RESUMO
BACKGROUND: VERVE-101 is an investigational in vivo CRISPR base-editing medicine designed to alter a single DNA base in the PCSK9 gene, permanently turn off hepatic protein production, and thereby durably lower low-density lipoprotein cholesterol. We test the efficacy, durability, tolerability, and potential for germline editing of VERVE-101 in studies of nonhuman primates and a murine F1 progeny study. METHODS: Cynomolgus monkeys were given a single intravenous infusion of a vehicle control (n=10) or VERVE-101 at a dose of 0.75 mg/kg (n=4) or 1.5 mg/kg (n=22) with subsequent follow-up up to 476 days. Two studies assessed the potential for germline editing, including sequencing sperm samples from sexually mature male nonhuman primates treated with VERVE-101 and genotyping offspring from female mice treated with the murine surrogate of VERVE-101 (VERVE-101mu). RESULTS: Liver biopsies 14 days after dosing noted mean PCSK9 editing of 46% and 70% in monkeys treated with VERVE-101 at 0.75 and 1.5 mg/kg, respectively. This translated into mean reductions in blood PCSK9 (proprotein convertase subtilisin/kexin type 9) of 67% and 83% and reductions of low-density lipoprotein cholesterol of 49% and 69% at the 0.75 and 1.5 mg/kg doses, respectively, assessed as time-weighted average change from baseline between day 28 and up to 476 days after dosing. Liver safety monitoring noted a transient rise in alanine aminotransferase and aspartate aminotransferase concentrations after infusion that fully resolved by day 14 with no accompanying change in total bilirubin. In a subset of monkeys necropsied 1 year after dosing, no findings related to VERVE-101 were identified on macroscopic and histopathologic assessment of the liver and other organs. In the study to assess potential germline editing of male nonhuman primates, sperm samples collected after VERVE-101 dosing showed no evidence of PCSK9 editing. Among 436 offspring of female mice treated with a saturating dose of VERVE-101mu, the PCSK9 edit was transmitted in 0 of 436 animals. CONCLUSIONS: VERVE-101 was well tolerated in nonhuman primates and led to 83% lower blood PCSK9 protein and 69% lower low-density lipoprotein cholesterol with durable effects up to 476 days after dosing. These results have supported the initiation of a first-in-human clinical trial in patients with heterozygous familial hypercholesterolemia and atherosclerotic cardiovascular disease.
Assuntos
Edição de Genes , Pró-Proteína Convertase 9 , Animais , Feminino , Humanos , Masculino , Camundongos , LDL-Colesterol/genética , LDL-Colesterol/metabolismo , Primatas/genética , Primatas/metabolismo , Pró-Proteína Convertase 9/genética , Pró-Proteína Convertase 9/uso terapêutico , Sêmen/metabolismo , Edição de Genes/métodos , Sistemas CRISPR-Cas , Terapia Genética/métodos , Hiperlipoproteinemia Tipo II/genética , Hiperlipoproteinemia Tipo II/terapia , Aterosclerose/genética , Aterosclerose/terapiaRESUMO
Mitochondrial diseases are a group of inherited diseases with highly varied and complex clinical presentations. Here, we report four individuals, including two siblings, affected by a progressive mitochondrial encephalopathy with biallelic variants in the cardiolipin biosynthesis gene CRLS1. Three affected individuals had a similar infantile presentation comprising progressive encephalopathy, bull's eye maculopathy, auditory neuropathy, diabetes insipidus, autonomic instability, cardiac defects and early death. The fourth affected individual presented with chronic encephalopathy with neurodevelopmental regression, congenital nystagmus with decreased vision, sensorineural hearing loss, failure to thrive and acquired microcephaly. Using patient-derived fibroblasts, we characterized cardiolipin synthase 1 (CRLS1) dysfunction that impaired mitochondrial morphology and biogenesis, providing functional evidence that the CRLS1 variants cause mitochondrial disease. Lipid profiling in fibroblasts from two patients further confirmed the functional defect demonstrating reduced cardiolipin levels, altered acyl-chain composition and significantly increased levels of phosphatidylglycerol, the substrate of CRLS1. Proteomic profiling of patient cells and mouse Crls1 knockout cell lines identified both endoplasmic reticular and mitochondrial stress responses, and key features that distinguish between varying degrees of cardiolipin insufficiency. These findings support that deleterious variants in CRLS1 cause an autosomal recessive mitochondrial disease, presenting as a severe encephalopathy with multi-systemic involvement. Furthermore, we identify key signatures in cardiolipin and proteome profiles across various degrees of cardiolipin loss, facilitating the use of omics technologies to guide future diagnosis of mitochondrial diseases.
