RESUMEN
In response to a meal, insulin drives hepatic glycogen synthesis to help regulate systemic glucose homeostasis. The mechanistic target of rapamycin complex 1 (mTORC1) is a well-established insulin target and contributes to the postprandial control of liver lipid metabolism, autophagy, and protein synthesis. However, its role in hepatic glucose metabolism is less understood. Here, we used metabolomics, isotope tracing, and mouse genetics to define a role for liver mTORC1 signaling in the control of postprandial glycolytic intermediates and glycogen deposition. We show that mTORC1 is required for glycogen synthase activity and glycogenesis. Mechanistically, hepatic mTORC1 activity promotes the feeding-dependent induction of Ppp1r3b, a gene encoding a phosphatase important for glycogen synthase activity whose polymorphisms are linked to human diabetes. Reexpression of Ppp1r3b in livers lacking mTORC1 signaling enhances glycogen synthase activity and restores postprandial glycogen content. mTORC1-dependent transcriptional control of Ppp1r3b is facilitated by FOXO1, a well characterized transcriptional regulator involved in the hepatic response to nutrient intake. Collectively, we identify a role for mTORC1 signaling in the transcriptional regulation of Ppp1r3b and the subsequent induction of postprandial hepatic glycogen synthesis.
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Glucógeno Sintasa , Glucógeno Hepático , Diana Mecanicista del Complejo 1 de la Rapamicina , Proteína Fosfatasa 1 , Animales , Humanos , Ratones , Glucógeno/genética , Glucógeno/metabolismo , Glucógeno Sintasa/metabolismo , Insulina/metabolismo , Hígado/metabolismo , Glucógeno Hepático/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Proteína Fosfatasa 1/metabolismo , Periodo PosprandialRESUMEN
AAV gene transfer is a promising treatment for many patients with life-threatening genetic diseases. However, host immune response to the vector poses a significant challenge for the durability and safety of AAV-mediated gene therapy. Here, we characterize the innate immune response to AAV in human whole blood. We identified neutrophils, monocyte-related dendritic cells, and monocytes as the most prevalent cell subsets able to internalize AAV particles, while conventional dendritic cells were the most activated in terms of the CD86 co-stimulatory molecule upregulation. Although low titers (≤1:10) of AAV neutralizing antibodies (NAb) in blood did not have profound effects on the innate immune response to AAV, higher NAb titers (≥1:100) significantly increased pro-inflammatory cytokine/chemokine secretion, vector uptake by antigen presenting cells (APCs) and complement activation. Interestingly, both full and empty viral particles were equally potent in inducing complement activation and cytokine secretion. By using a compstatin-based C3 and C3b inhibitor, APL-9, we demonstrated that complement pathway inhibition lowered CD86 levels on APCs, AAV uptake, and cytokine/chemokine secretion in response to AAV. Together these results suggest that the pre-existing humoral immunity to AAV may contribute to trigger adverse immune responses observed in AAV-based gene therapy, and that blockade of complement pathway may warrant further investigation as a potential strategy for decreasing immunogenicity of AAV-based therapeutics.
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Dependovirus , Vectores Genéticos , Anticuerpos Neutralizantes , Citocinas/genética , Dependovirus/genética , Vectores Genéticos/genética , Humanos , Inmunidad HumoralRESUMEN
Adeno-associated virus (AAV) vector-mediated gene transfer is lessening the impact of monogenetic disorders. Human AAV gene therapy recipients commonly mount immune responses to AAV or the encoded therapeutic protein, which requires transient immunosuppression. Most efforts to date have focused on blunting AAV capsid-specific T cell responses, which have been implicated in elimination of AAV-transduced cells. Here, we explore the use of immunosuppressants, rapamycin given alone or in combination with ibrutinib to inhibit AAV vector- or transgene product-specific antibody responses. Our results show that rapamycin or ibrutinib given alone reduces primary antibody responses against AAV capsid, but the combination of rapamycin and ibrutinib is more effective, blunts recall responses, and reduces numbers of circulating antibody-secreting plasma cells. The drugs fail to lower B cell memory formation or to reduce the inhibitory effects of pre-existing AAV capsid-specific antibodies on transduction efficiency.
