MAP2 Isoforms in Developing Cat Cerebral Cortex and Corpus Callosum.

The microtubule-associated protein MAP2 was studied in the developing cat visual cortex and corpus callosum. Biochemically, no MAP2a was detectable in either structure during the first postnatal month; adult cortex revealed small amounts of MAP2a. MAP2b was abundant in cortical tissue during the first postnatal month and decreased in concentration towards adulthood; it was barely detectable in corpus callosum at all ages. MAP2c was present in cortex and corpus callosum at birth; in cortex it consisted of three proteins of similar molecular weights between 65 and 70 kD. The two larger, phosphorylated forms disappeared after postnatal day 28, the smaller form after day 39. In corpus callosum, MAP2c changed from a phosphorylated to an unphosphorylated variant during the first postnatal month and then disappeared. Immunocytochemical experiments revealed MAP2 in cell bodies and dendrites of neurons in all cortical layers, from birth onwards. In corpus callosum, in the first month after birth, a little MAP2, possibly MAP2c, was detectable in axons. The present data indicate that MAP2 isoforms differ in their cellular distribution, temporal appearance and structural association, and that their composition undergoes profound changes during the period of axonal stabilization and dendritic maturation.

Feasibility of direct mapping of cerebral fluorodeoxy-D-glucose metabolism in situ at subcellular resolution using soft X-ray fluorescence.

Glucose metabolism is difficult to image with cellular resolution in mammalian brain tissue, particularly with (18) fluorodeoxy-D-glucose (FDG) positron emission tomography (PET). To this end, we explored the potential of synchrotron-based low-energy X-ray fluorescence (LEXRF) to image the stable isotope of fluorine (F) in phosphorylated FDG (DG-6P) at 1 μm(2) spatial resolution in 3-μm-thick brain slices. The excitation-dependent fluorescence F signal at 676 eV varied linearly with FDG concentration between 0.5 and 10 mM, whereas the endogenous background F signal was undetectable in brain. To validate LEXRF mapping of fluorine, FDG was administered in vitro and in vivo, and the fluorine LEXRF signal from intracellular trapped FDG-6P over selected brain areas rich in radial glia was spectrally quantitated at 1 μm(2) resolution. The subsequent generation of spatial LEXRF maps of F reproduced the expected localization and gradients of glucose metabolism in retinal Müller glia. In addition, FDG uptake was localized to periventricular hypothalamic tanycytes, whose morphological features were imaged simultaneously by X-ray absorption. We conclude that the high specificity of photon emission from F and its spatial mapping at ≤1 μm resolution demonstrates the ability to identify glucose uptake at subcellular resolution and holds remarkable potential for imaging glucose metabolism in biological tissue. © 2012 Wiley Periodicals, Inc.

J-shaped association between serum glucose and functional outcome in acute ischemic stroke.

BACKGROUND AND PURPOSE: Hyperglycemia after stroke is associated with larger infarct volume and poorer functional outcome. In an animal stroke model, the association between serum glucose and infarct volume is described by a U-shaped curve with a nadir ≈7 mmol/L. However, a similar curve in human studies was never reported. The objective of the present study is to investigate the association between serum glucose levels and functional outcome in patients with acute ischemic stroke. METHODS: We analyzed 1446 consecutive patients with acute ischemic stroke. Serum glucose was measured on admission at the emergency department together with multiple other metabolic, clinical, and radiological parameters. National Institutes of Health Stroke Scale (NIHSS) score was recorded at 24 hours, and Rankin score was recorded at 3 and 12 months. The association between serum glucose and favorable outcome (Rankin score ≤2) was explored in univariate and multivariate analysis. The model was further analyzed in a robust regression model based on fractional polynomial (-2-2) functions. RESULTS: Serum glucose is independently correlated with functional outcome at 12 months (OR, 1.15; P=0.01). Other predictors of outcome include admission NIHSS score (OR, 1.18; P<0001), age (OR, 1.06; P<0.001), prestroke Rankin score (OR, 20.8; P=0.004), and leukoaraiosis (OR, 2.21; P=0.016). Using these factors in multiple logistic regression analysis, the area under the receiver-operator characteristic curve is 0.869. The association between serum glucose and Rankin score at 12 months is described by a J-shaped curve with a nadir of 5 mmol/L. Glucose values between 3.7 and 7.3 mmol/L are associated with favorable outcome. A similar curve was generated for the association of glucose and 24-hour NIHSS score, for which glucose values between 4.0 and 7.2 mmol/L are associated with a NIHSS score <7. Discussion-Both hypoglycemia and hyperglycemia are dangerous in acute ischemic stroke as shown by a J-shaped association between serum glucose and 24-hour and 12-month outcome. Initial serum glucose values between 3.7 and 7.3 mmol/L are associated with favorable outcome.

