Localization and regulation of GLUTx1 glucose transporter in the hippocampus of streptozotocin diabetic rats

We describe the localization of the recently identified glucose transporter GLUTx1 and the regulation of GLUTx1 in the hippocampus of diabetic and control rats. GLUTx1 mRNA and protein exhibit a unique distribution when compared with other glucose transporter isoforms expressed in the rat hippocampus. In particular, GLUTx1 mRNA was detected in hippocampal pyramidal neurons and granule neurons of the dentate gyrus as well as in nonprincipal neurons. With immunohistochemistry, GLUTx1 protein expression is limited to neuronal cell bodies and the most proximal dendrites, unlike GLUT3 expression that is observed throughout the neuropil. Immunoblot analysis of hippocampal membrane fractions revealed that GLUTx1 protein expression is primarily localized to the intracellular compartment and exhibits limited association with the plasma membrane. In streptozotocin diabetic rats compared with vehicle-treated controls, quantitative autoradiography showed increased GLUTx1 mRNA levels in pyramidal neurons and granule neurons; up-regulation of GLUTx1 mRNA also was found in nonprincipal cells, as shown by single-cell emulsion autoradiography. In contrast, diabetic and control rats expressed similar levels of hippocampal GLUTx1 protein. These results indicate that GLUTx1 mRNA and protein have a unique expression pattern in rat hippocampus and suggest that streptozotocin diabetes increases steady-state mRNA levels in the absence of concomitant increases in GLUTx1 protein expression.

Combined transcriptome and proteome analysis as a powerful approach to study genes under glucose repression in Bacillus subtilis

We used 2D protein gel electrophoresis and DNA microarray technologies to systematically analyze genes under glucose repression in Bacillus subtilis. In particular, we focused on genes expressed after the shift from glycolytic to gluconeogenic at the middle logarithmic phase of growth in a nutrient sporulation medium, which remained repressed by the addition of glucose. We also examined whether or not glucose repression of these genes was mediated by CcpA, the catabolite control protein of this bacterium. The wild-type and ccpA1 cells were grown with and without glucose, and their proteomes and transcriptomes were compared. 2D gel electrophoresis allowed us to identify 11 proteins, the synthesis of which was under glucose repression. Of these proteins, the synthesis of four (IolA, I, S and PckA) was under CcpA-independent control. Microarray analysis enabled us to detect 66 glucose-repressive genes, 22 of which (glmS, acoA, C, yisS, speD, gapB, pckA, yvdR, yxeF, iolA, B, C, D, E, F, G, H, I, J, R, S and yxbF ) were at least partially under CcpA-independent control. Furthermore, we found that CcpA and IolR, a repressor of the iol divergon, were involved in the glucose repression of the synthesis of inositol dehydrogenase encoded by iolG included in the above list. The CcpA-independent glucose repression of the iol genes appeared to be explained by inducer exclusion.

Restoration of insulin-sensitive glucose transporter (GLUT4) gene expression in muscle cells by the transcriptional coactivator PGC-1

Muscle tissue is the major site for insulin-stimulated glucose uptake in vivo, due primarily to the recruitment of the insulin-sensitive glucose transporter (GLUT4) to the plasma membrane. Surprisingly, virtually all cultured muscle cells express little or no GLUT4. We show here that adenovirus-mediated expression of the transcriptional coactivator PGC-1, which is expressed in muscle in vivo but is also deficient in cultured muscle cells, causes the total restoration of GLUT4 mRNA levels to those observed in vivo. This increased GLUT4 expression correlates with a 3-fold increase in glucose transport, although much of this protein is transported to the plasma membrane even in the absence of insulin. PGC-1 mediates this increased GLUT4 expression, in large part, by binding to and coactivating the muscle-selective transcription factor MEF2C. These data indicate that PGC-1 is a coactivator of MEF2C and can control the level of endogenous GLUT4 gene expression in muscle.

