RESUMEN

BACKGROUND: Aberrant gluconeogenesis is considered among primary drivers of hyperglycemia under insulin resistant conditions, with multiple studies pointing towards epigenetic dysregulation. Here we examine the role of miR-721 and effect of epigenetic modulator laccaic acid on the regulation of gluconeogenesis under high fat diet induced insulin resistance. RESULTS: Reanalysis of miRNA profiling data of high-fat diet-induced insulin-resistant mice model, GEO dataset (GSE94799) revealed a significant upregulation of miR-721, which was further validated in invivo insulin resistance in mice and invitro insulin resistance in Hepa 1-6 cells. Interestingly, miR-721 mimic increased glucose production in Hepa 1-6 cells via activation of FOXO1 regulated gluconeogenic program. Concomitantly, inhibition of miR-721 reduced glucose production in palmitate induced insulin resistant Hepa 1-6 cells by blunting the FOXO1 induced gluconeogenesis. Intriguingly, at epigenetic level, enrichment of the transcriptional activation mark H3K36me2 got decreased around the FOXO1 promoter. Additionally, identifying targets of miR-721 using miRDB.org showed H3K36me2 demethylase KDM2A as a potential target. Notably, miR-721 inhibitor enhanced KDM2A expression which correlated with H3K36me2 enrichment around FOXO1 promoter and the downstream activation of the gluconeogenic pathway. Furthermore, inhibition of miR-721 in high-fat diet-induced insulin-resistant mice resulted in restoration of KDM2A levels, concomitantly reducing FOXO1, PCK1, and G6PC expression, attenuating gluconeogenesis, hyperglycemia, and improving glucose tolerance. Interestingly, the epigenetic modulator laccaic acid also reduced the hepatic miR-721 expression and improved KDM2A expression, supporting our earlier report that laccaic acid attenuates insulin resistance by reducing gluconeogenesis. CONCLUSION: Our study unveils the role of miR-721 in regulating gluconeogenesis through KDM2A and FOXO1 under insulin resistance, pointing towards significant clinical and therapeutic implications for metabolic disorders. Moreover, the promising impact of laccaic acid highlights its potential as a valuable intervention in managing insulin resistance-associated metabolic diseases.

Asunto(s)

Gluconeogénesis , Resistencia a la Insulina , Histona Demetilasas con Dominio de Jumonji , MicroARNs , Animales , Masculino , Ratones , Dieta Alta en Grasa , Epigénesis Genética , Proteína Forkhead Box O1/metabolismo , Proteína Forkhead Box O1/genética , Gluconeogénesis/genética , Gluconeogénesis/fisiología , Resistencia a la Insulina/fisiología , Histona Demetilasas con Dominio de Jumonji/metabolismo , Histona Demetilasas con Dominio de Jumonji/genética , Ratones Endogámicos C57BL , MicroARNs/metabolismo , MicroARNs/genética

RESUMEN

Trichophyton rubrum is the primary causative agent of dermatophytosis worldwide. This fungus colonizes keratinized tissues and uses keratin as a nutritional source during infection. In T. rubrum-host interactions, sensing a hostile environment triggers the adaptation of its metabolic machinery to ensure its survival. The glyoxylate cycle has emerged as an alternative metabolic pathway when glucose availability is limited; this enables the conversion of simple carbon compounds into glucose via gluconeogenesis. In this study, we investigated the impact of stuA deletion on the response of glyoxylate cycle enzymes during fungal growth under varying culture conditions in conjunction with post-transcriptional regulation through alternative splicing of the genes encoding these enzymes. We revealed that the ΔstuA mutant downregulated the malate synthase and isocitrate lyase genes in a keratin-containing medium or when co-cultured with human keratinocytes. Alternative splicing of an isocitrate lyase gene yielded a new isoform. Enzymatic activity assays showed specific instances where isocitrate lyase and malate synthase activities were affected in the mutant strain compared to the wild type strain. Taken together, our results indicate a relevant balance in transcriptional regulation that has distinct effects on the enzymatic activities of malate synthase and isocitrate lyase.

