RESUMEN
Bromodomain-containing protein 7 (BRD7) has emerged as a player in the regulation of glucose homeostasis. Hepatic BRD7 levels are decreased in obese mice, and the reinstatement of hepatic BRD7 in obese mice has been shown to establish euglycemia and improve glucose homeostasis. Of note, the upregulation of hepatic BRD7 levels activates the AKT cascade in response to insulin without enhancing the sensitivity of the insulin receptor (InsR)-insulin receptor substrate (IRS) axis. In this report, we provide evidence for the existence of an alternative insulin signaling pathway that operates independently of IRS proteins and demonstrate the involvement of BRD7 in this pathway. To investigate the involvement of BRD7 as a downstream component of InsR, we utilized liver-specific InsR knockout mice. Additionally, we employed liver-specific IRS1/2 knockout mice to examine the requirement of IRS1/2 for the action of BRD7. Our investigation of glucose metabolism parameters and insulin signaling unveiled the significance of InsR activation in mediating BRD7's effect on glucose homeostasis in the liver. Moreover, we identified an interaction between BRD7 and InsR. Notably, our findings indicate that IRS1/2 is not necessary for BRD7's regulation of glucose metabolism, particularly in the context of obesity. The upregulation of hepatic BRD7 significantly reduces blood glucose levels and restores glucose homeostasis in high-fat diet-challenged liver-specific IRS1/2 knockout mice. These findings highlight the presence of an alternative insulin signaling pathway that operates independently of IRS1/2 and offer novel insights into the mechanisms of a previously unknown insulin signaling in obesity.
Asunto(s)
Resistencia a la Insulina , Receptor de Insulina , Animales , Ratones , Glucosa/metabolismo , Homeostasis/genética , Insulina/metabolismo , Proteínas Sustrato del Receptor de Insulina/genética , Proteínas Sustrato del Receptor de Insulina/metabolismo , Hígado/metabolismo , Ratones Noqueados , Ratones Obesos , Obesidad/genética , Obesidad/metabolismo , Receptor de Insulina/metabolismo , Factores de Transcripción/metabolismoRESUMEN
OBJECTIVE: Body weight change and obesity follow the variance of excess energy input balanced against tightly controlled EE (energy expenditure). Since insulin resistance can reduce energy storage, we investigated whether genetic disruption of hepatic insulin signaling reduced adipose mass with increased EE. METHODS: Insulin signaling was disrupted by genetic inactivation of Irs1 (Insulin receptor substrate 1) and Irs2 in hepatocytes of LDKO mice (Irs1L/L·Irs2L/L·CreAlb), creating a state of complete hepatic insulin resistance. We inactivated FoxO1 or the FoxO1-regulated hepatokine Fst (Follistatin) in the liver of LDKO mice by intercrossing LDKO mice with FoxO1L/L or FstL/L mice. We used DEXA (dual-energy X-ray absorptiometry) to assess total lean mass, fat mass and fat percentage, and metabolic cages to measure EE (energy expenditure) and estimate basal metabolic rate (BMR). High-fat diet was used to induce obesity. RESULTS: Hepatic disruption of Irs1 and Irs2 (LDKO mice) attenuated HFD (high-fat diet)-induced obesity and increased whole-body EE in a FoxO1-dependent manner. Hepatic disruption of the FoxO1-regulated hepatokine Fst normalized EE in LDKO mice and restored adipose mass during HFD consumption; moreover, hepatic Fst disruption alone increased fat mass accumulation, whereas hepatic overexpression of Fst reduced HFD-induced obesity. Excess circulating Fst in overexpressing mice neutralized Mstn (Myostatin), activating mTORC1-promoted pathways of nutrient uptake and EE in skeletal muscle. Similar to Fst overexpression, direct activation of muscle mTORC1 also reduced adipose mass. CONCLUSIONS: Thus, complete hepatic insulin resistance in LDKO mice fed a HFD revealed Fst-mediated communication between the liver and muscle, which might go unnoticed during ordinary hepatic insulin resistance as a mechanism to increase muscle EE and constrain obesity.
