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Plasma metabolite concentrations reflect the activity of tissue metabolic pathways and their quantitative determination may be informative about pathogenic conditions. We searched for plasma lipid species whose concentrations correlate with various parameters of glucose homeostasis and susceptibility to type 2 diabetes (T2D). Shotgun lipidomic analysis of the plasma of mice from different genetic backgrounds, which develop a pre-diabetic state at different rates when metabolically stressed, led to the identification of a group of sphingolipids correlated with glucose tolerance and insulin secretion. Quantitative analysis of these and closely related lipids in the plasma of individuals from two population-based prospective cohorts revealed that specific long-chain fatty-acid-containing dihydroceramides were significantly elevated in the plasma of individuals who will progress to diabetes up to 9 years before disease onset. These lipids may serve as early biomarkers of, and help identify, metabolic deregulation in the pathogenesis of T2D.

The deleterious effect of chronic activation of the IL-1β system on type 2 diabetes and other metabolic diseases is well documented. However, a possible physiological role for IL-1β in glucose metabolism has remained unexplored. Here we found that feeding induced a physiological increase in the number of peritoneal macrophages that secreted IL-1β, in a glucose-dependent manner. Subsequently, IL-1β contributed to the postprandial stimulation of insulin secretion. Accordingly, lack of endogenous IL-1β signaling in mice during refeeding and obesity diminished the concentration of insulin in plasma. IL-1β and insulin increased the uptake of glucose into macrophages, and insulin reinforced a pro-inflammatory pattern via the insulin receptor, glucose metabolism, production of reactive oxygen species, and secretion of IL-1β mediated by the NLRP3 inflammasome. Postprandial inflammation might be limited by normalization of glycemia, since it was prevented by inhibition of the sodium–glucose cotransporter SGLT2. Our findings identify a physiological role for IL-1β and insulin in the regulation of both metabolism and immunity.

The counterregulatory response to hypoglycemia, which restores normal blood glucose levels to ensure sufficient provision of glucose to the brain, is critical for survival. To discover underlying brain regulatory systems, we performed a genetic screen in recombinant inbred mice for quantitative trait loci (QTL) controlling glucagon secretion in response to neuroglucopenia. We identified a QTL on the distal part of chromosome 7 and combined this genetic information with transcriptomic analysis of hypothalami. This revealed Fgf15as the strongest candidate to control the glucagon response. Fgf15was expressed by neurons of the dorsomedial hypothalamus and the perifornical area. Intracerebroventricular injection of FGF19, the human ortholog of Fgf15, reduced activation by neuroglucopenia of dorsal vagal complex neurons, of the parasympathetic nerve, and lowered glucagon secretion. In contrast, silencing Fgf15in the dorsomedial hypothalamus increased neuroglucopenia-induced glucagon secretion. These data identify hypothalamic Fgf15 as a regulator of glucagon secretion.

Feeding is controlled by hedonic cues that may override the homeostatic needs to eat, causing obesity. Labouèbe et al. have identified hypoglycemia-activated neurons in the paraventricular thalamus that increase motivated sucrose-seeking behavior. As their activity is not suppressed by fructose or sweeteners, these cells may contribute to sugar overconsumption and diabetes.

Specific metabolic pathways are activated by different nutrients to adapt the organism to available resources. Although essential, these mechanisms are incompletely defined. Here, we report that medium-chain fatty acids contained in coconut oil, a major source of dietary fat, induce the liver ω-oxidation genes Cyp4a10and Cyp4a14to increase the production of dicarboxylic fatty acids. Furthermore, these activate all ω- and β-oxidation pathways through peroxisome proliferator activated receptor (PPAR) α and PPARγ, an activation loop normally kept under control by dicarboxylic fatty acid degradation by the peroxisomal enzyme L-PBE. Indeed, L-pbe−/−mice fed coconut oil overaccumulate dicarboxylic fatty acids, which activate all fatty acid oxidation pathways and lead to liver inflammation, fibrosis, and death. Thus, the correct homeostasis of dicarboxylic fatty acids is a means to regulate the efficient utilization of ingested medium-chain fatty acids, and its deregulation exemplifies the intricate relationship between impaired metabolism and inflammation.

Plasma urate levels are higher in humans than rodents (240–360 vs. ∼30 μM) because humans lack the liver enzyme uricase. High uricemia in humans may protect against oxidative stress, but hyperuricemia also associates with the metabolic syndrome, and urate and uric acid can crystallize to cause gout and renal dysfunctions. Thus, hyperuricemic animal models to study urate-induced pathologies are needed. We recently generated mice with liver-specific ablation of Glut9, a urate transporter providing access of urate to uricase (LG9KO mice). LG9KO mice had moderately high uricemia (∼120 μM). To further increase their uricemia, here we gavaged LG9KO mice for 3 days with inosine, a urate precursor; this treatment was applied in both chow- and high-fat-fed mice. In chow-fed LG9KO mice, uricemia peaked at 300 μM 2 h after the first gavage and normalized 24 h after the last gavage. In contrast, in high-fat-fed LG9KO mice, uricemia further rose to 500 μM. Plasma creatinine strongly increased, indicating acute renal failure. Kidneys showed tubule dilation, macrophage infiltration, and urate and uric acid crystals, associated with a more acidic urine. Six weeks after inosine gavage, plasma urate and creatinine had normalized. However, renal inflammation, fibrosis, and organ remodeling had developed despite the disappearance of urate and uric acid crystals. Thus, hyperuricemia and high-fat diet feeding combined to induce acute renal failure. Furthermore, a sterile inflammation caused by the initial crystal-induced lesions developed despite the disappearance of urate and uric acid crystals.

