RESUMO
Aging induces alterations in bone structure and strength through a multitude of processes, exacerbating common aging- related diseases like osteoporosis and osteoarthritis. Cellular hallmarks of aging are examined, as related to bone and the marrow microenvironment, and ways in which these might contribute to a variety of age-related perturbations in osteoblasts, osteocytes, marrow adipocytes, chondrocytes, osteoclasts, and their respective progenitors. Cellular senescence, stem cell exhaustion, mitochondrial dysfunction, epigenetic and intracellular communication changes are central pathways and recognized as associated and potentially causal in aging. We focus on these in musculoskeletal system and highlight knowledge gaps in the literature regarding cellular and tissue crosstalk in bone, cartilage, and the bone marrow niche. While senolytics have been utilized to target aging pathways, here we propose non-pharmacologic, exercise-based interventions as prospective "senolytics" against aging effects on the skeleton. Increased bone mass and delayed onset or progression of osteoporosis and osteoarthritis are some of the recognized benefits of regular exercise across the lifespan. Further investigation is needed to delineate how cellular indicators of aging manifest in bone and the marrow niche and how altered cellular and tissue crosstalk impact disease progression, as well as consideration of exercise as a therapeutic modality, as a means to enhance discovery of bone-targeted therapies.
Assuntos
Osteoartrite , Osteoporose , Adipócitos , Idoso , Envelhecimento , Exercício Físico , Humanos , Osteoartrite/terapia , Osteoblastos , Estudos ProspectivosRESUMO
Loss of long-chain acyl-CoA synthetase isoform-1 (ACSL1) in mouse skeletal muscle (Acsl1M-/-) severely reduces acyl-CoA synthetase activity and fatty acid oxidation. However, the effects of decreased fatty acid oxidation on skeletal muscle function, histology, use of alternative fuels, and mitochondrial function and morphology are unclear. We observed that Acsl1M-/- mice have impaired voluntary running capacity and muscle grip strength and that their gastrocnemius muscle contains myocytes with central nuclei, indicating muscle regeneration. We also found that plasma creatine kinase and aspartate aminotransferase levels in Acsl1M-/- mice are 3.4- and 1.5-fold greater, respectively, than in control mice (Acsl1flox/flox ), indicating muscle damage, even without exercise, in the Acsl1M-/- mice. Moreover, caspase-3 protein expression exclusively in Acsl1M-/- skeletal muscle and the presence of cleaved caspase-3 suggested myocyte apoptosis. Mitochondria in Acsl1M-/- skeletal muscle were swollen with abnormal cristae, and mitochondrial biogenesis was increased. Glucose uptake did not increase in Acsl1M-/- skeletal muscle, and pyruvate oxidation was similar in gastrocnemius homogenates from Acsl1M-/- and control mice. The rate of protein synthesis in Acsl1M-/- glycolytic muscle was 2.1-fold greater 30 min after exercise than in the controls, suggesting resynthesis of proteins catabolized for fuel during the exercise. At this time, mTOR complex 1 was activated, and autophagy was blocked. These results suggest that fatty acid oxidation is critical for normal skeletal muscle homeostasis during both rest and exercise. We conclude that ACSL1 deficiency produces an overall defect in muscle fuel metabolism that increases protein catabolism, resulting in exercise intolerance, muscle weakness, and myocyte apoptosis.
Assuntos
Aminoácidos/metabolismo , Coenzima A Ligases/genética , Ácidos Graxos/metabolismo , Músculo Esquelético/metabolismo , Animais , Apoptose , Aspartato Aminotransferases/metabolismo , Caspase 3/metabolismo , Coenzima A Ligases/deficiência , Creatina Quinase/metabolismo , Metabolismo dos Lipídeos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Músculo Esquelético/patologia , Oxirredução , Condicionamento Físico Animal , Regulação para CimaRESUMO
Fatty acid channeling into oxidation or storage modes depends on physiological conditions and hormonal signaling. However, the directionality of this channeling may also depend on the association of each of the five acyl-CoA synthetase isoforms with specific protein partners. Long-chain acyl-CoA synthetases (ACSLs) catalyze the conversion of long-chain fatty acids to fatty acyl-CoAs, which are then either oxidized or used in esterification reactions. In highly oxidative tissues, ACSL1 is located on the outer mitochondrial membrane (OMM) and directs fatty acids into mitochondria for ß-oxidation. In the liver, however, about 50% of ACSL1 is located on the endoplasmic reticulum (ER) where its metabolic function is unclear. Because hepatic fatty acid partitioning is likely to require the interaction of ACSL1 with other specific proteins, we used an unbiased protein interaction technique, BioID, to discover ACSL1-binding partners in hepatocytes. We targeted ACSL1 either to the ER or to the OMM of Hepa 1-6 cells as a fusion protein with the Escherichia coli biotin ligase, BirA*. Proteomic analysis identified 98 proteins that specifically interacted with ACSL1 at the ER, 55 at the OMM, and 43 common to both subcellular locations. We found subsets of peroxisomal and lipid droplet proteins, tethering proteins, and vesicle proteins, uncovering a dynamic role for ACSL1 in organelle and lipid droplet interactions. Proteins involved in lipid metabolism were also identified, including acyl-CoA-binding proteins and ceramide synthase isoforms 2 and 5. Our results provide fundamental and detailed insights into protein interaction networks that control fatty acid metabolism.