Assuntos
Encefalopatias , Doenças Mitocondriais , Animais , Camundongos , Encefalopatias/genética , Encefalopatias/metabolismo , Cardiolipinas/genética , Cardiolipinas/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Doenças Mitocondriais/genética , Doenças Mitocondriais/metabolismo , ProteômicaRESUMO
Mitochondria maintain their own specialized protein synthesis machinery, which in mammals is used exclusively for the synthesis of the membrane proteins responsible for oxidative phosphorylation1,2. The initiation of protein synthesis in mitochondria differs substantially from bacterial or cytosolic translation systems. Mitochondrial translation initiation lacks initiation factor 1, which is essential in all other translation systems from bacteria to mammals3,4. Furthermore, only one type of methionyl transfer RNA (tRNAMet) is used for both initiation and elongation4,5, necessitating that the initiation factor specifically recognizes the formylated version of tRNAMet (fMet-tRNAMet). Lastly, most mitochondrial mRNAs do not possess 5' leader sequences to promote mRNA binding to the ribosome2. There is currently little mechanistic insight into mammalian mitochondrial translation initiation, and it is not clear how mRNA engagement, initiator-tRNA recruitment and start-codon selection occur. Here we determine the cryo-EM structure of the complete translation initiation complex from mammalian mitochondria at 3.2 Å. We describe the function of an additional domain insertion that is present in the mammalian mitochondrial initiation factor 2 (mtIF2). By closing the decoding centre, this insertion stabilizes the binding of leaderless mRNAs and induces conformational changes in the rRNA nucleotides involved in decoding. We identify unique features of mtIF2 that are required for specific recognition of fMet-tRNAMet and regulation of its GTPase activity. Finally, we observe that the ribosomal tunnel in the initiating ribosome is blocked by insertion of the N-terminal portion of mitochondrial protein mL45, which becomes exposed as the ribosome switches to elongation mode and may have an additional role in targeting of mitochondrial ribosomes to the protein-conducting pore in the inner mitochondrial membrane.
Assuntos
Microscopia Crioeletrônica , Mamíferos , Mitocôndrias/ultraestrutura , Iniciação Traducional da Cadeia Peptídica , Animais , Códon de Iniciação/genética , Fatores de Iniciação em Eucariotos/química , Fatores de Iniciação em Eucariotos/genética , Fatores de Iniciação em Eucariotos/metabolismo , Fatores de Iniciação em Eucariotos/ultraestrutura , Mitocôndrias/química , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/ultraestrutura , Modelos Moleculares , RNA Mitocondrial/química , RNA Mitocondrial/genética , RNA Mitocondrial/metabolismo , RNA Mitocondrial/ultraestrutura , RNA de Transferência de Metionina/genética , RNA de Transferência de Metionina/metabolismo , RNA de Transferência de Metionina/ultraestruturaRESUMO
The mitochondrial inner membrane contains a unique phospholipid known as cardiolipin (CL), which stabilises the protein complexes embedded in the membrane and supports its overall structure. Recent evidence indicates that the mitochondrial ribosome may associate with the inner membrane to facilitate co-translational insertion of the hydrophobic oxidative phosphorylation (OXPHOS) proteins into the inner membrane. We generated three mutant knockout cell lines for the CL biosynthesis gene Crls1 to investigate the effects of CL loss on mitochondrial protein synthesis. Reduced CL levels caused altered mitochondrial morphology and transcriptome-wide changes that were accompanied by uncoordinated mitochondrial translation rates and impaired respiratory chain supercomplex formation. Aberrant protein synthesis was caused by impaired formation and distribution of mitochondrial ribosomes. Reduction or loss of CL resulted in divergent mitochondrial and endoplasmic reticulum stress responses. We show that CL is required to stabilise the interaction of the mitochondrial ribosome with the membrane via its association with OXA1 (also known as OXA1L) during active translation. This interaction facilitates insertion of newly synthesised mitochondrial proteins into the inner membrane and stabilises the respiratory supercomplexes.
Assuntos
Cardiolipinas , Ribossomos Mitocondriais , Cardiolipinas/metabolismo , Mitocôndrias/genética , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismoRESUMO
[Figure: see text].
Assuntos
Anticorpos Neutralizantes/farmacologia , Aterosclerose/prevenção & controle , Moléculas de Adesão Celular Neuronais/antagonistas & inibidores , Adesão Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Proteínas da Matriz Extracelular/antagonistas & inibidores , Migração e Rolagem de Leucócitos/efeitos dos fármacos , Leucócitos/efeitos dos fármacos , Proteínas do Tecido Nervoso/antagonistas & inibidores , Oligonucleotídeos Antissenso/farmacologia , Animais , Aterosclerose/genética , Aterosclerose/imunologia , Aterosclerose/metabolismo , Receptor 1 de Quimiocina CX3C/genética , Moléculas de Adesão Celular Neuronais/deficiência , Moléculas de Adesão Celular Neuronais/genética , Técnicas de Cocultura , Modelos Animais de Doenças , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Proteínas da Matriz Extracelular/deficiência , Proteínas da Matriz Extracelular/genética , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteínas Relacionadas a Receptor de LDL/metabolismo , Leucócitos/imunologia , Leucócitos/metabolismo , Masculino , Camundongos Transgênicos , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Placa Aterosclerótica , Receptores de LDL/deficiência , Receptores de LDL/genética , Proteína Reelina , Serina Endopeptidases/deficiência , Serina Endopeptidases/genética , Transdução de Sinais , Células U937RESUMO
Supported by an abundance of experimental and genetic evidence, angiopoietin-like protein 3 (ANGPTL3) has emerged as a promising therapeutic target for cardiovascular disease. ANGPTL3 is primarily produced by the liver and is a potent modulator of plasma lipids and lipoproteins. Experimental models and subjects with loss-of-function Angptl3 mutations typically present with lower levels of HDL-C than noncarriers. The effect of ANGPTL3 on HDL-C is typically attributed to its function as an inhibitor of the enzyme endothelial lipase. The ability to facilitate reverse cholesterol transport (RCT), the transport of cholesterol from peripheral tissues back to the liver, is a proposed antiatherogenic property of HDL. However, the effect of ANGPTL3 inhibition on RCT remains unclear. Here, we performed a series of dose-response and RCT studies using an Angptl3 antisense oligonucleotide (ASO) in mouse models with varying plasma lipid profiles ranging from moderately to severely hyperlipidemic. Angptl3 ASO-mediated reduction in HDL-C was limited to the model with moderate lipidemia, where the majority of plasma cholesterol was associated with HDL. Surprisingly, regardless of the effect on HDL-C, treatment with the Angptl3 ASO enhanced RCT in all models tested. The observations from the RCT assays were confirmed in HDL clearance studies, where mice treated with the Angptl3 ASO displayed increased plasma clearance and hepatic uptake of labeled HDL. The results from our studies suggest that inhibition of ANGPTL3 not only reduces levels of proatherogenic lipids but also improves HDL-mediated RCT.
