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AIMS/HYPOTHESIS: Chronic hyperglycaemia and recurrent hypoglycaemia are independently associated with accelerated cognitive decline in type 1 diabetes. Recurrent hypoglycaemia in rodent models of chemically induced (streptozotocin [STZ]) diabetes leads to cognitive impairment in memory-related tasks associated with hippocampal oxidative damage. This study examined the hypothesis that post-hypoglycaemic hyperglycaemia in STZ-diabetes exacerbates hippocampal oxidative stress and explored potential contributory mechanisms. METHODS: The hyperinsulinaemic glucose clamp technique was used to induce equivalent hypoglycaemia and to control post-hypoglycaemic glucose levels in mice with and without STZ-diabetes and Nrf2-/- mice (lacking Nrf2 [also known as Nfe2l2]). Subsequently, quantitative proteomics based on stable isotope labelling by amino acids in cell culture and biochemical approaches were used to assess oxidative damage and explore contributory pathways. RESULTS: Evidence of hippocampal oxidative damage was most marked in mice with STZ-diabetes exposed to post-hypoglycaemic hyperglycaemia; these mice also showed induction of Nrf2 and the Nrf2 transcriptional targets Sod2 and Hmox-1. In this group, hypoglycaemia induced a significant upregulation of proteins involved in alternative fuel provision, reductive biosynthesis and degradation of damaged proteins, and a significant downregulation of proteins mediating the stress response. Key differences emerged between mice with and without STZ-diabetes following recovery from hypoglycaemia in proteins mediating the stress response and reductive biosynthesis. CONCLUSIONS/INTERPRETATION: There is a disruption of the cellular response to a hypoglycaemic challenge in mice with STZ-induced diabetes that is not seen in wild-type non-diabetic animals. The chronic hyperglycaemia of diabetes and post-hypoglycaemic hyperglycaemia act synergistically to induce oxidative stress and damage in the hippocampus, possibly leading to irreversible damage/modification to proteins or synapses between cells. In conclusion, recurrent hypoglycaemia in sub-optimally controlled diabetes may contribute, at least in part, to accelerated cognitive decline through amplifying oxidative damage in key brain regions, such as the hippocampus. DATA AVAILABILITY: The datasets generated during and/or analysed during the current study are available in ProteomeXchange, accession no. 1-20220824-173727 ( www.proteomexchange.org ). Additional datasets generated during and/or analysed during the present study are available from the corresponding author upon reasonable request.

© 2014 International Society of Nephrology. Urate is the metabolic end point of purines in humans. Although supra physiological plasma urate levels are associated with obesity insulin resistance dyslipidemia and hypertension a causative role is debated. We previously established a mouse model of hyperuricemia by liver specific deletion of Glut9 a urate transporter that provides urate to the hepatocyte enzyme uricase. These LG9 knockout mice show mild hyperuricemia (120 µmol/l) which can be further increased by the urate precursor inosine. Here we explored the role of progressive hyperuricemia on the cardiovascular function. Arterial blood pressure and heart rate were periodically measured by telemetry over 6 months in LG9 knockout mice supplemented with incremental amounts of inosine in a normal chow diet. This long term inosine treatment elicited a progressive increase in uricemia up to 300 µmol/l; however it did not modify heart rate or mean arterial blood pressure in LG9 knockout compared with control mice. Inosine treatment did not alter cardiac morphology or function measured by ultrasound echocardiography. However it did induce mild renal dysfunction as revealed by higher plasma creatinine levels lower glomerular filtration rate and histological signs of chronic inflammation and fibrosis. Thus in LG9 knockout mice inosine induced hyperuricemia was not associated with hypertension despite partial renal deficiency. This does not support a direct role of urate in the control of blood pressure.

