The rate-limiting step for glucose transport into the hypothalamus is across the blood-hypothalamus interface.

Specialized glucosensing neurons are present in the hypothalamus, some of which neighbor the median eminence, where the blood-brain barrier has been reported leaky. A leaky blood-brain barrier implies high tissue glucose levels and obviates a role for endothelial glucose transporters in the control of hypothalamic glucose concentration, important in understanding the mechanisms of glucose sensing We therefore addressed the question of blood-brain barrier integrity at the hypothalamus for glucose transport by examining the brain tissue-to-plasma glucose ratio in the hypothalamus relative to other brain regions. We also examined glycogenolysis in hypothalamus because its occurrence is unlikely in the potential absence of a hypothalamus-blood interface. Across all regions the concentration of glucose was comparable at a given plasma glucose concentration and was a near linear function of plasma glucose. At steady-state, hypothalamic glucose concentration was similar to the extracellular hypothalamic glucose concentration reported by others. Hypothalamic glycogen fell at a rate of approximately 1.5 micromol/g/h and remained present in substantial amounts. We conclude for the hypothalamus, a putative primary site of brain glucose sensing that: the rate-limiting step for glucose transport into brain cells is at the blood-hypothalamus interface, and that glycogenolysis is consistent with a substantial blood -to- intracellular glucose concentration gradient.

The role of SGLT1 and GLUT2 in intestinal glucose transport and sensing.

Intestinal glucose absorption is mediated by SGLT1 whereas GLUT2 is considered to provide basolateral exit. Recently, it was proposed that GLUT2 can be recruited into the apical membrane after a high luminal glucose bolus allowing bulk absorption of glucose by facilitated diffusion. Moreover, SGLT1 and GLUT2 are suggested to play an important role in intestinal glucose sensing and incretin secretion. In mice that lack either SGLT1 or GLUT2 we re-assessed the role of these transporters in intestinal glucose uptake after radiotracer glucose gavage and performed Western blot analysis for transporter abundance in apical membrane fractions in a comparative approach. Moreover, we examined the contribution of these transporters to glucose-induced changes in plasma GIP, GLP-1 and insulin levels. In mice lacking SGLT1, tissue retention of tracer glucose was drastically reduced throughout the entire small intestine whereas GLUT2-deficient animals exhibited higher tracer contents in tissue samples than wild type animals. Deletion of SGLT1 resulted also in reduced blood glucose elevations and abolished GIP and GLP-1 secretion in response to glucose. In mice lacking GLUT2, glucose-induced insulin but not incretin secretion was impaired. Western blot analysis revealed unchanged protein levels of SGLT1 after glucose gavage. GLUT2 detected in apical membrane fractions mainly resulted from contamination with basolateral membranes but did not change in density after glucose administration. SGLT1 is unequivocally the prime intestinal glucose transporter even at high luminal glucose concentrations. Moreover, SGLT1 mediates glucose-induced incretin secretion. Our studies do not provide evidence for GLUT2 playing any role in either apical glucose influx or incretin secretion.

Glucose transporters in the regulation of intestinal, renal, and liver glucose fluxes.

Five functional mammalian facilitated hexose carriers (GLUTs) have been characterized by molecular cloning. By functional expression in heterologous systems, their specificity and affinity for different hexoses have been defined. There are three high-affinity transporters (GLUT-1, GLUT-3 and GLUT-4) and one low-affinity transporter (GLUT-2), and GLUT-5 is primarily a fructose carrier. Because their Michaelis constants (Km) are below the normal blood glucose concentration, the high-affinity transporters function at rates close to maximal velocity. Thus their level of cell surface expression greatly influences the rate of glucose uptake into the cells. In contrast, the rate of glucose uptake by GLUT-2 (Km = 17 mM) increases in parallel with the rise in blood glucose over the physiological concentration range. High-affinity transporters are found in almost every tissue, but their expression is higher in cells with high glycolytic activity. Glut-2, however, is found in tissues carrying large glucose fluxes, such as intestine, kidney, and liver. As an adaptive response to variations in metabolic conditions, the expression of these transporters is regulated by glucose and different hormones. Thus, because of their specific characteristics and regulated expression, the facilitated glucose transporters control fundamental aspects of glucose homeostasis. I review data pertaining to the structure and regulated expression of the glucose carriers present in intestine, kidney, and liver and discuss their role in the control of glucose flux into or out of these different tissues.

