RESUMO
BACKGROUND: Large-scale human and mechanistic mouse studies indicate a strong relationship between the microbiome-dependent metabolite trimethylamine N-oxide (TMAO) and several cardiometabolic diseases. This study aims to investigate the role of TMAO in the pathogenesis of abdominal aortic aneurysm (AAA) and target its parent microbes as a potential pharmacological intervention. METHODS: TMAO and choline metabolites were examined in plasma samples, with associated clinical data, from 2 independent patient cohorts (N=2129 total). Mice were fed a high-choline diet and underwent 2 murine AAA models, angiotensin II infusion in low-density lipoprotein receptor-deficient (Ldlr-/-) mice or topical porcine pancreatic elastase in C57BL/6J mice. Gut microbial production of TMAO was inhibited through broad-spectrum antibiotics, targeted inhibition of the gut microbial choline TMA lyase (CutC/D) with fluoromethylcholine, or the use of mice genetically deficient in flavin monooxygenase 3 (Fmo3-/-). Finally, RNA sequencing of in vitro human vascular smooth muscle cells and in vivo mouse aortas was used to investigate how TMAO affects AAA. RESULTS: Elevated TMAO was associated with increased AAA incidence and growth in both patient cohorts studied. Dietary choline supplementation augmented plasma TMAO and aortic diameter in both mouse models of AAA, which was suppressed with poorly absorbed oral broad-spectrum antibiotics. Treatment with fluoromethylcholine ablated TMAO production, attenuated choline-augmented aneurysm initiation, and halted progression of an established aneurysm model. In addition, Fmo3-/- mice had reduced plasma TMAO and aortic diameters and were protected from AAA rupture compared with wild-type mice. RNA sequencing and functional analyses revealed choline supplementation in mice or TMAO treatment of human vascular smooth muscle cells-augmented gene pathways associated with the endoplasmic reticulum stress response, specifically the endoplasmic reticulum stress kinase PERK. CONCLUSIONS: These results define a role for gut microbiota-generated TMAO in AAA formation through upregulation of endoplasmic reticulum stress-related pathways in the aortic wall. In addition, inhibition of microbiome-derived TMAO may serve as a novel therapeutic approach for AAA treatment where none currently exist.
Assuntos
Aneurisma da Aorta Abdominal , Microbioma Gastrointestinal , Humanos , Camundongos , Animais , Suínos , Camundongos Endogâmicos C57BL , Colina , Aneurisma da Aorta Abdominal/induzido quimicamente , Aneurisma da Aorta Abdominal/genética , Aneurisma da Aorta Abdominal/prevenção & controleRESUMO
BACKGROUND: The risk of cardiovascular disease in type 1 diabetes remains extremely high, despite marked advances in blood glucose control and even the widespread use of cholesterol synthesis inhibitors. Thus, a deeper understanding of insulin regulation of cholesterol metabolism, and its disruption in type 1 diabetes, could reveal better treatment strategies. METHODS: To define the mechanisms by which insulin controls plasma cholesterol levels, we knocked down the insulin receptor, FoxO1, and the key bile acid synthesis enzyme, CYP8B1. We measured bile acid composition, cholesterol absorption, and plasma cholesterol. In parallel, we measured markers of cholesterol absorption and synthesis in humans with type 1 diabetes treated with ezetimibe and simvastatin in a double-blind crossover study. RESULTS: Mice with hepatic deletion of the insulin receptor showed marked increases in 12α-hydroxylated bile acids, cholesterol absorption, and plasma cholesterol. This phenotype was entirely reversed by hepatic deletion of FoxO1. FoxO1 is inhibited by insulin and required for the production of 12α-hydroxylated bile acids, which promote intestinal cholesterol absorption and suppress hepatic cholesterol synthesis. Knockdown of Cyp8b1 normalized 12α-hydroxylated bile acid levels and completely prevented hypercholesterolemia in mice with hepatic deletion of the insulin receptor (n=5-30), as well as mouse models of type 1 diabetes (n=5-22). In parallel, the cholesterol absorption inhibitor, ezetimibe, normalized cholesterol absorption and low-density lipoprotein cholesterol in patients with type 1 diabetes as well as, or better than, the cholesterol synthesis inhibitor, simvastatin (n=20). CONCLUSIONS: Insulin, by inhibiting FoxO1 in the liver, reduces 12α-hydroxylated bile acids, cholesterol absorption, and plasma cholesterol levels. Thus, type 1 diabetes leads to a unique set of derangements in cholesterol metabolism, with increased absorption rather than synthesis. These derangements are reversed by ezetimibe, but not statins, which are currently the first line of lipid-lowering treatment in type 1 diabetes. Taken together, these data suggest that a personalized approach to lipid lowering in type 1 diabetes may be more effective and highlight the need for further studies specifically in this group of patients.