Carbohydrate and Amino Acid Metabolism in the Eucalyptus globulus-Pisolithus tinctorius Ectomycorrhiza during Glucose Utilization1

The metabolism of [1-13C]glucose in Pisolithus tinctorius cv Coker & Couch, in uninoculated seedlings of Eucalyptus globulus bicostata ex Maiden cv Kirkp., and in the E. globulus-P. tinctorius ectomycorrhiza was studied using nuclear magnetic resonance spectroscopy. In roots of uninoculated seedlings, the 13C label was mainly incorporated into sucrose and glutamine. The ratio (13C3 + 13C2)/13C4 of glutamine was approximately 1.0 during the time-course experiment, indicating equivalent contributions of phosphoenolpyruvate carboxylase and pyruvate dehydrogenase to the production of α-ketoglutarate used for synthesis of this amino acid. In free-living P. tinctorius, most of the 13C label was incorporated into mannitol, trehalose, glutamine, and alanine, whereas arabitol, erythritol, and glutamate were weakly labeled. Amino acid biosynthesis was an important sink of assimilated 13C (43%), and anaplerotic CO2 fixation contributed 42% of the C flux entering the Krebs cycle. In ectomycorrhizae, sucrose accumulation was decreased in the colonized roots compared with uninoculated control plants, whereas 13C incorporation into arabitol and erythritol was nearly 4-fold higher in the symbiotic mycelium than in the free-living fungus. It appears that fungal utilization of glucose in the symbiotic state is altered and oriented toward the synthesis of short-chain polyols.

31P NMR magnetization transfer study of the control of ATP turnover in Saccharomyces cerevisiae.

31P NMR magnetization transfer measurements have been used to measure the steady state flux between Pi and ATP in yeast cells genetically modified to overexpress an adenine nucleotide translocase isoform. An increase in Pi -> ATP flux and apparent ratio of moles of ATP synthesized/atoms of oxygen consumed (P:O ratio), when these cells were incubated with glucose, demonstrated that the reactions catalyzed by the translocase and F1F0 ATP synthase were readily reversible in vivo. However, when the same cells were incubated with ethanol alone, translocase overexpression had no effect on the measured Pi -> ATP flux or apparent P:O ratio, suggesting that the synthase was now operating irreversibly. This change was accompanied by an increase in the intracellular ADP concentration. These observations are consistent with a model proposed for the kinetic control of mitochondrial ATP synthesis, which was based on isotope exchange measurements with isolated mammalian mitochondria [LaNoue, K. F., Jeffries, F. M. H. & Radda, G. K. (1986) Biochemistry 25, 7667-7675].

Glucose stimulation of insulin release in the absence of extracellular Ca2+ and in the absence of any increase in intracellular Ca2+ in rat pancreatic islets.

Insulin secretion has been studied in isolated rat pancreatic islets under stringent Ca(2+)-depleted, Ca(2+)-free conditions. Under these conditions, the effect of 16.7 mM glucose to stimulate insulin release was abolished. Forskolin, which activates adenylyl cyclase, also failed to stimulate release in the presence of either low or high glucose concentrations. A phorbol ester (phorbol 12-myristate 13-acetate; PMA) increased the release rate slightly and this was further increased by 16.7 mM glucose. Remarkably, in the presence of both forskolin and PMA, 16.7 mM glucose strongly augmented insulin release. The augmentation was concentration dependent and monophasic and had a temporal profile similar to the "second phase" of glucose-stimulated insulin release, which is seen under normal conditions when Ca2+ is present. Metabolism is required for the effect because mannoheptulose abolished the glucose response. Other nutrient secretagogues, alpha-ketoisocaproate, and the combination of leucine and glutamine augmented release under the same conditions. Norepinephrine, a physiological inhibitor of insulin secretion, totally blocked the stimulation of release by forskolin and PMA and the augmentation of release by glucose. Thus, under the stringent Ca(2+)-free conditions imposed, the stimulation of insulin release by forskolin and PMA, as well as the augmentation of release by glucose, is under normal physiological control. As no increase in intracellular [Ca2+] was observed, the results demonstrate that glucose can increase the rate of exocytosis and insulin release by pancreatic islets in a Ca(2+)-independent manner. This interesting pathway of stimulus-secretion coupling for glucose appears to exert its effect at a site beyond the usual elevation of intracellular [Ca2+] and is not due to an activation by glucose of protein kinase A or C.

