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Acyl-CoA thioesterase 1 (ACOT1) catalyzes the hydrolysis of long-chain acyl-CoAs to free fatty acids and CoA and is typically upregulated in obesity. Whether targeting ACOT1 in the setting of high-fat diet-induced (HFD-induced) obesity would be metabolically beneficial is not known. Here we report that male and female ACOT1KO mice are partially protected from HFD-induced obesity, an effect associated with increased energy expenditure without alterations in physical activity or food intake. In males, ACOT1 deficiency increased mitochondrial uncoupling protein-2 (UCP2) protein abundance while reducing 4-hydroxynonenal, a marker of oxidative stress, in white adipose tissue and liver of HFD-fed mice. Moreover, concurrent knockdown (KD) of UCP2 with ACOT1 in hepatocytes prevented increases in oxygen consumption observed with ACOT1 KD during high lipid loading, suggesting that UCP2-induced uncoupling may increase energy expenditure to attenuate weight gain. Together, these data indicate that targeting ACOT1 may be effective for obesity prevention during caloric excess by increasing energy expenditure.
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Dieta Alta en Grasa , Obesidad , Animales , Femenino , Masculino , Ratones , Metabolismo Energético , Hígado/metabolismo , Obesidad/metabolismo , Aumento de PesoRESUMEN
Since interventions such as caloric restriction or fasting robustly promote lipid catabolism and improve aging-related phenotypical markers, we investigated the direct effect of increased lipid catabolism via overexpression of bmm (brummer, FBgn0036449), the major triglyceride hydrolase in Drosophila, on lifespan and physiological fitness. Comprehensive characterization was carried out using RNA-seq, lipidomics and metabolomics analysis. Global overexpression of bmm strongly promoted numerous markers of physiological fitness, including increased female fecundity, fertility maintenance, preserved locomotion activity, increased mitochondrial biogenesis and oxidative metabolism. Increased bmm robustly upregulated the heat shock protein 70 (Hsp70) family of proteins, which equipped the flies with higher resistance to heat, cold, and ER stress via improved proteostasis. Despite improved physiological fitness, bmm overexpression did not extend lifespan. Taken together, these data show that bmm overexpression has broad beneficial effects on physiological fitness, but these effects did not impact lifespan.
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Proteínas de Drosophila , Drosophila melanogaster , Animales , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Femenino , Lipólisis , Longevidad , Triglicéridos/metabolismoRESUMEN
Lipid droplets (LDs) provide a reservoir for triacylglycerol storage and are a central hub for fatty acid trafficking and signaling in cells. Lipolysis promotes mitochondrial biogenesis and oxidative metabolism via a SIRT1/PGC-1α/PPARα-dependent pathway through an unknown mechanism. Herein, we identify that monounsaturated fatty acids (MUFAs) allosterically activate SIRT1 toward select peptide-substrates such as PGC-1α. MUFAs enhance PGC-1α/PPARα signaling and promote oxidative metabolism in cells and animal models in a SIRT1-dependent manner. Moreover, we characterize the LD protein perilipin 5 (PLIN5), which is known to enhance mitochondrial biogenesis and function, to be a fatty-acid-binding protein that preferentially binds LD-derived monounsaturated fatty acids and traffics them to the nucleus following cAMP/PKA-mediated lipolytic stimulation. Thus, these studies identify the first-known endogenous allosteric modulators of SIRT1 and characterize a LD-nuclear signaling axis that underlies the known metabolic benefits of MUFAs and PLIN5.
