Glucokinase and glucokinase regulatory proteins are functionally coexpressed before birth in the rat brain.

Our previous description of functional glucokinase (GK) isoforms and their interactions with glucokinase regulatory protein (GKRP) in adult rat and human brains suggested that both participate in glucose sensing in the central nervous system. To determine whether both proteins are coexpressed and active before birth or during early post-natal life, we characterised these molecules in the brains of foetal and post-natal pup rats. We found GK and GKRP mRNAs that were similar to those previously reported in the adult rat brain. Likewise, GK and GKRP gene expression gave rise to proteins of 52 and 69 kDa, respectively. Immunohistochemistry experiments showed the colocalisation of both GK and GKRP proteins in the same brain cells of 21-day-old rat foetuses. Furthermore, coprecipitation of GK and GKRP in the presence of fructose 6-phosphate suggests interactions between both proteins. The presence of GK phosphorylating activity was detected in different brain areas of 21-day-old foetuses with a contribution to the total glucose-phosphorylating activity of between 17.2 +/- 1.7% and 12.4 +/- 3.7%, with the hypothalamus being the region of maximum activity. The hypothalamic GK activity in 21-day-old foetuses has a high apparent K(m) for glucose and no product inhibition by glucose 6-phosphate. Our findings indicate that both proteins may be functionally active before birth and that they can act within a glucose sensor system involved in controlling food intake.

Peripheral versus central effects of glucagon-like peptide-1 receptor agonists on satiety and body weight loss in Zucker obese rats.

The present study explores the potential utility of peripheral versus central administration of glucagon-like peptide-1 (GLP-1) receptor agonists in the regulation of feeding behavior in Wistar and Zucker obese rats. Acute central (intracerebroventricular [i.c.v.]) and peripheral (subcutaneous [s.c.]) administration of both GLP-1 (7-36) amide and exendin-4 resulted in a reduction in food intake for at least 4 hours, exendin-4 being much more potent than GLP-1 (7-36) amide, especially after peripheral administration. Both Zucker obese rats (fa/fa) and their lean littermates (Fa/-) responded to acute central and peripheral administration of exendin-4. Moreover, in situ hybridization revealed specific labeling for the mRNA for GLP-1 receptors in several brain areas of both the obese and lean rats. The presence of this receptor was also detected by affinity cross-linking assays. Long-term s.c. administration of exendin-4 (1 single injection per day, 1 hour prior to the onset of the dark phase of the cycle) decreased daily food intake and practically blocked weight gain in obese rats. In contrast to previous studies, these findings show that peripheral (s.c.) administration of both GLP-1 receptor agonists also induces satiety and weight loss in rats, and suggest the potential usefulness of exendin-4 as a therapeutic tool for the treatment of diabetes and/or obesity.

Functional glucokinase isoforms are expressed in rat brain.

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.

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.

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

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 brain-stem. First-strand cDNA was prepared by using RNA from hypothalamus, brainstem, and R1Nm5F 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 olfactory 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.

Effect of oxodipine on calcium transport in isolated hepatocytes and rat liver mitochondria at different ages.

Electron microscopy and biochemical studies were carried out in hepatocytes and mitochondria isolated from livers of young (3-6 month) and old (20-24 month) rats to know under which conditions oxodipine, a calcium antagonist, affects calcium metabolism, the mitochondrial morphology and the differences due to the animals age. Under steady-state conditions, calcium transport through the membrane of isolated hepatocytes was not affected by the presence of the drug in either group of animals studied. By contrast, a clear age-dependent inhibitory effect on the uptake and accumulation of calcium was observed in mitochondria. In the young animals, evidence was observed for a complex mechanism related to cellular respiration and to a possible specific action of the drug. In older animals, this second mechanism appears to be very weak or inexistent. Quantitative morphological studies also point to a deterioration in the mitochondria due to the effect of the drug at the concentrations at which cellular respiration was affected.

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.

Changes induced by caffeine, theophylline and theobromine on calcium uptake, respiration and ATP levels in rat-liver mitochondria.

1. The inhibition of calcium uptake and cellular respiration depend on the concentration of the compounds as shown by the concentration-effect curves. 2. The concentrations at which 50% inhibition of the transport of calcium takes place (caffeine 45 mM, theophylline 12 mM, theobromine, 4 mM) do not coincide exactly with those that produce the same effect on cellular respiration (caffeine 60 mM, theophylline 22 mM, theobromine 8 mM). 3. ATP concentrations under different conditions were also determined; a decrease in their value induced by the drugs was observed. No significant differences were observed, however, between the effect produced by the methyl-xanthines. 4. These findings suggest that these compounds are able to affect in some way the maintenance of energy gradients linked to the effects studied.

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.

Effects of salicylate on insulin and glucagon secretion by the isolated and perfused rat pancreas.

The effects of sodium salicylate, a prostaglandin synthesis inhibitor, on glucose-induced secretion of insulin and glucagon by the isolated perfused rat pancreas have been studied. Sodium salicylate inhibited both basal (2.8 mM glucose) and stimulated (16.7 mM glucose) insulin release in a dose dependent manner (1, 5 and 10 mM). This inhibition is not interpretable in terms of a simple inhibition of cyclooxygenase by sodium salicylate. Basal glucagon release was not changed by 1 mM sodium salicylate but the latter partially blocked its inhibition by 16.7 mM glucose. Higher doses of sodium salicylate (5 and 10 mM) inhibited basal glucagon secretion without affecting its response to 16.7 mM glucose. These findings suggest a predominant stimulatory action of endogenous prostaglandins on glucagon release.