GLUT9 (SLC2A9) is a newly described urate transporter whose function, characteristics, and localization have just started to be elucidated. Some transport properties of human GLUT9 have been studied in the Xenopus laevisoocyte expression system, but the type of transport (uniport, coupled transport system, stoichiometry … .) is still largely unknown. We used the same experimental system to characterize in more detail the transport properties of mouse GLUT9, its sensitivity to several uricosuric drugs, and the specificities of two splice variants, mGLUT9a and mGLUT9b. [14C]urate uptake measurements show that both splice variants are high-capacity urate transporters and have a Kmof ∼650 μM. The well-known uricosuric agents benzbromarone (500 μM) and losartan (1 mM) inhibit GLUT9-mediated urate uptake by 90 and 50%, respectively. Surprisingly, phloretin, a glucose-transporter blocker, inhibits [14C]urate uptake by ∼50% at 1 mM. Electrophysiological measurements suggest that urate transport by mouse GLUT9 is electrogenic and voltage dependent, but independent of the Na+and Cl−transmembrane gradients. Taken together, our results suggest that GLUT9 works as a urate (anion) uniporter. Finally, we show by RT-PCR performed on RNA from mouse kidney microdissected tubules that GLUT9a is expressed at low levels in proximal tubules, while GLUT9b is specifically expressed in distal convoluted and connecting tubules. Expression of mouse GLUT9 in the kidney differs from that of human GLUT9, which could account for species differences in urate handling.

The liver secretes triglyceride-rich VLDLs, and the triglycerides in these particles are taken up by peripheral tissues, mainly heart, skeletal muscle, and adipose tissue. Blocking hepatic VLDL secretion interferes with the delivery of liver-derived triglycerides to peripheral tissues and results in an accumulation of triglycerides in the liver. However, it is unclear how interfering with hepatic triglyceride secretion affects adiposity, muscle triglyceride stores, and insulin sensitivity. To explore these issues, we examined mice that cannot secrete VLDL [due to the absence of microsomal triglyceride transfer protein (Mttp) in the liver]. These mice exhibit markedly reduced levels of apolipoprotein B-100 in the plasma, along with reduced levels of triglycerides in the plasma. Despite the low plasma triglyceride levels, triglyceride levels in skeletal muscle were unaffected. Adiposity and adipose tissue triglyceride synthesis rates were also normal, and body weight curves were unaffected. Even though the blockade of VLDL secretion caused hepatic steatosis accompanied by increased ceramides and diacylglycerols in the liver, the mice exhibited normal glucose tolerance and were sensitive to insulin at the whole-body level, as judged by hyperinsulinemic euglycemic clamp studies. Normal hepatic glucose production and insulin signaling were also maintained in the fatty liver induced by Mttp deletion. Thus, blocking VLDL secretion causes hepatic steatosis without insulin resistance, and there is little effect on muscle triglyceride stores or adiposity.

The liver secretes triglyceride-rich VLDLs, and the triglycerides in these particles are taken up by peripheral tissues, mainly heart, skeletal muscle, and adipose tissue. Blocking hepatic VLDL secretion interferes with the delivery of liver-derived triglycerides to peripheral tissues and results in an accumulation of triglycerides in the liver. However, it is unclear how interfering with hepatic triglyceride secretion affects adiposity, muscle triglyceride stores, and insulin sensitivity. To explore these issues, we examined mice that cannot secrete VLDL [due to the absence of microsomal triglyceride transfer protein (Mttp) in the liver]. These mice exhibit markedly reduced levels of apolipoprotein B-100 in the plasma, along with reduced levels of triglycerides in the plasma. Despite the low plasma triglyceride levels, triglyceride levels in skeletal muscle were unaffected. Adiposity and adipose tissue triglyceride synthesis rates were also normal, and body weight curves were unaffected. Even though the blockade of VLDL secretion caused hepatic steatosis accompanied by increased ceramides and diacylglycerols in the liver, the mice exhibited normal glucose tolerance and were sensitive to insulin at the whole-body level, as judged by hyperinsulinemic euglycemic clamp studies. Normal hepatic glucose production and insulin signaling were also maintained in the fatty liver induced by Mttpdeletion. Thus, blocking VLDL secretion causes hepatic steatosis without insulin resistance, and there is little effect on muscle triglyceride stores or adiposity.

