Modulatory effect of linoleic and oleic acid on cell proliferation and lipid metabolism gene expressions in primary bovine satellite cells

This study was performed to elucidate the effects of linoleic acid (LA), oleic acid (OA) and their combination (LA?+?OA) on cell proliferation, apoptosis, necrosis, and the lipid metabolism related gene expression in bovine satellite cells (BSCs), isolated from bovine muscles. Cell viability was significantly increased with the OA and LA treatment. Furthermore, LA?+?OA enhanced cell proliferation in a dose-dependent manner (10 to 100?µM), whereas it lowered at 250?µM. In addition, a cell-cycle analysis showed that 100?µM of LA and OA markedly decreased the G0/G1 phase proportion (62.58% and 61.33%, respectively), compared to controls (68.02%), whereas the S-phase cells’ proportion was increased. The ratio of G2/M phase cells was not significantly different among the groups. Moreover, analyses with AO/EtBr staining showed that no apoptosis occurred. Necrosis were determined by flow cytometry using Annexin V-FITC/PI staining which revealed no early apoptosis in the cells pretreated with LA or OA, but occurred in the LA?+?OA group. We also analyzed the mRNA expression of lipid metabolizing genes such as peroxisome proliferator receptor alfa (PPARα), peroxisome proliferator receptor gamma (PPARγ), acyl-CoA oxidase (ACOX), lipoprotein lipase (LPL), carnitine palmitoyl transferase (CPT-1), and fatty-acid binding protein4 (FABP4), which were upregulated in LA or OA treated cells compared to the control group. In essence, LA and OA alone promote the cell proliferation without any apoptosis and necrosis, which might upregulate the lipid metabolism related gene expressions, and increase fatty-acid oxidation in the BSCs’ lipid metabolism.     

Omega-3 and Omega-6 polyunsaturated fatty acids stimulate vascular differentiation of mouse embryonic stem cells

Polyunsaturated fatty acids (PUFAs) and their metabolites may influence cell fate regulation. Herein, we investigated the effects of linoleic acid (LA) as ω-6 PUFA, eicosapentaenoic acid (EPA) as ω-3 PUFA and palmitic acid (PA) on vasculogenesis of embryonic stem (ES) cells. LA and EPA increased vascular structure formation and protein expression of the endothelial-specific markers fetal liver kinase-1, CD31 as well as VE-cadherin, whereas PA was without effect. LA and EPA increased reactive oxygen species (ROS) and nitric oxide (NO), activated endothelial NO synthase (eNOS) and raised intracellular calcium. The calcium response was inhibited by the intracellular calcium chelator BAPTA, sulfo-N-succinimidyl oleate which is an antagonist of CD36, the scavenger receptor for fatty acid uptake as well as by a CD36 blocking antibody. Prevention of ROS generation by radical scavengers or the NADPH oxidase inhibitor VAS2870 and inhibition of eNOS by L-NAME blunted vasculogenesis. PUFAs stimulated AMP activated protein kinase-α (AMPK-α) as well as peroxisome proliferator-activated receptor-α (PPAR-α). AMPK activation was abolished by calcium chelation as well as inhibition of ROS and NO generation. Moreover, PUFA-induced vasculogenesis was blunted by the PPAR-α inhibitor GW6471. In conclusion, ω-3 and ω-6 PUFAs stimulate vascular differentiation of ES cells via mechanisms involving calcium, ROS and NO, which regulate function of the energy sensors AMPK and PPAR-α and determine the metabolic signature of vascular cell differentiation.     

Regulation of human lymphocyte proliferation by fatty acids

In the present study, the effect of increasing concentrations of palmitic (PA, C16:0), stearic (SA, C18:0), oleic (OA, C18:1, n-9), linoleic (LA, C18:2n-6), docosahexaenoic (DHA, C22:6 n-3) and eicosapentaenoic (EPA, C20:5 n-3) acids on lymphocyte proliferation was investigated. The maximal non-toxic concentrations of these fatty acids for human lymphocytes in vitro were determined. It was also evaluated whether these fatty acids at non-toxic concentrations affect IL-2 induced lymphocyte proliferation and cell cycle progression. OA and LA at 25 microM increased lymphocyte proliferation and at higher concentrations (75 microM and 100 microM) inhibited it. Both fatty acids promoted cell death at 200 microM concentration. PA and SA decreased lymphocyte proliferation at 50 microM and promoted cell death at concentrations of 100 microM and above. EPA and DHA decreased lymphocyte proliferation at 25 and 50 microM being toxic at 50 and 100 microM, respectively. PA, SA, DHA and EPA decreased the stimulatory effect of IL-2 on lymphocyte proliferation, increasing the percentage of cells in G1 phase and decreasing the proportion of cells in S and G2/M phases. OA and LA caused an even greater pronounced effect. The treatment with all fatty acids increased neutral lipid accumulation in the cells but the effect was more pronounced with PA and DHA. In conclusion, PA, SA, DHA and EPA decreased lymphocyte proliferation, whereas OA and LA stimulated it at non-toxic concentrations.     

