This study characterizes the high-fat diet-fed mouse as a model for impaired glucose tolerance (IGT) and type 2 diabetes. Female C57BL/6J mice were fed a high-fat diet (58% energy by fat) or a normal diet (11% fat). Body weight was higher in mice fed the high-fat diet already after the first week, due to higher dietary intake in combination with lower metabolic efficiency. Circulating glucose increased after 1 week on high-fat diet and remained elevated at a level of ϳ1 mmol/l throughout the 12-month study period. In contrast, circulating insulin increased progressively by time. Intravenous glucose challenge revealed a severely compromised insulin response in association with marked glucose intolerance already after 1 week. To illustrate the usefulness of this model for the development of new treatment, mice were fed an orally active inhibitor of dipeptidyl peptidase-IV (LAF237) in the drinking water (0.3 mg/ ml) for 4 weeks. This normalized glucose tolerance, as judged by an oral glucose tolerance test, in association with augmented insulin secretion. We conclude that the high-fat diet-fed C57BL/6J mouse model is a robust model for IGT and early type 2 diabetes, which may be used for studies on pathophysiology and development of new treatment. Diabetes 53 (Suppl. 3):S215-S219, 2004 T here is a need for new treatment modalities of type 2 diabetes in view of the progressive deterioration of metabolic control that occurs in spite of intense treatment with existing modalities (1). New treatment should aim at normalizing the basic defects in the disease, which are islet dysfunction in combination with insulin resistance (2). There is, however, also a need for more knowledge of the molecular mechanisms underlying these basic defects. These two needs require reliable and clinically relevant experimental models. Most animal models do not, however, fulfill such requirements, since they are based on monogenic disorders of little relevance for human diabetes (3-5) or on chemical destruction of ␤-cells, which is also of less clinical relevance (6,7). An important and relevant model, however, is the high-fat diet-fed C57BL/6J mouse model. This model was originally introduced by Surwit et al. in 1988 (8). The model has shown to be accompanied by insulin resistance, as determined by intravenous glucose tolerance tests, and of insufficient islet compensation to the insulin resistance (9). The model has, accordingly, been used in studies on pathophysiology of impaired glucose tolerance (IGT) and type 2 diabetes (10 -12) and for development of new treatments (13-16). Here we report the characteristics of this model and illustrate its relevance in studies on developing new treatment modes by showing beneficial influences of a novel and efficient orally active inhibitor of dipeptidyl peptidase-IV (DPP-IV), which is a new mode for treating type 2 diabetes by preventing the degradation of glucagon-like peptide-1 (GLP-1) (17-19). RESEARCH DESIGN AND METHODSFemale C57BL/6J mice were purchased from Taconic (Skensved, Denmark). The ani...

and glucose-dependent insulinotropic polypeptide (GIP) regulate islet function after carbohydrate ingestion. Whether incretin hormones are of importance for islet function after ingestion of noncarbohydrate macronutrients is not known. This study therefore examined integrated incretin and islet hormone responses to ingestion of pure fat (oleic acid; 0.88 g/kg) or protein (milk and egg protein; 2 g/kg) over 5 h in healthy men, aged 20 -25 yr (n ϭ 12); plain water ingestion served as control. Both intact (active) and total GLP-1 and GIP levels were determined as was plasma activity of dipeptidyl peptidase-4 (DPP-4). Following water ingestion, glucose, insulin, glucagon, GLP-1, and GIP levels and DPP-4 activity were stable during the 5-h study period. Both fat and protein ingestion increased insulin, glucagon, GIP, and GLP-1 levels without affecting glucose levels or DPP-4 activity. The GLP-1 responses were similar after protein and fat, whereas the early (30 min) GIP response was higher after protein than after fat ingestion (P Ͻ 0.001). This was associated with sevenfold higher insulin and glucagon responses compared with fat ingestion (both P Ͻ 0.001). After protein, the early GIP, but not GLP-1, responses correlated to insulin (r 2 ϭ 0.86; P ϭ 0.0001) but not glucagon responses. In contrast, after fat ingestion, GLP-1 and GIP did not correlate to islet hormones. We conclude that, whereas protein and fat release both incretin and islet hormones, the early GIP secretion after protein ingestion may be of primary importance to islet hormone secretion.insulin; glucagon; glucagon-like peptide-1; glucose-dependent insulinotropic polypeptide; incretins; man THE INTEGRATED ENDOCRINE RESPONSES TO FOOD INGESTION are dependent on both the size and the composition of a meal and include the postprandial release of the incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) and the islet hormones insulin and glucagon (3,5,21,32). Most studies have focused on responses to an oral glucose tolerance test, after which levels of GIP, GLP-1, and insulin rise, whereas glucagon levels are suppressed (4,18,20, 24). It is also known that fat and protein ingestion stimulate GLP-1 and GIP secretion (10,14,20,27). Less is known, however, regarding relationships between the incretin responses and changes in insulin and glucagon levels after meal or noncarbohydrate macronutrient ingestions.GLP-1 and GIP are rapidly degraded by dipeptidyl peptidase-4 (DPP-4), which cleaves the two NH 2 -terminal amino acids of the peptides, making them largely inactive (9). Accurate estimation of the relationship between incretin hormone secretion and islet hormones therefore requires measurement of both the total and the active intact forms of the two incretins. How this is related to macronutrient ingestion is not known. Indeed, we recently showed in mice that protein ingestion increased intact incretin hormone levels compared with carbohydrate ingestion, and this was associated with reduced intestinal DPP-4...

