The transCampus Metabolic Training Programme Explores the Link of SARS-CoV-2 Virus to Metabolic Disease.

Currently, we are experiencing a true pandemic of a communicable disease by the virus SARS-CoV-2 holding the whole world firmly in its grasp. Amazingly and unfortunately, this virus uses a metabolic and endocrine pathway via ACE2 to enter our cells causing damage and disease. Our international research training programme funded by the German Research Foundation has a clear mission to train the best students wherever they may come from to learn to tackle the enormous challenges of diabetes and its complications for our society. A modern training programme in diabetes and metabolism does not only involve a thorough understanding of classical physiology, biology and clinical diabetology but has to bring together an interdisciplinary team. With the arrival of the coronavirus pandemic, this prestigious and unique metabolic training programme is facing new challenges but also new opportunities. The consortium of the training programme has recognized early on the need for a guidance and for practical recommendations to cope with the COVID-19 pandemic for the community of patients with metabolic disease, obesity and diabetes. This involves the optimal management from surgical obesity programmes to medications and insulin replacement. We also established a global registry analyzing the dimension and role of metabolic disease including new onset diabetes potentially triggered by the virus. We have involved experts of infectious disease and virology to our faculty with this metabolic training programme to offer the full breadth and scope of expertise needed to meet these scientific challenges. We have all learned that this pandemic does not respect or heed any national borders and that we have to work together as a global community. We believe that this transCampus metabolic training programme provides a prime example how an international team of established experts in the field of metabolism can work together with students from all over the world to address a new pandemic.

Identifying barriers to the use of ultrasound in the perioperative period: a survey of southwestern Ontario anesthesiologists.

BACKGROUND: Ultrasound (US) can be used for many perioperative procedures, but evidence is lacking as to its frequency of use and barrier of application. The objectives of this survey were to determine i) how often US guidance was used perioperatively for vascular access placement, nerve blocks, and heart and lung assessment, and ii) to identify the barriers and the limitations of using US amongst anesthesiologists in southwestern Ontario. METHODS: We conducted a web-based survey in over 40 academic or community hospitals at southwestern Ontario. RESULTS: Of 266 surveys sent, 66 complete surveys were obtained (response rate of 25%). Most respondents (> 80%) reported that US was commonly used for central venous catheter (CVC) insertion, followed by regional blocks; the uses were less frequent for neuraxial blockade and cardiopulmonary assessment. Most respondents wanted to use US more frequently as part of their practice and felt that they already had adequate US training. However, most respondents (59%) reported limited access to US machines in their working institutes as being the major barrier to incorporating US in their daily practice. CONCLUSION: The most common uses of US in anesthesia practice in southwestern Ontario were for CVC insertion and regional blocks. Most anesthesiologists in southwestern Ontario are interested to incorporate US in their daily practice but most were limited by the lack of US resources. Apparently, only providing knowledge and skills teaching may not be sufficient to further improve the US utilization in our region; a matched administrative effort appears to be the next challenge.

Onset of labour epidural analgesia with low-dose bupivacaine and different doses of fentanyl.

This study investigated the effects of different doses of epidural fentanyl on the time to onset of epidural analgesia in women in early labour. We hypothesised that onset of epidural labour analgesia (the primary outcome defined as time in minutes from completion of epidural bolus to the first uterine contraction with a numeric pain rating scale [NPRS] score ≤ 3) would be faster with 100 µg of fentanyl epidural bolus compared with 20 µg or 50 µg. Epidural labour analgesia was initiated with 20 µg of fentanyl (F20 group), 50 µg (F50 group) or 100 µg (F100 group) along with 10 ml bupivacaine 0.08% as the loading dose. We randomly allocated 105 patients, with 35 patients in each group. Median (IQR [range]) time to achieve NPRS ≤ 3 was 18 (11-30 [6-20]) min in F20, 10 (8-19 [4-30]) min in F50 and 10 (6-16 [3-30]) min in F100 groups. There was a significant difference in onset times comparing F100 with F20 (p < 0.001) and F50 with F20 (p = 0.007), but not significantly different comparing F100 with F50 (p = 0.19). The median (IQR [range]) time from the epidural loading dose to first patient controlled epidural analgesia bolus was 61 min (20-165 [20-420]) in F20, 118 min (66-176 [20-396]) in F50 and 150 min (66-214 [30-764]) in F100 groups. This was not statistically significant (p = 0.16) comparing the F20 with the F100 group. There were no significant differences in maternal side-effects, mode of delivery, patient satisfaction scores or neonatal Apgar scores between all groups. We conclude that the 50 µg and 100 µg fentanyl doses were associated with reduced onset times to effective analgesia compared with the 20 µg dose.