Early revascularization of pancreatic islet cells after transplantation is crucial for engraftment, and it has been suggested that vascular endothelial growth factor-A (VEGF-A) plays a significant role in this process. Although VEGF gene therapy can improve angiogenesis, uncontrolled VEGF secretion can lead to vascular tumor formation. Here we have explored the role of temporal VEGF expression, controlled by a tetracycline (TC)-regulated promoter, on revascularization and engraftment of genetically modified beta cells following transplantation. To this end, we modified the CDM3D beta cell line using a lentiviral vector to promote secretion of VEGF-A either in a TC-regulated (TET cells) or a constitutive (PGK cells) manner. VEGF secretion, angiogenesis, cell proliferation, and stimulated insulin secretion were assessed in vitro. VEGF secretion was increased in TET and PGK cells, and VEGF delivery resulted in angiogenesis, whereas addition of TC inhibited these processes. Insulin secretion by the three cell types was similar. We used a syngeneic mouse model of transplantation to assess the effects of this controlled VEGF expression in vivo. Time to normoglycemia, intraperitoneal glucose tolerance test, graft vascular density, and cellular mass were evaluated. Increased expression of VEGF resulted in significantly better revascularization and engraftment after transplantation when compared to control cells. In vivo, there was a significant increase in vascular density in grafted TET and PGK cells versus control cells. Moreover, the time for diabetic mice to return to normoglycemia and the stimulated plasma glucose clearance were also significantly accelerated in mice transplanted with TET and PGK cells when compared to control cells. VEGF was only needed during the first 2–3 weeks after transplantation; when removed, normoglycemia and graft vascularization were maintained. TC-treated mice grafted with TC-treated cells failed to restore normoglycemia. This approach allowed us to switch off VEGF secretion when the desired effects had been achieved. TC-regulated temporal expression of VEGF using a gene therapy approach presents a novel way to improve early revascularization and engraftment after islet cell transplantation.

The control of glucose and energy homeostasis, including feeding behaviour, is tightly regulated by gut-derived peptidic and nonpeptidic endocrine mediators, autonomic nervous signals, as well as nutrients such as glucose. We will review recent findings on the role of the gastrointestinal tract innervation and of portal vein glucose sensors; we will review selected data on the action of gastrointestinally released hormones.

L’ingestion de nourriture déclenche une réponse hormonale complexe, dont le but est notamment de minimiser les oscillations de la glycémie grâce à une stimulation appropriée du captage de glucose par le foie et les tissus musculaire et adipeux. L’activité insulinosécrétoire des cellules β du pancréas joue un rôle majeur dans cette réponse. Bien que l’augmentation du glucose sanguin soit le stimulus principal de l’insulinosécrétion, des hormones intestinales, libérées par des cellules endocrines de l’épithélium intestinal lors du passage de nutriments, jouent un rôle important de potentialisation de l’effet du glucose sur les cellules β pancréatiques. Ces hormones, appelées gluco-incrétines, sont principalement le GLP-1 (glucagon like peptide-1) et le GIP (glucose- dependent insulinotropic polypeptide). Elles agissent au niveau des cellules β pancréatiques en se liant à des récepteurs heptahélicaux couplés à l’activation de l’adénylate-cyclase. Outre leur capacité de potentialiser la sécrétion d’insuline induite par le glucose, ces hormones stimulent également la production d’insuline par des effets transcriptionnels et post-transcriptionnels, et ont également un effet positif sur le contrôle de la masse cellulaire β. L’activité insulinotrope, dépendante du glucose, du GLP-1 étant préservée au cours du diabète de type 2, ce peptide est considéré comme un médicament potentiel pour le traitement de cette maladie.

Streptozotocin injection in animals destroys pancreatic β cells, leading to insulinopenic diabetes. Here, we evaluated the toxic effect of streptozotocin (STZ) in GLUT2−/− mice reexpressing either GLUT1 or GLUT2 in their β cells under the rat insulin promoter (RIPG1 × G2−/− and RIPG2 × G2−/− mice, respectively). We demonstrated that injection of STZ into RIPG2 × G2−/− mice induced hyperglycemia (>20 mM) and an ∼80% reduction in pancreatic insulin content. In vitro, the viability of RIPG2 × G2−/− islets was also strongly reduced. In contrast, STZ did not induce hyperglycemia in RIPG1 × G2−/− mice and did not reduce pancreatic insulin content. The viability of in vitro cultured RIPG1 × G2−/− islets was also unaffected by STZ. As islets from each type of transgenic mice were functionally indistinguishable, these data strongly support the notion that STZ toxicity toward β cells depends on the expression of GLUT2.

In this paper we explore the possibility of improving, by genetic engineering, the resistance of insulin-secreting cells to the metabolic and inflammatory stresses that are anticipated to limit their function and survival when encapsulated and transplanted in a type 1 diabetic environment. We show that transfer of the Bcl-2 antiapoptotic gene, and of genes specifically interfering with cytokine intracellular signaling pathways, greatly improves resistance of the cells to metabolic limitations and inflammatory stresses.