Asunto(s)
Resistencia a la Insulina , Ratones , Animales , Resistencia a la Insulina/fisiología , Metabolismo Basal , Folistatina/metabolismo , Obesidad/metabolismo , Hígado/metabolismo , Insulina/metabolismo , Dieta Alta en Grasa/efectos adversosRESUMEN
Chronic endoplasmic reticulum (ER) stress and sustained activation of unfolded protein response (UPR) signaling contribute to the development of type 2 diabetes in obesity. UPR signaling is a complex signaling pathway, which is still being explored in many different cellular processes. Here, we demonstrate that FK506-binding protein 11 (FKBP11), which is transcriptionally regulated by XBP1s, is severely reduced in the livers of obese mice. Restoring hepatic FKBP11 expression in obese mice initiates an atypical UPR signaling pathway marked by rewiring of PERK signaling toward NRF2, away from the eIF2α-ATF4 axis of the UPR. This alteration in UPR signaling establishes glucose homeostasis without changing hepatic ER stress, food consumption, or body weight. We conclude that ER stress during obesity can be beneficially rewired to promote glucose homeostasis. These findings may uncover possible new avenues in the development of novel approaches to treat diseases marked by ER stress.
Asunto(s)
Diabetes Mellitus Tipo 2 , Glucosa , Obesidad , Proteínas de Unión a Tacrolimus , Respuesta de Proteína Desplegada , Animales , Diabetes Mellitus Tipo 2/metabolismo , Glucosa/metabolismo , Homeostasis , Ratones , Ratones Obesos , Obesidad/metabolismo , Transducción de Señal , Proteínas de Unión a Tacrolimus/metabolismoRESUMEN
The hepatokine follistatin is elevated in patients with type 2 diabetes (T2D) and promotes hyperglycemia in mice. Here we explore the relationship of plasma follistatin levels with incident T2D and mechanisms involved. Adjusted hazard ratio (HR) per standard deviation (SD) increase in follistatin levels for T2D is 1.24 (CI: 1.04-1.47, p < 0.05) during 19-year follow-up (n = 4060, Sweden); and 1.31 (CI: 1.09-1.58, p < 0.01) during 4-year follow-up (n = 883, Finland). High circulating follistatin associates with adipose tissue insulin resistance and non-alcoholic fatty liver disease (n = 210, Germany). In human adipocytes, follistatin dose-dependently increases free fatty acid release. In genome-wide association study (GWAS), variation in the glucokinase regulatory protein gene (GCKR) associates with plasma follistatin levels (n = 4239, Sweden; n = 885, UK, Italy and Sweden) and GCKR regulates follistatin secretion in hepatocytes in vitro. Our findings suggest that GCKR regulates follistatin secretion and that elevated circulating follistatin associates with an increased risk of T2D by inducing adipose tissue insulin resistance.
Asunto(s)
Diabetes Mellitus Tipo 2/sangre , Folistatina/sangre , Proteínas Adaptadoras Transductoras de Señales/sangre , Tejido Adiposo/metabolismo , Estudio de Asociación del Genoma Completo , Hepatocitos/metabolismo , Humanos , Resistencia a la Insulina/fisiología , Persona de Mediana Edad , Enfermedad del Hígado Graso no Alcohólico/sangreRESUMEN
Fgf21 (fibroblast growth factor 21) is a regulatory hepatokine that, in pharmacologic form, powerfully promotes weight loss and glucose homeostasis. Although "Fgf21 resistance" is inferred from higher plasma Fgf21 levels in insulin-resistant mice and humans, diminished Fgf21 function is understood primarily via Fgf21 knockout mice. By contrast, we show that modestly reduced Fgf21-owing to cell-autonomous suppression by hepatic FoxO1-contributes to dysregulated metabolism in LDKO mice (Irs1L/Lâ Irs2L/Lâ CreAlb), a model of severe hepatic insulin resistance caused by deletion of hepatic Irs1 (insulin receptor substrate 1) and Irs2. Knockout of hepatic Foxo1 in LDKO mice or direct restoration of Fgf21 by adenoviral infection restored glucose utilization by BAT (brown adipose tissue) and skeletal muscle, normalized thermogenic gene expression in LDKO BAT, and corrected acute cold intolerance of LDKO mice. These studies highlight the Fgf21-dependent plasticity and importance of BAT function to metabolic health during hepatic insulin resistance.