Equilibrative nucleoside transporter 3 depletion in ß-cells impairs mitochondrial function and promotes apoptosis: Relationship to pigmented hypertrichotic dermatosis with insulin-dependent diabetes.

Loss of function recessive mutations in the SLC29A3 gene that encodes human equilibrative nucleoside transporter 3 (ENT3) have been identified in patients with pigmented hypertrichotic dermatosis with insulin-dependent diabetes (PHID). ENT3 is a member of the equilibrative nucleoside transporter (ENT) family whose primary function is mediating transport of nucleosides and nucleobases. The aims of this study were to characterise ENT3 expression in islet ß-cells and identify the effects of its depletion on ß-cell mitochondrial activity and apoptosis. RT-PCR amplification identified ENT3 expression in human and mouse islets and exocrine pancreas, and in MIN6 ß-cells. Immunohistochemistry using human and mouse pancreas sections exhibited extensive ENT3 immunostaining of ß-cells, which was confirmed by co-staining with an anti-insulin antibody. In addition, exposure of dispersed human islet cells and MIN6 ß-cells to MitoTracker and an ENT3 antibody showed co-localisation of ENT3 to ß-cell mitochondria. Consistent with this, Western blot analysis confirmed enhanced ENT3 immunoreactivity in ß-cell mitochondria-enriched fractions. Furthermore, ENT3 depletion in ß-cells increased mitochondrial DNA content and promoted an energy crisis characterised by enhanced ATP-linked respiration and proton leak. Finally, inhibition of ENT3 activity by dypridamole and depletion of ENT3 by siRNA-induced knockdown resulted in increased caspase 3/7 activities in ß-cells. These observations demonstrate that ENT3 is predominantly expressed by islet ß-cells where it co-localises with mitochondria. Depletion of ENT3 causes mitochondrial dysfunction which is associated with enhanced ß-cell apoptosis. Thus, apoptotic loss of islet ß-cells may contribute to the occurrence of autoantibody-negative insulin-dependent diabetes in individuals with non-functional ENT3 mutations.

Reduced nuclear protein 1 expression improves insulin sensitivity and protects against diet-induced glucose intolerance through up-regulation of heat shock protein 70.

We recently reported that deletion of the stress-regulated nuclear protein 1 (Nupr1) protected against obesity-associated metabolic alterations due to increased beta cell mass, but complete Nupr1 ablation was not advantageous since it led to insulin resistance on a normal diet. The current study used Nupr1 haplodeficient mice to investigate whether a partial reduction in Nupr1 expression conferred beneficial effects on glucose homeostasis. Islet number, morphology and area, assessed by immunofluorescence and morphometric analyses, were not altered in Nupr1 haplodeficient mice under normal diet conditions and nor was beta cell BrdU incorporation. Glucose and insulin tolerance tests indicated that there were no significant changes in in vivo insulin secretion and glucose clearance in Nupr1 haplodeficient mice, and beta cell function in vitro was normal. However, reduced Nupr1 expression decreased visceral fat deposition and significantly increased insulin sensitivity in vivo. In contrast to wild type animals, high fat diet-fed Nupr1 haplodeficient mice were not hyperinsulinaemic or glucose intolerant, and their sustained insulin sensitivity was demonstrated by appropriate insulin-induced Akt phosphorylation, as determined by Western blotting. At the molecular level, measurements of gene expression levels and promoter activities identified Nupr1-dependent inhibition of heat shock factor-1-induced heat shock protein 70 (Hsp70) expression as a mechanism through which Nupr1 regulates insulin sensitivity. We have shown for the first time that Nupr1 plays a central role in inhibiting Hsp70 expression in tissues regulating glucose homeostasis, and reductions in Nupr1 expression could be used to protect against the metabolic defects associated with obesity-induced insulin resistance.