Adipose cells produce and secrete several physiologically important proteins, such as lipoprotein lipase (LPL), leptin, adipsin, Acrp30, etc. However, secretory pathways in adipocytes have not been characterized, and vesicular carriers responsible for the accumulation and transport of secreted proteins have not been identified. We have compared the intracellular localization of two proteins secreted from adipose cells: leptin and LPL. Adipocytes accumulate large amounts of both proteins, suggesting that neither of them is targeted to the constitutive secretory pathway. By means of velocity centrifugation in sucrose gradients, equilibrium density centrifugation in iodixanol gradients, and immunofluorescence confocal microscopy, we determined that LPL and leptin were localized in different membrane structures. LPL was found mainly in the endoplasmic reticulum with a small pool being present in low density membrane vesicles that may represent a secretory compartment in adipose cells. Virtually all intracellular leptin was localized in these low density secretory vesicles. Insulin-sensitive Glut4 vesicles did not contain either LPL or leptin. Thus, secretion from adipose cells is controlled both at the exit from the endoplasmic reticulum as well as at the level of “downstream” secretory vesicles.

In the pathogenesis of type I diabetes mellitus, activated leukocytes infiltrate pancreatic islets and induce β cell dysfunction and destruction. Interferon (IFN)-γ, tumor necrosis factor-α and interleukin (IL)-1β play important, although not completely defined, roles in these mechanisms. Here, using the highly differentiated βTc-Tet insulin-secreting cell line, we showed that IFN-γ dose- and time-dependently suppressed insulin synthesis and glucose-stimulated secretion. As described previously IFN-γ, in combination with IL-1β, also induces inducible NO synthase expression and apoptosis (Dupraz, P., Cottet, S., Hamburger, F., Dolci, W., Felley-Bosco, E., and Thorens, B. (2000) J. Biol. Chem.275, 37672–37678). To assess the role of the Janus kinase/signal transducer and activator of transcription (STAT) pathway in IFN-γ intracellular signaling, we stably overexpressed SOCS-1 (suppressor of cytokinesignaling-1) in the β cell line. We demonstrated that SOCS-1 suppressed cytokine-induced STAT-1 phosphorylation and increased cellular accumulation. This was accompanied by a suppression of the effect of IFN-γ on: (i) reduction in insulin promoter-luciferase reporter gene transcription, (ii) decrease in insulin mRNA and peptide content, and (iii) suppression of glucose-stimulated insulin secretion. Furthermore, SOCS-1 also suppressed the cellular effects that require the combined presence of IL-1β and IFN-γ: induction of nitric oxide production and apoptosis. Together our data demonstrate that IFN-γ is responsible for the cytokine-induced defect in insulin gene expression and secretion and that this effect can be completely blocked by constitutive inhibition of the Janus kinase/STAT pathway.

Insulin-dependent diabetes mellitus is an autoimmune disease in which pancreatic islet β cells are destroyed by a combination of immunological and inflammatory mechanisms. In particular, cytokine-induced production of nitric oxide has been shown to correlate with β cell apoptosis and/or inhibition of insulin secretion. In the present study, we investigated whether the interleukin (IL)-1β intracellular signal transduction pathway could be blocked by overexpression of dominant negative forms of the IL-1 receptor interacting protein MyD88. We show that overexpression of the Toll domain or the lpr mutant of MyD88 in βTc-Tet cells decreased nuclear factor κB (NF-κB) activation upon IL-1β and IL-1β/interferon (IFN)-γ stimulation. Inducible nitric oxide synthase mRNA accumulation and nitrite production, which required the simultaneous presence of IL-1β and IFN-γ, were also suppressed by ∼70%, and these cells were more resistant to cytokine-induced apoptosis as compared with parental cells. The decrease in glucose-stimulated insulin secretion induced by IL-1β and IFN-γ was however not prevented. This was because these dysfunctions were induced by IFN-γ alone, which decreased cellular insulin content and stimulated insulin exocytosis. These results demonstrate that IL-1β is involved in inducible nitric oxide synthase gene expression and induction of apoptosis in mouse β cells but does not contribute to impaired glucose-stimulated insulin secretion. Furthermore, our data show that IL-1β cellular actions can be blocked by expression of MyD88 dominant negative proteins and, finally, that cytokine-induced β cell secretory dysfunctions are due to the action of IFN-γ.

GLUT2-null mice are hyperglycemic, hypoinsulinemic, hyperglucagonemic, and glycosuric and die within the first 3 weeks of life. Their endocrine pancreas shows a loss of first phase glucose-stimulated insulin secretion (GSIS) and inverse α to β cell ratio. Here we show that reexpression by transgenesis of either GLUT1 or GLUT2 in the pancreatic β cells of these mice allowed mouse survival and breeding. The rescued mice had normal-fed glycemia but fasted hypoglycemia, glycosuria, and an elevated glucagon to insulin ratio. Glucose tolerance was, however, normal. In vivo, insulin secretion assessed following hyperglycemic clamps was normal. In vitro, islet perifusion studies revealed that first phase of insulin secretion was restored as well by GLUT1 or GLUT2, and this was accompanied by normalization of the glucose utilization rate. The ratio of pancreatic insulin to glucagon and volume densities of α to β cells were, however, not corrected. These data demonstrate that 1) reexpression of GLUT1 or GLUT2 in β cells is sufficient to rescue GLUT2-null mice from lethality, 2) GLUT1 as well as GLUT2 can restore normal GSIS, 3) restoration of GSIS does not correct the abnormal composition of the endocrine pancreas. Thus, normal GSIS does not depend on transporter affinity but on the rate of uptake at stimulatory glucose concentrations.

