 diabetes.

The intestinal microbiota plays an important role in host metabolism via production of dietary metabolites. Microbiota imbalances are linked to type 2 diabetes (T2D), but dietary modification of the microbiota may promote glycemic control. Using a rodent model of T2D and an gut model system, this study investigated whether differences in gut microbiota between control mice and mice fed a high-fat, high-fructose (HFHFr) diet influenced the production of phenolic acid metabolites following fermentation of wholegrain (WW) and control wheat (CW).

The glucose tolerance test in mice: Sex, drugs and protocol.

Aim: To establish the impact of sex, dosing route, fasting duration and acute habituation stress on glucose tolerance test (GTT) measurements used in the preclinical evaluation of potential glucose-modulating therapeutics.

Methods: Adult male and female C57Bl/6J mice, implanted with HD-XG glucose telemetry devices, were fasted for 16 hours or 6 hours following acute habituation stress due to whole cage change, cage change with retention of used bedding or no cage change prior to intraperitoneal (IP) GTTs. To evaluate protocol refinement and sex on the ability of the GTT to detect drug effects, we administered 250 mg/kg oral metformin or 10 nmol/kg IP exendin-4 using optimized protocols.

Impaired Nociception in the Diabetic Mouse.

The mechanisms responsible for painful and insensate diabetic neuropathy are not completely understood. Here, we have investigated sensory neuropathy in the mouse, a hereditary model of diabetes. Akita mice become diabetic soon after weaning, and we show that this is accompanied by an impaired mechanical and thermal nociception and a significant loss of intraepidermal nerve fibers.

Imatinib prevents beta cell death in vitro but does not improve islet transplantation outcome.

Introduction Improving islet transplantation outcome could not only bring benefits to individual patients but also widen the patient pool to which this life-changing treatment is available. Imatinib has previously been shown to protect beta cells from apoptosis in a variety of in vitro and in vivo models. The aim of this study was to investigate whether imatinib could be used to improve islet transplantation outcome.

Annexin A1 Is a Key Modulator of Mesenchymal Stromal Cell-Mediated Improvements in Islet Function.

We have previously demonstrated that coculture of islets with mesenchymal stromal cells (MSCs) enhanced islet insulin secretory capacity in vitro, correlating with improved graft function in vivo. To identify factors that contribute to MSC-mediated improvements in islet function, we have used an unbiased quantitative RT-PCR screening approach to identify MSC-derived peptide ligands of G-protein-coupled receptors that are expressed by islets cells. We demonstrated high expression of annexin A1 (ANXA1) mRNA by MSCs and confirmed expression at the protein level in lysates and MSC-conditioned media by Western blot analysis and ELISA.

Assembly of bioactive multilayered nanocoatings on pancreatic islet cells: incorporation of α1-antitrypsin into the coatings.

A spontaneous multilayer deposition approach for presenting therapeutic proteins onto pancreatic islet surfaces, using a heparin polyaldehyde and glycol chitosan alternating layering scheme, has been developed to enable the nanoscale engineering of a microenvironment for transplanted cells. The nanocoating incorporating α1-antitrypsin, an anti-inflammatory protein, exhibited effective anti-coagulant activities in vitro.

Preculturing Islets With Adipose-Derived Mesenchymal Stromal Cells Is an Effective Strategy for Improving Transplantation Efficiency at the Clinically Preferred Intraportal Site.

We have recently shown that preculturing islets with kidney-derived mesenchymal stromal cells (MSCs) improves transplantation outcome in streptozotocin-diabetic mice implanted with a minimal mass of islets beneath the kidney capsule. In the present study, we have extended our previous observations to investigate whether preculturing islets with MSCs can also be used to enhance islet function at the clinically used intraportal site. We have used MSCs derived from adipose tissue, which are more readily accessible than alternative sources in human subjects and can be expanded to clinically efficacious numbers, to preculture islets throughout this study.