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Dependovirus , Vectores Genéticos , Adenina/análogos & derivados , Formación de Anticuerpos , Proteínas de la Cápside/genética , Dependovirus/metabolismo , Técnicas de Transferencia de Gen , Terapia Genética , Vectores Genéticos/genética , Humanos , Piperidinas , Sirolimus/farmacología , Sirolimus/uso terapéuticoRESUMEN
Pompe disease (PD) is a severe neuromuscular disorder caused by deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). PD is currently treated with enzyme replacement therapy (ERT) with intravenous infusions of recombinant human GAA (rhGAA). Although the introduction of ERT represents a breakthrough in the management of PD, the approach suffers from several shortcomings. Here, we developed a mouse model of PD to compare the efficacy of hepatic gene transfer with adeno-associated virus (AAV) vectors expressing secretable GAA with long-term ERT. Liver expression of GAA results in enhanced pharmacokinetics and uptake of the enzyme in peripheral tissues compared to ERT. Combination of gene transfer with pharmacological chaperones boosts GAA bioavailability, resulting in improved rescue of the PD phenotype. Scale-up of hepatic gene transfer to non-human primates also successfully results in enzyme secretion in blood and uptake in key target tissues, supporting the ongoing clinical translation of the approach.
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Enfermedad del Almacenamiento de Glucógeno Tipo II/enzimología , alfa-Glucosidasas/metabolismo , Animales , Autofagia , Terapia de Reemplazo Enzimático , Femenino , Enfermedad del Almacenamiento de Glucógeno Tipo II/terapia , Hígado/enzimología , Masculino , Ratones , alfa-Glucosidasas/genéticaRESUMEN
Low-protein diets promote metabolic health in rodents and humans, and the benefits of low-protein diets are recapitulated by specifically reducing dietary levels of the three branched-chain amino acids (BCAAs), leucine, isoleucine, and valine. Here, we demonstrate that each BCAA has distinct metabolic effects. A low isoleucine diet reprograms liver and adipose metabolism, increasing hepatic insulin sensitivity and ketogenesis and increasing energy expenditure, activating the FGF21-UCP1 axis. Reducing valine induces similar but more modest metabolic effects, whereas these effects are absent with low leucine. Reducing isoleucine or valine rapidly restores metabolic health to diet-induced obese mice. Finally, we demonstrate that variation in dietary isoleucine levels helps explain body mass index differences in humans. Our results reveal isoleucine as a key regulator of metabolic health and the adverse metabolic response to dietary BCAAs and suggest reducing dietary isoleucine as a new approach to treating and preventing obesity and diabetes.
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Aminoácidos de Cadena Ramificada/metabolismo , Dieta , Isoleucina/metabolismo , Valina/metabolismo , Tejido Adiposo Blanco/metabolismo , Animales , Índice de Masa Corporal , Dieta/veterinaria , Metabolismo Energético , Factores de Crecimiento de Fibroblastos/deficiencia , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Humanos , Hígado/metabolismo , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad/metabolismo , Obesidad/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismoRESUMEN
Youth in military families are frequently challenged by the adjustment demands associated with the deployment and reintegration of a parent. A positive youth development approach was undertaken by the Boys and Girls Clubs of America to develop and implement a Military Teen Ambassadors (MTA) training for youth in military families that would facilitate knowledge of resiliency and reintegration and foster leadership skills to build assets for themselves as well as their peers within their local communities. To determine if MTA was functioning as intended and to refine future programming, this preliminary formative study assessed perceived participant learning outcomes associated with MTA on variables pertaining to knowledge acquisition, perceived skill acquisition, and community needs awareness. Data were collected prior to the training, immediately following the training, and 6 months after the training. Repeated measures analysis indicated significant mean increases over time in knowledge and awareness of resiliency and reintegration; perceived leadership skills; and community awareness. Qualitative findings provided triangulation in the aforementioned areas. These findings strengthen the body of knowledge on resiliency by demonstrating that the 7 Cs model may be an effective strategy to incorporate into leadership development programs seeking to build knowledge of resiliency among military youth. Study limitations, lessons learned, and recommendations for further research are delineated.
