RESUMEN
Olfactory receptors (ORs) are G protein-coupled receptors that mediate olfactory chemosensation, leading to the perception of smell. ORs are expressed in many tissues, but their functions are largely unknown. Here, we show that the olfactory receptor Olfr15 is highly and selectively expressed in both mouse pancreatic ß-cells and MIN6 cells. In addition, octanoic acid (OA), a medium-chain fatty acid, potentiates glucose-stimulated insulin secretion (GSIS). The OA-induced enhancement of GSIS was inhibited by Olfr15 knockdown. Treatment with a PLC inhibitor or an Ins(1,4,5)P3 receptor (IP3R) antagonist also blocked the OA-induced enhancement of GSIS. These results suggest that OA potentiates GSIS via Olfr15 though the PLC-IP3 pathway. Furthermore, long-term treatment with OA increased cellular glucose uptake in MIN6 cells by up-regulating the expression of glucokinase (GK). Moreover, this process was blocked by an IP3R antagonist and a Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) inhibitor. Similarly, OA stimulated GK promoter activity, while either Olfr15 or CaMKIV knockdown blocked the stimulatory effect of OA on GK promoter activity. These results suggest that long-term treatment of OA induces GK promoter activity via Olfr15 through the IP3-CaMKK/CaMKIV pathway. In islets from type 2 diabetic mice, the expression level of Olfr15 and the OA-induced enhancement of GSIS were strongly reduced. Collectively, our results highlight the crucial role of the olfactory receptor Olfr15 in potentiating GSIS in pancreatic ß-cells, suggesting that Olfr15 may be an important therapeutic target in type 2 diabetes.
Asunto(s)
Caprilatos/metabolismo , Glucoquinasa/metabolismo , Glucosa/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Receptores Odorantes/metabolismo , Animales , Caprilatos/análisis , Caprilatos/farmacología , Línea Celular , Células Cultivadas , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/terapia , Alimentos Funcionales/análisis , Regulación de la Expresión Génica/efectos de los fármacos , Glucoquinasa/análisis , Glucoquinasa/genética , Células Secretoras de Insulina/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores Odorantes/análisis , Receptores Odorantes/genética , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND: Fructose administration rapidly induces oxidative stress that triggers compensatory hepatic metabolic changes. We evaluated the effect of an antioxidant, R/S-α-lipoic acid on fructose-induced oxidative stress and carbohydrate metabolism changes. METHODS: Wistar rats were fed a standard commercial diet, the same diet plus 10% fructose in drinking water, or injected with R/S-α-lipoic acid (35mg/kg, i.p.) (control+L and fructose+L). Three weeks thereafter, blood samples were drawn to measure glucose, triglycerides, insulin, and the homeostasis model assessment-insulin resistance (HOMA-IR) and Matsuda indices. In the liver, we measured gene expression, protein content and activity of several enzymes, and metabolite concentration. RESULTS: Comparable body weight changes and calorie intake were recorded in all groups after the treatments. Fructose fed rats had hyperinsulinemia, hypertriglyceridemia, higher HOMA-IR and lower Matsuda indices compared to control animals. Fructose fed rats showed increased fructokinase gene expression, protein content and activity, glucokinase and glucose-6-phosphatase gene expression and activity, glycogen storage, glucose-6-phosphate dehydrogenase mRNA and enzyme activity, NAD(P)H oxidase subunits (gp91(phox) and p22(phox)) gene expression and protein concentration and phosphofructokinase-2 protein content than control rats. All these changes were prevented by R/S-α-lipoic acid co-administration. CONCLUSIONS: Fructose induces hepatic metabolic changes that presumably begin with increased fructose phosphorylation by fructokinase, followed by adaptive changes that attempt to switch the substrate flow from mitochondrial metabolism to energy storage. These changes can be effectively prevented by R/S-α-lipoic acid co-administration. GENERAL SIGNIFICANCE: Control of oxidative stress could be a useful strategy to prevent the transition from impaired glucose tolerance to type 2 diabetes.
Asunto(s)
Metabolismo de los Hidratos de Carbono/efectos de los fármacos , Fructosa/farmacología , Hígado/metabolismo , Estrés Oxidativo , Ácido Tióctico/farmacología , Animales , Glucoquinasa/análisis , Glucoquinasa/genética , Masculino , NADPH Oxidasas/análisis , NADPH Oxidasas/genética , Ratas , Ratas WistarRESUMEN
A hexokinase, with a low Michaelis constant, not previously described, has been found in extracts of human and dog liver but not of rat liver. Earlier reports are contradicted in that glucokinase occurs in extracts of liver from well-nourished humans and dogs; it is absent, or almost so, during states of poor nutrition.