Assuntos
Coenzima A Ligases/fisiologia , Retículo Endoplasmático/metabolismo , Ácidos Graxos/metabolismo , Fígado/metabolismo , Mitocôndrias/metabolismo , Domínios e Motivos de Interação entre Proteínas , Animais , Feminino , Fígado/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Because the signaling eicosanoids, epoxyeicosatrienoic acids (EETs) and HETEs, are esterified to membrane phospholipids, we asked which long-chain acyl-CoA synthetase (ACSL) isoforms would activate these molecules and whether the apparent FA substrate preferences of each ACSL isoform might differ depending on whether it was assayed in mammalian cell membranes or as a purified bacterial recombinant protein. We found that all five ACSL isoforms were able to use EETs and HETEs as substrates and showed by LC-MS/MS that ACSLs produce EET-CoAs. We found differences in substrate preference between ACS assays performed in COS7 cell membranes and recombinant purified proteins. Similarly, preferences and Michaelis-Menten kinetics for long-chain FAs were distinctive. Substrate preferences identified for the purified ACSLs did not correspond to those observed in ACSL-deficient mouse models. Taken together, these data support the concept that each ACSL isoform exhibits a distinct substrate preference, but apparent substrate specificities depend upon multiple factors including membrane character, coactivators, inhibitors, protein interactions, and posttranslational modification.
Assuntos
Coenzima A Ligases/metabolismo , Eicosanoides/química , Eicosanoides/metabolismo , Animais , Células COS , Chlorocebus aethiops , Isoenzimas/metabolismo , Ratos , Especificidade por SubstratoRESUMO
Long-chain acyl-CoA esters are important intermediates in lipid metabolism and are synthesized from fatty acids by long-chain acyl-CoA synthetases (ACSL). The hematophagous insect Rhodnius prolixus, a vector of Chagas' disease, produces glycerolipids in the midgut after a blood meal, which are stored as triacylglycerol in the fat body and eggs. We identified twenty acyl-CoA synthetase genes in R. prolixus, two encoding ACSL isoforms (RhoprAcsl1 and RhoprAcsl2). RhoprAcsl1 transcripts increased in posterior midgut on the second day after feeding, and RhoprAcsl2 was highly transcribed on the tenth day. Both enzymes were expressed in Escherichia coli. Recombinant RhoprACSL1 and RhoprACSL2 had broad pH optima (7.5-9.5 and 6.5-9.5, respectively), were inhibited by triacsin C, and were rosiglitazone-insensitive. Both showed similar apparent Km for palmitic and oleic acid (2-6 µM), but different Km for arachidonic acid (0.5 and 6 µM for RhoprACSL1-Flag and RhoprACSL2-Flag, respectively). The knockdown of RhoprAcsl1 did not result in noticeable phenotypes. However, RhoprACSL2 deficient insects exhibited a 2.5-fold increase in triacylglycerol content in the fat body, and 90% decrease in fatty acid ß-oxidation. RhoprAcsl2 knockdown also resulted in 20% increase in lifespan, delayed digestion, 30% reduced oviposition, and 50% reduction in egg hatching. Laid eggs and hatched nymphs showed remarkable alterations in morphology. In summary, R. prolixus ACSL isoforms have distinct roles on lipid metabolism. Although RhoprACSL1 functions remain unclear, we propose that RhoprACSL2 is the main contributor for the formation of the intracellular acyl-CoA pool channeled for ß-oxidation in the fat body, and is also required for normal reproduction.
