RESUMO
OBJECTIVE: Freshly isolated endothelial cells from both conduit arteries and microvasculature were used to test the hypothesis that eNOS protein content and nitric oxide production in coronary endothelial cells increases with vessel radius. METHODS: Porcine hearts were obtained from a local abattoir. Large and small arteries as well as arterioles were dissected free of myocardium and homogenized as whole vessels. Additionally, endothelial cells were isolated from both conduit arteries and left ventricular myocardium by tissue digestion with collagenase, followed by endothelial cell isolation using biotinylated-anti-CD31 and streptavidin-coated paramagnetic beads. Purity of isolated endothelial cells was confirmed by immunofluorescence and immunoblot. RESULTS: In whole vessel lysate, immunoblot analysis revealed that protein content for eNOS was greater in arterioles compared to small and large arteries. Nitric oxide metabolites (nitrite plus nitrate; NOx) levels measured from whole vessel lysate decreased as vessel size increased, with both arterioles and small arteries displaying significantly greater NOx content than conduit. Consistent with our hypothesis, both eNOS protein level and NOx were significantly greater in endothelial cells isolated from conduit arteries compared with those from coronary microvasculature. Furthermore, confocal microscopy revealed that eNOS protein was present in all conduit and microvascular endothelial cells, although eNOS staining was less intense in microvascular cells than those of conduit artery. CONCLUSIONS: These findings demonstrate increased eNOS protein and NOx content in endothelial cells of conduit arteries compared with the microcirculation and underscore the importance of comparing endothelial-specific molecules in freshly isolated endothelial cells, rather than whole lysate of different sized vessels.
Assuntos
Arteríolas/enzimologia , Vasos Coronários/enzimologia , Células Endoteliais/enzimologia , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico/metabolismo , Animais , Arteríolas/citologia , Vasos Coronários/citologia , Nitratos/metabolismo , Nitritos/metabolismo , Sus scrofaRESUMO
Phytocannabinoids, such as Δ9-tetrahydrocannabinol (THC), bind and activate cannabinoid (CB) receptors, thereby "piggy-backing" on the same pathway's endogenous endocannabinoids (ECs). The recent discovery that liver fatty acid binding protein-1 (FABP1) is the major cytosolic "chaperone" protein with high affinity for both Δ9-THC and ECs suggests that Δ9-THC may alter hepatic EC levels. Therefore, the impact of Δ9-THC or EC treatment on the levels of endogenous ECs, such as N-arachidonoylethanolamide (AEA) and 2-arachidonoylglycerol (2-AG), was examined in cultured primary mouse hepatocytes from WT and Fabp1 gene-ablated (LKO) mice. Δ9-THC alone or 2-AG alone significantly increased AEA and especially 2-AG levels in WT hepatocytes. LKO alone markedly increased AEA and 2-AG levels. However, LKO blocked/diminished the ability of Δ9-THC to further increase both AEA and 2-AG. In contrast, LKO potentiated the ability of exogenous 2-AG to increase the hepatocyte level of AEA and 2-AG. These and other data suggest that Δ9-THC increases hepatocyte EC levels, at least in part, by upregulating endogenous AEA and 2-AG levels. This may arise from Δ9-THC competing with AEA and 2-AG binding to FABP1, thereby decreasing targeting of bound AEA and 2-AG to the degradative enzymes, fatty acid amide hydrolase and monoacylglyceride lipase, to decrease hydrolysis within hepatocytes.
Assuntos
Dronabinol/efeitos adversos , Endocanabinoides/metabolismo , Proteínas de Ligação a Ácido Graxo/deficiência , Proteínas de Ligação a Ácido Graxo/genética , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Animais , Dronabinol/farmacologia , Proteínas de Ligação a Ácido Graxo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Although serum Δ9-tetrahydrocannabinol (Δ9-THC) undergoes rapid hepatic clearance and metabolism, almost nothing is known regarding the mechanism(s) whereby this highly lipophilic phytocannabinoid is transported for metabolism/excretion. A novel NBD-arachidonoylethanolamide (NBD-AEA) fluorescence displacement assay showed that liver fatty acid binding protein (FABP1), the major hepatic endocannabinoid (EC) binding protein, binds the first major metabolite of Δ9-THC (Δ9-THC-OH) as well as Δ9-THC itself. Circular dichroism (CD) confirmed that not only Δ9-THC and Δ9-THC-OH but also downstream metabolites Δ9-THC-COOH and Δ9-THC-CO-glucuronide directly interact with FABP1. Δ9-THC and metabolite interaction differentially altered the FABP1 secondary structure, increasing total α-helix (all), decreasing total ß-sheet (Δ9-THC-COOH, Δ9-THC-CO-glucuronide), increasing turns (Δ9-THC-OH, Δ9-THC-COOH, Δ9-THC-CO-glucuronide), and decreasing unordered structure (Δ9-THC, Δ9-THC-OH). Cultured primary hepatocytes from wild-type (WT) mice took up and converted Δ9-THC to the above metabolites. Fabp1 gene ablation (LKO) dramatically increased hepatocyte accumulation of Δ9-THC and even more so its primary metabolites Δ9-THC-OH and Δ9-THC-COOH. Concomitantly, rtPCR and Western blotting indicated that LKO significantly increased Δ9-THC's ability to regulate downstream nuclear receptor transcription of genes important in both EC ( Napepld > Daglb > Dagla, Naaa, Cnr1) and lipid ( Cpt1A > Fasn, FATP4) metabolism. Taken together, the data indicated that FABP1 may play important roles in Δ9-THC uptake and elimination as well as Δ9-THC induction of genes regulating hepatic EC levels and downstream targets in lipid metabolism.