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Liderazgo , Personal Militar , Adolescente , Femenino , Humanos , Masculino , Grupo Paritario , Evaluación de Programas y Proyectos de SaludRESUMEN
Immune cell function is influenced by metabolic conditions. Low-glucose, high-lactate environments, such as the placenta, gastrointestinal tract, and the tumor microenvironment, are immunosuppressive, especially for glycolysis-dependent effector T cells. We report that nicotinamide adenine dinucleotide (NAD+), which is reduced to NADH by lactate dehydrogenase in lactate-rich conditions, is a key point of metabolic control in T cells. Reduced NADH is not available for NAD+-dependent enzymatic reactions involving glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and 3-phosphoglycerate dehydrogenase (PGDH). We show that increased lactate leads to a block at GAPDH and PGDH, leading to the depletion of post-GAPDH glycolytic intermediates, as well as the 3-phosphoglycerate derivative serine that is known to be important for T cell proliferation. Supplementing serine rescues the ability of T cells to proliferate in the presence of lactate-induced reductive stress. Directly targeting the redox state may be a useful approach for developing novel immunotherapies in cancer and therapeutic immunosuppression.
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Ácido Láctico/metabolismo , NAD/metabolismo , Proliferación Celular , Humanos , Oxidación-ReducciónRESUMEN
The Pro47Ser variant of p53 (S47) exists in African-descent populations and is associated with increased cancer risk in humans and mice. Due to impaired repression of the cystine importer Slc7a11, S47 cells show increased glutathione (GSH) accumulation compared to cells with wild -type p53. We show that mice containing the S47 variant display increased mTOR activity and oxidative metabolism, as well as larger size, improved metabolic efficiency, and signs of superior fitness. Mechanistically, we show that mTOR and its positive regulator Rheb display increased association in S47 cells; this is due to an altered redox state of GAPDH in S47 cells that inhibits its ability to bind and sequester Rheb. Compounds that decrease glutathione normalize GAPDH-Rheb complexes and mTOR activity in S47 cells. This study reveals a novel layer of regulation of mTOR by p53, and raises the possibility that this variant may have been selected for in early Africa.
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Serina-Treonina Quinasas TOR/metabolismo , Proteína p53 Supresora de Tumor/genética , Sustitución de Aminoácidos/genética , Animales , Población Negra/genética , Línea Celular , Glutatión/metabolismo , Glucólisis , Humanos , Mitocondrias/metabolismo , Oxidación-Reducción , Serina-Treonina Quinasas TOR/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Lack of interleukin 15 receptor alpha (IL15RA) increases spontaneous activity, exercise capacity and protects from diet-induced obesity by enhancing muscle energy metabolism, suggesting a role as exercise mimetic for IL15RA antagonists. Using controlled in vivo muscle stimulation mimicking moderate exercise in normal and Il15ra-/- mice, we mapped and contrasted the metabolic pathways activated upon stimulation or deletion of IL15RA. Stimulation caused the differential regulation of 123 out of the 321 detected metabolites (FDR ≤ 0.05 and fold change ≥ ±1.5). The main energy pathways activated were fatty acid oxidation, nucleotide metabolism, and anaplerotic reactions. Notably, resting Il15ra-/- muscles were primed in a semi-exercised state, characterized by higher pool sizes of fatty acids oxidized to support muscle activity. These studies identify the role of IL15RA in the system-wide metabolic response to exercise and should enable translational studies to harness the potential of IL15RA blockade as a novel exercise mimetic strategy.