© 2014 by The American Society for Biochemistry and Molecular Biology Inc. IGF2 is an autocrine ligand for the beta cell IGF1R receptor and GLP 1 increases the activity of this autocrine loop by enhancing IGF1R expression a mechanism that mediates the trophic effects of GLP 1 on beta cell mass and function. Here we investigated the regulation of IGF2 biosynthesis and secretion. We showed that glutamine rapidly and strongly induced IGF2 mRNA translation using reporter constructs transduced in MIN6 cells and primary islet cells. This was followed by rapid secretion of IGF2 via the regulated pathway as revealed by the presence of mature IGF2 in insulin granule fractions and by inhibition of secretion by nimodipine and diazoxide. When maximally stimulated by glutamine the amount of secreted IGF2 rapidly exceeded its initial intracellular pool and tolbutamide and high K+ increased IGF2 secretion only marginally. This indicates that the intracellular pool of IGF2 is small and that sustained secretion requires de novo synthesis. The stimulatory effect of glutamine necessitates its metabolism but not mTOR activation. Finally exposure of insulinomas or beta cells to glutamine induced Akt phosphorylation an effect that was dependent on IGF2 secretion and reduced cytokine induced apoptosis. Thus glutamine controls the activity of the beta cell IGF2/IGF1R autocrine loop by increasing the biosynthesis and secretion of IGF2. This autocrine loop can thus integrate changes in feeding and metabolic state to adapt beta cell mass and function.

© The Author 2014. Type 2 diabetes (T2D) is characterized by ß cell dysfunction and loss. Single nucleotide polymorphisms in the T cell factor 7 like 2 (TCF7L2) gene associated with T2D by genome wide association studies lead to impaired ß cell function. While deletion of the homologous murine Tcf7l2 gene throughout the developing pancreas leads to impaired glucose tolerance deletion in the ß cell in adult mice reportedly has more modest effects. To inactivate Tcf7l2 highly selectively in ß cells from the earliest expression of the Ins1 gene (E11.5) we have therefore used a Cre recombinase introduced at the Ins1 locus. Tcfl2fl/fl::Ins1Cre mice display impaired oral and intraperitoneal glucose tolerance by 8 and 16 weeks respectively and defective responses to the GLP 1 analogue liraglutide at 8 weeks. Tcfl2fl/fl::Ins1Cre islets displayed defective glucose and GLP 1 stimulated insulin secretion and the expression of both the Ins2 (20) and Glp1r (40) genes were significantly reduced. Glucose and GLP 1 induced intracellular free Ca2+ increases and connectivity between individual ß cellswere both lowered by Tcf7l2 deletion in islets from mice maintained on a high (60) fat diet. Finally analysis by optical projection tomography revealed 30 decrease in ß cell mass in pancreata from Tcfl2fl/fl::Ins1Cre mice. These data demonstrate that Tcf7l2 plays a cell autonomous role in the control of ß cell function and mass serving as an important regulator of gene expression and islet cell coordination. The possible relevance of these findings for the action of TCF7L2 polymorphisms associated with Type 2 diabetes in man is discussed.

Aims/hypothesis Pancreatic beta cells play a central role in the control of glucose homeostasis by secreting insulin to stimulate glucose uptake by peripheral tissues. Understanding the molecular mechanisms that control beta cell function and plasticity has critical implications for the pathophysiology and therapy of major forms of diabetes. Selective gene inactivation in pancreatic beta cells, using the Cre-lox system, is a powerful approach to assess the role of particular genes in beta cells and their impact on whole body glucose homeostasis. Several Cre recombinase (Cre) deleter mice have been established to allow inactivation of genes in beta cells, but many show non-specific recombination in other cell types, often in the brain. Methods We describe the generation of Ins1Cre and Ins1CreERT2 mice in which the Cre or Cre-oestrogen receptor fusion protein (CreERT2) recombinases have been introduced at the initiation codon of the Ins1 gene. Results We show that Ins1Cre mice induce efficient and selective recombination of floxed genes in beta cells from the time of birth, with no recombination in the central nervous system. These mice have normal body weight and glucose homeostasis. Furthermore, we show that tamoxifen treatment of adult Ins1CreERT2 mice crossed with Rosa26-tdTomato mice induces efficient recombination in beta cells. Conclusions/interpretation These two strains of deleter mice are useful new resources to investigate the molecular physiology of pancreatic beta cells. Electronic supplementary material The online version of this article (doi:10.1007/s00125-014-3468-5) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

International audience; Pregnancy and obesity are frequently associated with diminished insulin sensitivity, which is normally compensated for by an expansion of the functional β cell mass that prevents chronic hyperglycemia and development of diabetes mellitus. The molecular basis underlying compensatory β cell mass expansion is largely unknown. We found in rodents that β cell mass expansion during pregnancy and obesity is associated with changes in the expression of several islet microRNAs, including miR-338-3p. In isolated pancreatic islets, we recapitulated the decreased miR-338-3p level observed in gestation and obesity by activating the G protein-coupled estrogen receptor GPR30 and the glucagon-like peptide 1 (GLP1) receptor. Blockade of miR-338-3p in β cells using specific anti-miR molecules mimicked gene expression changes occurring during β cell mass expansion and resulted in increased proliferation and improved survival both in vitro and in vivo. These findings point to a major role for miR-338-3p in compensatory β cell mass expansion occurring under different insulin resistance states.