Assuntos
Proteína 3 Semelhante a Angiopoietina/metabolismo , HDL-Colesterol/metabolismo , Colesterol/metabolismo , Proteína 3 Semelhante a Angiopoietina/antagonistas & inibidores , Animais , Transporte Biológico , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oligonucleotídeos Antissenso/farmacologiaRESUMO
The etiology of diabetic nephropathy in type 2 diabetes is multifactorial. Sustained hyperglycemia is a major contributor, but additional contributions come from the hypertension, obesity, and hyperlipidemia that are also commonly present in patients with type 2 diabetes and nephropathy. The leptin deficient BTBR ob/ob mouse is a model of type 2 diabetic nephropathy in which hyperglycemia, obesity, and hyperlipidemia, but not hypertension, are present. We have shown that reversal of the constellation of these metabolic abnormalities with leptin replacement can reverse the morphologic and functional manifestations of diabetic nephropathy. Here we tested the hypothesis that reversal specifically of the hypertriglyceridemia, using an antisense oligonucleotide directed against ApoC-III, an apolipoprotein that regulates the interactions of VLDL (very low density lipoproteins) with the LDL receptor, is sufficient to ameliorate the nephropathy of Type 2 diabetes. Antisense treatment resulted in reduction of circulating ApoC-III protein levels and resulted in substantial lowering of triglycerides to near-normal levels in diabetic mice versus controls. Antisense treatment did not ameliorate proteinuria or pathologic manifestations of diabetic nephropathy, including podocyte loss. These studies indicate that pathologic manifestations of diabetic nephropathy are unlikely to be reduced by lipid-lowering therapeutics alone, but does not preclude a role for such interventions to be used in conjunction with other therapeutics commonly employed in the treatment of diabetes and its complications.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Nefropatias Diabéticas/metabolismo , Hipertrigliceridemia/metabolismo , Animais , Apolipoproteína C-III/genética , Apolipoproteína C-III/metabolismo , Diabetes Mellitus Experimental/metabolismo , Feminino , Leptina/genética , Masculino , Camundongos , Camundongos Obesos , Oligonucleotídeos Antissenso , Podócitos/metabolismo , Podócitos/patologiaRESUMO
Enhancing the functional uptake of antisense oligonucleotide (ASO) in the muscle will be beneficial for developing ASO therapeutics targeting genes expressed in the muscle. We hypothesized that improving albumin binding will facilitate traversal of ASO from the blood compartment to the interstitium of the muscle tissues to enhance ASO functional uptake. We synthesized structurally diverse saturated and unsaturated fatty acid conjugated ASOs with a range of hydrophobicity. The binding affinity of ASO fatty acid conjugates to plasma proteins improved with fatty acid chain length and highest binding affinity was observed with ASO conjugates containing fatty acid chain length from 16 to 22 carbons. The degree of unsaturation or conformation of double bond appears to have no influence on protein binding or activity of ASO fatty acid conjugates. Activity of fatty acid ASO conjugates correlated with the affinity to albumin and the tightest albumin binder exhibited the highest activity improvement in muscle. Palmitic acid conjugation increases ASO plasma Cmax and improved delivery of ASO to interstitial space of mouse muscle. Conjugation of palmitic acid improved potency of DMPK, Cav3, CD36 and Malat-1 ASOs (3- to 7-fold) in mouse muscle. Our approach provides a foundation for developing more effective therapeutic ASOs for muscle disorders.