RESUMEN
Mitochondrial Ca2+ uniporter (MCU)-mediated Ca2+ uptake promotes the buildup of reducing equivalents that fuel oxidative phosphorylation for cellular metabolism. Although MCU modulates mitochondrial bioenergetics, its function in energy homeostasis in vivo remains elusive. Here we demonstrate that deletion of the Mcu gene in mouse liver (MCUΔhep) and in Danio rerio by CRISPR/Cas9 inhibits mitochondrial Ca2+ (mCa2+) uptake, delays cytosolic Ca2+ (cCa2+) clearance, reduces oxidative phosphorylation, and leads to increased lipid accumulation. Elevated hepatic lipids in MCUΔhep were a direct result of extramitochondrial Ca2+-dependent protein phosphatase-4 (PP4) activity, which dephosphorylates AMPK. Loss of AMPK recapitulates hepatic lipid accumulation without changes in MCU-mediated Ca2+ uptake. Furthermore, reconstitution of active AMPK, or PP4 knockdown, enhances lipid clearance in MCUΔhep hepatocytes. Conversely, gain-of-function MCU promotes rapid mCa2+ uptake, decreases PP4 levels, and reduces hepatic lipid accumulation. Thus, our work uncovers an MCU/PP4/AMPK molecular cascade that links Ca2+ dynamics to hepatic lipid metabolism.
Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Hepatocitos/metabolismo , Metabolismo de los Lípidos , Proteínas Mitocondriales/metabolismo , Quinasas de la Proteína-Quinasa Activada por el AMP , Animales , Canales de Calcio/genética , Células Cultivadas , Femenino , Células Hep G2 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias Hepáticas/metabolismo , Proteínas Mitocondriales/genética , Fosfoproteínas Fosfatasas/metabolismo , Proteínas Quinasas/metabolismo , Pez CebraRESUMEN
Alterations in the synthesis and bioavailability of NO are central to the pathogenesis of cardiovascular and metabolic disorders. Although endothelial NO synthase-derived (eNOS-derived) NO affects mitochondrial long-chain fatty acid ß-oxidation, the pathophysiological significance of this regulation remains unclear. Accordingly, we determined the contributions of eNOS/NO signaling in the adaptive metabolic responses to fasting and in age-induced metabolic dysfunction. Four-month-old eNOS-/- mice are glucose intolerant and exhibit serum dyslipidemia and decreased capacity to oxidize fatty acids. However, during fasting, eNOS-/- mice redirect acetyl-CoA to ketogenesis to elevate circulating levels of ß-hydroxybutyrate similar to wild-type mice. Treatment of 4-month-old eNOS-/- mice with nitrite for 10 days corrected the hypertension and serum hyperlipidemia and normalized the rate of fatty acid oxidation. Fourteen-month-old eNOS-/- mice exhibited metabolic derangements, resulting in reduced utilization of fat to generate energy, lower resting metabolic activity, and diminished physical activity. Seven-month administration of nitrite to eNOS-/- mice reversed the age-dependent metabolic derangements and restored physical activity. While the eNOS/NO signaling is not essential for the metabolic adaptation to fasting, it is critical for regulating systemic metabolic homeostasis in aging. The development of age-dependent metabolic disorder is prevented by low-dose replenishment of bioactive NO.
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Envejecimiento/metabolismo , Homeostasis/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo III/deficiencia , Nitrito de Sodio/administración & dosificación , Administración Oral , Envejecimiento/efectos de los fármacos , Animales , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Ayuno/metabolismo , Humanos , Hiperlipidemias/tratamiento farmacológico , Hiperlipidemias/genética , Hiperlipidemias/metabolismo , Hipertensión/tratamiento farmacológico , Hipertensión/genética , Hipertensión/metabolismo , Masculino , Ratones , Ratones Noqueados , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo III/genética , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Resultado del TratamientoRESUMEN
The redox cofactor nicotinamide adenine dinucleotide (NAD) plays a central role in metabolism and is a substrate for signaling enzymes including poly-ADP-ribose-polymerases (PARPs) and sirtuins. NAD concentration falls during aging, which has triggered intense interest in strategies to boost NAD levels. A limitation in understanding NAD metabolism has been reliance on concentration measurements. Here, we present isotope-tracer methods for NAD flux quantitation. In cell lines, NAD was made from nicotinamide and consumed largely by PARPs and sirtuins. In vivo, NAD was made from tryptophan selectively in the liver, which then excreted nicotinamide. NAD fluxes varied widely across tissues, with high flux in the small intestine and spleen and low flux in the skeletal muscle. Intravenous administration of nicotinamide riboside or mononucleotide delivered intact molecules to multiple tissues, but the same agents given orally were metabolized to nicotinamide in the liver. Thus, flux analysis can reveal tissue-specific NAD metabolism.