Assuntos
Diabetes Mellitus Tipo 1 , Hipercolesterolemia , Hiperlipidemias , Animais , Ácidos e Sais Biliares/metabolismo , LDL-Colesterol , Estudos Cross-Over , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/prevenção & controle , Ezetimiba/farmacologia , Ezetimiba/uso terapêutico , Humanos , Hipercolesterolemia/tratamento farmacológico , Hipercolesterolemia/genética , Insulina , Fígado/metabolismo , Camundongos , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Sinvastatina/farmacologia , Sinvastatina/uso terapêutico , Esteroide 12-alfa-Hidroxilase/genética , Esteroide 12-alfa-Hidroxilase/metabolismoRESUMO
BACKGROUND: Changes in cholesterol absorption and cholesterol synthesis may promote dyslipidemia and cardiovascular disease in individuals with type 2 diabetes mellitus (T2DM). OBJECTIVE: To assess cholesterol synthesis and absorption in lean individuals, obese individuals, and individuals with T2DM. METHODS: We measured lathosterol and lanosterol (markers of cholesterol synthesis) as well as campesterol and ß-sitosterol (markers of cholesterol absorption) in the serum of 15 to 26 years old individuals with T2DM (n = 95), as well as their lean (n = 98) and obese (n = 92) controls. RESULTS: Individuals with T2DM showed a 51% increase in lathosterol and a 65% increase in lanosterol compared to lean controls. Similarly, obese individuals showed a 31% increase in lathosterol compared to lean controls. Lathosterol and lanosterol were positively correlated with body mass index, fasting insulin and glucose, serum triglycerides, and C-reactive protein, and negatively correlated with HDL-cholesterol. In contrast, campesterol and ß-sitosterol were not altered in individuals with T2DM. Moreover, campesterol and ß-sitosterol were negatively correlated with body mass index, fasting insulin, and C-reactive protein and were positively correlated with HDL-cholesterol. CONCLUSIONS: Adolescents and young adults with T2DM show evidence of increased cholesterol synthesis compared to non-diabetic lean controls. These findings suggest that T2DM may promote cardiovascular disease by increasing cholesterol synthesis, and provide additional rationale for the use of cholesterol synthesis inhibitors in this group.
Assuntos
Colesterol/metabolismo , Diabetes Mellitus Tipo 2/sangue , Adolescente , Adulto , Biomarcadores , Índice de Massa Corporal , Estudos de Casos e Controles , Colesterol/análogos & derivados , Colesterol/sangue , Diabetes Mellitus Tipo 2/complicações , Humanos , Obesidade/sangue , Obesidade/complicações , Fitosteróis/sangue , Sitosteroides/sangue , Adulto JovemRESUMO
Long-term dietary intake influences the structure and activity of the trillions of microorganisms residing in the human gut, but it remains unclear how rapidly and reproducibly the human gut microbiome responds to short-term macronutrient change. Here we show that the short-term consumption of diets composed entirely of animal or plant products alters microbial community structure and overwhelms inter-individual differences in microbial gene expression. The animal-based diet increased the abundance of bile-tolerant microorganisms (Alistipes, Bilophila and Bacteroides) and decreased the levels of Firmicutes that metabolize dietary plant polysaccharides (Roseburia, Eubacterium rectale and Ruminococcus bromii). Microbial activity mirrored differences between herbivorous and carnivorous mammals, reflecting trade-offs between carbohydrate and protein fermentation. Foodborne microbes from both diets transiently colonized the gut, including bacteria, fungi and even viruses. Finally, increases in the abundance and activity of Bilophila wadsworthia on the animal-based diet support a link between dietary fat, bile acids and the outgrowth of microorganisms capable of triggering inflammatory bowel disease. In concert, these results demonstrate that the gut microbiome can rapidly respond to altered diet, potentially facilitating the diversity of human dietary lifestyles.