Asunto(s)
Tejido Adiposo/enzimología , Glucoquinasa/análisis , Hexoquinasa/análisis , Isoenzimas/análisis , Hígado/enzimología , Animales , Perros , Electroforesis , HumanosRESUMEN
The enzyme activities involved in fructose metabolism were measured in samples of human liver. On the basis of U/g of wet-weight the following results were found: ketohexokinase, 1.23; aldolase (substrate, fructose-1-phosphate), 2.08; aldolase (substrate, fructose-1,6-diphosphate), 3.46; triokinase, 2.07; aldehyde dehydrogenase (substrate, D-glyceraldehyde), 1.04; D-glycerate kinase, 0.13; alcohol dehydrogenase (nicotinamide adenine dinucleotide [NAD]) substrate, D-glyceraldehyde), 3.1; alcohol dehydrogenase (nicotinamide adenine dinucleotide phosphate [NADP]) (substrate, D-glyceraldehyde), 3.6; and glycerol kinase, 0.62. Sorbitol dehydrogenases (25.0 U/g), hexosediphosphatase (4.06 U/g), hexokinase (0.23 U/g), and glucokinase (0.08 U/g) were also measured. Comparing these results with those of the rat liver it becomes clear that the activities of alcohol dehydrogenases (NAD and NADP) in rat liver are higher than those in human liver, and that the values of ketohexokinase, sorbitol dehydrogenases, and hexosediphosphatase in human liver are lower than those values found in rat liver. Human liver contains only traces of glycerate kinase. The rate of fructose uptake from the blood, as described by other investigators, can be based on the activity of ketohexokinase reported in the present paper. In human liver, ketohexokinase is present in a four-fold activity of glucokinase and hexokinase. This result may explain the well-known fact that fructose is metabolized faster than glucose.
Asunto(s)
Fructosa/metabolismo , Hígado/enzimología , Oxidorreductasas de Alcohol/análisis , Animales , Femenino , Fructosa-Bifosfatasa/análisis , Glucoquinasa/análisis , Hexoquinasa/análisis , Humanos , Masculino , NAD/análisis , NADP/análisis , Fosfotransferasas/análisis , RatasRESUMEN
Maternal protein restriction is a model of fetal programming of adult glucose intolerance. Perfused livers of 48-h- starved adult offspring of rat dams fed 8% protein diets during pregnancy and lactation produced more glucose from 6 mM lactate than did control livers from rats whose dams were fed 20% protein. In control livers, a mean of 24% of the glucose formed from lactate in the periportal region of the lobule was taken up by the most distal perivenous cells; this distal perivenous uptake was greatly diminished in maternal low protein (MLP) livers, accounting for a major fraction of the increased glucose output of MLP livers. In control livers, the distal perivenous cells contained 40% of the total glucokinase of the liver; this perivenous concentration of glucokinase was greatly reduced in MLP livers. Intralobular distribution of phosphenolpyruvate carboxykinase was unaltered, though overall increased activity could have contributed to the elevated glucose output. Hepatic lobular volume in MLP livers was twice that in control livers, indicating that MLP livers had half the normal number of lobules. Fetal programming of adult glucose metabolism may operate partly through structural alterations and changes in glucokinase expression in the immediate perivenous region.
Asunto(s)
Intolerancia a la Glucosa/etiología , Lactancia , Hígado/metabolismo , Complicaciones del Embarazo , Efectos Tardíos de la Exposición Prenatal , Deficiencia de Proteína/complicaciones , Animales , Digitonina/farmacología , Modelos Animales de Enfermedad , Femenino , Glucoquinasa/análisis , Gluconeogénesis , Glucosa/metabolismo , Técnicas In Vitro , Hígado/efectos de los fármacos , Hígado/patología , Perfusión , Fosfoenolpiruvato Carboxiquinasa (GTP) , Embarazo , Ratas , Ratas WistarRESUMEN
Glucokinase activity is increased in pancreatic islets during pregnancy and in vitro by prolactin (PRL). The underlying mechanisms that lead to increased glucokinase have not been resolved. Since glucose itself regulates glucokinase activity in beta-cells, it was unclear whether the lactogen effects are direct or occur through changes in glucose metabolism. To clarify the roles of glucose metabolism in this process, we examined the interactions between glucose and PRL on glucose metabolism, insulin secretion, and glucokinase expression in insulin 1 (INS-1) cells and rat islets. Although the PRL-induced changes were more pronounced after culture at higher glucose concentrations, an increase in glucose metabolism, insulin secretion, and glucokinase expression occurred even in the absence of glucose. The presence of comparable levels of insulin secretion at similar rates of glucose metabolism from both control and PRL-treated INS-1 cells suggests the PRL-induced increase in glucose metabolism is responsible for the increase in insulin secretion. Similarly, increases in other known PRL responsive genes (e.g. the PRL receptor, glucose transporter-2, and insulin) were also detected after culture without glucose. We show that the upstream glucokinase promoter contains multiple STAT5 binding sequences with increased binding in response to PRL. Corresponding increases in glucokinase mRNA and protein synthesis were also detected. This suggests the PRL-induced increase in glucokinase mRNA and its translation are sufficient to account for the elevated glucokinase activity in beta-cells with lactogens. Importantly, the increase in islet glucokinase observed with PRL is in line with that observed in islets during pregnancy.
