A. Barber

Distribution of the long leptin receptor isoform in brush border, basolateral membrane, and cytoplasm of enterocytes

Background and aim: Leptin, a hormone mainly produced by fat cells, acts primarily on the hypothalamus regulating energy expenditure and food intake. Leptin receptors are expressed in several tissues and the possible physiological role of leptin is being extensively investigated, with the result that important peripheral actions of the hormone in the organism are being discovered. Recent studies have demonstrated leptin and leptin receptor expression in gastric epithelial cells. In the present study, we report the presence of the long leptin receptor isoform (OB-Rb) in human, rat, and mouse small intestine, supporting the hypothesis of leptin as a hormone involved in gastrointestinal function. Methods: The presence of the leptin receptor was determined by immunocytochemical methods using antibodies against the peptide corresponding to the carboxy terminus of the long isoform of the leptin receptor. Human duodenal biopsies from normal individuals undergoing gastrointestinal endoscopy, and intestinal fragments of Wistar rats and Swiss mice were processed for the study. Results: Immunoreactivity for the long leptin receptor isoform was observed in the three studied species. Staining was located throughout the cytoplasm of the enterocytes, of both villi and crypts, and in the basolateral plasma membrane. Immunolabelling for OB-Rb protein was also found in the brush border of human enterocytes of formol and paraformaldehyde fixed samples. Conclusion: This report demonstrates the presence of the long leptin receptor isoform in the absorptive cells of rat, mouse, and human small intestine, suggesting that leptin could have a physiological role in the regulation of nutrient absorption.

Presence of leptin receptors in rat small intestine and leptin effect on sugar absorption

Leptin is involved in food intake and thermogenesis regulation. Since leptin receptor expression has been found in several tissues including small intestine, a possible role of leptin in sugar absorption by the intestine was investigated. Leptin inhibited d‐galactose uptake by rat small intestinal rings 33% after 5 min of incubation. The inhibition increased to 56% after 30 min. However, neither at 5 min nor at 30 min did leptin prevent intracellular galactose accumulation. This leptin effect was accompanied by a decrease of the active sugar transport apparent V max (20 vs. 4.8 μmol/g wet weight 5 min) and apparent K m (15.8 vs. 5.3 mM) without any change in the phlorizin‐resistant component. On the other hand, immunohistochemical experiments using anti‐leptin monoclonal antibodies recognized leptin receptors in the plasma membrane of immune cells located in the lamina propria. These results indicate for the first time that leptin has a rapid inhibitory effect on sugar absorption and demonstrate the presence of leptin receptors in the intestinal mucosa.

Alterations in carbohydrate and lipid metabolism induced by a diet rich in coconut oil and cholesterol in a rat model

OBJECTIVE The type of dietary fat as well as the amount of cholesterol occurring in the diet have been associated with several metabolic disorders. Thus, the aim of the present study was to investigate the influence of a hypercholesterolemic diet enriched with coconut oil and cholesterol on carbohydrate and lipid metabolism in a rat model. METHODS Twenty male Wistar rats weighing about 190 g were assigned to two dietary groups. One group received a semipurified control diet and the other was given a diet enriched in coconut oil (25% by weight) and cholesterol (1% by weight) for 26 days. RESULTS Our results indicated a significant increase in serum total cholesterol (+285%; p<0.001), low-density lipoproteins (+154%; p<0.01), liver cholesterol (+1509%; p<0.001), as well as a significant increase in liver weight (+46%; p<0.001) in those rats fed the hypercholesterolemia-inducing diet as compared to controls. Moreover, a significant decrease in serum high-density lipoproteins (-67%; p<0.001), triacylglycerols levels (-33%; p<0.05), and abdominal fat weight (-39%; p<0.01) were found. The observed alterations in serum lipid and lipoprotein profile resembled a situation of type IIa hyperlipidemia in humans. Measurement of several enzymes concerned with lipid utilization revealed a significant increase in 3-hydroxy-3-methylglutaryl-CoA reductase activity (+68%; p<0.01) in the liver of animals fed the hypercholesterolemic diet, while a significant reduction in plasma lecithin-cholesterol acyltransferase activity (-66%; p<0.001) was found. The situation of hypoglycemia (-18%; p<0.05) was accompanied by lower levels of serum insulin (-45%; p<0.01) and liver glycogen (-30%; p<0.05) in the hypercholesterolemic rats. Furthermore, glucose utilization was altered since lower glucose-6-Pase (-33%; p<0.05) and increased glucokinase (+212%; p<0.001) activities in the liver were found in the rat model of hypercholesterolemia. CONCLUSION These results provide new evidence that a diet-induced hypercholesterolemia in rats is associated with several adaptative changes in carbohydrate metabolism. These findings may be of importance not only considering the role of western diets on cholesterogenesis, but also in other metabolic disturbances involving lipid and carbohydrate metabolism.