Asunto(s)

Arthrodermataceae , Factores de Transcripción , Humanos , Isocitratoliasa/genética , Malato Sintasa/genética , Gluconeogénesis/genética , Empalme Alternativo , Carbono , Glucosa , Queratinas , Glioxilatos

RESUMEN

To investigate the role of the transient receptor potential channel vanilloid type 1 (TRPV1) in hepatic glucose metabolism, we analyzed genes related to the clock system and glucose/lipid metabolism and performed glycogen measurements at ZT8 and ZT20 in the liver of C57Bl/6J (WT) and Trpv1 KO mice. To identify molecular clues associated with metabolic changes, we performed proteomics analysis at ZT8. Liver from Trpv1 KO mice exhibited reduced Per1 expression and increased Pparα, Pparγ, Glut2, G6pc1 (G6pase), Pck1 (Pepck), Akt, and Gsk3b expression at ZT8. Liver from Trpv1 KO mice also showed reduced glycogen storage at ZT8 but not at ZT20 and significant proteomics changes consistent with enhanced glycogenolysis, as well as increased gluconeogenesis and inflammatory features. The network propagation approach evidenced that the TRPV1 channel is an intrinsic component of the glucagon signaling pathway, and its loss seems to be associated with increased gluconeogenesis through PKA signaling. In this sense, the differentially identified kinases and phosphatases in WT and Trpv1 KO liver proteomes show that the PP2A phosphatase complex and PKA may be major players in glycogenolysis in Trpv1 KO mice.

Asunto(s)

Gluconeogénesis , Proteoma , Canales Catiónicos TRPV , Animales , Expresión Génica , Gluconeogénesis/genética , Glucosa/metabolismo , Glucógeno/metabolismo , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteoma/metabolismo , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/metabolismo

RESUMEN

The white shrimp Penaeus (Litopenaeus) vannamei is the most economically important crustacean species cultivated in the Western Hemisphere. This crustacean shifts its metabolism to survive under extreme environmental conditions such as hypoxia, although for a limited time. Glucose-6-phosphatase (G6Pase) is a key enzyme contributing to maintain blood glucose homeostasis through gluconeogenesis and glycogenolysis. To our knowledge, there are no current detailed studies about cDNA or gene sequences of G6Pase from any crustacean reported. Herein we report the shrimp P. (L.) vannamei cDNA and gene sequences. The gene contains seven exons interrupted by six introns. The deduced amino acid sequence has 35% identity to other homolog proteins, with the catalytic amino acids conserved and phylogenetically close to the corresponding invertebrate homologs. Protein molecular modeling predicted eight transmembrane helices with the catalytic site oriented towards the lumen of the endoplasmic reticulum. G6Pase expression under normoxic conditions was evaluated in hepatopancreas, gills, and muscle and the highest transcript abundance was detected in hepatopancreas. In response to different times of hypoxia, G6Pase mRNA expression did not change in hepatopancreas and became undetectable in muscle; however, in gills, its expression increased after 3 h and 24 h of oxygen limitation, indicating its essential role to maintain glycemic control in these conditions.

Asunto(s)

Clonación Molecular/métodos , Branquias/metabolismo , Gluconeogénesis/genética , Glucosa-6-Fosfatasa/metabolismo , Hepatopáncreas/metabolismo , Animales , Glucosa-6-Fosfatasa/genética , Penaeidae