PPARβ/δ protects against obesity by reducing dyslipidemia and insulin resistance via effects in muscle, adipose tissue, and liver. However, its function in pancreas remains ill defined. To gain insight into its hypothesized role in β cell function, we specifically deleted Pparb/d in the epithelial compartment of the mouse pancreas. Mutant animals presented increased numbers of islets and, more importantly, enhanced insulin secretion, causing hyperinsulinemia. Gene expression profiling of pancreatic β cells indicated a broad repressive function of PPARβ/δ affecting the vesicular and granular compartment as well as the actin cytoskeleton. Analyses of insulin release from isolated PPARβ/δ-deficient islets revealed an accelerated second phase of glucose-stimulated insulin secretion. These effects in PPARβ/δ-deficient islets correlated with increased filamentous actin (F-actin) disassembly and an elevation in protein kinase D activity that altered Golgi organization. Taken together, these results provide evidence for a repressive role for PPARβ/δ in β cell mass and insulin exocytosis, and shed a new light on PPARβ/δ metabolic action.

Type 2 diabetes (T2D) is characterized by β cell dysfunction and loss. Single nucleotide polymorphisms in the T-cell factor 7-like 2 (TCF7L2) gene, associated with T2D by genome-wide association studies, lead to impaired β cell function. While deletion of the homologous murine Tcf7l2 gene throughout the developing pancreas leads to impaired glucose tolerance, deletion in the β cell in adult mice reportedly has more modest effects. To inactivate Tcf7l2 highly selectively in β cells from the earliest expression of the Ins1 gene (∼E11.5) we have therefore used a Cre recombinase introduced at the Ins1 locus. Tcfl2(fl/fl)::Ins1Cre mice display impaired oral and intraperitoneal glucose tolerance by 8 and 16 weeks, respectively, and defective responses to the GLP-1 analogue liraglutide at 8 weeks. Tcfl2(fl/fl)::Ins1Cre islets displayed defective glucose- and GLP-1-stimulated insulin secretion and the expression of both the Ins2 (∼20%) and Glp1r (∼40%) genes were significantly reduced. Glucose- and GLP-1-induced intracellular free Ca(2+) increases, and connectivity between individual β cells, were both lowered by Tcf7l2 deletion in islets from mice maintained on a high (60%) fat diet. Finally, analysis by optical projection tomography revealed ∼30% decrease in β cell mass in pancreata from Tcfl2(fl/fl)::Ins1Cre mice. These data demonstrate that Tcf7l2 plays a cell autonomous role in the control of β cell function and mass, serving as an important regulator of gene expression and islet cell coordination. The possible relevance of these findings for the action of TCF7L2 polymorphisms associated with Type 2 diabetes in man is discussed.

NHA2 is a sodium/hydrogen exchanger with unknown physiological function. Here we show that NHA2 is present in rodent and human β-cells, as well as β-cell lines. In vivo, two different strains of NHA2-deficient mice displayed a pathological glucose tolerance with impaired insulin secretion but normal peripheral insulin sensitivity. In vitro, islets of NHA2-deficient and heterozygous mice, NHA2-depleted Min6 cells, or islets treated with an NHA2 inhibitor exhibited reduced sulfonylurea- and secretagogue-induced insulin secretion. The secretory deficit could be rescued by overexpression of a wild-type, but not a functionally dead, NHA2 transporter. NHA2 deficiency did not affect insulin synthesis or maturation and had no impact on basal or glucose-induced intracellular Ca 2+ homeostasis in islets. Subcellular fractionation and imaging studies demonstrated that NHA2 resides in transferrin-positive endosomes and synaptic-like microvesicles but not in insulin-containing large dense core vesicles in β-cells. Loss of NHA2 inhibited clathrin-dependent, but not clathrin-independent, endocytosis in Min6 and primary β-cells, suggesting defective endo–exocytosis coupling as the underlying mechanism for the secretory deficit. Collectively, our in vitro and in vivo studies reveal the sodium/proton exchanger NHA2 as a critical player for insulin secretion in the β-cell. In addition, our study sheds light on the biological function of a member of this recently cloned family of transporters.