Asunto(s)
Glucoquinasa/metabolismo , Islotes Pancreáticos/metabolismo , Preñez/fisiología , Prolactina/fisiología , Animales , Western Blotting/métodos , Línea Celular , Ensayo de Cambio de Movilidad Electroforética , Femenino , Glucoquinasa/análisis , Glucosa/genética , Glucosa/metabolismo , Glucosa/farmacología , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/química , Embarazo , Prolactina/farmacología , Regiones Promotoras Genéticas , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
In an attempt to study the role of glucokinase (GK) and the effects of glucose and peptides on GK gene expression and on the activity of this enzyme in the hypothalamus, we used two kinds of biological models: hypothalamic GT1-7 cells and rat hypothalamic slices. The expression of the GK gene in GT1-7 cells was reduced by insulin (INS) and was not modified by different glucose concentrations, while GK enzyme activities were significantly reduced by the different peptides. Interestingly, a distinctive pattern of GK activities between the ventromedial hypothalamus (VMH) and lateral hypothalamus (LH) were found, with higher enzyme activities in the VMH as the glucose concentrations rose, while LH enzyme activities decreased at 2.8 and 20 mM glucose, the latter effect being prevented by incubation with INS. These effects were produced only by d-glucose and the modifications found were due to GK, but not to other hexokinases. In addition, GK activities in the VMH and the LH were reduced by glucagon-like peptide 1, leptin, orexin B, INS, and neuropeptide Y (NPY), but this effect was only statistically significant for NPY in LH. Our results indicate that the effects of both glucose and peptides occur on GK enzyme activities rather than on GK gene transcription. Moreover, the effects of glucose and INS on GK activity suggest that in the brain GK behaves in a manner opposite to that in the liver, which might facilitate its role in glucose sensing. Finally, hypothalamic slices seem to offer a good physiological model to discriminate the effects between different areas.
Asunto(s)
Glucoquinasa/metabolismo , Glucosa/farmacología , Hipoglucemiantes/farmacología , Hipotálamo/enzimología , Insulina/farmacología , Animales , Western Blotting/métodos , Línea Celular , Activación Enzimática/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Péptido 1 Similar al Glucagón/farmacología , Glucoquinasa/análisis , Glucoquinasa/genética , Área Hipotalámica Lateral/efectos de los fármacos , Área Hipotalámica Lateral/enzimología , Hipotálamo/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/farmacología , Leptina/farmacología , Masculino , Neuropéptido Y/farmacología , Neuropéptidos/farmacología , Orexinas , Regiones Promotoras Genéticas , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Técnicas de Cultivo de Tejidos , Núcleo Hipotalámico Ventromedial/efectos de los fármacos , Núcleo Hipotalámico Ventromedial/enzimologíaRESUMEN
Detection and quantification of unmethylated circulating insulin (INS) DNA presumably released from ß cells has been previously used for assessing their destruction. As the targets within the INS gene suffer from suboptimal specificity, we sought to improve the assay parameters by using the glucokinase gene (GCK) tissue-specific pancreatic promoter. The amount of methylated and unmethylated GCK DNA was measured using a droplet polymerase chain reaction assay and compared with the previously published INS-targeted assay. The method was tested using synthetic target sequences and DNA from pancreatic islets, blood, brain, kidney, large intestine, liver, lung, small intestine, and stomach. Circulating serum DNA was obtained from children with recent-onset type 1 diabetes (T1D) (n = 25), autoantibody-positive first-degree relatives of T1D patients (n = 14), and healthy controls (n = 20). The unmethylated GCK DNA was found to be more islet specific than unmethylated INS DNA. The proportion of the unmethylated GCK DNA was lower than INS in all tested extrapancreatic tissues, except kidney. Although the amounts of methylated DNA measured by the two assays were similar, the INS assay detected considerably more unmethylated DNA. Whereas none of the assays showed significant increase in the amount of unmethylated DNA, the ratio of unmethylated/methylated GCK DNA was borderline significantly increased in autoantibody-positive relatives compared with T1D patients (P = 0.04) and controls (P = 0.06). Targeting the assay into the GCK gene improved analytical parameters of the assay. As the amount of unmethylated target DNA in properly treated samples is very low, the clinical utility of this method remains to be evaluated.