RESUMEN

The phosphoenolpyruvate-pyruvate-oxaloacetate node is a major branch within the central carbon metabolism and acts as a connection point between glycolysis, gluconeogenesis, and the TCA cycle. Phosphoenolpyruvate carboxylase, pyruvate carboxylase, phosphoenolpyruvate carboxykinase, malic enzymes, and pyruvate kinase, among others, are enzymes included in this node. We determined the mRNA levels and specific activity profiles of some of these genes and enzymes in Streptomyces coelicolor M-145. The results obtained in the presence of glucose demonstrated that all genes studied of the phosphoenolpyruvate-pyruvate-oxaloacetate node were expressed, although at different levels, with 10- to 100-fold differences. SCO3127 (phosphoenolpyruvate carboxylase gene) and SCO5261 (NADP+-dependent malic enzyme gene) showed the highest expression in the rapid growth phase, and the mRNA levels corresponding to SCO5896 (phosphoenolpyruvate-utilizing enzyme gene), and SCO0546 (pyruvate carboxylase gene) increased 5- to 10-fold towards the stationary phase. In casamino acids, in general mRNA levels of S. coelicolor were lower than in glucose, however, results showed greater mRNA expression of SCO4979 (PEP carboxykinase), SCO0208 (pyruvate phosphate dikinase gene), and SCO5261 (NADP+-dependent malic enzyme). These results suggest that PEP carboxylase (SCO3127) is an important enzyme during glucose catabolism and oxaloacetate replenishment. On the other hand, phosphoenolpyruvate carboxykinase, pyruvate phosphate dikinase, and NADP+-malic enzyme could have an important role in gluconeogenesis in S. coelicolor.

Asunto(s)

Gluconeogénesis/genética , Glucosa/metabolismo , Streptomyces coelicolor/metabolismo , Ciclo del Ácido Cítrico/genética , Metabolismo Energético , Expresión Génica , Genes Bacterianos , Malato Deshidrogenasa/genética , Malato Deshidrogenasa/metabolismo , Fosfoenolpiruvato Carboxiquinasa (ATP)/genética , Fosfoenolpiruvato Carboxiquinasa (ATP)/metabolismo , Piruvato Carboxilasa/genética , Piruvato Carboxilasa/metabolismo , Streptomyces coelicolor/genética

RESUMEN

Nutritional recovery of early malnutrition with a soybean diet reduces liver glycogen stores in the fed state and produces liver insulin resistance. We investigated whether nutritional recovery on a soybean flour diet alters hepatic gluconeogenesis in the adult offspring of rats deprived of protein during pregnancy and lactation. Male rats from mothers that were fed either 17% (C) or 6% (L) protein during pregnancy and lactation were maintained on a 17% casein (CC, n = 16 and LC, n = 17), 17% soybean flour (CS, n = 10 and LS, n = 10), or 6% casein (LL, n = 10) diet after weaning. The soybean diet reduced basal serum glucose (soybean diet, 5.6 ± 0.6 mmol/L vs. casein diet, 6.2 ± 0.6 mmol/L; p < 0.05) but increased alanine aminotransferase mRNA/GAPDH (soybean diet, 0.062 ± 0.038 vs. casein diet, 0.024 ± 0.011; p < 0.01), phosphoenolpyruvate carboxykinase mRNA/GAPDH (soybean diet, 1.53 ± 0.52 vs. casein diet, 0.95 ± 0.43; p < 0.05), and glycerokinase protein content (soybean diet, 0.86 ± 0.08 vs. casein diet, 0.75 ± 0.11; p < 0.05). The serum glucose concentration (recovered groups, 5.6 ± 0.5 mmol/L vs. control groups, 6.2 ± 0.7 mmol/L; p < 0.05) and phosphoenolpyruvate carboxykinase activity (recovered groups, 2.8 ± 0.6 µU/mg vs. control groups, 3.6 ± 0.6 µU/mg; p < 0.05) were decreased in rats subjected to protein restriction in early life. The glucose area under the curve during the pyruvate tolerance test did not differ among groups, whereas glucose area under the curve after glucagon infusion was reduced by early malnutrition (recovered groups, 4210 ± 572 mg/dL·40 min vs. control groups, 4493 ± 688 mg/dL·40 min; p < 0.001) and by the soybean diet (soybean diet, 3995 ± 500 mg/dL·40 min vs. casein diet, 4686 ± 576 mg/dL·40 min; p < 0.05). Thus, the soybean diet impaired the response to glucagon but did not alter gluconeogenesis.