Assuntos
Dronabinol , Proteínas de Ligação a Ácido Graxo , Hepatócitos/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Animais , Dicroísmo Circular , Dronabinol/farmacocinética , Dronabinol/farmacologia , Proteínas de Ligação a Ácido Graxo/química , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Humanos , Camundongos , Camundongos Knockout , Estrutura Secundária de ProteínaRESUMO
Although singly ablating Fabp1 or Scp2/Scpx genes may exacerbate the impact of high fat diet (HFD) on whole body phenotype and non-alcoholic fatty liver disease (NAFLD), concomitant upregulation of the non-ablated gene, preference for ad libitum fed HFD, and sex differences complicate interpretation. Therefore, these issues were addressed in male and female mice ablated in both genes (Fabp1/Scp2/Scpx null or TKO) and pair-fed HFD. Wild-type (WT) males gained more body weight as fat tissue mass (FTM) and exhibited higher hepatic lipid accumulation than WT females. The greater hepatic lipid accumulation in WT males was associated with higher hepatic expression of enzymes in glyceride synthesis, higher hepatic bile acids, and upregulation of transporters involved in hepatic reuptake of serum bile acids. While TKO had little effect on whole body phenotype and hepatic bile acid accumulation in either sex, TKO increased hepatic accumulation of lipids in both, specifically phospholipid and cholesteryl esters in males and females and free cholesterol in females. TKO-induced increases in glycerides were attributed not only to complete loss of FABP1, SCP2 and SCPx, but also in part to sex-dependent upregulation of hepatic lipogenic enzymes. These data with WT and TKO mice pair-fed HFD indicate that: i) Sex significantly impacted the ability of HFD to increase body weight, induce hepatic lipid accumulation and increase hepatic bile acids; and ii) TKO exacerbated the HFD ability to induce hepatic lipid accumulation, regardless of sex, but did not significantly alter whole body phenotype in either sex.
Assuntos
Proteínas de Transporte/metabolismo , Colesterol/metabolismo , Gorduras na Dieta/efeitos adversos , Proteínas de Ligação a Ácido Graxo/metabolismo , Fígado/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Fosfolipídeos/metabolismo , Animais , Proteínas de Transporte/genética , Colesterol/genética , Gorduras na Dieta/farmacologia , Proteínas de Ligação a Ácido Graxo/genética , Feminino , Fígado/patologia , Masculino , Camundongos , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/induzido quimicamente , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologia , Fosfolipídeos/genéticaRESUMO
Dysregulation of the hepatic endocannabinoid (EC) system and high fat diet (HFD) are associated with non-alcoholic fatty liver disease. Liver cytosol contains high levels of two novel endocannabinoid binding proteins-liver fatty acid binding protein (FABP1) and sterol carrier protein-2 (SCP-2). While Fabp1 gene ablation significantly increases hepatic levels of arachidonic acid (ARA)-containing EC and sex-dependent response to pair-fed high fat diet (HFD), the presence of SCP-2 complicates interpretation. These issues were addressed by ablating Scp-2/Scp-x in Fabp1 null mice (TKO). In control-fed mice, TKO increased hepatic levels of arachidonoylethanolamide (AEA) in both sexes. HFD impacted hepatic EC levels by decreasing AEA in TKO females and decreasing 2-arachidonoyl glycerol (2-AG) in WT of both sexes. Only TKO males on HFD had increased hepatic 2-AG levels. Hepatic ARA levels were decreased in control-fed TKO of both sexes. Changes in hepatic AEA/2-AG levels were not associated with altered amounts of hepatic proteins involved in AEA/2-AG synthesis or degradation. These findings suggested that ablation of the Scp-2/Scp-x gene in Fabp1 null mice exacerbated hepatic EC accumulation and antagonized the impact of HFD on hepatic EC levels-suggesting both proteins play important roles in regulating the hepatic EC system.
