José A. Zueco

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.

Coexpression of glucagon-like peptide-1 (GLP-1) receptor, vasopressin, and oxytocin mRNAs in neurons of the rat hypothalamic supraoptic and paraventricular nuclei: effect of GLP-1(7-36)amide on vasopressin and oxytocin release.

Abstract : This study was designed to gain better insight into the relationship between glucagon‐like peptide‐1 (GLP‐1) (7‐36) amide and vasopressin (AVP) and oxytocin (OX). In situ hybridization histochemistry revealed colocalization of the mRNAs for GLP‐1 receptor, AVP, and OX in neurons of the hypothalamic supraoptic and paraventricular nuclei. To determine whether GLP‐1(7‐36)amide alters AVP and/or OX release, both in vivo and in vitro experimental study designs were used. In vivo, intravenous administration of 1 μg of GLP‐1(7‐36)amide into the jugular vein significantly decreased plasma AVP and OX concentrations. In vitro incubation of the neurohypophysis with either 0.1 or 1 μg of GLP‐1(7‐36)amide did not modify the release of AVP. However, addition of 1 μg of GLP‐1(7‐36)amide to the incubation medium increased slightly the secretion of OX. The coexpression of GLP‐1 receptor and AVP mRNAs in hypothalamic supraoptic and paraventricular nuclei gives further support to the already reported central effects of GLP‐1(7‐36)amide on AVP. Our findings also suggest a dual secretory response of AVP and OX to the effect of GLP‐1(7‐36)amide, which most likely is related to the amount and/or the route of peptide administration.

Evidence That Circadian Variations of Circulating Melatonin Levels in Fetal and Suckling Rats Are Dependent on Maternal Melatonin Transfer

Although the circadian variation of melatonin content in the pineal gland appears during the 3rd week of extrauterine life, recent studies suggest that the fetus perceives the day length through maternal melatonin transfer. Accordingly, we determined serum melatonin concentrations in pregnant and lactating rats and in their offsprings during the day (D) and at night (N). As compared with nonpregnant adult female rats (D: 6.0 +/- 0.5 and N: 112.0 +/- 5.0 pg/ml), significant increases of serum melatonin concentrations, both during the day and at night, were observed in 21-day pregnant (D: 21.0 +/- 2.6 and N: 222.0 +/- 7.2 pg/ml) and in 10-day (D: 20.0 +/- 2.6 and N: 145.0 +/- 12.3 pg/ml) and 20-day (D: 19.0 +/- 1.2 and N: 140.0 +/- 8.0 pg/ml) lactating animals, while a profound decrease was found in 5-day (D: 13.0 +/- 1.0 and N: 50.0 +/- 5.6 pg/ml) lactating rats. In addition, melatonin levels were significantly higher during the night than during the day in all the experimental groups. High levels and circadian variation of serum melatonin were detected also in 21-day-old fetuses (D: 13.0 +/- 0.8 and N: 108.0 +/- 4.8 pg/ml) and in 5- and 10-day-old suckling (in 5-day-old, D: 13.0 +/- 0.6 and N: 71.0 +/- 3.1 pg/ml; in 10-day-old, D: 17.0 +/- 1.4 and N: 63.0 +/- 3.8 pg/ml) rats.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Cyclosporin A induces apoptosis in rat hepatocytes in culture.

Abstract Cyclosporin A (CsA) at concentrations up to 1 μM induced apoptosis in a dose-dependent manner in cultured rat hepatocytes for 48 h in the presence of insulin and epidermal growth factor (EGF). The effect of CsA was evidenced by the DNA fragmentation pattern constituted by fragments of multiples of 180–200 base pairs, which is a characteristic of programmed cell death. The metabolic activity did not change significantly in the presence of 0.1 μM CsA and diminished to 49% of control in the presence of 1 μM CsA. Changes in the metabolic activity were correlated with a decrease in both [methyl-3H]thymidine uptake and DNA content, which reflects a decrease in the cell number. The treatment of cells with CsA (1 μM) decreased the metabolic activity/DNA content ratio by 24% with respect to dimethyl sulphoxide (DMSO) control, which also suggests, under these conditions, that the necrosis achieved is at most only 24%. In addition, the changes observed (apoptotic process, arrest of the cell cycle and apparition of a necrotic process) were correlated with an increase in the high-affinity guanosine triphosphatase (GTPase) enzymes.