Assuntos
Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/farmacocinética , Ácido Palmítico/química , Animais , Proteínas Sanguíneas/metabolismo , Antígenos CD36/genética , Caveolina 3/genética , Ácidos Graxos/química , Ácidos Graxos Insaturados/química , Masculino , Camundongos Endogâmicos C57BL , Miotonina Proteína Quinase/genética , Oligonucleotídeos Antissenso/síntese química , Oligonucleotídeos Antissenso/metabolismo , RNA Longo não Codificante/metabolismo , Relação Estrutura-AtividadeRESUMO
Antisense oligonucleotides (ASOs) have demonstrated variation of efficacy in patient populations. This has prompted our investigation into the contribution of genetic architecture to ASO pharmacokinetics (PK) and pharmacodynamics (PD). Genome wide association (GWA) and transcriptomic analysis in a hybrid mouse diversity panel (HMDP) were used to identify and validate novel genes involved in the uptake and efficacy of a single dose of a Malat1 constrained ethyl (cEt) modified ASO. The GWA of the HMDP identified two significant associations on chromosomes 4 and 10 with hepatic Malat1 ASO concentrations. Stabilin 2 (Stab2) and vesicle associated membrane protein 3 (Vamp3) were identified by cis-eQTL analysis. HMDP strains with lower Stab2 expression and Stab2 KO mice displayed significantly lower PK than strains with higher Stab2 expression and the wild type (WT) animals respectively, confirming the role of Stab2 in regulating hepatic Malat1 ASO uptake. GWA examining ASO efficacy uncovered three loci associated with Malat1 potency: Small Subunit Processome Component (Utp11l) on chromosome 4, Rho associated coiled-coil containing protein kinase 2 (Rock2) and Aci-reductone dioxygenase (Adi1) on chromosome 12. Our results demonstrate the utility of mouse GWAS using the HMDP in detecting genes capable of impacting the uptake of ASOs, and identifies genes critical for the activity of ASOs in vivo.
Assuntos
Oligonucleotídeos Antissenso/farmacocinética , RNA Longo não Codificante/genética , RNA Longo não Codificante/fisiologia , Animais , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Perfilação da Expressão Gênica/métodos , Variação Genética , Estudo de Associação Genômica Ampla , Fígado/metabolismo , Camundongos , Camundongos Knockout , Oligonucleotídeos Antissenso/genética , RNA Mensageiro/metabolismo , Proteína 3 Associada à Membrana da Vesícula/genética , Proteína 3 Associada à Membrana da Vesícula/metabolismoRESUMO
BACKGROUND: Epidemiologic and genomewide association studies have linked loss-of-function variants in ANGPTL3, encoding angiopoietin-like 3, with low levels of plasma lipoproteins. METHODS: We evaluated antisense oligonucleotides (ASOs) targeting Angptl3 messenger RNA (mRNA) for effects on plasma lipid levels, triglyceride clearance, liver triglyceride content, insulin sensitivity, and atherosclerosis in mice. Subsequently, 44 human participants (with triglyceride levels of either 90 to 150 mg per deciliter [1.0 to 1.7 mmol per liter] or >150 mg per deciliter, depending on the dose group) were randomly assigned to receive subcutaneous injections of placebo or an antisense oligonucleotide targeting ANGPTL3 mRNA in a single dose (20, 40, or 80 mg) or multiple doses (10, 20, 40, or 60 mg per week for 6 weeks). The main end points were safety, side-effect profile, pharmacokinetic and pharmacodynamic measures, and changes in levels of lipids and lipoproteins. RESULTS: The treated mice had dose-dependent reductions in levels of hepatic Angptl3 mRNA, Angptl3 protein, triglycerides, and low-density lipoprotein (LDL) cholesterol, as well as reductions in liver triglyceride content and atherosclerosis progression and increases in insulin sensitivity. After 6 weeks of treatment, persons in the multiple-dose groups had reductions in levels of ANGPTL3 protein (reductions of 46.6 to 84.5% from baseline, P<0.01 for all doses vs. placebo) and in levels of triglycerides (reductions of 33.2 to 63.1%), LDL cholesterol (1.3 to 32.9%), very-low-density lipoprotein cholesterol (27.9 to 60.0%), non-high-density lipoprotein cholesterol (10.0 to 36.6%), apolipoprotein B (3.4 to 25.7%), and apolipoprotein C-III (18.9 to 58.8%). Three participants who received the antisense oligonucleotide and three who received placebo reported dizziness or headache. There were no serious adverse events. CONCLUSIONS: Oligonucleotides targeting mouse Angptl3 retarded the progression of atherosclerosis and reduced levels of atherogenic lipoproteins in mice. Use of the same strategy to target human ANGPTL3 reduced levels of atherogenic lipoproteins in humans. (Funded by Ionis Pharmaceuticals; ClinicalTrials.gov number, NCT02709850 .).