Portal vein glucose sensors detect variations in glycemia to induce a nervous signal that influences food intake and glucose homeostasis. Previous experiments using high infusions of glucose suggested a metabolic sensing involving glucose transporter 2 (GLUT2). Here we evaluated the afferent route for the signal and candidate molecules for detecting low glucose fluxes. Common hepatic branch vagotomy did not abolish the anorectic effect of portal glucose indicating dorsal transmission. GLUT2 null mice reduced their food intake in response to portal glucose signal initiated by protein enriched diet. A similar response of Trpm5 null mice and portal infusions of sweeteners also excluded sugar taste receptors. Conversely infusions of alpha methylglucose but not 3 O methylglucose decreased food intake while phlorizin prevented the effect of glucose. This suggested sensing through SGLT3 which was expressed in the portal area. From these results we propose a finely tuned dual mechanism for portal glucose sensing that responds to different physiological conditions. © 2012 Elsevier GmbH.

The hypothalamus plays an essential role in the central nervous system of mammals by among others regulating glucose homeostasis, food intake, temperature, and to some extent blood pressure. Assessments of hypothalamic metabolism using, e.g. (1)H MRS in mouse models can provide important insights into its function. To date, direct in vivo (1)H MRS measurements of hypothalamus have not been reported. Here, we report that in vivo single voxel measurements of mouse hypothalamus are feasible using (1)H MRS at 14.1T. Localized (1)H MR spectra from hypothalamus were obtained unilaterally (2-2.2 microL, VOI) and bilaterally (4-4.4 microL) with a quality comparable to that of hippocampus (3-3.5 microL). Using LCModel, a neurochemical profile consisting of 21 metabolites was quantified for both hypothalamus and hippocampus with most of the Cramér-Rao lower bounds within 20%. Relative to the hippocampus, the hypothalamus was characterized by high gamma-aminobutryric acid and myo-inositol, and low taurine concentrations. When studying transgenic mice with no glucose transporter isoform 8 expressed, small metabolic changes were observed, yet glucose homeostasis was well maintained. We conclude that a specific neurochemical profile of mouse hypothalamus can be measured by (1)H MRS which will allow identifying and following metabolic alterations longitudinally in the hypothalamus of genetic modified models.

We performed 1H MRS at 14.1T in the liver of GLUT2-/- and wild-type (WT) mice. Improved spectral resolution allowed identifying peaks from the lipid resonances, choline containing compounds, taurine and glycogen in WT mice. Performing the same measurements in GLUT2-/- mice revealed a strong contribution from carbohydrates, notably glucose, to the 1H MR liver spectrum. Also a reduced lipid content was detected by 1H MRS in those mice. The characterization of the hepatic profile by 1H MRS in GLUT2-/- mice is of great value to address changes in the main energy fuels in the liver longitudinally.

AIMS/HYPOTHESIS: Recurrent hypoglycaemia in people with diabetes leads to progressive suppression of counterregulatory hormonal responses to subsequent hypoglycaemia. Recently it has been proposed that the mechanism underpinning this is a form of adaptive memory referred to as habituation. To test this hypothesis, we use two different durations of cold exposure to examine whether rodents exposed to recurrent hypoglycaemia exhibit two characteristic features of habituation, namely stimulus generalisation and dishabituation. METHODS: In the first study (stimulus generalisation study), hyperinsulinaemic-hypoglycaemic (2.8 mmol/l) glucose clamps were performed in non-diabetic rodents exposed to prior moderate-duration cold (4°C for 3 h) or control conditions. In the second study (dishabituation study), rodents exposed to prior recurrent hypoglycaemia or saline (154 mmol/l NaCl) injections over 4 weeks underwent a longer-duration cold (4°C for 4.5 h) exposure followed 24 h later by a hyperinsulinaemic-hypoglycaemic (2.8 mmol/l) glucose clamp. Output measures were counterregulatory hormone responses during experimental hypoglycaemia. RESULTS: Moderate-duration cold exposure blunted the adrenaline (epinephrine) response (15,266 ± 1920 vs 7981 ± 1258 pmol/l, Control vs Cold; p < 0.05) to next day hypoglycaemia in healthy non-diabetic rodents. In contrast, the suppressed adrenaline response (Control 5912 ± 1417 vs recurrent hypoglycaemia 1836 ± 736 pmol/l; p < 0.05) that is associated with recurrent hypoglycaemia was restored following longer-duration cold exposure (recurrent hypoglycaemia + Cold 4756 ± 826 pmol/l; not significant vs Control). CONCLUSIONS/INTERPRETATION: Non-diabetic rodents exhibit two cardinal features of habituation, namely stimulus generalisation and dishabituation. These findings provide further support for the hypothesis that suppressed counterregulatory responses following exposure to recurrent hypoglycaemia in diabetes result from habituation.