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Ácidos Grasos Monoinsaturados/metabolismo , Gotas Lipídicas/química , Perilipina-5/metabolismo , Sirtuina 1/metabolismo , Regulación Alostérica , Animales , Transporte Biológico , Línea Celular , Células Cultivadas , Dieta , Ácidos Grasos/metabolismo , Lipasa/metabolismo , Masculino , Ratones Endogámicos C57BL , Aceite de Oliva , Perilipina-5/fisiología , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Transcripción GenéticaRESUMEN
Lipid droplets (LDs) are energy-storage organelles that are coated with hundreds of proteins, including members of the perilipin (PLIN) family. PLIN5 is highly expressed in oxidative tissues, including the liver, and is thought to play a key role in uncoupling LD accumulation from lipotoxicity; however, the mechanisms behind this action are incompletely defined. We investigated the role of hepatic PLIN5 in inflammation and lipotoxicity in a murine model under both fasting and refeeding conditions and in hepatocyte cultures. PLIN5 ablation with antisense oligonucleotides triggered a pro-inflammatory response in livers from mice only under fasting conditions. Similarly, PLIN5 mitigated lipopolysaccharide- or palmitic acid-induced inflammatory responses in hepatocytes. During fasting, PLIN5 was also required for the induction of autophagy, which contributed to its anti-inflammatory effects. The ability of PLIN5 to promote autophagy and prevent inflammation were dependent upon signaling through sirtuin 1 (SIRT1), which is known to be activated in response to nuclear PLIN5 under fasting conditions. Taken together, these data show that PLIN5 signals via SIRT1 to promote autophagy and prevent FA-induced inflammation as a means to maintain hepatocyte homeostasis during periods of fasting and FA mobilization.
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Autofagia , Ayuno , Inflamación/metabolismo , Hígado/química , Perilipina-5/metabolismo , Sirtuina 1/metabolismo , Animales , Células Cultivadas , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Transducción de SeñalRESUMEN
During normal proliferation, hepatocytes accumulate triglycerides (TGs) in lipid droplets (LDs), but the underlying mechanisms and functional significance of this steatosis are unknown. In the current study, we examined the coordinated regulation of cell cycle progression and LD accumulation. As previously shown, hepatocytes develop increased LD content after mitogen stimulation. Cyclin D1, in addition to regulating proliferation, was both necessary and sufficient to promote LD accumulation in response to mitogens. Interestingly, cyclin D1 promotes LD accumulation by inhibiting the breakdown of TGs by lipolysis through a mechanism involving decreased lipophagy, the autophagic degradation of LDs. To examine whether inhibition of lipolysis is important for cell cycle progression, we overexpressed adipose TG lipase (ATGL), a key enzyme involved in TG breakdown. As expected, ATGL reduced LD content but also markedly inhibited hepatocyte proliferation, suggesting that lipolysis regulates a previously uncharacterized cell cycle checkpoint. Consistent with this, in mitogen-stimulated cells with small interfering RNA-mediated depletion of cyclin D1 (which inhibits proliferation and stimulates lipolysis), concurrent ATGL knockdown restored progression into S phase. Following partial hepatectomy, a model of robust hepatocyte proliferation in vivo, ATGL overexpression led to decreased LD content, cell cycle inhibition, and marked liver injury, further indicating that down-regulation of lipolysis is important for normal hepatocyte proliferation. Conclusion: We suggest a new relationship between steatosis and proliferation in hepatocytes: cyclin D1 inhibits lipolysis, resulting in LD accumulation, and suppression of lipolysis is necessary for cell cycle progression.
RESUMEN
Hepatic lipid droplet (LD) catabolism is thought to occur via cytosolic lipases such as adipose triglyceride lipase (ATGL) or through autophagy of LDs, a process known as lipophagy. We tested the potential interplay between these metabolic processes and its effects on hepatic lipid metabolism. We show that hepatic ATGL is both necessary and sufficient to induce both autophagy and lipophagy. Moreover, lipophagy is required for ATGL to promote LD catabolism and the subsequent oxidation of hydrolyzed fatty acids (FAs). Following previous work showing that ATGL promotes sirtuin 1 (SIRT1) activity, studies in liver-specific SIRT1-/- mice and in primary hepatocytes reveal that SIRT1 is required for ATGL-mediated induction of autophagy and lipophagy. Taken together, these studies show that ATGL-mediated signaling via SIRT1 promotes autophagy/lipophagy as a primary means to control hepatic LD catabolism and FA oxidation.
