Simon J. Dunmore

Glucose induces and leptin decreases expression of uncoupling protein-2 mRNA in human islets

Elevated islet uncoupling protein‐2 (UCP‐2) impairs β‐cell function and UCP‐2 may be increased in clinical obesity and diabetes. We investigated the effects of glucose and leptin on UCP‐2 expression in isolated human islets. Human islets were incubated for 24 h with glucose (5.5–22 mmol/l)±leptin (0–10 nmol/l). Some islet batches were incubated at high (22 mmol/l), and subsequently lower (5.5 mmol/l), glucose to assess reversibility of effects. Leptin effects on insulin release were also measured. Glucose dose‐dependently increased UCP‐2 expression in all islet batches, maximally by three‐fold. This was not fully reversed by subsequently reduced glucose levels. Leptin decreased UCP‐2 expression by up to 75%, and maximally inhibited insulin release by 47%, at 22 mmol/l glucose. This is the first report of UCP‐2 expression in human islets and provides novel evidence of its role in the loss of β‐cell function in diabetes.

Leptin decreases apoptosis and alters BCL-2: bax ratio in clonal rodent pancreatic beta-cells

The adipocyte derived peptide hormone leptin is known to regulate apoptosis and cell viability in several cells and tissues, as well as having several pancreatic islet beta‐cell specific effects such as inhibition of glucose‐stimulated insulin secretion. This study investigated the effects of leptin upon apoptosis induced by serum depletion and on expression of the apoptotic regulators B‐cell leukaemia 2 gene product (BCL‐2) and BCL2‐associated X protein (Bax) in the glucose‐responsive BRIN‐BD11 beta‐cell line.

Regulation of beta-cell viability and gene expression by distinct agonist fragments of adiponectin.

Obesity is an established risk factor for type 2 diabetes. Activation of the adiponectin receptors has a clear role in improving insulin resistance although conflicting evidence exists for its effects on pancreatic beta-cells. Previous reports have identified both adiponectin receptors (ADR-1 and ADR-2) in the beta-cell. Recent evidence has suggested that two distinct regions of the adiponectin molecule, the globular domain and a small N-terminal region, have agonist properties. This study investigates the effects of two agonist regions of adiponectin on insulin secretion, gene expression, cell viability and cell signalling in the rat beta-cell line BRIN-BD11, as well as investigating the expression levels of adiponectin receptors (ADRs) in these cells. Cells were treated with globular adiponectin and adiponectin (15-36) +/-leptin to investigate cell viability, expression of key beta-cell genes and ERK1/2 activation. Both globular adiponectin and adiponectin (15-36) caused significant ERK1/2 dependent increases in cell viability. Leptin co-incubation attenuated adiponectin (15-36) but not globular adiponectin induced cell viability. Globular adiponectin, but not adiponectin (15-36), caused a significant 450% increase in PDX-1 expression and a 45% decrease in LPL expression. ADR-1 was expressed at a higher level than ADR-2, and ADR mRNA levels were differentially regulated by non-esterified fatty acids and peroxisome-proliferator-activated receptor agonists. These data provide evidence of roles for two distinct adiponectin agonist domains in the beta-cell and confirm the potentially important role of adiponectin receptor agonism in maintaining beta-cell mass.

Resistin down-regulates insulin receptor expression, and modulates cell viability in rodent pancreatic beta-cells

The adipokine resistin is known to induce insulin resistance in rodent tissues. Increases in adipose tissue mass are known to have a negative effect on pancreatic beta‐cell function, although the mechanisms are poorly understood. This study investigated the effects of resistin on insulin secretion, insulin receptor expression and cell viability in pancreatic beta‐cells. BTC‐6 or BRIN‐BD11 cells were treated for 24 h with resistin, and insulin receptor expression, insulin secretion and cell viability were measured. Incubation with 40 ng/ml resistin caused significant decreases in insulin receptor mRNA and protein expression, but did not affect insulin secretion. At low concentrations, resistin caused significant increases in cell viability. These data implicate resistin as a factor that may regulate beta‐cell function/viability, and suggests a potential mechanism by which increased adiposity causes beta‐cell dysfunction.