Assuntos
Proteínas de Transporte/genética , Dieta Hiperlipídica , Gorduras na Dieta/metabolismo , Endocanabinoides/metabolismo , Proteínas de Ligação a Ácido Graxo/genética , Fígado/metabolismo , Animais , Proteínas de Transporte/metabolismo , Proteínas de Ligação a Ácido Graxo/metabolismo , Feminino , Deleção de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Using recombinant human wild-type fatty acid binding protein 1 (WT FABP1 T94T) and a variant (FABP1 T94A) protein, fluorescence binding assays, and circular dichroism, it was shown for the first time that WT FABP1 and the T94A variant each have a single, relatively hydrophobic site for binding fluorescent NBD-labeled analogues of N-arachidonoylethanolamide and 2-arachidonoylglycerol with high affinity. Most native N-acylethanolamides (NAEs) but only one 2-monoacylglycerol [i.e., 2-arachidonoylglycerol (2-AG)] displaced WT FABP1-bound fluorescently labeled endocannabinoids (ECs). While the T94A variant did not differ in affinity for AEA and most other NAEs, it exhibited a modestly higher affinity for OEA, as well as a higher affinity for 2-AG. Binding of AEA and 2-AG altered WT FABP1's secondary structure more extensively than any other previously examined ligand did. The T94A variant without a ligand was more susceptible to temperature-induced unfolding. While the T94A variant was much less sensitive to ligand (i.e., AEA or 2-AG)-induced conformational change, nevertheless binding of AEA and 2-AG significantly stabilized the T94A structure to thermal unfolding. These data provide the first evidence that ECs not only bind to but also alter the secondary structure of the human FABP1, with the latter markedly impacted by the T94A substitution, a variant strongly associated with hepatic accumulation of lipids and non-alcoholic fatty liver disease (NAFLD). Importantly, NAFLD has been associated with elevated hepatic levels of ECs and FABP1.
Assuntos
Endocanabinoides/metabolismo , Proteínas de Ligação a Ácido Graxo/química , Proteínas de Ligação a Ácido Graxo/metabolismo , Animais , Ácidos Araquidônicos/metabolismo , Sítios de Ligação , Dicroísmo Circular , Endocanabinoides/química , Proteínas de Ligação a Ácido Graxo/genética , Fluorescência , Glicerídeos/metabolismo , Humanos , Alcamidas Poli-Insaturadas/metabolismo , Estrutura Secundária de Proteína , Ratos , TemperaturaRESUMO
Studies in vitro have suggested that both sterol carrier protein-2/sterol carrier protein-x (Scp-2/Scp-x) and liver fatty acid binding protein [Fabp1 (L-FABP)] gene products facilitate hepatic uptake and metabolism of lipotoxic dietary phytol. However, interpretation of physiological function in mice singly gene ablated in the Scp-2/Scp-x has been complicated by concomitant upregulation of FABP1. The work presented herein provides several novel insights: i) An 8-anilino-1-naphthalenesulfonic acid displacement assay showed that neither SCP-2 nor L-FABP bound phytol, but both had high affinity for its metabolite, phytanic acid; ii) GC-MS studies with phytol-fed WT and Fabp1/Scp-2/SCP-x gene ablated [triple KO (TKO)] mice showed that TKO exacerbated hepatic accumulation of phytol metabolites in vivo in females and less so in males. Concomitantly, dietary phytol increased hepatic levels of total long-chain fatty acids (LCFAs) in both male and female WT and TKO mice. Moreover, in both WT and TKO female mice, dietary phytol increased hepatic ratios of saturated/unsaturated and polyunsaturated/monounsaturated LCFAs, while decreasing the peroxidizability index. However, in male mice, dietary phytol selectively increased the saturated/unsaturated ratio only in TKO mice, while decreasing the peroxidizability index in both WT and TKO mice. These findings suggested that: 1) SCP-2 and FABP1 both facilitated phytol metabolism after its conversion to phytanic acid; and 2) SCP-2/SCP-x had a greater impact on hepatic phytol metabolism than FABP1.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ligação a Ácido Graxo/deficiência , Proteínas de Ligação a Ácido Graxo/genética , Técnicas de Inativação de Genes , Fígado/metabolismo , Fitol/metabolismo , Animais , Retículo Endoplasmático/metabolismo , Feminino , Masculino , Camundongos , Peroxissomos/metabolismo , Ácido Fitânico/metabolismo , Especificidade por SubstratoRESUMO
Upregulation of the hepatic endocannabinoid (EC) receptor [cannabinoid receptor-1 (CB1)] and arachidonoylethanolamide (AEA) is associated with nonalcoholic fatty liver disease (NAFLD). Male mice fed high-fat diet (HFD) ad libitum also exhibit NAFLD, increased hepatic AEA, and obesity. But, preference for HFD complicates interpretation and almost nothing is known about these effects in females. These issues were addressed by pair-feeding HFD. Similarly to ad libitum-fed HFD, pair-fed HFD also increased WT male and female mouse fat tissue mass (FTM), but preferentially at the expense of lean tissue mass. In contrast, pair-fed HFD did not elicit NAFLD in WT mice regardless of sex. Concomitantly, pair-fed HFD oppositely impacted hepatic AEA, 2-arachidonoyl glycerol, and/or CB1 in WT males versus females. In pair-fed HFD mice, liver FA binding protein-1 (Fabp1) gene ablation (LKO): i) exacerbated FTM in both sexes; ii) did not elicit liver neutral lipid accumulation in males and only slightly in females; iii) increased liver AEA in males, but decreased it in females; and iv) decreased CB1 only in males. Thus, pair-fed HFD selectively impacted hepatic ECs more in females, but did not elicit NAFLD in either sex. These effects were modified by LKO consistent with FABP1's ability to impact EC and FA metabolism.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Endocanabinoides/metabolismo , Proteínas de Ligação a Ácido Graxo/deficiência , Proteínas de Ligação a Ácido Graxo/genética , Técnicas de Inativação de Genes , Fígado/efeitos dos fármacos , Fígado/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/genética , Biomarcadores/sangue , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Citosol/efeitos dos fármacos , Citosol/metabolismo , Ácidos Graxos/metabolismo , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/genética , Lipogênese/efeitos dos fármacos , Lipogênese/genética , Fígado/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Tamanho do Órgão/efeitos dos fármacos , Tamanho do Órgão/genética , Fenótipo , Proteína de Ligação a Elemento Regulador de Esterol 1/genéticaRESUMO