Assuntos
Angiopoietinas/antagonistas & inibidores , Aterosclerose/tratamento farmacológico , Doença da Artéria Coronariana/genética , Dislipidemias/tratamento farmacológico , Lipídeos/sangue , Oligonucleotídeos Antissenso/uso terapêutico , Oligonucleotídeos/uso terapêutico , Adulto , Idoso , Proteína 3 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina , Angiopoietinas/genética , Animais , Aterosclerose/metabolismo , Aterosclerose/prevenção & controle , Doença da Artéria Coronariana/metabolismo , Modelos Animais de Doenças , Método Duplo-Cego , Dislipidemias/sangue , Feminino , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos , Pessoa de Meia-Idade , Oligonucleotídeos/farmacologia , Oligonucleotídeos Antissenso/farmacologia , RNA Mensageiro/antagonistas & inibidoresRESUMO
OBJECTIVE: ApoC-III (apolipoprotein C-III) glycosylation can predict cardiovascular disease risk. Higher abundance of disialylated (apoC-III2) over monosialylated (apoC-III1) glycoforms is associated with lower plasma triglyceride levels. Yet, it remains unclear whether apoC-III glycosylation impacts TRL (triglyceride-rich lipoprotein) clearance and whether apoC-III antisense therapy (volanesorsen) affects distribution of apoC-III glycoforms. Approach and Results: To measure the abundance of human apoC-III glycoforms in plasma over time, human TRLs were injected into wild-type mice and mice lacking hepatic TRL clearance receptors, namely HSPGs (heparan sulfate proteoglycans) or both LDLR (low-density lipoprotein receptor) and LRP1 (LDLR-related protein 1). ApoC-III was more rapidly cleared in the absence of HSPG (t1/2=25.4 minutes) than in wild-type animals (t1/2=55.1 minutes). In contrast, deficiency of LDLR and LRP1 (t1/2=56.1 minutes) did not affect clearance of apoC-III. After injection, a significant increase in the relative abundance of apoC-III2 was observed in HSPG-deficient mice, whereas the opposite was observed in mice lacking LDLR and LRP1. In patients, abundance of plasma apoC-III glycoforms was assessed after placebo or volanesorsen administration. Volanesorsen treatment correlated with a statistically significant 1.4-fold increase in the relative abundance of apoC-III2 and a 15% decrease in that of apoC-III1. The decrease in relative apoC-III1 abundance was strongly correlated with decreased plasma triglyceride levels in patients. CONCLUSIONS: Our results indicate that HSPGs preferentially clear apoC-III2. In contrast, apoC-III1 is more effectively cleared by LDLR/LRP1. Clinically, the increase in the apoC-III2/apoC-III1 ratio on antisense lowering of apoC-III might reflect faster clearance of apoC-III1 because this metabolic shift associates with improved triglyceride levels.
Assuntos
Apolipoproteína C-III/sangue , Hipertrigliceridemia/tratamento farmacológico , Lipoproteínas HDL3/metabolismo , Oligonucleotídeos/administração & dosagem , Receptores de LDL/metabolismo , Animais , Apolipoproteína C-III/efeitos dos fármacos , Modelos Animais de Doenças , Glicosilação/efeitos dos fármacos , Humanos , Hipertrigliceridemia/sangue , Masculino , Camundongos , Terapia de Alvo Molecular/métodos , Receptores de LDL/efeitos dos fármacos , Valores de ReferênciaRESUMO
OBJECTIVE: The role of hepatocyte Abca1 (ATP binding cassette transporter A1) in trafficking hepatic free cholesterol (FC) into plasma versus bile for reverse cholesterol transport (RCT) is poorly understood. We hypothesized that hepatocyte Abca1 recycles plasma HDL-C (high-density lipoprotein cholesterol) taken up by the liver back into plasma, maintaining the plasma HDL-C pool, and decreasing HDL-mediated RCT into feces. Approach and Results: Chow-fed hepatocyte-specific Abca1 knockout (HSKO) and control mice were injected with human HDL radiolabeled with 125I-tyramine cellobiose (125I-TC; protein) and 3H-cholesteryl oleate (3H-CO). 125I-TC and 3H-CO plasma decay, plasma HDL 3H-CO selective clearance (ie, 3H-125I fractional catabolic rate), liver radiolabel uptake, and fecal 3H-sterol were significantly greater in HSKO versus control mice, supporting increased plasma HDL RCT. Twenty-four hours after 3H-CO-HDL injection, HSKO mice had reduced total hepatic 3H-FC (ie, 3H-CO hydrolyzed to 3H-FC in liver) resecretion into plasma, demonstrating Abca1 recycled HDL-derived hepatic 3H-FC back into plasma. Despite similar liver LDLr (low-density lipoprotein receptor) expression between genotypes, HSKO mice treated with LDLr-targeting versus control antisense oligonucleotide had slower plasma 3H-CO-HDL decay, reduced selective 3H-CO clearance, and decreased fecal 3H-sterol excretion that was indistinguishable from control mice. Increased RCT in HSKO mice was selective for 3H-CO-HDL, since macrophage RCT was similar between genotypes. CONCLUSIONS: Hepatocyte Abca1 deletion unmasks a novel and selective FC trafficking pathway that requires LDLr expression, accelerating plasma HDL-selective CE uptake by the liver and promoting HDL RCT into feces, consequently reducing HDL-derived hepatic FC recycling into plasma.