Asunto(s)
Diabetes Mellitus Tipo 1/diagnóstico , Glucoquinasa/genética , Células Secretoras de Insulina/fisiología , Insulina/genética , Adolescente , Adulto , Estudios de Casos y Controles , Muerte Celular/genética , Niño , Preescolar , Metilación de ADN , Análisis Mutacional de ADN/métodos , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/patología , Femenino , Glucoquinasa/análisis , Humanos , Lactante , Insulina/análisis , Células Secretoras de Insulina/patología , Masculino , Reacción en Cadena de la Polimerasa , Valor Predictivo de las Pruebas , Adulto JovenRESUMEN
Administration of a sucrose-rich diet (SRD) to normal hamsters induces an insulin-resistant state and a significant increase of insulin secretion and beta-cell mass. Islets isolated from these animals had a marked increase in glucose metabolism and glucose-induced insulin secretion, at both low and high glucose concentrations. They also presented increased hexokinase (HK) activity, without measurable changes in glucokinase (GK) activity. In this study we measured HK and GK activity in homogenates of islets isolated from normal control and SRD-fed hamsters, as well as in their particulate and cytosolic fractions. We also measured transcription rate (mRNA by reverse transcriptase PCR) and expression levels (Western blotting) of both enzymes in these islets. We found an increase in HK activity and expression levels, without measurable changes in HK mRNA level in SRD-fed animals. Whereas a similar GK activity was measured in homogenates of islets isolated from both groups, such activity was significantly higher in the cytosolic fraction of SRD islets. On the other hand, GK transcription rate and expression level were similar in both experimental groups. Our results suggest that the increased beta-cell secretory response to low glucose can be partly ascribed to an increased activity of islet HK consecutive to an enhanced expression of the enzyme, while the enhanced response to high glucose could be due to changes in GK compartmentalization.
Asunto(s)
Glucoquinasa/metabolismo , Hexoquinasa/metabolismo , Islotes Pancreáticos/metabolismo , Sacarosa/administración & dosificación , Animales , Glucemia/análisis , Western Blotting/métodos , Peso Corporal/fisiología , Cricetinae , Citosol/metabolismo , Dieta , Ingestión de Líquidos/fisiología , Expresión Génica/genética , Glucoquinasa/análisis , Glucoquinasa/genética , Hexoquinasa/análisis , Hexoquinasa/genética , Insulina/sangre , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Masculino , Mesocricetus , Fosforilación , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Transcripción Genética/genéticaRESUMEN
Activities of key carbohydrate-metabolizing enzymes in biopsied human tissues of hepatocellular carcinoma and related conditions were determined by established methods. Among the enzymes analyzed, fetal-type liver enzymes (low-Km hexokinase, glucose 6-phosphate dehydrogenase, and pyruvate kinase-M2) showed increased activities, and adult-type liver enzymes [glucose 6-phosphatase, fructose 1,6-bisphosphatase, high-Km hexokinase (or glucokinase), and pyruvate kinase-L] showed decreased activities, resulting in undifferentiated enzyme patterns not only in fetal livers and hepatocellular carcinomas but also in livers of acute and chronic hepatitis and liver cirrhosis with or without tumors. Hepatocellular carcinomas showed a general tendency of having greater enzyme deviations than hepatitic and cirrhotic livers. The extent of the enzyme deviation in hepatocellular carcinomas varied considerably from one enzyme to another for each tumor tissue as compared with that in the benign liver diseases. Thus, the phenotypic heterogeneity was important for discriminating between the neoplastic and inflammatory changes in differentiation markers. The enzyme patterns of tumors and their corresponding host cirrhotic livers were unrelated, suggesting that the cirrhotic liver has a significance as preneoplastic state only in terms of having a high incidence of evolving hepatocellular carcinoma.
