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1.
J Physiol ; 600(5): 1135-1154, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-33932956

RESUMO

The prevalence of non-alcoholic fatty liver disease (NAFLD) is increasing worldwide, and new treatments are sorely needed. Nicotinamide adenine dinucleotide (NAD+ ) has been proposed as a potential target to prevent and reverse NAFLD. NAD+ is an important redox factor for energy metabolism and is used as a substrate by a range of enzymes, including sirtuins (SIRT), which regulates histone acetylation, transcription factor activity and mitochondrial function. NAD+ is also a precursor for reduced nicotinamide adenine dinucleotide phosphate (NADPH), which is an important component of the antioxidant defense system. NAD+ precursors such as nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN) are available as over-the-counter dietary supplements, and oral supplementation with these precursors increases hepatic NAD+ levels and prevents hepatic lipid accumulation in pre-clinical models of NAFLD. NAD+ precursors have also been found to improve hepatic mitochondrial function and decrease oxidative stress in pre-clinical NAFLD models. NAD+ repletion also prevents NAFLD progression to non-alcoholic steatohepatitis (NASH), as NAD+ precursor supplementation is associated with decreased hepatic stellate cell activation, and decreased fibrosis. However, initial clinical trials have only shown modest effects when NAD+ precursors were administrated to people with obesity. We review the available pre-clinical investigations of NAD+ supplementation for targeting NAFLD, and discuss how data from the first clinical trials can be reconciled with observations from preclinical research.


Assuntos
Hepatopatia Gordurosa não Alcoólica , Animais , Suplementos Nutricionais , Humanos , Camundongos , Camundongos Endogâmicos C57BL , NAD/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo
2.
J Biol Chem ; 297(6): 101388, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34762911

RESUMO

Nicotinamide phosphoribosyltransferase (NAMPT) converts nicotinamide to NAD+. As low hepatic NAD+ levels have been linked to the development of nonalcoholic fatty liver disease, we hypothesized that ablation of hepatic Nampt would affect susceptibility to liver injury in response to diet-induced metabolic stress. Following 3 weeks on a low-methionine and choline-free 60% high-fat diet, hepatocyte-specific Nampt knockout (HNKO) mice accumulated less triglyceride than WT littermates but had increased histological scores for liver inflammation, necrosis, and fibrosis. Surprisingly, liver injury was also observed in HNKO mice on the purified control diet. This HNKO phenotype was associated with decreased abundance of mitochondrial proteins, especially proteins involved in oxidoreductase activity. High-resolution respirometry revealed lower respiratory capacity in purified control diet-fed HNKO liver. In addition, fibrotic area in HNKO liver sections correlated negatively with hepatic NAD+, and liver injury was prevented by supplementation with NAD+ precursors nicotinamide riboside and nicotinic acid. MS-based proteomic analysis revealed that nicotinamide riboside supplementation rescued hepatic levels of oxidoreductase and OXPHOS proteins. Finally, single-nucleus RNA-Seq showed that transcriptional changes in the HNKO liver mainly occurred in hepatocytes, and changes in the hepatocyte transcriptome were associated with liver necrosis. In conclusion, HNKO livers have reduced respiratory capacity, decreased abundance of mitochondrial proteins, and are susceptible to fibrosis because of low NAD+ levels. Our data suggest a critical threshold level of hepatic NAD+ that determines the predisposition to liver injury and supports that NAD+ precursor supplementation can prevent liver injury and nonalcoholic fatty liver disease progression.