Lessons from aviation - the role of checklists in minimally invasive cardiac surgery.

We describe an adverse event during minimally invasive cardiac surgery that resulted in a multi-disciplinary review of intra-operative errors and the creation of a procedural checklist. This checklist aims to prevent errors of omission and communication failures that result in increased morbidity and mortality. We discuss the application of the aviation - led "threats and errors model" to medical practice and the role of checklists and other strategies aimed at reducing medical errors.

The role of non-placental signals in the adaptation of islets to pregnancy.

It is well established that the maternal ß-cell mass increases during pregnancy in both humans and rodents to compensate insulin resistance and increased metabolic demand, and rapidly returns to normal levels post-partum. However, the mechanisms underlying this adaptation are not well understood. It is established that this process is driven partly by placental signals, but the contribution of non-placental signals is still unclear. This study aimed to differentiate between the role of placental and non-placental signals in regulating the ß-cell mass and glucose homeostasis during and after pregnancy. Pseudopregnant, pregnant and lactating mice were used to study the effects of maternal hormones on ß-cell function during early pregnancy, mid-to-late pregnancy and post-partum, respectively. Pseudopregnant mice, with circulating hormone levels mirroring those during pregnancy but lacking placental signals, had significantly increased ß-cell proliferation compared to non-pregnant controls but no change in glucose homeostasis, suggesting a role for non-placental hormones in increasing ß-cell mass. The rate of ß-cell proliferation rate dropped immediately after parturition, but lactating mice still had a significantly higher rate of ß-cell proliferation compared to non-lactating post-partum mice, suggesting that lactation-related hormones play a role in the controlled involution of ß-cell mass post-partum. These results implicate a role for both non-placental and placental signals in regulating ß-cell mass during and after pregnancy.

ALK5 inhibition maintains islet endothelial cell survival but does not enhance islet graft revascularisation or function.

Islet transplantation is a potential treatment for Type 1 diabetes but long term graft function is suboptimal. The rich supply of intraislet endothelial cells diminishes rapidly after islet isolation and culture, which affects the revascularisation rate of islets after transplantation. The ALK5 pathway inhibits endothelial cell proliferation and thus inhibiting ALK5 is a potential target for improving endothelial cell survival. The aim of the study was to establish whether ALK5 inhibition prevents the loss of intraislet endothelial cells during islet culture and thus improves the functional survival of transplanted islets by enhancing their subsequent revascularisation after implantation. Islets were cultured for 48 h in the absence or presence of 2 different ALK inhibitors: SB-431542 or A-83-01. Their vascular density after culture was analysed using immunohistochemistry. Islets pre-cultured with the ALK5 inhibitors were implanted into streptozotocin-diabetic mice for either 3 or 7 days and blood glucose concentrations were monitored and vascular densities of the grafts were analysed. Islets cultured with ALK5 inhibitors had higher vascular densities than control-cultured islets. Three days after implantation, endothelial cell numbers in islet grafts were minimal, irrespective of treatment during culture. Seven days after implantation, endothelial cells were evident within the islet grafts but there was no difference between control-cultured islets and islets pre-treated with an ALK5 inhibitor. Blood glucose concentrations were no different between the treatment groups. In conclusion, inhibition of ALK5 improved intraislet endothelial cell numbers after islet culture, but this effect was lost in the early post-transplantation period.