Asunto(s)

Alimentación Animal , Glucagón/metabolismo , Gluconeogénesis , Glycine max/metabolismo , Hígado/metabolismo , Efectos Tardíos de la Exposición Prenatal , Desnutrición Proteico-Calórica/dietoterapia , Factores de Edad , Animales , Dieta con Restricción de Proteínas , Modelos Animales de Enfermedad , Femenino , Regulación Enzimológica de la Expresión Génica , Gluconeogénesis/genética , Lactancia , Hígado/enzimología , Masculino , Estado Nutricional , Embarazo , Fenómenos Fisiologicos de la Nutrición Prenatal , Desnutrición Proteico-Calórica/genética , Desnutrición Proteico-Calórica/metabolismo , Desnutrición Proteico-Calórica/fisiopatología , Ratas Wistar

RESUMEN

Because of the paucity of information regarding metabolic effects of advanced glycation end products (AGEs) on liver, we evaluated effects of AGEs chronic administration in (1) insulin sensitivity; (2) hepatic expression of genes involved in AGEs, glucose and fat metabolism, oxidative stress and inflammation and; (3) hepatic morphology and glycogen content. Rats received intraperitoneally albumin modified (AlbAGE) or not by advanced glycation for 12 weeks. AlbAGE induced whole-body insulin resistance concomitantly with increased hepatic insulin sensitivity, evidenced by activation of AKT, inactivation of GSK3, increased hepatic glycogen content, and decreased expression of gluconeogenesis genes. Additionally there was reduction in hepatic fat content, in expression of lipogenic, pro-inflamatory and pro-oxidative genes and increase in reactive oxygen species and in nuclear expression of NRF2, a transcription factor essential to cytoprotective response. Although considered toxic, AGEs become protective when administered chronically, stimulating AKT signaling, which is involved in cellular defense and insulin sensitivity.

Asunto(s)

Productos Finales de Glicación Avanzada/farmacología , Hormesis/efectos de los fármacos , Resistencia a la Insulina , Hígado/metabolismo , Albúminas/farmacología , Animales , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Gluconeogénesis/efectos de los fármacos , Gluconeogénesis/genética , Productos Finales de Glicación Avanzada/administración & dosificación , Glucógeno/metabolismo , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Proteína HMGB1/metabolismo , Mediadores de Inflamación/metabolismo , Inyecciones Intraperitoneales , Lipogénesis/efectos de los fármacos , Lipogénesis/genética , Hígado/efectos de los fármacos , Masculino , Modelos Biológicos , Factor 2 Relacionado con NF-E2/metabolismo , Oxidación-Reducción , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo

RESUMEN

KEY MESSAGE: The sunflower transcription factor HaWRKY10 stimulates reserves mobilization in Arabidopsis. Gene expression and enzymes activity assays indicated that lipolysis and gluconeogenesis were increased. Microarray results suggested a parallelism in sunflower. Germinating oilseeds converts stored lipids into sugars, and thereafter in metabolic energy that is used in seedling growth and establishment. During germination, the induced lipolysis linked to the glyoxylate pathway and gluconeogenesis produces sucrose, which is then transported to the embryo and driven through catabolic routes. Herein, we report that the sunflower transcription factor HaWRKY10 regulates carbon partitioning by reducing carbohydrate catabolism and increasing lipolysis and gluconeogenesis. HaWRKY10 was regulated by abscisic acid and gibberellins in the embryo leaves 48 h after seed imbibition and highly expressed during sunflower seed germination and seedling growth, concomitantly with lipid mobilization. Sunflower leaf disks overexpressing HaWRKY10 showed repressed expression of genes related to sucrose cleavage and glycolysis compared with controls. Moreover, HaWRKY10 constitutive expression in Arabidopsis seeds produced higher decrease in lipid reserves, whereas starch and sucrose were more preserved compared with wild type. Gene transcripts abundance and enzyme activities involved in stored lipid mobilization and gluconeogenesis increased more in transgenic than in wild type seeds 36 h after imbibition, whereas the negative regulator of lipid mobilization, ABI4, was repressed. Altogether, the results point out a functional parallelism between tissues and plant species, and reveal HaWRKY10 as a positive regulator of storage reserve mobilization in sunflower.