Assuntos
Hepatócitos/metabolismo , Mitocôndrias Hepáticas/metabolismo , NAD/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Animais , Citocinas/deficiência , Citocinas/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias Hepáticas/genética , NAD/genética , Nicotinamida Fosforribosiltransferase/deficiência , Nicotinamida Fosforribosiltransferase/metabolismo , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologia , Fosforilação Oxidativa , Fenótipo
3.
Biochem J ; 478(15): 2977-2997, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34259310

RESUMO

SBI-0206965, originally identified as an inhibitor of the autophagy initiator kinase ULK1, has recently been reported as a more potent and selective AMP-activated protein kinase (AMPK) inhibitor relative to the widely used, but promiscuous inhibitor Compound C/Dorsomorphin. Here, we studied the effects of SBI-0206965 on AMPK signalling and metabolic readouts in multiple cell types, including hepatocytes, skeletal muscle cells and adipocytes. We observed SBI-0206965 dose dependently attenuated AMPK activator (991)-stimulated ACC phosphorylation and inhibition of lipogenesis in hepatocytes. SBI-0206965 (≥25 µM) modestly inhibited AMPK signalling in C2C12 myotubes, but also inhibited insulin signalling, insulin-mediated/AMPK-independent glucose uptake, and AICA-riboside uptake. We performed an extended screen of SBI-0206965 against a panel of 140 human protein kinases in vitro, which showed SBI-0206965 inhibits several kinases, including members of AMPK-related kinases (NUAK1, MARK3/4), equally or more potently than AMPK or ULK1. This screen, together with molecular modelling, revealed that most SBI-0206965-sensitive kinases contain a large gatekeeper residue with a preference for methionine at this position. We observed that mutation of the gatekeeper methionine to a smaller side chain amino acid (threonine) rendered AMPK and ULK1 resistant to SBI-0206965 inhibition. These results demonstrate that although SBI-0206965 has utility for delineating AMPK or ULK1 signalling and cellular functions, the compound potently inhibits several other kinases and critical cellular functions such as glucose and nucleoside uptake. Our study demonstrates a role for the gatekeeper residue as a determinant of the inhibitor sensitivity and inhibitor-resistant mutant forms could be exploited as potential controls to probe specific cellular effects of SBI-0206965.


Assuntos
Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/antagonistas & inibidores , Benzamidas/farmacologia , Pirimidinas/farmacologia , Proteínas Recombinantes/metabolismo , Células 3T3-L1 , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Benzamidas/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Mutação de Sentido Incorreto , Ligação Proteica/efeitos dos fármacos , Multimerização Proteica , Pirimidinas/metabolismo , Ratos Sprague-Dawley , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
4.
FASEB J ; 35(5): e21450, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33788980

RESUMO

Agouti-related protein (AgRP) neurons in the arcuate nucleus of the hypothalamus regulates food intake and whole-body metabolism. NAD+ regulates multiple cellular processes controlling energy metabolism. Yet, its role in hypothalamic AgRP neurons to control food intake is poorly understood. Here, we aimed to assess whether genetic deletion of nicotinamide phosphoribosyltransferase (Nampt), a rate-limiting enzyme in NAD+ production, affects AgRP neuronal function to impact whole-body metabolism and food intake. Metabolic parameters during fed and fasted states, and upon systemic ghrelin and leptin administration were studied in AgRP-specific Nampt knockout (ARNKO) mice. We monitored neuropeptide expression levels and density of AgRP neurons in ARNKO mice from embryonic to adult age. NPY cells were used to determine effects of NAMPT inhibition on neuronal viability, energy status, and oxidative stress in vitro. In these cells, NAD+ depletion reduced ATP levels, increased oxidative stress, and promoted cell death. Agrp expression in the hypothalamus of ARNKO mice gradually decreased after weaning due to progressive AgRP neuron degeneration. Adult ARNKO mice had normal glucose and insulin tolerance, but exhibited an elevated respiratory exchange ratio (RER) when fasted. Remarkably, fasting-induced food intake was unaffected in ARNKO mice when evaluated in metabolic cages, but fasting- and ghrelin-induced feeding and body weight gain decreased in ARNKO mice when evaluated outside metabolic cages. Collectively, deletion of Nampt in AgRP neurons causes progressive neurodegeneration and impairs fasting and ghrelin responses in a context-dependent manner. Our data highlight an essential role of Nampt in AgRP neuron function and viability.