Assuntos
Coenzima A Ligases/genética , Corpo Adiposo/metabolismo , Ácidos Graxos/metabolismo , Oogênese/genética , Rhodnius/genética , Triglicerídeos/biossíntese , Sequência de Aminoácidos , Animais , Coenzima A Ligases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Proteínas de Insetos , Isoenzimas/genética , Isoenzimas/metabolismo , Dados de Sequência Molecular , Oxirredução , Filogenia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reprodução/genética , Rhodnius/classificação , Alinhamento de Sequência , Transcrição Gênica , Triazenos , Zigoto/metabolismoRESUMO
Glycerol-3-phosphate acyltransferases (GPAT) catalyze the initial and rate-limiting step for the de novo synthesis of triacylglycerol (TAG). Four mammalian GPAT isoforms have been identified: the mitochondria-associated GPAT1 and 2, and the endoplasmic reticulum (ER)-associated GPAT3 and 4. In the insect Rhodnius prolixus, a vector of Chagas' disease, we previously predicted a mitochondrial-like isoform (RhoprGPAT1) from genomic data. In the current study, we clone the RhoprGPAT1 coding sequence and identify an ER-associated GPAT (RhoprGPAT4) as the second isoform in the insect. RhoprGPAT1 contributes 15% of the total GPAT activity in anterior midgut, 50% in posterior midgut and fat body, and 70% in the ovary. The RhoprGpat1 gene is the predominant transcript in the midgut and fat body. To evaluate the physiological relevance of RhoprGPAT1, we generate RhoprGPAT1-deficient insects. The knockdown of RhoprGpat1 results in 50% and 65% decrease in TAG content in the posterior midgut and fat body, respectively. RhoprGpat1-deficient insects also exhibits impaired lipid droplet expansion and a 2-fold increase in fatty acid ß-oxidation rates in the fat body. We propose that the RhoprGPAT1 mitochondrial-like isoform is required to channel fatty acyl chains towards TAG synthesis and away from ß-oxidation. Such a process is crucial for the insect lipid homeostasis.
Assuntos
Corpo Adiposo/metabolismo , Ácidos Graxos/metabolismo , Glicerol-3-Fosfato O-Aciltransferase/metabolismo , Insetos/metabolismo , Rhodnius/metabolismo , Triglicerídeos/metabolismo , Animais , Retículo Endoplasmático/metabolismo , Gotículas Lipídicas/metabolismo , Mitocôndrias/metabolismo , OxirreduçãoRESUMO
Glycerol-3-phosphate acyltransferase-4 (GPAT4) null pups grew poorly during the suckling period and, as adults, were protected from high fat diet-induced obesity. To determine why Gpat4(-/-) mice failed to gain weight during these two periods of high fat feeding, we examined energy metabolism. Compared with controls, the metabolic rate of Gpat4(-/-) mice fed a 45% fat diet was 12% higher. Core body temperature was 1 ºC higher after high fat feeding. Food intake, fat absorption, and activity were similar in both genotypes. Impaired weight gain in Gpat4(-/-) mice did not result from increased heat loss, because both cold tolerance and response to a ß3-adrenergic agonist were similar in both genotypes. Because GPAT4 comprises 65% of the total GPAT activity in brown adipose tissue (BAT), we characterized BAT function. A 45% fat diet increased the Gpat4(-/-) BAT expression of peroxisome proliferator-activated receptor α (PPAR) target genes, Cpt1α, Pgc1α, and Ucp1, and BAT mitochondria oxidized oleate and pyruvate at higher rates than controls, suggesting that fatty acid signaling and flux through the TCA cycle were enhanced. To assess the role of GPAT4 directly, neonatal BAT preadipocytes were differentiated to adipocytes. Compared with controls, Gpat4(-/-) brown adipocytes incorporated 33% less fatty acid into triacylglycerol and 46% more into the pathway of ß-oxidation. The increased oxidation rate was due solely to an increase in the oxidation of exogenous fatty acids. These data suggest that in the absence of cold exposure, GPAT4 limits excessive fatty acid oxidation and the detrimental induction of a hypermetabolic state.