Asunto(s)

Germinación , Helianthus/crecimiento & desarrollo , Helianthus/metabolismo , Proteínas de Plantas/metabolismo , Semillas/metabolismo , Factores de Transcripción/metabolismo , Ácido Abscísico/metabolismo , Arabidopsis/genética , Metabolismo de los Hidratos de Carbono/genética , Carbono/metabolismo , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Germinación/genética , Giberelinas/metabolismo , Gluconeogénesis/genética , Helianthus/genética , Metabolismo de los Lípidos/genética , Modelos Biológicos , Anotación de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Hojas de la Planta/metabolismo , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Plantones/metabolismo , Semillas/genética , Semillas/crecimiento & desarrollo

RESUMEN

Thiamine is one of several essential cofactors for ATP generation. Its deficiency, like in beriberi and in the Wernicke-Korsakoff syndrome, has been studied for many decades. However, its mechanism of action is still not completely understood at the cellular and molecular levels. Since it acts as a coenzyme for dehydrogenases of pyruvate, branched-chain keto acids, and ketoglutarate, its nutritional privation is partly a phenocopy of inborn errors of metabolism, among them maple syrup urine disease. In the present paper, we report metabolic and genomic findings in mice deprived of thiamine. They are similar to the ones we have previously found in biotin deficiency, another ATP generation cofactor. Here we show that thiamine deficiency substantially reduced the energy state in the liver and activated the energy sensor AMP-activated kinase. With this vitamin deficiency, several metabolic parameters changed: blood glucose was diminished and serum lactate was increased, but insulin, triglycerides, and cholesterol, as well as liver glycogen, were reduced. These results indicate a severe change in the energy status of the whole organism. Our findings were associated with modified hepatic levels of the mRNAs of several carbon metabolism genes: a reduction of transcripts for liver glucokinase and fatty acid synthase and augmentation of those for carnitine palmitoyl transferase 1 and phosphoenolpyruvate carboxykinase as markers for glycolysis, fatty acid synthesis, beta-oxidation, and gluconeogenesis, respectively. Glucose tolerance was initially increased, suggesting augmented insulin sensitivity, as we had found in biotin deficiency; however, in the case of thiamine, it was diminished from the 3rd week on, when the deficient animals became undernourished, and paralleled the changes in AKT and mTOR, 2 main proteins in the insulin signaling pathway. Since many of the metabolic and gene expression effects on mice deprived of thiamine are similar to those in biotin deficiency, it may be that they result from a more general impairment of oxidative phosphorylation due to a shortage of ATP generation cofactors. These findings may be relevant to energy-related disorders, among them several inborn errors of metabolism, as well as common energy disorders like obesity, diabetes, and neurodegenerative illnesses.

Asunto(s)

Adenosina Trifosfato/metabolismo , Deficiencia de Biotinidasa , Metabolismo Energético , Hígado/metabolismo , Enfermedades Metabólicas/etiología , Deficiencia de Tiamina/genética , Deficiencia de Tiamina/metabolismo , Adenosina Trifosfato/deficiencia , Animales , Deficiencia de Biotinidasa/genética , Deficiencia de Biotinidasa/metabolismo , Metabolismo Energético/efectos de los fármacos , Metabolismo Energético/genética , Interacción Gen-Ambiente , Genoma/efectos de los fármacos , Gluconeogénesis/efectos de los fármacos , Gluconeogénesis/genética , Hígado/efectos de los fármacos , Masculino , Enfermedades Metabólicas/genética , Enfermedades Metabólicas/metabolismo , Ratones , Ratones Endogámicos C57BL , Tiamina/farmacología