The endocannabinoid system shifts energy balance toward storage and fat accumulation, especially in the context of diet-induced obesity. Relatively little is known about factors outside the central nervous system that may mediate the effect of high-fat diet (HFD) on brain endocannabinoid levels. One candidate is the liver fatty acid binding protein (FABP1), a cytosolic protein highly prevalent in liver, but not detected in brain, which facilitates hepatic clearance of fatty acids. The impact of Fabp1 gene ablation (LKO) on the effect of high-fat diet (HFD) on brain and plasma endocannabinoid levels was examined and data expressed for each parameter as the ratio of high-fat diet/control diet. In male wild-type mice, HFD markedly increased brain N-acylethanolamides, but not 2-monoacylglycerols. LKO blocked these effects of HFD in male mice. In female wild-type mice, HFD slightly decreased or did not alter these endocannabinoids as compared with male wild type. LKO did not block the HFD effects in female mice. The HFD-induced increase in brain arachidonic acid-derived arachidonoylethanolamide in males correlated with increased brain-free and total arachidonic acid. The ability of LKO to block the HFD-induced increase in brain arachidonoylethanolamide correlated with reduced ability of HFD to increase brain-free and total arachidonic acid in males. In females, brain-free and total arachidonic acid levels were much less affected by either HFD or LKO in the context of HFD. These data showed that LKO markedly diminished the impact of HFD on brain endocannabinoid levels, especially in male mice.
Assuntos
Encéfalo/metabolismo , Endocanabinoides/metabolismo , Metabolismo Energético/fisiologia , Proteínas de Ligação a Ácido Graxo/metabolismo , Animais , Ácidos Araquidônicos/farmacologia , Dieta Hiperlipídica , Endocanabinoides/farmacologia , Feminino , Resistência à Insulina/fisiologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Obesidade/metabolismo , Alcamidas Poli-Insaturadas/farmacologia , Receptor CB1 de Canabinoide/metabolismoRESUMO
In vitro studies suggest that liver fatty acid binding protein (L-FABP) and sterol carrier protein-2/sterol carrier protein-x (SCP2/SCPx) gene products facilitate uptake and metabolism and detoxification of dietary-derived phytol in mammals. However, concomitant upregulation of L-FABP in SCP2/SCPx null mice complicates interpretation of their physiological phenotype. Therefore, the impact of ablating both the L-FABP gene and SCP2/SCPx gene (L-FABP/SCP2/SCPx null or TKO) was examined in phytol-fed female wild-type (WT) and TKO mice. TKO increased hepatic total lipid accumulation, primarily phospholipid, by mechanisms involving increased hepatic levels of proteins in the phospholipid synthetic pathway. Concomitantly, TKO reduced expression of proteins in targeting fatty acids towards the triacylglycerol synthetic pathway. Increased hepatic lipid accumulation was not associated with any concomitant upregulation of membrane fatty acid transport/translocase proteins involved in fatty acid uptake (FATP2, FATP4, FATP5 or GOT) or cytosolic proteins involved in fatty acid intracellular targeting (ACBP). In addition, TKO exacerbated dietary phytol-induced whole body weight loss, especially lean tissue mass. Since individually ablating SCPx or SCP2/SCPx elicited concomitant upregulation of L-FABP, these findings with TKO mice help to resolve the contributions of SCP2/SCPx gene ablation on dietary phytol-induced whole body and hepatic lipid phenotype independent of concomitant upregulation of L-FABP.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ligação a Ácido Graxo/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Fitol/administração & dosagem , Animais , Dieta/métodos , Ácidos Graxos/metabolismo , Feminino , Lipídeos/fisiologia , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfolipídeos/metabolismo , Triglicerídeos/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
While prior studies focusing on male mice suggest a role for sterol carrier protein-2/sterol carrier protein-x (SCP-2/SCP-x; DKO) on hepatic phytol metabolism, its role in females is unresolved. This issue was addressed using female and male wild-type (WT) and DKO mice fed a phytoestrogen-free diet without or with 0.5% phytol. GC/MS showed that hepatic: i) phytol was absent and its branched-chain fatty acid (BCFA) metabolites were barely detectable in WT control-fed mice; ii) accumulation of phytol as well as its peroxisomal metabolite BCFAs (phytanic acid ¼ pristanic and 2,3-pristenic acids) was increased by dietary phytol in WT females, but only slightly in WT males; iii) accumulation of phytol and BCFA was further increased by DKO in phytol-fed females, but much more markedly in males. Livers of phytol-fed WT female mice as well as phytol-fed DKO female and male mice also accumulated increased proportion of saturated straight-chain fatty acids (LCFA) at the expense of unsaturated LCFA. Liver phytol accumulation was not due to increased SCP-2 binding/transport of phytol since SCP-2 bound phytanic acid, but not its precursor phytol. Thus, the loss of Scp-2/Scp-x contributed to a sex-dependent hepatic accumulation of dietary phytol and BCFA.
