RESUMEN
Reduced expression of the plasma membrane citrate transporter INDY (acronym I'm Not Dead, Yet) extends life span in lower organisms. Deletion of the mammalian Indy (mIndy) gene in rodents improves metabolism via mechanisms akin to caloric restriction, known to lower blood pressure (BP) by sympathoadrenal inhibition. We hypothesized that mIndy deletion attenuates sympathoadrenal support of BP. Continuous arterial BP and heart rate (HR) were reduced in mINDY-KO mice. Concomitantly, urinary catecholamine content was lower, and the decreases in BP and HR by mIndy deletion were attenuated after autonomic ganglionic blockade. Catecholamine biosynthesis pathways were reduced in mINDY-KO adrenals using unbiased microarray analysis. Citrate, the main mINDY substrate, increased catecholamine content in pheochromocytoma cells, while pharmacological inhibition of citrate uptake blunted the effect. Our data suggest that deletion of mIndy reduces sympathoadrenal support of BP and HR by attenuating catecholamine biosynthesis. Deletion of mIndy recapitulates beneficial cardiovascular and metabolic responses to caloric restriction, making it an attractive therapeutic target.
Asunto(s)
Presión Sanguínea/genética , Presión Sanguínea/fisiología , Transportadores de Ácidos Dicarboxílicos/genética , Transportadores de Ácidos Dicarboxílicos/fisiología , Sistema Simpatoadrenal/fisiología , Simportadores/genética , Simportadores/fisiología , Glándulas Suprarrenales/anatomía & histología , Glándulas Suprarrenales/fisiología , Animales , Restricción Calórica , Catecolaminas/biosíntesis , Línea Celular , Células Cromafines/fisiología , Transportadores de Ácidos Dicarboxílicos/deficiencia , Expresión Génica , Frecuencia Cardíaca/genética , Frecuencia Cardíaca/fisiología , Longevidad/genética , Longevidad/fisiología , Malatos/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Cardiovasculares , Actividad Motora/genética , Actividad Motora/fisiología , Piridinas/farmacología , Simportadores/deficienciaRESUMEN
Context: Depending on its lipolytic activity, glucagon plays a promising role in obesity treatment. Glucagon-induced growth hormone (GH) release can promote its effect on lipid metabolism, although the underlying mechanisms have not been well-defined. Objective: The present study highlights the glucagon effect on the GH/insulinlike growth factor 1 (IGF-1)/IGF-binding protein (IGFBP) axis in vivo and in vitro, taking into consideration insulin as a confounding factor. Materials and Methods: In a double-blind, placebo-controlled study, we investigated changes in GH, IGFBP, and IGF-1 bioactivity after intramuscular glucagon administration in 13 lean controls, 11 obese participants, and 13 patients with type 1 diabetes mellitus (T1DM). The effect of glucagon on the transcription factor forkhead box protein O1 (FOXO1) translocation, the transcription of GH/IGF-1 system members, and phosphorylation of protein kinase B (Akt) was further investigated in vitro. Results: Despite unchanged total IGF-1 and IGFBP-3 levels, glucagon decreased IGF-1 bioactivity in all study groups by increasing IGFBP-1 and IGFBP-2. The reduction in IGF-1 bioactivity occurred before the glucagon-induced surge in GH. In contrast to the transient increase in circulating insulin in obese and lean participants, no change was observed in those with T1DM. In vitro, glucagon dose dependently induced a substantial nuclear translocation of FOXO1 in human osteosarcoma cells and tended to increase IGFBP-1 and IGFBP-2 gene expression in mouse primary hepatocytes, despite absent Akt phosphorylation. Conclusions: Our data point to the glucagon-induced decrease in bioactive IGF-1 levels as a mechanism through which glucagon induces GH secretion. This insulin-independent reduction is related to increased IGFBP-1 and IGFBP-2 levels, which are most likely mediated via activation of the FOXO/mTOR (mechanistic target of rapamycin) pathway.