RESUMEN

The objective of this study was to investigate the impact of elevated tissue omega-3 (n-3) polyunsaturated fatty acids (PUFA) status on age-related glucose intolerance utilizing the fat-1 transgenic mouse model, which can endogenously synthesize n-3 PUFA from omega-6 (n-6) PUFA. Fat-1 and wild-type mice, maintained on the same dietary regime of a 10% corn oil diet, were tested at two different ages (2 months old and 8 months old) for various glucose homeostasis parameters and related gene expression. The older wild-type mice exhibited significantly increased levels of blood insulin, fasting blood glucose, liver triglycerides, and glucose intolerance, compared to the younger mice, indicating an age-related impairment of glucose homeostasis. In contrast, these age-related changes in glucose metabolism were largely prevented in the older fat-1 mice. Compared to the older wild-type mice, the older fat-1 mice also displayed a lower capacity for gluconeogenesis, as measured by pyruvate tolerance testing (PTT) and hepatic gene expression of phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6 phosphatase (G6Pase). Furthermore, the older fat-1 mice showed a significant decrease in body weight, epididymal fat mass, inflammatory activity (NFκ-B and p-IκB expression), and hepatic lipogenesis (acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) expression), as well as increased peroxisomal activity (70-kDa peroxisomal membrane protein (PMP70) and acyl-CoA oxidase1 (ACOX1) expression). Altogether, the older fat-1 mice exhibit improved glucose homeostasis in comparison to the older wild-type mice. These findings support the beneficial effects of elevated tissue n-3 fatty acid status in the prevention and treatment of age-related chronic metabolic diseases.

Asunto(s)

Proteínas de Caenorhabditis elegans/metabolismo , Ácido Graso Desaturasas/metabolismo , Ácidos Grasos Omega-3/metabolismo , Intolerancia a la Glucosa/metabolismo , Acetil-CoA Carboxilasa/genética , Acetil-CoA Carboxilasa/metabolismo , Factores de Edad , Animales , Glucemia/metabolismo , Proteínas de Caenorhabditis elegans/genética , Ácido Graso Desaturasas/genética , Ácido Graso Sintasas/genética , Ácido Graso Sintasas/metabolismo , Expresión Génica , Gluconeogénesis/genética , Glucosa/metabolismo , Intolerancia a la Glucosa/genética , Glucosa-6-Fosfatasa/genética , Glucosa-6-Fosfatasa/metabolismo , Homeostasis/genética , Immunoblotting , Insulina/sangre , Lipogénesis/genética , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fosfoenolpiruvato Carboxiquinasa (ATP)/genética , Fosfoenolpiruvato Carboxiquinasa (ATP)/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

The adaptation of the liver to periods of negative energy balance is largely unknown in beef cattle on grazing systems. We evaluated liver transcriptome throughout gestation and early lactation of purebred and crossbred beef cows [Angus, Hereford, and their F1 crossbreeds (CR)], grazing high or low herbage allowances (HA) of native grasslands (4 and 2.5 kg dry matter/kg body wt annual mean; n = 16) using an Agilent 4 × 44k bovine array. A total of 4,661 transcripts were affected by days [272 ≥ 2.5-fold difference, false discovery rate (FDR) ≤ 0.10] and 47 pathways were altered during winter gestation (-165 to -15 days relative to calving), when cows experienced decreased body condition score, decreased insulin, and increased nonesterified fatty acid concentrations. Gluconeogenesis and fatty acid oxidation pathways were upregulated, while cell growth, DNA replication, and transcription pathways were downregulated (FDR ≤ 0.25). We observed only small changes in the liver transcriptome during early lactation (+15 to +60 days). A total of 225 genes were differentially expressed (47 ≥ 2-fold difference, FDR ≤ 0.10) between HA. The majority of those were related to glucose and pyruvate metabolism and were upregulated in high HA, reflecting their better metabolic status. Two genes were upregulated in CR cows, but 148 transcripts (74 ≥ 2-fold change difference, FDR ≤ 0.10) were affected by the HA and cow genotype interaction. The transcriptional changes observed indicated a complex and previously unrecognized, hepatic adaptive program of grazing beef cows in different nutritional environments. Novel target candidate genes, metabolic pathways, and regulatory mechanisms were reported.