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Autofagia , Lipasa/metabolismo , Gotas Lipídicas/metabolismo , Metabolismo de los Lípidos , Hígado/metabolismo , Sirtuina 1/metabolismo , Análisis de Varianza , Animales , Células Cultivadas , Ácidos Grasos/metabolismo , Hepatocitos/metabolismo , Lipasa/genética , Gotas Lipídicas/química , Lipólisis , Hígado/anatomía & histología , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Oxidación-Reducción , Sirtuina 1/genéticaRESUMEN
Cyclin D1 is a cell cycle protein that promotes proliferation by mediating progression through key checkpoints in G1 phase. It is also a proto-oncogene that is commonly overexpressed in human cancers. In addition to its canonical role in controlling cell cycle progression, cyclin D1 affects other aspects of cell physiology, in part through transcriptional regulation. In this study, we find that cyclin D1 inhibits the activity of a key metabolic transcription factor, peroxisome proliferator-activated receptor α (PPARα), a member of nuclear receptor family that induces fatty acid oxidation and may play an anti-neoplastic role. In primary hepatocytes, cyclin D1 inhibits PPARα transcriptional activity and target gene expression in a cdk4-independent manner. In liver and breast cancer cells, knockdown of cyclin D1 leads to increased PPARα transcriptional activity, expression of PPARα target genes, and fatty acid oxidation. Similarly, cyclin D1 depletion enhances binding of PPARα to target sequences by chromatin immunoprecipitation. In proliferating hepatocytes and regenerating liver in vivo, induction of endogenous cyclin D1 is associated with diminished PPARα activity. Cyclin D1 expression is both necessary and sufficient for growth factor-mediated repression of fatty acid oxidation in proliferating hepatocytes. These studies indicate that in addition to playing a pivotal role in cell cycle progression, cyclin D1 represses PPARα activity and inhibits fatty acid oxidation. Our findings establish a new link between cyclin D1 and metabolism in both tumor cells and physiologic hepatocyte proliferation.
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Ciclina D1/metabolismo , Ácidos Grasos/metabolismo , Neoplasias Hepáticas Experimentales/metabolismo , PPAR alfa/metabolismo , Animales , Línea Celular Tumoral , Células Hep G2 , Humanos , Metabolismo de los Lípidos , Hígado/metabolismo , Hígado/patología , Neoplasias Hepáticas Experimentales/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Oxidación-Reducción , Proto-Oncogenes Mas , Ratas , Ratas Sprague-Dawley , TransfecciónRESUMEN
Metabolism is a highly integrated process that is coordinately regulated between tissues and within individual cells. FoxO proteins are major targets of insulin action and contribute to the regulation of gluconeogenesis, glycolysis, and lipogenesis in the liver. However, the mechanisms by which FoxO proteins exert these diverse effects in an integrated fashion remain poorly understood. We report that FoxO proteins also exert important effects on intrahepatic lipolysis and fatty acid oxidation via the regulation of adipose triacylglycerol lipase (ATGL), which mediates the first step in lipolysis, and its inhibitor, the G0/S1 switch 2 gene (G0S2). We also find that ATGL-dependent lipolysis plays a critical role in mediating diverse effects of FoxO proteins in the liver, including effects on gluconeogenic, glycolytic, and lipogenic gene expression and metabolism. These results indicate that intrahepatic lipolysis plays a critical role in mediating and integrating the regulation of glucose and lipid metabolism downstream of FoxO proteins.
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Tejido Adiposo/metabolismo , Proteína Forkhead Box O1/metabolismo , Glucosa/metabolismo , Lipasa/metabolismo , Metabolismo de los Lípidos , Hígado/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Ácidos Grasos/metabolismo , Regulación de la Expresión Génica , Hepatocitos/metabolismo , Homeostasis , Humanos , Lipasa/genética , Metabolismo de los Lípidos/genética , Lipogénesis , Masculino , Ratones Transgénicos , Modelos Biológicos , Oxidación-ReducciónRESUMEN
Sirtuin 1 (SIRT1), an NAD(+)-dependent protein deacetylase, regulates a host of target proteins, including peroxisome proliferator-activated receptor (PPAR)-γ coactivator-1α (PGC-1α), a transcriptional coregulator that binds to numerous transcription factors in response to deacetylation to promote mitochondrial biogenesis and oxidative metabolism. Our laboratory and others have shown that adipose triglyceride lipase (ATGL) increases the activity of the nuclear receptor PPAR-α, a PGC-1α binding partner, to promote fatty acid oxidation. Fatty acids bind and activate PPAR-α; therefore, it has been presumed that fatty acids derived from ATGL-catalyzed lipolysis act as PPAR-α ligands. We provide an alternate mechanism that links ATGL to PPAR-α signaling. We show that SIRT1 deacetylase activity is positively regulated by ATGL to promote PGC-1α signaling. In addition, ATGL mediates the effects of ß-adrenergic signaling on SIRT1 activity, and PGC-1α and PPAR-α target gene expression independent of changes in NAD(+). Moreover, SIRT1 is required for the induction of PGC-1α/PPAR-α target genes and oxidative metabolism in response to increased ATGL-mediated lipolysis. Taken together, this work identifies SIRT1 as a critical node that links ß-adrenergic signaling and lipolysis to changes in the transcriptional regulation of oxidative metabolism.