Development of our TAVI protocol for emergency initiation of cardiopulmonary bypass.

All transcatheter aortic valve implantation (TAVI) cases are done in our hybrid operating room with a multidisciplinary team and a primed cardiopulmonary bypass (CPB) circuit on pump stand-by. We decided that we would resuscitate all patients undergoing a TAVI procedure via a transfemoral, transapical or transaortic approach, if required. Perfusion plays an essential role in providing rescue CPB for patient salvage when catastrophic complications occur. To coordinate the multidisciplinary effort, we have developed a written safety checklist that assigns a pre-determined role for team members for the rapid sequence initiation of CPB. Although many TAVI patients are not candidates for conventional aortic valve replacements, we feel strongly that rescue CPB should be offered to all TAVI patients to allow the correction of potentially reversible complications. This protocol is included in every surgical "Time Out" involving a TAVI procedure (Figure 1). The protocol has led to rapid and safe CPB initiation in less than five minutes of cardiac arrest. It has also led to a coordinated and consistent team, with pre-specified roles and improved communication. We discuss a case series of four TAVI patients who required emergent use of CPB. The first few cases did not have a written protocol. The experience from these cases led to the development of our protocol. We identified a lack of coordination, wasted movements, unnecessary delayed resuscitation and overall chaos, each of which was targeted for correction with the protocol. We will discuss the merits of the protocol in two recent TAVI cases which required emergent CPB.

Blurring the boundaries: decays of multiparticle isomers at the proton drip line.

A multiparticle spin-trap isomer has been discovered in the proton-unbound nucleus (73)(158)Ta85 . The isomer mainly decays by γ-ray emission with a half-life of 6.1(1) µs. Analysis of the γ-ray data shows that the isomer lies 2668 keV above the known 9+ state and has a spin 10ℏ higher and negative parity. This 19- isomer also has an 8644(11) keV, 1.4(2)% α-decay branch that populates the 9+ state in (154)Lu. No proton-decay branch from the isomer was identified, despite the isomer being unbound to proton emission by 3261(14) keV. This remarkable stability against proton emission is compared with theoretical predictions, and the implications for the extent of observable nuclides are considered.

Effect of hyperglycaemia on muscarinic M3 receptor expression and secretory sensitivity to cholinergic receptor activation in islets.

AIMS: Islets are innervated by parasympathetic nerves which release acetylcholine (ACh) to amplify glucose-induced insulin secretion, primarily via muscarinic M3 receptors (M3R). Here we investigate the consequence of chronic hyperglycaemia on islet M3R expression and secretory sensitivity of mouse islets to cholinergic receptor activation. METHODS: The impact of hyperglycaemia was studied in (i) islets isolated from ob/ob mice, (ii) alginate-encapsulated mouse islets transplanted intraperitoneally into streptozotocin-induced diabetic mice and (iii) mouse and human islets maintained in vitro at 5.5 or 16 mmol/l glucose. Blood glucose levels were assessed by a commercial glucose meter, insulin content by RIA and M3R expression by qPCR and immunohistochemistry. RESULTS: M3R mRNA expression was reduced in both ob/ob islets and islets maintained at 16 mmol/l glucose for 3 days (68 and 50% control, respectively). In all three models of hyperglycaemia the secretory sensitivity to the cholinergic receptor agonist, carbachol, was reduced by 60-70% compared to control islets. Treatment for 72 h with the irreversible PKC activator, PMA, or the PKC inhibitor, Gö6983, did not alter islet M3R mRNA expression nor did incubation with the PI3K-inhibitor, LY294002, although enhancement of glucose-induced insulin secretion by LY294002 was reduced in islets maintained at 16 mmol/l glucose, as was mRNA expression of the PI3K regulatory subunit, p85α. CONCLUSIONS: Cholinergic regulation of insulin release is impaired in three experimental islet models of hyperglycaemia consistent with reduced expression of M3 receptors. Our data suggest that the receptor downregulation is a PKC- and PI3K-independent consequence of the hyperglycaemic environment, and they imply that M3 receptors could be potential targets in the treatment of type 2 diabetes.