Assuntos
Transportador 1 de Cassete de Ligação de ATP/fisiologia , Colesterol/metabolismo , Hepatócitos/fisiologia , Lipoproteínas HDL/sangue , Receptores de LDL/fisiologia , Animais , Transporte Biológico , Feminino , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
OBJECTIVE: Human genetic variants near the FADS (fatty acid desaturase) gene cluster (FADS1-2-3) are strongly associated with cardiometabolic traits including dyslipidemia, fatty liver, type 2 diabetes mellitus, and coronary artery disease. However, mechanisms underlying these genetic associations are unclear. APPROACH AND RESULTS: Here, we specifically investigated the physiological role of the Δ-5 desaturase FADS1 in regulating diet-induced cardiometabolic phenotypes by treating hyperlipidemic LDLR (low-density lipoprotein receptor)-null mice with antisense oligonucleotides targeting the selective knockdown of Fads1. Fads1 knockdown resulted in striking reorganization of both ω-6 and ω-3 polyunsaturated fatty acid levels and their associated proinflammatory and proresolving lipid mediators in a highly diet-specific manner. Loss of Fads1 activity promoted hepatic inflammation and atherosclerosis, yet was associated with suppression of hepatic lipogenesis. Fads1 knockdown in isolated macrophages promoted classic M1 activation, whereas suppressing alternative M2 activation programs, and also altered systemic and tissue inflammatory responses in vivo. Finally, the ability of Fads1 to reciprocally regulate lipogenesis and inflammation may rely in part on its role as an effector of liver X receptor signaling. CONCLUSIONS: These results position Fads1 as an underappreciated regulator of inflammation initiation and resolution, and suggest that endogenously synthesized arachidonic acid and eicosapentaenoic acid are key determinates of inflammatory disease progression and liver X receptor signaling.
Assuntos
Aorta/enzimologia , Doenças da Aorta/enzimologia , Aterosclerose/enzimologia , Dislipidemias/enzimologia , Ácidos Graxos Dessaturases/metabolismo , Mediadores da Inflamação/metabolismo , Inflamação/enzimologia , Lipogênese , Animais , Aorta/patologia , Doenças da Aorta/genética , Doenças da Aorta/patologia , Ácido Araquidônico/metabolismo , Aterosclerose/genética , Aterosclerose/patologia , Células Cultivadas , Dessaturase de Ácido Graxo Delta-5 , Modelos Animais de Doenças , Dislipidemias/genética , Dislipidemias/patologia , Ácido Eicosapentaenoico/metabolismo , Ácidos Graxos Dessaturases/genética , Inflamação/genética , Inflamação/patologia , Fígado/metabolismo , Receptores X do Fígado/metabolismo , Ativação de Macrófagos , Macrófagos Peritoneais/enzimologia , Macrófagos Peritoneais/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Placa Aterosclerótica , Receptores de LDL/deficiência , Receptores de LDL/genéticaRESUMO
Mitochondrial biogenesis is intimately dependent on the coordinated expression of the nuclear and mitochondrial genomes that is necessary for the assembly and function of the respiratory complexes to produce most of the energy required by cells. Although highly compacted in animals, the mitochondrial genome and its expression are essential for survival, development, and optimal energy production. The machinery that regulates gene expression within mitochondria is localised within the same compartment and, like in their ancestors, the bacteria, this machinery does not use membrane-based compartmentalisation to order the gene expression pathway. Therefore, the lifecycle of mitochondrial RNAs from transcription through processing, maturation, translation to turnover is mediated by a gamut of RNA-binding proteins (RBPs), all contained within the mitochondrial matrix milieu. Recent discoveries indicate that multiple processes regulating RNA metabolism occur at once but since mitochondria have a new complement of RBPs, many evolved de novo from nuclear genes, we are left wondering how co-ordinated are these processes? Here, we review recently identified examples of the co-ordinated and stochastic processes that govern the mitochondrial transcriptome. These new discoveries reveal the complexity of mitochondrial gene expression and the need for its in-depth exploration to understand how these organelles can respond to the energy demands of the cell.
Assuntos
Regulação da Expressão Gênica , Genes Mitocondriais , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Animais , Núcleo Celular/metabolismo , Perfilação da Expressão Gênica , Genoma Mitocondrial , Mitocôndrias/genética , Proteínas Mitocondriais/genética , RNA/análise , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Processos Estocásticos , Fatores de Transcrição/metabolismo , TranscriptomaRESUMO
Nonalcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease. NAFLD progresses from benign steatosis to steatohepatitis (NASH) to cirrhosis and is linked to hepatocellular carcinoma. No targeted treatment is currently approved for NAFLD/NASH. We previously showed that fat-specific protein 27 (FSP27), a lipid droplet-associated protein that controls triglyceride turnover in the hepatocyte, is required for fasting- and diet-induced triglyceride accumulation in the liver. However, silencing Fsp27 with antisense oligonucleotides (ASOs) did not improve hepatosteatosis in genetic nor nutritional mouse models of obesity. Herein, we tested the therapeutic potential of ASO-Fsp27 when used in combination with the PPARα agonist fenofibrate. C57BL/6 mice were fed a high-trans-fat, high-cholesterol, high-fructose diet for eight weeks to establish NASH, then kept on diet for six additional weeks while dosed with ASOs and fenofibrate, alone or in combination. Data show that ASO-Fsp27 and fenofibrate synergize to promote resistance to diet-induced obesity and hypertriglyceridemia and to reverse hepatic steatosis, inflammation, oxidative stress, and fibrosis. This multifactorial improvement of liver disease noted when combining both drugs suggests that a course of treatment that includes both reduced FSP27 activity and activation of PPARα could provide therapeutic benefit to patients with NAFLD/NASH.