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Lipasa/metabolismo , PPAR alfa/metabolismo , Transducción de Señal/fisiología , Sirtuina 1/metabolismo , Factores de Transcripción/metabolismo , Animales , Células Cultivadas , Regulación de la Expresión Génica , Hepatocitos/metabolismo , Lipasa/genética , Lipólisis/fisiología , Masculino , Ratones , PPAR alfa/genética , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Sirtuina 1/genética , Factores de Transcripción/genéticaRESUMEN
How endurance training alters muscle lipid metabolism while preserving insulin sensitivity remains unclear. Because acute free fatty acid (FFA) elevation by lipid infusion reduces insulin sensitivity, we hypothesized that training status would alter accumulation of muscle triacylglycerol (TAG), diacylglycerol (DAG), ceramide, and acylcarnitine during acute FFA elevation. Trained (n = 15) and sedentary (n = 13) participants matched for age, sex, and BMI received either a 6-h infusion of lipid (20% Intralipid at 90 ml/h) or glycerol (2.25 g/100 ml at 90 ml/h) during a hyperinsulinemic euglycemic clamp. Muscle biopsies were taken at 0, 120, and 360 min after infusion initiation to measure intramyocellular concentrations of TAG, DAG, ceramides, and acylcarnitines by liquid chromatography-tandem mass spectrometry. Trained participants had a higher Vo2 max and insulin sensitivity than sedentary participants. The lipid infusion produced a comparable elevation of FFA (594 ± 90 µmol/l in trained, 721 ± 30 µmol/l in sedentary, P = 0.4) and a decline in insulin sensitivity (-44.7% trained vs. -47.2% sedentary, P = 0.89). In both groups, lipid infusion increased the linoleic and linolenic acid content of TAG without changing total TAG. In the sedentary group, lipid infusion increased total, oleic, and linoleic acid and linolenic acid content of DAG. Regardless of training status, lipid infusion did not alter total ceramide, saturated ceramide, palmitoyl-carnitine, or oleoyl-carnitine. We conclude that during acute FFA elevation, trained adults have a similar decline in insulin sensitivity with less accumulation of muscle DAG than sedentary adults, suggesting that lipid-induced insulin resistance can occur without elevation of total muscle DAG.
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Diglicéridos/metabolismo , Ejercicio Físico/fisiología , Ácidos Grasos no Esterificados/sangre , Músculo Esquelético/fisiología , Acondicionamiento Físico Humano/métodos , Resistencia Física/fisiología , Aptitud Física/fisiología , Adulto , Humanos , Masculino , Adulto JovenRESUMEN
Adipose TG lipase (ATGL) catalyzes the rate-limiting step in TG hydrolysis in most tissues. We have shown that hepatic ATGL preferentially channels hydrolyzed FAs to ß-oxidation and induces PPAR-α signaling. Previous studies have suggested that liver FA binding protein (L-FABP) transports FAs from lipid droplets to the nucleus for ligand delivery and to the mitochondria for ß-oxidation. To determine if L-FABP is involved in ATGL-mediated FA channeling, we used adenovirus-mediated suppression or overexpression of hepatic ATGL in either WT or L-FABP KO mice. Hepatic ATGL knockdown increased liver weight and TG content of overnight fasted mice regardless of genotype. L-FABP deletion did not impair the effects of ATGL overexpression on the oxidation of hydrolyzed FAs in primary hepatocyte cultures or on serum ß-hydroxybutyrate concentrations in vivo. Moreover, L-FABP deletion did not influence the effects of ATGL knockdown or overexpression on PPAR-α target gene expression. Taken together, we conclude that L-FABP is not required to channel ATGL-hydrolyzed FAs to mitochondria for ß-oxidation or the nucleus for PPAR-α regulation.