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Hígado/metabolismo , NAD/análisis , NAD/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Sirtuinas/metabolismo , Triptófano/metabolismo , Animales , Femenino , Células HCT116 , Células Hep G2 , Humanos , Intestino Delgado/metabolismo , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/metabolismo , NAD/biosíntesis , Niacinamida/administración & dosificación , Niacinamida/farmacocinética , Bazo/metabolismoRESUMEN
Liver triacylglycerol (TAG) synthesis and secretion are closely linked to nutrient availability. After a meal, hepatic TAG formation from fatty acids is decreased, largely due to a reduction in circulating free fatty acids (FFA). Despite the postprandial decrease in FFA-driven esterification and oxidation, VLDL-TAG secretion is maintained to support peripheral lipid delivery and metabolism. The regulatory mechanisms underlying the postprandial control of VLDL-TAG secretion remain unclear. Here, we demonstrated that the mTOR complex 1 (mTORC1) is essential for this sustained VLDL-TAG secretion and lipid homeostasis. In murine models, the absence of hepatic mTORC1 reduced circulating TAG, despite hepatosteatosis, while activation of mTORC1 depleted liver TAG stores. Additionally, mTORC1 promoted TAG secretion by regulating phosphocholine cytidylyltransferase α (CCTα), the rate-limiting enzyme involved in the synthesis of phosphatidylcholine (PC). Increasing PC synthesis in mice lacking mTORC1 rescued hepatosteatosis and restored TAG secretion. These data identify mTORC1 as a major regulator of phospholipid biosynthesis and subsequent VLDL-TAG secretion, leading to increased postprandial TAG secretion.
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Lipogénesis , Diana Mecanicista del Complejo 1 de la Rapamicina/fisiología , Fosfatidilcolinas/biosíntesis , Triglicéridos/metabolismo , Animales , Células Cultivadas , Hígado Graso/metabolismo , Hepatocitos/metabolismo , Hígado/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Fosfatidilcolinas/metabolismo , Triglicéridos/biosíntesisRESUMEN
Immune cells function in diverse metabolic environments. Tissues with low glucose and high lactate concentrations, such as the intestinal tract or ischemic tissues, frequently require immune responses to be more pro-tolerant, avoiding unwanted reactions against self-antigens or commensal bacteria. T-regulatory cells (Tregs) maintain peripheral tolerance, but how Tregs function in low-glucose, lactate-rich environments is unknown. We report that the Treg transcription factor Foxp3 reprograms T cell metabolism by suppressing Myc and glycolysis, enhancing oxidative phosphorylation, and increasing nicotinamide adenine dinucleotide oxidation. These adaptations allow Tregs a metabolic advantage in low-glucose, lactate-rich environments; they resist lactate-mediated suppression of T cell function and proliferation. This metabolic phenotype may explain how Tregs promote peripheral immune tolerance during tissue injury but also how cancer cells evade immune destruction in the tumor microenvironment. Understanding Treg metabolism may therefore lead to novel approaches for selective immune modulation in cancer and autoimmune diseases.
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Microambiente Celular/inmunología , Reprogramación Celular/inmunología , Factores de Transcripción Forkhead/inmunología , Glucosa/inmunología , Ácido Láctico/inmunología , Linfocitos T Reguladores/inmunología , Animales , Línea Celular , Microambiente Celular/genética , Reprogramación Celular/genética , Factores de Transcripción Forkhead/genética , Glucosa/genética , Glucólisis/genética , Glucólisis/inmunología , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Fosforilación Oxidativa , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/inmunologíaRESUMEN
Long-lived plasma cells (PCs) in the bone marrow (BM) are a critical source of antibodies after infection or vaccination, but questions remain about the factors that control PCs. We found that systemic infection alters the BM, greatly reducing PCs and regulatory T (Treg) cells, a population that contributes to immune privilege in the BM. The use of intravital imaging revealed that BM Treg cells display a distinct behavior characterized by sustained co-localization with PCs and CD11c-YFP+ cells. Gene expression profiling indicated that BM Treg cells express high levels of Treg effector molecules, and CTLA-4 deletion in these cells resulted in elevated PCs. Furthermore, preservation of Treg cells during systemic infection prevents PC loss, while Treg cell depletion in uninfected mice reduced PC populations. These studies suggest a role for Treg cells in PC biology and provide a potential target for the modulation of PCs during vaccine-induced humoral responses or autoimmunity.