Assuntos
Dieta/efeitos adversos , Fenofibrato/farmacologia , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/genética , Oligonucleotídeos Antissenso/genética , Proteínas/genética , Animais , Sinergismo Farmacológico , Fenofibrato/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Obesidade/tratamento farmacológico , Obesidade/genética , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , RiscoRESUMO
Obesity is a component of the metabolic syndrome, mechanistically linked to diabetes, fatty liver disease, and cardiovascular disease. Proteins that regulate the metabolic fate of intracellular lipid droplets are potential therapeutic candidates to treat obesity and its related consequences. CIDEC (cell death-inducing DFFA-like effector C), also known in mice as Fsp27 (fat-specific protein 27), is a lipid droplet-associated protein that prevents lipid mobilization and promotes intracellular lipid storage. The consequences of complete loss of FSP27 on hepatic metabolism and on insulin resistance are controversial, as both healthy and deleterious lipodystrophic phenotypes have been reported in Fsp27-/- mice. To test whether therapeutic silencing of Fsp27 might be useful to improve obesity, fatty liver, and glycemic control, we used antisense oligonucleotides (ASOs) in both nutritional (high-fat diet) and genetic (leptin-deficient ob/ob) mouse models of obesity, hyperglycemia, and hepatosteatosis. We show that partial silencing Fsp27 in either model results in the robust decrease in visceral fat, improved insulin sensitivity and whole-body glycemic control, and tissue-specific changes in transcripts controlling lipid oxidation and synthesis. These data suggest that partial reduction of FSP27 activity (e.g., using ASOs) might be exploited therapeutically in insulin-resistant obese or overweight patients.
Assuntos
Diabetes Mellitus/terapia , Fígado Gorduroso/terapia , Obesidade/terapia , Oligonucleotídeos Antissenso/administração & dosagem , Proteínas/genética , Animais , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Dieta Hiperlipídica , Modelos Animais de Doenças , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Humanos , Resistência à Insulina/genética , Gotículas Lipídicas/metabolismo , Gotículas Lipídicas/patologia , Metabolismo dos Lipídeos/genética , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Obesos , Obesidade/genética , Oligonucleotídeos Antissenso/genética , Proteínas/antagonistas & inibidoresRESUMO
Hepatitis C virus (HCV) is an enveloped RNA virus responsible for 170 million cases of viral hepatitis worldwide. Over 50% of chronically infected HCV patients develop hepatic steatosis, and steatosis can be induced by expression of HCV core protein (core) alone. Additionally, core must associate with cytoplasmic lipid droplets (LDs) for steatosis development and viral particle assembly. Due to the importance of the LD as a key component of hepatic lipid storage and as a platform for HCV particle assembly, it seems this dynamic subcellular organelle is a gatekeeper in the pathogenesis of viral hepatitis. Here, we hypothesized that core requires the host LD scaffold protein, perilipin (PLIN)3, to induce hepatic steatosis. To test our hypothesis in vivo, we have studied core-induced hepatic steatosis in the absence or presence of antisense oligonucleotide-mediated knockdown of PLIN3. PLIN3 knockdown blunted HCV core-induced steatosis in transgenic mice fed either chow or a moderate fat diet. Collectively, our studies demonstrate that the LD scaffold protein, PLIN3, is essential for HCV core-induced hepatic steatosis and provide new insights into the pathogenesis of HCV.
Assuntos
Fígado Gorduroso/genética , Hepatite C/metabolismo , Fígado/metabolismo , Perilipina-3/genética , Animais , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Técnicas de Silenciamento de Genes , Genótipo , Hepacivirus , Hepatite C/genética , Hepatite C/patologia , Hepatite C/virologia , Humanos , Gotículas Lipídicas/metabolismo , Gotículas Lipídicas/patologia , Gotículas Lipídicas/virologia , Metabolismo dos Lipídeos/genética , Fígado/patologia , Fígado/virologia , Camundongos , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/genética , Perilipina-3/antagonistas & inibidoresRESUMO
IκB kinase ß (IKKß), a central coordinator of inflammation through activation of nuclear factor-κB, has been identified as a potential therapeutic target for the treatment of obesity-associated metabolic dysfunctions. In this study, we evaluated an antisense oligonucleotide (ASO) inhibitor of IKKß and found that IKKß ASO ameliorated diet-induced metabolic dysfunctions in mice. Interestingly, IKKß ASO also inhibited adipocyte differentiation and reduced adiposity in high-fat (HF)-fed mice, indicating an important role of IKKß signaling in the regulation of adipocyte differentiation. Indeed, CRISPR/Cas9-mediated genomic deletion of IKKß in 3T3-L1 preadipocytes blocked these cells differentiating into adipocytes. To further elucidate the role of adipose progenitor IKKß signaling in diet-induced obesity, we generated mice that selectively lack IKKß in the white adipose lineage and confirmed the essential role of IKKß in mediating adipocyte differentiation in vivo. Deficiency of IKKß decreased HF-elicited adipogenesis in addition to reducing inflammation and protected mice from diet-induced obesity and insulin resistance. Further, pharmacological inhibition of IKKß also blocked human adipose stem cell differentiation. Our findings establish IKKß as a pivotal regulator of adipogenesis and suggest that overnutrition-mediated IKKß activation serves as an initial signal that triggers adipose progenitor cell differentiation in response to HF feeding. Inhibition of IKKß with antisense therapy may represent as a novel therapeutic approach to combat obesity and metabolic dysfunctions. Stem Cells 2016;34:1883-1895.