Apelin is the endogenous ligand of the G-protein coupled apj receptor. Apelin is expressed in the brain, the hypothalamus and the stomach and was recently shown also to be an adipokine secreted from the adipocytes. Although apelin has been suggested to be involved in the regulation of food intake, it is not known whether the peptide affects islet function and glucose homeostasis. We show here that the apj receptor is expressed in pancreatic islets and that intravenous administration of full-length apelin-36 (2 nmol/kg) inhibits the rapid insulin response to intravenous glucose (1g/kg) by 35% in C57BL/6J mice. Thus, the acute (1-5 min) insulin response to intravenous glucose was 682±23 pmol/l after glucose alone (n=17) and 445±58 pmol/l after glucose plus apelin-36 (n=18; P=0.017). This was associated with impaired glucose elimination (the 5-20 min glucose elimination was 2.9±0.1%/min after glucose alone versus 2.3±0.2%/min after glucose plus apelin-36, P=0.008). Apelin (2nmol/kg) also inhibited the insulin response to intravenous glucose in obese insulin resistant high-fat fed C57BL/6J mice (P=0.041). After 60 min incubation of isolated islets from normal mice, insulin secretion in the presence of 16.7 mmol/l glucose was inhibited by apelin-36 at 1 µmol/l, whereas apelin-36 did not significantly affect insulin secretion at 2.8 or 8.3 mmol/l glucose or after stimulation of insulin secretion by KCl. Islet glucose oxidation at 16.7 mmol/l was not affected by apelin-36. We conclude that the apj receptor is expressed in pancreatic islets and that apelin-36 inhibits glucose-stimulated insulin secretion both in vivo and in vitro.This may suggest that the islet beta-cells are targets for apelin-36.

Aims/hypothesisThe NEFA-responsive G-protein coupled receptor 120 (GPR120) has been implicated in the regulation of inflammation, in the control of incretin secretion and as a predisposing factor influencing the development of type 2 diabetes by regulation of islet cell apoptosis. However, there is still considerable controversy about the tissue distribution of GPR120 and, in particular, it remains unclear which islet cell types express this molecule. In the present study, we have addressed this issue by constructing a Gpr120-knockout/β-galactosidase (LacZ) knock-in (KO/KI) mouse to examine the distribution and functional role of GPR120 in the endocrine pancreas.MethodsA KO/KI mouse was generated in which exon 1 of the Gpr120 gene (also known as Ffar4) was replaced in frame by LacZ, thereby allowing for regulated expression of β-galactosidase under the control of the endogenous GPR120 promoter. The distribution of GPR120 was inferred from expression studies detecting β-galactosidase activity and protein production. Islet hormone secretion was measured from isolated mouse islets treated with selective GPR120 agonists.Resultsβ-galactosidase activity was detected as a surrogate for GPR120 expression exclusively in a small population of islet endocrine cells located peripherally within the islet mantle. Immunofluorescence analysis revealed co-localisation with somatostatin suggesting that GPR120 is preferentially produced in islet delta cells. In confirmation of this, glucose-induced somatostatin secretion was inhibited by a range of selective GPR120 agonists. This response was lost in GPR120-knockout mice.Conclusions/interpretationThe results imply that GPR120 is selectively present within the delta cells of murine islets and that it regulates somatostatin secretion.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-014-3213-0) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Islet function is regulated by a number of different signals. A main signal is generated by glucose, which stimulates insulin secretion and inhibits glucagon secretion. The glucose effects are modulated by many factors, including hormones, neurotransmitters and nutrients. Several of these factors signal through guanine nucleotide-binding protein (G-protein) coupled receptors (GPCRs). Examples of islet GPCRs are GPR40 and GPR119, which are GPCRs with fatty acids as ligands, the receptors for the incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), the receptors for the islet hormones glucagon and somatostatin, the receptors for the classical neurotransmittors

