RESUMO
BACKGROUND & AIMS: Molecular mechanisms underlying the different susceptibility of men and women to non-alcoholic fatty liver disease (NAFLD) are poorly understood. The TTC39B locus encodes a scaffolding protein, associates with gynecological disorders and its deletion protects mice from diet-induced steatohepatitis. This study aimed to elucidate the molecular mechanisms linking TTC39B (T39) to the expression of lipogenic genes and to explore sex-specific effects. METHODS: Co-expression in HEK293A cells validated the novel T39/pRb interaction predicted by a protein-protein interaction algorithm. T39 was knocked down using an antisense oligonucleotide (ASO) in mice with dietary NAFLD and a genetic deficiency of pRb or its downstream effector E2F1, as well as in primary human hepatocytes. RESULTS: T39 interacts with pRb via its C-terminal TPR domain and promotes its proteasomal degradation. In female mice, T39 deficiency reduces the mRNA of lipogenic genes, especially Pnpla3, in a pRb- and E2F1-dependent manner. In contrast, in male mice, T39 deficiency results in a much smaller reduction in lipogenic gene expression that is independent of pRb/E2F1. T39 also interacts with VAPB via an N-terminal FFAT motif and stabilizes the interaction of VAPB with SCAP. Ovariectomy abolishes the effect of T39 knockdown on the hepatic pRb/E2F1/Pnpla3 axis. In both sexes T39 knockdown reduces SCAP independently of pRb. In primary human hepatocytes, T39 knockdown reduces expression of PNPLA3 and other lipogenic genes in women but not men. CONCLUSIONS: We have uncovered a conserved sexual dimorphism in the regulation of hepatic lipogenic genes, with effects of T39 mediated through pRb/E2F1 in females and VAPB/SCAP in both sexes. T39 inhibition could be a novel strategy to downregulate PNPLA3 and treat NAFLD in women. LAY SUMMARY: In females, the protein TTC39B degrades a tumor suppressor in the liver to promote the synthesis of new fat and the expression of a major genetic risk factor for non-alcoholic fatty liver disease. TTC39B is a potential therapeutic target for non-alcoholic fatty liver disease, especially in women.
Assuntos
Lipoproteínas HDL/efeitos adversos , Proteínas de Neoplasias/efeitos adversos , Proteína do Retinoblastoma/efeitos dos fármacos , Fatores Sexuais , Animais , Modelos Animais de Doenças , Expressão Gênica/genética , Expressão Gênica/fisiologia , Lipogênese/efeitos dos fármacos , Lipogênese/genética , Camundongos , Camundongos Endogâmicos C57BL/metabolismoRESUMO
OBJECTIVE: In mouse models, deficiency of TTC39B (T39) decreases hepatic lipogenic gene expression and protects against diet-induced steatohepatitis. While assessing the therapeutic potential of antisense oligonucleotides (ASOs) targeting T39, we discovered an unexpected weight loss phenotype. The objective of this study was to determine the mechanism of the resistance to diet-induced obesity. METHODS: To assess therapeutic potential, we used antisense oligonucleotides (ASO) to knock down T39 expression in a Western or high-fat, high-cholesterol, high-sucrose-diet-fed Ldlr-/- or wild-type mice. RESULTS: T39 ASO treatment led to decreased hepatic lipogenic gene expression and decreased hepatic triglycerides. Unexpectedly, T39 ASO treatment protected against diet-induced obesity. The reduced weight gain was seen with two different ASOs that decreased T39 mRNA in adipose tissue macrophages (ATMs), but not with a liver-targeted GalNac-ASO. Mice treated with the T39 ASO displayed increased browning of gonadal white adipose tissue (gWAT) and evidence of increased lipolysis. However, T39 knockout mice displayed a similar weight loss response when treated with T39 ASO, indicating an off-target effect. RNA-seq analysis of gWAT showed a widespread increase in type I interferon (IFN)-responsive genes, and knockout of the IFN receptor abolished the weight loss phenotype induced by the T39 ASO. Some human T39 ASOs and ASOs with different modifications targeting LDLR also induced a type I IFN response in THP1 macrophages. CONCLUSION: Our data suggest that extrahepatic targeting of T39 by ASOs in ATMs produced an off-target type 1 IFN response, leading to activation of lipolysis, browning of WAT, and weight loss. While our findings suggest that ASOs may induce off-target type 1 IFN response more commonly than previously thought, they also suggest that therapeutic induction of type 1 IFN selectively in ATMs could potentially represent a novel approach to the treatment of obesity.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Interferon Tipo I/biossíntese , Obesidade/induzido quimicamente , Obesidade/tratamento farmacológico , Oligonucleotídeos Antissenso/farmacologia , Animais , Feminino , Injeções Subcutâneas , Interferon Tipo I/deficiência , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/metabolismo , Obesidade/prevenção & controle , Oligonucleotídeos Antissenso/administração & dosagemRESUMO