Requirement of glucose metabolism for regulation of glucose transporter type 2 (GLUT2) gene expression in liver.

Previous studies have shown that glucose increases the glucose transporter (GLUT2) mRNA expression in the liver in vivo and in vitro. Here we report an analysis of the effects of glucose metabolism on GLUT2 gene expression. GLUT2 mRNA accumulation by glucose was not due to stabilization of its transcript but rather was a direct effect on gene transcription. A proximal fragment of the 5' regulatory region of the mouse GLUT2 gene linked to a reporter gene was transiently transfected into liver GLUT2-expressing cells. Glucose stimulated reporter gene expression in these cells, suggesting that glucose-responsive elements were included within the proximal region of the promoter. A dose-dependent effect of glucose on GLUT2 expression was observed over 10 mM glucose irrespective of the hexokinase isozyme (glucokinase K(m) 16 mM; hexokinase I K(m) 0.01 mM) present in the cell type used. This suggests that the correlation between extracellular glucose and GLUT2 mRNA concentrations is simply a reflection of an activation of glucose metabolism. The mediators and the mechanism responsible for this response remain to be determined. In conclusion, glucose metabolism is required for the proper induction of the GLUT2 gene in the liver and this effect is transcriptionally regulated.

Deep thiopental anesthesia alters steady-state glucose homeostasis but not the neurochemical profile of rat cortex.

Barbiturates are regularly used as an anesthetic for animal experimentation and clinical procedures and are frequently provided with solubilizing compounds, such as ethanol and propylene glycol, which have been reported to affect brain function and, in the case of (1)H NMR experiments, originate undesired resonances in spectra affecting the quantification. As an alternative, thiopental can be administrated without any solubilizing agents. The aim of the study was to investigate the effect of deep thiopental anesthesia on the neurochemical profile consisting of 19 metabolites and on glucose transport kinetics in vivo in rat cortex compared with alpha-chloralose using localized (1)H NMR spectroscopy. Thiopental was devoid of effects on the neurochemical profile, except for the elevated glucose at a given plasma glucose level resulting from thiopental-induced depression of glucose consumption at isoelectrical condition. Over the entire range of plasma glucose levels, steady-state glucose concentrations were increased on average by 48% +/- 8%, implying that an effect of deep thiopental anesthesia on the transport rate relative to cerebral glucose consumption ratio was increased by 47% +/- 8% compared with light alpha-chloralose-anesthetized rats. We conclude that the thiopental-induced isoelectrical condition in rat cortex significantly affected glucose contents by depressing brain metabolism, which remained substantial at isoelectricity.

Regulation of glucagon secretion by glucose transporter type 2 (glut2) and astrocyte-dependent glucose sensors.

Ripglut1;glut2-/- mice have no endogenous glucose transporter type 2 (glut2) gene expression but rescue glucose-regulated insulin secretion. Control of glucagon plasma levels is, however, abnormal, with fed hyperglucagonemia and insensitivity to physiological hypo- or hyperglycemia, indicating that GLUT2-dependent sensors control glucagon secretion. Here, we evaluated whether these sensors were located centrally and whether GLUT2 was expressed in glial cells or in neurons. We showed that ripglut1;glut2-/- mice failed to increase plasma glucagon levels following glucoprivation induced either by i.p. or intracerebroventricular 2-deoxy-D-glucose injections. This was accompanied by failure of 2-deoxy-D-glucose injections to activate c-Fos-like immunoreactivity in the nucleus of the tractus solitarius and the dorsal motor nucleus of the vagus. When glut2 was expressed by transgenesis in glial cells but not in neurons of ripglut1;glut2-/- mice, stimulated glucagon secretion was restored as was c-Fos-like immunoreactive labeling in the brainstem. When ripglut1;glut2-/- mice were backcrossed into the C57BL/6 genetic background, fed plasma glucagon levels were also elevated due to abnormal autonomic input to the alpha cells; glucagon secretion was, however, stimulated by hypoglycemic stimuli to levels similar to those in control mice. These studies identify the existence of central glucose sensors requiring glut2 expression in glial cells and therefore functional coupling between glial cells and neurons. These sensors may be activated at different glycemic levels depending on the genetic background.