Asunto(s)
Metabolismo de los Hidratos de Carbono , Carcinoma Hepatocelular/enzimología , Neoplasias Hepáticas/enzimología , Hígado/enzimología , Lesiones Precancerosas/enzimología , Femenino , Glucoquinasa/análisis , Glucosafosfato Deshidrogenasa/análisis , Hexoquinasa/análisis , Humanos , Isoenzimas/análisis , Cirrosis Hepática/enzimología , Masculino , Piruvato Quinasa/análisisRESUMEN
AIMS/INTRODUCTION: Src, a non-receptor tyrosine kinase, regulates a wide range of cellular functions, and hyperactivity of Src is involved in impaired glucose metabolism in pancreatic ß-cells. However, the physiological role of Src in glucose metabolism in normal, unstressed ß-cells remains unclear. In the present study, we investigated the role of Src in insulin secretion and glucose metabolism. MATERIALS AND METHODS: Src was downregulated using small interfering ribonucleic acid in INS-1 cells, and glucose-induced insulin secretion, adenosine triphosphate content, intracellular calcium concentration, glucose utilization and glucokinase activity were measured. Expression levels of messenger ribonucleic acid and protein of glucokinase were examined by semiquantitative real-time polymerase chain reaction and immunoblotting, respectively. Cells were fractionated by digitonin treatment, and subcellular localization of glucokinase was examined by immunoblotting. Interaction between glucokinase and neuronal nitric oxide synthase was estimated by immunoprecipitation. RESULTS: In Src downregulated INS-1 cells, glucose-induced insulin secretion was impaired, whereas insulin secretion induced by high K(+) was not affected. Intracellular adenosine triphosphate content and elevation of intracellular calcium concentration by glucose stimulation were suppressed by Src downregulation. Src downregulation reduced glucose utilization in the presence of high glucose, which was accompanied by a reduction in glucokinase activity without affecting its expression. However, Src downregulation reduced glucokinase in soluble, cytoplasmic fraction, and increased it in pellet containing intaracellular organelles. In addition, interaction between glucokinase and neuronal nitric oxide synthase was facilitated by Src downregulation. CONCLUSIONS: Src plays an important role in glucose-induced insulin secretion in pancreatic ß-cells through maintaining subcellular localization and activity of glucokinase.
Asunto(s)
Glucoquinasa/metabolismo , Glucosa/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Familia-src Quinasas/fisiología , Adenosina Trifosfato/metabolismo , Animales , Línea Celular , Regulación hacia Abajo , Glucoquinasa/análisis , Transportador de Glucosa de Tipo 2/metabolismo , Secreción de Insulina , Mitocondrias/metabolismo , Óxido Nítrico Sintasa de Tipo I/metabolismo , Transporte de Proteínas , Ratas , Especies Reactivas de Oxígeno/metabolismo , Familia-src Quinasas/genéticaRESUMEN
Assays in which the detection of a biological phenomenon is coupled to the production of bioluminescence by luciferase have gained widespread use. As firefly luciferases (FLuc) and kinases share a common substrate (ATP), coupling of a kinase to FLuc allows for the amount of ATP remaining following a kinase reaction to be assessed by quantitating the amount of luminescence produced. Alternatively, the amount of ADP produced by the kinase reaction can be coupled to FLuc through a two-step process. This chapter describes the bioluminescent assays that were developed for three classes of kinases (lipid, protein, and metabolic kinases) and miniaturized to 1536-well format, enabling their use for quantitative high-throughput (qHTS) of small-molecule libraries.
Asunto(s)
Glucoquinasa/análisis , Ensayos Analíticos de Alto Rendimiento/métodos , Mediciones Luminiscentes/métodos , Fosfotransferasas (Aceptor de Grupo Alcohol)/análisis , Proteínas Proto-Oncogénicas c-yes/análisis , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adenosina Difosfato/análisis , Adenosina Trifosfato/análisis , Glucoquinasa/metabolismo , Ensayos Analíticos de Alto Rendimiento/instrumentación , Humanos , Indicadores y Reactivos , Luciferasas de Luciérnaga/metabolismo , Mediciones Luminiscentes/instrumentación , Unión Proteica , Especificidad por SustratoRESUMEN
The activities of hexokinase isoenzymes I-IV (EC 2.7.1.1) and of N-acetylglucosamine kinase (EC 2.7.1.59) were determined in normal human liver and in alcoholic liver disease and primary biliary cirrhosis after FPLC fractionation of high-speed supernatants on Mono-Q with a linear NaCl gradient. In control human liver the hexokinase activities were: I, 3.6; II, 0.7; III, 3.5, IV, 4.8 (mUnits/mg supernatant protein). The activity of N-acetylglucosamine kinase was 8 mU/mg of protein. In alcoholic liver disease and primary biliary cirrhosis, the activity of hexokinase IV (glucokinase) was suppressed to less than 10% of control activity and the activity of hexokinase I was increased 3-fold. The activity of hexokinase II was increased approximately 7-fold in alcoholic liver disease. The activities of hexokinase III and N-acetylglucosamine kinase were unchanged in cirrhosis. Hexokinase III showed 50% substrate inhibition at 100 mM glucose as compared with 0.5mM glucose. The high activity of hexokinase III in human liver (approximately 50% of the low-Km activity and 70% of glucokinase activity) results in a significant underestimation of glucokinase activity as determined by the conventional spectrometric assay while the activity of N-acetylglucosamine kinase may contribute to an overestimation of glucokinase activity in the radiochemical assay. Furthermore glucokinase is dramatically suppressed in liver disease, which although partly compensated for by the increase in hexokinase I (and II), accounts in part for the well-known glucose intolerance of liver cirrhosis.
