Enrique Blázquez

Colocalization of glucagon-like peptide-1 (GLP-1) receptors, glucose transporter GLUT-2, and glucokinase mRNAs in rat hypothalamic cells : Evidence for a role of GLP-1 receptor agonists as an inhibitory signal for food and water intake

Abstract: This study was designed to determine the possible role of brain glucagon‐like peptide‐1 (GLP‐1) receptors in feeding behavior. In situ hybridization showed colocalization of the mRNAs for GLP‐1 receptors, glucokinase, and GLUT‐2 in the third ventricle wall and adjacent arcuate nucleus, median eminence, and supraoptic nucleus. These brain areas are considered to contain glucose‐sensitive neurons mediating feeding behavior. Because GLP‐1 receptors, GLUT‐2, and glucokinase are proteins involved in the multistep process of glucose sensing in pancreatic β cells, the colocalization of specific GLP‐1 receptors and glucose sensing‐related proteins in hypothalamic neurons supports a role of this peptide in the hypothalamic regulation of macronutrient and water intake. This hypothesis was confirmed by analyzing the effects of both systemic and central administration of GLP‐1 receptor ligands. Acute or subchronic intraperitoneal administration of GLP‐1 (7–36) amide did not modify food and water intake, although a dose‐dependent loss of body weight gain was observed 24 h after acute administration of the higher dose of the peptide. By contrast, the intracerebroventricular (i.c.v.) administration of GLP‐1 (7–36) amide produced a biphasic effect on food intake characterized by an increase in the amount of food intake after acute i.c.v. delivery of 100 ng of the peptide. There was a marked reduction of food ingestion with the 1,000 and 2,000 ng doses of the peptide, which also produced a significant decrease of water intake. These effects seemed to be specific because i.c.v. administration of GLP‐1 (1–37), a peptide with lower biological activity than GLP‐1 (7–36) amide, did not change feeding behavior in food‐deprived animals. Exendin‐4, when given by i.c.v. administration in a broad range of doses (0.2, 1, 5, 25, 100, and 500 ng), proved to be a potent agonist of GLP‐1 (7–36) amide. It decreased, in a dose‐dependent manner, both food and water intake, starting at the dose of 25 ng per injection. Pretreatment with an i.c.v. dose of a GLP‐1 receptor antagonist [exendin (9–39); 2,500 ng] reversed the inhibitory effects of GLP‐1 (7–36) amide (1,000 ng dose) and exendin‐4 (25 ng dose) on food and water ingestion. These findings suggest that GLP‐1 (7–36) amide may modulate both food and drink intake in the rat through a central mechanism.

Expression of the glucagon-like peptide-1 receptor gene in rat brain

Abstract: Evidence that glucagon‐like peptide‐1 (GLP‐1) (7–36) amide functions as a novel neuropeptide prompted us to study the gene expression of its receptor in rat brain. Northern blot analysis showed transcripts of similar size in RINm5F cells, hypothalamus, and brainstem. First‐strand cDNA was prepared by using RNA from hypothalamus, brainstem, and RINm5F cells and subsequently amplified by PCR. Southern blot analysis of the PCR products showed a major 1.4‐kb band in all these preparations. PCR products amplified from hypothalamus were cloned, and the nucleotide sequence of one strand was identical to that described in rat pancreatic islets. In situ hybridization studies showed specific labeling in both neurons and glia of the thalamus, hypothalamus, hippocampus, primary olfatory cortex, choroid plexus, and pituitary gland. In the hypothalamus, ventromedial nuclei cells were highly labeled. These findings indicate that GLP‐1 receptors are actually synthesized in rat brain. In addition, the colocalization of GLP‐1 receptors, glucokinase, and GLUT‐2 in the same areas supports the idea that these cells play an important role in glucose sensing in the brain.