Asunto(s)

Perfilación de la Expresión Génica/veterinaria , Regulación de la Expresión Génica/genética , Lactancia/metabolismo , Hígado/metabolismo , Embarazo/metabolismo , Análisis de Varianza , Animales , Constitución Corporal/fisiología , Bovinos , Ácidos Grasos/metabolismo , Femenino , Gluconeogénesis/genética , Gluconeogénesis/fisiología , Insulina/sangre , Análisis por Micromatrices/veterinaria , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Uruguay

RESUMEN

Hepatic insulin resistance is the major contributor to fasting hyperglycemia in type 2 diabetes. The protein kinase Akt plays a central role in the suppression of gluconeogenesis involving forkhead box O1 (Foxo1) and peroxisome proliferator-activated receptor gamma co-activator 1 alpha (PGC-1α), and in the control of glycogen synthesis involving the glycogen synthase kinase beta (GSK3ß) in the liver. It has been demonstrated that endosomal adaptor protein APPL1 interacts with Akt and blocks the association of Akt with its endogenous inhibitor, tribbles-related protein 3 (TRB3), improving the action of insulin in the liver. Here, we demonstrated that chronic exercise increased the basal levels and insulin-induced Akt serine phosphorylation in the liver of diet-induced obese mice. Endurance training was able to increase APPL1 expression and the interaction between APPL1 and Akt. Conversely, training reduced both TRB3 expression and TRB3 and Akt association. The positive effects of exercise on insulin action are reinforced by our findings that showed that trained mice presented an increase in Foxo1 phosphorylation and Foxo1/PGC-1α association, which was accompanied by a reduction in gluconeogenic gene expressions (PEPCK and G6Pase). Finally, exercised animals demonstrated increased at basal and insulin-induced GSK3ß phosphorylation levels and glycogen content at 24 h after the last session of exercise. Our findings demonstrate that exercise increases insulin action, at least in part, through the enhancement of APPL1 and the reduction of TRB3 expression in the liver of obese mice, independently of weight loss.

Asunto(s)

Insulina/metabolismo , Hígado/metabolismo , Obesidad/metabolismo , Condicionamiento Físico Animal/fisiología , Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatología , Dieta , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Gluconeogénesis/genética , Gluconeogénesis/fisiología , Glucosa-6-Fosfatasa/genética , Glucosa-6-Fosfatasa/metabolismo , Glucógeno/genética , Glucógeno/metabolismo , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Insulina/genética , Masculino , Ratones , Ratones Obesos , Obesidad/etiología , Obesidad/genética , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Fosforilación , Resistencia Física/fisiología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción , Pérdida de Peso/fisiología

RESUMEN

Small neutral amino acid transporter 2 (SNAT2) is the most abundant and ubiquitous transporter for zwitterionic short-chain amino acids. The activity of this amino acid transporter is stimulated in vivo or in vitro by glucagon or cAMP analogs. However, it is not known whether the increase in activity at the protein level is due to an increase in SNAT2 gene transcription. Thus, the aim of the present work was to study whether cAMP was able to stimulate SNAT2 gene expression and to localize and characterize the presence of cAMP response elements (CRE) in the promoter that controls the expression of the rat SNAT2 gene. We found that consumption of a high-protein diet that increased serum glucagon concentration or the administration of glucagon or incubation of hepatocytes with forskolin increased the SNAT2 mRNA level. We then isolated the 5' regulatory region of the SNAT2 gene and determined that the transcriptional start site was located 970 bp upstream of the translation start codon. We identified two potential CRE sites located at -354 and -48 bp. Our results, using deletion analysis of the 5' regulatory region of the SNAT2 gene, revealed that the CRE site located at -48 bp was fully responsible for SNAT2 regulation by cAMP. This evidence was strongly supported by mutation of the CRE site and EMSA and ChIP analysis. Alignment of rat, mouse, and human sequences revealed that this CRE site is highly conserved among species, indicating its essential role in the regulation of SNAT2 gene expression.