Assuntos
Proteínas de Transporte/metabolismo , Metabolismo dos Lipídeos/fisiologia , Fígado/metabolismo , Ácido Fitânico/análogos & derivados , Fitol/farmacocinética , Administração Oral , Animais , Proteínas de Transporte/genética , Feminino , Inativação Gênica , Masculino , Taxa de Depuração Metabólica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ácido Fitânico/metabolismo , Fitol/administração & dosagem , Fatores SexuaisRESUMO
Endocannabinoids (ECs) and cannabinoids are very lipophilic molecules requiring the presence of cytosolic binding proteins that chaperone these molecules to intracellular targets. While three different fatty acid binding proteins (FABP3, -5, and -7) serve this function in brain, relatively little is known about how such hydrophobic ECs and cannabinoids are transported within the liver. The most prominent hepatic FABP, liver fatty acid binding protein (FABP1 or L-FABP), has high affinity for arachidonic acid (ARA) and ARA-CoA, suggesting that FABP1 may also bind ARA-derived ECs (AEA and 2-AG). Indeed, FABP1 bound ECs with high affinity as shown by displacement of FABP1-bound fluorescent ligands and by quenching of FABP1 intrinsic tyrosine fluorescence. FABP1 also had high affinity for most non-ARA-containing ECs, FABP1 inhibitors, EC uptake/hydrolysis inhibitors, and phytocannabinoids and less so for synthetic cannabinoid receptor (CBR) agonists and antagonists. The physiological impact was examined with liver from wild-type (WT) versus FABP1 gene-ablated (LKO) male mice. As shown by liquid chromatography and mass spectrometry, FABP1 gene ablation significantly increased hepatic levels of AEA, 2-AG, and 2-OG. These increases were not due to increased protein levels of EC synthetic enzymes (NAPEPLD and DAGL) or a decreased level of EC degradative enzyme (FAAH) but correlated with complete loss of FABP1, a decreased level of SCP2 (8-fold less prevalent than FABP1, but also binds ECs), and a decreased level of degradative enzymes (NAAA and MAGL). These data indicated that FABP1 not only is the most prominent endocannabinoid and cannabinoid binding protein but also impacts hepatic endocannabinoid levels.
Assuntos
Endocanabinoides/metabolismo , Proteínas de Ligação a Ácido Graxo/metabolismo , Receptores de Canabinoides/metabolismo , Animais , Feminino , Corantes Fluorescentes , Humanos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Although expression of the human liver fatty acid binding protein (FABP1) T94A variant alters serum lipoprotein cholesterol levels in human subjects, nothing is known whereby the variant elicits these effects. This issue was addressed by in vitro cholesterol binding assays using purified recombinant wild-type (WT) FABP1 T94T and T94A variant proteins and in cultured primary human hepatocytes expressing the FABP1 T94T (genotyped as TT) or T94A (genotyped as CC) proteins. The human FABP1 T94A variant protein had 3-fold higher cholesterol-binding affinity than the WT FABP1 T94T as shown by NBD-cholesterol fluorescence binding assays and by cholesterol isothermal titration microcalorimetry (ITC) binding assays. CC variant hepatocytes also exhibited 30% higher total FABP1 protein. HDL- and LDL-mediated NBD-cholesterol uptake was faster in CC variant than TT WT human hepatocytes. VLDL-mediated uptake of NBD-cholesterol did not differ between CC and TT human hepatocytes. The increased HDL- and LDL-mediated NBD-cholesterol uptake was not associated with any significant change in mRNA levels of SCARB1, LDLR, CETP, and LCAT encoding the key proteins in lipoprotein cholesterol uptake. Thus, the increased HDL- and LDL-mediated NBD-cholesterol uptake by CC hepatocytes may be associated with higher affinity of T94A protein for cholesterol and/or increased total T94A protein level.