Objective- To assess the role of HDL (high-density lipoprotein)-mediated cholesterol mass efflux capacity (CMEC) in incident cardiovascular disease and carotid plaque progression. Approach and Results- We measured CMEC in 2 cohorts aged 45 to 84 years at baseline derived from the MESA (Multi-Ethnic Study of Atherosclerosis). Cohort 1 comprised 465 cases with incident cardiovascular disease events during 10 years of follow-up and 465 age- and sex-matched controls; cohort 2 comprised 407 cases with progression of carotid plaque measured by ultrasonography at 2 exams >10 years and 407 similarly matched controls. Covariates and outcome events were ascertained according to the MESA protocol. CMEC level was modestly correlated with HDL cholesterol ( R=0.13; P<0.001) but was not associated with age, sex, race/ethnicity, body mass index, diabetes mellitus, alcohol use, smoking status, or statin use. Higher CMEC level was significantly associated with lower odds of cardiovascular disease (odds ratio, 0.82 per SD of CMEC [95% CI, 0.69-0.98; P=0.031] in the fully adjusted model) in cohort 1 but higher odds of carotid plaque progression (odds ratio, 1.24 per SD of CMEC [95% CI, 1.04-1.48; P=0.018] in the fully adjusted model) in cohort 2 but without dose-response effect. In subgroup analysis within cohort 1, higher CMEC was associated with lower risk of incident coronary heart disease events (odds ratio, 0.72 per SD of CMEC (95% CI, 0.5-0.91; P=0.007) while no association was found with stroke events. Conclusions- These findings support a role for HDL-mediated cholesterol efflux in an atheroprotective mechanism for coronary heart disease but not stroke.
Assuntos
Doenças Cardiovasculares/metabolismo , Doenças das Artérias Carótidas/etiologia , HDL-Colesterol/fisiologia , Colesterol/metabolismo , Placa Aterosclerótica/etiologia , Idoso , Idoso de 80 Anos ou mais , Doença das Coronárias/complicações , Doença das Coronárias/metabolismo , Progressão da Doença , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
OBJECTIVE: The mechanisms underlying the cardiovascular benefit of the anti-diabetic drug metformin are poorly understood. Recent studies have suggested metformin may upregulate macrophage reverse cholesterol transport. The final steps of reverse cholesterol transport are mediated by the sterol transporters, ABCG5 (ATP-binding cassette transporter G5) and ABCG8 (ATP-binding cassette transporter G8), which facilitate hepato-biliary transport of cholesterol. This study was undertaken to assess the possibility that metformin induces Abcg5 and Abcg8 expression in liver and to elucidate the underlying mechanisms. APPROACH AND RESULTS: Metformin-treated mouse or human primary hepatocytes showed increased expression of Abcg5/8 and the bile salt export pump, Bsep. Administration of metformin to Western-type diet-fed mice showed significant upregulation of Abcg5/8 and Bsep. This resulted in increased initial clearance of 3H-cholesteryl ester HDL (high-density lipoprotein) from plasma. However, fecal 3H-cholesterol output was only marginally increased, possibly reflecting increased hepatic Ldlr (low-density lipoprotein receptor) expression, which would increase nonradiolabeled cholesterol uptake. Abcg5/8 undergo strong circadian variation. Available chromatin immunoprecipitation-Seq data suggested multiple binding sites for Period 2, a transcriptional repressor, within the Abcg5/8 locus. Addition of AMPK (5' adenosine monophosphate-activated protein kinase) agonists decreased Period 2 occupancy, suggesting derepression of Abcg5/8. Inhibition of ATP citrate lyase, which generates acetyl-CoA from citrate, also decreased Period 2 occupancy, with concomitant upregulation of Abcg5/8. This suggests a mechanistic link between feeding-induced acetyl-CoA production and decreased cholesterol excretion via Period 2, resulting in inhibition of Abcg5/8 expression. CONCLUSIONS: Our findings provide partial support for the concept that metformin may provide cardiovascular benefit via increased reverse cholesterol transport but also indicate increased Ldlr expression as a potential additional mechanism. AMPK activation or ATP citrate lyase inhibition may mediate antiatherogenic effects through increased ABCG5/8 expression.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Membro 5 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Membro 8 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Colesterol/sangue , Hepatócitos/efeitos dos fármacos , Lipoproteínas/metabolismo , Metformina/farmacologia , Proteínas Quinases Ativadas por AMP/deficiência , Proteínas Quinases Ativadas por AMP/genética , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/metabolismo , Membro 5 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Membro 8 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , ATP Citrato (pro-S)-Liase/metabolismo , Animais , Ativação Enzimática , Células HEK293 , Hepatócitos/enzimologia , Humanos , Lipoproteínas/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Circadianas Period/metabolismo , Cultura Primária de Células , Receptores de LDL/metabolismo , Regulação para CimaRESUMO
