Jaione Barrenetxe

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.

Leptin resistance and diet-induced obesity: central and peripheral actions of leptin

Obesity is a chronic disease that represents one of the most serious global health burdens associated to an excess of body fat resulting from an imbalance between energy intake and expenditure, which is regulated by environmental and genetic interactions. The adipose-derived hormone leptin acts via a specific receptor in the brain to regulate energy balance and body weight, although this protein can also elicit a myriad of actions in peripheral tissues. Obese individuals, rather than be leptin deficient, have in most cases, high levels of circulating leptin. The failure of these high levels to control body weight suggests the presence of a resistance process to the hormone that could be partly responsible of disturbances on body weight regulation. Furthermore, leptin resistance can impair physiological peripheral functions of leptin such as lipid and carbohydrate metabolism and nutrient intestinal utilization. The present document summarizes those findings regarding leptin resistance development and the role of this hormone in the development and maintenance of an obese state. Thus, we focused on the effect of the impaired leptin action on adipose tissue, liver, skeletal muscle and intestinal function and the accompanying relationships with diet-induced obesity. The involvement of some inflammatory mediators implicated in the development of obesity and their roles in leptin resistance development are also discussed.

Physiological and metabolic functions of melatonin

Melatonin is a lipophilic hormone, mainly produced and secreted at night by the pineal gland. Melatonin synthesis is under the control of postganglionic sympathetic fibers that innervates the pineal gland. Melatonin acts via high affinity G protein-coupled membrane receptors. To date, three different receptor subtypes have been identified in mammals: MT1 (Mel 1a) and MT2 (Mel 1b) and a putative binding site called MT3. The chronobiotic properties of the hormone for resynchronization of sleep and circadian rhythms disturbances has been demonstrated both in animal models or in clinical trials. Several other physiological effects of melatonin in diffrent peripheral tissues have been described in the past years. In this way, it has been demonstrated that the hormone is involved in the regulation of seasonal reproduction, body weight and energy balance. This contribution has been focused to review some of the physiological functions of melatonin as well as the role of the hormone in the regulation of energy balance and its possible involvement in the development of obesity.ResumenLa melatonina es una hormona producida fundamentalmente durante la noche por la glándula pineal. La síntesis de melatonina está controlada por las fibras simpáticas postglanglionares que inervan la glándula pineal. La melatonina actúa a través de receptores de membrana de alta afinidad acoplados a proteína G. Hasta la fecha, tres diferentes subtipos de receptores han sido identificados en mamíferos: MT1 (Mel 1a), MT2 (Mel 1b) y un posible sitio de unión denominado MT3. Las propiedades cronobióticas de esta hormona se han descrito tanto en modelos animales como en ensayos con humanos, participando en la sincronización del sueño y los ritmos circadianos. También se han observado en los últimos años otros efectos fisiológicos de la melatonina en diferentes tejidos periféricos. Así, se ha comprobado que esta hormona está implicada en la regulación de la reproducción estacional y en el control tanto del peso corporal como del balance energético. Esta revisión supone una actualización de las functiones fisiológicas de la melatonina así como el papel de esta hormona en la regulación de la homeostasis energética y su posible implicación en la obesidad.

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.

Helichrysum and grapefruit extracts inhibit carbohydrate digestion and absorption, improving postprandial glucose levels and hyperinsulinemia in rats.

Several plant extracts rich in flavonoids have been reported to improve hyperglycemia by inhibiting digestive enzyme activities and SGLT1-mediated glucose uptake. In this study, helichrysum ( Helichrysum italicum ) and grapefruit ( Citrus × paradisi ) extracts inhibited in vitro enzyme activities. The helichrysum extract showed higher inhibitory activity of α-glucosidase (IC50 = 0.19 mg/mL) than α-amylase (IC50 = 0.83 mg/mL), whereas the grapefruit extract presented similar α-amylase and α-glucosidase inhibitory activities (IC50 = 0.42 mg/mL and IC50 = 0.41 mg/mL, respectively). Both extracts reduced maltose digestion in noneverted intestinal sacs (57% with helichrysum and 46% with grapefruit). Likewise, both extracts inhibited SGLT1-mediated methylglucoside uptake in Caco-2 cells in the presence of Na(+) (56% of inhibition with helichrysum and 54% with grapefruit). In vivo studies demonstrated that helichrysum decreased blood glucose levels after an oral maltose tolerance test (OMTT), and both extracts reduced postprandial glucose levels after the oral starch tolerance test (OSTT). Finally, both extracts improved hyperinsulinemia (31% with helichrysum and 50% with grapefruit) and HOMA index (47% with helichrysum and 54% with grapefruit) in a dietary model of insulin resistance in rats. In summary, helichrysum and grapefruit extracts improve postprandial glycemic control in rats, possibly by inhibiting α-glucosidase and α-amylase enzyme activities and decreasing SGLT1-mediated glucose uptake.