Reduction of insulin gene transcription in HIT-T15 beta cells chronically exposed to a supraphysiologic glucose concentration is associated with loss of STF-1 transcription factor expression.

Chronic exposure of HIT-T15 beta cells to elevated glucose concentrations leads to decreased insulin gene transcription. The reduction in expression is accompanied by diminished binding of a glucose-sensitive transcription factor (termed GSTF) that interacts with two (A+T)-rich elements within the 5' flanking control region of the insulin gene. In this study we examined whether GSTF corresponds to the recently cloned insulin gene transcription factor STF-1, a homeodomain protein whose expression is restricted to the nucleus of endodermal cells of the duodenum and pancreas. We found that an affinity-purified antibody recognizing STF-1 supershifted the GSTF activator complex formed from HIT-T15 extracts. In addition, we demonstrated a reduction in STF-1 mRNA and protein levels that closely correlated with the change in GSTF binding in HIT-T15 cells chronically cultured under supraphysiologic glucose concentrations. The reduction in STF-1 expression in these cells could be accounted for by a change in the rate of STF-1 gene transcription, suggesting a posttranscriptional control mechanism. In support of this hypothesis, no STF-1 mRNA accumulated in HIT-T15 cells passaged in 11.1 mM glucose. The only RNA species detected was a 6.4-kb STF-1 RNA species that hybridized with 5' and 3' STF-1-specific cDNA probes. We suggest that the 6.4-kb RNA represents an STF-1 mRNA precursor and that splicing of this RNA is defective in these cells. Overall, this study suggests that reduced expression of a key transcriptional regulatory factor, STF-1, contributes to the decrease in insulin gene transcription in HIT-T15 cells chronically cultured in supraphysiologic glucose concentration.

In vivo regulation of muscle glycogen synthase and the control of glycogen synthesis.

The activity of glycogen synthase (GSase; EC 2.4.1.11) is regulated by covalent phosphorylation. Because of this regulation, GSase has generally been considered to control the rate of glycogen synthesis. This hypothesis is examined in light of recent in vivo NMR experiments on rat and human muscle and is found to be quantitatively inconsistent with the data under conditions of glycogen synthesis. Our first experiments showed that muscle glycogen synthesis was slower in non-insulin-dependent diabetics compared to normals and that their defect was in the glucose transporter/hexokinase (GT/HK) part of the pathway. From these and other in vivo NMR results a quantitative model is proposed in which the GT/HK steps control the rate of glycogen synthesis in normal humans and rat muscle. The flux through GSase is regulated to match the proximal steps by "feed forward" to glucose 6-phosphate, which is a positive allosteric effector of all forms of GSase. Recent in vivo NMR experiments specifically designed to test the model are analyzed by metabolic control theory and it is shown quantitatively that the GT/HK step controls the rate of glycogen synthesis. Preliminary evidence favors the transporter step. Several conclusions are significant: (i) glucose transport/hexokinase controls the glycogen synthesis flux; (ii) the role of covalent phosphorylation of GSase is to adapt the activity of the enzyme to the flux and to control the metabolite levels not the flux; (iii) the quantitative data needed for inferring and testing the present model of flux control depended upon advances of in vivo NMR methods that accurately measured the concentration of glucose 6-phosphate and the rate of glycogen synthesis.

Herpes simplex virus vectors overexpressing the glucose transporter gene protect against seizure-induced neuron loss.

We have generated herpes simplex virus (HSV) vectors vIE1GT and v alpha 4GT bearing the GLUT-1 isoform of the rat brain glucose transporter (GT) under the control of the human cytomegalovirus ie1 and HSV alpha 4 promoters, respectively. We previously reported that such vectors enhance glucose uptake in hippocampal cultures and the hippocampus. In this study we demonstrate that such vectors can maintain neuronal metabolism and reduce the extent of neuron loss in cultures after a period of hypoglycemia. Microinfusion of GT vectors into the rat hippocampus also reduces kainic acid-induced seizure damage in the CA3 cell field. Furthermore, delivery of the vector even after onset of the seizure is protective, suggesting that HSV-mediated gene transfer for neuroprotection need not be carried out in anticipation of neurologic crises. Using the bicistronic vector v alpha 22 beta gal alpha 4GT, which coexpresses both GT and the Escherichia coli lacZ marker gene, we further demonstrate an inverse correlation between the extent of vector expression in the dentate and the amount of CA3 damage resulting from the simultaneous delivery of kainic acid.

