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Zhi Z.-L.,King's College London | Liu B.,Diabetes Research Group | Jones P.M.,Diabetes Research Group | Pickup J.C.,King's College London
Biomacromolecules | Year: 2010

This paper describes the use of a layer-by-layer nanocoating technique for the encapsulation of insulin-producing pancreatic β-cell spheroids (pseudoislets) within chitosan/alginate multilayers. We used pseudoislets self-organized from a population of the insulinoma cell line MIN6, derived from a transgenic mouse expressing the large T-antigen of S V40 in pancreatic β-cells, as an experimental model for the study of cell nanoencapsulation. The maintenance of spheroid morphology and retention of cell viability and metabolic functionality was demonstrated postencapsulation. By depositing an additional protein-repelling phosphorylcholine-modified chondroitin-4-sulfate layer, the coatings were found to shield effectively access of large molecules of the immune systems to the antigenpresenting cell surfaces. Transmission electron microscopy analysis of the encapsulated pseudoislets revealed that the coating did not damage the cell structure. In addition, nanoencapsulation permits the cells to respond to changes in extracellular glucose and other insulin secretagogues by releasing insulin with a profile similar to that of nonencapsulated cells. These results suggest that this nanofilm encapsulation technique has the characteristics required for the efficient transplantation of cellular engineered β-cells as a cell replacement therapy for type 1 diabetes. This encapsulation method is general in scope and has implications for use in a variety of cellular therapeutics employing engineered tissues from cells generated in vitro from various sources, including those using genetic and cellular engineering techniques. © 2010 American Chemical Society.


Kieswich J.,Queen Mary, University of London | Sayers S.R.,Diabetes Research Group | Silvestre M.F.,University of Lisbon | Silvestre M.F.,University of Auckland | And 3 more authors.
Diabetologia | Year: 2016

Aims/hypothesis: Serum extracellular nicotinamide phosphoribosyltransferase (eNAMPT) concentrations are elevated in type 2 diabetes. However, the relationship between abnormally elevated serum eNAMPT and type 2 diabetes pathophysiology is unclear. eNAMPT circulates in functionally and structurally distinct monomeric and dimeric forms. Dimeric eNAMPT promotes NAD biosynthesis. The role of eNAMPT-monomer is unclear but it may have NAD-independent proinflammatory effects. However, studies of eNAMPT in type 2 diabetes have not distinguished between monomeric and dimeric forms. Since type 2 diabetes is characterised by chronic inflammation, we hypothesised a selective NAD-independent role for eNAMPT-monomer in type 2 diabetes. Methods: Two mouse models were used to examine the role of eNAMPT-monomer in type 2 diabetes; (1) a mouse model of diabetes fed a high-fat diet (HFD) for 10 weeks received i.p. injections with an anti-monomeric-eNAMPT antibody; and (2) lean non-diabetic mice received i.p. injections with recombinant monomeric eNAMPT daily for 14 days. Results: Serum monomeric eNAMPT levels were elevated in HFD-fed mouse models of diabetes, whilst eNAMPT-dimer levels were unchanged. eNAMPT-monomer neutralisation in HFD-fed mice resulted in lower blood glucose levels, amelioration of impaired glucose tolerance (IGT) and whole-body insulin resistance, improved pancreatic islet function, and reduced inflammation. These effects were maintained for at least 3 weeks post-treatment. eNAMPT-monomer administration induced a diabetic phenotype in mice, characterised by elevated blood glucose, IGT, impaired pancreatic insulin secretion and the presence of systemic and tissue inflammation, without changes in NAD levels. Conclusions/interpretation: We demonstrate that elevation of monomeric-eNAMPT plays an important role in the pathogenesis of diet-induced diabetes via proinflammatory mechanisms. These data provide proof-of-concept evidence that the eNAMPT-monomer represents a potential therapeutic target for type 2 diabetes. © 2016 The Author(s)


Stadler M.,Diabetes Research Group | Frohlich-Reiterer E.,Medical University of Graz | Prager R.,3 Medizinische Abteilung For Stoffwechselerkrankungen Und Nephrologie Und Karl Landsteiner Institute For Stoffwechselerkrankungen Und Nephrologie
Wiener Klinische Wochenschrift | Year: 2016

The prevalence of diabetes is increasing in westernized countries. In addition, about half of all patients suffering from diabetes are not diagnosed. The current article represents the recommendations of the Austrian Diabetes Association for the screening and prevention of type 2 diabetes, based on currently available evidence. © 2016, The Author(s).