Assuntos
Bactérias/genética , Bactérias/isolamento & purificação , Dieta , Trato Gastrointestinal/microbiologia , Metagenoma , Microbiota , Adulto , Bactérias/efeitos dos fármacos , Bacteroides/efeitos dos fármacos , Bacteroides/genética , Bacteroides/isolamento & purificação , Ácidos e Sais Biliares/análise , Ácidos e Sais Biliares/metabolismo , Bilophila/efeitos dos fármacos , Bilophila/genética , Bilophila/isolamento & purificação , Carnivoridade , Dieta/efeitos adversos , Dieta Vegetariana , Gorduras na Dieta/efeitos adversos , Gorduras na Dieta/farmacologia , Fezes/química , Fezes/microbiologia , Feminino , Fermentação/efeitos dos fármacos , Microbiologia de Alimentos , Trato Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/virologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Herbivoria , Humanos , Doenças Inflamatórias Intestinais/microbiologia , Masculino , Metagenoma/efeitos dos fármacos , Metagenoma/genética , Microbiota/efeitos dos fármacos , Microbiota/genética , Fatores de Tempo , Adulto JovemRESUMO
MicroRNAs (miRNAs) are important posttranscriptional regulators of metabolism and energy homeostasis. Dysregulation of certain miRNAs in the liver has been shown to contribute to the pathogenesis of Type 2 diabetes (T2D), in part by impairing hepatic insulin sensitivity. By small RNA-sequencing analysis, we identified seven hepatic miRNAs (including miR-29b) that are consistently aberrantly expressed across five different rodent models of metabolic dysfunction that share the feature of insulin resistance (IR). We also showed that hepatic miR-29b exhibits persistent dysregulation during disease progression in a rat model of diabetes, UCD-T2DM. Furthermore, we observed that hepatic levels of miR-29 family members are attenuated by interventions known to improve IR in rodent and rhesus macaque models. To examine the function of the miR-29 family in modulating insulin sensitivity, we used locked nucleic acid (LNA) technology and demonstrated that acute in vivo suppression of the miR-29 family in adult mice leads to significant reduction of fasting blood glucose (in both chow-fed lean and high-fat diet-fed obese mice) and improvement in insulin sensitivity (in chow-fed lean mice). We carried out whole transcriptome studies and uncovered candidate mechanisms, including regulation of DNA methyltransferase 3a (Dnmt3a) and the hormone-encoding gene Energy homeostasis associated (Enho). In sum, we showed that IR/T2D is linked to dysregulation of hepatic miR-29b across numerous models and that acute suppression of the miR-29 family in adult mice leads to improved glycemic control. Future studies should investigate the therapeutic utility of miR-29 suppression in different metabolic disease states.Enho; insulin resistance; liver; microRNA-29 (miR-29); UCD-T2DM.
Assuntos
Glicemia/análise , Diabetes Mellitus Tipo 2/genética , Resistência à Insulina/genética , Fígado/metabolismo , MicroRNAs/antagonistas & inibidores , Oligonucleotídeos/farmacologia , Animais , Sequência de Bases/genética , Glicemia/efeitos dos fármacos , DNA Metiltransferase 3A , Células HEK293 , Humanos , Macaca mulatta , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , MicroRNAs/genética , Oligonucleotídeos/administração & dosagem , Ratos , Ratos ZuckerRESUMO
Trimethylamine-N-oxide (TMAO), a microbial choline metabolism byproduct that is processed in the liver and excreted into circulation, is associated with increased atherosclerotic lesion formation and cardiovascular disease risk. Genetic regulators of TMAO levels are largely unknown. In the present study, we used 288 mice from a genetically heterogeneous mouse population [Diversity Outbred (DO)] to determine hepatic microRNA associations with TMAO in the context of an atherogenic diet. We also validated findings in two additional animal models of atherosclerosis: liver-specific insulin receptor knockout mice fed a chow diet (LIRKO) and African green monkeys fed high-fat/high-cholesterol diet. Small RNA-sequencing analysis in DO mice, LIRKO mice, and African green monkeys identified only one hepatic microRNA (miR-146a-5p) that is aberrantly expressed across all three models. Moreover, miR-146a-5p levels are associated with circulating TMAO after atherogenic diet in each of these models. We also performed high-resolution genetic mapping and identified a novel quantitative trait locus on Chromosome 12 for TMAO levels. This interval includes two genes, Numb and Dlst, which are inversely correlated with both miR-146a and TMAO and are predicted targets of miR-146a. Both of these genes have been validated as direct targets of miR-146a, though in other cellular contexts. This is the first report to our knowledge of a link between miR-146 and TMAO. Our findings suggest that miR-146-5p, as well as one or more genes at the Chromosome 12 QTL (possibly Numb or Dlst), is strongly linked to TMAO levels and likely involved in the control of atherosclerosis.
Assuntos
Aterosclerose/genética , Aterosclerose/metabolismo , Hipercolesterolemia/genética , Hipercolesterolemia/metabolismo , Metilaminas/metabolismo , MicroRNAs/genética , Animais , Chlorocebus aethiops , Colina/metabolismo , Estudos de Coortes , Camundongos de Cruzamento Colaborativo , Dieta Aterogênica , Dieta Hiperlipídica , Modelos Animais de Doenças , Feminino , Técnicas de Inativação de Genes , Fígado/metabolismo , Camundongos , Camundongos Knockout , MicroRNAs/metabolismo , NF-kappa B/metabolismo , RNA-Seq , Receptor de Insulina/genética , Fatores de RiscoRESUMO
Although calorically equivalent to glucose, fructose appears to be more lipogenic, promoting dyslipidemia, fatty liver disease, cardiovascular disease, and diabetes. To better understand how fructose induces lipogenesis, we compared the effects of fructose and glucose on mammalian target of rapamycin complex 1 (mTORC1), which appeared to have both positive and negative effects on lipogenic gene expression. We found that fructose acutely and transiently suppressed mTORC1 signaling in vitro and in vivo The constitutive activation of mTORC1 reduced hepatic lipogenic gene expression and produced hypotriglyceridemia after 1 week of fructose feeding. In contrast, glucose did not suppress mTORC1, and the constitutive activation of mTORC1 failed to suppress plasma triglycerides after 1 week of glucose feeding. Thus, these data reveal fundamental differences in the signaling pathways used by fructose and glucose to regulate lipid metabolism.