Assuntos
Adipócitos/metabolismo , Tecido Adiposo Marrom/metabolismo , Ácidos Graxos/metabolismo , Glicerol-3-Fosfato O-Aciltransferase/metabolismo , Adipócitos/enzimologia , Tecido Adiposo Marrom/citologia , Tecido Adiposo Marrom/enzimologia , Animais , Gorduras na Dieta/administração & dosagem , Glicerol-3-Fosfato O-Aciltransferase/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxirredução , Termogênese/genética , Triglicerídeos/metabolismo , Aumento de PesoRESUMO
Meeting the complex physiological demands of mammalian life requires strict control of the metabolism of long-chain fatty acyl-CoAs because of the multiplicity of their cellular functions. Acyl-CoAs are substrates for energy production; stored within lipid droplets as triacylglycerol, cholesterol esters, and retinol esters; esterified to form membrane phospholipids; or used to activate transcriptional and signaling pathways. Indirect evidence suggests that acyl-CoAs do not wander freely within cells, but instead, are channeled into specific pathways. In this review, we will discuss the evidence for acyl-CoA compartmentalization, highlight the key modes of acyl-CoA regulation, and diagram potential mechanisms for controlling acyl-CoA partitioning.
Assuntos
Acil Coenzima A/metabolismo , Compartimento Celular , Acilação , Animais , Meios de Cultura , Homeostase , Camundongos , Camundongos Knockout , Transdução de Sinais , Especificidade por Substrato , TermogêneseRESUMO
Long-chain fatty acyl-coenzyme As (CoAs) are critical regulatory molecules and metabolic intermediates. The initial step in their synthesis is the activation of fatty acids by one of 13 long-chain acyl-CoA synthetase isoforms. These isoforms are regulated independently and have different tissue expression patterns and subcellular locations. Their acyl-CoA products regulate metabolic enzymes and signaling pathways, become oxidized to provide cellular energy, and are incorporated into acylated proteins and complex lipids such as triacylglycerol, phospholipids, and cholesterol esters. Their differing metabolic fates are determined by a network of proteins that channel the acyl-CoAs toward or away from specific metabolic pathways and serve as the basis for partitioning. This review evaluates the evidence for acyl-CoA partitioning by reviewing experimental data on proteins that are believed to contribute to acyl-CoA channeling, the metabolic consequences of loss of these proteins, and the potential role of maladaptive acyl-CoA partitioning in the pathogenesis of metabolic disease and carcinogenesis.
Assuntos
Acil Coenzima A/metabolismo , Membrana Celular/metabolismo , Coenzima A Ligases/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Transporte de Ácido Graxo/metabolismo , Metabolismo dos Lipídeos , Modelos Biológicos , Animais , Membrana Celular/enzimologia , Coenzima A Ligases/genética , Retículo Endoplasmático/enzimologia , Proteínas de Transporte de Ácido Graxo/genética , Regulação da Expressão Gênica , Humanos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte ProteicoRESUMO
Glucose-stimulated insulin secretion (GSIS) in pancreatic beta-cells is potentiated by fatty acids (FA). The initial step in the metabolism of intracellular FA is the conversion to acyl-CoA by long chain acyl-CoA synthetases (Acsls). Because the predominantly expressed Acsl isoforms in INS 832/13 cells are Acsl4 and -5, we characterized the role of these Acsls in beta-cell function by using siRNA to knock down Acsl4 or Acsl5. Compared with control cells, an 80% suppression of Acsl4 decreased GSIS and FA-potentiated GSIS by 32 and 54%, respectively. Knockdown of Acsl5 did not alter GSIS. Acsl4 knockdown did not alter FA oxidation or long chain acyl-CoA levels. With Acsl4 knockdown, incubation with 17 mm glucose increased media epoxyeicosatrienoic acids (EETs) and reduced cell membrane levels of EETs. Further, exogenous EETs reduced GSIS in INS 832/13 cells, and in Acsl4 knockdown cells, an EET receptor antagonist partially rescued GSIS. These results strongly suggest that Acsl4 activates EETs to form EET-CoAs that are incorporated into glycerophospholipids, thereby sequestering EETs. Exposing INS 832/13 cells to arachidonate or linoleate reduced Acsl4 mRNA and protein expression and reduced GSIS. These data indicate that Acsl4 modulates GSIS by regulating the levels of unesterified EETs and that arachidonate controls the expression of its activator Acsl4.
