Isabel Roncero

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.

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.

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.

Effects of novel maturity-onset diabetes of the young (MODY)-associated mutations on glucokinase activity and protein stability

Glucokinase acts as the pancreatic glucose sensor and plays a critical role in the regulation of insulin secretion by the beta-cell. Heterozygous mutations in the glucokinase-encoding GCK gene, which result in a reduction of the enzymatic activity, cause the monogenic form of diabetes, MODY2 (maturity-onset diabetes of the young 2). We have identified and functionally characterized missense mutations in the GCK gene in diabetic families that result in protein mutations Leu165-->Phe, Glu265-->Lys and Thr206-->Met. The first two are novel GCK mutations that co-segregate with the diabetes phenotype in their respective families and are not found in more than 50 healthy control individuals. In order to measure the biochemical effects of these missense mutations on glucokinase activity, we bacterially expressed and affinity-purified islet human glucokinase proteins carrying the respective mutations and fused to GST (glutathione S-transferase). Enzymatic assays on the recombinant proteins revealed that mutations Thr206-->Met and Leu165-->Phe strongly affect the kinetic parameters of glucokinase, in agreement with the localization of both residues close to the active site of the enzyme. In contrast, mutation Glu265-->Lys, which has a weaker effect on the kinetics of glucokinase, strongly affects the protein stability, suggesting a possible structural defect of this mutant protein. Finally, none of the mutations tested appears to affect the interaction of gluco-kinase with the glucokinase regulatory protein in the yeast two-hybrid system.

Glucagon-like peptide-1 (7-36) amide as a novel neuropeptide.

Although earlier studies indicated that GLP-1 (7–36) amide was an intestinal peptide with a potent effect on glucose-dependent insulin secretion, later on it was found that several biological effects of this peptide occur in the brain, rather than in peripheral tissues. Thus, proglucagon is expressed in pancreas, intestine, and brain, but post translational processing of the precursor yields different products in these organs, glucagon-like peptide-1 (7–36) amide being one of the forms produced in the brain. Also, GLP-1 receptor cDNA from human and rat brains has been cloned and sequenced, and the deduced amino acid sequences are the same as those found in pancreatic islets. Through these receptors, GLP-1 (7–36) amide from gut or brain sources induces its effects on the release of neurotransmitters from selective brain nuclei, the inhibition of gastric secretion and motility, the regulation of food and drink intake, thermoregulation, and arterial blood pressure. Central administration (icv) of GLP-1 (7–36) amide produces a marked reduction in food and water intake, and the colocalization of the GLP-1 receptor, GLUT-2, and glucokinase mRNAs in hypothalamic neurons involved in glucose sensing suggests that these cells may be involved in the transduction of signals needed to produce a state of fullness. In addition, GLP-1 (7–36) amide inhibits gastric acid secretion and gastric emptying, but these effects are not found in vagotomized subjects, suggesting a centrally mediated effect. Similar results have been found with the action of this peptide on arterial blood pressure and heart rate in rats. Synthesis of GLP-1 (7–36) amide and its own receptors in the brain together with its abovementioned central physiological effects imply that this peptide may be considered a neuropeptide. Also, the presence of GLP-1 (7–36) amide in the synaptosome fraction and its calcium-dependent release by potassium stimulation, suggest that the peptide may act as a neurotransmitter although further electrophysiological and ultrastructural studies are needed to confirm this possibility.

Effects of glucose and insulin on glucokinase activity in rat hypothalamus

In an attempt to study the role of glucokinase (GK) and the effects of glucose and peptides on GK gene expression and on the activity of this enzyme in the hypothalamus, we used two kinds of biological models: hypothalamic GT1-7 cells and rat hypothalamic slices. The expression of the GK gene in GT1-7 cells was reduced by insulin (INS) and was not modified by different glucose concentrations, while GK enzyme activities were significantly reduced by the different peptides. Interestingly, a distinctive pattern of GK activities between the ventromedial hypothalamus (VMH) and lateral hypothalamus (LH) were found, with higher enzyme activities in the VMH as the glucose concentrations rose, while LH enzyme activities decreased at 2.8 and 20 mM glucose, the latter effect being prevented by incubation with INS. These effects were produced only by d-glucose and the modifications found were due to GK, but not to other hexokinases. In addition, GK activities in the VMH and the LH were reduced by glucagon-like peptide 1, leptin, orexin B, INS, and neuropeptide Y (NPY), but this effect was only statistically significant for NPY in LH. Our results indicate that the effects of both glucose and peptides occur on GK enzyme activities rather than on GK gene transcription. Moreover, the effects of glucose and INS on GK activity suggest that in the brain GK behaves in a manner opposite to that in the liver, which might facilitate its role in glucose sensing. Finally, hypothalamic slices seem to offer a good physiological model to discriminate the effects between different areas.

Effects of aging on respiration, ATP levels and calcium transport in rat liver mitochondria. Response to theophylline.

The concept that aging results in an impairment of mitochondrial biochemistry has been tested on organelles isolated from the liver of 3-4-month-old and 24-month-old rats of the Wistar strain. Our data suggest that aging results in significant decreases in succinate-supported respiration, ATP levels and calcium uptake. When theophylline was added to the incubation mixture, both respiration and calcium uptake were depressed in approximately the same proportion in the mitochondria from old rats, although the mitochondrial ATP of young animals was significantly decreased by this substance.

Morphological and functional studies during aging at mitochondrial level action of drugs

1. Mitochondria show morphological changes on aging: the volume density decreases and the total surface area or volume increase. 2. Biochemical studies indicated a decrease in the rate of oxygen consumption and ATP levels for increasing age. Measurements of calcium transport across the mitochondrial membrane revealed a decrease in accumulated calcium and an alteration in the uptake kinetics of the ion in older animals. 3. Theophylline and calcitonin action were also studied. In both age groups (young and old) theophylline shows an inhibitory effect on all the parameters studied (both morphological and biochemical). 4. Calcitonin showed no effect on morphological and respiratory parameters, although it increased calcium uptake into the mitochondrion to a lesser extent in the 24 month old animals.