The expression of GLP‐1 receptor mRNA and protein allows the effect of GLP‐1 on glucose metabolism in the human hypothalamus and brainstem

In the present work, several experimental approaches were used to determine the presence of the glucagon‐like peptide‐1 receptor (GLP‐1R) and the biological actions of its ligand in the human brain. In situ hybridization histochemistry revealed specific labelling for GLP‐1 receptor mRNA in several brain areas. In addition, GLP‐1R, glucose transporter isoform (GLUT‐2) and glucokinase (GK) mRNAs were identified in the same cells, especially in areas of the hypothalamus involved in feeding behaviour. GLP‐1R gene expression in the human brain gave rise to a protein of 56 kDa as determined by affinity cross‐linking assays. Specific binding of 125I‐GLP‐1(7–36) amide to the GLP‐1R was detected in several brain areas and was inhibited by unlabelled GLP‐1(7–36) amide, exendin‐4 and exendin (9–39). A further aim of this work was to evaluate cerebral‐glucose metabolism in control subjects by positron emission tomography (PET), using 2‐[F‐18] deoxy‐d‐glucose (FDG). Statistical analysis of the PET studies revealed that the administration of GLP‐1(7–36) amide significantly reduced (p < 0.001) cerebral glucose metabolism in hypothalamus and brainstem. Because FDG‐6‐phosphate is not a substrate for subsequent metabolic reactions, the lower activity observed in these areas after peptide administration may be due to reduction of the glucose transport and/or glucose phosphorylation, which should modulate the glucose sensing process in the GLUT‐2‐ and GK‐containing cells.

Neural contribution to the effect of glucagon-like peptide-1-(7—36) amide on arterial blood pressure in rats

This study was designed to determine the contribution of the central nervous system (CNS) to the effects of glucagon-like peptide-1-(7-36) amide (tGLP-1) on arterial blood pressure and heart rate in rats. Accordingly, intracerebroventricular administration of the peptide produced an increase in cardiovascular parameters, which was blocked by previous administration of exendin-(9-39) through the same route, but not when it was intravenously injected. Intravenous administration of tGLP-1 produced a significant increase in arterial blood pressure and heart rate, which was blocked by the previous intracerebroventricular or intravenous administration of exendin-(9-39). Bilateral vagotomy blocked the stimulating effect of intracerebroventricular tGLP-1 administration on arterial blood pressure and heart rate. Also, bilateral vagotomy prevented the blocking effect of intracerebroventricular but not of intravenous exendin-(9-39) on cardiovascular parameters after intravenous administration of tGLP-1. These findings suggest that the action of tGLP-1 on cardiovascular parameters is under a dual control generated in the CNS and in peripheral structures and that the neural information emerging in the brain is transmitted to the periphery through the vagus nerve.This study was designed to determine the contribution of the central nervous system (CNS) to the effects of glucagon-like peptide-1-(7-36) amide (tGLP-1) on arterial blood pressure and heart rate in rats. Accordingly, intracerebroventricular administration of the peptide produced an increase in cardiovascular parameters, which was blocked by previous administration of exendin-(9-39) through the same route, but not when it was intravenously injected. Intravenous administration of tGLP-1 produced a significant increase in arterial blood pressure and heart rate, which was blocked by the previous intracerebroventricular or intravenous administration of exendin-(9-39). Bilateral vagotomy blocked the stimulating effect of intracerebroventricular tGLP-1 administration on arterial blood pressure and heart rate. Also, bilateral vagotomy prevented the blocking effect of intracerebroventricular but not of intravenous exendin-(9-39) on cardiovascular parameters after intravenous administration of tGLP-1. These findings suggest that the action of tGLP-1 on cardiovascular parameters is under a dual control generated in the CNS and in peripheral structures and that the neural information emerging in the brain is transmitted to the periphery through the vagus nerve.