Cellular mechanisms that mediate steatohepatitis, an increasingly prevalent condition in the Western world for which no therapies are available, are poorly understood. Despite the fact that its synthetic agonists induce fatty liver, the liver X receptor (LXR) transcription factor remains a target of interest because of its anti-atherogenic, cholesterol removal, and anti-inflammatory activities. Here we show that tetratricopeptide repeat domain protein 39B (Ttc39b, C9orf52) (T39), a high-density lipoprotein gene discovered in human genome-wide association studies, promotes the ubiquitination and degradation of LXR. Chow-fed mice lacking T39 (T39(-/-)) display increased high-density lipoprotein cholesterol levels associated with increased enterocyte ATP-binding cassette transporter A1 (Abca1) expression and increased LXR protein without change in LXR messenger RNA. When challenged with a high fat/high cholesterol/bile salt diet, T39(-/-) mice or mice with hepatocyte-specific T39 deficiency show increased hepatic LXR protein and target gene expression, and unexpectedly protection from steatohepatitis and death. Mice fed a Western-type diet and lacking low-density lipoprotein receptor (Ldlr(-/-)T39(-/-)) show decreased fatty liver, increased high-density lipoprotein, decreased low-density lipoprotein, and reduced atherosclerosis. In addition to increasing hepatic Abcg5/8 expression and limiting dietary cholesterol absorption, T39 deficiency inhibits hepatic sterol regulatory element-binding protein 1 (SREBP-1, ADD1) processing. This is explained by an increase in microsomal phospholipids containing polyunsaturated fatty acids, linked to an LXRα-dependent increase in expression of enzymes mediating phosphatidylcholine biosynthesis and incorporation of polyunsaturated fatty acids into phospholipids. The preservation of endogenous LXR protein activates a beneficial profile of gene expression that promotes cholesterol removal and inhibits lipogenesis. T39 inhibition could be an effective strategy for reducing both steatohepatitis and atherosclerosis.
Assuntos
Aterosclerose/genética , Fígado Gorduroso/genética , Lipoproteínas HDL/deficiência , Lipoproteínas HDL/genética , Receptores Nucleares Órfãos/metabolismo , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Membro 5 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Membro 8 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Aterosclerose/prevenção & controle , Aterosclerose/terapia , Ácidos e Sais Biliares/metabolismo , Colesterol na Dieta/metabolismo , HDL-Colesterol/metabolismo , Dieta Hiperlipídica , Ácidos Graxos Insaturados/metabolismo , Fígado Gorduroso/prevenção & controle , Fígado Gorduroso/terapia , Feminino , Regulação da Expressão Gênica , Hepatócitos/metabolismo , Ligantes , Lipogênese/genética , Lipoproteínas/metabolismo , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/metabolismo , Receptores X do Fígado , Masculino , Camundongos , Receptores Nucleares Órfãos/genética , Fosfatidilcolinas/biossíntese , Fosfatidilcolinas/metabolismo , Estabilidade Proteica , Proteólise , Receptores de LDL/deficiência , Receptores de LDL/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , UbiquitinaçãoRESUMO
RATIONALE: Human genome-wide association studies have revealed novel genetic loci that are associated with coronary heart disease. One such locus resides in LNK/SH2B3, which in mice is expressed in hematopoietic cells and suppresses thrombopoietin signaling via its receptor myeloproliferative leukemia virus oncogene. However, the mechanisms underlying the association of LNK single-nucleotide polymorphisms with coronary heart disease are poorly understood. OBJECTIVE: To understand the functional effects of LNK single-nucleotide polymorphisms and explore the mechanisms whereby LNK loss of function impacts atherosclerosis and thrombosis. METHODS AND RESULTS: Using human cord blood, we show that the common TT risk genotype (R262W) of LNK is associated with expansion of hematopoietic stem cells and enhanced megakaryopoiesis, demonstrating reduced LNK function and increased myeloproliferative leukemia virus oncogene signaling. In mice, hematopoietic Lnk deficiency leads to accelerated arterial thrombosis and atherosclerosis, but only in the setting of hypercholesterolemia. Hypercholesterolemia acts synergistically with LNK deficiency to increase interleukin 3/granulocyte-macrophage colony-stimulating factor receptor signaling in bone marrow myeloid progenitors, whereas in platelets cholesterol loading combines with Lnk deficiency to increase activation. Platelet LNK deficiency increases myeloproliferative leukemia virus oncogene signaling and AKT activation, whereas cholesterol loading decreases SHIP-1 phosphorylation, acting convergently to increase AKT and platelet activation. Together with increased myelopoiesis, platelet activation promotes prothrombotic and proatherogenic platelet/leukocyte aggregate formation. CONCLUSIONS: LNK (R262W) is a loss-of-function variant that promotes thrombopoietin/myeloproliferative leukemia virus oncogene signaling and platelet and leukocyte production. In mice, LNK deficiency is associated with both increased platelet production and activation. Hypercholesterolemia acts in platelets and hematopoietic progenitors to exacerbate thrombosis and atherosclerosis associated with LNK deficiency.