Metabolic fate of fructose ingested with and without glucose in a mixed meal.

Ingestion of pure fructose stimulates de novo lipogenesis and gluconeogenesis. This may however not be relevant to typical nutritional situations, where fructose is invariably ingested with glucose. We therefore assessed the metabolic fate of fructose incorporated in a mixed meal without or with glucose in eight healthy volunteers. Each participant was studied over six hours after the ingestion of liquid meals containing either 13C-labelled fructose, unlabeled glucose, lipids and protein (Fr + G) or 13C-labelled fructose, lipids and protein, but without glucose (Fr), or protein and lipids alone (ProLip). After Fr + G, plasma 13C-glucose production accounted for 19.0% ± 1.5% and 13CO2 production for 32.2% ± 1.3% of 13C-fructose carbons. After Fr, 13C-glucose production (26.5% ± 1.4%) and 13CO2 production (36.6% ± 1.9%) were higher (p < 0.05) than with Fr + G. 13C-lactate concentration and very low density lipoprotein VLDL 13C-palmitate concentrations increased to the same extent with Fr + G and Fr, while chylomicron 13C-palmitate tended to increase more with Fr + G. These data indicate that gluconeogenesis, lactic acid production and both intestinal and hepatic de novo lipogenesis contributed to the disposal of fructose carbons ingested together with a mixed meal. Co-ingestion of glucose decreased fructose oxidation and gluconeogenesis and tended to increase 13C-pamitate concentration in gut-derived chylomicrons, but not in hepatic-borne VLDL-triacylglycerol (TG). This trial was approved by clinicaltrial. gov. Identifier is NCT01792089.

Organ-specific lymphangiectasia, arrested lymphatic sprouting, and maturation defects resulting from gene-targeting of the PI3K regulatory isoforms p85alpha, p55alpha, and p50alpha.

The phosphoinositide 3-kinase (PI3K) family has multiple vascular functions, but the specific regulatory isoform supporting lymphangiogenesis remains unidentified. Here, we report that deletion of the Pik3r1 gene, encoding the regulatory subunits p85alpha, p55alpha, and p50alpha impairs lymphatic sprouting and maturation, and causes abnormal lymphatic morphology, without major impact on blood vessels. Pik3r1 deletion had the most severe consequences among gut and diaphragm lymphatics, which share the retroperitoneal anlage, initially suggesting that the Pik3r1 role in this vasculature is anlage-dependent. However, whereas lymphatic sprouting toward the diaphragm was arrested, lymphatics invaded the gut, where remodeling and valve formation were impaired. Thus, cell-origin fails to explain the phenotype. Only the gut showed lymphangiectasia, lymphatic up-regulation of the transforming growth factor-beta co-receptor endoglin, and reduced levels of mature vascular endothelial growth factor-C protein. Our data suggest that Pik3r1 isoforms are required for distinct steps of embryonic lymphangiogenesis in different organ microenvironments, whereas they are largely dispensable for hemangiogenesis.

MRS glucose mapping and PET joining forces: re-evaluation of the lumped constant in the rat brain under isoflurane anaesthesia.