Expression of an uncleavable N-terminal RasGAP fragment in insulin-secreting cells increases their resistance toward apoptotic stimuli without affecting their glucose-induced insulin secretion.

Apoptosis of pancreatic beta cells is implicated in the onset of type 1 and type 2 diabetes. Consequently, strategies aimed at increasing the resistance of beta cells toward apoptosis could be beneficial in the treatment of diabetes. RasGAP, a regulator of Ras and Rho GTPases, is an atypical caspase substrate, since it inhibits, rather than favors, apoptosis when it is partially cleaved by caspase-3 at position 455. The antiapoptotic signal generated by the partial processing of RasGAP is mediated by the N-terminal fragment (fragment N) in a Ras-phosphatidylinositol 3-kinase-Akt-dependent, but NF-kappaB-independent, manner. Further cleavage of fragment N at position 157 abrogates its antiapoptotic properties. Here we demonstrate that an uncleavable form of fragment N activates Akt, represses NF-kappaB activity, and protects the conditionally immortalized pancreatic insulinoma betaTC-tet cell line against various insults, including exposure to genotoxins, trophic support withdrawal, and incubation with inflammatory cytokines. Fragment N also induced Akt activity and protection against cytokine-induced apoptosis in primary pancreatic islet cells. Fragment N did not alter insulin cell content and insulin secretion in response to glucose. These data indicate that fragment N protects beta cells without affecting their function. The pathways regulated by fragment N are therefore promising targets for antidiabetogenic therapy.

Futile cycling of intermediates of fatty acid biosynthesis toward peroxisomal beta-oxidation in Saccharomyces cerevisiae.

The flux of fatty acids toward beta-oxidation was analyzed in Saccharomyces cerevisiae by monitoring polyhydroxyalkanoate synthesis in the peroxisome from the polymerization, by a bacterial polyhydroxyalkanoate synthase, of the beta-oxidation intermediates 3-hydroxyacyl-CoAs. Synthesis of polyhydroxyalkanoate was dependent on the beta-oxidation enzymes acyl-CoA oxidase and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase multifunctional protein, which are involved in generating 3-hydroxyacyl-CoAs, and on the peroxin PEX5, which is involved in the import of proteins into the peroxisome. In wild type cells grown in media containing fatty acids, the polyhydroxyalkanoate monomer composition was largely influenced by the nature of the external fatty acid, such that even-chain monomers are generated from oleic acid and odd-chain monomers are generated from heptadecenoic acid. In contrast, polyhydroxyalkanoate containing predominantly 3-hydroxyoctanoate, 3-hydroxydecanoate, and 3-hydroxydodecanoate was synthesized in a mutant deficient in the peroxisomal 3-ketothiolase (fox3 Delta 0) growing either on oleic acid or heptadecenoic acid as well as in wild type and fox3 Delta 0 mutants grown on glucose or raffinose, indicating that 3-hydroxyacyl-CoAs used for polyhydroxyalkanoate synthesis were generated from the degradation of intracellular short- and medium-chain fatty acids by the beta-oxidation cycle. Inhibition of fatty acid biosynthesis with cerulenin blocked the synthesis of polyhydroxyalkanoate from intracellular fatty acids but still enabled the use of extracellular fatty acids for polymer production. Mutants affected in the synthesis of lipoic acid showed normal polyhydroxyalkanoate synthesis capacity. Together, these results uncovered the existence of a substantial futile cycle whereby short- and medium-chain intermediates of the cytoplasmic fatty acid biosynthetic pathway are directed toward the peroxisomal beta-oxidation pathway.

Phosphatidyl inositol 3-kinase signaling in hypothalamic proopiomelanocortin neurons contributes to the regulation of glucose homeostasis.