Asunto(s)

Sistemas de Transporte de Aminoácidos/biosíntesis , Sistemas de Transporte de Aminoácidos/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/fisiología , Sistema de Transporte de Aminoácidos A , Animales , Células Cultivadas , Inmunoprecipitación de Cromatina , Clonación Molecular , Simulación por Computador , AMP Cíclico/fisiología , Dieta , Proteínas en la Dieta/farmacología , Ensayo de Cambio de Movilidad Electroforética , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/fisiología , Glucagón/sangre , Glucagón/farmacología , Gluconeogénesis/genética , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Homeostasis/fisiología , Humanos , Informática , Masculino , Ratones , Mutagénesis Sitio-Dirigida , Regiones Promotoras Genéticas , ARN , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Especificidad de la Especie , Regulación hacia Arriba

RESUMEN

Strains of Escherichia coli bearing different forms of phosphofructokinase were used to assess the occurrence of futile cycling in cell resuspensions supplied with glycerol as gluconeogenic carbon source. A model was used to simulate results of different kinds of experiments for different levels of futile cycle. The main predictions of the model were experimentally confirmed in a strain with a mutant phosphofructokinase-2 (phosphofructokinase-2*) which is not inhibited by MgATP. The intracellular fructose 1, 6-bisphosphate concentration reaches significantly higher levels in the mutant-bearing strain than in strains with either phosphofructokinase-1 or -2. Also, this strain showed a higher rate and level of in vivo radioactive labelling of fructose 1, 6-bisphosphate, from a trace of [U-14C]glucose supplied during gluconeogenesis, indicating higher kinase activity in these conditions. Cell resuspensions of the mutant-bearing strain produced higher levels of radioactively labelled CO2 when supplied with [U-14C]glycerol as the only carbon source. Simultaneously, fewer glycerol carbons were incorporated into HClO4-insoluble macromolecules. Finally, radioactive CO2 output was measured in resuspensions supplied with glycerol as the major carbon source with traces of either [1-14C]glucose or [6-14C]glucose. It was found that, whereas in the strains with either of the wild-type phosphofructokinase isoenzymes, radioactive CO2 output from [1-14C]glucose was higher than with [6-14C]glucose, the reverse is found for the strain with phosphofructokinase-2*. This result also agrees with the corresponding prediction of the model. Using the radioactivity flux rates predicted by the model, an explanation linking the futile cycle to the differential labelling of CO2 is advanced. Finally, on the basis of these results it is proposed that strains bearing phosphofructokinase-2* sustain higher rates of futile cycling during gluconeogenesis than strains bearing either of the wild-type isoforms of phosphofructokinase. The kinetic equations and parameter values used for the model simulations are given in Supplementary Publication SUP 50183 (8 pages), which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1997) 321, 8.

Asunto(s)

Escherichia coli/metabolismo , Gluconeogénesis/fisiología , Isoenzimas/metabolismo , Mutación , Fosfofructoquinasa-1/metabolismo , Ciclo del Sustrato/fisiología , Dióxido de Carbono/metabolismo , Radioisótopos de Carbono , Escherichia coli/enzimología , Escherichia coli/genética , Fructosadifosfatos/metabolismo , Gluconeogénesis/genética , Glucosa/metabolismo , Glicerol/metabolismo , Glucólisis/genética , Glucólisis/fisiología , Líquido Intracelular/metabolismo , Isoenzimas/genética , Modelos Biológicos , Fosfofructoquinasa-1/genética , Ciclo del Sustrato/genética