Asunto(s)
Adaptación Fisiológica , Frío , Factores de Crecimiento de Fibroblastos/metabolismo , Proteína Forkhead Box O1/metabolismo , Glucosa/metabolismo , Resistencia a la Insulina , Insulina/metabolismo , Hígado/metabolismo , Adipocitos Marrones/metabolismo , Tejido Adiposo Pardo/metabolismo , Animales , Glucemia/metabolismo , Peso Corporal , Dieta Alta en Grasa , Regulación de la Expresión Génica , Homeostasis , Proteínas Sustrato del Receptor de Insulina/metabolismo , Metabolismo de los Lípidos , Ratones Noqueados , Especificidad de Órganos , Oxidación-Reducción , Termogénesis/genéticaRESUMEN
Skeletal muscle insulin resistance, decreased phosphatidylinositol 3-kinase (PI3K) activation and altered mitochondrial function are hallmarks of type 2 diabetes. To determine the relationship between these abnormalities, we created mice with muscle-specific knockout of the p110α or p110ß catalytic subunits of PI3K. We find that mice with muscle-specific knockout of p110α, but not p110ß, display impaired insulin signaling and reduced muscle size due to enhanced proteasomal and autophagic activity. Despite insulin resistance and muscle atrophy, M-p110αKO mice show decreased serum myostatin, increased mitochondrial mass, increased mitochondrial fusion, and increased PGC1α expression, especially PCG1α2 and PCG1α3. This leads to enhanced mitochondrial oxidative capacity, increased muscle NADH content, and higher muscle free radical release measured in vivo using pMitoTimer reporter. Thus, p110α is the dominant catalytic isoform of PI3K in muscle in control of insulin sensitivity and muscle mass, and has a unique role in mitochondrial homeostasis in skeletal muscle.
Asunto(s)
Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Mitocondrias/enzimología , Músculo Esquelético/enzimología , Animales , Fosfatidilinositol 3-Quinasa Clase I/genética , Homeostasis , Insulina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/genética , Mitocondrias/metabolismo , Músculo Esquelético/metabolismo , NAD/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismoRESUMEN
In the version of this article originally published, the y axis labels in Fig. 4b,d were incorrect. In Fig. 4b, the unit on the label was (ng mg-1). This should have been (ng/ml). In Fig. 4d, the y axis label was Serum Fst (ng ml-1). It should have been Serum insulin (ng/ml). The errors have been corrected in the HTML and PDF versions of this article.
RESUMEN
Unsuppressed hepatic glucose production (HGP) contributes substantially to glucose intolerance and diabetes, which can be modeled by the genetic inactivation of hepatic insulin receptor substrate 1 (Irs1) and Irs2 (LDKO mice). We previously showed that glucose intolerance in LDKO mice is resolved by hepatic inactivation of the transcription factor FoxO1 (that is, LTKO mice)-even though the liver remains insensitive to insulin. Here, we report that insulin sensitivity in the white adipose tissue of LDKO mice is also impaired but is restored in LTKO mice in conjunction with normal suppression of HGP by insulin. To establish the mechanism by which white adipose tissue insulin signaling and HGP was regulated by hepatic FoxO1, we identified putative hepatokines-including excess follistatin (Fst)-that were dysregulated in LDKO mice but normalized in LTKO mice. Knockdown of hepatic Fst in the LDKO mouse liver restored glucose tolerance, white adipose tissue insulin signaling and the suppression of HGP by insulin; however, the expression of Fst in the liver of healthy LTKO mice had the opposite effect. Of potential clinical significance, knockdown of Fst also improved glucose tolerance in high-fat-fed obese mice, and the level of serum Fst was reduced in parallel with glycated hemoglobin in obese individuals with diabetes who underwent therapeutic gastric bypass surgery. We conclude that Fst is a pathological hepatokine that might be targeted for diabetes therapy during hepatic insulin resistance.