TNFα regulates sugar transporters in the human intestinal epithelial cell line Caco-2

PURPOSE During intestinal inflammation TNFα levels are increased and as a consequence malabsorption of nutrients may occur. We have previously demonstrated that TNFα inhibits galactose, fructose and leucine intestinal absorption in animal models. In continuation with our work, the purpose of the present study was to investigate in the human intestinal epithelial cell line Caco-2, the effect of TNFα on sugar transport and to identify the intracellular mechanisms involved. METHODS Caco-2 cells were grown on culture plates and pre-incubated during different periods with various TNFα concentrations before measuring the apical uptake of galactose, α-methyl-glucoside (MG) or fructose for 15 min. To elucidate the signaling pathway implicated, cells were pre-incubated for 30min with the PKA inhibitor H-89 or the PKC inhibitor chelerythrine, before measuring the sugar uptake. The expression in the apical membrane of the transporters implicated in the sugars uptake process (SGLT1 and GLUT5) was determined by Western blot. RESULTS TNFα inhibited 0.1mM MG uptake after pre-incubation of the cells for 6-48h with the cytokine and in the absence of cytokine pre-incubation. In contrast, 5mM fructose uptake was stimulated by TNFα only after long pre-incubation times (24 and 48 h). These effects were mediated by the binding of the cytokine to its specific receptor TNFR1, present in the apical membrane of the Caco-2 cells. Analysis of the expression of the MG and fructose transporters at the brush border membrane of the cells, after 24h pre-incubation with the cytokine, revealed decrease on the amount of SGLT1 and increase on the amount of GLUT5 proteins. Short-term inhibition of MG transport by TNFα was not modified by H-89 but was blocked by chelerythrine. CONCLUSIONS SGLT1 and GLUT5 expression in the plasma membrane is regulated by TNFα in the human epithelial cell line Caco-2 cells, leading to alteration on sugars transport, suggesting that TNFα could be considered as a physiological local regulator of nutrients absorption in response to an intestinal inflammatory status.

The facilitative glucose transporter GLUT12: what do we know and what would we like to know?

Glucose, one of the most abundant molecules in nature, is used by most of the mammalian cells as their main energy source. Obtained from the diet, glucose is absorbed in the small intestine, incorporated into the circulating blood and stored as glycogen, mainly in the liver and muscle. Due to its hydrophilic nature, glucose cannot cross the plasma membrane by simple diffusion; instead, glucose enters the cell by specific membrane transporters. There are two families of glucose transporters, distinguished by their functional and structural properties: the Na/glucose cotransporter family SGLT/SLC5A [47] and the facilitative glucose transporter family GLUT/SLC2A [11, 22]. GLUT protein family members transport monosaccharides across the plasma membrane without energetic requirement, using the favourable concentration gradient of the hexose generated in some physiological situations. GLUTs share a common structural feature of 12 transmembrane domains, with both amino and carboxy terminal domains located on the cytosolic side, and an N-linked oligosaccharide site present either on the first or on the fifth extracellular loop [37]. At present, 14 different members of this family, divided in three classes according to its sequence homology, have been identified [11]. Class I is constituted by the well-characterized GLUT1, GLUT2, GLUT3, GLUT4 and GLUT14 (gene duplication of GLUT3); class II comprises the fructose transporter GLUT5, and GLUT7, GLUT9 and GLUT11; and class III includes GLUT6, GLUT8, GLUT10, GLUT12 and GLUT13 (HMIT) [12]. In relation to this classification, a recent phylogenetic analysis proposes that the proteins belonging to class III could be separated into three different groups and, therefore, suggests five structurally and/or functionally distinct GLUT classes (Fig. 1) [43]. Location, expression and regulation of the GLUT transporters are specific for each tissue and cellular type and are related to the cell metabolic needs. Inmany cases, the upor downregulation of the GLUT proteins is directly linked to the development of diseases (Table 1). GLUT1, 2, 3, 4 and 5 were the first members of the GLUT family cloned and their physiological function has been well characterized. GLUT9 seems to be a urate transporter, newly described as electrogenic [44], while GLUT13 (HMIT) is a H-dependent myoinositol cotransporter [36]. However, the physiological significance of the rest of GLUT transporters of class II and III still needs to be elucidated. Therefore, it is important to continue investigating the location and cellular function of recently discovered GLUT transporters, not only to determine their function in the organism, but also to J Physiol Biochem (2013) 69:325–333 DOI 10.1007/s13105-012-0213-8

Cytokine effect on intestinal galactose absorption.

