Elvira Alvarez

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.

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.

Direct Evidence of Imbalanced Seagrass (Posidonia oceanica) Shoot Population Dynamics in the Spanish Mediterranean

Direct census of shoots tagged in permanent plots was used to assess the present (2000–2002)Posidonia oceanica population dynamics in 25 meadows along the Spanish Mediterranean Coast. Shoot density ranged from 154±8 to 1,551±454 shoots m−2, absolute shoot mortality from 5±0 to 249±53 shoots m−2 yr−1, and absolute shoot recruitment from <5 ±1 to 62±42 shoots m−2yr−1. Specific shoot mortality and recruitment rates, which are mathematically and statistically (p>0.05) independent of shoot density, varied from 0.015±0.006 to 0.282±0.138 yr−1 and 0.018±0.005 to 0.302±0.093 yr−1, respectively. Absolute shoot mortality rate was scaled to shoot density (Pearson correlation, r=0.78, p<0.0001), and variability in specific shoot recruitment rate was partially due to differences in the percentage of growing apexes, which produce most of the recruits within the population (Pearson correlation, r=0.50, p<0.001), demonstrating the existence of structural constraints on shoot demography. Shoot half-life was estimated to range from 2.5 to 60.4 yr and meadow turnover times between 6.7 yr and more than a century, provided current estimates of shoot mortality, recruitment rates, and density remain uniform. There were differences in shoot mortality and recruitment at the regional scale, with the meadows developing along the coast of the Spanish mainland experiencing the highest shoot mortality (Tukey test, p<0.05) and tending to exhibit the highest shoot recruitment. The low shoot recruitment did not balance shoot mortality in most (60%) of the meadows, showing a prevalence of declining populations among the 25 meadows studied (Wilcoxon ranked sign test, p<0.0005). This study demonstrates the power of direct census of seagrass shoots in permanent plots to evaluate the present status of seagrass meadows, to detect on-going population decline, and to provide some insight onto the possible factors involved. The incorporation of direct census of seagrass meadows to monitoring programs will help provide the early-warning signals necessary to support management decisions to conserve seagrass meadows.

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.

Benthic input rates predict seagrass (Posidonia oceanica) fish farm-induced decline

Fish farms represent a growing source of anthropogenic disturbance to benthic communities, and efficient predictors of such impacts are urgently needed. We explored the effects of fish farm benthic organic and nutrient inputs on the population dynamics of a key seagrass species (Posidonia oceanica) in four Mediterranean deep meadows adjacent to sea bream and sea bass farms. We performed two annual plant censuses on permanent plots at increasing distance from farms and measured benthic sedimentation rates around plots. High shoot mortality rates were recorded near the cages, up to 20 times greater than at control sites. Recruitment rates increased in variability but could not compensate mortality, leading to rapid seagrass decline within the first 100 m from cages. Seagrass mortality increased with total sedimentation rates (K=0.55, p<0.0002), and with organic matter (K=0.50, p=0.001), total nitrogen (K=0.46, p=0.002) and total phosphorus (K=0.56, p<3.10(-5)) inputs. P. oceanica decline accelerated above a phosphorus loading threshold of 50mg m(-2)day(-1). Phosphorus benthic sedimentation rate seems a powerful predictor of seagrass mortality from fish farming. Coupling direct measurements of benthic sedimentation rates with dynamics of key benthic species is proposed as an efficient strategy to predict fish farm impacts to benthic communities.

Expression of glucose transporter isoform GLUT-2 and glucokinase genes in human brain

The glucose transporter isoform‐2 (GLUT‐2) and glucokinase are considered to be components of a glucose sensor system controlling several key processes, and hence may modulate feeding behaviour. We have found GLUT‐2 and glucokinase mRNAs in several brain regions, including the ventromedial and arcuate nuclei of the hypothalamus. GLUT‐2, glucokinase and glucokinase regulatory protein mRNAs and proteins were present in these areas as determined by biochemical approaches. In addition, glucose‐phosphorylating activity with a high apparent Km for glucose that displayed no product inhibition by glucose‐6‐phosphate was observed. Increased glycaemia after meals may be recognized by specific hypothalamic neurones due to the high Km of GLUT‐2 and glucokinase. This enzyme is considered to be the true glucose sensor because it catalyses the rate‐limiting step of glucose catabolism its activity being regulated by interaction with glucokinase regulatory protein, that functions as a metabolic sensor.

Effect of Pinealectomy on Liver Insulin and Glucagon Receptor Concentrations in the Rat

The studies described here were undertaken to characterize the hepatic insulin and glucagon receptors of control (C), pinealectomized (Pn), and melatonin‐treated pinealectomized (Pn + Mel) rats. Compared with C rats, an increase in plasma glucose and glucagon levels and a reduction in circulating concentrations of insulin in Pn animals were observed. Melatonin treatment of Pn rats reverses all three parameters toward the normal values. In liver membranes, insulin binding was lower in Pn than in C rats, and glucagon binding was greater in Pn than in C animals; in Pn + Mel rats both insul in and glucagon binding reverse toward the normal values that were observed in C rats. The modifications in hormone binding reflect changes in the number of receptors but not in the affinity constants. The time courses of hormone association and dissociation from liver membranes were similar in all three experimental groups. The degradation of both hormones by liver membranes was similar in all three groups. Insulin receptor degradation also was similar in the three groups, while glucagon receptor degradation was similar in the liver membranes of C and Pn rats but smaller in Pn + Mel animals. These findings suggest that the pineal gland may modulate the circulating levels and liver receptor concentrations of insulin and glucagon. In addition, our results indicate that insulin and glucagon did not induce a down‐regulation of liver receptors in Pn rats.

Increased glucagon-like peptide-1 receptor expression in glia after mechanical lesion of the rat brain

Glucagon-like peptide-1 (GLP-1)(7-36) amide, a member of the glucagon and related peptides family, and its receptor have an anatomically specific expression in the brain. Furthermore, the GLP-1 receptor is expressed in both neurons and glia. Because after a penetrating injury a large population of astrocytes become activated and augment their expression of numerous substances, we have used in situ hybridization to determine whether the expression of the GLP-1 receptor increases in response to a penetrating injury. We have found that GLP-1 receptor expression increases dramatically along the border of the injury. Furthermore, this expression can be colocalized to glial fibrillary acidic protein (GFAP) and non-GFAP mRNA containing cells, suggesting that at least part of this increase is due to an increase in GLP-1 receptor expression in glial cells.