We investigated the impact of GLUT2 gene inactivation on the regulation of hepatic glucose metabolism during the fed to fast transition. In control and GLUT2-null mice, fasting was accompanied by a ∼10-fold increase in plasma glucagon to insulin ratio, a similar activation of liver glycogen phosphorylase and inhibition of glycogen synthase and the same elevation in phosphoenolpyruvate carboxykinase and glucose-6-phosphatase mRNAs. In GLUT2-null mice, mobilization of glycogen stores was, however, strongly impaired. This was correlated with glucose-6-phosphate (G6P) levels, which remained at the fed values, indicating an important allosteric stimulation of glycogen synthase by G6P. These G6P levels were also accompanied by a paradoxical elevation of the mRNAs forl-pyruvate kinase. Re-expression of GLUT2 in liver corrected the abnormal regulation of glycogen andl-pyruvate kinase gene expression. Interestingly, GLUT2-null livers were hyperplasic, as revealed by a 40% increase in liver mass and 30% increase in liver DNA content. Together, these data indicate that in the absence of GLUT2, the G6P levels cannot decrease during a fasting period. This may be due to neosynthesized glucose entering the cytosol, being unable to diffuse into the extracellular space, and being phosphorylated back to G6P. Because hepatic glucose production is nevertheless quantitatively normal, glucose produced in the endoplasmic reticulum may also be exported out of the cell through an alternative, membrane traffic-based pathway, as previously reported (Guillam, M.-T., Burcelin, R., and Thorens, B. (1998) Proc. Natl. Acad. Sci. U. S. A.95, 12317–12321). Therefore, in fasting, GLUT2 is not required for quantitative normal glucose output but is necessary to equilibrate cytosolic glucose with the extracellular space. In the absence of this equilibration, the control of hepatic glucose metabolism by G6P is dominant over that by plasma hormone concentrations.

Based on homology with GLUT1–5, we have isolated a cDNA for a novel glucose transporter, GLUTX1. This cDNA encodes a protein of 478 amino acids that shows between 29 and 32% identity with rat GLUT1–5 and 32–36% identity with plant and bacterial hexose transporters. Unlike GLUT1–5, GLUTX1 has a short extracellular loop between transmembrane domain (TM) 1 and TM2 and a long extracellular loop between TM9 and TM10 that contains the onlyN-glycosylation site. When expressed in Xenopusoocytes, GLUTX1 showed strong transport activity only after suppression of a dileucine internalization motif present in the amino-terminal region. Transport activity was inhibited by cytochalasin B and partly competed by d-fructose and d-galactose. The Michaelis-Menten constant for glucose was approximately 2 mm. When translated in reticulocytes lysates, GLUTX1 migrates as a 35-kDa protein that becomes glycosylated in the presence of microsomal membranes. Western blot analysis of GLUTX1 transiently expressed in HEK293T cells revealed a diffuse band with a molecular mass of 37–50 kDa that could be converted to a ∼35-kDa polypeptide following enzymatic deglycosylation. Immunofluorescence microscopy detection of GLUTX1 transfected into HEK293T cells showed an intracellular staining. Mutation of the dileucine internalization motif induced expression of GLUTX1 at the cell surface. GLUTX1 mRNA was detected in testis, hypothalamus, cerebellum, brainstem, hippocampus, and adrenal gland. We hypothesize that, in a similar fashion to GLUT4,in vivocell surface expression of GLUTX1 may be inducible by a hormonal or other stimulus.

ABSTRACT: Non‐insulin‐dependent, or type II, diabetes mellitus is characterized by a progressive impairment of glucose‐induced insulin secretion by pancreatic β cells and by a relative decreased sensitivity of target tissues to the action of this hormone. About one third of type II diabetic patients are treated with oral hypoglycemic agents to stimulate insulin secretion. These drugs however risk inducing hypoglycemia and, over time, lose their efficacy. An alternative treatment is the use of glucagon‐like peptide‐1 (GLP‐1), a gut peptidic hormone with a strong insulinotropic activity. Its activity depends of the presence of normal blood glucose concentrations and therefore does not risk inducing hypoglycemia. GLP‐1 can correct hyperglycemia in diabetic patients, even in those no longer responding to hypoglycemic agents. Because it is a peptide, GLP‐1 must be administered by injection; this may prevent its wide therapeutic use. Here we propose to use cell lines genetically engineered to secrete a mutant form of GLP‐1 which has a longer half‐life in vivobut which is as potent as the wild‐type peptide. The genetically engineered cells are then encapsulated in semi‐permeable hollow fibers for implantation in diabetic hosts for constant, long‐term, in situdelivery of the peptide. This approach may be a novel therapy for type II diabetes.