Joshi M.,Kings College Hospital | Choudhary P.,Diabetes Research Group
Current Diabetes Reports | Year: 2015

Achieving optimal glucose control with minimal hypoglycemia and minimizing the impact of diabetes on quality of life are the aims of management of type 1 diabetes. The main therapeutic options for patients include multiple daily injections (MDI) and continuous subcutaneous insulin therapy (CSII). It is important to differentiate fixed dose MDI with more flexible use, based on carbohydrate counting and structured education programmes, often termed functional insulin therapy (FIT), shown to deliver better outcomes. A significant proportion of patients can achieve optimal glucose control with either therapy, and for those who are unable to achieve desired glucose control with MDI, there is a large body of observational data showing CSII enables them to reduce HbA1c and hypoglycemia, with associated improvements in diabetes-related quality of life. However, in many healthcare systems, guidelines restrict the use of CSII on the basis of cost, with only 20–35 % of patients with type 1 diabetes across Europe using CSII. Although data support improved glucose control and quality of life with CSII, we must recognize that insulin pump therapy is not for everyone and has some downsides such as being attached to a device or issues with cannulas. When we sit down with our patients, we have a responsibility to support those patients with the therapeutic strategy that is best suited to them. In this paper, we review some of the literature that informs this decision-making, highlighting areas where CSII offers clear benefits and also some areas where it may not be appropriate. © 2015, Springer Science+Business Media New York.


Li C.,Diabetes Research Group | Vilches-Flores A.,Diabetes Research Group | Zhao M.,Diabetes Research Group | Amiel S.A.,Diabetes Research Group | And 2 more authors.
Cellular Physiology and Biochemistry | Year: 2012

Elements of the endocannabinoid system (ECS) are expressed by islet endocrine cells and activation of CB1 and CB2 cannabinoid receptors regulates insulin secretion from mouse and human β-cells. The current study aimed to investigate the expression and function, in mouse and human β-cells, of monoacylglycerol lipase (MGL), an enzyme that facilitates degradation of the endocannabinoid 2-arachidonoylglycerol (2-AG). We found that MGL mRNA is expressed by MIN6 β-cells, mouse islets, human islets and enriched human islet β-cells, and immunohistochemistry indicated that MGL localisation in human islets is consistent with its expression by some β-and-α-cells. Blockade of MGL activity with the pharmacological inhibitor URB602 led to increased [Ca2+]iand enhanced insulin secretion from MIN6 β-cells, and MGL inhibition also elevated insulin and glucagon secretion from isolated human islets in vitro. These data imply a stimulatory role for endogenous 2-AG in islets that is amplified when its degradation is blocked. © 2012 S. Karger AG, Basel.


This study was designed to determine the effects of 10 and 13 amino acid forms of kisspeptin on dynamic insulin secretion from mammalian islets since it is not clear from published data whether the shorter peptide is stimulatory while the longer peptide inhibits insulin release. Insulin secretion was measured by radioimmunoassay following perifusion of human, pig, rat and mouse isolated islets with kisspeptin-10 or kisspeptin-13 in the presence of 20 mM glucose. Both peptides stimulated rapid, reversible potentiation of glucose-stimulated insulin secretion from islets of all species tested. These data indicate that both kisspeptin-10 and kisspeptin-13, which is an extension of kisspeptin-10 by three amino acids, act directly at islet β-cells of various species to potentiate insulin secretion, and suggest that inhibitory effects reported in earlier studies may reflect differences in experimental protocols.