Assuntos
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Coenzima A Ligases/metabolismo , Glucose/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Insulina/metabolismo , Ácido 8,11,14-Eicosatrienoico/metabolismo , Acil Coenzima A/metabolismo , Animais , Western Blotting , Linhagem Celular Tumoral , Coenzima A Ligases/genética , Ácidos Graxos/metabolismo , Ácidos Graxos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glicerofosfolipídeos/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Insulinoma/genética , Insulinoma/metabolismo , Insulinoma/patologia , Lipídeos de Membrana/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Oxirredução , Interferência de RNA , Ratos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Glycerol-3-phosphate acyltransferase (GPAT) activity is highly induced in obese individuals with insulin resistance, suggesting a correlation between GPAT function, triacylglycerol accumulation, and insulin resistance. We asked whether microsomal GPAT4, an isoform regulated by insulin, might contribute to the development of hepatic insulin resistance. Compared with control mice fed a high fat diet, Gpat4(-/-) mice were more glucose tolerant and were protected from insulin resistance. Overexpression of GPAT4 in mouse hepatocytes impaired insulin-suppressed gluconeogenesis and insulin-stimulated glycogen synthesis. Impaired glucose homeostasis was coupled to inhibited insulin-stimulated phosphorylation of Akt(Ser47³) and Akt(Thr³°8). GPAT4 overexpression inhibited rictor's association with the mammalian target of rapamycin (mTOR), and mTOR complex 2 (mTORC2) activity. Compared with overexpressed GPAT3 in mouse hepatocytes, GPAT4 overexpression increased phosphatidic acid (PA), especially di16:0-PA. Conversely, in Gpat4(-/-) hepatocytes, both mTOR/rictor association and mTORC2 activity increased, and the content of PA in Gpat4(-/-) hepatocytes was lower than in controls, with the greatest decrease in 16:0-PA species. Compared with controls, liver and skeletal muscle from Gpat4(-/-)-deficient mice fed a high-fat diet were more insulin sensitive and had a lower hepatic content of di16:0-PA. Taken together, these data demonstrate that a GPAT4-derived lipid signal, likely di16:0-PA, impairs insulin signaling in mouse liver and contributes to hepatic insulin resistance.
Assuntos
Proteínas de Transporte/metabolismo , Glicerol-3-Fosfato O-Aciltransferase/metabolismo , Hepatócitos/efeitos dos fármacos , Hipoglicemiantes/farmacologia , Resistência à Insulina , Insulina/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Animais , Células Cultivadas , Dieta Hiperlipídica/efeitos adversos , Feminino , Glicerol-3-Fosfato O-Aciltransferase/genética , Hepatócitos/citologia , Hepatócitos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/etiologia , Obesidade/metabolismo , Ácidos Fosfatídicos/metabolismo , Proteína Companheira de mTOR Insensível à Rapamicina , Proteínas Recombinantes/metabolismo , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
Cells adjust their metabolism by remodeling membrane contact sites that channel metabolites to different fates. Lipid droplet (LD)-mitochondria contacts change in response to fasting, cold exposure, and exercise. However, their function and mechanism of formation have remained controversial. We focused on perilipin 5 (PLIN5), an LD protein that tethers mitochondria, to probe the function and regulation of LD-mitochondria contacts. We demonstrate that efficient LD-to-mitochondria fatty acid (FA) trafficking and ß-oxidation during starvation of myoblasts are promoted by phosphorylation of PLIN5 and require an intact PLIN5 mitochondrial tethering domain. Using human and murine cells, we further identified the acyl-CoA synthetase, FATP4 (ACSVL4), as a mitochondrial interactor of PLIN5. The C-terminal domains of PLIN5 and FATP4 constitute a minimal protein interaction capable of inducing organelle contacts. Our work suggests that starvation leads to phosphorylation of PLIN5, lipolysis, and subsequent channeling of FAs from LDs to FATP4 on mitochondria for conversion to fatty-acyl-CoAs and subsequent oxidation.
Assuntos
Gotículas Lipídicas , Perilipina-5 , Animais , Humanos , Camundongos , Proteínas de Transporte/metabolismo , Ácidos Graxos/metabolismo , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos , Mitocôndrias/metabolismo , Perilipina-5/metabolismoRESUMO
Although the underlying causes of insulin resistance have not been completely delineated, in most analyses, a recurring theme is dysfunctional metabolism of fatty acids. Because the conversion of fatty acids to activated acyl-CoAs is the first and essential step in the metabolism of long-chain fatty acid metabolism, interest has grown in the synthesis of acyl-CoAs, their contribution to the formation of signaling molecules like ceramide and diacylglycerol, and their direct effects on cell function. In this review, we cover the evidence for the involvement of acyl-CoAs in what has been termed lipotoxicity, the regulation of the acyl-CoA synthetases, and the emerging functional roles of acyl-CoAs in the major tissues that contribute to insulin resistance and lipotoxicity, adipose, liver, heart and pancreas.