Glucagon-like peptide-1(7–37) (GLP-1) is the most potent insulinotropic hormone characterized thus far. Because its activity is preserved in non-insulin-dependent diabetes mellitus (NIDDM) patients, it is considered a potential new drug for the treatment of this disease. One limitation in its therapeutic use is a short half-life in vivo (5 minutes), due in part to a fast degradation by the endoprotease dipeptidylpeptidase IV (DPPIV). Recently, it was reported that GLP-1 became resistant to DPPIV when the alanine residue at position 8 was replaced by a glycine (GLP-1-Gly8). We report here that this change slightly decreased the affinity of the peptide for its receptor (IC50, 0.41 ± 0.14 and 1.39 ± 0.61 nmol/L for GLP-1 and GLP-1-Gly8, respectively) but did not change the efficiency to stimulate accumulation of intracellular cyclic adenosine monophosphate (cAMP) (EC50, 0.25 ± 0.05 and 0.36 ± 0.06 nmol/L for GLP-1 and GLP-1-Gly8, respectively). Second, we demonstrate for the first time that this mutant has an improved insulinotropic activity compared with the wild-type peptide when tested in vivo in an animal model of diabetes. A single injection of 0.1 nmol GLP-1-Gly8 in diabetic mice fed a high-fat diet can correct fasting hyperglycemia and glucose intolerance for several hours, whereas the activity of 1 nmol GLP-1 vanishes a few minutes after injection. These actions were correlated with increased insulin and decreased glucagon levels. Interestingly, normoglycemia was maintained over a period that was longer than the predicted peptide half-life, suggesting a yet undescribed long-term effect of GLP-1-Gly8. GLP-1-Gly8 thus has a markedly improved therapeutic potential compared with GLP-1, since it can be used at much lower doses and with a more flexible schedule of administration.

We have investigated in vitro, the effects of glucagon-like peptide-1-(7-36) amide), oxyntomodulin and glucagon on two rabbit parietal cell-enriched fractions (F3, F3n), with parietal cell contents of 60% and 88%, respectively. Histamine (10 M) stimulated [ 14 C]aminopyrine accumulation to an amount of 850% in excess of the basal level, whereas GLP-1-(7-36) amide (10 M) and oxyntomodulin (10 M) induced increases of 50% and 30%, respectively. With a histamine concentration of 10 M, [ 14 C]aminopyrine accumulation was stimulated to 498% in excess of the basal level; GLP-1-(7-36) amide (10 M) and oxyntomodulin (10 M) induced increases of 50% and 15%, respectively. With these parameters, oxyntomodulin[19-37] and glucagon were without effect. Specific binding of [ 125 I]GLP-1-(7-36) amide to parietal cell plasma membranes was inhibited dose-dependently by GLP-1-(7-36) amide, oxyntomodulin and glucagon with inhibitory concentrations of 0.25 nM, 65 nM and 800 nM, respectively. No specific binding of [ 125 I]oxyntomodulin or [ 125 I]glucagon was detectable. GLP-1-(7-36) amide receptor mRNA was only detected in parietal cell-enriched fractions. GLP-1-(7-36) amide, oxyntomodulin and glucagon stimulated parietal cell cAMP production to similar maximal levels with median values close to 0.28 nM, 10.5 nM and 331.7 nM, whereas oxyntomodulin[19-37] had no effect. The maximal cAMP production induced by GLP-1-(7-36) amide, oxyntomodulin or glucagon was additive to that induced by histamine. Our data demonstrate the presence of a GLP-1-(7-36) amide receptor in rabbit parietal cell coupled to the cAMP pathway and the interaction of oxyntomodulin and glucagon with this receptor. The results argue in favor of a cAMP compartment coupled to the GLP-1-(7-36) amide receptor, different from that involved in histamine-induced [ 14 C]aminopyrine accumulation.

Glucokinase is an enzyme that catalyzes the transformation of glucose into glucose-6-phosphate in liver, pancreatic β cells, and other neuroendocrine cells present in the gut and central nervous system. Kinetically, it is characterized by a relatively high Kmfor glucose of about 6 to 8 mmol/L, ie, in the range of normal blood glucose concentration. In pancreatic β cells, this reaction is the rate-limiting step in glycolysis and is thereby a control point in the glucose-signaling pathway leading to insulin secretion. The importance of glucokinase in glucose homeostasis was further demonstrated by the discovery that maturity-onset diabetes of the young (MODY)-2, a form of non-insulin dependent diabetes mellitus with an autosomal dominant mode of inheritance, results from mutations in the gene for this enzyme. Recent biochemical studies of mutant forms of glucokinase have provided information on the amino acid residues involved in the catalytic activity and allosteric regulation of this enzyme. Transgenic mice with tissue-specific reduction in glucokinase expression have revealed that MODY-2 can be induced by reduction of glucokinase activity solely in pancreatic β cells, but that overt non-insulin dependent diabetes mellitus requires the combined impairment of both insulin secretion and insulin action. Of interest, glucokinase is also expressed in pancreatic a cells and specific hypothalamic neurons. Therefore, by controlling the glucose-dependent activity of many different neuroendocrine cells, glucokinase may have a broader physiologic role than originally thought.