Assuntos
Proteína Relacionada com Agouti/metabolismo , Citocinas/fisiologia , Ingestão de Alimentos , Jejum , Grelina/farmacologia , Doenças Neurodegenerativas/patologia , Neurônios/metabolismo , Nicotinamida Fosforribosiltransferase/fisiologia , Proteína Relacionada com Agouti/genética , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Doenças Neurodegenerativas/etiologia , Doenças Neurodegenerativas/metabolismo
5.
Mol Metab ; 42: 101080, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32937194

RESUMO

OBJECTIVE: Glucagon is well known to regulate blood glucose but may be equally important for amino acid metabolism. Plasma levels of amino acids are regulated by glucagon-dependent mechanism(s), while amino acids stimulate glucagon secretion from alpha cells, completing the recently described liver-alpha cell axis. The mechanisms underlying the cycle and the possible impact of hepatic steatosis are unclear. METHODS: We assessed amino acid clearance in vivo in mice treated with a glucagon receptor antagonist (GRA), transgenic mice with 95% reduction in alpha cells, and mice with hepatic steatosis. In addition, we evaluated urea formation in primary hepatocytes from ob/ob mice and humans, and we studied acute metabolic effects of glucagon in perfused rat livers. We also performed RNA sequencing on livers from glucagon receptor knock-out mice and mice with hepatic steatosis. Finally, we measured individual plasma amino acids and glucagon in healthy controls and in two independent cohorts of patients with biopsy-verified non-alcoholic fatty liver disease (NAFLD). RESULTS: Amino acid clearance was reduced in mice treated with GRA and mice lacking endogenous glucagon (loss of alpha cells) concomitantly with reduced production of urea. Glucagon administration markedly changed the secretion of rat liver metabolites and within minutes increased urea formation in mice, in perfused rat liver, and in primary human hepatocytes. Transcriptomic analyses revealed that three genes responsible for amino acid catabolism (Cps1, Slc7a2, and Slc38a2) were downregulated both in mice with hepatic steatosis and in mice with deletion of the glucagon receptor. Cultured ob/ob hepatocytes produced less urea upon stimulation with mixed amino acids, and amino acid clearance was lower in mice with hepatic steatosis. Glucagon-induced ureagenesis was impaired in perfused rat livers with hepatic steatosis. Patients with NAFLD had hyperglucagonemia and increased levels of glucagonotropic amino acids, including alanine in particular. Both glucagon and alanine levels were reduced after diet-induced reduction in Homeostatic Model Assessment for Insulin Resistance (HOMA-IR, a marker of hepatic steatosis). CONCLUSIONS: Glucagon regulates amino acid metabolism both non-transcriptionally and transcriptionally. Hepatic steatosis may impair glucagon-dependent enhancement of amino acid catabolism.


Assuntos
Aminoácidos/metabolismo , Fígado Gorduroso/fisiopatologia , Glucagon/metabolismo , Adulto , Animais , Glicemia/metabolismo , Fígado Gorduroso/metabolismo , Feminino , Glucagon/fisiologia , Células Secretoras de Glucagon/metabolismo , Glucose/metabolismo , Hepatócitos/metabolismo , Humanos , Insulina/metabolismo , Resistência à Insulina/fisiologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/metabolismo , Ratos , Ratos Wistar , Receptores de Glucagon/antagonistas & inibidores , Receptores de Glucagon/metabolismo , Ureia/metabolismo
6.
Acta Physiol (Oxf) ; 228(4): e13437, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31900990