Assuntos
Frutose/metabolismo , Regulação da Expressão Gênica , Glucose/metabolismo , Lipogênese , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Animais , Fígado/metabolismo , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais , Triglicerídeos/metabolismoRESUMO
Treatment of nonalcoholic fatty liver disease (NAFLD) focuses on the underlying metabolic syndrome, and Roux-en-Y gastric bypass surgery (RYGB) remains one of the most effective options. In rodents and human patients, RYGB induces an increase in the gene and protein expression levels of the M2 isoenzyme of pyruvate kinase (PKM2) in the jejunum. Since PKM2 can be secreted in the circulation, our hypothesis was that the circulating levels of PKM2 increase after RYGB. Our data, however, revealed an unexpected finding and a potential new role of PKM2 for the natural history of metabolic syndrome and NAFLD. Contrary to our initial hypothesis, RYGB-treated patients had decreased PKM2 blood levels compared with a well-matched group of patients with severe obesity before RYGB. Interestingly, PKM2 serum concentration correlated with body mass index before but not after the surgery. This prompted us to evaluate other potential mechanisms and sites of PKM2 regulation by the metabolic syndrome and RYGB. We found that in patients with NAFLD and nonalcoholic steatohepatitis (NASH), the liver had increased PKM2 expression levels, and the enzyme appears to be specifically localized in Kupffer cells. The study of murine models of metabolic syndrome and NASH replicated this pattern of expression, further suggesting a metabolic link between hepatic PKM2 and NAFLD. Therefore, we conclude that PKM2 serum and hepatic levels increase in both metabolic syndrome and NAFLD and decrease after RYGB. Thus, PKM2 may represent a new target for monitoring and treatment of NAFLD.
Assuntos
Proteínas de Transporte/metabolismo , Derivação Gástrica , Jejuno/metabolismo , Proteínas de Membrana/metabolismo , Síndrome Metabólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Obesidade/metabolismo , Piruvato Quinase/metabolismo , Hormônios Tireóideos/metabolismo , Adulto , Animais , Modelos Animais de Doenças , Feminino , Humanos , Fígado/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Obesidade/cirurgia , Proteínas de Ligação a Hormônio da TireoideRESUMO
Cell growth and proliferation are tightly coupled to metabolism, and dissecting the signaling molecules which link these processes is an important step toward understanding development, regeneration, and cancer. The transcriptional regulator Yes-associated protein 1 (YAP) is a key regulator of liver size, development, and function. We now show that YAP can also suppress gluconeogenic gene expression. Yap deletion in primary hepatocytes potentiates the gluconeogenic gene response to glucagon and dexamethasone, whereas constitutively active YAP suppresses it. The effects of YAP are mediated by the transcriptional coactivator peroxisome proliferator-activated receptor-gamma coactivator 1. YAP inhibits its ability to bind to and activate transcription from the promoters of its gluconeogenic targets, and the effects of YAP are blunted upon its knockdown. In vivo, constitutively active YAP lowers plasma glucose levels and increases liver size. CONCLUSION: YAP appears to reprogram cellular metabolism, diverting substrates away from the energy-consuming process of gluconeogenesis and toward the anabolic process of growth. (Hepatology 2017;66:2029-2041).
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Regulação da Expressão Gênica , Gluconeogênese/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Fosfoproteínas/metabolismo , Animais , Carcinoma Hepatocelular/metabolismo , Proteínas de Ciclo Celular , Glucose-6-Fosfatase/metabolismo , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Cultura Primária de Células , Distribuição Aleatória , Fatores de Transcrição , Proteínas de Sinalização YAPRESUMO
Diabetes is characterized by increased lipogenesis as well as increased endoplasmic reticulum (ER) stress and inflammation. The nuclear hormone receptor liver X receptor (LXR) is induced by insulin and is a key regulator of lipid metabolism. It promotes lipogenesis and cholesterol efflux, but suppresses endoplasmic reticulum stress and inflammation. The goal of these studies was to dissect the effects of insulin on LXR action. We used antisense oligonucleotides to knock down Lxrα in mice with hepatocyte-specific deletion of the insulin receptor and their controls. We found, surprisingly, that knock-out of the insulin receptor and knockdown of Lxrα produced equivalent, non-additive effects on the lipogenic genes. Thus, insulin was unable to induce the lipogenic genes in the absence of Lxrα, and LXRα was unable to induce the lipogenic genes in the absence of insulin. However, insulin was not required for LXRα to modulate the phospholipid profile, or to suppress genes in the ER stress or inflammation pathways. These data show that insulin is required specifically for the lipogenic effects of LXRα and that manipulation of the insulin signaling pathway could dissociate the beneficial effects of LXR on cholesterol efflux, inflammation, and ER stress from the negative effects on lipogenesis.