Assuntos
Acil Coenzima A/metabolismo , Coenzima A Ligases/metabolismo , Metabolismo dos Lipídeos , Animais , Humanos , Resistência à InsulinaRESUMO
The consumption of linoleic acid (LA, ω-6 18:2), the most common ω-6 polyunsaturated fatty acid (PUFA) in the Modern Western diet (MWD), has significantly increased over the last century in tandem with unprecedented incidence of chronic metabolic diseases like obesity and type 2 diabetes mellitus (T2DM). Although an essential fatty acid for health, LA was a very rare fatty acid in the diet of humans during their evolution. While the intake of other dietary macronutrients (carbohydrates like fructose) has also risen, diets rich in ω-6 PUFAs have been promoted in an effort to reduce cardiovascular disease despite unclear evidence as to how increased dietary LA consumption could promote a proinflammatory state and affect glucose metabolism. Current evidence suggests that sex, genetics, environmental factors, and disease status can differentially modulate how LA influences insulin sensitivity and peripheral glucose uptake as well as insulin secretion and pancreatic beta-cell function. Therefore, the aim of this review will be to summarize recent additions to our knowledge to refine the unique physiological and pathophysiological roles of LA in the regulation of glucose homeostasis.
Assuntos
Glucose/metabolismo , Resistência à Insulina , Secreção de Insulina/efeitos dos fármacos , Ácido Linoleico/administração & dosagem , Dieta Ocidental , Feminino , Homeostase , Humanos , Ácido Linoleico/farmacologia , Masculino , Fatores de Risco , Caracteres SexuaisRESUMO
Exercise, typically beneficial for skeletal health, has not yet been studied in lipodystrophy, a condition characterized by paucity of white adipose tissue, with eventual diabetes, and steatosis. We applied a mouse model of global deficiency of Bscl2 (SEIPIN), required for lipid droplet formation. Male twelve-week-old B6 knockouts (KO) and wild type (WT) littermates were assigned six-weeks of voluntary, running exercise (E) versus non-exercise (N=5-8). KO weighed 14% less than WT (p=0.01) and exhibited an absence of epididymal adipose tissue; KO liver Plin1 via qPCR was 9-fold that of WT (p=0.04), consistent with steatosis. Bone marrow adipose tissue (BMAT), unlike white adipose, was measurable, although 40.5% lower in KO vs WT (p=0.0003) via 9.4T MRI/advanced image analysis. SEIPIN ablation's most notable effect marrow adiposity was in the proximal femoral diaphysis (-56% KO vs WT, p=0.005), with relative preservation in KO-distal-femur. Bone via µCT was preserved in SEIPIN KO, though some quality parameters were attenuated. Running distance, speed, and time were comparable in KO and WT. Exercise reduced weight (-24% WT-E vs WT p<0.001) but not in KO. Notably, exercise increased trabecular BV/TV in both (+31%, KO-E vs KO, p=0.004; +14%, WT-E vs WT, p=0.006). The presence and distribution of BMAT in SEIPIN KO, though lower than WT, is unexpected and points to a uniqueness of this depot. That trabecular bone increases were achievable in both KO and WT, despite a difference in BMAT quantity/distribution, points to potential metabolic flexibility during exercise-induced skeletal anabolism.
Assuntos
Tecido Adiposo/metabolismo , Medula Óssea/metabolismo , Osso Esponjoso/metabolismo , Fêmur/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/genética , Lipodistrofia/metabolismo , Condicionamento Físico Animal , Tecido Adiposo/diagnóstico por imagem , Tecido Adiposo/patologia , Animais , Peso Corporal , Medula Óssea/diagnóstico por imagem , Medula Óssea/patologia , Osso Esponjoso/diagnóstico por imagem , Diáfises/diagnóstico por imagem , Modelos Animais de Doenças , Epididimo/metabolismo , Epididimo/patologia , Fêmur/diagnóstico por imagem , Lipodistrofia/diagnóstico por imagem , Lipodistrofia/genética , Lipodistrofia/patologia , Masculino , Camundongos , Camundongos Knockout , Tamanho do Órgão , Perilipina-1/genética , Microtomografia por Raio-XRESUMO