Asunto(s)
Glucoquinasa/metabolismo , Hexoquinasa/metabolismo , Cirrosis Hepática/enzimología , Supresión Genética/genética , Cromatografía , Inhibidores Enzimáticos/farmacología , Glucoquinasa/análisis , Glucosa/farmacología , Hexoquinasa/análisis , Humanos , Isoenzimas/análisis , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Cirrosis Hepática Biliar/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/análisis , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismoRESUMEN
This article reviews evidence for a pivotal role of glucokinase as glucose sensor of the pancreatic beta-cells. Glucokinase explains the capacity, hexose specificity, affinities, sigmoidicity, and anomeric preference of pancreatic islet glycolysis, and because stimulation of glucose metabolism is a prerequisite of glucose stimulation of insulin release, glucokinase also explains many characteristics of this beta-cell function. Glucokinase of the beta-cell is induced or activated by glucose in contrast to liver glucokinase, which is regulated by insulin. Tissue-specific regulation corresponds with observations that liver and pancreatic beta-cell glucokinase are structurally distinct. Glucokinase could play a glucose-sensor role in hepatocytes as well, and certain forms of diabetes mellitus might be due to glucokinase deficiencies in pancreatic beta-cells, hepatocytes, or both.
Asunto(s)
Glucemia/análisis , Glucoquinasa/análisis , Islotes Pancreáticos/metabolismo , Hígado/metabolismo , Transducción de Señal , Animales , Diabetes Mellitus Tipo 2/fisiopatología , Humanos , Islotes Pancreáticos/citología , Hígado/citología , Fenómenos Fisiológicos de la Nutrición/fisiologíaRESUMEN
In the Goto-Kakizaki rat, a new genetic model of NIDDM, insulin response to glucose is selectively impaired. To elucidate the mechanism of this abnormality, we studied the properties of ATP-sensitive K+ channels, the inhibition of which is a key step of insulin secretion induced by fuel substrates, using the patch-clamp technique. The glucose-sensitivity of KATP channels was considerably reduced in GK rats. However, the inhibitory effects of ATP on channel activity and unitary conductance were not significantly different between control and GK rats. Thus, it appears that the impaired insulinotropic action of glucose in beta-cells of GK rats is attributable to insufficient closure of the KATP channels, probably because of deficient ATP production by impaired glucose metabolism. KATP-channel activities in both control and diabetic beta-cells were found to be equally suppressed by glyceraldehyde and 2-ketoisocaproate. These results strongly suggest that the step responsible for the metabolic dysfunction of diabetic beta-cells is located within the glycolytic pathway before glyceraldehyde-3-phosphate or in the glycerol phosphate shuttle.
Asunto(s)
Adenosina Trifosfato/farmacología , Diabetes Mellitus Tipo 2/fisiopatología , Glucosa/farmacología , Islotes Pancreáticos/fisiopatología , Canales de Potasio/fisiología , Animales , Glucemia/análisis , Peso Corporal/efectos de los fármacos , Errores Innatos del Metabolismo de los Carbohidratos/genética , Diabetes Mellitus Tipo 2/enzimología , Diabetes Mellitus Tipo 2/genética , Modelos Animales de Enfermedad , Glucoquinasa/análisis , Gliceraldehído/farmacología , Insulina/sangre , Islotes Pancreáticos/enzimología , Islotes Pancreáticos/patología , Cetoácidos/farmacología , Masculino , Canales de Potasio/efectos de los fármacos , Ratas , Ratas WistarRESUMEN
In the accompanying article, we describe the creation of novel cell lines derived from RIN 1046-38 rat insulinoma cells by stable transfection with combinations of genes encoding human insulin, GLUT2, and glucokinase. Herein we describe the regulation of insulin secretion and glucose metabolism in these new cell lines. A cell line (betaG I/17) expressing only the human proinsulin transgene exhibits a clear increase in basal insulin production (measured in the absence of secretagogues) relative to parental RIN 1046-38 cells. betaG I/17 cells engineered for high levels of GLUT2 expression and a twofold increase in glucokinase activity (betaG 49/206) or engineered for a 10-fold increase in glucokinase activity alone (betaG 40/110) exhibit a 66% and 80% suppression in basal insulin secretion relative to betaG I/17 cells, respectively. As a result, betaG 49/206 and betaG 40/110 cells exhibit potent insulin-secretory responses to glucose alone (6.1- and 7.6-fold, respectively) or to glucose plus isobutylmethylxanthine (10.8- and 15.1-fold, respectively) that are clearly larger than the corresponding responses of betaG I/17 or parental RIN 1046-38 cells. betaG 49/206 and betaG 40/110 cells also exhibit a rapid and sustained response to glucose plus isobutyl-methylxanthine in perifusion studies that is clearly larger in magnitude than that of the two control lines. Glucose dose-response studies show that both engineered and non-engineered lines respond maximally to submillimolar concentrations of glucose and that betaG 49/206 cells are the most sensitive to low concentrations of the hexose, consistent with their clearly elevated rate of [5-3H]glucose usage. Finally, 5-thioglucose, a potent inhibitor of low-K(m) hexokinases, most effectively normalizes glucose concentration dependence for insulin secretion in the cell line with highest glucokinase expression (betaG 40/110). We conclude that GLUT2 and/or glucokinase expression imposes tight regulation of basal insulin secretion in cell lines that overexpress human proinsulin, allowing a marked improvement in the range of secretagogue responsiveness in such cells.