Assuntos
Estresse do Retículo Endoplasmático , Regulação da Expressão Gênica , Hepatite/metabolismo , Insulina/metabolismo , Lipogênese , Fígado/metabolismo , Receptores Nucleares Órfãos/agonistas , Animais , Cruzamentos Genéticos , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/imunologia , Diabetes Mellitus Tipo 2/metabolismo , Feminino , Regulação Enzimológica da Expressão Gênica , Técnicas de Silenciamento de Genes , Hepatite/complicações , Hepatite/enzimologia , Hepatite/imunologia , Resistência à Insulina , Fígado/enzimologia , Fígado/imunologia , Receptores X do Fígado , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Receptores Nucleares Órfãos/antagonistas & inibidores , Receptores Nucleares Órfãos/genética , Receptores Nucleares Órfãos/metabolismo , Fosfolipídeos/metabolismo , Receptor de Insulina/agonistas , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: In nephrotic syndrome, damage to the podocytes of the kidney produces severe hypercholesterolemia for which novel treatments are urgently needed. PCSK9 (proprotein convertase subtilisin/kexin type 9) has emerged as an important regulator of plasma cholesterol levels and therapeutic target. Here, we tested the role of PCSK9 in mediating the hypercholesterolemia of nephrotic syndrome. METHODS: PCSK9 and plasma lipids were studied in nephrotic syndrome patients before and after remission of disease, mice with genetic ablation of the podocyte (Podocyte Apoptosis Through Targeted Activation of Caspase-8, Pod-ATTAC mice) and mice treated with nephrotoxic serum (NTS), which triggers immune-mediated podocyte damage. In addition, mice with hepatic deletion of Pcsk9 were treated with NTS to determine the contribution of PCSK9 to the dyslipidemia of nephrotic syndrome. RESULTS: Patients with nephrotic syndrome showed a decrease in plasma cholesterol and plasma PCSK9 on remission of their disease (P<0.05, n=47-50). Conversely, Pod-ATTAC mice and NTS-treated mice showed hypercholesterolemia and a 7- to 24-fold induction in plasma PCSK9. The induction of plasma PCSK9 appeared to be attributable to increased secretion of PCSK9 from the hepatocyte coupled with decreased clearance. Interestingly, knockout of Pcsk9ameliorated the effects of NTS on plasma lipids. Thus, in the presence of NTS, mice lacking hepatic Pcsk9 showed a 40% to 50% decrease in plasma cholesterol and triglycerides. Moreover, the ability of NTS treatment to increase the percentage of low-density lipoprotein-associated cholesterol (from 9% in vehicle-treated Flox mice to 47% after NTS treatment), was lost in mice with hepatic deletion of Pcsk9 (5% in both the presence and absence of NTS). CONCLUSIONS: Podocyte damage triggers marked inductions in plasma PCSK9, and knockout of Pcsk9 ameliorates dyslipidemia in a mouse model of nephrotic syndrome. These data suggest that PCSK9 inhibitors may be beneficial in patients with nephrotic syndrome-associated hypercholesterolemia.
Assuntos
Hipercolesterolemia/etiologia , Síndrome Nefrótica/complicações , Pró-Proteína Convertase 9/fisiologia , Animais , Humanos , Hipercolesterolemia/enzimologia , Lipídeos/sangue , Fígado/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Síndrome Nefrótica/sangue , Síndrome Nefrótica/enzimologia , Podócitos/patologia , Pró-Proteína Convertase 9/deficiência , Pró-Proteína Convertase 9/genética , Pró-Proteína Convertase 9/uso terapêutico , Proteínas Recombinantes/uso terapêuticoRESUMO
BACKGROUND: Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a key regulator of low-density lipoprotein cholesterol and cardiovascular disease risk, and is an emerging therapeutic target. OBJECTIVE: We compared serum PCSK9 levels in young adults, with and without type 2 diabetes. SUBJECTS AND METHODS: Cross-sectional analysis was conducted in a cohort, aged 15 to 26 years, in Cincinnati, OH, from 2005 to 2010. Serum PCSK9 levels were measured in 94 youth with type 2 diabetes, 93 obese control subjects, and 99 lean control subjects. Correlative analyses were conducted to determine significant covariates of PCSK9 by group and sex, and multivariate linear regression models were used to study the independent determinants of PCSK9. RESULTS: In females, PCSK9 levels were significantly increased in the obese and type 2 diabetes subjects relative to the lean controls (P < .01). Moreover, PCSK9 was positively correlated with multiple metabolic parameters in females: body mass index, systolic blood pressure, fasting glucose, fasting insulin, and C-reactive protein levels (P ≤ .02). In males, PCSK9 levels were decreased overall compared with females (P = .03), and did not differ between the lean, obese, or type 2 diabetes groups. CONCLUSIONS: Obesity and type 2 diabetes were associated with significantly higher levels of PCSK9 in young women, but not in young men. These data suggest that sex could modify the effects of obesity and diabetes on PCSK9 in young adults.