Insulin in the Brain: Its Pathophysiological Implications for States Related with Central Insulin Resistance, Type 2 Diabetes and Alzheimer’s Disease

Although the brain has been considered an insulin-insensitive organ, recent reports on the location of insulin and its receptors in the brain have introduced new ways of considering this hormone responsible for several functions. The origin of insulin in the brain has been explained from peripheral or central sources, or both. Regardless of whether insulin is of peripheral origin or produced in the brain, this hormone may act through its own receptors present in the brain. The molecular events through which insulin functions in the brain are the same as those operating in the periphery. However, certain insulin actions are different in the central nervous system, such as hormone-induced glucose uptake due to a low insulin-sensitive GLUT-4 activity, and because of the predominant presence of GLUT-1 and GLUT-3. In addition, insulin in the brain contributes to the control of nutrient homeostasis, reproduction, cognition, and memory, as well as to neurotrophic, neuromodulatory, and neuroprotective effects. Alterations of these functional activities may contribute to the manifestation of several clinical entities, such as central insulin resistance, type 2 diabetes mellitus (T2DM), and Alzheimer’s disease (AD). A close association between T2DM and AD has been reported, to the extent that AD is twice more frequent in diabetic patients, and some authors have proposed the name “type 3 diabetes” for this association. There are links between AD and T2DM through mitochondrial alterations and oxidative stress, altered energy and glucose metabolism, cholesterol modifications, dysfunctional protein O-GlcNAcylation, formation of amyloid plaques, altered Aβ metabolism, and tau hyperphosphorylation. Advances in the knowledge of preclinical AD and T2DM may be a major stimulus for the development of treatment for preventing the pathogenic events of these disorders, mainly those focused on reducing brain insulin resistance, which is seems to be a common ground for both pathological entities.

Interactions of exendin-(9–39) with the effects of glucagon-like peptide-1-(7–36) amide and of exendin-4 on arterial blood pressure and heart rate in rats

This study was designed to determine the interactions of peptide exendin-(9-39) with the effect of glucagon-like peptide-1-(7-36) (GLP-1 (7-36)) amide and of exendin-4 on arterial blood pressure and heart rate in the rat. Both GLP-1 (7-36) amide and exendin-4 produced a dose-dependent increase in systolic, diastolic and mean arterial blood pressure, as well as in heart rate, although the effect of exendin-4 was more prolonged. These data indicate a longer functional half-life in vivo for exendin-4 as compared to GLP-1 (7-36) amide, which may have therapeutical applications. The antagonist effect of exendin-(9-39) on these cardiovascular parameters was also tested with 3000 ng of exendin-(9-39) intravenously administered 5 min before i.v. injection of 10 ng of either GLP-1 (7-36) amide or exendin-4. Under these experimental conditions the effect of the latter two peptides on arterial blood pressure and heart rate was blocked. By contrast, single administration of exendin-(9-39) did not modify cardiovascular parameters. These findings indicate that exendin-4 is an agonist and that exendin-(9-39) is an antagonist of the action of GLP-1 (7-36) amide on arterial blood pressure and heart rate. Therefore, the action of GLP-1 (7-36) amide on these parameters seems to be mediated through its own receptors.

Functional Glucokinase Isoforms Are Expressed in Rat Brain

Abstract: Recently, the description of glucokinase mRNA in certain neuroendocrine cells has opened new ways to characterize this enzyme in the rat brain. In this study, we found glucokinase mRNA and a similar RNA splicing pattern of the glucokinase gene product in rat hypothalamus and pancreatic islets; the mRNA that codes for B1 isoform was the most abundant, with minor amounts of those coding for the B2, P1, P2, P1/B2, and P2/B2 isoforms. Glucokinase gene expression in rat brain gave rise to a protein of 52 kDa with a high apparent Km for glucose and no product inhibition by glucose 6‐phosphate, with a contribution to the total glucose phosphorylating activity of between 40 and 14%; the hypothalamus and cerebral cortex were the regions of maximal activity. Low and high Km hexokinases were characterized by several criteria. Also, using RT‐PCR analysis we found a glucokinase regulatory protein mRNA similar to that previously reported in liver. These findings indicate that the glucokinase present in rat brain should facilitate the adaptation of this organ to fluctuations in blood glucose concentrations, and the expression of glucokinase and GLUT‐2 in the same hypothalamic neurons suggests a role in glucose sensing.