Asunto(s)
Regulación de la Expresión Génica/genética , Glucosa/metabolismo , Insulina/metabolismo , Insulinoma/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , 1-Metil-3-Isobutilxantina/farmacología , Animales , Relación Dosis-Respuesta a Droga , Glucoquinasa/análisis , Glucosa/análisis , Glucosa/farmacología , Transportador de Glucosa de Tipo 2 , Humanos , Immunoblotting , Insulina/análisis , Insulina/genética , Secreción de Insulina , Insulinoma/genética , Insulinoma/patología , Proteínas de Transporte de Monosacáridos/análisis , Neoplasias Pancreáticas/patología , Inhibidores de Fosfodiesterasa/farmacología , Fosforilación , Ratas , Tritio , Células Tumorales CultivadasRESUMEN
In mice, diabetes can be induced by multiple low doses of streptozotocin (MLD-STZ), i.e., 40 mg/kg body wt on each of 5 consecutive days. In this model, diabetes develops only when STZ induces both beta-cell toxicity and T-cell-dependent immune reactions. The target molecule(s) of MLD-STZ-induced beta-cell toxicity are not known, however. In this study, we report that GLUT2 is a target molecule for MLD-STZ toxicity. Ex vivo, a gradual decrement of both GLUT2 protein and mRNA expression was found in pancreatic islets isolated from MLD-STZ-treated C57BL/6 male mice, whereas mRNA expression of beta-actin, glucokinase, and proinsulin remained unaffected. Significant reduction of both GLUT2 protein and mRNA expression was first noted 1 day after the third STZ injection, clearly preceding the onset of hyperglycemia. The extent of reduction increased with the number of STZ injections administered and increased over time, after the last, i.e., fifth, STZ injection. The STZ-induced reduction of GLUT2 protein and mRNA was not due to an essential loss of beta-cells, because ex vivo, not only the total RNA yield and protein content in isolated islets, but also proinsulin mRNA expression, failed to differ significantly in the differently treated groups. Furthermore, islets isolated from MLD-STZ-treated donors responded to the nonglucose secretagogue arginine in a pattern similar to that of solvent-treated donors. Interestingly, the MLD-STZ-induced reduction of both GLUT2 protein and mRNA was prevented by preinjecting mice with 5-thio-D-glucose before each STZ injection. Apparently, GLUT2 is a crucial target molecule of MLD-STZ toxicity, and this toxicity seems to precede the immune reactions against beta-cells.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Islotes Pancreáticos/química , Proteínas de Transporte de Monosacáridos/análisis , Estreptozocina/administración & dosificación , Actinas/análisis , Actinas/genética , Actinas/metabolismo , Animales , Secuencia de Bases , Glucemia/análisis , Glucemia/metabolismo , Western Blotting , Células Cultivadas , ADN Complementario/análisis , ADN Complementario/química , ADN Complementario/genética , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/patología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica , Glucoquinasa/análisis , Glucoquinasa/genética , Glucoquinasa/metabolismo , Glucosa/análogos & derivados , Glucosa/farmacología , Transportador de Glucosa de Tipo 2 , Islotes Pancreáticos/embriología , Islotes Pancreáticos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Proteínas de Transporte de Monosacáridos/genética , Proteínas de Transporte de Monosacáridos/metabolismo , Reacción en Cadena de la Polimerasa , Proinsulina/análisis , Proinsulina/genética , Proinsulina/metabolismo , ARN Mensajero/análisis , ARN Mensajero/química , ARN Mensajero/genética , RadioinmunoensayoRESUMEN
Identification of the fundamental polypeptide difference between yellow (A(y)/-, A(vy)/-) and non-yellow mice is important for biomedical research because of the influence of the yellow genotype on normal and neoplastic growth and obesity. The complexity of the "yellow mouse syndrome" makes attainment of this objective dependent on the separation of those pleiotropic enzyme differences which are secondary, and depend on the background genome, from those which are primary, and depend primarily on the agouti locus genotype.