Assuntos
Diabetes Mellitus Tipo 2/sangue , Obesidade/sangue , Pró-Proteína Convertase 9/sangue , Estudos de Coortes , Estudos Transversais , Diabetes Mellitus Tipo 2/complicações , Feminino , Humanos , Masculino , Obesidade/complicações , Caracteres Sexuais , Adulto JovemRESUMO
OBJECTIVE: Proprotein convertase subtilisin/kexin type 9 (PCSK9), which binds the low-density lipoprotein receptor and targets it for degradation, has emerged as an important regulator of serum cholesterol levels and cardiovascular disease risk. Although much work is currently focused on developing therapies for inhibiting PCSK9, the endogenous regulation of PCSK9, particularly by insulin, remains unclear. The objective of these studies was to determine the effects of insulin on PCSK9 in vitro and in vivo. APPROACH AND RESULTS: Using rat hepatoma cells and primary rat hepatocytes, we found that insulin increased PCSK9 expression and increased low-density lipoprotein receptor degradation in a PCSK9-dependent manner. In parallel, hepatic Pcsk9 mRNA and plasma PCSK9 protein levels were reduced by 55% to 75% in mice with liver-specific knockout of the insulin receptor; 75% to 88% in mice made insulin-deficient with streptozotocin; and 65% in ob/ob mice treated with antisense oligonucleotides against the insulin receptor. However, antisense oligonucleotide-mediated knockdown of insulin receptor in lean, wild-type mice had little effect. In addition, we found that fasting was able to reduce PCSK9 expression by 80% even in mice that lack hepatic insulin signaling. CONCLUSIONS: Taken together, these data indicate that although insulin induces PCSK9 expression, it is not the sole or even dominant regulator of PCSK9 under all conditions.
Assuntos
Insulina/farmacologia , Insulina/fisiologia , Serina Endopeptidases/metabolismo , Animais , Carcinoma Hepatocelular , Linhagem Celular , Diabetes Mellitus Experimental/metabolismo , Meia-Vida , Hepatócitos/metabolismo , Camundongos Knockout , Camundongos Obesos , Pró-Proteína Convertase 9 , RNA Mensageiro/metabolismo , Ratos , Receptores de LDL/metabolismo , Serina Endopeptidases/efeitos dos fármacosRESUMO
Sirtuins are NAD(+)-dependent protein deacetylases. They mediate adaptive responses to a variety of stresses, including calorie restriction and metabolic stress. Sirtuin 3 (SIRT3) is localized in the mitochondrial matrix, where it regulates the acetylation levels of metabolic enzymes, including acetyl coenzyme A synthetase 2 (refs 1, 2). Mice lacking both Sirt3 alleles appear phenotypically normal under basal conditions, but show marked hyperacetylation of several mitochondrial proteins. Here we report that SIRT3 expression is upregulated during fasting in liver and brown adipose tissues. During fasting, livers from mice lacking SIRT3 had higher levels of fatty-acid oxidation intermediate products and triglycerides, associated with decreased levels of fatty-acid oxidation, compared to livers from wild-type mice. Mass spectrometry of mitochondrial proteins shows that long-chain acyl coenzyme A dehydrogenase (LCAD) is hyperacetylated at lysine 42 in the absence of SIRT3. LCAD is deacetylated in wild-type mice under fasted conditions and by SIRT3 in vitro and in vivo; and hyperacetylation of LCAD reduces its enzymatic activity. Mice lacking SIRT3 exhibit hallmarks of fatty-acid oxidation disorders during fasting, including reduced ATP levels and intolerance to cold exposure. These findings identify acetylation as a novel regulatory mechanism for mitochondrial fatty-acid oxidation and demonstrate that SIRT3 modulates mitochondrial intermediary metabolism and fatty-acid use during fasting.