Here we evaluated the effect of leptin on glucose-induced insulin secretion by normal rat pancreatic islets. We show in perifusion experiments that leptin had no acute effect on the secretory activity of β-cells. However, following preexposure to leptin a pronounced time- and dose-dependent inhibition of both first and second phases of secretion was observed. Maximum inhibition was obtained at 24 h and with 100 nM leptin. This inhibition did not involve a decrease in cellular insulin content. It was also not observed with islets from fa/fa rats. Leptin thus inhibits insulin secretion by a mechanism which requires long-term preexposure to the hormone and which may involve alteration in β-cell gene expression.

Glut-2 is a low-affinity transporter present in the plasma membrane of pancreatic (β-cells, hepatocytes and intestine and kidney absorptive epithelial cells of mice1. In β-cells, Glut-2 has been proposed to be active in the control of glucose-stimulated insulin secretion (GSIS; ref. 2), and its expression is strongly reduced in glucose-unresponsive islets from different animal models of diabetes2–4. However, recent investigations have yielded conflicting data on the possible role of Glut-2 in GSIS. Whereas some reports have supported a specific role for Glut-2 (refs 5,6), others have suggested that GSIS could proceed normally even in the presence of low7or almost undetectable8levels of this transporter. Here we show that homozygous, but not heterozygous, mice deficient in Glut-2 are hyperglycaemic and relatively hypo-insulinaemic and have elevated plasma levels of glucagon, free fatty acids and β-hydroxybutyrate. In vivo, their glucose tolerance is abnormal. In vitro, (β-cells display loss of control of insulin gene expression by glucose and impaired GSIS with a loss of first phase but preserved second phase of secretion, while the secretory response to non-glucidic nutrients or to D-glyceraldehyde is normal. This is accompanied by alterations in the postnatal development of pancreatic islets, evidenced by an inversion of the α- to (β-cell ratio. Glut-2 is thus required to maintain normal glucose homeostasis and normal function and development of the endocrine pancreas. Its absence leads to symptoms characteristic of non-insulin-dependent diabetes mellitus.

Two general mechanisms mediate glucose transport, one is a sodium-coupled glucose transporter found in the apical border of intestinal and kidney epithelia, while the other is a sodium-independent transport system. Of the latter, several facilitated transporters have been identified, including GLUTl (erythrocyte/brain), GLUT2 (liver) and GLUT4 (adipose/muscle) isoforms. In this study, we used Western-blot analysis and high resolution immunoelectron microscopy (IEM) to investigate the stage-related expression and cellular localization of GLUTl, 2 and 4. The Western blot results demonstrate that GLUTl is detectable in the oocyte and throughout preimplantation development. GLUT2 isoforms were not detectable until the blastocyst stage, while the GLUT4 isoform was undetectable in the oocyte through blastocyst stages. The present findings confirm previous studies at the molecular level which demon-strated that mRNAs encoding the same GLUT isoforms are detectable at corresponding developmental stages. GLUTl and GLUT2 display different cellular distributions at the blastocyst stage as shown by IEM studies. GLUTl has a widespread distribution in both trophecto-derm and inner cell mass cells, while GLUT2 is located on trophectoderm membranes facing the blastocyst cavity. This observation suggests a different functional significance for these isoforms during mouse dereimplanttation development.

The glucose transporter (GLUT) isoforms responsible for glucose uptake in early mouse embryos have been identified. GLUT 1, the isoform present in nearly every tissue examined including adult brain and erythrocytes, is expressed throughout preimplantation development. GLUT 2, which is normally present in adult liver, kidney, intestine and pancreatic β cells is expressed from the 8-cell stage onward. GLUT 4, an insulin-recruitable isoform, which is expressed in adult fat and muscle, is not expressed at any stage of preimplantation develop- ment or in early postimplantation stage embryos. Genetic mapping studies of glucose transporters in the mouse show that Glut-1 is located on chromosome 4, Glut-2 on chromosome 3, Glut-3 on chromosome 6, and Glut-4 on chromosome 11.

Glucagon-like peptide-1 stimulates glucose-induced insulin secretion by binding to a specific G protein-coupled receptor that activates the adenylyl cyclase pathway. We previously demonstrated that heterologous desensitization of the receptor by protein kinase C correlated with phosphorylation in a 33-amino acid-long segment of the receptor carboxyl-terminal cytoplasmic tail. Here, we determined that the in vivosites of phosphorylation are four serine doublets present at positions 431/432, 441/442, 444/445, and 451/452. In vitrophosphorylation of fusion proteins containing mutant receptor C-tails, however, indicated that whereas serines at position 431/432 were good substrates for protein kinase C (PKC), serines 444/445 and 451/452 were poor substrates, and serines 441/442 were not substrates. In addition, serine 416 was phosphorylated on fusion protein but not in intact cells. This indicated that in vivoa different PKC isoform or a PKC-activated kinase may phosphorylate the receptor. The role of phosphorylation on receptor desensitization was assessed using receptor mutants expressed in COS cells or Chinese hamster lung fibroblasts. Mutation of any single serine doublet to alanines reduced the extent of phorbol 12-myristate 13-acetate-induced desensitization, whereas substitution of any combination of two serine doublets suppressed it. Our data thus show that the glucagon-like peptide-1 receptor can be phosphorylated in response to phorbol 12-myristate 13-acetate on four different sites within the cytoplasmic tail. Furthermore, phosphorylation of at least three sites was required for desensitization, although maximal desensitization was only achieved when all four sites were phosphorylated.