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Proteínas de Unión a Ácidos Grasos/metabolismo , Ácidos Grasos/metabolismo , Lipasa/metabolismo , Hígado/citología , Hígado/enzimología , PPAR alfa/metabolismo , Transducción de Señal , Ácido 3-Hidroxibutírico/sangre , Adenoviridae/genética , Animales , Ayuno , Proteínas de Unión a Ácidos Grasos/deficiencia , Proteínas de Unión a Ácidos Grasos/genética , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Técnicas de Inactivación de Genes , Lipasa/deficiencia , Lipasa/genética , Hígado/crecimiento & desarrollo , Hígado/metabolismo , Masculino , Ratones , Tamaño de los Órganos , Oxidación-Reducción , ARN Interferente Pequeño/genética , Triglicéridos/metabolismoRESUMEN
The use of microalgae for biofuel production will be beneficial to society if we can produce biofuels at large scales with minimal mechanical energy input in the production process. Understanding micro-algal physiological responses under variable environmental conditions in bioreactors is essential for the optimization of biofuel production. We demonstrate that measuring micro-algal swimming speed provides information on culture health and total fatty acid accumulation. Three strains of Chlamydomonas reinhardtii were grown heterotrophically on acetate and subjected to various levels of nitrogen starvation. Other nutrient levels were explored to determine their effect on micro-algal kinetics. Swimming velocities were measured with two-dimensional micro-particle tracking velocimetry. The results show an inverse linear relationship between normalized total fatty acid mass versus swimming speed of micro-algal cells. Analysis of RNA sequencing data confirms these results by demonstrating that the biological processes of cell motion and the generation of energy precursors are significantly down-regulated. Experiments demonstrate that changes in nutrient concentration in the surrounding media also affect swimming speed. The findings have the potential for the in situ and indirect assessment of lipid content by measuring micro-algal swimming kinetics.
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Biocombustibles , Movimiento Celular/fisiología , Chlamydomonas reinhardtii/metabolismo , Ácidos Grasos/metabolismo , Microalgas/metabolismo , Análisis de Varianza , Chlamydomonas reinhardtii/fisiología , Ácidos Grasos/análisis , Flagelos/fisiología , Microalgas/fisiología , Nitrógeno/metabolismo , Reología , Estrés FisiológicoRESUMEN
Adipose triglyceride lipase (ATGL) is the predominant triacylglycerol (TAG) hydrolase in mammals; however, the tissue-specific effects of ATGL outside of adipose tissue have not been well characterized. Hence, we tested the contribution of hepatic ATGL on mediating glucose tolerance and insulin action. Glucose or insulin tolerance tests and insulin signaling were performed in C57BL/6 mice administered control (nongene specific shRNA) or Atgl shRNA adenoviruses. Glucose and lipid metabolism assays were conducted in primary hepatocytes isolated from mice transduced with control or Atgl shRNA adenoviruses. Knocking down hepatic ATGL completely abrogated the increase in serum insulin following either 1 or 12 wk of feeding a high-fat (HF) diet despite higher hepatic TAG content. Glucose tolerance tests demonstrated that ATGL knockdown normalized glucose tolerance in HF-diet-fed mice. The observed improvements in glucose tolerance were present despite unaltered hepatic insulin signaling and increased liver TAG. Mice with suppressed hepatic ATGL had reduced hepatic glucose production in vivo, and hepatocytes isolated from Atgl shRNA-treated mice displayed a 26% decrease in glucose production and a 38% increase in glucose oxidation compared to control cells. Taken together, these data suggest that hepatic ATGL knockdown enhances glucose tolerance by increasing hepatic glucose utilization and uncouples impairments in insulin action from hepatic TAG accumulation.
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Prueba de Tolerancia a la Glucosa , Lipasa/fisiología , Hígado/metabolismo , Triglicéridos/metabolismo , Animales , Glucemia/análisis , Células Cultivadas , Hígado Graso/genética , Insulina/sangre , Insulina/metabolismo , Lipasa/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de SeñalRESUMEN
AMP-activated protein kinase (AMPK) is an essential sensor of cellular energy status. Defects in the α2 catalytic subunit of AMPK (AMPKα1) are associated with metabolic syndrome. The current study investigated the role AMPKα1 in the pathogenesis of obesity and inflammation using male AMPKα1-deficent (AMPKα1(-/-)) mice and their wild-type (WT) littermates. After being fed a high-fat diet (HFD), global AMPKα1(-/-) mice gained more body weight and greater adiposity and exhibited systemic insulin resistance and metabolic dysfunction with increased severity in their adipose tissues compared with their WT littermates. Interestingly, upon HFD feeding, irradiated WT mice that received the bone marrow of AMPKα1(-/-) mice showed increased insulin resistance but not obesity, whereas irradiated AMPKα1(-/-) mice with WT bone marrow had a phenotype of metabolic dysregulation that was similar to that of global AMPKα1(-/-) mice. AMPKα1 deficiency in macrophages markedly increased the macrophage proinflammatory status. In addition, AMPKα1 knockdown enhanced adipocyte lipid accumulation and exacerbated the inflammatory response and insulin resistance. Together, these data show that AMPKα1 protects mice from diet-induced obesity and insulin resistance, demonstrating that AMPKα1 is a promising therapeutic target in the treatment of the metabolic syndrome.
