RESUMEN
AIMS/HYPOTHESIS: Plasma glucagon concentrations rise sharply during the early postnatal period. This condition is associated with increased alpha cell mass. However, the trophic factors that regulate alpha cell turnover during the perinatal period have not been studied. Macrophage infiltrations are present in the neonatal pancreas, and this cell type releases cytokines such as IL-6. Alpha cells have been identified as a primary target of IL-6 actions. We therefore investigated the physiological relevance of IL-6 to neonatal pancreatic alpha cell maturation. METHODS: Histochemical analyses were performed to quantify alpha cell mass, replication and apoptosis. Pancreatic Il6 expression was determined by quantitative RT-PCR. The biological effect of IL-6 was tested in two in vivo rat models of IL-6 blockade and chronic undernutrition. RESULTS: Alpha cell mass increased sharply shortly after birth but decreased significantly after weaning. Pancreatic alpha cell proliferation was as high as 2.5% at the beginning of suckling but diminished with time to 1.2% in adulthood. Similarly, alpha cell neoformation was remarkably high on postnatal day (PN) 4, whereas alpha cell apoptosis was low throughout the neonatal period. Moreover, Il6 mRNA exhibited developmental upregulation in the pancreas of suckling rats, with the highest expression on PN2. Neutralisation of IL-6 reduced alpha cell mass expansion and glucagon production. IL-6 staining was detected within the islets, mainly in the alpha cells. Finally, undernourished neonates showed altered alpha cell number and function and delayed appearance of IL-6 in the pancreas. CONCLUSIONS/INTERPRETATION: These data point to a potential role for local IL-6 in the regulation of alpha cell growth and function during suckling.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Células Secretoras de Glucagón/metabolismo , Glucagón/metabolismo , Interleucina-6/metabolismo , Páncreas/crecimiento & desarrollo , Animales , Animales Recién Nacidos , Animales Lactantes , Apoptosis , Proliferación Celular , Células Cultivadas , Femenino , Glucagón/genética , Células Secretoras de Glucagón/citología , Células Secretoras de Glucagón/inmunología , Interleucina-6/antagonistas & inhibidores , Interleucina-6/genética , Masculino , Desnutrición/inmunología , Desnutrición/metabolismo , Desnutrición/patología , Fenómenos Fisiologicos Nutricionales Maternos , Páncreas/inmunología , Páncreas/metabolismo , Embarazo , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptores de Glucagón/genética , Receptores de Glucagón/metabolismo , Transducción de Señal , Técnicas de Cultivo de TejidosRESUMEN
Previous comparative studies of fumarate hydratase (FH) protein density revealed that the enzyme was overexpressed in the striatum of rodents that are less influenced by rewarding stimuli, from cocaine to food. Therefore, we recently proposed FH as a potential striatal biomarker of brain reward deficiency and addiction vulnerability. This work has been focused to investigate FH activity in the Nucleus Accumbens (NAc) of undernourished rats, taking into account that malnutrition has been related to increased responsiveness to food and drug reward. To this end, we have studied adult female Wistar rats severely food restricted from the 16th day of intrauterine life until adulthood. Animals were sacrificed to dissect the NAc and obtain mitochondrial and cytosolic fractions after homogenisation and centrifugation. FH activity was measured by conversion of malate to fumarate, and protein levels were compared by Western blot analysis when fractions showed differences in activity. Undernutrition did not change cytosolic FH activity but led to a marked increase of mitochondrial FH activity (72 %) and protein content (50 %) in the NAc. This change was in the opposite direction that one would predict if it was related to addiction vulnerability of some kind, but strongly suggests that mitochondrial FH needs to be at some optimal level for normal reward responsiveness.