Although numerous positron emission tomography (PET) studies with (18) F-fluoro-deoxyglucose (FDG) have reported quantitative results on cerebral glucose kinetics and consumption, there is a large variation between the absolute values found in the literature. One of the underlying causes is the inconsistent use of the lumped constants (LCs), the derivation of which is often based on multiple assumptions that render absolute numbers imprecise and errors hard to quantify. We combined a kinetic FDG-PET study with magnetic resonance spectroscopic imaging (MRSI) of glucose dynamics in Sprague-Dawley rats to obtain a more comprehensive view of brain glucose kinetics and determine a reliable value for the LC under isoflurane anaesthesia. Maps of Tmax /CMRglc derived from MRSI data and Tmax determined from PET kinetic modelling allowed to obtain an LC-independent CMRglc . The LC was estimated to range from 0.33 ± 0.07 in retrosplenial cortex to 0.44 ± 0.05 in hippocampus, yielding CMRglc between 62 ± 14 and 54 ± 11 μmol/min/100 g, respectively. These newly determined LCs for four distinct areas in the rat brain under isoflurane anaesthesia provide means of comparing the growing amount of FDG-PET data available from translational studies.

Sex differences in lipid and glucose kinetics after ingestion of an acute oral fructose load.

The increase in VLDL TAG concentration after ingestion of a high-fructose diet is more pronounced in men than in pre-menopausal women. We hypothesised that this may be due to a lower fructose-induced stimulation of de novo lipogenesis (DNL) in pre-menopausal women. To evaluate this hypothesis, nine healthy male and nine healthy female subjects were studied after ingestion of oral loads of fructose enriched with 13C6 fructose. Incorporation of 13C into breath CO2, plasma glucose and plasma VLDL palmitate was monitored to evaluate total fructose oxidation, gluconeogenesis and hepatic DNL, respectively. Substrate oxidation was assessed by indirect calorimetry. After 13C fructose ingestion, 44.0 (sd 3.2)% of labelled carbons were recovered in plasma glucose in males v. 41.9 (sd 2.3)% in females (NS), and 42.9 (sd 3.7)% of labelled carbons were recovered in breath CO2 in males v. 43.0 (sd 4.5)% in females (NS), indicating similar gluconeogenesis from fructose and total fructose oxidation in males and females. The area under the curve for 13C VLDL palmitate tracer-to-tracee ratio was four times lower in females (P < 0.05), indicating a lower DNL. Furthermore, lipid oxidation was significantly suppressed in males (by 16.4 (sd 5.2), P < 0.05), but it was not suppressed in females ( -1.3 (sd 4.7)%). These results support the hypothesis that females may be protected against fructose-induced hypertriglyceridaemia because of a lower stimulation of DNL and a lower suppression of lipid oxidation.

Evidence of systematic and proportional error in a widely used glucose oxidase analyser: Impact for clinical research?

Real time glycemia is a cornerstone for metabolic research, particularly when performing oral glucose tolerance tests (OGTT) or glucose clamps. From 1965 to 2009, the gold standard device for real time plasma glucose assessment was the Beckman glucose analyzer 2 (Beckman Instruments, Fullerton, CA), which technology couples glucose oxidase enzymatic assay with oxygen sensors. Since its discontinuation in 2009, today's researchers are left with few choices that utilize glucose oxidase technology. The first one is the YSI 2300 (Yellow Springs Instruments Corp., Yellow Springs, OH), known to be as accurate as the Beckman(1). The YSI has been used extensively for clinical research studies and is used to validate other glucose monitoring devices(2). The major drawback of the YSI is that it is relatively slow and requires high maintenance. The Analox GM9 (Analox instruments, London), more recent and faster, is increasingly used in clinical research(3) as well as in basic sciences(4) (e.g. 23 papers in Diabetes or 21 in Diabetologia). This article is protected by copyright. All rights reserved.

Thermogenesis induced by five different intravenous glucose/insulin infusions in healthy young men.

