RESUMO
OBJECTIVE: An increased ω6/ω3-polyunsaturated fatty acid ratio in the current Western diet is regarded as a critical epigenetic nutritional factor in the pathogenesis of several human lifestyle diseases, metabolic syndrome, cardiovascular disease, the central nervous system and the female and male reproductive systems. The impact of nutrient ω3-and ω6-PUFAs in the pathogenesis of dyslipoproteinemia and atherosclerosis has been a topic of intense efforts for several decades. Cellular homeostasis of the ω3-and ω6- PUFA pool is maintained by the synthesis of ω3-and ω6-PUFAs from essential fatty acids (EFA) (linoleic and α-linolenic acid) and their dietary supply. In this study, we used the auxotrophic Δ6-fatty acid desaturase- (FADS2) deficient mouse (fads2-/-), an unbiased model congenial for stringent feeding experiments, to investigate the molecular basis of the proposed protective role of dietary ω3-and ω6-PUFAs (Western diet) in the pathogenesis of multifactorial dyslipoproteinemia and atherosclerosis. We focused on the metabolic axis-liver endoplasmic reticulum (ER), serum lipoprotein system (Lp) and aorta vessel wall. Furthermore, we addressed the impact of the inactivated fads2-locus with inactivated PUFA synthesis on the development and progression of extended atherosclerosis in two different mouse mutants with disrupted cholesterol homeostasis, using the apoe-/- and ldlr-/- mutants and the fads2-/- x apoe-/- and fads2-/- x ldlr-/- double mutants. METHODS: Cohorts of +/+ and fads2-/- mice underwent two long-term dietary regimens: a) a PUFA-free standard chow diet containing only EFAs, essential for viability, and b) a high fat/high cholesterol (HFHC) diet, a mimicry of the human atherogenic "Western" diet. c) To study the molecular impact of PUFA synthesis deficiency on the development and progression of atherosclerosis in the hypercholesterolemic apoe-/- and ldlr-/- mouse models fed PUFA-free regular and sustained HFHC diets, we generated the fads2-/- x apoe-/- and the fads2-/- x ldlr-/- double knockout mutants. We assessed essential molecular, biochemical and cell biological links between the diet-induced modified lipidomes of the membrane systems of the endoplasmic reticulum/Golgi complex, the site of lipid synthesis, the PL monolayer and neutral lipid core of LD and serum-Lp profiles and cellular reactions in the aortic wall. RESULTS: ω3-and ω6-PUFA synthesis deficiency in the fads2-/- mouse causes a) hypocholesterolemia and hypotriglyceridemia, b) dyslipoproteinemia with a shift of high-density lipoprotein (HDL) to very low-density lipoprotein (VLDL)-enriched Lp-pattern and c) altered liver lipid droplet structures. d) Long-term HFHC diet does not trigger atherosclerotic plaque formation in the aortic arc, the thoracic and abdominal aorta of PUFA-deficient fads2-/- mice. Inactivation of the fads2-/- locus, abolishing systemic PUFA synthesis in the fads2-/- x apoe-/- and fads2-/- x ldlr-/- double knockout mouse lines. CONCLUSIONS: Deficiency of ω3-and ω6-PUFA in the fads2-/- mutant perturbs liver lipid metabolism, causes hypocholesterolemia and hypotriglyceridemia and renders the fads2-/- mutant resistant to sustained atherogenic HFHC diet. Neither PUFA-free regular nor long-term HFHC-diet impacts the apoe- and LDL-receptor deficiency-provoked hypercholesterolemia and atherosclerotic plaque formation, size and distribution in the aorta. Our study strongly suggests that the absence of PUFAs as highly vulnerable chemical targets of autoxidation attenuates inflammatory responses and the formation of atherosclerotic lesions. The cumulative data and insight into the molecular basis of the pleiotropic functions of PUFAs challenge a differentiated view of PUFAs as culprits or benefactors during a lifespan, pivotal for legitimate dietary recommendations.