Assuntos
Colesterol/metabolismo , Proteínas de Ligação a Ácido Graxo/genética , Hepatócitos/metabolismo , Mutação de Sentido Incorreto , Alanina/genética , Substituição de Aminoácidos , Animais , Transporte Biológico/genética , Células Cultivadas , Proteínas de Ligação a Ácido Graxo/metabolismo , Feminino , Humanos , Metabolismo dos Lipídeos/genética , Pessoa de Meia-Idade , Treonina/genéticaRESUMO
Liver fatty acid-binding protein (FABP1, L-FABP) has high affinity for and enhances uptake of arachidonic acid (ARA, C20:4, n-6) which, when esterified to phospholipids, is the requisite precursor for synthesis of endocannabinoids (EC) such as arachidonoylethanolamide (AEA) and 2-arachidonoylglycerol (2-AG). The brain derives most of its ARA from plasma, taking up ARA and transporting it intracellularly via cytosolic fatty acid-binding proteins (FABPs 3,5, and 7) localized within the brain. In contrast, the much more prevalent cytosolic FABP1 is not detectable in the brain but is instead highly expressed in the liver. Therefore, the possibility that FABP1 outside the central nervous system may regulate brain AEA and 2-AG was examined in wild-type (WT) and FABP1 null (LKO) male mice. LKO increased brain levels of AA-containing EC (AEA, 2-AG), correlating with increased free and total ARA in brain and serum. LKO also increased brain levels of non-ARA that contain potentiating endocannabinoids (EC*) such as oleoyl ethanolamide (OEA), PEA, 2-OG, and 2-PG. Concomitantly, LKO decreased serum total ARA-containing EC, but not non-ARA endocannabinoids. LKO did not elicit these changes in the brain EC and EC* as a result of compensatory up-regulation of brain protein levels of enzymes in EC synthesis (NAPEPLD, DAGLα) or cytosolic EC chaperone proteins (FABPs 3, 5, 7, SCP-2, HSP70), or cannabinoid receptors (CB1, TRVP1). These data show for the first time that the non-CNS fatty acid-binding protein FABP1 markedly affected brain levels of both ARA-containing endocannabinoids (AEA, 2-AG) as well as their non-ARA potentiating endocannabinoids. Fatty acid-binding protein-1 (FABP-1) is not detectable in brain but instead is highly expressed in liver. The possibility that FABP1 outside the central nervous system may regulate brain endocannabinoids arachidonoylethanolamide (AEA) and 2-arachidonoylglycerol (2-AG) was examined in wild-type (WT) and FABP-1 null (LKO) male mice. LKO increased brain levels of arachidonic acid-containing endocannabinoids (AEA, 2-AG), correlating with increased free and total arachidonic acid in brain and serum. Read the Editorial Highlight for this article on page 371.
Assuntos
Ácidos Araquidônicos/metabolismo , Encéfalo/metabolismo , Endocanabinoides/metabolismo , Proteínas de Ligação a Ácido Graxo/genética , Fígado/metabolismo , Ácidos Oleicos/metabolismo , Alcamidas Poli-Insaturadas/metabolismo , Animais , Ácidos Araquidônicos/genética , Encéfalo/efeitos dos fármacos , Endocanabinoides/genética , Glicerídeos/metabolismo , Fígado/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
While a high-cholesterol diet induces hepatic steatosis, the role of intracellular sterol carrier protein-2/sterol carrier protein-x (SCP-2/SCP-x) proteins is unknown. We hypothesized that ablating SCP-2/SCP-x [double knockout (DKO)] would impact hepatic lipids (cholesterol and cholesteryl ester), especially in high-cholesterol-fed mice. DKO did not alter food consumption, and body weight (BW) gain decreased especially in females, concomitant with hepatic steatosis in females and less so in males. DKO-induced steatosis in control-fed wild-type (WT) mice was associated with 1) loss of SCP-2; 2) upregulation of liver fatty acid binding protein (L-FABP); 3) increased mRNA and/or protein levels of sterol regulatory element binding proteins (SREBP1 and SREBP2) as well as increased expression of target genes of cholesterol synthesis (Hmgcs1 and Hmgcr) and fatty acid synthesis (Acc1 and Fas); and 4) cholesteryl ester accumulation was also associated with increased acyl-CoA cholesterol acyltransferase-2 (ACAT2) in males. DKO exacerbated the high-cholesterol diet-induced hepatic cholesterol and glyceride accumulation, without further increasing SREBP1, SREBP2, or target genes. This exacerbation was associated both with loss of SCP-2 and concomitant downregulation of Ceh/Hsl, apolipoprotein B (ApoB), MTP, and/or L-FABP protein expression. DKO diminished the ability to secrete excess cholesterol into bile and oxidize cholesterol to bile acid for biliary excretion, especially in females. This suggested that SCP-2/SCP-x affects cholesterol transport to particular intracellular compartments, with ablation resulting in less to the endoplasmic reticulum for SREBP regulation, making more available for cholesteryl ester synthesis, for cholesteryl-ester storage in lipid droplets, and for bile salt synthesis and/or secretion. These alterations are significant findings, since they affect key processes in regulation of sterol metabolism.