Asunto(s)
Fumarato Hidratasa/metabolismo , Desnutrición/enzimología , Núcleo Accumbens/enzimología , Animales , Femenino , Ratas , Ratas Wistar , Recompensa , Regulación hacia ArribaRESUMEN
AIM/HYPOTHESIS: AMP-activated protein kinase (AMPK), encoded by Prkaa genes, is emerging as a key regulator of overall energy homeostasis and the control of insulin secretion and action. We sought here to investigate the role of AMPK in controlling glucagon secretion from pancreatic islet alpha cells. METHODS: AMPK activity was modulated in vitro in clonal alphaTC1-9 cells and isolated mouse pancreatic islets using pharmacological agents and adenoviruses encoding constitutively active or dominant negative forms of AMPK. Glucagon secretion was measured during static incubation by radioimmunoassay. AMPK activity was assessed by both direct phosphotransfer assay and by western (immuno-)blotting of the phosphorylated AMPK α subunits and the downstream target acetyl-CoA carboxylase 1. Intracellular free [Ca²(+)] was measured using Fura-Red. RESULTS: Increasing glucose concentrations strongly inhibited AMPK activity in clonal pancreatic alpha cells. Forced increases in AMPK activity in alphaTC1-9 cells, achieved through the use of pharmacological agents including metformin, phenformin and A-769662, or via adenoviral transduction, resulted in stimulation of glucagon secretion at both low and high glucose concentrations, whereas AMPK inactivation inhibited both [Ca²(+)](i) increases and glucagon secretion at low glucose. Transduction of isolated mouse islets with an adenovirus encoding AMPK-CA under the control of the preproglucagon promoter increased glucagon secretion selectively at elevated glucose concentrations. CONCLUSIONS/INTERPRETATION: AMPK is strongly regulated by glucose in pancreatic alpha cells, and increases in AMPK activity are sufficient and necessary for the stimulation of glucagon release in vitro. Modulation of AMPK activity in alpha cells may therefore provide a novel approach to controlling blood glucose concentrations.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Células Secretoras de Glucagón/enzimología , Células Secretoras de Glucagón/metabolismo , Glucagón/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Acetiltransferasas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Compuestos de Bifenilo , Western Blotting , Calcio/metabolismo , Línea Celular , Activación Enzimática/efectos de los fármacos , Femenino , Células Secretoras de Glucagón/efectos de los fármacos , Glucosa/metabolismo , Hipoglucemiantes/farmacología , Inmunohistoquímica , Metformina/farmacología , Ratones , Fenformina/farmacología , Fosforilación/efectos de los fármacos , Pironas/farmacología , Tiofenos/farmacologíaRESUMEN
Nutritional restriction early in life followed by catch-up growth has been associated with increased risk of metabolic syndrome in adulthood. To elucidate whether altered gut colonization underlies the mechanisms responsible of this predisposition gut microbiome was studied before or afterwards catch-up growth. Offspring of dams fed ad libitum (C) or undernourished during pregnancy and suckling (U), were weaned onto high-fat diet (HFD) for 22 weeks (CHF and UHF, respectively) or continued on their diet. HF-feeding induced glucose intolerance (P<.05), insulin resistance (P<.001), and white adipose tissue inflammation (P<.001) in UHF rats compared to CHF. Analyses of gut microbial composition before catch-up growth revealed reduced F/B ratio and significant expansion of the mucolytic genera Akkermansia (P<.05) and Desulfovibrio (P<.05) in U pups. Although relative abundance of Akkermansia remained elevated to adulthood in U rats, HFD normalized its levels to C and CHF. Food-restriction increased intestinal permeability causing disorganization on the tight-junction proteins of colonic epithelium, Zonula Occludens-1 (ZO-1) and occludin, and reducing the mucus thickness layer in U adult rats. The levels of ZO-1 and occludin were not recovered in U rats after HF-feeding. This event was correlated with increased circulating levels of bacterial lipopolysaccharides in both U and UHF adult rats. Even more, serum lipopolysaccharides were already elevated in U rats compared to C group (P<.001) at weaning. Thus, gut dysbiosis and chronic endotoxemia observed in U rats, even before catch-up growth, might anticipate a pro-inflammatory milieu promoting metabolic diseases when fed hyperlipidic diets.