PCT No. PCT/GB96/00256 Sec. 371 Date Mar. 3, 1998 Sec. 102(e) Date Mar. 3, 1998 PCT Filed Feb. 7, 1996 PCT Pub. No. WO96/25487 PCT Pub. Date Aug. 22, 1996This application relates to cell lines transfected with (a) nucleic acid encoding one or more hormone receptors, the receptors being capable of transmitting a signal, and (b) nucleic acid encoding a desired polypeptide, the nucleic acid encoding the polypeptide being under the control of a promoter containing regulatory elements responsive to the signal transmitted by the receptor(s) so that transcription of the nucleic acid encoding the desired polypeptide is modulated by the level of the hormone(s) in the patient. Preferably, the receptors are the GIP or GLP-1 receptors controlling the production of insulin in the cells in response to changes in the metabolic status of a patient. The application also relates to compositions comprising these cells and their use in methods of medical treatment.

Introduction: Whole-body energy balance is strongly dependent on the hepatic handling of metabolic fuels, which is affected by the hepatic lipid content (HLC). With in vivo nuclear magnetic resonance spectroscopy (MRS) HLC can be measured non-invasively and longitudinally. The study of mice is relevant for the characterization of transgenic models that provide insight into disease mechanisms but comparing with bigger subjects, the reduced sample size remains a limiting factor for in vivo MRS experiments. To overcome this sensitivity issue, we performed liver MRS in vivo at high field (14.1T) in wild-type C57BL/6J mice (WT) and GLUT2-/- mice that display altered whole-body energy balance. Methods: WT and GLUT2-/- mice reexpressing GLUT1 in the pancreatic β-cells to allow for survival and normal glucose-stimulated insulin secretion were studied at 4 months and 1 year of age. Non-fasted mice under isofluorane anesthesia were scanned in the supine position with a 1H quadrature surface coil over the abdomen. MRS measurements were performed in a horizontal bore 14.1T-26 cm magnet. Multi-slice gradient echo images were acquired for anatomical identification of the liver. Localized, respiration-gated 1H-MR spectra were acquired from a 10-15 µl voxel with STEAM with and without water suppression. HLC was estimated as the T2-corrected area of 1.3 ppm-lipid resonance relative to that of the water plus 1.3 ppm-lipid. Results/Discussion: Highly sensitive MRS at 14.1T allowed to accurately quantifying HLC in short experiments in mice. Suppression of the water signal revealed fatty-acyl resonances reflecting the lipid saturation profile. At 4 months, HLC was similar between WT and GLUT2-/-. HLC increased significantly in aged WT mice but remained low in aged GLUT2-/-. Also, the body weight of aged GLUT2-/- was slightly lower than that of WT. Low HLC in aged GLUT2-/- mice probably results from a defect on intra-hepatic pathway fluxes due to GLUT2 ablation in the liver. In addition, it may reflect altered utilization of metabolic substrates due to inadequate whole-body blood glucose sensing.

In the nervous system, NMDA receptors (NMDARs) participate in neurotransmission and modulate the viability of neurons. In contrast, little is known about the role of NMDARs in pancreatic islets and the insulin-secreting beta cells whose functional impairment contributes to diabetes mellitus. Here we found that inhibition of NMDARs in mouse and human islets enhanced their glucose-stimulated insulin secretion (GSIS) and survival of islet cells. Further, NMDAR inhibition prolonged the amount of time that glucose-stimulated beta cells spent in a depolarized state with high cytosolic Ca(2+) concentrations. We also noticed that, in vivo, the NMDAR antagonist dextromethorphan (DXM) enhanced glucose tolerance in mice, and that in vitro dextrorphan, the main metabolite of DXM, amplified the stimulatory effect of exendin-4 on GSIS. In a mouse model of type 2 diabetes mellitus (T2DM), long-term treatment with DXM improved islet insulin content, islet cell mass and blood glucose control. Further, in a small clinical trial we found that individuals with T2DM treated with DXM showed enhanced serum insulin concentrations and glucose tolerance. Our data highlight the possibility that antagonists of NMDARs may provide a useful adjunct treatment for diabetes.