Insulin induces a similar reduction in the concentrations of its own receptor and of an insulin‐sensitive glycosyl‐phosphatidylinositol in isolated rat hepatocytes

We have used isolated rat hepatocytes to study whether the insulin‐induced reduction of its own receptors may modify the transduction of hormone signals by changes in the content of a glycosyl‐phosphatidylinositol. Both subsequent insulin binding and glycosyl‐phosphatidylinositol concentrations markedly decreased as a function of time and insulin concentration during preincubation of hepatocytes with insulin. The modifications observed in insulin binding were due to changes in receptor concentration. These results show that insulin regulates both the number of its own receptors and glycosyl‐phosphatidylinositol concentrations in target cells, which may be of interest in many pathophysiological situations.

Insulin Does Not Induce the Hydrolysis of a Glycosyl Phosphatidylinositol in Rat Fetal Hepatocytes

An inositol phosphoglycan that is the polar head group of a glycosyl phosphatidylinositol has been considered as a putative mediator of insulin action. To gain insight into the functions of this hormone during development, the relationships between insulin, insulin receptors, glycosyl phosphatidylinositol, and inositol phosphoglycan were studied. Glycosyl phosphatidylinositol was isolated and characterized in fetal liver as early as day 15 of intrauterine life. In isolated hepatocytes from fetal and adult rats labeled with [3H]glucosamine, [3H]galactose, or [3H]myo-inositol, these molecules were incorporated into glycosyl phosphatidylinositol. In hepatocytes labeled with [3H]glucosamine and then allowed to react with [1-14C]IAI, the [3H]glycosyl phosphatidylinositol was purified as the 14C-labeled amidinated lipid. Glycosyl phosphatidylinositol molecules from fetal and adult cells were sensitive to hydrolysis by a phosphatidylinositol-specific phospholipase C from B. cereus. The product of this hydrolysis inhibits the activity of a cAMP-dependent protein kinase, whereas this effect was abolished by nitrous acid deamination. In isolated hepatocytes from adult animals, an inverse correlation between extracellular insulin and the number of insulin receptors and the cellular content of glycosyl phosphatidylinositol was observed. However, in fetal hepatocytes insulin failed to reduce the glycosyl-phosphatidylinositol content when labeled either with [1-14C]isethionyl acetimidate or [3H]glucosamine, whereas insulin-like growth factor I produced a significant hydrolysis of glycosyl phosphatidylinositol. Fetal and adult hepatocytes were incubated with insulin or inositol phosphoglycan after which glycogen phosphorylase activities were determined. Inositol phosphoglycan mimicked the action of insulin on both forms of the enzyme from adult hepatocytes, whereas in fetal cells insulin did not change, and purified inositol phosphoglycan reduced the activities of glycogen phosphorylase. These findings suggest a dissociation between insulin receptor occupancy and the expected hormonal effects in fetal hepatocytes. This could be related to alterations at a postreceptor level.

Inhibitory Effect of Cyclosporin A Peptide on Rat Hepatocytes Proliferation Induced by Mitogens

Treatment of cultured rat hepatocytes with cyclosporin A (0.01-1 microM) for 24, 48, or 72 h in the presence of insulin and epidermal growth factor induced an inhibition on cell proliferation in a time- and concentration-dependent manner, with an IC50 = 0.05 microM CsA corresponding to 48-h treatment. The inhibitory effect of CsA at < or = 0.1 microM doses for 48 h on [3H]thymidine uptake was reversed after withdrawal of the drug and subsequent addition of insulin plus EGF or serum; however, at 1 microM CsA the effect was irreversible and numerous bright small vesicles were observed. The molecular mechanism involved in CsA action in hepatocytes seems to be independent on cAMP and pertussis-toxin sensitive G proteins.