Asunto(s)
Folistatina/metabolismo , Hiperglucemia/patología , Hígado/metabolismo , Células 3T3-L1 , Tejido Adiposo Blanco/metabolismo , Animales , Cirugía Bariátrica , Regulación hacia Abajo/genética , Proteína Forkhead Box O1/metabolismo , Técnicas de Silenciamiento del Gen , Glucosa/metabolismo , Intolerancia a la Glucosa/complicaciones , Intolerancia a la Glucosa/patología , Humanos , Hiperglucemia/complicaciones , Resistencia a la Insulina , Hígado/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de SeñalRESUMEN
AIMS/HYPOTHESIS: Sirtuin 6 (SIRT6) has been implicated in ageing, DNA repair and metabolism; however, its function in pancreatic beta cells is unclear. The aim of this study is to elucidate the role of SIRT6 in pancreatic beta cells. METHODS: To investigate the function of SIRT6 in pancreatic beta cells, we performed Sirt6 gene knockdown in MIN6 cells and generated pancreatic- and beta cell-specific Sirt6 knockout mice. Islet morphology and glucose-stimulated insulin secretion (GSIS) were analysed. Glycolysis and oxygen consumption rates in SIRT6-deficient beta cells were measured. Cytosolic calcium was monitored using the Fura-2-AM fluorescent probe (Invitrogen, Grand Island, NY, USA). Mitochondria were analysed by immunoblots and electron microscopy. RESULTS: Sirt6 knockdown in MIN6 beta cells led to a significant decrease in GSIS. Pancreatic beta cell Sirt6 knockout mice showed a ~50% decrease in GSIS. The knockout mouse islets had lower ATP levels compared with the wild-type controls. Mitochondrial oxygen consumption rates were significantly decreased in the SIRT6-deficient beta cells. Cytosolic calcium dynamics in response to glucose or potassium chloride were attenuated in the Sirt6 knockout islets. Numbers of damaged mitochondria were increased and mitochondrial complex levels were decreased in the SIRT6-deficient islets. CONCLUSIONS/INTERPRETATION: These data suggest that SIRT6 is important for GSIS from pancreatic beta cells and activation of SIRT6 may be useful to improve insulin secretion in diabetes.
Asunto(s)
Envejecimiento , Reparación del ADN , Regulación de la Expresión Génica , Células Secretoras de Insulina/citología , Sirtuinas/genética , Sirtuinas/fisiología , Animales , Calcio/metabolismo , Cruzamientos Genéticos , Eliminación de Gen , Glucosa/metabolismo , Prueba de Tolerancia a la Glucosa , Glucólisis , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/citología , Masculino , Ratones , Ratones Noqueados , Microscopía Electrónica , Mitocondrias/metabolismo , Oxígeno/química , Consumo de OxígenoRESUMEN
Sestrin proteins have been implicated in multiple biological processes including resistance to oxidative and genotoxic stresses, protection against aging-related pathologies, and promotion of metabolic homeostasis; however, the underlying mechanisms are incompletely understood. Some evidence suggests that sestrins may inhibit mTORC1 (mechanistic target of rapamycin complex 1) through inhibition of RagA/B GTPases or activation of AMPK; however, whether sestrins are also involved in mTORC2 regulation and function is unclear. To investigate the functions and mechanisms of Sestrin 3 (Sesn3), we generated Sesn3 liver-specific transgenic and knockout mice. Our data show that Sesn3 liver-specific knockout mice exhibit insulin resistance and glucose intolerance, and Sesn3 transgenic mice were protected against insulin resistance induced by a high-fat diet. Using AMPK liver-specific knockout mice, we demonstrate that the Sesn3 insulin-sensitizing effect is largely independent of AMPK. Biochemical analysis reveals that Sesn3 interacts with and activates mTORC2 and subsequently stimulates Akt phosphorylation at Ser473. These findings suggest that Sesn3 can activate Akt via mTORC2 to regulate hepatic insulin sensitivity and glucose metabolism.