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Proteínas Quinasas Activadas por AMP/metabolismo , Dieta Alta en Grasa/efectos adversos , Resistencia a la Insulina/fisiología , Obesidad/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Animales , Western Blotting , Células Cultivadas , Citometría de Flujo , Inmunohistoquímica , Resistencia a la Insulina/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad/genética , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Following acute hepatic injury, the metabolic capacity of the liver is altered during the process of compensatory hepatocyte proliferation by undefined mechanisms. In this study, we examined the regulation of de novo lipogenesis by cyclin D1, a key mediator of hepatocyte cell cycle progression. In primary hepatocytes, cyclin D1 significantly impaired lipogenesis in response to glucose stimulation. Cyclin D1 inhibited the glucose-mediated induction of key lipogenic genes, and similar effects were seen using a mutant (D1-KE) that does not activate cdk4 or induce cell cycle progression. Cyclin D1 (but not D1-KE) inhibited the activity of the carbohydrate response element-binding protein (ChREBP) by regulating the glucose-sensing motif of this transcription factor. Because changes in ChREBP activity could not fully explain the effect of cyclin D1, we examined hepatocyte nuclear factor 4α (HNF4α), which regulates numerous differentiated functions in the liver including lipid metabolism. We found that both cyclins D1 and D1-KE bound to HNF4α and significantly inhibited its recruitment to the promoter region of lipogenic genes in hepatocytes. Conversely, knockdown of cyclin D1 in the AML12 hepatocyte cell line promoted HNF4α activity and lipogenesis. In mouse liver, HNF4α bound to a central domain of cyclin D1 involved in transcriptional repression. Cyclin D1 inhibited lipogenic gene expression in the liver following carbohydrate feeding. Similar findings were observed in the setting of physiologic cyclin D1 expression in the regenerating liver. In conclusion, these studies demonstrate that cyclin D1 represses ChREBP and HNF4α function in hepatocytes via Cdk4-dependent and -independent mechanisms. These findings provide a direct link between the cell cycle machinery and the transcriptional control of metabolic function of the liver.
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Ciclina D1/metabolismo , Glucosa/farmacología , Factor Nuclear 4 del Hepatocito/metabolismo , Lipogénesis/efectos de los fármacos , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Células Cultivadas , Ciclina D1/antagonistas & inhibidores , Ciclina D1/genética , Quinasa 4 Dependiente de la Ciclina/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Hepatocitos/citología , Hepatocitos/metabolismo , Metabolismo de los Lípidos/efectos de los fármacos , Ratones , Proteínas Nucleares/genética , Unión Proteica , Estructura Terciaria de Proteína , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Factores de Transcripción/genéticaRESUMEN
CONTEXT: Both training and normal body mass index are associated with high insulin sensitivity, but the mechanism may be different. OBJECTIVE: The aim of the study was to examine whether lean trained humans may be protected from acute free fatty acid (FFA)-induced insulin resistance compared with lean sedentary humans. DESIGN AND SETTING: We conducted an interventional trial using either a 6-h lipid (20% Intralipid at 90 ml/h) or glycerol (2.25 g/100 ml at 90 ml/h) infusion along with a concurrent hyperinsulinemic-euglycemic clamp and serial muscle biopsies (0, 120, 360 min) at a clinical research unit at the University of Minnesota. PATIENTS OR PARTICIPANTS: The study included lean endurance-trained (n = 14) and sedentary (n = 14) individuals matched for age, gender, and body mass index. MAIN OUTCOME MEASURES: We measured the decline in glucose infusion rate (GIR) during the hyperinsulinemic-euglycemic clamp. RESULTS: The trained group had higher baseline mitochondrial DNA copy number, mRNA of cytochrome C oxidase subunit 3, and insulin sensitivity (as measured by GIR) compared with the sedentary group. When FFA was acutely elevated to the upper physiological range (0.6-0.7 mEq/liter) by lipid infusion, the GIR in both activity groups declined similarly compared with their respective glycerol controls, although insulin signaling, as measured by Ser 473 pAKT/AKT, remained comparable. Specific to the trained group, the stimulatory effect of hyperinsulinemia on mitochondrial mRNA levels during the glycerol infusion was absent during the lipid infusion. CONCLUSIONS: Elevated FFA had similar effects in reducing insulin sensitivity in trained and sedentary humans. In trained participants, this decline was associated with alterations in the skeletal muscle mitochondrial mRNA response to hyperinsulinemia.