Liu B.,Diabetes Research Group | Hassan Z.,Diabetes Research Group | Amisten S.,Diabetes Research Group | King A.J.,Diabetes Research Group | And 4 more authors.
Diabetologia | Year: 2013

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. In some experiments GPR75 was downregulated by transient transfection with small interfering RNA. Real-time changes in intracellular calcium were determined by single-cell microfluorimetry. Dynamic insulin secretion from perifused islets was quantified by radioimmunoassay. Glucose homeostasis in lean and obese mice was determined by measuring glucose and insulin tolerance, and insulin secretion in vivo. Results: Mouse and human islets express GPR75 and its ligand CCL5. Exogenous CCL5 reversibly increased intracellular calcium in beta cells via GPR75, this phenomenon being dependent on phospholipase C activation and calcium influx. CCL5 also stimulated insulin secretion from mouse and human islets in vitro, and improved glucose tolerance in lean mice and in a mouse model of hyperglycaemia and insulin resistance (ob/ob). The improvement in glucose tolerance was associated with enhanced insulin secretion in vivo, without changes in insulin sensitivity. Conclusions/interpretation: Although CCL5 is implicated in the pathogenesis of diabetes through activation of CCRs, it has beneficial effects on beta cells through GPR75 activation. © 2013 Springer-Verlag Berlin Heidelberg.


Persaud S.J.,Diabetes Research Group | Liu B.,Diabetes Research Group | Sampaio H.B.,Diabetes Research Group | Jones P.M.,Diabetes Research Group | Muller D.S.,Diabetes Research Group
Diabetologia | Year: 2011

Aims/hypothesis: Irs2, which is upregulated by glucose, is important for beta cell plasticity. Cyclic AMP response element-binding protein (CREB) stimulates beta cell Irs2 expression and is a major calcium/calmodulin-dependent kinase (CaMK)IV target in neurons. We therefore hypothesised that CaMKIV mediates glucose-induced Irs2 expression in beta cells via CREB activation. Methods: The functions of CaMKIV and CREB were investigated in MIN6 beta cells and mouse islets using the CaMK inhibitor KN62, the calcium chelator bapta-(AM) and the voltage-dependent calcium channel inhibitor nifedipine. Small interfering RNAs were used to silence endogenous CaMKIV production and expression vectors to overproduce constitutively active and dominant negative forms of CaMKIV and CREB. Irs1 and Irs2 expression were determined by quantitative PCR and Western blotting, and the role of CREB was also investigated by assessing its phosphorylation on serine 133. Results: Increasing the glucose concentration from 2.5 to 25 mmol/l stimulated CREB phosphorylation on serine 133 and specifically stimulated Irs2 but not Irs1 expression. Similarly, overproduction of a constitutively active form of CaMKIV promoted sustained CREB phosphorylation and a significant increase in Irs2 but not Irs1 expression. In contrast, these stimulatory effects of glucose were all suppressed by overproducing an inactive CaMKIV mutant. Inhibition of glucose-induced calcium influx with nifedipine or chelation of intracellular calcium with bapta-(AM), as well as silencing of CaMKIV or inhibition of its activity with KN62 resulted in similar observations. Finally, overproduction of a dominant negative form of CREB completely suppressed glucose and CaMKIV stimulation of Irs2 expression. Conclusions/ interpretation: Our results suggest that the Ca2+/CaMK IV/CREB cascade plays a critical role in the regulation of Irs2 expression in beta cells. © 2011 Springer-Verlag.