The permissive effects of glucose on receptor-operated potentiation of insulin secretion from mouse islets: a role for ERK1/2 activation and cytoskeletal remodelling.

AIMS/HYPOTHESIS: Glucose plays two distinct roles in regulating insulin secretion from beta cells--an initiatory role, and a permissive role enabling receptor-operated secretagogues to potentiate glucose-induced insulin secretion. The molecular mechanisms underlying the permissive effects of glucose on receptor-operated insulin secretion remain uncertain. We have investigated the role of extracellular signal-regulated kinase 1/2 (ERK1/2) activation and consequent cytoskeletal remodelling in this process. METHODS: Insulin release was measured from groups of isolated mouse islets using static incubation experiments and subsequent radioimmunoassay of samples. ERK1/2 activation was measured by western blotting of islet protein samples for both phosphorylated and total ERK1/2. Rhodamine-phalloidin staining was used to measure filamentous actin in dispersed primary beta cells. RESULTS: Inhibition of ERK1/2 blocked potentiation of glucose-induced insulin release by the receptor-operated secretagogues kisspeptin, A568, exendin-4 and JWH015, although the agonists alone had minimal effects on ERK1/2 activation, suggesting a permissive rather than causal role for ERK1/2 activation in receptor-operated insulin release. Following pharmacological activation of ERK1/2 all agonists caused a significant increase in insulin release from islets incubated with sub-stimulatory levels of glucose. ERK1/2 inhibition significantly reduced the glucose-dependent decreases in filamentous actin observed in primary beta cells, while pharmacological dissociation of actin filaments enabled all receptor-operated secretagogues tested to significantly stimulate insulin release from islets at a sub-stimulatory glucose concentration. CONCLUSIONS/INTERPRETATION: Glucose-induced ERK1/2 activation in beta cells mediates the permissive effects of stimulatory glucose concentrations on receptor-operated insulin secretagogues, at least in part through effects on actin depolymerisation and cytoskeletal remodelling.

Prevention of post-dural puncture headache in parturients: a systematic review and meta-analysis.

Post-dural puncture headaches (PDPHs) present an important clinical problem. We assessed methods to decrease accidental dural punctures (ADPs) and interventions to reduce PDPH following ADP. Multiple electronic databases were searched for randomised clinical trials (RCTs) of parturients having labour epidurals, in which the studied intervention could plausibly affect ADP or PDPH, and the incidence of at least one of these was recorded. Forty RCTs (n = 11,536 epidural insertions) were included, studying combined spinal-epidurals (CSEs), loss of resistance medium, prophylactic epidural blood patches, needle bevel orientation, ultrasound-guided insertion, epidural morphine, Special Sprotte needles, acoustic-guided insertion, administration of cosyntropin, and continuous spinal analgesia. The RCTs for CSE, loss of resistance medium, and prophylactic epidural blood patches were meta-analysed. Five methods reduced PDPH: prophylactic epidural blood patch {four trials, median quality score = 2, risk difference = -0.48 [95% confidence interval (CI): -0.88 to -0.086]}, lateral positioning of the epidural needle bevel upon insertion (one trial, quality score = 1), Special Sprotte needles [one trial, quality score = 5, risk difference = -0.44 (95% CI: -0.67 to -0.21)], epidural morphine [one trial, quality score = 4, risk difference = -0.36 (95% CI -0.59 to -0.13)], and cosyntropin [one trial, quality score = 5, risk difference = -0.36 (95% CI -0.55 to -0.16)]. Several methods potentially reduce PDPH. Special Sprotte needles, epidural morphine, and cosyntropin are thus far each supported by a single, albeit good quality trial. Prophylactic blood patches are supported by three trials, but these had flawed methodology. Mostly, trials were of limited quality, and further well-conducted, large studies are needed.