Assuntos
Proteínas de Transporte/metabolismo , Colesterol na Dieta/farmacologia , Metabolismo dos Lipídeos , Fígado/metabolismo , Acetiltransferases/genética , Acetiltransferases/metabolismo , Animais , Apolipoproteína B-100 , Apolipoproteínas B/genética , Apolipoproteínas B/metabolismo , Proteínas de Transporte/genética , Colesterol na Dieta/metabolismo , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Feminino , Hidroximetilglutaril-CoA Redutases/genética , Hidroximetilglutaril-CoA Redutases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Esterol O-Aciltransferase/genética , Esterol O-Aciltransferase/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Receptor fas/genética , Receptor fas/metabolismo , Esterol O-Aciltransferase 2RESUMO
Although roles for both sterol carrier protein-2/sterol carrier protein-x (SCP-2/SCP-x) and liver fatty acid binding protein (L-FABP) have been proposed in hepatic lipid accumulation, individually ablating these genes has been complicated by concomitant alterations in the other gene product(s). For example, ablating SCP2/SCP-x induces upregulation of L-FABP in female mice. Therefore, the impact of ablating SCP-2/SCP-x (DKO) or L-FABP (LKO) individually or both together (TKO) was examined in female mice. Loss of SCP-2/SCP-x (DKO, TKO) more so than loss of L-FABP alone (LKO) increased hepatic total lipid and total cholesterol content, especially cholesteryl ester. Hepatic accumulation of nonesterified long chain fatty acids (LCFA) and phospholipids occurred only in DKO and TKO mice. Loss of SCP-2/SCP-x (DKO, TKO) increased serum total lipid primarily by increasing triglycerides. Altered hepatic level of proteins involved in cholesterol uptake, efflux, and/or secretion was observed, but did not compensate for the loss of L-FABP, SCP-2/SCP-x or both. However, synergistic responses were not seen with the combinatorial knock out animals-suggesting that inhibiting SCP-2/SCP-x is more correlative with hepatic dysfunction than L-FABP. The DKO- and TKO-induced hepatic accumulation of cholesterol and long chain fatty acids shared significant phenotypic similarities with non-alcoholic fatty liver disease (NAFLD).
Assuntos
Proteínas de Transporte/genética , Proteínas de Ligação a Ácido Graxo/genética , Metabolismo dos Lipídeos/genética , Fígado/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Animais , Proteínas de Transporte/metabolismo , Colesterol/sangue , Ésteres do Colesterol/sangue , Modelos Animais de Doenças , Proteínas de Ligação a Ácido Graxo/deficiência , Ácidos Graxos não Esterificados/sangue , Feminino , Deleção de Genes , Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologia , Fosfolipídeos/sangue , Triglicerídeos/sangueRESUMO
Both sterol carrier protein-2/sterol carrier protein-x (SCP-2/SCP-x) and liver fatty acid binding protein (L-FABP) have been proposed to function in hepatobiliary bile acid metabolism/accumulation. To begin to address this issue, the impact of ablating L-FABP (LKO) or SCP-2/SCP-x (DKO) individually or both together (TKO) was examined in female mice. Biliary bile acid levels were decreased in LKO, DKO, and TKO mice; however, hepatic bile acid concentration was decreased in LKO mice only. In contrast, biliary phospholipid level was decreased only in TKO mice, while biliary cholesterol levels were unaltered regardless of phenotype. The loss of either or both genes increased hepatic expression of the major bile acid synthetic enzymes (CYP7A1 and/or CYP27A1). Loss of L-FABP and/or SCP-2/SCP-x genes significantly altered the molecular composition of biliary bile acids, but not the proportion of conjugated/unconjugated bile acids or overall bile acid hydrophobicity index. These data suggested that L-FABP was more important in hepatic retention of bile acids, while SCP-2/SCP-x more broadly affected biliary bile acid and phospholipid levels.
Assuntos
Sistema Biliar/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ligação a Ácido Graxo/metabolismo , Fígado/metabolismo , Animais , Ácidos e Sais Biliares/química , Ácidos e Sais Biliares/metabolismo , Proteínas de Transporte/genética , Colesterol/metabolismo , Proteínas de Ligação a Ácido Graxo/deficiência , Proteínas de Ligação a Ácido Graxo/genética , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Fosfolipídeos/metabolismoRESUMO
Although liver fatty acid binding protein (L-FABP) binds fibrates and PPARα in vitro and enhances fibrate induction of PPARα in transformed cells, the functional significance of these findings is unclear, especially in normal hepatocytes. Studies with cultured primary mouse hepatocytes show that: 1) At physiological (6mM) glucose, fibrates (bezafibrate, fenofibrate) only weakly activated PPARα transcription of genes in LCFA ß-oxidation; 2) High (11-20mM) glucose, but not maltose (osmotic control), significantly potentiated fibrate-induction of mRNA of these and other PPARα target genes to increase LCFA ß-oxidation. These effects were associated with fibrate-mediated redistribution of L-FABP into nuclei-an effect prolonged by high glucose-but not with increased de novo fatty acid synthesis from glucose; 3) Potentiation of bezafibrate action by high glucose required an intact L-FABP/PPARα signaling pathway as shown with L-FABP null, PPARα null, PPARα inhibitor-treated WT, or PPARα-specific fenofibrate-treated WT hepatocytes. High glucose alone in the absence of fibrate was ineffective. Thus, high glucose potentiation of PPARα occurred through FABP/PPARα rather than indirectly through other PPARs or glucose induced signaling pathways. These data indicated L-FABP's importance in fibrate-induction of hepatic PPARα LCFA ß-oxidative genes, especially in the context of high glucose levels.