Ajaz S.,Diabetes Research Group | Czajka A.,Diabetes Research Group | Malik A.,Diabetes Research Group
Methods in Molecular Biology | Year: 2015

We describe a protocol to accurately measure the amount of human mitochondrial DNA (MtDNA) in peripheral blood samples which can be modified to quantify MtDNA from other body fluids, human cells, and tissues. This protocol is based on the use of real-time quantitative PCR (qPCR) to quantify the amount of MtDNA relative to nuclear DNA (designated the Mt/N ratio). In the last decade, there have been increasing numbers of studies describing altered MtDNA or Mt/N in circulation in common nongenetic diseases where mitochondrial dysfunction may play a role (for review see Malik and Czajka, Mitochondrion 13:481–492, 2013). These studies are distinct from those looking at genetic mitochondrial disease and are attempting to identify acquired changes in circulating MtDNA content as an indicator of mitochondrial function. However, the methodology being used is not always specific and reproducible. As more than 95% of the human mitochondrial genome is duplicated in the human nuclear genome, it is important to avoid co-amplification of nuclear pseudogenes. Furthermore, template preparation protocols can also affect the results because of the size and structural differences between the mitochondrial and nuclear genomes. Here we describe how to (1) prepare DNA from blood samples; (2) pretreat the DNA to prevent dilution bias; (3) prepare dilution standards for absolute quantification using the unique primers human mitochondrial genome forward primer (hMitoF3) and human mitochondrial genome reverse primer(hMitoR3) for the mitochondrial genome, and human nuclear genome forward primer (hB2MF1) and human nuclear genome reverse primer (hB2MR1) primers for the human nuclear genome; (4) carry out qPCR for either relative or absolute quantification from test samples; (5) analyze qPCR data; and (6) calculate the sample size to adequately power studies. The protocol presented here is suitable for high-throughput use. © Springer Science+Business Media New York 2015.


PubMed | Diabetes Research Group, Childrens Hospital Research Institute of Manitoba and University of Manitoba
Type: Journal Article | Journal: International journal of obesity (2005) | Year: 2016

The chronic effects of high-intensity endurance training on metabolic health outcomes in overweight adolescents remains poorly understood.To test the hypothesis that high-intensity endurance training (ET) is superior to moderate-intensity ET for improving risk factors for type 2 diabetes in overweight adolescents.In this randomized trial, 106 overweight and obese adolescents (15.2 years; 76% female; 62% Caucasian) were randomly assigned to high-intensity ET (70-85% of heart rate reserve, n=38), moderate-intensity ET (40-55% heart rate reserve; n=32) or control for 6 months (n=36). The primary and secondary outcome measures were insulin sensitivity assessed using a frequently sampled intravenous glucose tolerance test and hepatic triglyceride content with magnetic resonance spectroscopy. Exploratory outcomes were cardiorespiratory fitness, physical activity and MRI and dual x-ray absorptiometry-derived measures of adiposity.The study had 96% retention and attendance was 6121% and 5524% in the high- and moderate-intensity ET arms. Intention-to-treat analyses revealed that, at follow-up, insulin sensitivity was not different between high-intensity (-1.0mUkg(-1)min(-1); 95% confidence interval (CI): -1.6, +1.4mUkg(-1)min(-1)) and moderate-intensity (+0.26mUkg(-1)min(-1); 95% CI: -1.3, +1.8mUkg(-1)min(-1)) ET arms compared with controls (interaction, P=0.97). Similarly, hepatic triglyceride at follow-up was not different in high-intensity (-1.7% fat/water (F/W); 95% CI: -7.0, +3.6% F/W) and moderate-intensity (-0.40% FW; 95% CI: -6.0, +5.3% F/W) ET compared with controls. Both high intensity (+4.4ml per kg-FFM (fat-free mass) per minute; 95% CI: 1.7, 7.1mlkg-FFM(-1)min(-1)) and moderate intensity (+4.4mlkg-FFM(-1)min(-1); 95% CI: 1.6, 7.3mlkg-FFM(-1)min(-1)) increased cardiorespiratory fitness, relative to controls (interaction P<0.001).ET improves cardiorespiratory fitness among obese adolescents; however, owing to lack of compliance, the influence of exercise intensity on insulin sensitivity and hepatic triglycerides remains unclear.

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