Asunto(s)
Proteínas de Choque Térmico/metabolismo , Insulina/metabolismo , Hígado/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Adenilato Quinasa/metabolismo , Animales , Dieta Alta en Grasa , Glucosa/metabolismo , Intolerancia a la Glucosa/metabolismo , Proteínas de Choque Térmico/genética , Homeostasis/fisiología , Resistencia a la Insulina/genética , Diana Mecanicista del Complejo 2 de la Rapamicina , Ratones , Ratones Transgénicos , Fosforilación , Transducción de Señal/genéticaRESUMEN
Natural product Hypericum perforatum L. has been used in folk medicine to improve mental performance. However, the effect of H. perforatum L. on metabolism is still unknown. In order to test whether H. perforatum L. extract (EHP) has an effect on metabolic syndrome, we treated diet induced obese (DIO) C57BL/6J mice with the extract. The chemical characters of EHP were investigated with thin-layer chromatography, ultraviolet, high-performance liquid chromatography (HPLC), and HPLC-mass spectrometry fingerprint analysis. Oral glucose tolerance test (OGTT), insulin tolerance test (ITT), and the glucose infusion rate (GIR) in hyperinsulinemic-euglycemic clamp test were performed to evaluate the glucose metabolism and insulin sensitivity. Skeletal muscle was examined for lipid metabolism. The results suggest that EHP can significantly improve the glucose and lipid metabolism in DIO mice. In vitro, EHP inhibited the catalytic activity of recombinant human protein tyrosine phosphatase 1B (PTP1B) and reduced the protein and mRNA levels of PTP1B in the skeletal muscle. Moreover, expressions of genes related to fatty acid uptake and oxidation were changed by EHP in the skeletal muscle. These results suggest that EHP may improve insulin resistance and lipid metabolism in DIO mice.
Asunto(s)
Hypericum/química , Resistencia a la Insulina , Metabolismo de los Lípidos/efectos de los fármacos , Síndrome Metabólico/metabolismo , Extractos Vegetales/farmacología , Animales , Dieta Alta en Grasa/efectos adversos , Ácidos Grasos/metabolismo , Glucosa/metabolismo , Prueba de Tolerancia a la Glucosa , Humanos , Masculino , Síndrome Metabólico/tratamiento farmacológico , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Músculo Esquelético/metabolismo , Obesidad/tratamiento farmacológico , Obesidad/metabolismo , Componentes Aéreos de las Plantas/química , Proteína Tirosina Fosfatasa no Receptora Tipo 1 , Proteínas Recombinantes/metabolismoRESUMEN
Sestrins (Sesns) are a family of stress-sensitive genes that have been suggested to regulate lipid metabolism. Chronic ethanol feeding is known to cause lipid accumulation in hepatocytes. This study was designed to investigate the role of Sesn3 in the pathogenesis of alcohol-induced hepatic steatosis. We demonstrated that ethanol inhibited the expression of Sesn3 in VL-17A cells. Overexpression of Sesn3 ameliorated triglyceride accumulation; downregulation using short hairpin RNA significantly deteriorated triglyceride accumulation in these cells. The expression of Sesn3 was also reduced in mice fed with ethanol for 4 wk. Overexpression of Sesn3 prevented hepatic steatosis, whereas knockdown of Sesn3 worsened hepatic steatosis in ethanol-fed mice. Overexpression of Sesn3 significantly reduced the expression of genes encoding for lipid synthesis through AMPK pathway. Overexpression of Sesn3 augmented the effect of ethanol on phospho-p70 S6 kinase. The levels of hepatic light chain 3, a marker for autophagy, expression were significantly decreased in ethanol-fed mice after Sesn3 gene was knocked down. Our findings suggest that inhibitory effect of ethanol on Sesn3 may play an important role in the development of ethanol-induced fatty liver.