The thermogenic response induced by glucose/insulin administered intravenously was examined in 22 healthy male volunteers using indirect calorimetry in combination with the euglycaemic insulin clamp technique. Five increasing steady state levels of insulinaemia (62 muU/ml to 1132 muU/ml) were achieved by means of continuous infusions of insulin at 5 rates ranging from 0.5 mU/kg.min to 10 mU/kg.min. Euglycaemia was maintained at each insulin level by infusing glucose at different rates ranging from steady state values of 0.41 g/min to 0.77 g/min. These glucose/insulin infusions resulted in a significant net rise in resting energy expenditure from 0.33 kJ/min to 0.94 kJ/min over preinfusion baseline values for the lowest and the highest doses respectively. There was a highly significant relationship (r = 0.93, p<0.001, n = 42) between the amount of glucose infused and the net increase in energy expenditure over preinfusion baseline values. Intravenous glucose induced thermogenesis (GIT(iv)) was calculated as incremental values of energy expenditure related to step changes in glucose infusion rates. GIT(iv) was found to be approximately 5.5% a physiological plasma insulin levels (i.e. below 200 muU/ml) whereas at supraphysiological levels (i.e.>400 muU/ml) GIT(iv) was increased up to 8%. It was concluded that: 1. the magnitude of the GIT(iv) at physiological insulinaemia was similar to that found by other investigators who have administered glucose per os; 2. the elevated thermogenesis observed at high doses of glucose/insulin infusion is consistent with recent clinical findings showing a markedly increased energy expenditure in patients supported by large quantities of intravenous glucose (TPN).

Quette place pour l'automesure glycémique dans la prise en charge du diabète de type 2 [Which role for self-monitoring of blood glucose in type 2 diabetes?]

There is a sustained controversy in the literature about the role and utility of self-monitoring of blood glucose (SMBG) in type 2 diabetes. The study results in this field do not provide really useful clues for the integration of SMBG in the follow-up of the individual patient, because they are based on a misconception of SMBG. It is studied as if it was a medical treatment whose effect on glycemic control is to be isolated. However, SMBG has no such intrinsic effect. It gains its purpose only as an inseparable component of a comprehensive and structured educational strategy. To be appropriate this strategy cannot be based on the health care professionals' view on diabetes only. It rather has to be tailored to the individual patient's needs through an ongoing process of shared reflection with him.

Comparison of thermogenic effect of fructose and glucose in normal humans.

After nutrient ingestion there is an increase in energy expenditure that has been referred to as dietary-induced thermogenesis. In the present study we have employed indirect calorimetry to compare the increment in energy expenditure after the ingestion of 75 g of glucose or fructose in 17 healthy volunteers. During the 4 h after glucose ingestion the plasma insulin concentration increased by 33 +/- 4 microU/ml and this was associated with a significant increase in carbohydrate oxidation and decrement in lipid oxidation. Energy expenditure increased by 0.08 +/- 0.01 kcal/min. When fructose was ingested, the plasma insulin concentration increased by only 8 +/- 2 microU/ml vs. glucose. Nonetheless, the increments in carbohydrate oxidation and decrement in lipid oxidation were significantly greater than with glucose. The increment in energy expenditure was also greater with fructose. When the mean increment in plasma insulin concentration after fructose was reproduced using the insulin clamp technique, the increase in carbohydrate oxidation and decrement in lipid oxidation were markedly reduced compared with the fructose-ingestion study; energy expenditure failed to increase above basal levels. To examine the role of the adrenergic nervous system in fructose-induced thermogenesis, fructose ingestion was also performed during beta-adrenergic blockade with propranolol. The increase in energy expenditure during fructose plus propranolol was lower than with fructose ingestion alone. These results indicate that the stimulation of thermogenesis after carbohydrate ingestion is related to an augmentation of cellular metabolism and is not dependent on an increase in the plasma insulin concentration per se.(ABSTRACT TRUNCATED AT 250 WORDS)

Brain glucose sensing and neural regulation of insulin and glucagon secretion.