Assuntos
Acil-CoA Desidrogenase de Cadeia Longa/metabolismo , Ácidos Graxos/metabolismo , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Sirtuína 3/metabolismo , Acetilação , Acil-CoA Desidrogenase de Cadeia Longa/química , Trifosfato de Adenosina/biossíntese , Trifosfato de Adenosina/metabolismo , Tecido Adiposo Marrom/enzimologia , Tecido Adiposo Marrom/metabolismo , Animais , Regulação da Temperatura Corporal , Restrição Calórica , Carnitina/análogos & derivados , Carnitina/metabolismo , Linhagem Celular , Temperatura Baixa , Jejum/metabolismo , Humanos , Hipoglicemia/metabolismo , Fígado/enzimologia , Fígado/metabolismo , Masculino , Espectrometria de Massas , Camundongos , Oxirredução , Sirtuína 3/deficiência , Sirtuína 3/genética , Triglicerídeos/metabolismo , Regulação para CimaRESUMO
The liver plays a central role in metabolism and mediating insulin action. To dissect the effects of insulin on the liver in vivo, we have studied liver insulin receptor knockout (LIRKO) mice. Because LIRKO livers lack insulin receptors, they are unable to respond to insulin. Surprisingly, the most profound derangement observed in LIRKO livers by microarray analysis is a suppression of the cholesterologenic genes. Sterol regulatory element binding protein (SREBP)-2 promotes cholesterologenic gene transcription, and is inhibited by intracellular cholesterol. LIRKO livers show a slight increase in hepatic cholesterol, a 40% decrease in Srebp-2, and a 50-90% decrease in the cholesterologenic genes at the mRNA and protein levels. In control mice, SREBP-2 and cholesterologenic gene expression are suppressed by fasting and restored by refeeding; in LIRKO mice, this response is abolished. Similarly, the ability of statins to induce Srebp-2 and the cholesterologenic genes is lost in LIRKO livers. In contrast, ezetimibe treatment robustly induces Srepb-2 and its targets in LIRKO livers, raising the possibility that insulin may regulate SREBP-2 indirectly, by altering the accumulation or distribution of cholesterol within the hepatocyte. Taken together, these data indicate that cholesterol synthesis is a key target of insulin action in the liver.
Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Fígado/metabolismo , Lovastatina/farmacologia , Receptor de Insulina/deficiência , Proteína de Ligação a Elemento Regulador de Esterol 2/fisiologia , Animais , Azetidinas/farmacologia , Vias Biossintéticas/genética , Colesterol/biossíntese , Ezetimiba , Jejum , Expressão Gênica/efeitos dos fármacos , Lipogênese/efeitos dos fármacos , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos , Receptor de Insulina/genética , Ativação Transcricional/efeitos dos fármacos , TranscriptomaRESUMO
AIMS/HYPOTHESIS: Insulin resistance, a major contributor to the pathogenesis of type 2 diabetes, leads to increased hepatic glucose production (HGP) owing to an impaired ability of insulin to suppress hepatic gluconeogenesis. Nuclear receptor oestrogen-related receptor γ (ERRγ) is a major transcriptional regulator of hepatic gluconeogenesis. In this study, we investigated insulin-dependent post-translational modifications (PTMs) altering the transcriptional activity of ERRγ for the regulation of hepatic gluconeogenesis. METHODS: We examined insulin-dependent phosphorylation and subcellular localisation of ERRγ in cultured cells and in the liver of C57/BL6, leptin receptor-deficient (db/db), liver-specific insulin receptor knockout (LIRKO) and protein kinase B (PKB) ß-deficient (Pkbß (-/-)) mice. To demonstrate the role of ERRγ in the inhibitory action of insulin on hepatic gluconeogenesis, we carried out an insulin tolerance test in C57/BL6 mice expressing wild-type or phosphorylation-deficient mutant ERRγ. RESULTS: We demonstrated that insulin suppressed the transcriptional activity of ERRγ by promoting PKB/Akt-mediated phosphorylation of ERRγ at S179 and by eliciting translocation of ERRγ from the nucleus to the cytoplasm through interaction with 14-3-3, impairing its ability to promote hepatic gluconeogenesis. In addition, db/db, LIRKO and Pkbß (-/-) mice displayed enhanced ERRγ transcriptional activity due to a block in PKBß-mediated ERRγ phosphorylation during refeeding. Finally, the phosphorylation-deficient mutant ERRγ S179A was resistant to the inhibitory action of insulin on HGP. CONCLUSIONS/INTERPRETATION: These results suggest that ERRγ is a major contributor to insulin action in maintaining hepatic glucose homeostasis.
Assuntos
Gluconeogênese/efeitos dos fármacos , Insulina/farmacologia , Fígado/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Estrogênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Diabetes Mellitus Tipo 2/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Gluconeogênese/fisiologia , Fígado/metabolismo , Camundongos , Camundongos Knockout , Fosforilação/efeitos dos fármacos , Receptor de Insulina/genética , Receptor de Insulina/metabolismoRESUMO
Insulin resistance plays a central role in the development of the metabolic syndrome, but how it relates to cardiovascular disease remains controversial. Liver insulin receptor knockout (LIRKO) mice have pure hepatic insulin resistance. On a standard chow diet, LIRKO mice have a proatherogenic lipoprotein profile with reduced high-density lipoprotein (HDL) cholesterol and very low-density lipoprotein (VLDL) particles that are markedly enriched in cholesterol. This is due to increased secretion and decreased clearance of apolipoprotein B-containing lipoproteins, coupled with decreased triglyceride secretion secondary to increased expression of Pgc-1 beta (Ppargc-1b), which promotes VLDL secretion, but decreased expression of Srebp-1c (Srebf1), Srebp-2 (Srebf2), and their targets, the lipogenic enzymes and the LDL receptor. Within 12 weeks on an atherogenic diet, LIRKO mice show marked hypercholesterolemia, and 100% of LIRKO mice, but 0% of controls, develop severe atherosclerosis. Thus, insulin resistance at the level of the liver is sufficient to produce the dyslipidemia and increased risk of atherosclerosis associated with the metabolic syndrome.