Asunto(s)
Etanol , Hígado Graso Alcohólico/metabolismo , Proteínas de Choque Térmico/metabolismo , Hígado/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Modelos Animales de Enfermedad , Regulación hacia Abajo , Hígado Graso Alcohólico/etiología , Hígado Graso Alcohólico/genética , Hígado Graso Alcohólico/patología , Hígado Graso Alcohólico/prevención & control , Proteínas de Choque Térmico/genética , Células Hep G2 , Humanos , Lipogénesis , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Asociadas a Microtúbulos/metabolismo , Interferencia de ARN , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Factores de Tiempo , Transfección , Triglicéridos/metabolismoRESUMEN
Forkhead transcription factors FoxO1/3/4 have pleiotrophic functions including anti-oxidative stress and metabolism. With regard to glucose metabolism, most studies have been focused on FoxO1. To further investigate their hepatic functions, we generated liver-specific FoxO1/3/4 knockout mice (LTKO) and examined their collective impacts on glucose homeostasis under physiological and pathological conditions. As compared to wild-type mice, LTKO mice had lower blood glucose levels under both fasting and non-fasting conditions and they manifested better glucose and pyruvate tolerance on regular chow diet. After challenged by a high-fat diet, wild-type mice developed type 2 diabetes, but LTKO mice remained euglycemic and insulin-sensitive. To understand the underlying mechanisms, we examined the roles of SIRT6 (Sirtuin 6) and Gck (glucokinase) in the FoxO-mediated glucose metabolism. Interestingly, ectopic expression of SIRT6 in the liver only reduced gluconeogenesis in wild-type but not LTKO mice whereas knockdown of Gck caused glucose intolerance in both wild-type and LTKO mice. The data suggest that both decreased gluconeogenesis and increased glycolysis may contribute to the overall glucose phenotype in the LTKO mice. Collectively, FoxO1/3/4 transcription factors play important roles in hepatic glucose homeostasis.
Asunto(s)
Factores de Transcripción Forkhead/metabolismo , Gluconeogénesis/fisiología , Glucosa/metabolismo , Glucólisis/fisiología , Hígado/metabolismo , Animales , Proteínas de Ciclo Celular , Grasas de la Dieta/farmacología , Proteína Forkhead Box O1 , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/genética , Eliminación de Gen , Glucoquinasa/genética , Glucoquinasa/metabolismo , Gluconeogénesis/efectos de los fármacos , Glucosa/genética , Glucólisis/efectos de los fármacos , Homeostasis/efectos de los fármacos , Homeostasis/fisiología , Ratones , Ratones Noqueados , Especificidad de Órganos/genética , Sirtuinas/genética , Sirtuinas/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
Pyruvate dehydrogenase kinases (PDK1-4) play a critical role in the inhibition of the mitochondrial pyruvate dehydrogenase complex especially when blood glucose levels are low and pyruvate can be conserved for gluconeogenesis. Under diabetic conditions, the Pdk genes, particularly Pdk4, are often induced, and the elevation of the Pdk4 gene expression has been implicated in the increased gluconeogenesis in the liver and the decreased glucose utilization in the peripheral tissues. However, there is no direct evidence yet to show to what extent that the dysregulation of hepatic Pdk genes attributes to hyperglycemia and insulin resistance in vivo. To address this question, we crossed Pdk2 or Pdk4 null mice with a diabetic model that is deficient in hepatic insulin receptor substrates 1 and 2 (Irs1/2). Metabolic analyses reveal that deletion of the Pdk4 gene had better improvement in hyperglycemia and glucose tolerance than knockout of the Pdk2 gene whereas the Pdk2 gene deletion showed better insulin tolerance as compared to the Pdk4 gene inactivation on the Irs1/2 knockout genetic background. To examine the specific hepatic effects of Pdks on diabetes, we also knocked down the Pdk2 or Pdk4 gene using specific shRNAs. The data also indicate that the Pdk4 gene knockdown led to better glucose tolerance than the Pdk2 gene knockdown. In conclusion, our data suggest that hepatic Pdk4 may be critically involved in the pathogenesis of diabetes.