GLUT2 expression is strongly decreased in glucose-unresponsive pancreatic β cells of diabetic rodents. This decreased expression is due to circulating factors distinct from insulin or glucose. Here we evaluated the effect of palmitic acid and the synthetic glucocorticoid dexamethasone on GLUT2 expression by in vitrocultured rat pancreatic islets. Palmitic acid induced a 40% decrease in GLUT2 mRNA levels with, however, no consistent effect on protein expression. Dexamethasone, in contrast, had no effect on GLUT2 mRNA, but decreased GLUT2 protein by about 65%. The effect of dexamethasone was more pronounced at high glucose concentrations and was inhibited by the glucocorticoid antagonist RU-486. Biosynthetic labeling experiments revealed that GLUT2 translation rate was only minimally affected by dexamethasone, but that its half-life was decreased by 50%, indicating that glucocorticoids activated a posttranslational degradation mechanism. This degradation mechanism was not affecting all membrane proteins, since the α subunit of the Na+/K+-ATPase was unaffected. Glucose-induced insulin secretion was strongly decreased by treatment with palmitic acid and/or dexamethasone. The insulin content was decreased (∼55 percent) in the presence of palmitic acid, but increased (∼180%) in the presence of dexamethasone. We conclude that a combination of elevated fatty acids and glucocorticoids can induce two common features observed in diabetic β cells, decreased GLUT2 expression, and loss of glucose-induced insulin secretion.

In adipocytes and muscle cells, the GLUT4 glucose trans-porter isoform is present in intracellular vesicles which continuously recycle between an intracytoplasmic location and the plasma membrane. It is not clear whether the GLUT4-vesicles represent a specific kind of vesicle or resemble typical secretory granules or synaptic-like microvesicles. To approach this question, we expressed GLUT4 in the β cell line RINm5F and determined its intra-cellular localization by subcellular fractionation and by immunofluorescence and immunoelectron microscopy. GLUT4 was not found in insulin granules but was associated with a subpopulation of smooth-surface vesicles present in the trans-Golgi region and in vesicular structures adjacent to the plasma membrane. In the trans-Golgi region, GLUT4 did not colocalize with synaptophysin or TGN38. Incubation of the cells with horseradish peroxidase (HRP) led to colocalization of HRP and GLUT4 in some endosomal structures adjacent to the plasma membrane and in occasional trans-Golgi region vesicles. When cells were incubated in the presence of Bafilomycin A, analysis by confocal microscopy revealed GLUT4 in numerous large spots present throughout the cytoplasm, many of which costained for TGN38 and synaptophysin. By immunoelectron microscopy, numerous endosomes were observed which stained strongly for GLUT4. Together our data demonstrate that ectopic expression of GLUT4 in insulinoma cells reveals the presence of a subset of vesicular structures distinct from synaptic-like vesicles and insulin secretory granules. Furthermore, they indicate that GLUT4 constitutively recycles between the plasma membrane and its intracellular location by an endocytic route also taken by TGN38 and synaptophysin.

Glucose exerts inverse effects upon the secretory function of islet α- and β-cells, suppressing glucagon release and increasing insulin release. This diverse action may result from differences in glucose transport and metabolism between the two cell types. The present study compares glucose transport in rat α- and β-cells. β-Cells transcribed GLUT2 and, to a lesser extent, GLUT 1; α-cells contained GLUT1 but no GLUT2 mRNA. No other GLUT-like sequences were found among cDNAs from α- or β-cells. Both cell types expressed 43-kDa GLUT1 protein which was enhanced by culture. The 62-kDa β-cell GLUT2 protein was converted to a 58-kDa protein after trypsin treatment of the cells without detectable consequences upon glucose transport kinetics. In β-cells, the rates of glucose transport were 10-fold higher than in α-cells. In both cell types, glucose uptake exceeded the rates of glucose utilization by a factor of 10 or more. Glycolytic flux, measured as D-[53H]glucose utilization, was comparable in α- and β-cells between 1 and 10 mmol/liter substrate. In conclusion, differences in glucose transporter gene expression between α- and β-cells can be correlated with differences in glucose transport kinetics but not with different glucose utilization rates.

Here we evaluated the effect of leptin on glucose‐induced insulin secretion by normal rat pancreatic islets. We show in perifusion experiments that leptin had no acute effect on the secretory activity of β‐cells. However, following preexposure to leptin a pronounced time‐ and dose‐dependent inhibition of both first and second phases of secretion was observed. Maximum inhibition was obtained at 24 h and with 100 nM leptin. This inhibition did not involve a decrease in cellular insulin content. It was also not observed with islets from fa/fa rats. Leptin thus inhibits insulin secretion by a mechanism which requires long‐term preexposure to the hormone and which may involve alteration in β‐cell gene expression.