Glucose homeostasis requires the tight regulation of glucose utilization by liver, muscle and white or brown fat, and glucose production and release in the blood by liver. The major goal of maintaining glycemia at ∼ 5 mM is to ensure a sufficient flux of glucose to the brain, which depends mostly on this nutrient as a source of metabolic energy. This homeostatic process is controlled by hormones, mainly glucagon and insulin, and by autonomic nervous activities that control the metabolic state of liver, muscle and fat tissue but also the secretory activity of the endocrine pancreas. Activation or inhibition of the sympathetic or parasympathetic branches of the autonomic nervous systems are controlled by glucose-excited or glucose-inhibited neurons located at different anatomical sites, mainly in the brainstem and the hypothalamus. Activation of these neurons by hyper- or hypoglycemia represents a critical aspect of the control of glucose homeostasis, and loss of glucose sensing by these cells as well as by pancreatic β-cells is a hallmark of type 2 diabetes. In this article, aspects of the brain-endocrine pancreas axis are reviewed, highlighting the importance of central glucose sensing in the control of counterregulation to hypoglycemia but also mentioning the role of the neural control in β-cell mass and function. Overall, the conclusions of these studies is that impaired glucose homeostasis, such as associated with type 2 diabetes, but also defective counterregulation to hypoglycemia, may be caused by initial defects in glucose sensing.

Glucagon-like peptide-1 increases beta-cell glucose competence and proliferation by translational induction of insulin-like growth factor-1 receptor expression.

Glucagon-like peptide-1 (GLP-1) protects beta-cells against apoptosis, increases their glucose competence, and induces their proliferation. We previously demonstrated that the anti-apoptotic effect was mediated by an increase in insulin-like growth factor-1 receptor (IGF-1R) expression and signaling, which was dependent on autocrine secretion of insulin-like growth factor 2 (IGF-2). Here, we further investigated how GLP-1 induces IGF-1R expression and whether the IGF-2/IGF-1R autocrine loop is also involved in mediating GLP-1-increase in glucose competence and proliferation. We show that GLP-1 up-regulated IGF-1R expression by a protein kinase A-dependent translational control mechanism, whereas isobutylmethylxanthine, which led to higher intracellular accumulation of cAMP than GLP-1, increased both IGF-1R transcription and translation. We then demonstrated, using MIN6 cells and primary islets, that the glucose competence of these cells was dependent on the level of IGF-1R expression and on IGF-2 secretion. We showed that GLP-1-induced primary beta-cell proliferation was suppressed by Igf-1r gene inactivation and by IGF-2 immunoneutralization or knockdown. Together our data show that regulation of beta-cell number and function by GLP-1 depends on the cAMP/protein kinase A mediated-induction of IGF-1R expression and the increased activity of an IGF-2/IGF-1R autocrine loop.

Metabolism of oral glucose in children born small for gestational age : evidence for an impaired whole body glucose oxidation