Asunto(s)
Diabetes Mellitus Experimental/genética , Resistencia a la Insulina/genética , Hígado/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Animales , Diabetes Mellitus Experimental/metabolismo , Regulación Enzimológica de la Expresión Génica , Silenciador del Gen , Intolerancia a la Glucosa/genética , Prueba de Tolerancia a la Glucosa , Proteínas Sustrato del Receptor de Insulina/genética , Ratones , Ratones Noqueados , Especificidad de Órganos/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Piruvato Deshidrogenasa Quinasa Acetil-TransferidoraRESUMEN
Elevated LDL-cholesterol is a risk factor for the development of cardiovascular disease. Thus, proper control of LDL-cholesterol homeostasis is critical for organismal health. Genetic analysis has identified PCSK9 (proprotein convertase subtilisin/kexin type 9) as a crucial gene in the regulation of LDL-cholesterol via control of LDL receptor degradation. Although biochemical characteristics and clinical implications of PCSK9 have been extensively investigated, epigenetic regulation of this gene is largely unknown. In this work we have discovered that Sirt6, an NAD(+)-dependent histone deacetylase, plays a critical role in the regulation of the Pcsk9 gene expression in mice. Hepatic Sirt6 deficiency leads to elevated Pcsk9 gene expression and LDL-cholesterol as well. Mechanistically, we have demonstrated that Sirt6 can be recruited by forkhead transcription factor FoxO3 to the proximal promoter region of the Pcsk9 gene and deacetylates histone H3 at lysines 9 and 56, thereby suppressing the gene expression. Also remarkably, overexpression of Sirt6 in high fat diet-fed mice lowers LDL-cholesterol. Overall, our data suggest that FoxO3 and Sirt6, two longevity genes, can reduce LDL-cholesterol levels through regulation of the Pcsk9 gene.
Asunto(s)
LDL-Colesterol/metabolismo , Factores de Transcripción Forkhead/metabolismo , Homeostasis , Proproteína Convertasas/metabolismo , Serina Endopeptidasas/metabolismo , Sirtuinas/metabolismo , Acetilación , Animales , Western Blotting , LDL-Colesterol/sangre , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/genética , Regulación de la Expresión Génica , Histonas/metabolismo , Hígado/metabolismo , Lisina/metabolismo , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Regiones Promotoras Genéticas/genética , Proproteína Convertasa 9 , Proproteína Convertasas/genética , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Serina Endopeptidasas/genética , Sirtuinas/genéticaRESUMEN
Cholesterol homeostasis is crucial for cellular function and organismal health. The key regulator for the cholesterol biosynthesis is sterol-regulatory element binding protein (SREBP)-2. The biochemical process and physiological function of SREBP-2 have been well characterized; however, it is not clear how this gene is epigenetically regulated. Here we have identified sirtuin (Sirt)6 as a critical factor for Srebp2 gene regulation. Hepatic deficiency of Sirt6 in mice leads to elevated cholesterol levels. On the mechanistic level, Sirt6 is recruited by forkhead box O (FoxO)3 to the Srebp2 gene promoter where Sirt6 deacetylates histone H3 at lysines 9 and 56, thereby promoting a repressive chromatin state. Remarkably, Sirt6 or FoxO3 overexpression improves hypercholesterolemia in diet-induced or genetically obese mice. In summary, our data suggest an important role of hepatic Sirt6 and FoxO3 in the regulation of cholesterol homeostasis.
Asunto(s)
Colesterol/biosíntesis , Factores de Transcripción Forkhead/fisiología , Hígado/metabolismo , Sirtuinas/fisiología , Proteína 2 de Unión a Elementos Reguladores de Esteroles/biosíntesis , Acetilación , Animales , Secuencia de Bases , Colesterol/sangre , Epigénesis Genética , Proteína Forkhead Box O3 , Expresión Génica , Técnicas de Inactivación de Genes , Células HEK293 , Histonas/metabolismo , Homeostasis , Humanos , Hipercolesterolemia/metabolismo , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Regiones Promotoras Genéticas , Procesamiento Proteico-PostraduccionalRESUMEN
Protein tyrosine phosphatases (PTPs) constitute a large family of signaling enzymes that control the cellular levels of protein tyrosine phosphorylation. A detailed understanding of PTP functions in normal physiology and in pathogenic conditions has been hampered by the absence of PTP-specific, cell-permeable small-molecule agents. We present a stepwise focused library approach that transforms a weak and general non-hydrolyzable pTyr mimetic (F(2)Pmp, phosphonodifluoromethyl phenylalanine) into a highly potent and selective inhibitor of PTP-MEG2, an antagonist of hepatic insulin signaling. The crystal structures of the PTP-MEG2-inhibitor complexes provide direct evidence that potent and selective PTP inhibitors can be obtained by introducing molecular diversity into the F(2)Pmp scaffold to engage both the active site and unique nearby peripheral binding pockets. Importantly, the PTP-MEG2 inhibitor possesses highly efficacious cellular activity and is capable of augmenting insulin signaling and improving insulin sensitivity and glucose homeostasis in diet-induced obese mice. The results indicate that F(2)Pmp can be converted into highly potent and selective PTP inhibitory agents with excellent in vivo efficacy. Given the general nature of the approach, this strategy should be applicable to other members of the PTP superfamily.