A novel extract of Gymnema sylvestre improves glucose tolerance in vivo and stimulates insulin secretion and synthesis in vitro.

Herbal medicines, especially plant-derived extracts, have been used to treat Type 2 diabetes mellitus (T2DM) for many centuries, and offer the potential of cheap and readily available alternatives to conventional pharmaceuticals in developing countries. Extracts of Gymnema sylvestre (GS) have anti-diabetic activities and have been used as a folk medicine in India for centuries. We have investigated the effects of a novel high molecular weight GS extract termed OSA® on glucose tolerance in insulin-resistant ob/ob mice, and on insulin secretion and synthesis by isolated mouse islets. Single administration of OSA® (500 mg/kg) to ob/ob mice 30 min before an intraperitoneal glucose load improved their abnormal glucose tolerance. In vitro studies indicated that OSA® (0.25 mg/ml) initiated rapid and reversible increases in insulin secretion from isolated mouse islets at substimulatory (2 mM) and stimulatory (20 mM) glucose concentrations. In addition, prolonged treatment (24-48 h) of mouse islets with OSA® elevated the expression of preproinsulin mRNA and maintained the total insulin content of mouse islets in the presence of stimulated insulin secretion. These effects of OSA® are consistent with its potential use as a therapy for the hyperglycemia associated with obesity-related T2DM.

Delta cell secretory responses to insulin secretagogues are not mediated indirectly by insulin.

AIMS/HYPOTHESIS: Somatostatin from islet delta cells inhibits insulin and glucagon secretion, but knowledge of the regulation of pancreatic somatostatin release is limited. Some insulin secretagogues stimulate somatostatin secretion, and here we investigated whether delta cell secretory responses are indirectly regulated in a paracrine manner by insulin released from beta cells. METHODS: Hormone release from static incubations of primary mouse islets or somatostatin-secreting TGP52 cells was measured by RIA. mRNA expression was assessed by RT-PCR. RESULTS: Glucose and a range of other physiological and pharmacological agents stimulated insulin and somatostatin release, and insulin receptor mRNA was expressed in islets, MIN6 beta cells and TGP52 cells. However, exogenous insulin did not modulate basal or glucose-induced somatostatin secretion from islets, nor did pre-incubation with an antibody against the insulin receptor or with the insulin receptor tyrosine kinase inhibitor, HNMPA(AM)(3). Glucose and tolbutamide stimulated somatostatin release from TGP52 cells, whereas a range of receptor-operating agents had no effect, the latter being consistent with a lack of corresponding receptor mRNA expression in these cells. Parasympathetic activation stimulated insulin, but inhibited somatostatin release from mouse islets in accordance with differences in muscarinic receptor mRNA expression in islets, MIN6 and TGP52 cells. The inhibitory effect on somatostatin secretion was reversed by pertussis toxin or the muscarinic receptor 2 antagonist, methoctramine. CONCLUSIONS/INTERPRETATIONS: A number of insulin secretagogues have analogous effects on insulin and somatostatin release, but this similarity of response is not mediated by an indirect, paracrine action of insulin on delta cells.

Nano-scale encapsulation enhances allograft survival and function of islets transplanted in a mouse model of diabetes.