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Ácidos Grasos no Esterificados/metabolismo , Resistencia a la Insulina , Resistencia Física/fisiología , Adulto , Femenino , Técnica de Clampeo de la Glucosa , Humanos , Insulina/sangre , Masculino , Músculo Esquelético/metabolismo , Consumo de Oxígeno , ARN Mensajero/análisis , Adulto JovenRESUMEN
UNLABELLED: Despite advances in our understanding of the ways in which nutrient oversupply and triacylglycerol (TAG) anabolism contribute to hepatic steatosis, little is known about the lipases responsible for regulating hepatic TAG turnover. Recent studies have identified adipose triglyceride lipase (ATGL) as a major lipase in adipose tissue, although its role in the liver is largely unknown. Thus, we tested the contribution of ATGL to hepatic lipid metabolism and signaling. Adenovirus-mediated knockdown of hepatic ATGL resulted in steatosis in mice and decreased hydrolysis of TAG in primary hepatocyte cultures and in vitro assays. In addition to altering TAG hydrolysis, ATGL was shown to play a significant role in partitioning hydrolyzed fatty acids between metabolic pathways. Although ATGL gain and loss of function did not alter hepatic TAG secretion, fatty acid oxidation was increased by ATGL overexpression and decreased by ATGL knockdown. The effects on fatty acid oxidation coincided with decreased expression of peroxisome proliferator-activated receptor α (PPAR-α) and its target genes in mice with suppressed hepatic ATGL expression. However, PPAR-α agonism was unable to normalize the effects of ATGL knockdown on PPAR-α target gene expression, and this suggests that ATGL influences PPAR-α activity independently of ligand-induced activation. CONCLUSION: Taken together, these data show that ATGL is a major hepatic TAG lipase that plays an integral role in fatty acid partitioning and signaling to control energy metabolism.
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Tejido Adiposo/enzimología , Lipasa/metabolismo , Hígado/enzimología , Triglicéridos/metabolismo , Animales , Ácidos Grasos/metabolismo , Hígado Graso/etiología , Masculino , Ratones , Ratones Endogámicos C57BL , PPAR alfa/agonistas , PPAR alfa/metabolismo , Transducción de SeñalRESUMEN
Long chain acyl-CoA synthetases (ACSL) and fatty acid transport proteins (FATP) activate fatty acids to acyl-CoAs in the initial step of fatty acid metabolism. Numerous isoforms of ACSL and FATP exist with different tissue distribution patterns, intracellular locations, and substrate preferences, suggesting that each isoform has distinct functions in channeling fatty acids into different metabolic pathways. Because fatty acids, acyl-CoAs, and downstream lipid metabolites regulate various transcription factors that control hepatic energy metabolism, we hypothesized that ACSL or FATP isoforms differentially regulate hepatic gene expression. Using small interference RNA (siRNA), we knocked down each liver-specific ACSL and FATP isoform in rat primary hepatocyte cultures and subsequently analyzed reporter gene activity of numerous transcription factors and performed quantitative mRNA analysis of their target genes. Compared with control cells, which were transfected with control siRNA, knockdown of acyl-CoA synthetase 3 (ACSL3) significantly decreased reporter gene activity of several lipogenic transcription factors such as peroxisome proliferator activation receptor-gamma, carbohydrate-responsive element-binding protein, sterol regulatory element-binding protein-1c, and liver X receptor-alpha and the expression of their target genes. These findings were further supported by metabolic labeling studies that showed [1-(14)C]acetate incorporation into lipid extracts was decreased in cells treated with ACSL3 siRNAs and that ACSL3 expression is up-regulated in ob/ob mice and mice fed a high sucrose diet. ACSL3 knockdown decreased total acyl-CoA synthetase activity without substantially altering the expression of other ACSL isoforms. In summary, these results identify a novel role for ACSL3 in mediating transcriptional control of hepatic lipogenesis.