-Four of nine hepatic enzyme activities assayed simultaneously differed between eight-week-old yellow (A(y)/-, A(vy)/-) and non-yellow (A/-, a/a) male inbred and F(1) hybrid mice. Among these four, only cytoplasmic malic enzyme activity was elevated in all yellow mice, as compared with the non-yellow sibs, regardless of background genome. Glucokinase, serine dehydratase, and tyrosine alpha-ketoglutarate transaminase activities were also changed in yellow mice, but these alterations depended on the background genome.-The ratio of malic enzyme activity to citrate-cleavage enzyme activity, possibly related to the altered fat metabolism of yellow mice, was influenced by background genome as well as by the yellow genotype.--Significant deviations of enzyme activities from mid-parent values among F(1) hybrids were associated with particular background genomes; the number of such deviations was larger among yellow mice than among non-yellows and this difference was greater among C3H F(1) hybrids than among C57BL/6 F(1) hybrids.
Asunto(s)
Cromosomas , Genotipo , Ratones Endogámicos/enzimología , Amoníaco-Liasas/análisis , Animales , Femenino , Glucoquinasa/análisis , Hexoquinasa/análisis , L-Serina Deshidratasa/análisis , Hígado/enzimología , Malato Deshidrogenasa/análisis , Masculino , Ratones , Obesidad/genética , Ornitina , Oxo-Ácido-Liasas/análisis , Fosfogluconato Deshidrogenasa/análisis , Pigmentación , Transaminasas/análisis , Tirosina Transaminasa/análisisRESUMEN
BACKGROUND: Preliminary clinical and experimental results suggest that iron can modify hepatocytes' insulin sensitivity by interfering with insulin receptor and intracellular insulin signalling. AIM: To evaluate in vivo the influence of iron on insulin resistance and insulin release in patients with non-alcoholic fatty liver disease and in vitro the interaction between iron and insulin sensitivity by analysing the effect of iron manipulation on insulin receptor expression in hepatoblastoma HepG2 cell line. RESULTS: Insulin resistance evaluated by homeostatis model assessment (HOMA)-insulin resistance significantly decreased after diet, and a further reduction was observed after phlebotomies. Iron depletion by desferrioxamine increased by twofold the 125I-insulin-specific binding, whereas iron addition reduced insulin binding, similarly to cells exposed to high glucose concentration. CONCLUSION: Iron status affects insulin sensitivity by modulating the transcription and membrane expression/affinity of insulin receptor expression in hepatocytes and influencing insulin-dependent gene expression suggesting that increased insulin clearance and decreased insulin resistance may contribute to the positive effect of iron depletion in patients with non-alcoholic fatty liver disease.
Asunto(s)
Hígado Graso/metabolismo , Resistencia a la Insulina , Hierro/metabolismo , Línea Celular Tumoral , Deferoxamina/metabolismo , Dieta , Ingestión de Energía , Femenino , Expresión Génica , Glucoquinasa/análisis , Humanos , Insulina/metabolismo , Hierro/administración & dosificación , Masculino , Persona de Mediana Edad , ARN Mensajero/análisis , Receptor de Insulina/metabolismoRESUMEN
Chickens are more hyperglycaemic and insulin-resistant than mammals, and in efforts to understand their glucose metabolism we investigated whether glucokinase (GK) is present in chicken liver or pancreas. This enzyme plays a major role in glucose-sensing in mammals and we have examined whether it also contributes to glucose homeostasis in chickens. Using RT-PCR, we cloned and sequenced a partial cDNA fragment (750 bp) from liver and pancreas that showed a high degree of identity with mammalian GK. Using antibodies directed towards human GK, we immunodetected a 50 kDa band in chicken liver and pancreas. The molecular mass of the band and its specific interaction with the antibody suggest that this protein corresponds to a chicken homologue of human GK. We also determined by spectrophotometry a glucokinase-like activity in crude liver homogenates with an apparent half saturating concentration for glucose of 8.6 mM. GK gene and protein expression did not differ between fed and 24 h fasted states but GK-like activity was significantly increased in fed chickens. In conclusion, our study provides evidence for the presence of GK gene and protein in chicken liver and pancreas and shows that the liver enzyme is active.