Asunto(s)
Diabetes Mellitus Tipo 1/metabolismo , Glucagón/administración & dosificación , Hormona del Crecimiento/efectos de los fármacos , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/efectos de los fármacos , Factor I del Crecimiento Similar a la Insulina/efectos de los fármacos , Adulto , Western Blotting , Diabetes Mellitus Tipo 1/diagnóstico , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Esquema de Medicación , Ensayo de Inmunoadsorción Enzimática , Femenino , Proteína Forkhead Box O1/efectos de los fármacos , Proteína Forkhead Box O1/metabolismo , Hormona del Crecimiento/metabolismo , Humanos , Inyecciones Intramusculares , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Masculino , Obesidad/metabolismo , Obesidad/fisiopatología , Estadísticas no ParamétricasRESUMEN
Reduced expression of the Indy ("I am Not Dead, Yet") gene in lower organisms promotes longevity in a manner akin to caloric restriction. Deletion of the mammalian homolog of Indy (mIndy, Slc13a5) encoding for a plasma membrane-associated citrate transporter expressed highly in the liver, protects mice from high-fat diet-induced and aging-induced obesity and hepatic fat accumulation through a mechanism resembling caloric restriction. We studied a possible role of mIndy in human hepatic fat metabolism. In obese, insulin-resistant patients with nonalcoholic fatty liver disease, hepatic mIndy expression was increased and mIndy expression was also independently associated with hepatic steatosis. In nonhuman primates, a 2-year high-fat, high-sucrose diet increased hepatic mIndy expression. Liver microarray analysis showed that high mIndy expression was associated with pathways involved in hepatic lipid metabolism and immunological processes. Interleukin-6 (IL-6) was identified as a regulator of mIndy by binding to its cognate receptor. Studies in human primary hepatocytes confirmed that IL-6 markedly induced mIndy transcription through the IL-6 receptor and activation of the transcription factor signal transducer and activator of transcription 3, and a putative start site of the human mIndy promoter was determined. Activation of the IL-6-signal transducer and activator of transcription 3 pathway stimulated mIndy expression, enhanced cytoplasmic citrate influx, and augmented hepatic lipogenesis in vivo. In contrast, deletion of mIndy completely prevented the stimulating effect of IL-6 on citrate uptake and reduced hepatic lipogenesis. These data show that mIndy is increased in liver of obese humans and nonhuman primates with NALFD. Moreover, our data identify mIndy as a target gene of IL-6 and determine novel functions of IL-6 through mINDY. CONCLUSION: Targeting human mINDY may have therapeutic potential in obese patients with nonalcoholic fatty liver disease. German Clinical Trials Register: DRKS00005450. (Hepatology 2017;66:616-630).
Asunto(s)
Enzimas Desubicuitinizantes/genética , Hígado Graso/metabolismo , Regulación de la Expresión Génica , Interleucina-6/metabolismo , Metabolismo de los Lípidos/genética , Longevidad/genética , Animales , Biopsia con Aguja , Células Cultivadas , Hígado Graso/patología , Hepatocitos/citología , Hepatocitos/metabolismo , Humanos , Inmunohistoquímica , Interleucina-6/farmacología , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Mutación , ARN Mensajero/genética , MuestreoRESUMEN
AIMS: Molecular adaptations in human non-alcoholic fatty liver disease (NAFLD) are incompletely understood. This study investigated the main gene categories related to hepatic de novo lipogenesis and lipid oxidation capacity. METHODS: Liver specimens of 48 subjects were histologically classified according to steatosis severity. In-depth analyses were undertaken using real-time polymerase chain reaction, immunoblotting, and immunohistochemistry. Lipid profiles were analyzed by gas chromatography/flame ionization detection, and effects of key fatty acids were studied in primary human hepatocytes. RESULTS: Real-time polymerase chain reaction, immunoblotting, and immunohistochemistry indicated 5'AMP-activated protein kinase (AMPK) to be increased with steatosis score ≥ 2 (all P < 0.05), including various markers of de novo lipogenesis and lipid degradation (all P < 0.05). Regarding endoplasmic reticulum stress, X-Box binding protein-1 (XBP1) was upregulated in steatosis score ≥ 2 (P = 0.029) and correlated with plasma palmitate (r = 0.34; P = 0.035). Palmitate incubation of primary human hepatocytes increased XBP1 and downstream stearoyl CoA desaturase-1 mRNA expression (both P < 0.05). Moreover, plasma and liver tissue exposed a NAFLD-related lipid profile with reduced polyunsaturated/saturated fatty acid ratio, increased palmitate and palmitoleate, and elevated lipogenesis and desaturation indices with steatosis score ≥ 2 (all P < 0.05). CONCLUSION: In humans with advanced fatty liver disease, hepatic AMPK protein is upregulated, potentially in a compensatory manner. Moreover, pathways of lipid synthesis and degradation are co-activated in subjects with advanced steatosis. Palmitate may drive lipogenesis by activating XBP1-mediated endoplasmic reticulum stress and represent a target for future dietary or pharmacological intervention.