Résumé Les études épidémiologiques indiquent que la restriction intra-utérine confère un risque accru de développement de diabète de type 2 au cours de la vie. Certaines études ont documenté la présence d'une résistance à l'insuline chez les jeunes adultes ou les adolescents nés petits pour l'âge gestationnel. Comme la plupart des études ont impliqués des individus post-pubères et comme la puberté influence de manière marquée le métabolisme énergétique, nous avons évalué le devenir du glucose administré oralement dans un groupe incluant essentiellement des enfants pré-pubères ou en début de puberté avec restriction intra-utérine, et chez des enfants matchés pour l'âge et pour le poids. Tous les enfants ont eu une évaluation de leur composition corporelle par mesure des plis cutanés. Ils ont ensuite été étudiés dans des conditions standardisées et ont reçu 4 charges consécutives orales de glucose à raison de 180 mg/kg de poids corporel jusqu'à atteindre un état d'équilibre relatif. La dépense énergétique et l'oxydation des substrats ont été évaluées durant la quatrième heure par calorimétrie indirecte. Comparativement avec les enfants matchés pour l'âge et le poids, les enfants nés petits pour l'âge gestationnel avaient une plus petite stature. Leur dépense énergétique n'était pas significativement abaissée, mais leur oxydation du glucose était plus basse. Ces résultats indiquent que des altérations métaboliques sont présentes précocement chez les enfants nés petits pour l'âge gestationnel, et qu'elles sont possiblement reliées à des altérations de la composition corporelle. Abstract: Epidemiological studies indicate that intrauterine growth restriction confers an increased risk of developing type 2 diabetes mellitus in subsequent life. Several studies have further documented the presence of insulin resistance in young adults or adolescent children born small for gestational age. Since most studies addressed postpubertal individuals, and since puberty markedly affects energy metabolism, we evaluated the disposal of oral glucose in a group including mainly prepubertal and early pubertal children with intrauterine growth restriction and in healthy age- and weight-matched control children. All children had an evaluation of their body composition by skinfold thickness measurements. They were then studied in standardized conditions and received 4 consecutive hourly loads of 180 mg glucose/kg body weight to reach a near steady state. Energy expenditure and substrate oxidation were evaluated during the fourth hour by indirect calorimetry. Compared to both age- and weight-matched children, children born small for gestational age had lower stature. Their energy expenditure was not significantly decreased, but they had lower glucose oxidation rates. These results indicate that metabolic alterations are present early in children born small for gestational age, and are possibly related to alterations of body composition.

Peripheral Nerve Neuropathy is Associated with a Glial Reaction in the Gracile Nucleus Associated with a regulation of the GABA transporter GAT-1

Allodynia (pain in response to normally non painful stimulation) and paresthesia (erroneous sensory experience) are two debilitating symptoms of neuropathic pain. These stem, at least partly, from profound changes in the non-nociceptive sensory pathway that comprises large myelinated neuronal afferents terminating in the gracile and cuneate nuclei. Further than neuronal changes, well admitted evidence indicates that glial cells (especially in the spinal cord) are key actors in neuropathic pain, in particular the possible alteration in astrocytic capacity to reuptake neurotransmitters (glutamate and GABA). Yet, the possibility of such a changed astrocytic scavenging capacity remains unexplored in the dorsal column pathway. The present study was therefore undertaken to assess whether peripheral nerve injury (spared nerve injury model, SNI) could trigger a glial reaction, and especially changes in glutamate and GABA transporters, in the gracile nucleus. SNI surgery was performed on male Sprague-Dawley rats. Seven days after surgery, rats were used for immunofluorescence (fixation and brain slicing), western-blot (fresh brain freezing and protein extraction) or GABA reuptake on synaptosomes. We found that SNI results in a profound glial reaction in the ipsilateral gracile nucleus. This reaction was characterized by an enhanced immunolabelling for microglial marker Iba1 as well as astrocytic protein GFAP (further confirmed by western-blot, p <0.05, n = 7). These changes were not observed in sham animals. Immunofluorescence and western-blot analysis shows that the GABA transporter GAT-1 is upregulated in the ipsilateral gracile nucleus (p <0.001; n = 7), with no detectable change in GAT-3 or glutamate transporters EAAT-1 and EAAT-2. Double immunoflurescence shows that GAT-1 and GFAP colocalize within the same cells. Furthermore, the upregulation of GFAP and GAT-1 were shown to occur all along the rostrocaudal axis of the gracile nucleus. Finally, synaptosomes from ipsilateral gracile nucleus show an increased capacity to reuptake GABA. Together, the data presented herein show that glial cells in the gracile nucleus react to neuropathic lesion, in particular through an upregulation of the GABA transporter GAT-1. Hence, this study points to role of an increased GABA transport in the dorsal column nuclei in neuropathic pain, calling attention to GAT-1 as a putative future pharmacological target to treat allodynia and paresthesia.