Assuntos
Aterosclerose/genética , Polimorfismo de Nucleotídeo Único/genética , Proteínas/genética , Trombose/genética , Proteínas Adaptadoras de Transdução de Sinal , Animais , Aterosclerose/metabolismo , Aterosclerose/patologia , Feminino , Sangue Fetal/metabolismo , Estudo de Associação Genômica Ampla/métodos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ativação Plaquetária/fisiologia , Proteínas/metabolismo , Trombose/metabolismo , Trombose/patologiaRESUMO
Protein kinase A (PKA) regulates osteoblast cell function in vitro and is activated by important bone mass modulating agents. We determined whether PKA activation in osteoblasts is sufficient to mediate a bone anabolic response. Thus, a mouse model conditionally expressing a constitutively active PKA (CA-PKA) in osteoblasts (CA-PKA-OB mouse) was developed by crossing a 2.3-kb α1 (I)-collagen promoter-Cre mouse with a floxed-CA-PKA mouse. Primary osteoblasts from the CA-PKA-OB mice exhibited higher basal PKA activity than those from control mice. Microcomputed tomographic analysis revealed that CA-PKA-OB female mice had an 8.6-fold increase in femoral but only 1.16-fold increase in lumbar 5 vertebral bone volume/total volume. Femur cortical thickness and volume were also higher in the CA-PKA-OB mice. In contrast, alterations in many femoral microcomputed tomographic parameters in male CA-PKA-OB mice were modest. Interestingly, the 3-dimensional structure model index was substantially lower both in femur and lumbar 5 of male and female CA-PKA-OB mice, reflecting an increase in the plate to rod-like structure ratio. In agreement, femurs from female CA-PKA-OB mice had greater load to failure and were stiffer compared with those of control mice. Furthermore, the CA-PKA-OB mice had higher levels of serum bone turnover markers and increased osteoblast and osteoclast numbers per total tissue area compared with control animals. In summary, constitutive activation of PKA in osteoblasts is sufficient to increase bone mass and favorably modify bone architecture and improve mechanical properties. PKA activation in mature osteoblasts is, therefore, an important target for designing anabolic drugs for treating diseases with bone loss.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Fêmur/citologia , Vértebras Lombares/citologia , Osteócitos/enzimologia , Osteogênese , Mutação Puntual , Regulação para Cima , Proteínas Quinases Ativadas por AMP/genética , Animais , Densidade Óssea , Células Cultivadas , Metabolismo Energético , Ativação Enzimática , Feminino , Fêmur/diagnóstico por imagem , Fêmur/metabolismo , Vértebras Lombares/diagnóstico por imagem , Vértebras Lombares/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Osteoblastos/citologia , Osteoblastos/enzimologia , Osteoblastos/metabolismo , Osteócitos/citologia , Osteócitos/metabolismo , Regiões Promotoras Genéticas , Caracteres Sexuais , Microtomografia por Raio-XRESUMO
PACAP exerts neuroprotective effects during development, especially in the cerebellum where PAC1 receptor and ligand are both expressed. However, while previous studies using PACAP injections in postnatal animals defined trophic effects of exogenous peptide, the role of endogenous PACAP remains unexplored. Here, we used PAC1(-/-) mice to investigate the role of PACAP receptor signaling in postnatal day 7 cerebellum. There was no difference in DNA synthesis in the cerebellar EGL of PAC1(-/-) compared to wild type animals, assessed using thymidine incorporation and BrdU immunohistochemistry. In contrast, we found that a significant proportion of newly generated neurons were eliminated before they successfully differentiated in the granule cell layer. In aggregate, these results suggest that endogenous PACAP plays an important role in cell survival during cerebellar development, through the activation of the PAC1 receptor.