Evidence that glucokinase regulatory protein is expressed and interacts with glucokinase in rat brain

Our previous description of functional glucokinase isoforms in the rat brain has opened new questions concerning the presence of glucokinase regulatory protein in the brain and the functional role of its interactions with glucokinase. In this study, we found glucokinase regulatory protein mRNA in rat brain, pancreatic islets and liver. In addition, we found two other variant splicing isoforms, both identified in hypothalamus, pancreatic islets and liver. In situ hybridization studies revealed the presence of glucokinase regulatory protein mRNA, the highest number of positive cells being found in the paraventricular nucleus of the hypothalamus. Glucokinase regulatory protein gene expression gave rise to a protein of 69 kDa mainly in nuclear and soluble cell fractions. Glutathione S‐transferase protein fused either to rat liver or human pancreatic islet glucokinase were able to precipitate glucokinase regulatory protein from liver or hypothalamic extracts in the presence of fructose‐6‐phosphate, the amount of protein co‐precipitated being decreased with fructose‐1‐phosphate. These findings suggest that the presence of glucokinase and glucokinase regulatory protein in the rat brain would facilitate the adaptation of this organ to fluctuations in blood glucose concentrations, and both proteins may participate in glucose‐sensing and metabolic regulation in the central nervous system.

Autoradiographic localization of receptors for glucagon-like peptide-1(7-36) amide in rat brain

Glucagon-like peptide-1 (GLP-1) has a sparse but well defined distribution in the rat brain where it is co-localized with glucagon-like immunoreactivity due to other fragments of the glucagon precursor. We have investigated the localization of GLP-1 receptors in rat brain using mono-125I-iodinated GLP-1(7-36) amide, the biologically active form of the peptide that occurs in brain, as the tracer for binding and autoradiographic studies of tissue sections. Displaceable binding of the label was sharply localized to discrete areas, being high in mamillary nuclei, the arcuate nucleus, nucleus of the solitary tract and the pretectal area, intermediate in the lateral septal nuclei, olfactory bulb, dorsal tegmental nuclei and the interpenduncular nucleus, and low in other regions. These results indicate areas where GLP-1(7-36) amide may have a role as a neurotransmitter or neuromodulator.

Signaling and biological effects of glucagon-like peptide 1 on the differentiation of mesenchymal stem cells from human bone marrow

Glucagon-like peptide 1 (GLP-1) functions as an incretin hormone with antidiabetogenic properties. However, the role of GLP-1 in human bone marrow-derived mesenchymal stem cells (hMSCs), if any, remains unknown. The effects of GLP-1 on hMSCs were tested with regard to cell proliferation, cytoprotection, and cell differentiation into adipocytes. The signaling pathways involved in these processes were also analyzed. Cells were characterized with biochemical and morphological approaches before and after being induced to differentiate into adipocytes. PCNA protein levels were used as a proliferation index, whereas cell apoptosis was studied by deprivation of fetal bovine serum. Isolated hMSCs expressed stem cell markers as well as mRNA and GLP-1 receptor protein. GLP-1 increased the proliferation of hMSCs, which decreased when they were induced to differentiate into adipocytes. This process produced biochemical and morphological changes in cells expressing PPARgamma, C/EBPbeta, AP2, and LPL in a time-dependent pattern. Notably, GLP-1 significantly reduced the expression of PPARgamma, C/EBPbeta, and LPL. These effects were exerted at least through the MEK and PKC signaling pathways. In addition, GLP-1 significantly reduced cell apoptosis. Our data indicate that, in hMSCs, GLP-1 promotes cellular proliferation and cytoprotection and prevents cell differentiation into adipocytes. These latter findings underscore the potential therapeutic role of GLP-1 in preventing the adipocyte hyperplasia associated with obesity and, additionally, could bolster the maintenance of hMSC stores by promoting the proliferation and cytoprotection of undifferentiated hMSC.