RESUMO

AIM: Neurons in the arcuate nucleus of the hypothalamus are involved in regulation of food intake and energy expenditure, and dysregulation of signalling in these neurons promotes development of obesity. The role of the rate-limiting enzyme in the NAD+ salvage pathway, nicotinamide phosphoribosyltransferase (NAMPT), for regulation energy homeostasis by the hypothalamus has not been extensively studied. METHODS: We determined whether Nampt mRNA or protein levels in the hypothalamus of mice were affected by diet-induced obesity, by fasting and re-feeding, and by leptin and ghrelin treatment. Primary hypothalamic neurons were treated with FK866, a selective inhibitor of NAMPT, or rAAV carrying shRNA directed against Nampt, and levels of reactive oxygen species (ROS) and mitochondrial respiration were assessed. Fasting and ghrelin-induced food intake was measured in mice in metabolic cages after intracerebroventricular (ICV)-mediated FK866 administration. RESULTS: NAMPT levels in the hypothalamus were elevated by administration of ghrelin and leptin. In diet-induced obese mice, both protein and mRNA levels of NAMPT decreased in the hypothalamus. NAMPT inhibition in primary hypothalamic neurons significantly reduced levels of NAD+ , increased levels of ROS, and affected the expression of Agrp, Pomc and genes related to mitochondrial function. Finally, ICV-induced NAMPT inhibition by FK866 did not cause malaise or anhedonia, but completely ablated fasting- and ghrelin-induced increases in food intake. CONCLUSION: Our findings indicate that regulation of NAMPT levels in hypothalamic neurons is important for the control of fasting- and ghrelin-induced food intake.


Assuntos
Jejum/metabolismo , Grelina/metabolismo , Hipotálamo/metabolismo , Nicotinamida Fosforribosiltransferase/metabolismo , Acrilamidas/administração & dosagem , Proteína Relacionada com Agouti/genética , Proteína Relacionada com Agouti/metabolismo , Animais , Linhagem Celular , Ingestão de Alimentos , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Piperidinas/administração & dosagem , Pró-Opiomelanocortina/genética , Pró-Opiomelanocortina/metabolismo
7.
J Biol Chem ; 294(36): 13304-13326, 2019 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-31320478

RESUMO

Supplementation with NAD precursors such as nicotinamide riboside (NR) has been shown to enhance mitochondrial function in the liver and to prevent hepatic lipid accumulation in high-fat diet (HFD)-fed rodents. Hepatocyte-specific knockout of the NAD+-synthesizing enzyme nicotinamide phosphoribosyltransferase (NAMPT) reduces liver NAD+ levels, but the metabolic phenotype of Nampt-deficient hepatocytes in mice is unknown. Here, we assessed Nampt's role in maintaining mitochondrial and metabolic functions in the mouse liver. Using the Cre-LoxP system, we generated hepatocyte-specific Nampt knockout (HNKO) mice, having a 50% reduction of liver NAD+ levels. We screened the HNKO mice for signs of metabolic dysfunction following 60% HFD feeding for 20 weeks ± NR supplementation and found that NR increases hepatic NAD+ levels without affecting fat mass or glucose tolerance in HNKO or WT animals. High-resolution respirometry revealed that NR supplementation of the HNKO mice did not increase state III respiration, which was observed in WT mice following NR supplementation. Mitochondrial oxygen consumption and fatty-acid oxidation were unaltered in primary HNKO hepatocytes. Mitochondria isolated from whole-HNKO livers had only a 20% reduction in NAD+, suggesting that the mitochondrial NAD+ pool is less affected by HNKO than the whole-tissue pool. When stimulated with tryptophan in the presence of [15N]glutamine, HNKO hepatocytes had a higher [15N]NAD+ enrichment than WT hepatocytes, indicating that HNKO mice compensate through de novo NAD+ synthesis. We conclude that NAMPT-deficient hepatocytes can maintain substantial NAD+ levels and that the Nampt knockout has only minor consequences for mitochondrial function in the mouse liver.


Assuntos
Hepatócitos/metabolismo , Mitocôndrias/metabolismo , NAD/metabolismo , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Tumorais Cultivadas
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