Assuntos
Aterosclerose/metabolismo , Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos Ômega-3/biossíntese , Ácidos Graxos Ômega-6/biossíntese , Receptores de LDL/metabolismo , Animais , Colesterol na Dieta/efeitos adversos , Dieta Hiperlipídica/efeitos adversos , Ácidos Graxos Dessaturases/deficiência , Ácidos Graxos Dessaturases/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de LDL/deficiênciaRESUMO
OBJECTIVE: Polyunsaturated fatty acids (PUFAs), including essential fatty acids linoleic and α-linolenic acid and derived long chain and very long chain ω3-and ω6-polyunsaturated fatty acids, are vital structures in mammalian membrane systems and signaling molecules, pivotal in brain development, lipid, and energy metabolism and in female and male fertility during human evolution. Numerous nutritional studies suggest imbalance of PUFA metabolism as a critical factor in the pathogenesis of several human lifestyle diseases: dyslipoproteinemia, obesity, cardiovascular and neurodegenerative diseases, and infertility. The lack of unbiased animal models impedes molecular interpretation of the role of synthesized and dietary supplied PUFAs in these conditions. In this study, we used a Δ6 fatty acid desaturase (FADS2) deficient mouse mutant lacking key enzyme activity in the biosynthesis of ω3-and ω6-PUFAs from EFAs to address the molecular role of PUFAs in female and male fertility. Infertility is a hallmark of the pleiotropic but auxotrophic fads2-/- phenotype and is therefore helpful for stringent dietary studies on the role of individual PUFAs. METHODS: Feeding regimens: Age- and gender-matched infertile fads2-/- mice were maintained on defined diets, normal diet containing essential fatty acids, and supplemented with ω6-arachidonic acid, ω3-docosahexaenoic acid, and arachidonic/docosahexaenoic acid, starting (a) after weaning and (b) initiated in 4-month-old female and male fads2-/- mice. Phospho- and sphingolipidomes of ovarian and testicular membrane lipid bilayers in each cohort were established and the impact on the expression and topology of membrane marker proteins, membrane morphology, germ cell development, and female and male fertility in the respective cohorts was elaborated. RESULTS: PUFA synthesis deficiency caused a halt to folliculogenesis, atresia of oocytes, and infertility of fads2-/- female mice. A PUFA-deficient membrane lipid bilayer core structure led to the disassembly of the gap junction network of the follicular granulosa cells. In fads2-/- testis, the blood-testis barrier was disrupted and spermatogenesis arrested, leading to infertility. Sustained supply of combined AA and DHA remodeled the PUFA-deficient ovarian and testicular membrane lipidomes, facilitating the reassembly of the functional gap junction network for regular ovarian cycles and the reconstitution of the blood-testis barrier in Sertoli cells, reconstituting fertility not only in developing newborns, but surprisingly also in adult infertile fads2-/- mice. CONCLUSIONS: These findings demonstrate the previously unrecognized membrane structure-based molecular link between nutrient ω3-and ω6-PUFAs, gonadal membrane structures, and female and male fertility and might foster studies of the pivotal role of dietary PUFAs in human fertility.
Assuntos
Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos Insaturados/metabolismo , Fertilidade/fisiologia , Animais , Dieta , Suplementos Nutricionais , Ácidos Graxos Dessaturases/deficiência , Ácidos Graxos Ômega-3/metabolismo , Ácidos Graxos Ômega-6/metabolismo , Feminino , Gônadas/efeitos dos fármacos , Gônadas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , ObesidadeRESUMO
Mammalian ω3- and ω6-PUFAs are synthesized from essential fatty acids (EFAs) or supplied by the diet. PUFAs are constitutive elements of membrane architecture and precursors of lipid signaling molecules. EFAs and long-chain (LC)-PUFAs are precursors in the synthesis of endocannabinoid ligands of Gi/o protein-coupled cannabinoid receptor (CB)1 and CB2 in the endocannabinoid system, which critically regulate energy homeostasis as the metabolic signaling system in hypothalamic neuronal circuits and behavioral parameters. We utilized the auxotrophic fatty acid desaturase 2-deficient (fads2-/-) mouse, deficient in LC-PUFA synthesis, to follow the age-dependent dynamics of the PUFA pattern in the CNS-phospholipidome in unbiased dietary studies of three cohorts on sustained LC-PUFA-free ω6-arachidonic acid- and DHA-supplemented diets and their impact on the precursor pool of CB1 ligands. We discovered the transformation of eicosa-all cis-5,11,14-trienoic acid, uncommon in mammalian lipidomes, into two novel endocannabinoids, 20:35,11,14-ethanolamide and 2-20:35,11,14-glycerol. Their function as ligands of CB1 has been characterized in HEK293 cells. Labeling experiments excluded Δ8-desaturase activity and proved the position specificity of FADS2. The fads2-/- mutant might serve as an unbiased model in vivo in the development of novel CB1 agonists and antagonists.
Assuntos
Endocanabinoides/metabolismo , Ácidos Graxos Ômega-3/deficiência , Ácidos Graxos Ômega-6/deficiência , Receptor CB1 de Canabinoide/agonistas , Animais , Endocanabinoides/genética , Ácidos Graxos Dessaturases/deficiência , Ácidos Graxos Ômega-3/farmacologia , Ácidos Graxos Ômega-6/farmacologia , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Receptor CB1 de Canabinoide/genética , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/agonistas , Receptor CB2 de Canabinoide/genética , Receptor CB2 de Canabinoide/metabolismoRESUMO
Δ-6-fatty acid desaturase (FADS2) is the key enzyme in the biosynthesis of polyunsaturated fatty acids (PUFAs), the essential structural determinants of mammalian membrane lipid-bilayers. We developed the auxotrophic fads2(-/-) mouse mutant to assess the enigmatic role of ω3- and ω6-PUFAs in lipid homeostasis, membrane structure and function. Obesity resistance is another major phenotype of the fads2(-/-) mutant, the molecular basis of which is unknown. Phospholipidomic profiling of membrane systems of fads2(-/-)mice revealed diacylglycerol-structures, deprived of PUFAs but substituted with surrogate eicosa-5,11,14-trienoic acid. ω6-Arachidonic (AA) and ω3-docosahexaenoic acid (DHA) supplemented diets transformed fads2(-/-) into AA-fads2(-/-) and DHA-fads2(-/-) mutants. Severely altered phospholipid-bilayer structures of subcellular membranes of fads2(-/-) liver specifically interfered with maturation of transcription factor sterol-regulatory-element-binding protein, the key regulator of lipogenesis and lipid homeostasis. This study strengthens the concept that specific PUFA-substituted membrane phospholipid species are critical constituents of the structural platform operative in lipid homeostasis in normal and disease conditions.