Metabolic phenotyping guidelines: assessing glucose homeostasis in rodent models.

The pathophysiology of diabetes as a disease is characterised by an inability to maintain normal glucose homeostasis. In type 1 diabetes, this is due to autoimmune destruction of the pancreatic β-cells and subsequent lack of insulin production, and in type 2 diabetes it is due to a combination of both insulin resistance and an inability of the β-cells to compensate adequately with increased insulin release. Animal models, in particular genetically modified mice, are increasingly being used to elucidate the mechanisms underlying both type 1 and type 2 diabetes, and as such the ability to study glucose homeostasis in vivo has become an essential tool.

Sustained NF-κB activation and inhibition in β-cells have minimal effects on function and islet transplant outcomes.

The activation of the transcription factor NF-κB leads to changes in expression of many genes in pancreatic β-cells. However, the role of NF-κB activation in islet transplantation has not been fully elucidated. The aim of the present study was to investigate whether the state of NF-κB activation would influence the outcome of islet transplantation.

The novel chemokine receptor, G-protein-coupled receptor 75, is expressed by islets and is coupled to stimulation of insulin secretion and improved glucose homeostasis.

Aims/hypothesis: Chemokine (C-C motif) ligand 5 (CCL5) acts at C-C chemokine receptors (CCRs) to promote immune cell recruitment to sites of inflammation, but is also an agonist at G-protein-coupled receptor 75 (GPR75), which has very limited homology with CCRs. GPR75 is coupled to Gq to elevate intracellular calcium, so we investigated whether islets express this receptor and whether its activation by CCL5 increases beta cell calcium levels and insulin secretion.

Methods: Islet CCL5 receptor mRNA expression was measured by quantitative RT-PCR and GPR75 was detected in islets by western blotting and immunohistochemistry.

Nupr1 deletion protects against glucose intolerance by increasing beta cell mass.

Aims/hypothesis: The stress-activated nuclear protein transcription regulator 1 (NUPR1) is induced in response to glucose and TNF-α, both of which are elevated in type 2 diabetes, and Nupr1 has been implicated in cell proliferation and apoptosis cascades. We used Nupr1(-/-) mice to study the role of Nupr1 in glucose homeostasis under normal conditions and following maintenance on a high-fat diet (HFD).

Methods: Glucose homeostasis in vivo was determined by measuring glucose tolerance, insulin sensitivity and insulin secretion.

Maintenance of islet morphology is beneficial for transplantation outcome in diabetic mice.

We have previously shown that co-transplantation of islets and Mesenchymal Stem Cells (MSCs) improves islet graft function and revascularisation, which was associated with the maintenance of normal islet morphology. The aim of the current study was to determine whether maintaining islet morphology in the absence of additional islet-helper cells would improve transplantation outcome in diabetic mice. Islets were isolated from C57BL/6 mice.

Pre-culturing islets with mesenchymal stromal cells using a direct contact configuration is beneficial for transplantation outcome in diabetic mice.

Background Aims: We recently showed that co-transplantation of mesenchymal stromal cells (MSCs) improves islet function and revascularization in vivo. Pre-transplant islet culture is associated with the loss of islet cells. MSCs may enhance islet cell survival or function by direct cell contact mechanisms and soluble mediators.

Co-transplantation of islets with mesenchymal stem cells in microcapsules demonstrates graft outcome can be improved in an isolated-graft model of islet transplantation in mice.

Background Aims: Co-transplantation of islets with mesenchymal stem cells (MSCs) has been shown to improve graft outcome in mice, which has been partially attributed to the effects of MSCs on revascularization and preservation of islet morphology. Microencapsulation of islets provides an isolated-graft model of islet transplantation that is non-vascularized and prevents islet aggregation to preserve islet morphology. The aim of this study was to investigate whether MSCs could improve graft outcome in a microencapsulated/isolated-graft model of islet transplantation.