Pseudoislets as primary islet replacements for research : report on a symposium at King's College London, London UK

Laboratory-based research aimed at understanding processes regulating insulin secretion and mechanisms underlying β-cell dysfunction and loss in diabetes often makes use of rodents, as these processes are in many respects similar between rats/mice and humans. Indeed, a rough calculation suggests that islets have been isolated from as many as 150,000 rodents to generate the data contained within papers published in 2009 and the first four months of 2010. Rodent use for islet isolation has been mitigated, to a certain extent, by the availability of a variety of insulin-secreting cell lines that are used by researchers world-wide. However, when maintained as monolayers the cell lines do not replicate the robust, sustained secretory responses of primary islets which limits their usefulness as islet surrogates. On the other hand, there have been several reports that configuration of MIN6 β-cells, derived from a mouse insulinoma, as three-dimensional cell clusters termed ‘pseudoislets’ largely recapitulates the function of primary islet β-cells. The Diabetes Research Group at King’s College London has been using the MIN6 pseudoislet model for over a decade and they hosted a symposium on “Pseudoislets as primary islet replacements for research”, which was funded by the UK National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs), in London on 15th and 16th April 2010. This small, focused meeting was conceived as an opportunity to consolidate information on experiences of working with pseudoislets between different UK labs, and to introduce the theory and practice of pseudoislet culture to laboratories working with islets and/or β-cell lines but who do not currently use pseudoislets. This short review summarizes the background to the development of the cell line-derived pseudoislet model, the key messages arising from the symposium and emerging themes for future pseudoislet research.

B-type natriuretic peptide in reversible myocardial ischaemia

Background: Coronary heart disease is associated with increased B-type natriuretic peptides (BNPs), and, although controversial, may cause exaggerated exercise-induced BNP secretion. We investigated BNP in relation to reversible myocardial ischaemia. Materials and methods: Serum N-terminal proBNP (NT-proBNP) was measured before and after an exercise electrocardiogram test (ETT) in 14 patients with and 45 patients without exercise-induced myocardial ischaemia. Statistical analysis was carried out on logarithmically transformed data. Results, however, are pre-transformed data. Results: NT-proBNP increased with exercise both in ETT-positive patients (mean (SD) 71.4 (41.2) v 76.8 (44.0) ng/l; p<0.001) and ETT-negative patients (54.0 (61.2) v 60.1 (69.0) ng/l; p<0.001). Pre-exercise and post-exercise NT-proBNP were higher (p<0.05) in ETT-positive than in ETT-negative patients. Incremental NT-proBNP was similar in ETT-positive (4.7 (4.2) ng/l) and ETT-negative (6.2 (8.6) ng/l) patients. Conclusion: Serum NT-proBNP concentrations are higher in patients with exercise-induced myocardial ischaemia than in those without. Exercise-induced electrocardiographic myocardial ischaemia, however, is not associated with exaggerated BNP secretion.

Lipopolysaccharide induces nitric oxide synthase expression and platelet-activating factor increases nitric oxide production in human fetal membranes in culture

BackgroundPlatelet-activating factor and nitric oxide may be involved in the initiation of human labour as inflammatory mediators. The aim of this study was to test whether platelet-activating factor and lipopolysaccharide were able to induce nitric oxide synthase expression and stimulate the production of nitric oxide in human fetal membrane explants in culture.MethodsFetal membranes were collected from Caesarean sections at term. RNA was extracted from membranes and subjected to a qualitative RT-PCR to assess the baseline expression of iNOS. Discs of fetal membranes were cultured for 24 hours in the presence of platelet-activating factor at a dose range of 0.1 nanomolar – 1 micomolar or 1 microgram/ml lipopolysaccharide. Nitric oxide production was measured via nitrite ions in the culture medium and mRNA for iNOS was detected by RT-PCR.ResultsCulturing the membrane discs in medium containing serum induced nitric oxide synthase expression and platelet-activating factor significantly stimulated the production of nitric oxide under these conditions. When cultured without serum inducible nitric oxide synthase expression was induced by lipopolysaccharide, but not by platelet-activating factor.ConclusionPlatelet-activating factor may have a role in the initiation of labour, at term or preterm, via the increased local production of nitric oxide as an inflammatory mediator. In this model of intrauterine infection, lipopolysaccharide was found to induce iNOS expression by fetal membranes, and this mechanism could be involved in preterm labour.

Evidence that Differences in Fructosamine-3-Kinase Activity May be Associated with the Glycation Gap in Human Diabetes

The phenomenon of a discrepancy between glycated hemoglobin levels and other indicators of average glycemia may be due to many factors but can be measured as the glycation gap (GGap). This GGap is associated with differences in complications in patients with diabetes and may possibly be explained by dissimilarities in deglycation in turn leading to altered production of advanced glycation end products (AGEs). We hypothesized that variations in the level of the deglycating enzyme fructosamine-3-kinase (FN3K) might be associated with the GGap. We measured erythrocyte FN3K concentrations and enzyme activity in a population dichotomized for a large positive or negative GGap. FN3K protein was higher and we found a striking threefold greater activity (323%) at any given FN3K protein level in the erythrocytes of the negative-GGap group compared with the positive-GGap group. This was associated with lower AGE levels in the negative-GGap group (79%), lower proinflammatory adipokines (leptin-to-adiponectin ratio) (73%), and much lower prothrombotic PAI-1 levels (19%). We conclude that FN3K may play a key role in the GGap and thus diabetes complications such that FN3K may be a potential predictor of the risk of diabetes complications. Pharmacological modifications of its activity may provide a novel approach to their prevention.

