James E. Bowe

Differential role of corticotrophin-releasing factor receptor types 1 and 2 in stress-induced suppression of pulsatile luteinising hormone secretion in the female rat.

Corticotrophin‐releasing factor (CRF) plays a pivotal role in stress‐induced suppression of the gonadotrophin‐releasing hormone pulse generator. We have previously shown that type 2 CRF receptors (CRF2) mediate restraint stress‐induced suppression of luteinising hormone (LH) pulses in the rat. The present study aimed: (i) to determine whether type 1 CRF receptors (CRF1) are also involved in this response to restraint and (ii) to investigate the differential involvement of CRF1 and CRF2 in the suppression of LH pulses in response to the metabolic perturbation of insulin‐induced hypoglycemia and the innate immunological challenge of lipopolysaccharide (LPS). Ovariectomised rats with oestrogen replacement were implanted with intracerebroventricular (i.c.v.) and intravenous (i.v.) cannulae. Blood samples (25 µl) were collected every 5 min for 5 h for LH measurement. After 2 h of controlled blood sampling, rats were either exposed to restraint (1 h) or injected intravenously with insulin (0.25 IU/kg) or LPS (5 µg/kg). All three stressors suppressed LH pulses. The CRF1 antagonist SSR125543Q (11.5 µmol/rat i.v., 30 min before stressor) blocked the inhibitory response to restraint, but not hypoglycaemia or LPS stress. In addition to its effect on restraint, the CRF2 antagonist astressin2‐B (28 nmol/rat i.c.v., 10 min before insulin or LPS) blocked hypoglycaemia or LPS stress‐induced suppression of LH pulses. These results suggest that hypoglycaemia and LPS stress‐induced LH suppression involves activation of CRF2 while restraint stress‐induced inhibition of LH pulses involves both CRF1 and CRF2.

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. Several techniques exist for measuring different aspects of glucose tolerance and each of these methods has distinct advantages and disadvantages. Thus the appropriate methodology may vary from study to study depending on the desired end-points, the animal model, and other practical considerations. This review outlines the most commonly used techniques for assessing glucose tolerance in rodents and details the factors that should be taken into account in their use. Representative scenarios illustrating some of the practical considerations of designing in vivo experiments for the measurement of glucose homeostasis are also discussed.

Expression and function of cannabinoid receptors in mouse islets.

The endocannabinoid system plays a key role in energy homeostasis, with agonists and antagonists of CB1 receptors acting centrally to stimulate and inhibit food intake, respectively. In addition to their established effects on the central nervous system, cannabinoid receptor agonists also exert peripheral effects by modulating cellular cyclic AMP and calcium levels and there have been reports that they regulate β-cell function. However, the few reports to date on islet expression of cannabinoid receptors and effects of agonists on insulin secretion have failed to reach a consensus. We have therefore investigated cannabinoid receptor expression by mouse islet β-and α-cells and the effects of selective receptor agonists on cyclic AMP and calcium levels, and on dynamic insulin secretory responses. CB1 and CB2 mRNA and protein expression by islets was detected by RT-PCR and western blotting respectively, and cellular location of the receptors was identified by immunohistochemistry with insulin and glucagon antibody co-staining. Cyclic AMP generation was quantified by enzyme immunoassay and changes in calcium levels were measured by microfluorimetry of Fura-2-loaded mouse islet cells. Dynamic insulin secretion was quantified by radioimmunoassay after perifusion of isolated islets. We found that mouse islets expressed both CB1 and CB2 receptors, and they were localised to β-cells. Activation of mouse β-cell CB1 and CB2 receptors resulted in decreased cyclic AMP, increased calcium and potentiation of glucose-stimulated insulin secretion. Thus, activation of islet cannabinoid receptors by locally produced endocannabinoids such as 2-aminoglycerol may be another regulatory pathway by which islets stimulate insulin secretion to maintain glucose homeostasis.

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.