Assuntos
Adipócitos/patologia , Linhagem da Célula , Quinase I-kappa B/metabolismo , Síndrome Metabólica/tratamento farmacológico , Terapia de Alvo Molecular , Obesidade/tratamento farmacológico , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Adipogenia/efeitos dos fármacos , Animais , Linhagem da Célula/efeitos dos fármacos , Dieta , Fígado Gorduroso/patologia , Deleção de Genes , Técnicas de Silenciamento de Genes , Humanos , Quinase I-kappa B/deficiência , Insulina/farmacologia , Masculino , Síndrome Metabólica/patologia , Camundongos Endogâmicos C57BL , Obesidade/patologia , Oligonucleotídeos Antissenso/farmacologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismoRESUMO
BACKGROUND & AIMS: Aberrantly high expression of TRIM24 occurs in human cancers, including hepatocellular carcinoma. In contrast, TRIM24 in the mouse is reportedly a liver-specific tumour suppressor. To address this dichotomy and to uncover direct regulatory functions of TRIM24 in vivo, we developed a new mouse model that lacks expression of all Trim24 isoforms, as the previous model expressed normal levels of Trim24 lacking only exon 4. METHODS: To produce germline-deleted Trim24(dlE1) mice, deletion of the promoter and exon 1 of Trim24 was induced in Trim24(LoxP) mice by crossing with a zona pellucida 3-Cre line for global deletion. Liver-specific deletion (Trim24(hep)) was achieved by crossing with an albumin-Cre line. Phenotypic analyses were complemented by protein, gene-specific and global RNA expression analyses and quantitative chromatin immunoprecipitation. RESULTS: Global loss of Trim24 disrupted hepatic homeostasis in 100% of mice with highly significant, decreased expression of oxidation/reduction, steroid, fatty acid, and lipid metabolism genes, as well as increased expression of genes involved in unfolded protein response, endoplasmic reticulum stress and cell cycle pathways. Trim24(dlE1/dlE1) mice have markedly depleted visceral fat and, like Trim24(hep/hep) mice, spontaneously develop hepatic lipid-filled lesions, steatosis, hepatic injury, fibrosis and hepatocellular carcinoma. CONCLUSIONS: TRIM24, an epigenetic co-regulator of transcription, directly and indirectly represses hepatic lipid accumulation, inflammation, fibrosis and damage in the murine liver. Complete loss of Trim24 offers a model of human non-alcoholic fatty liver disease, steatosis, fibrosis and development of hepatocellular carcinoma in the absence of high-fat diet or obesity.
Assuntos
Carcinoma Hepatocelular/genética , Fígado Gorduroso/genética , Regulação Neoplásica da Expressão Gênica , Lipídeos/análise , Neoplasias Hepáticas Experimentais/genética , Proteínas Nucleares/genética , RNA Neoplásico/genética , Fatores de Transcrição/genética , Animais , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Progressão da Doença , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Humanos , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Camundongos , Camundongos Knockout , Proteínas Nucleares/biossíntese , Reação em Cadeia da Polimerase , Fatores de Transcrição/biossínteseRESUMO
RATIONALE: Elevated plasma triglyceride levels have been recognized as a risk factor for the development of coronary heart disease. Apolipoprotein C-III (apoC-III) represents both an independent risk factor and a key regulatory factor of plasma triglyceride concentrations. Furthermore, elevated apoC-III levels have been associated with metabolic syndrome and type 2 diabetes mellitus. To date, no selective apoC-III therapeutic agent has been evaluated in the clinic. OBJECTIVE: To test the hypothesis that selective inhibition of apoC-III with antisense drugs in preclinical models and in healthy volunteers would reduce plasma apoC-III and triglyceride levels. METHODS AND RESULTS: Rodent- and human-specific second-generation antisense oligonucleotides were identified and evaluated in preclinical models, including rats, mice, human apoC-III transgenic mice, and nonhuman primates. We demonstrated the selective reduction of both apoC-III and triglyceride in all preclinical pharmacological evaluations. We also showed that inhibition of apoC-III was well tolerated and not associated with increased liver triglyceride deposition or hepatotoxicity. A double-blind, placebo-controlled, phase I clinical study was performed in healthy subjects. Administration of the human apoC-III antisense drug resulted in dose-dependent reductions in plasma apoC-III, concomitant lowering of triglyceride levels, and produced no clinically meaningful signals in the safety evaluations. CONCLUSIONS: Antisense inhibition of apoC-III in preclinical models and in a phase I clinical trial with healthy subjects produced potent, selective reductions in plasma apoC-III and triglyceride, 2 known risk factors for cardiovascular disease. This compelling pharmacological profile supports further clinical investigations in hypertriglyceridemic subjects.