Functional anatomy of human eyeblink conditioning determined with regional cerebral glucose metabolism and positron-emission tomography.

Relative cerebral glucose metabolism was examined with positron-emission tomography (PET) as a measure of neuronal activation during performance of the classically conditioned eyeblink response in 12 young adult subjects. Each subject received three sessions: (i) a control session with PET scan in which unpaired presentations of the tone conditioned stimulus and corneal airpuff unconditioned stimulus were administered, (ii) a paired training session to allow associative learning to occur, and (iii) a paired test session with PET scan. Brain regions exhibiting learning-related activation were identified as those areas that showed significant differences in glucose metabolism between the unpaired control condition and well-trained state in the 9 subjects who met the learning criterion. Areas showing significant activation included bilateral sites in the inferior cerebellar cortex/deep nuclei, anterior cerebellar vermis, contralateral cerebellar cortex and pontine tegmentum, ipsilateral inferior thalamus/red nucleus, ipsilateral hippocampal formation, ipsilateral lateral temporal cortex, and bilateral ventral striatum. Among all subjects, including those who did not meet the learning criterion, metabolic changes in ipsilateral cerebellar nuclei, bilateral cerebellar cortex, anterior vermis, contralateral pontine tegmentum, ipsilateral hippocampal formation, and bilateral striatum correlated with degree of learning. The localization to cerebellum and its associated brainstem circuitry is consistent with neurobiological studies in the rabbit model of eyeblink classical conditioning and neuropsychological studies in brain-damaged humans. In addition, these data support a role for the hippocampus in conditioning and suggest that the ventral striatum may also be involved.

Cyclin-dependent protein kinase and cyclin homologs SSN3 and SSN8 contribute to transcriptional control in yeast.

The SSN3 and SSN8 genes of Saccharomyces cerevisiae were identified by mutations that suppress a defect in SNF1, a protein kinase required for release from glucose repression. Mutations in SSN3 and SSN8 also act synergistically with a mutation of the MIG1 repressor protein to relieve glucose repression. We have cloned the SSN3 and SSN8 genes. SSN3 encodes a cyclin-dependent protein kinase (cdk) homolog and is identical to UME5. SSN8 encodes a cyclin homolog 35% identical to human cyclin C. SSN3 and SSN8 fusion proteins interact in the two-hybrid system and coimmunoprecipitate from yeast cell extracts. Using an immune complex assay, we detected protein kinase activity that depends on both SSN3 and SSN8. Thus, the two SSN proteins are likely to function as a cdk-cyclin pair. Genetic analysis indicates that the SSN3-SSN8 complex contributes to transcriptional repression of diversely regulated genes and also affects induction of the GAL1 promoter.

High fat diet-induced hyperglycemia: prevention by low level expression of a glucose transporter (GLUT4) minigene in transgenic mice.

High-fat intake leading to obesity contributes to the development of non-insulin-dependent diabetes mellitus (NIDDM, type 2). Similarly, mice fed a high-fat (safflower oil) diet develop defective glycemic control, hyperglycemia, and obesity. To assess the effect of a modest increase in the expression of GLUT4 (the insulin-responsive glucose transporter) on impaired glycemic control caused by fat feeding, transgenic mice harboring a GLUT4 minigene were fed a high-fat diet. Low-level tissue-specific (heart, skeletal muscle, and adipose tissue) expression of the GLUT4 minigene in transgenic mice prevented the impairment of glycemic control and accompanying hyperglycemia, but not obesity, caused by fat feeding. Thus, a small increase (< or = 2-fold) in the tissue level of GLUT4 prevents a primary symptom of the diabetic state in a mouse model, suggesting a possible target for intervention in the treatment of NIDDM.