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. We investigated the capacity of MSCs to improve islet cell survival or β-cell function in vitro using direct and indirect contact islet-MSC configurations. We also investigated whether pre-culturing islets with MSCs improves islet transplantation outcome. METHODS The effect of pre-culturing islets with MSCs on islet function in vitro was investigated by measuring glucose-stimulated insulin secretion. The endothelial cell density of fresh islets and islets cultured with or without MSCs was determined by immunohistochemistry. The efficacy of transplanted islets was tested in vivo using a syngeneic streptozotocin-diabetic minimal islet mass model. Graft function was investigated by monitoring blood glucose concentrations. RESULTS Indirect islet-MSC co-culture configurations did not improve islet function in vitro. Pre-culturing islets using a direct contact MSC monolayer configuration improved glucose-stimulated insulin secretion in vitro, which correlated with superior islet graft function in vivo. MSC pre-culture had no effect on islet endothelial cell number in vitro or in vivo. CONCLUSIONS Pre-culturing islets with MSCs using a direct contact configuration maintains functional β-cell mass in vitro and the capacity of cultured islets to reverse hyperglycemia in diabetic mice.

Animal models for diabetes: Understanding the pathogenesis and finding new treatments

Diabetes mellitus is a lifelong, metabolic disease that is characterised by an inability to maintain normal glucose homeostasis. There are several different forms of diabetes, however the two most common are Type 1 and Type 2 diabetes. Type 1 diabetes is caused by the autoimmune destruction of pancreatic beta cells and a subsequent lack of insulin production, whilst Type 2 diabetes is due to a combination of both insulin resistance and an inability of the beta cells to compensate adequately with increased insulin release. Animal models are increasingly being used to elucidate the mechanisms underlying both Type 1 and Type 2 diabetes as well as to identify and refine novel treatments. However, a wide range of different animal models are currently in use. The majority of these models are suited to addressing certain specific aspects of diabetes research, but may be of little use in other studies. All have pros and cons, and selecting an appropriate model for addressing a specific question is not always a trivial task and will influence the study results and their interpretation. Thus, as the number of available animal models increases it is important to consider the potential roles of these models in the many different aspects of diabetes research. This review gathers information on the currently used experimental animal models of both Type 1 and Type 2 diabetes and evaluates their advantages and disadvantages for research purposes and details the factors that should be taken into account in their use.

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/hypothesisChemokine (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.MethodsIslet 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.ResultsMouse 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/interpretationAlthough CCL5 is implicated in the pathogenesis of diabetes through activation of CCRs, it has beneficial effects on beta cells through GPR75 activation.

Calcitonin gene-related peptide-induced suppression of luteinizing hormone pulses in the rat: the role of endogenous opioid peptides

Calcitonin gene‐related peptide (CGRP) is involved in a variety of stress responses in the rat. Central administration of CGRP activates the hypothalamo–pituitary–adrenal axis resulting in increased corticosterone secretion. We have previously shown that central CGRP suppresses the gonadotrophin‐releasing hormone (GnRH) pulse generator, specifically LH pulses. Endogenous opioid peptides (EOPs) have been shown to play an important role in stress‐induced suppression of the reproductive axis. The aim of the present study was to test the hypothesis that EOPs mediate CGRP‐induced suppression of pulsatile LH secretion. Ovariectomized rats were implanted with intracerebroventricular (i.c.v.) and i.v. cannulae. Intravenous administration of the opioid antagonist naloxone (250 μg) completely blocked the suppression of LH pulses induced by 1.5 μg i.c.v. CGRP and significantly attenuated the suppression of pulsatile LH secretion induced by 5 μg i.c.v. CGRP. Furthermore, intravenous administration of naloxone was found to immediately restore normal LH pulse frequency in animals treated 90 min earlier with 1.5 μg i.c.v. CGRP. Co‐administration (i.c.v.) of CGRP (1.5 μg) with the μ and κ opioid receptor‐specific antagonists naloxone (10 μg) and norbinaltorphimine (5 μg), respectively, blocked the CGRP‐induced suppression of LH pulses, whilst i.c.v. co‐administration of CGRP (1.5 μg) with the δ opioid receptor‐specific antagonist naltrindole (5 μg) did not. These data provide evidence that EOPs play a pivotal role in mediating the inhibitory effects of CGRP on pulsatile LH secretion in the rat. They also suggest that the μ and κ, but not the δ, opioid receptors may be responsible for mediating the effects of CGRP on LH pulses.