RESUMEN
Non-alcoholic fatty liver disease is a growing problem in industrialized and developing countries. Hepatic lipid accumulation is the result of an imbalance between fatty acid uptake, fatty acid de novo synthesis, ß-oxidation and secretion of triglyceride-rich lipoproteins from the hepatocyte. A central regulator of hepatic lipid metabolism is cytosolic citrate that can either be derived from the mitochondrium or be taken up from the blood via the plasma membrane sodium citrate transporter NaCT, the product of the mammalian INDY gene (SLC13A5). mINDY ablation protects against diet-induced steatosis whereas mINDY expression is increased in patients with hepatic steatosis. Diet-induced hepatic steatosis is also enhanced by activation of the arylhyrocarbon receptor (AhR) both in humans and animal models. Therefore, the hypothesis was tested whether the mINDY gene might be a target of the AhR. In accordance with such a hypothesis, the AhR activator benzo[a]pyrene induced the mINDY expression in primary cultures of rat hepatocytes in an AhR-dependent manner. This induction resulted in an increased citrate uptake and citrate incorporation into lipids which probably was further enhanced by the benzo[a]pyrene-dependent induction of key enzymes of fatty acid synthesis. A potential AhR binding site was identified in the mINDY promoter that appears to be conserved in the human promoter. Elimination or mutation of this site largely abolished the activation of the mINDY promoter by benzo[a]pyrene. This study thus identified the mINDY as an AhR target gene. AhR-dependent induction of the mINDY gene might contribute to the development of hepatic steatosis.
Asunto(s)
Benzo(a)pireno/toxicidad , Carcinógenos/toxicidad , Hepatocitos/efectos de los fármacos , Metabolismo de los Lípidos/efectos de los fármacos , Receptores de Hidrocarburo de Aril/fisiología , Simportadores/fisiología , Animales , Translocador Nuclear del Receptor de Aril Hidrocarburo/biosíntesis , Translocador Nuclear del Receptor de Aril Hidrocarburo/genética , Inmunoprecipitación de Cromatina , Citratos/metabolismo , Células HEK293 , Humanos , Masculino , Enfermedad del Hígado Graso no Alcohólico/inducido químicamente , Enfermedad del Hígado Graso no Alcohólico/patología , Cultivo Primario de Células , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Ratas Wistar , Simportadores/biosíntesis , Simportadores/genéticaRESUMEN
Reduced expression of the INDY (I'm not dead yet) tricarboxylate carrier increased the life span in different species by mechanisms akin to caloric restriction. Mammalian INDY homolog (mIndy, SLC13A5) gene expression seems to be regulated by hormonal and/or nutritional factors. The underlying mechanisms are still unknown. The current study revealed that mIndy expression and [(14)C]-citrate uptake was induced by physiological concentrations of glucagon via a cAMP-dependent and cAMP-responsive element-binding protein (CREB)-dependent mechanism in primary rat hepatocytes. The promoter sequence of mIndy located upstream of the most frequent transcription start site was determined by 5'-rapid amplification of cDNA ends. In silico analysis identified a CREB-binding site within this promoter fragment of mIndy. Functional relevance for the CREB-binding site was demonstrated with reporter gene constructs that were induced by CREB activation when under the control of a fragment of a wild-type promoter, whereas promoter activity was lost after site-directed mutagenesis of the CREB-binding site. Moreover, CREB binding to this promoter element was confirmed by chromatin immunoprecipitation in rat liver. In vivo studies revealed that mIndy was induced in livers of fasted as well as in high-fat-diet-streptozotocin diabetic rats, in which CREB is constitutively activated. mIndy induction was completely prevented when CREB was depleted in these rats by antisense oligonucleotides. Together, these data suggest that mIndy is a CREB-dependent glucagon target gene that is induced in fasting and in type 2 diabetes. Increased mIndy expression might contribute to the metabolic consequences of diabetes in the liver.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/fisiología , Diabetes Mellitus Tipo 2/metabolismo , Regulación de la Expresión Génica , Simportadores/genética , Animales , Inmunoprecipitación de Cromatina , AMP Cíclico/fisiología , Glucagón/farmacología , Células Hep G2 , Hepatocitos/metabolismo , Humanos , Masculino , Regiones Promotoras Genéticas , Ratas , Ratas WistarRESUMEN
Cardiac natriuretic peptides (NP) are major activators of human fat cell lipolysis and have recently been shown to control brown fat thermogenesis. Here, we investigated the physiological role of NP on the oxidative metabolism of human skeletal muscle. NP receptor type A (NPRA) gene expression was positively correlated to mRNA levels of PPARγ coactivator-1α (PGC1A) and several oxidative phosphorylation (OXPHOS) genes in human skeletal muscle. Further, the expression of NPRA, PGC1A, and OXPHOS genes was coordinately upregulated in response to aerobic exercise training in human skeletal muscle. In human myotubes, NP induced PGC-1α and mitochondrial OXPHOS gene expression in a cyclic GMP-dependent manner. NP treatment increased OXPHOS protein expression, fat oxidation, and maximal respiration independent of substantial changes in mitochondrial proliferation and mass. Treatment of myotubes with NP recapitulated the effect of exercise training on muscle fat oxidative capacity in vivo. Collectively, these data show that activation of NP signaling in human skeletal muscle enhances mitochondrial oxidative metabolism and fat oxidation. We propose that NP could contribute to exercise training-induced improvement in skeletal muscle fat oxidative capacity in humans.