OBJECTIVE: Regulation of fatty acid (FA) metabolism is central to adipocyte dysfunction during diet-induced obesity (DIO). Long-chain acyl-CoA synthetase-4 (ACSL4) has been hypothesized to modulate the metabolic fates of polyunsaturated FA (PUFA), including arachidonic acid (AA), but the in vivo actions of ACSL4 are unknown. The purpose of our studies was to determine the in vivo role of adipocyte ACSL4 in regulating obesity-associated adipocyte dysfunction. METHODS: We developed a novel mouse model with adipocyte-specific ablation of ACSL4 (Ad-KO) using loxP Cre recombinase technology. Metabolic phenotyping of Ad-KO mice relative to their floxed littermates (ACSL4floxed) was performed, including body weight and body composition over time; insulin and glucose tolerance tests; and energy expenditure, activity, and food intake in metabolic cages. Adipocytes were isolated for ex vivo adipocyte oxygen consumption by Clark electrode and lipidomics analysis. In vitro adipocyte analysis including oxygen consumption by Seahorse and real-time PCR analysis were performed to confirm our in vivo findings. RESULTS: Ad-KO mice were protected against DIO, adipocyte death, and metabolic dysfunction. Adipocytes from Ad-KO mice fed high-fat diet (HFD) had reduced incorporation of AA into phospholipids (PL), free AA, and levels of the AA lipid peroxidation product 4-hydroxynonenal (4-HNE). Additionally, adipocytes from Ad-KO mice fed HFD had reduced p53 activation and increased adipocyte oxygen consumption (OCR), which we demonstrated are direct effects of 4-HNE on adipocytes in vitro. CONCLUSION: These studies are the first to elucidate ACSL4's in vivo actions to regulate the incorporation of AA into PL and downstream effects on DIO-associated adipocyte dysfunction. By reducing the incorporation of AA into PL and free fatty acid pools in adipocytes, Ad-KO mice were significantly protected against HFD-induced increases in adipose and liver fat accumulation, adipocyte death, gonadal white adipose tissue (gWAT) inflammation, and insulin resistance (IR). Additionally, deficiency of adipocyte ACSL4 expression in mice fed a HFD resulted in increased gWAT adipocyte OCR and whole body energy expenditure (EE).
Assuntos
Adipócitos/metabolismo , Coenzima A Ligases/genética , Obesidade/metabolismo , Células 3T3 , Adipócitos/patologia , Adiposidade , Animais , Células Cultivadas , Coenzima A Ligases/metabolismo , Dieta Hiperlipídica/efeitos adversos , Feminino , Resistência à Insulina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/etiologia , Obesidade/patologia , Consumo de Oxigênio , Fosfolipídeos/metabolismoRESUMO
BACKGROUND: Mutations in either of two genes comprising the STSL locus, ATP-binding cassette (ABC)-transporters ABCG5 (encoding sterolin-1) and ABCG8 (encoding sterolin-2), result in sitosterolemia, a rare autosomal recessive disorder of sterol trafficking characterized by increased plasma plant sterol levels. Based upon the genetics of sitosterolemia, ABCG5/sterolin-1 and ABCG8/sterolin-2 are hypothesized to function as obligate heterodimers. No phenotypic difference has yet been described in humans with complete defects in either ABCG5 or ABCG8. These proteins, based upon the defects in humans, are responsible for regulating dietary sterol entry and biliary sterol secretion. METHODS: In order to mimic the human disease, we created, by a targeted disruption, a mouse model of sitosterolemia resulting in Abcg8/sterolin-2 deficiency alone. Homozygous knockout mice are viable and exhibit sitosterolemia. RESULTS: Mice deficient in Abcg8 have significantly increased plasma and tissue plant sterol levels (sitosterol and campesterol) consistent with sitosterolemia. Interestingly, Abcg5/sterolin-1 was expressed in both liver and intestine in Abcg8/sterolin-2 deficient mice and continued to show an apical expression. Remarkably, Abcg8 deficient mice had an impaired ability to secrete cholesterol into bile, but still maintained the ability to secrete sitosterol. We also report an intermediate phenotype in the heterozygous Abcg8+/- mice that are not sitosterolemic, but have a decreased level of biliary sterol secretion relative to wild-type mice. CONCLUSION: These data indicate that Abcg8/sterolin-2 is necessary for biliary sterol secretion and that loss of Abcg8/sterolin-2 has a more profound effect upon biliary cholesterol secretion than sitosterol. Since biliary sitosterol secretion is preserved, although not elevated in the sitosterolemic mice, this observation suggests that mechanisms other than by Abcg8/sterolin-2 may be responsible for its secretion into bile.