Asunto(s)
Diabetes Mellitus Tipo 2/tratamiento farmacológico , Fenilalanina/análogos & derivados , Proteínas Tirosina Fosfatasas no Receptoras/antagonistas & inhibidores , Animales , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Modelos Moleculares , Estructura Molecular , Fenilalanina/síntesis química , Fenilalanina/química , Fenilalanina/farmacología , Proteínas Tirosina Fosfatasas no Receptoras/química , Proteínas Tirosina Fosfatasas no Receptoras/metabolismoRESUMEN
Hepatic steatosis often accompanies obesity and insulin resistance. The cornerstones of steatosis treatment include reducing body weight and dietary fat intake, which are marginally successful over the long term. Ad36, a human adenovirus, may offer a template to overcome these limitations. In vitro and in vivo studies collectively indicate that via its E4orf1 protein, Ad36 improves hyperglycemia, and attenuates hepatic steatosis, despite a high fat diet and without weight loss. Considering that hepatic insulin sensitivity, or the synthesis, oxidation, or export of fatty acid by hepatocytes are the key determinant of hepatic lipid storage, we determined the role of E4orf1 protein in modulating these physiological pathways. For this study, HepG2 cells, or mouse primary hepatocytes were transfected with E4orf1 or the null vector. Glucose output by hepatocytes was determined under gluconeogenic conditions (cAMP and dexamethasone, or glucagon exposure). Also, de-novo lipogenesis, palmitate oxidation, and lipid export as determined by apoB secretion were measured 48 h post transfection. Results show that compared to null vector transfected cells, E4orf1 significantly reduced glucose output in basal and gluconeogenic conditions. E4orf1 reduced de-novo lipogenesis by about 35%, increased complete fatty acid oxidation 2-fold (p<0.0001), and apoB secretion 1.5 fold(p<0.003). Response of key signaling molecules to E4orf1 transfection was in agreement with these findings. Thus, E4orf1 offers a valuable template to exogenously modulate hepatic glucose and lipid metabolism. Elucidating the underlying molecular mechanism may help develop therapeutic approaches for treating diabetes or non-alcoholic fatty liver disease(NAFLD).
Asunto(s)
Proteínas E4 de Adenovirus/metabolismo , Adenovirus Humanos/genética , Hígado Graso/metabolismo , Glucosa/metabolismo , Hiperglucemia/metabolismo , Metabolismo de los Lípidos/genética , Proteínas E4 de Adenovirus/genética , Animales , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Vectores Genéticos/genética , Células Hep G2 , Hepatocitos/metabolismo , Humanos , Ratones , TransfecciónRESUMEN
Autophagy plays a critical role in cell survival from prolonged starvation and recycling of aggregated proteins and damaged organelles. One of the essential genes involved in the autophagic initiation is autophagy-related 14 (Atg14), also called Barkor for Beclin 1-associated autophagy-related key regulator. Although its crucial role in the autophagic process has been reported, the gene regulation of Atg14 and its metabolic functions remain unclear. In this work we have identified that the Atg14 gene is regulated by forkhead box O (FoxO) transcription factors and circadian rhythms in the mouse liver. Luciferase reporter analyses and chromatin immunoprecipitation assays have revealed well conserved cis-elements for FoxOs and Clock/Bmal1 in the proximal promoter of the Atg14 gene. To examine the functions of hepatic Atg14, we have performed the gene knockdown and overexpression in the mouse livers. Remarkably, knockdown of Atg14 leads to elevated levels of triglycerides in the liver and serum as well. Conversely, overexpression of Atg14 improves hypertriglyceridemia in both high fat diet-treated wild-type mice and FoxO1/3/4 liver-specific knock-out mice. In summary, our data suggest that Atg14 is a new target gene of FoxOs and the core clock machinery, and this gene plays an important role in hepatic lipid metabolism.