Recent studies demonstrated a role for hypothalamic insulin and leptin action in the regulation of glucose homeostasis. This regulation involves proopiomelanocortin (POMC) neurons because suppression of phosphatidyl inositol 3-kinase (PI3K) signaling in these neurons blunts the acute effects of insulin and leptin on POMC neuronal activity. In the current study, we investigated whether disruption of PI3K signaling in POMC neurons alters normal glucose homeostasis using mouse models designed to both increase and decrease PI3K-mediated signaling in these neurons. We found that deleting p85alpha alone induced resistance to diet-induced obesity. In contrast, deletion of the p110alpha catalytic subunit of PI3K led to increased weight gain and adipose tissue along with reduced energy expenditure. Independent of these effects, increased PI3K activity in POMC neurons improved insulin sensitivity, whereas decreased PI3K signaling resulted in impaired glucose regulation. These studies show that activity of the PI3K pathway in POMC neurons is involved in not only normal energy regulation but also glucose homeostasis.

Glomerular hyperfiltration and increased proximal sodium reabsorption in subjects with type 2 diabetes or impaired fasting glucose in a population of the African region.

BACKGROUND. Glomerular hyperfiltration (GHF) is a well-recognized early renal alteration in diabetic patients. As the prevalence of GHF is largely unknown in populations in the African region with respect to normal fasting glucose (NFG), impaired fasting glucose (IFG) and type 2 diabetes [diabetes mellitus (DM)], we conducted a cross-sectional study in the Seychelles islands among families including at least one member with hypertension. METHODS. The glomerular filtration rate (GFR), effective renal plasma flow (ERPF) and proximal tubular sodium reabsorption were measured using inulin, p-aminohippurate (PAH) and endogenous lithium clearance, respectively. Twenty-four-hour urine was collected on the preceding day. RESULTS. Of the 363 participants (mean age 44.7 years), 6.6% had IFG, 9.9% had DM and 63.3% had hypertension. The prevalence of GHF, defined as a GFR >140 ml/min, was 17.2%, 29.2% and 52.8% in NFG, IFG and DM, respectively (P trend <0.001). Compared to NFG, the adjusted odds ratio for GHF was 1.99 [95% confidence interval (CI) 0.73-5.44] for IFG and 5.88 (2.39-14.45) for DM. Lithium clearance and fractional excretion of lithium were lower in DM and IFG than NFG (P < 0.001). CONCLUSION. In this population of African descent, subjects with impaired fasting glucose or type 2 diabetes had a high prevalence of GHF and enhanced proximal sodium reabsorption. These findings provide further insight on the elevated incidence of nephropathy reported among African diabetic individuals.

Evidence that extrapancreatic GLUT2-dependent glucose sensors control glucagon secretion.

GLUT2-/- mice reexpressing GLUT1 or GLUT2 in their beta-cells (RIPGLUT1 x GLUT2-/- or RIPGLUT2 x GLUT2-/- mice) have nearly normal glucose-stimulated insulin secretion but show high glucagonemia in the fed state. Because this suggested impaired control of glucagon secretion, we set out to directly evaluate the control of glucagonemia by variations in blood glucose concentrations. Using fasted RIPGLUT1 x GLUT2-/- mice, we showed that glucagonemia was no longer increased by hypoglycemic (2.5 mmol/l glucose) clamps or suppressed by hyperglycemic (10 and 20 mmol/l glucose) clamps. However, an increase in plasma glucagon levels was detected when glycemia was decreased to &lt; or =1 mmol/l, indicating preserved glucagon secretory ability, but of reduced sensitivity to glucopenia. To evaluate whether the high-fed glucagonemia could be due to an abnormally increased tone of the autonomic nervous system, fed mutant mice were injected with the ganglionic blockers hexamethonium and chlorisondamine. Both drugs lead to a rapid return of glucagonemia to the levels found in control fed mice. We conclude that 1) in the absence of GLUT2, there is an impaired control of glucagon secretion by low or high glucose; 2) this impaired glucagon secretory activity cannot be due to absence of GLUT2 from alpha-cells because these cells do not normally express this transporter; 3) this dysregulation may be due to inactivation of GLUT2-dependent glucose sensors located outside the endocrine pancreas and controlling glucagon secretion; and 4) because fed hyperglucagonemia is rapidly reversed by ganglionic blockers, this suggests that in the absence of GLUT2, there is an increased activity of the autonomic nervous system stimulating glucagon secretion during the fed state.

Glucose sensing and the pathogenesis of obesity and type 2 diabetes.