Activin receptor-like kinase 5 inhibition reverses impairment of endothelial cell viability by endogenous islet mesenchymal stromal cells.

Following islet transplantation, islet graft revascularization is compromised due to loss of endothelial cells (ECs) during islet culture. TGF-β signaling pathways are essential for vascular homeostasis but their importance for islet EC function is unclear. We have identified a population of multipotent mesenchymal stromal cells (MSCs) within islets and investigated how modulation of TGF-β signaling by these cells influences islet EC viability.

Nop-7-associated 2 (NSA2), a candidate gene for diabetic nephropathy, is involved in the TGFβ1 pathway.

We recently showed that Nop-7-associated 2 (NSA2) originally described in yeast as a nuclear protein involved in ribosomal biogenesis, is a hyperglycemia induced gene involved in diabetic nephropathy [Shahni et al., Elevated levels of renal and circulating Nop-7-associated 2 (NSA2) in rat and mouse models of diabetes, in mesangial cells in vitro and in patients with diabetic nephropathy. Diabetologia 2012;55(March(3)):825-34].

The use of animal models in diabetes research.

Diabetes is a disease characterized by a relative or absolute lack of insulin, leading to hyperglycaemia. There are two main types of diabetes: type 1 diabetes and type 2 diabetes. Type 1 diabetes is due to an autoimmune destruction of the insulin-producing pancreatic beta cells, and type 2 diabetes is caused by insulin resistance coupled by a failure of the beta cell to compensate.

In vivo laboratory practicals in research-led teaching: an example using glucose tolerance tests in lean and obese mice.

The use of animal models is an essential part of medical research and drug development. The essential skills required to be able to do such research includes experimental design, statistical analysis and the actual handling and treating of the animals (in vivo skills). The number of students in the U.

A role for islet somatostatin in mediating sympathetic regulation of glucagon secretion.

Aims/hypothesis: Somatostatin (SST) released from islet δ-cells inhibits both insulin and glucagon secretion but the role of this tonic inhibition is unclear. In this study we investigated whether δ-cell SST may facilitate sympathetic regulation of glucagon secretion as part of an 'accelerator/brake' mechanism.

Methods: The secretory characteristics of islets isolated from SST-deficient (Sst-/-) and control mouse islets were assessed in static incubation studies.

Somatostatin secreted by islet delta-cells fulfills multiple roles as a paracrine regulator of islet function.

Objective: Somatostatin (SST) is secreted by islet delta-cells and by extraislet neuroendocrine cells. SST receptors have been identified on alpha- and beta-cells, and exogenous SST inhibits insulin and glucagon secretion, consistent with a role for SST in regulating alpha- and beta-cell function. However, the specific intraislet function of delta-cell SST remains uncertain.

Anti-apoptotic effects of arachidonic acid and prostaglandin E2 in pancreatic beta-cells.

Background/aims: The polyunsaturated fatty acid arachidonic acid (AA) has been implicated in beta-cell defence mechanisms and prostaglandin (PG) products of cyclooxygenase (COX) 2 action confer resistance to alloxan-induced apoptosis in insulin-secreting RIN cells. We have now investigated the anti-apoptotic effects of AA and its metabolite, PGE(2), in the MIN6 mouse insulin-secreting beta-cell line and mouse islets.

Methods: Apoptosis was determined in MIN6 beta-cell and mouse islet extracts by measurement of capase-3 activity, and COX2 mRNA levels were quantified by real-time RT-PCR.

Normal relationship of beta- and non-beta-cells not needed for successful islet transplantation.

Islets are composed mostly of beta-cells, and therefore stem cell research has concentrated on generating purified beta-cells, neglecting the other endocrine cell types in the islet. We investigated the presence of endocrine non-beta-cells after islet transplantation. In addition, we studied whether the transplantation of pure beta-cells, in volumes similar to that used in islet transplantation, would suffice to reverse hyperglycemia in diabetic mice.