AIMS/HYPOTHESIS: The success of islet transplantation as a treatment for type 1 diabetes is currently hampered by post-transplantation loss of functional islets through adverse immune and non-immune reactions. We aimed to test whether early islet loss can be limited and transplant survival improved by the application of conformal nano-coating layers to islets. METHODS: Our novel coating protocol used alternate layers of phosphorylcholine-derived polysaccharides (chitosan or chondroitin-4-sulphate) and alginate as coating materials, with the binding based on electrostatic complexation. The in vitro function of encapsulated mouse islets was studied by analysing islet secretory function and cell viability. The in vivo function was evaluated using syngeneic and allogeneic transplantation in the streptozotocin-induced mouse model of diabetes. RESULTS: Nano-scale encapsulated islets retained appropriate islet secretory function in vitro and were less susceptible to complement- and cytokine-induced apoptosis than non-encapsulated control islets. In in vivo experiments using a syngeneic mouse transplantation model, no deleterious responses to the coatings were observed in host animals, and the encapsulated islet grafts were effective in reversing hyperglycaemia. Allo-transplantation of the nano-coated islets resulted in preserved islet function post-implantation in five of seven mice throughout the 1 month monitoring period. CONCLUSIONS/INTERPRETATION: Nano-scale encapsulation offers localised immune protection for implanted islets, and may be able to limit early allograft loss and extend survival of transplanted islets. This versatile coating scheme has the potential to be integrated with tolerance induction mechanisms, thereby achieving long-term success in islet transplantation.

Investigation of intracellular signalling cascades mediating stimulatory effect of a Gymnema sylvestre extract on insulin secretion from isolated mouse and human islets of Langerhans.

AIM: Traditional plant-based remedies such as Gymnema sylvestre (GS) extracts have been used to treat diabetes mellitus for many centuries. We have shown previously that a novel GS extract, OSA®, has a direct effect on insulin secretion but its mode of action has not been studied in detail Thus this study investigated the possible underlying mechanism(s) by which OSA® exerts its action. METHODS: The effects of OSA® on [Ca(2+)]i and K(+) conductances were assessed by Ca(2+) microfluorimetry and electrophysiology in dispersed mouse islets and MIN6 ß-cells, respectively. Isolated mouse (from 20 to 25 mice) and human (from 3 donors) islets, and MIN6 ß-cells, were used to investigate whether the stimulatory effect of OSA® on insulin secretion was dependent on the presence of extracellular calcium and protein kinase activation. RESULTS: OSA ®-induced insulin secretion from mouse islets and MIN6 ß-cells was inhibited by nifedipine, a voltage-gated Ca(2+) channel blocker, and by the removal of extracellular Ca(2+), respectively. OSA® did not affect the activities of KATP channels or voltage-dependent K(+) channels in MIN6 ß-cells but it caused an increase in intracellular Ca(2+) ([Ca(2+)]i) concentrations in Fura-2-loaded mouse islet cells. The insulin secretagogue effect of OSA® was dependent, in part, on protein kinase activation since incubating mouse or human islets with staurosporine, a general protein kinase inhibitor, resulted in partial inhibition of OSA®-induced insulin secretion. Experiments using permeabilized, Ca(2+)-clamped MIN6 ß-cells revealed a Ca(2+)-independent component action of OSA® at a late stage in the stimulus-response coupling pathway. OSA®-induced insulin secretion was unexpectedly associated with a decrease in intracellular cAMP levels. CONCLUSIONS: These data indicate that the GS isolate OSA® stimulates insulin secretion from mouse and human islets in vitro, at least in part as a consequence of Ca(2+) influx and protein kinase activation.

Calcium/calmodulin-dependent kinase IV controls glucose-induced Irs2 expression in mouse beta cells via activation of cAMP response element-binding protein.

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 CaMK(IV) mediates glucose-induced Irs2 expression in beta cells via CREB activation. METHODS: The functions of CaMK(IV) 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 CaMK(IV) production and expression vectors to overproduce constitutively active and dominant negative forms of CaMK(IV) 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 CaMK(IV) 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 CaMK(IV) mutant. Inhibition of glucose-induced calcium influx with nifedipine or chelation of intracellular calcium with bapta-(AM), as well as silencing of CaMK(IV) or inhibition of its activity with KN62 resulted in similar observations. Finally, overproduction of a dominant negative form of CREB completely suppressed glucose and CaMK(IV) stimulation of Irs2 expression. CONCLUSIONS/INTERPRETATION: Our results suggest that the Ca(2+)/CaMK(IV)/CREB cascade plays a critical role in the regulation of Irs2 expression in beta cells.