Clinical pathway intervention compliance and effectiveness when used in the treatment of patients with severe sepsis and septic shock at an Intensive Care Unit in Spain

The purpose of this quasi-experimental study was to assess levels of compliance with the intervention bundles contained in a clinical pathway used in the treatment of patients with severe sepsis and septic shock, and to analyze the pathway’s impact on survival and duration of hospital stays. We used data on 125 patients in an Intensive Care Unit, divided into a control group (N=84) and an intervention group (N=41). Levels of compliance increased from 13.1% to 29.3% in 5 resuscitation bundle interventions and from 14.3% to 22% in 3 monitoring bundle interventions. In-hospital mortality at 28 days decreased by 11.2% and the duration of hospital stay was reduced by 5 days. Although compliance was low, the intervention enhanced adherence to the instructions given in the clinical pathway and we observed a decline in mortality at 28 days and shorter hospital stays.

Control glucémico a través del ejercicio físico en pacientes con diabetes mellitus tipo 2; revisión sistemática

Introducción: En España, cerca del 14% de la población es diabética y el 95% corresponde a DM2. Un pobre control glucémico provoca un aumento de la morbilidad y mortalidad. Tres son los pilares en el tratamiento de la DM2: la dieta, la medicación y el ejercicio físico, sin embargo, el potencial de la prescripción de entrenamiento físico no ha sido totalmente explotado. Objetivo: Analizar el efecto de las distintas modalidades de ejercicio físico (AE, RT, Combo, INT) en el control glucémico en pacientes con diabetes mellitus tipo 2. Métodos: La búsqueda bibliográfica se realizó en 3 bases de datos electrónicas (Pubmed, Scopus y Proquest), incluyendo publicaciones desde enero de 2011 hasta mayo de 2014, que realizaran la intervención con AE, RT, Combo o INT, y que midieran la glucemia a través de la glucosa capilar, CGMS o HbA1c. Resultados: Del total de 386 artículos encontrados, 14 cumplieron los criterios de inclusión. Estos artículos fueron clasificados atendiendo a la modalidad de ejercicio físico de la intervención (AE, RT, Combo, INT), y en función de si analizaban el control glucémico como consecuencia del entrenamiento a largo plazo o tras una sesión de entrenamiento. Conclusiones: El AE, RT, Combo e INT muestran eficacia en el control glucémico tanto en el entrenamiento prolongado como en las 24-48h post-entrenamiento. Es necesaria la prescripción de un entrenamiento estructurado con una frecuencia, volumen e intensidad determinados para lograr beneficios en el control glucémico. El combo es la modalidad que obtiene mejores resultados a través del entrenamiento a largo plazo.

Nocardia infection in solid organ transplant recipients: a multicenter European case-control study.

BACKGROUND  Nocardiosis is a rare, life-threatening opportunistic infection, affecting 0.04% to 3.5% of patients after solid organ transplantation (SOT). The aim of this study was to identify risk factors for Nocardia infection after SOT and to describe the presentation of nocardiosis in these patients. METHODS  We performed a retrospective case-control study of adult patients diagnosed with nocardiosis after SOT between 2000 and 2014 in 36 European (France, Belgium, Switzerland, Netherlands, Spain) centers. Two control subjects per case were matched by institution, transplant date and transplanted organ. A multivariable analysis was performed using conditional logistic regression to identify risk factors for nocardiosis. RESULTS  One hundred and seventeen cases of nocardiosis and 234 control patients were included. Nocardiosis occurred at a median of 17.5 [range 2-244] months after transplantation. In multivariable analysis, high calcineurin inhibitor trough levels in the month before diagnosis (OR=6.11 [2.58-14.51]), use of tacrolimus (OR=2.65 [1.17-6.00]) and corticosteroid dose (OR=1.12 [1.03-1.22]) at the time of diagnosis, patient age (OR=1.04 [1.02-1.07]) and length of stay in intensive care unit after SOT (OR=1.04 [1.00-1.09]) were independently associated with development of nocardiosis; low-dose cotrimoxazole prophylaxis was not found to prevent nocardiosis. Nocardia farcinica was more frequently associated with brain, skin and subcutaneous tissue infections than were other Nocardia species. Among the 30 cases with central nervous system nocardiosis, 13 (43.3%) had no neurological symptoms. CONCLUSIONS  We identified five risk factors for nocardiosis after SOT. Low-dose cotrimoxazole was not found to prevent Nocardia infection. These findings may help improve management of transplant recipients.