The control of body weight and of blood glucose concentrations depends on the exquisite coordination of the function of several organs and tissues, in particular the liver, muscle and fat. These organs and tissues have major roles in the use and storage of nutrients in the form of glycogen or triglycerides and in the release of glucose or free fatty acids into the blood, in periods of metabolic needs. These mechanisms are tightly regulated by hormonal and nervous signals, which are generated by specialized cells that detect variations in blood glucose or lipid concentrations. The hormones insulin and glucagon not only regulate glycemic levels through their action on these organs and the sympathetic and parasympathetic branches of the autonomic nervous system, which are activated by glucose or lipid sensors, but also modulate pancreatic hormone secretion and liver, muscle and fat glucose and lipid metabolism. Other signaling molecules, such as the adipocyte hormones leptin and adiponectin, have circulating plasma concentrations that reflect the level of fat stored in adipocytes. These signals are integrated at the level of the hypothalamus by the melanocortin pathway, which produces orexigenic and anorexigenic neuropeptides to control feeding behavior, energy expenditure and glucose homeostasis. Work from several laboratories, including ours, has explored the physiological role of glucose as a signal that regulates these homeostatic processes and has tested the hypothesis that the mechanism of glucose sensing that controls insulin secretion by the pancreatic beta-cells is also used by other cell types. I discuss here evidence for these mechanisms, how they integrate signals from other nutrients such as lipids and how their deregulation may initiate metabolic diseases.

Macrophage migration inhibitory factor deficiency leads to age-dependent impairment of glucose homeostasis in mice.

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine produced by many cells and tissues including pancreatic beta-cells, liver, skeletal muscle, and adipocytes. This study investigates the potential role of MIF in carbohydrate homeostasis in a physiological setting outside of severe inflammation, utilizing Mif knockout (MIF-/-) mice. Compared with wild-type (WT) mice, MIF-/- mice had a lower body weight, from birth until 4 months of age, but subsequently gained weight faster, resulting in a higher body weight at 12 months of age. The lower weight in young mice was related to a higher energy expenditure, and the higher weight in older mice was related to an increased food intake and a higher fat mass. Fasting blood insulin level was higher in MIF-/- mice compared with WT mice at any age. After i.p. glucose injection, the elevation of blood insulin level was higher in MIF-/- mice compared with WT mice, at 2 months of age, but was lower in 12-month-old MIF-/- mice. As a result, the glucose clearance during intraperitoneal glucose tolerance tests was higher in MIF-/- mice compared with WT mice until 4 months of age, and was lower in 12-month-old MIF-/- mice. Insulin resistance was estimated (euglycemic-hyperinsulinemic clamp tests), and the phosphorylation activity of AKT was similar in MIF-/- mice and WT mice. In conclusion, this mouse model provides evidence for the role of MIF in the control of glucose homeostasis.

Fluoxetine regulates the expression of neurotrophic/growth factors and glucose metabolism in astrocytes.

Rationale The pharmacological actions of most antidepressants are ascribed to the modulation of serotonergic and/or noradrenergic transmission in the brain. During therapeutic treatment for major depression, fluoxetine, one of the most commonly prescribed selective serotonin reuptake inhibitor (SSRI) antidepressants, accumulates in the brain, suggesting that fluoxetine may interact with additional targets. In this context, there is increasing evidence that astrocytes are involved in the pathophysiology of major depression.Objectives The aim of this study was to examine the effects of fluoxetine on the expression of neurotrophic/growth factors that have antidepressant properties and on glucose metabolism in cultured cortical astrocytes.Results Treatment of astrocytes with fluoxetine and paroxetine, another SSRI antidepressant, upregulated brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), and VGF mRNA expression. In contrast, the tricyclic antidepressants desipramine and imipramine did not affect the expression of these neurotrophic/growth factors. Analysis of the effects of fluoxetine on glucose metabolism revealed that fluoxetine reduces glycogen levels and increases glucose utilization and lactate release by astrocytes. Similar data were obtained with paroxetine, whereas imipramine and desipramine did not regulate glucose metabolism in this glial cell population. Our results also indicate that the effects of fluoxetine and paroxetine on glucose utilization, lactate release, and expression of BDNF, VEGF, and VGF are not mediated by serotonin-dependent mechanisms.Conclusions These data suggest that, by increasing the expression of specific astrocyte-derived neurotrophic factors and lactate release from astrocytes, fluoxetine may contribute to normalize the trophic and metabolic support to neurons in major depression.