In this work, the effect of the cytokines TNF-a and IL-1b on the intestinal galactose absorption is investigated. The absorption studies were performed using everted rings of rat intestine which were incubated in galactose (C14)-containing saline solution for 5 or 30 min. Different concentrations of TNF-a (1-100 ng/ml) or IL-1b (0.5-25 ng/ml) were added to the incubation saline solution. At 10, 50 and 100 ng/ml concentrations, TNF-a inhibits galactose absorption by 30% and at 25 ng/ml, there is a reversal of the inhibition. This biphasic effect of TNF-a on galactose absorption is observed at both 5 and 30 min incubation. IL-1b inhibited galactose uptake at all tested concentrations after 30 min incubation, although the effect was not concentration dependent. Both TNF-a and IL-1b inhibits only the phloridzin-sensitive component of the intestinal galactose absorption, indicating that they alter SGLT1 functionality.

Functional expression of the short isoform of the murine leptin receptor Ob-Rc (muB1.219) in Xenopus laevis oocytes

Leptin, a hormone mainly secreted by the adipose tissue, acts on the hypothalamus to regulate food intake and thermogenesis. Six leptin receptor isoforms have been identified and localized in different tissues. While it is clear that leptin action in the brain occurs by binding to the long receptor isoform, several studies have shown that the short isoforms could be involved in the transcellular transport of the hormone from the blood to the brain. Based on these works, we decided to investigate whether the murine short leptin receptor isoform Ob-Rc (muB1.219) could transport leptin when expressed inXenopus laevis oocytes. MuB1.219 cRNA was injected into the oocytes and functional studies were performed by incubating the oocytes in the presence of 2.5 nM[125I]-leptin, under different conditions. Results showed that leptin binding to the injected oocytes was four to eight-fold higher than the binding to the non-injected oocytes. This was blocked by 250 nM of non-radiolabeled leptin, suggesting that the binding was specific. Leptin internalization was observed from 30 min incubation onwards. Coexpression of the human Na+/glucose cotransporter and the leptin receptor showed that leptin increased sugar uptake into the oocytes. These results demonstrate that the short leptin receptor Ob-Rc is able to mediate binding and internalization of the hormone when expressed in oocytes and that it may perform intracellular signaling.ResumenLa leptina es una hormona principalmente secretada por el tejido adiposo que actúa en el hipotálamo para regular la ingesta y la termogénesis. Se han identificado y localizado en diferentes tejidos seis isoformas del receptor de leptina. La acción de la leptina en el cerebro ocurre por unión a la isoforma larga del receptor, y varios estudios han mostrado que las isoformas cortas podrían estar implicadas en el transporte transcelular de la hormona desde la sangre hasta el cerebro. En base a esos trabajos, se decidió investigar si la isoforma corta del receptor de ratón Ob-Rc (muB1.219) podía transportar leptina cuando se expresaba en oocitos deXenopus laevis. Los oocitos se inyectaron con el cRNA del receptor muB1.219 y se realizaron estudios funcionales mediante la incubación de los oocitos en presencia de [125I]-leptina 2,5 nM bajo diferentes condiciones. Los resultados muestran que la unión de la leptina a los oocitos inyectados es de cuatro a ocho veces superior a la unión a los oocitos no inyectados. La unión es específica, pues se bloquea por 250 nM de leptina no radiactiva. La internalización de leptina se observa a partir de los 30 minutos de incubación de los oocitos. La coexpresión del cotransportador humano Na+/glucosa y el receptor de leptina determina aumento de la captación del azúcar en los oocitos en presencia de leptina. Estos resultados demuestran que el receptor corto de leptina Ob-Rc es capaz de mediar unión e internalización de la hormona cuando se expresa en oocitos y que puede dar lugar a señalización intracelular.