Abstract-Red blood cells may regulate tissue circulation and O 2 delivery by releasing the vasodilator ATP in response to hypoxia. When released extracellularly, ATP is rapidly degraded to ADP in the circulation by ectonucleotidases. In this study, we show that ADP acting on P2Y 13 receptors on red blood cells serves as a negative feedback pathway for the inhibition of ATP release. mRNA of the ADP receptor P2Y 13 was highly expressed in human red blood cells and reticulocytes. Key Words: ATP release Ⅲ cAMP Ⅲ P2 receptors Ⅲ microdialysis Ⅲ red blood cells I t has become increasingly clear that, in addition to functioning as an intracellular energy source, ATP and ADP can serve as important extracellular signaling molecules. 1,2 Extracellular ATP in the circulation is rapidly degraded into ADP, AMP, and adenosine by ectonucleotidases. 3 ATP and ADP activate P2 receptors on endothelium, platelets, 4,5 and other blood cells, 6 regulating several physiological responses including vascular tone, 7 platelet aggregation, and the release of endothelial factors. At least 15 nucleotide-activated cell surface receptors (7 of P2X and 8 of P2Y) have been found in man, with remarkably broad and varied physiological responses.The matching of oxygen supply with demand requires a mechanism that increases blood flow in response to decreased tissue oxygen levels. Several reports suggest that the red blood cell (RBC) acts as a sensor for hypoxia, and different mechanisms have been suggested by which the deoxygenated RBC stimulates vasodilatation. 7-10 RBCs contain millimolar amounts of ATP and possess the membrane-bound glycolytic enzymes necessary for its production. 11-13 ATP is released in response to reductions in oxygen tension and pH. 7,9 It has been shown in vitro that the vessels dilate in response to low O 2 levels only when blood vessels are perfused with RBCs. 14 Recently, in vivo studies in man demonstrated that ATP is released in working skeletal muscle circulation depending on the number of unoccupied hemoglobin O 2 binding sites. 7,9 The released ATP then binds to P2Y receptors on the endothelium and stimulates vasodilatation by the release of nitric oxide (NO), prostaglandins, 2 and endothelium-derived hyperpolarizing factor (EDHF). 15,16 Thus, the RBC functions as an O 2 sensor, contributing to the regulation of blood flow and O 2 delivery by releasing ATP depending on the oxygenation state of hemoglobin.Physiologically important signaling systems are usually regulated by negative feedback systems, for example, noradrenaline and ATP release from sympathetic nerves is inhibited by presynaptic ␣ 2 and P 1 (A1 subtype) receptors. 17 We hypothesized that ATP release from RBCs is regulated by a P2 receptor-mediated negative feedback pathway. Materials and MethodsThe studies were approved by the local Ethics Committee of the Lund University and were conducted according to the principles of the Declaration of Helsinki. All participants gave written consent for the study. Preparation of RBCs and ReticulocytesHuman RBCs were...

The early release of GLP-1 and GIP are more pronounced in the morning than in the afternoon. This may contribute to the more rapid early insulin response, more pronounced potentiation of beta-cell function, and lower glucose after the morning meal.

Lipids may serve as coupling factors in K ATP -independent glucose sensing in ␤-cells. We have previously demonstrated that ␤-cells harbor lipase activities, one of which is the hormone-sensitive lipase. Whether ␤-cell lipases are critical for glucose-stimulated insulin secretion (GSIS) by providing lipid-derived signals from endogenous lipids is unknown. Therefore, using a lipase inhibitor (orlistat), we examined whether lipase inhibition reduces insulin secretion. Islet lipolysis stimulated by glucose and diglyceride lipase activity was abolished by orlistat. Incubation of rat islets with orlistat dose dependently inhibited GSIS; this inhibition was reversed by 1 mmol/l palmitate, suggesting that orlistat acts via impaired formation of an acylglyceride-derived coupling signal. Orlistat inhibited the potentiating effect of forskolin on GSIS, an effect proposed to be due to activation of a lipase. In perifused islets, orlistat attenuated mainly the second phase of insulin secretion. Because the rise in islet ATP/ADP levels in response to glucose and oxidation of the sugar were unaffected by orlistat whereas the second phase of insulin secretion was reduced, it seems likely that a lipid coupling factor involved in K ATP -independent glucose sensing has been perturbed. Thus, ␤-cell lipase activity is involved in GSIS, emphasizing the important role of ␤-cell lipid metabolism for insulin secretion. Diabetes 53:122-128, 2004A large body of evidence suggests that lipids are required for appropriate glucose sensing in pancreatic ␤-cells. For instance, when triglycerides in pancreatic islets are depleted by hyperleptinemia, pancreatic ␤-cells fail to release insulin (1). Along similar lines, perfused pancreas from fasted rats is unresponsive to a rise in glucose (2). Importantly, in both cases, glucose responsiveness is reinstated by the addition of exogenous fatty acids.The current view holds that lipids serve as coupling factors in glucose-stimulated insulin secretion (GSIS) that does not rely on closure of the ATP-sensitive K ϩ channel (K ATP independent) (3). These putative mechanisms presumably drive the second phase of GSIS and accordingly have been termed as amplifying (4), and the coupling factors have been proposed to emanate from intermediary metabolism in the ␤-cell (5). Against this background, lipids have become attractive candidates for coupling factors in K ATP -independent glucose sensing. In fact, a model has been proposed to delineate the combined events in metabolism of glucose and lipids that result in a dose-dependent regulation of insulin secretion, the socalled long-chain acyl-CoA hypothesis (3). This model is based on the observations that an increase in glucose metabolism in ␤-cells results in decreased oxidation of fats while esterification of lipids increases. As a consequence, the levels of complex lipids rise, perhaps acyl-CoA moieties, which then act as a signaling molecules coupling stimulus to secretion in the ␤-cell. We have previously demonstrated that different preparations of ␤-cells...