Downregulation of aquaporin 3 inhibits cellular proliferation, migration and invasion in the MDA‑MB‑231 breast cancer cell line

Aquaporins are membrane proteins that regulate cellular water flow. Recently, aquaporins have been proposed as mediators of cancer cell biology. A subset of aquaporins, referred to as aquaglyceroporins are known to facilitate the transport of glycerol. The present study describes the effect of gene knockdown of the aquaglyceroporin AQP3 on MDA-MB-231 breast cancer cell proliferation, migration, invasion, adherence and response to the chemotherapeutic agent 5-fluorouracil. shRNA mediated AQP3 gene knockdown induced a 28% reduction in cellular proliferation (P<0.01), a 39% decrease in migration (P<0.0001), a 24% reduction in invasion (P<0.05) and a 25% increase in cell death at 100 µM 5-FU (P<0.01). Analysis of cell permeability to water and glycerol revealed that MDA-MB-231 cells with knocked down AQP3 demonstrated a modest decrease in water permeability (17%; P<0.05) but a more marked decrease in glycerol permeability (77%; P<0.001). These results suggest that AQP3 has a role in multiple aspects of breast cancer cell pathophysiology and therefore represents a novel target for therapeutic intervention.

Of fat mice and men: the rise of the adipokines.

The inexorable rise in obesity around the planet, particularly in developed countries but increasingly in the developing world, is accompanied by a parallel rise in co-morbid metabolic diseases including type 2 diabetes and vascular disease. Obesity, defined by an excess of adipose tissue, has complex aetiological factors that vary considerably between individual cases and across ethnic groupings but primarily involve genetic (and indeed epigenetic – see Drong et al. (2012)), environmental and behavioural contributions. New obesity genes are being identified regularly and range from those causing monogenic obesity such as mutations in the Lep gene to those that make a small but significant contribution to polygenic obesity such as the TCF7L2 and FTO genes. The cloning by Zhang et al. (1994), of the ob or Lep gene in mice, which produces a single-gene obesity, led to the identification of leptin, the first hormone-like product of adipose tissue to be identified and arguably one of the first ‘adipokines’ to be identified. This discovery underlined the increasingly apparent fact that adipose tissue is not merely an inert store of triacylglycerols but a highly metabolically active tissue with endocrine and other functions that allow it to influence many physiological processes ranging from appetite to metabolic control. A wide range of secretory factors from adipose tissue have been (and are still being) subsequently found, some of which are known cytokines (e.g. TNFa (Hotamisligil et al. 1994)) and others of which were novel peptides or proteins such as adiponectin (Scherer et al. 1995). The term ‘adipocytokine’ was subsequently applied to these factors; however, the contracted term ‘adipokine’ is perhaps more appropriate as not all these products are also cytokines and many have wider hormonal properties (and indeed the term ‘adipose-derived hormone’, or similar, may be more appropriate for many adipokines but has yet to be adopted); in addition, some products such as visfatin (Brown et al. 2010) have enzymatic properties and some are not even proteins – for example free fatty acids released from adipocytes may also be seen in some circumstances as adipokines. It is becoming increasingly apparent that adipokines form an important link between excessive adipose tissue (particularly in the visceral depots) in obesity and associated metabolic syndrome, type 2 diabetes and cardiovascular disease. The three thematic reviews in this issue of the Journal provide a timely survey of the evidence for adipokine involvement in these disorders – specifically, the contribution of adipokines to insulin resistance (Piya et al. 2012) and hence metabolic syndrome and type 2 diabetes, to b-cell failure (an essential element in the pathogenesis and progression of type 2 diabetes) (Dunmore & Brown 2012) and to cardiovascular disease (Mattu & Randeva 2012). In their review, Piya et al. emphasise the inflammatory nature of obesity and related diseases and discuss the role of adipokines and cytokines in the inflammatory processes that lead to the development of insulin resistance. Adipose tissue is itself an important site of inflammation in obesity, which leads to dysregulation of adipokines, particularly in the visceral depots but also in a specific s.c. depot – that of the abdominal area, which of course overlies the visceral depot – with these areas secreting many pro-inflammatory adipokines. The authors discuss the contribution of elevated glucose and free fatty acids to adipokine inflammation and insulin resistance, underlining the centrality of the NF-kB pathway to this process. While insulin resistance is a key contributor to the pathogenesis of type 2 diabetes, it is rarely of itself sufficient to initiate the disease process as most obese individuals are insulin resistant but are able to increase insulin secretion and produce a hyperinsulinaemic response that maintains euglycaemia – type 2 diabetes