Lipoic acid prevents body weight gain induced by a high fat diet in rats: Effects on intestinal sugar transport

Several studies have suggested that oxidative stress might cause and aggravate the inflammatory state associated with obesity and could be the link between excessive weight gain and its related disorders such as insulin resistance and cardiovascular diseases. Thus, antioxidant treatment has been proposed as a therapy to prevent and manage obesity and associated complications. Therefore, the aim of the present study was to investigate the effects of supplementation of a standard or high fat diet with the antioxidant lipoic acid (LA) during 56 days, on body weight gain, adiposity, feed efficiency and intestinal sugar absorption, in male Wistar rats. LA supplementation induced a lower body weight gain and adipose tissue size in both control or high fat fed rats accompanied by a reduction in food intake. The group fed on a high fat diet and treated with LA (OLIP group) showed a lower body weight gain than its corresponding Pair-Fed (PF) group (P<0.05), which received the same amount of food than LA-treated animals but with no LA. In fact, LA induced a reduction on feed efficiency and also significantly decreased intestinal α-methylglucoside (α-MG) absorption both in lean and obese rats. These results suggest that the beneficial effects of dietary supplementation with LA on body weight gain are mediated, at least in part, by the reduction observed in food intake and feed efficiency. Furthemore, the inhibitory action of LA on intestinal sugar transport could explain in part the lower feed efficiency observed in LA-treated animals and therefore, highlighting the beneficial effects of LA on obesity.ResumenVarios estudios han sugerido que el estrés oxidativo podría actuar como desencadenante y agravante del estado inflamatorio asociado a la obesidad y podría ser un potencial nexo de unión entre la excesiva ganancia de peso y las co-morbilidades asociadas. Así, se ha propuesto el tratamineto con antioxidantes naturales como posible terapia contra el desarrollo de obesidad así como sus complicaciones asociadas. Por ello, el objeto del presente trabajo fue investigar en ratas Wistar macho los efectos de la suplementación de una dieta estándar o alta en grasa con un antioxidante, el ácido lipoico (AL) (0,25g/ 100g de comida) durante 56 días sobre la ganancia de peso corporal, la adiposidad, la eficiencia metabólica y la absorción intestinal de azúcares. La suplementación de la dieta con AL indujo una menor ganacia de peso corporal y redujo el tamaño del tejido adiposo blanco total, tanto en ratas alimentadas con dieta control como alta en grasa. Además, disminuyó la ingesta. La ganancia de peso en el grupo alimentado con dieta alta en grasa y AL fue menor que la de su correspondiente grupoPair-Fed (P<0,05), el cual recibía la misma cantidad de comida que los animales tratados con AL pero sin este ácido. De hecho, la suplementación con ácido lipoico redujo la eficiencia metabólica y disminuyó significativamente la absorción intestinal de α-metilglucósido (α-MG) tanto en ratas control como obesas. Estos resultados sugieren que los efectos beneficiosos de la suplementación de la dieta con AL sobre la ganancia de peso corporal están mediados, al menos en parte, por la reducción observada en la ingesta de comida y en la eficiencia metabólica. Además, la acción inhibitoria del AL sobre el transporte intestinal de azúcares podría explicar, en parte, la menor eficiencia metabólica observada en los animales tratados con AL justificando, por consiguiente, los efectos beneficiosos del AL sobre la obesidad.

Luminal leptin inhibits intestinal sugar absorption in vivo.

Aim:  We have previously demonstrated that leptin inhibits galactose absorption in rat intestinal everted rings and that leptin receptors are present in the apical membrane of the enterocytes. This adipocyte‐derived hormone is also secreted by gastric mucosal cells and is able to reach the intestinal lumen. The goal of the present study was to prove whether luminal leptin acts on intestinal sugar absorption in vivo both at low (basal state) and high sugar concentration (post‐prandial state).

Pharmacological evaluation of IQM-95,333, a highly selective CCKA receptor antagonist with anxiolytic-like activity in animal models