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Células de la Médula Ósea/inmunología , Médula Ósea/inmunología , Células Plasmáticas/inmunología , Linfocitos T Reguladores/inmunología , Animales , Autoinmunidad/inmunología , Antígeno CTLA-4/inmunología , Inmunidad Humoral , Inmunofenotipificación/métodos , Ratones , Ratones Endogámicos C57BLRESUMEN
NAD is an obligate co-factor for the catabolism of metabolic fuels in all cell types. However, the availability of NAD in several tissues can become limited during genotoxic stress and the course of natural aging. The point at which NAD restriction imposes functional limitations on tissue physiology remains unknown. We examined this question in murine skeletal muscle by specifically depleting Nampt, an essential enzyme in the NAD salvage pathway. Knockout mice exhibited a dramatic 85% decline in intramuscular NAD content, accompanied by fiber degeneration and progressive loss of both muscle strength and treadmill endurance. Administration of the NAD precursor nicotinamide riboside rapidly ameliorated functional deficits and restored muscle mass despite having only a modest effect on the intramuscular NAD pool. Additionally, lifelong overexpression of Nampt preserved muscle NAD levels and exercise capacity in aged mice, supporting a critical role for tissue-autonomous NAD homeostasis in maintaining muscle mass and function.
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Homeostasis , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , NAD/metabolismo , Administración Oral , Envejecimiento/fisiología , Animales , Disponibilidad Biológica , Metabolismo Energético , Glucosa/metabolismo , Inflamación/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/metabolismo , Fuerza Muscular , Músculo Esquelético/enzimología , Músculo Esquelético/fisiopatología , Necrosis , Niacinamida/administración & dosificación , Niacinamida/análogos & derivados , Niacinamida/metabolismo , Niacinamida/farmacología , Nicotinamida Fosforribosiltransferasa/deficiencia , Nicotinamida Fosforribosiltransferasa/metabolismo , Tamaño de los Órganos , Condicionamiento Físico Animal , Compuestos de Piridinio , Transcripción GenéticaRESUMEN
During insulin-resistant states such as type II diabetes mellitus (T2DM), insulin fails to suppress hepatic glucose production (HGP) yet promotes lipid synthesis. This metabolic state has been termed "selective insulin resistance" to indicate a defect in one arm of the insulin-signaling cascade, potentially downstream of Akt. Here we demonstrate that Akt-dependent activation of mTORC1 and inhibition of Foxo1 are required and sufficient for de novo lipogenesis, suggesting that hepatic insulin signaling is likely to be intact in insulin-resistant states. Moreover, cell-nonautonomous suppression of HGP by insulin depends on a reduction of adipocyte lipolysis and serum FFAs but is independent of vagal efferents or glucagon signaling. These data are consistent with a model in which, during T2DM, intact liver insulin signaling drives enhanced lipogenesis while excess circulating FFAs become a dominant inducer of nonsuppressible HGP.