Islet neogenesis-associated protein signaling in neonatal pancreatic rat islets: involvement of the cholinergic pathway

Islet neogenesis associated protein (INGAP) increases islet mass and insulin secretion in neonatal and adult rat islets. In the present study, we measured the short- and long-term effects of INGAP-PP (a pentadecapeptide having the 104-118 amino acid sequence of INGAP) upon islet protein expression and phosphorylation of components of the PI3K, MAPK and cholinergic pathways, and on insulin secretion. Short-term exposure of neonatal islets to INGAP-PP (90 s, 5, 15, and 30 min) significantly increased Akt1(-Ser473) and MAPK3/1(-Thr202/Tyr204) phosphorylation and INGAP-PP also acutely increased insulin secretion from islets perifused with 2 and 20 mM glucose. Islets cultured for 4 days in the presence of INGAP-PP showed an increased expression of Akt1, Frap1, and Mapk1 mRNAs as well as of the muscarinic M3 receptor subtype, and phospholipase C (PLC)-beta2 proteins. These islets also showed increased Akt1 and MAPK3/1 protein phosphorylation. Brief exposure of INGAP-PP-treated islets to carbachol (Cch) significantly increased P70S6K(-Thr389) and MAPK3/1 phosphorylation and these islets released more insulin when challenged with Cch that was prevented by the M3 receptor antagonist 4-DAMP, in a concentration-dependent manner. In conclusion, these data indicate that short- and long-term exposure to INGAP-PP significantly affects the expression and the phosphorylation of proteins involved in islet PI3K and MAPK signaling pathways. The observations of INGAPP-PP-stimulated up-regulation of cholinergic M3 receptors and PLC-beta2 proteins, enhanced P70S6K and MAPK3/1 phosphorylation and Cch-induced insulin secretion suggest a participation of the cholinergic pathway in INGAP-PP-mediated effects.

Corticotrophin-releasing factor type 2 receptor-mediated suppression of gonadotrophin-releasing hormone mRNA expression in GT1-7 cells.

Corticotrophin-releasing factor (CRF) released during stress has been implicated in the suppression of the hypothalamo–pituitary–gonadal (HPG) axis, especially the gonadotrophin-releasing hormone (GnRH) pulse generator, the central neural regulator of pituitary LH and FSH secretion, resulting in reproductive dysfunction. The gonadal steroid 17β-oestradiol (E2) has been shown to enhance CRF- and stress-induced suppression of pulsatile LH secretion. In the present study, we investigated the potential direct action of CRF on GnRH neurones by using GT1-7 cells, an established GnRH cell line. Furthermore, we investigated the modulatory influence of E2 on the effects of CRF and expression of CRF type 2 receptors (CRF-R2). Expression of CRF-R2 in the GT1-7 cells was detected by reverse transcription-polymerase chain reaction (RT-PCR). CRF produced a dose-dependent suppression of GnRH mRNA expression, an effect reversed by the selective CRF-R2 antagonist, astressin2-B (Ast2-B). E2 combined with CRF resulted in a greater suppression of GnRH expression compared with either treatment alone. E2 also increased CRF-R2 expression. These results demonstrate for the first time expression of CRF-R2 in the GT1-7 cells and suggest that CRF may directly regulate GnRH gene expression, an effect mediated, at least in part, by CRF-R2. They also raise the possibility that up-regulation of CRF-R2 may contribute to the sensitising influence of E2 on CRF- and stress-induced suppression of the GnRH pulse generator.