Assuntos
Bezafibrato/farmacologia , Proteínas de Ligação a Ácido Graxo/metabolismo , Fenofibrato/farmacologia , Glucose/farmacologia , Hepatócitos/metabolismo , Hipolipemiantes/farmacologia , PPAR alfa/metabolismo , Edulcorantes/farmacologia , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas de Ligação a Ácido Graxo/genética , Ácidos Graxos/genética , Ácidos Graxos/metabolismo , Hepatócitos/citologia , Camundongos , Camundongos Mutantes , Oxirredução/efeitos dos fármacos , PPAR alfa/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
On the basis of their abilities to bind bile acids and/or cholesterol, the physiological role(s) of liver fatty acid-binding protein (L-FABP) and sterol carrier protein (SCP) 2/SCP-x (SCP-2/SCP-x) gene products in biliary bile acid and cholesterol formation was examined in gene-ablated male mice. L-FABP (LKO) or L-FABP/SCP-2/SCP-x [triple-knockout (TKO)] ablation markedly decreased hepatic bile acid concentration, while SCP-2/SCP-x [double-knockout (DKO)] ablation alone had no effect. In contrast, LKO increased biliary bile acid, while DKO and TKO had no effect on biliary bile acid levels. LKO and DKO also altered biliary bile acid composition to increase bile acid hydrophobicity. Furthermore, LKO and TKO decreased hepatic uptake and biliary secretion of high-density lipoprotein (HDL)-derived 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3ß-ol (NBD-cholesterol), while DKO alone had no effect. Finally, LKO and, to a lesser extent, DKO decreased most indexes contributing to cholesterol solubility in biliary bile. These results suggest different, but complementary, roles for L-FABP and SCP-2/SCP-x in biliary bile acid and cholesterol formation. L-FABP appears to function more in hepatic retention of bile acids as well as hepatic uptake and biliary secretion of HDL-cholesterol. Conversely, SCP-2/SCP-x may function more in formation and biliary secretion of bile acid, with less impact on hepatic uptake or biliary secretion of HDL-cholesterol.
Assuntos
Ácidos e Sais Biliares/metabolismo , Bile/metabolismo , Proteínas de Transporte/fisiologia , HDL-Colesterol/metabolismo , Proteínas de Ligação a Ácido Graxo/fisiologia , Animais , Proteínas de Transporte/genética , Colesterol/metabolismo , Proteínas de Ligação a Ácido Graxo/genética , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfolipídeos/metabolismoRESUMO
Although human liver fatty acid-binding protein (FABP1) T94A variant has been associated with nonalcoholic fatty liver disease and reduced ability of fenofibrate to lower serum triglycerides (TG) to target levels, molecular events leading to this phenotype are poorly understood. Cultured primary hepatocytes from female human subjects expressing the FABP1 T94A variant exhibited increased neutral lipid (TG, cholesteryl ester) accumulation associated with (1) upregulation of total FABP1, a key protein stimulating mitochondrial glycerol-3-phosphate acyltransferase (GPAM), the rate-limiting enzyme in lipogenesis; (2) increased mRNA expression of key enzymes in lipogenesis (GPAM, LPIN2) in heterozygotes; (3) decreased mRNA expression of microsomal triglyceride transfer protein; (4) increased secretion of ApoB100 but not TG; (5) decreased long-chain fatty acid (LCFA) ß-oxidation. TG accumulation was not due to any increase in LCFA uptake, de novo lipogenesis, or the alternate monoacylglycerol O-acyltransferase pathway in lipogenesis. Despite increased expression of total FABP1 mRNA and protein, fenofibrate-mediated FABP1 redistribution to nuclei and ligand-induced peroxisome proliferator-activated receptor (PPAR-α) transcription of LCFA ß-oxidative enzymes (carnitine palmitoyltransferase 1A, carnitine palmitoyltransferase 2, and acyl-coenzyme A oxidase 1, palmitoyl) were attenuated in FABP1 T94A hepatocytes. Although the phenotype of FABP1 T94A variant human hepatocytes exhibits some similarities to that of FABP1-null or PPAR-α-null hepatocytes and mice, expression of FABP1 T94A variant did not abolish or reduce ligand binding. Thus the FABP1 T94A variant represents an altered/reduced function mutation resulting in TG accumulation.