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Glucosa/biosíntesis , Hepatocitos/metabolismo , Insulina/metabolismo , Lipogénesis , Transducción de Señal , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/metabolismo , Animales , Dieta , Vías Eferentes/efectos de los fármacos , Vías Eferentes/metabolismo , Ácidos Grasos no Esterificados/metabolismo , Proteína Forkhead Box O1/metabolismo , Eliminación de Gen , Regulación de la Expresión Génica/efectos de los fármacos , Glucagón/metabolismo , Glucoquinasa/metabolismo , Gluconeogénesis/efectos de los fármacos , Gluconeogénesis/genética , Prueba de Tolerancia a la Glucosa , Heparina/farmacología , Hepatocitos/efectos de los fármacos , Insulina/farmacología , Resistencia a la Insulina , Lipogénesis/efectos de los fármacos , Lipogénesis/genética , Hígado/efectos de los fármacos , Hígado/inervación , Hígado/metabolismo , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones Noqueados , Complejos Multiproteicos/metabolismo , Periodo Posprandial/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Serina-Treonina Quinasas TOR/metabolismo , Nervio Vago/efectos de los fármacos , Nervio Vago/fisiologíaRESUMEN
In non-alcoholic fatty liver disease (NAFLD) and insulin resistance, hepatic de novo lipogenesis is often elevated, but the underlying mechanisms remain poorly understood. Recently, we show that CDK8 functions to suppress de novo lipogenesis. Here, we identify the mammalian target of rapamycin complex 1 (mTORC1) as a critical regulator of CDK8 and its activating partner CycC. Using pharmacologic and genetic approaches, we show that increased mTORC1 activation causes the reduction of the CDK8-CycC complex in vitro and in mouse liver in vivo. In addition, mTORC1 is more active in three mouse models of NAFLD, correlated with the lower abundance of the CDK8-CycC complex. Consistent with the inhibitory role of CDK8 on de novo lipogenesis, nuclear SREBP-1c proteins and lipogenic enzymes are accumulated in NAFLD models. Thus, our results suggest that mTORC1 activation in NAFLD and insulin resistance results in down-regulation of the CDK8-CycC complex and elevation of lipogenic protein expression.
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Ciclina C/biosíntesis , Quinasa 8 Dependiente de Ciclina/biosíntesis , Regulación hacia Abajo , Regulación Enzimológica de la Expresión Génica , Complejos Multiproteicos/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Animales , Ciclina C/genética , Quinasa 8 Dependiente de Ciclina/genética , Células HEK293 , Humanos , Lipogénesis/genética , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Noqueados , Ratones Obesos , Complejos Multiproteicos/genética , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Serina-Treonina Quinasas TOR/genéticaRESUMEN
OBJECTIVE: To determine the necessity for any individual BAFF receptor in the development of systemic lupus erythematosus (SLE). METHODS: Bcma-, Taci-, and Br3-null mutations were introgressed into NZM 2328 mice. NZM.Bcma-/-, NZM.Taci-/-, and NZM.Br3-/- mice were evaluated for lymphocyte phenotype and BAFF receptor expression by flow cytometry; for B cell responsiveness to BAFF by in vitro culture; for serum levels of BAFF and total IgG and IgG anti-double-stranded DNA (anti-dsDNA) by enzyme-linked immunosorbent assay; for renal immunopathology by immunofluorescence and histopathology; and for clinical disease. RESULTS: BCMA, TACI, and B lymphocyte stimulator receptor 3 (BR3) were not surface-expressed in NZM.Bcma-/-, NZM.Taci-/-, and NZM.Br3-/- mice, respectively. Transitional and follicular B cells from NZM.Br3-/- mice were much less responsive to BAFF than were the corresponding cells from wild-type, NZM.Bcma-/-, or NZM.Taci-/- mice. In comparison with wild-type mice, NZM.Bcma-/- and NZM.Taci-/- mice harbored an increased number of spleen B cells, T cells, and plasma cells, whereas serum levels of total IgG and IgG anti-dsDNA were similar to those in wild-type mice. Despite their paucity of B cells, NZM.Br3-/- mice had an increased number of T cells, and the numbers of plasma cells and levels of IgG anti-dsDNA were similar to those in wild-type mice. Serum levels of BAFF were increased in NZM.Taci-/- and NZM.Br3-/- mice but were decreased in NZM.Bcma-/- mice. Despite their phenotypic differences, NZM.Bcma-/-, NZM.Taci-/-, and NZM.Br3-/- mice had renal immunopathology and clinical disease that were at least as severe as that in wild-type mice. CONCLUSION: Any single BAFF receptor, including BR3, is dispensable for the development of SLE in NZM mice. Development of disease in NZM.Br3-/- mice demonstrates that BAFF-BCMA and/or BAFF-TACI interactions contribute to SLE, and that a profound, life-long reduction in the numbers of B cells does not guarantee protection against SLE.