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Coenzima A Ligasas/fisiología , Ácidos Grasos/biosíntesis , Hígado/metabolismo , Transcripción Genética , Animales , Células Cultivadas , Coenzima A Ligasas/antagonistas & inhibidores , Metabolismo Energético , Regulación de la Expresión Génica , Lipogénesis , Hígado/citología , Redes y Vías Metabólicas , Ratones , Ratones Obesos , ARN Mensajero/análisis , Ratas , Factores de TranscripciónRESUMEN
Recent evidence suggests that fatty acids generated from intracellular triacylglycerol (TAG) hydrolysis may have important roles in intracellular signaling. This study was conducted to determine if fatty acids liberated from TAG hydrolysis regulate peroxisome proliferator-activated receptor alpha (PPARalpha). Primary rat hepatocyte cultures were treated with adenoviruses overexpressing adipose differentiation-related protein (ADRP) or adipose triacylglycerol lipase (ATGL) or treated with short interfering RNA (siRNA) targeted against ADRP. Subsequent effects on TAG metabolism and PPARalpha activity and target gene expression were determined. Overexpressing ADRP attenuated TAG hydrolysis, whereas siRNA-mediated knockdown of ADRP or ATGL overexpression resulted in enhanced TAG hydrolysis. Results from PPARalpha reporter activity assays demonstrated that decreasing TAG hydrolysis by ADRP overexpression resulted in a 35-60% reduction in reporter activity under basal conditions or in the presence of fatty acids. As expected, PPARalpha target genes were also decreased in response to ADRP overexpression. However, the PPARalpha ligand, WY-14643, was able to restore PPARalpha activity following ADRP overexpression. Despite its effects on PPARalpha, overexpressing ADRP did not affect PPARgamma activity. Enhancing TAG hydrolysis through ADRP knockdown or ATGL overexpression increased PPARalpha activity. These results indicate that TAG hydrolysis and the consequential release of fatty acids regulate PPARalpha activity.
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Ácidos Grasos/metabolismo , Hepatocitos/metabolismo , PPAR alfa/metabolismo , Triglicéridos/metabolismo , Animales , Hidrolasas de Éster Carboxílico/genética , Hidrolasas de Éster Carboxílico/metabolismo , Células Cultivadas , Expresión Génica , Técnicas de Transferencia de Gen , Hepatocitos/química , Hidrólisis , Lipasa , Lípidos/análisis , Masculino , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , PPAR alfa/agonistas , PPAR alfa/genética , PPAR gamma/metabolismo , Perilipina-2 , Proliferadores de Peroxisomas/farmacología , Pirimidinas/farmacología , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de SeñalRESUMEN
The ubiquitin-proteasome pathway has emerged as an important regulatory mechanism governing the activity of several transcription factors. While estrogen receptor alpha (ERalpha) is also subjected to rapid ubiquitin-proteasome degradation, the relationship between proteolysis and transcriptional regulation is incompletely understood. Based on studies primarily focusing on the C-terminal ligand-binding and AF-2 transactivation domains, an assembly of an active transcriptional complex has been proposed to signal ERalpha proteolysis that is in turn necessary for its transcriptional activity. Here, we investigated the role of other regions of ERalpha and identified S118 within the N-terminal AF-1 transactivation domain as an additional element for regulating estrogen-induced ubiquitination and degradation of ERalpha. Significantly, different S118 mutants revealed that degradation and transcriptional activity of ERalpha are mechanistically separable functions of ERalpha. We find that proteolysis of ERalpha correlates with the ability of ERalpha mutants to recruit specific ubiquitin ligases regardless of the recruitment of other transcription-related factors to endogenous model target genes. Thus, our findings indicate that the AF-1 domain performs a previously unrecognized and important role in controlling ligand-induced receptor degradation which permits the uncoupling of estrogen-regulated ERalpha proteolysis and transcription.