Co-transplantation of mesenchymal stem cells maintains islet organisation and morphology in mice.

AIMS/HYPOTHESIS: Recent studies have shown that mesenchymal stem cells (MSCs) secrete several factors that improve survival and function of transplanted islets. Implantation of islets beneath the kidney capsule results in morphological changes, due to interactions of the graft with the host, thus impairing islet function. We co-transplanted MSCs with islets to determine their effects on the remodelling process and studied graft function in a mouse model of minimal islet mass. METHODS: Islets were syngeneically transplanted, either alone or with kidney-derived MSCs, underneath the kidney capsule of streptozotocin-induced diabetic C57Bl/6 mice. Blood glucose levels were monitored and intraperitoneal glucose tolerance tests carried out. Hormone contents of grafts and pancreas were assessed by radioimmunoassay. Graft morphology and vascularisation were evaluated by immunohistochemistry. RESULTS: MSCs improved the capacity of islet grafts to reverse hyperglycaemia, with 92% of mice co-transplanted with MSCs reverting to normoglycaemia, compared with 42% of those transplanted with islets alone. Average blood glucose concentrations were lower throughout the 1 month monitoring period in MSC co-transplanted mice. MSCs did not alter graft hormone content. Islets co-transplanted with MSCs maintained a morphology that more closely resembled that of islets in the endogenous pancreas, both in terms of size, and of endocrine and endothelial cell distribution. Vascular engraftment was superior in MSC co-transplanted mice, as shown by increased endothelial cell numbers within the endocrine tissue. CONCLUSIONS/INTERPRETATION: Co-transplantation of islets with MSCs had a profound impact on the remodelling process, maintaining islet organisation and improving islet revascularisation. MSCs also improved the capacity of islets to reverse hyperglycaemia.

Cannabinoid receptor agonists and antagonists stimulate insulin secretion from isolated human islets of Langerhans.

AIMS: The role of cannabinoid receptors in human islets of Langerhans has not been investigated in any detail, so the current study examined CB1 and CB2 receptor expression by human islets and the effects of pharmacological cannabinoid receptor agonists and antagonists on insulin secretion. METHODS: Human islets were isolated from pancreases retrieved from heart-beating organ donors. Messenger RNAs encoding human CB1 and CB2 receptors were amplified from human islet RNA by RT-PCR and receptor localization within islets was identified by immunohistochemistry. Dynamic insulin secretion from human islets perifused with buffers supplemented with CB1 and CB2 receptor agonists and antagonists was quantified by radioimmunoassay. RESULTS: RT-PCR showed that both CB1 and CB2 receptors are expressed by human islets and immunohistochemistry indicated that receptor expression co-localized with insulin-expressing ß-cells. Perifusion experiments using isolated human islets showed that insulin secretion was reversibly stimulated by both CB1 and CB2 receptor agonists, with CB1 receptor activation associated with increased basal secretion whereas CB2 receptors were coupled to initiation and potentiation of insulin secretion. Antagonists at CB1 (N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide) and CB2 (N-(1,3-Benzodioxol-5-ylmethyl)-1,2-dihydro-7-methoxy-2-oxo-8-(pentyloxy)-3-quinoline carboxamide) receptors failed to inhibit the stimulatory effects of the respective agonists and, unexpectedly, reversibly stimulated insulin secretion. CONCLUSIONS: These data confirm the expression of CB1 and CB2 receptors by human islets and indicate that both receptor subtypes are coupled to the stimulation of insulin secretion. They also implicate involvement of CB1/2 receptor-independent pathways in the antagonist-induced stimulatory effects.