Asunto(s)
Factor Natriurético Atrial/fisiología , Músculo Esquelético/metabolismo , Péptido Natriurético Encefálico/fisiología , Fosforilación Oxidativa , Receptores del Factor Natriurético Atrial/metabolismo , Adaptación Fisiológica , Adulto , Células Cultivadas , Regulación de la Expresión Génica , Genes Mitocondriales , Proteínas de Choque Térmico/metabolismo , Humanos , Metabolismo de los Lípidos , Masculino , Mitocondrias Musculares/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Obesidad , Oxidación-Reducción , Consumo de Oxígeno , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Cultivo Primario de Células , Receptores del Factor Natriurético Atrial/genética , Transducción de Señal , Factores de Transcripción/metabolismo , Regulación hacia ArribaRESUMEN
Reduced expression of the Indy (I'm Not Dead, Yet) gene in D. melanogaster and its homolog in C. elegans prolongs life span and in D. melanogaster augments mitochondrial biogenesis in a manner akin to caloric restriction. However, the cellular mechanism by which Indy does this is unknown. Here, we report on the knockout mouse model of the mammalian Indy (mIndy) homolog, SLC13A5. Deletion of mIndy in mice (mINDY(-/-) mice) reduces hepatocellular ATP/ADP ratio, activates hepatic AMPK, induces PGC-1α, inhibits ACC-2, and reduces SREBP-1c levels. This signaling network promotes hepatic mitochondrial biogenesis, lipid oxidation, and energy expenditure and attenuates hepatic de novo lipogenesis. Together, these traits protect mINDY(-/-) mice from the adiposity and insulin resistance that evolve with high-fat feeding and aging. Our studies demonstrate a profound effect of mIndy on mammalian energy metabolism and suggest that mINDY might be a therapeutic target for the treatment of obesity and type 2 diabetes.
Asunto(s)
Adiposidad/genética , Metabolismo Energético/genética , Resistencia a la Insulina/genética , Metabolismo de los Lípidos/genética , Simportadores/biosíntesis , Envejecimiento , Animales , Restricción Calórica , Transportadores de Ácidos Dicarboxílicos , Células HEK293 , Humanos , Ratones , Ratones Noqueados , Mitocondrias/genética , Mitocondrias/metabolismo , Obesidad/genética , Simportadores/deficiencia , Simportadores/genéticaRESUMEN
Recent studies have implicated endoplasmic reticulum (ER) stress in insulin resistance associated with caloric excess. In mice placed on a 3-day high fat diet, we find augmented eIF2α signaling, together with hepatic lipid accumulation and insulin resistance. To clarify the role of the liver ER stress-dependent phospho-eIF2α (eIF2α-P) pathway in response to acute caloric excess on liver and muscle glucose and lipid metabolism, we studied transgenic mice in which the hepatic ER stress-dependent eIF2α-P pathway was inhibited by overexpressing a constitutively active C-terminal fragment of GADD34/PPP1R15a, a regulatory subunit of phosphatase that terminates ER stress signaling by phospho-eIF2α. Inhibition of the eIF2α-P signaling in liver led to a decrease in hepatic glucose production in the basal and clamped state, which could be attributed to reduced gluconeogenic gene expression, resulting in reduced basal plasma glucose concentrations. Surprisingly, hepatic eIF2α inhibition also impaired insulin-stimulated muscle and adipose tissue insulin sensitivity. This latter effect could be attributed at least in part by an increase in circulating IGFBP-3 levels in the transgenic animals. In addition, infusion of insulin during a hyperinsulinemic-euglycemic clamp induced conspicuous ER stress in the 3-day high fat diet-fed mice, which was aggravated through continuous dephosphorylation of eIF2α. Together, these data imply that the hepatic ER stress eIF2α signaling pathway affects hepatic glucose production without altering hepatic insulin sensitivity. Moreover, hepatic ER stress-dependent eIF2α-P signaling is implicated in an unanticipated cross-talk between the liver and peripheral organs to influence insulin sensitivity, probably via IGFBP-3. Finally, eIF2α is crucial for proper resolution of insulin-induced ER stress.