Assuntos
Colesterol/análogos & derivados , Colesterol/metabolismo , Lipoproteínas/deficiência , Modelos Animais , Mutação/genética , Sitosteroides/sangue , 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/genética , Animais , Bile/metabolismo , Ácidos e Sais Biliares/metabolismo , Colesterol/sangue , Feminino , Expressão Gênica , Intestinos/química , Lipoproteínas/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fitosteróis/sangueRESUMO
BACKGROUND: The molecular mechanisms that regulate the entry of dietary sterols into the body and their removal via hepatobiliary secretion are now beginning to be defined. These processes are specifically disrupted in the rare autosomal recessive disease, Sitosterolemia (MIM 210250). Mutations in either, but not both, of two genes ABCG5 or ABCG8, comprising the STSL locus, are now known to cause this disease and their protein products are proposed to function as heterodimers. Under normal circumstances cholesterol, but not non-cholesterol sterols, is preferentially absorbed from the diet. Additionally, any small amounts of non-cholesterol sterols that are absorbed are rapidly taken up by the liver and preferentially excreted into bile. Based upon the defects in sitosterolemia, ABCG5 and ABCG8 serve specifically to exclude non-cholesterol sterol entry at the intestinal level and are involved in sterol excretion at the hepatobiliary level. METHODS: Here we report the biochemical and immuno-localization of ABCG5 and ABCG8 in human liver, gallbladder and intestine using cell fractionation and immunohistochemical analyses. RESULTS: We raised peptide antibodies against ABCG5 and ABCG8 proteins. Using human liver samples, cell fractionation studies showed both proteins are found in membrane fractions, but they did not co-localize with caveolin-rafts, ER, Golgi or mitochondrial markers. Although their distribution in the sub-fractions was similar, they were not completely contiguous. Immunohistochemical analyses showed that while both proteins were readily detectable in the liver, ABCG5 was found predominately lining canalicular membranes, whereas ABCG8 was found in association with bile duct epithelia. At the cellular level, ABCG5 appeared to be apically expressed, whereas ABCG8 had a more diffuse expression pattern. Both ABCG5 and ABCG8 appeared to localize apically as shown by co-localization with MRP2. The distribution patterns of ABCG5 and ABCG8 in the gallbladder were very similar to each other. In the small intestine both ABCG5 and ABCG8 appear to line the brush border. However, at the level of the enterocyte, the cellular distribution patterns of ABCG5 and ABCG8 differed, such that ABCG5 was more diffuse, but ABCG8 was principally apical. Using standard deglycosylation methods, ABCG5 and ABCG8 do not appear to be glycosylated, suggesting a difference between human and mouse proteins. CONCLUSION: We report the distribution patterns of ABCG5 and ABCG8 in human tissues. Cell fractionation studies showed that both proteins co-fractionated in general, but could also be found independent of each other. As predicted, they are expressed apically in both intestine and liver, although their intracellular expression patterns are not completely congruent. These studies support the concept of heterodimerization of ABCG5 and ABCG8, but also support the notion that these proteins may have an independent function.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Sistema Digestório/química , Sistema Digestório/metabolismo , Lipoproteínas/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/imunologia , Centrifugação com Gradiente de Concentração/métodos , Vesícula Biliar/química , Vesícula Biliar/metabolismo , Humanos , Imuno-Histoquímica/métodos , Intestino Delgado/química , Intestino Delgado/metabolismo , Iohexol/química , Lipoproteínas/imunologia , Fígado/química , Fígado/metabolismo , Extratos Hepáticos/química , Sacarose/químicaRESUMO
The metabolic syndrome, a cluster of metabolic derangements that include obesity, glucose intolerance, dyslipidemia and hypertension, is a major risk factor for cardiovascular disease. Insulin resistance has been proposed to be the common feature that links obesity to the metabolic syndrome, but the mechanism remains obscure. Although the excess content of triacylglycerol in muscle and liver is highly associated with insulin resistance in these tissues, triacylglycerol itself is not causal but merely a marker. Thus, attention has turned to the accumulation of cellular lipids known to have signaling roles. This review will discuss recent progress in understanding how glycerolipids and related lipid intermediates may impair insulin signaling.
RESUMO
The association of hepatic steatosis with hepatic insulin resistance and type 2 diabetes has prompted investigators to elucidate the underlying mechanism. In this review we focus on pathways of lipid metabolism, and we review recent data, primarily from mouse models, that link lipid intermediates with insulin resistance. Most of the studies that implicate acyl-CoA, lysophosphatidic acid, phosphatidic acid, diacylglycerol, or ceramide rely on indirect associations. Convincing data to support the hypothesis that specific lipid intermediates initiate pathways that alter insulin signaling will require studies in which the concentration of each purported signaling molecule can be manipulated independently.