Assuntos
Aterosclerose/etiologia , Dislipidemias/etiologia , Resistência à Insulina , Animais , Suscetibilidade a Doenças , Hipercolesterolemia/etiologia , Lipoproteínas/sangue , Hepatopatias , Camundongos , Camundongos Knockout , Receptor de Insulina/deficiênciaRESUMO
OBJECTIVE: Hypertriglyceridemia and fatty liver are common in patients with type 2 diabetes, but the factors connecting alterations in glucose metabolism with plasma and liver lipid metabolism remain unclear. Apolipoprotein CIII (apoCIII), a regulator of hepatic and plasma triglyceride metabolism, is elevated in type 2 diabetes. In this study, we analyzed whether apoCIII is affected by altered glucose metabolism. METHODS AND RESULTS: Liver-specific insulin receptor-deficient mice display lower hepatic apoCIII mRNA levels than controls, suggesting that factors other than insulin regulate apoCIII in vivo. Glucose induces apoCIII transcription in primary rat hepatocytes and immortalized human hepatocytes via a mechanism involving the transcription factors carbohydrate response element-binding protein and hepatocyte nuclear factor-4α. ApoCIII induction by glucose is blunted by treatment with agonists of farnesoid X receptor and peroxisome proliferator-activated receptor-α but not liver X receptor, ie, nuclear receptors controlling triglyceride metabolism. Moreover, in obese humans, plasma apoCIII protein correlates more closely with plasma fasting glucose and glucose excursion after oral glucose load than with insulin. CONCLUSIONS: Glucose induces apoCIII transcription, which may represent a mechanism linking hyperglycemia, hypertriglyceridemia, and cardiovascular disease in type 2 diabetes.
Assuntos
Apolipoproteína C-III/genética , Complicações do Diabetes/etiologia , Diabetes Mellitus Tipo 2/complicações , Dislipidemias/etiologia , Glucose/metabolismo , Hepatócitos/metabolismo , Ativação Transcricional , Adulto , Animais , Apolipoproteína C-III/sangue , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Glicemia/metabolismo , Células Cultivadas , Complicações do Diabetes/genética , Complicações do Diabetes/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Dislipidemias/genética , Dislipidemias/metabolismo , Proteínas de Choque Térmico/agonistas , Proteínas de Choque Térmico/metabolismo , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Humanos , Insulina/sangue , Receptores X do Fígado , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Obesidade/sangue , Receptores Nucleares Órfãos/genética , Receptores Nucleares Órfãos/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Regiões Promotoras Genéticas , Interferência de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/agonistas , Proteínas de Ligação a RNA/metabolismo , Ratos , Receptor de Insulina/deficiência , Receptor de Insulina/genética , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Tempo , Fatores de Transcrição/agonistas , Fatores de Transcrição/metabolismo , Transfecção , Regulação para CimaRESUMO
We investigate the extent to which human genetic data are incorporated into studies that hypothesize novel links between genes and metabolic disease. To lower the barriers to using genetic data, we present an approach to enable researchers to evaluate human genetic support for experimentally determined hypotheses.
Assuntos
Doenças Metabólicas , Genética Humana , Humanos , Doenças Metabólicas/genéticaRESUMO
Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) remain without effective therapies. The mechanistic target of rapamycin complex 1 (mTORC1) pathway is a potential therapeutic target, but conflicting interpretations have been proposed for how mTORC1 controls lipid homeostasis. We show that selective inhibition of mTORC1 signaling in mice, through deletion of the RagC/D guanosine triphosphatase-activating protein folliculin (FLCN), promotes activation of transcription factor E3 (TFE3) in the liver without affecting other mTORC1 targets and protects against NAFLD and NASH. Disease protection is mediated by TFE3, which both induces lipid consumption and suppresses anabolic lipogenesis. TFE3 inhibits lipogenesis by suppressing proteolytic processing and activation of sterol regulatory element-binding protein-1c (SREBP-1c) and by interacting with SREBP-1c on chromatin. Our data reconcile previously conflicting studies and identify selective inhibition of mTORC1 as a potential approach to treat NASH and NAFLD.