Asunto(s)
Disbiosis/metabolismo , Microbioma Gastrointestinal , Desnutrición/metabolismo , Síndrome Metabólico/metabolismo , Animales , Colon/metabolismo , Dieta Alta en Grasa/efectos adversos , Endotoxemia/metabolismo , Heces/microbiología , Femenino , Intolerancia a la Glucosa/etiología , Intolerancia a la Glucosa/metabolismo , Humanos , Inflamación/etiología , Inflamación/metabolismo , Resistencia a la Insulina , Mucosa Intestinal/metabolismo , Lipopolisacáridos/sangre , Masculino , Embarazo , RatasRESUMEN
Oxidative stress plays an important role in the development of beta-cell dysfunction and insulin resistance, two major pathophysiological abnormalities of type 2 diabetes. Expression levels of antioxidant enzymes in beta cells are very low, rendering them more susceptible to damage caused by reactive oxygen species (ROS). Although the antioxidant effects of glucagon-like peptide-1 (GLP-1) and its analogs have been previously reported, the exact mechanisms involved are still unclear. In this study, we demonstrated that GLP-1 was able to effectively inhibit oxidative stress and cell death of INS-1E beta cells induced by the pro-oxidant tert-butyl hydroperoxide (tert-BOOH). Incubation with GLP-1 enhanced cellular levels of glutathione and the activity of its related enzymes, glutathione-peroxidase (GPx) and -reductase (GR) in beta cells. However, inhibition of ERK, but not of the PI3K/AKT pathway abolished, at least in part, the antioxidant effect of GLP-1. Moreover, ERK activation seems to be protein kinase A (PKA)-dependent because inhibition of PKA with H-89 was sufficient to block the GLP-1-derived protective effect on beta cells. GLP-1 likewise increased the synthesis of GR and favored the translocation of the nuclear transcription factor erythroid 2p45-related factor (Nrf2), a transcription factor implicated in the expression of several antioxidant/detoxificant enzymes. Glucose-stimulated insulin secretion was also preserved in beta-cells challenged with tert-BOOH but pre-treated with GLP-1, probably through the down-regulation of the mitochondrial uncoupling-protein2 (UCP2). Thus, our results provide additional mechanisms of action of GLP-1 to prevent oxidative damage in beta cells through the modulation of signaling pathways involved in antioxidant enzyme regulation.
Asunto(s)
Diabetes Mellitus Tipo 2/genética , Péptido 1 Similar al Glucagón/genética , Factor 2 Relacionado con NF-E2/genética , Proteína Desacopladora 2/genética , Animales , Antioxidantes/metabolismo , Antioxidantes/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Quinasas MAP Reguladas por Señal Extracelular/genética , Péptido 1 Similar al Glucagón/metabolismo , Péptido 1 Similar al Glucagón/farmacología , Glucosa/metabolismo , Glutatión/biosíntesis , Glutatión Reductasa/biosíntesis , Glutatión Reductasa/genética , Humanos , Insulina/metabolismo , Resistencia a la Insulina/genética , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Isoquinolinas/farmacología , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo/genética , Ratas , Especies Reactivas de Oxígeno/metabolismo , Sulfonamidas/farmacología , Proteína Desacopladora 2/metabolismo , terc-Butilhidroperóxido/metabolismoRESUMEN
Catch-up growth has been associated with the appearance of metabolic dysfunctions such as obesity and type 2 diabetes in adulthood. Because the entero-insular axis is critical to glucose homeostasis control, we explored the relevance of the incretins glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) in the development of these pathologies. Offspring of rat dams fed ad libitum (control [C]) or 65% food-restricted during pregnancy and suckling time (undernourished [U]) were weaned onto a high-fat (HF) diet (CHF and UHF, respectively) to drive catch-up growth. Both male and female UHF rats showed an obese phenotype characterized by hyperphagy, visceral fat accumulation, and adipocyte hypertrophy. High-fat diet induced deterioration of glucose tolerance in a sex-dependent manner. Female UHF rats experienced much more severe glucose intolerance than males, which was not compensated by insulin hypersecretion, suggesting insulin resistance, as shown by homeostatic model assessment of insulin resistance values. Moreover, female, but not male, UHF rats displayed enhanced GIP but not GLP-1 secretion during oral glucose tolerance test. Administration of the GIP receptor antagonist (Pro3)GIP to UHF female rats over 21 days markedly reduced visceral fat mass and adipocyte hypertrophy without variations in food intake or body weight. These changes were accompanied by improvement of glucose tolerance and insulin sensitivity. In conclusion, the exacerbated production and secretion of GIP after the catch-up growth seems to represent the stimulus for insulin hypersecretion and insulin resistance, ultimately resulting in derangement of glucose homeostasis. Overall, these data evidence the role of GIP as a critical link between catch-up growth and the development of metabolic disturbances.