The pyridopyrimidine derivative IQM‐95,333 ((4aS,5R)‐2‐benzyl‐5‐[Nα‐tert‐butoxicarbonyl)L‐tryptophyl]amino‐1,3dioxoperhydropyrido[1,2‐c]pyrimidine), a new non‐peptide antagonist of cholecystokinin type A (CCKA) receptors, has been evaluated in vitro and in vivo in comparison with typical CCKA and CCKB receptor antagonists, such as devazepide, lorglumide, L‐365,260 and PD‐135,158. IQM‐95,333 displaced [3H]‐CCK‐8S binding to CCKA receptors from rat pancreas with a high potency in the nanomolar range. Conversely, the affinity of this new compound at brain CCKB receptors was negligible (IC50>10 μM). IQM‐95,333 was a more selective CCKA receptor ligand than devazepide and other CCKA receptor antagonists. Like devazepide, IQM‐95,333 was a more potent antagonist of CCK‐8S‐ than of CCK‐4‐induced contraction of the longitudinal muscle from guinea‐pig ileum, suggesting selective antagonism at CCKA receptors. IQM‐95,333 and devazepide were also potent inhibitors of CCK‐8S‐stimulated amylase release from isolated pancreatic acini, a CCKA receptor‐mediated effect. The drug concentrations required (IC50s around 20 nM) were higher than in binding studies to pancreas homogenates. Low doses (50–100 μg kg−1, i.p.) of IQM‐95,333 and devazepide, without any intrinsic effect on food intake or locomotion, blocked the hypophagia and the hypolocomotion induced by systemic administration of CCK‐8S, two effects associated with stimulation of peripheral CCKA receptors. IQM‐95,333 showed an anxiolytic‐like profile in the light/dark exploration test in mice over a wide dose range (10–5,000 μg kg−1). Typical CCKA and CCKB antagonists, devazepide and L‐365,260 respectively, were only effective within a more limited dose range. In a classical conflict paradigm for the study of anxiolytic drugs, the punished‐drinking test, IQM‐95,333, devazepide and L‐365,260 were effective within a narrow dose range. The dose‐response curve for the three drugs was biphasic, suggesting that other mechanisms are operative at higher doses. In conclusion, IQM‐95,333 is a potent and selective CCKA receptor antagonist both in vitro and in vivo with an anxiolytic‐like activity in two different animal models, which can only be attributed to blockade of this CCK receptor subtype.

Luminal leptin inhibits l-glutamine transport in rat small intestine: involvement of ASCT2 and B0AT1

L-glutamine is the primary metabolic fuel for enterocytes. Glutamine from the diet is transported into the absorptive cells by two sodium-dependent neutral amino acid transporters present at the apical membrane: ASCT2/SLC1A5 and B(0)AT1/SLC6A19. We have demonstrated that leptin is secreted into the stomach lumen after a meal and modulates the transport of sugars after binding to its receptors located at the brush border of the enterocytes. The present study was designed to address the effect of luminal leptin on Na(+)-dependent glutamine (Gln) transport in rat intestine and identify the transporters involved. We found that 0.2 nM leptin inhibited uptake of Gln and phenylalanine (Phe) (substrate of B(0)AT1) using everted intestinal rings. In Ussing chambers, 10 mM Gln absorption followed as Na(+)-induced short-circuit current was inhibited by leptin in a dose-dependent manner (maximum inhibition at 10 nM; I(C50) = approximately 0.1 nM). Phe absorption was also decreased by leptin. Western blot analysis after 3-min incubation of the intestinal loops with 10 mM Gln, showed marked increase of ASCT2 and B(0)AT1 protein in the brush-border membrane that was reduced by rapid preincubation of the intestinal lumen with 1 nM leptin. Similarly, the increase in ASCT2 and B(0)AT1 gene expression induced by 60-min incubation of the intestine with 10 mM Gln was strongly reduced after a short preincubation period with leptin. Altogether these data demonstrate that, in rat, leptin controls the active Gln entry through reduction of both B(0)AT1 and ASCT2 proteins traffic to the apical plasma membrane and modulation of their gene expression.

Leptin regulates sugar and amino acids transport in the human intestinal cell line Caco-2

Studies in rodents have shown that leptin controls sugars and glutamine entry in the enterocytes by regulating membrane transporters. Here, we have examined the effect of leptin on sugar and amino acids absorption in the human model of intestinal cells Caco‐2 and investigated the transporters involved.

Leptin effect on galactose absorption in mice jejunum.

Previous studies from our laboratory show that leptin inhibits sugar absorption in rat small intestine. In this work, the effect of the hormone on galactose absorption in mice jejunum is characterized. The experimentes were carried out in vitro by using everted jejunal rings. Leptin inhibits galactose uptake by the rings depending on the concentration of the hormone (0.20-78 nM) and time exposure. Maximal inhibition is obtained at 7.8 nM (2 min incubation) and at 0.39 nM (30 min). Leptin does not affect non mediated sugar uptake (measured in the presence of 1 mM phloridzin), indicating that hormone only alters SGLT1. Kinetic studies demonstrate that leptine increases the apparent affinity constant of the galactose transport without modifying the Vmax value. On the whole, the results indicate that leptin inhibits intestinal galactose absorption in mouse by decreasin affinity of SGLT1 for the sugar.

Na+ and pH dependence of proline and β-alanine absorption in rat small intestine

Aims:  Early characterization of intestinal absorption of imino acids in mammals has demonstrated the existence of a Na+‐dependent, Cl−‐independent transport system in rat small intestine, which is the only carrier for β‐alanine. Based on the substrate selectivity, it was proposed that the Proton Amino Acid Transporter 1 (PAT1) could be the same as this imino acid carrier. The present study characterizes the pH and Na+ dependence of proline and β‐alanine uptake in rat small intestine.