Differential effects of retinoic acid upon early and late events in adipose conversion of 3T3 preadipocytes

When confluent 3T3-F442A cell cultures (Day 0) were grown for 3 days in fetal calf serum-supplemented medium containing isobutyl methyl xanthine and dexamethasone (induction phase) and then shifted to serum-free hormone-defined medium (expression phase), they spontaneously exhibited a sharp rise in lipoprotein lipase activity (LPL); at the peak (Day 7) the LPL activity was about 25 times higher than in control cultures and was further enhanced by insulin. Although this expression of LPL activity was spontaneous, the emergence of glycerophosphate dehydrogenase (G3PDH) activity was completely dependent upon insulin as well as upon the expression of the differentiated phenotype. In committed cells, insulin elicited sustained DNA synthesis associated with limited cell proliferation. The addition of retinoic acid during the phase of expression inhibited insulin-dependent terminal differentiation (i.e., the emergence of G3PDH activity and acquisition of the differentiated phenotype). In addition, retinoic acid counteracted the stimulating effect of insulin upon LPL activity, but affected neither the mitotic process nor the spontaneous emergence of LPL activity. When added during the phase of induction, it prevented the overall process of adipogenic differentiation. Thus, the use of retinoic acid can indicate independent control of the mitogenic and lipogenic effects of insulin following commitment to adipogenic differentiation.     

Phospholipid fatty acid composition affects enzymatic antioxidant defenses in cultured Swiss 3T3 fibroblasts

Summary

The aim of this work was to study the adaptation of enzymatic antioxidant cell defense to the nature of the membrane polyunsaturated fatty acids (PUFA). 3T3 Swiss fibroblasts were grown for 5 days in a medium supplemented with 50 μM linoleic acid (LA) or eicosapentaenoic acid (EPA) and compared t control cells (C). The phospholipid fatty acid content was evaluated: LA were enriched in n-6 PUFA (27.8%) in comparison to C (6.7%) or EPA (5.6%); EPA were enriched in n-3 PUFA (26.2%) in comparison to LA (4.4%) or C (4.6%). The fatty acid double bond index (DBI) increased from C to LA and EPA. The activities of the three key enzymatic antioxidant defenses, SOD, GPx and GST, increased with the degree of unsaturation of the phospholipid fatty acids. In the cells with fatty acids that are very sensitive to oxidative stress, the higher activities of SOD and GPx might act to limit the initiation of lipid peroxidation and the higher activities of GST and GPx to decrease the toxic effects of the various species produced from lipid degradation.     

ATGL-mediated fat catabolism regulates cardiac mitochondrial function via PPAR-α and PGC-1

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that regulate genes involved in energy metabolism and inflammation. For biological activity, PPARs require cognate lipid ligands, heterodimerization with retinoic X receptors, and coactivation by PPAR-γ coactivator-1α or PPAR-γ coactivator-1β (PGC-1α or PGC-1β, encoded by Ppargc1a and Ppargc1b, respectively). Here we show that lipolysis of cellular triglycerides by adipose triglyceride lipase (patatin-like phospholipase domain containing protein 2, encoded by Pnpla2; hereafter referred to as Atgl) generates essential mediator(s) involved in the generation of lipid ligands for PPAR activation. Atgl deficiency in mice decreases mRNA levels of PPAR-α and PPAR-δ target genes. In the heart, this leads to decreased PGC-1α and PGC-1β expression and severely disrupted mitochondrial substrate oxidation and respiration; this is followed by excessive lipid accumulation, cardiac insufficiency and lethal cardiomyopathy. Reconstituting normal PPAR target gene expression by pharmacological treatment of Atgl-deficient mice with PPAR-α agonists completely reverses the mitochondrial defects, restores normal heart function and prevents premature death. These findings reveal a potential treatment for the excessive cardiac lipid accumulation and often-lethal cardiomyopathy in people with neutral lipid storage disease, a disease marked by reduced or absent ATGL activity.     

Impact of α-lipoic acid on liver peroxisome proliferator-activated receptor-α, vascular remodeling, and oxidative stress in insulin-resistant rats

This study sought to determine the impact of α-lipoic acid (LA) on superoxide anion (O(2)(?-)) production and peroxisome proliferator-activated receptor-α (PPARα) expression in liver tissue, plasma free fatty acids (FFA), and aortic remodeling in a rat model of insulin resistance. Sprague-Dawley rats (50-75 g) were given either tap water or a drinking solution containing 10% D-glucose for 14 weeks, combined with a diet with or without LA supplement. O(2)(?-) production was measured by lucigenin chemiluminescence, and PPAR-α expression by Western blotting. Cross-sectional area (CSA) of the aortic media and lumen and number of smooth muscle cells (SMC) were determined histologically. Glucose increased systolic blood pressure (SBP), plasma levels of glucose and insulin, and insulin resistance (HOMA index). All of these effects were attenuated by LA. Whereas glucose had no effect on liver PPAR-α protein level, it decreased plasma FFA. LA decreased the aortic and liver O(2)(?-) production, body weight, and plasma FFA levels in control and glucose-treated rats. Liver PPAR-α protein levels were increased by LA, and negatively correlated with plasma FFA. Medial CSA was reduced in all glucose-treated rats, and positively correlated with plasma FFA but not with SBP or aortic O(2)(?-) production. Glucose also reduced aortic lumen area, so that the media-to-lumen ratio remained unchanged. The ability of LA to lower plasma FFA appears to be mediated, in part, by increased hepatic PPAR-α expression, which may positively affect insulin resistance. Glucose-fed rats may serve as a unique model of aortic atrophic remodeling in hypertension and early metabolic syndrome.     

ATP敏感钾通道参与大鼠前脂肪细胞增殖和分化

为了探讨ATP敏感钾通道在前脂肪细胞增殖分化中作用,本实验用逆转录实时定量PCR方法检测大鼠前脂肪细胞和诱导5d获得的脂肪细胞中该通道磺脲类受体2（sulphonylurea receptor2,SUR2）mRNA表达,探讨该通道阻滞剂格列本脲和激动剂二氮嗪对前脂肪细胞中SUR2mRNA表达的影响;MTT检测前脂肪细胞增殖;流式细胞仪检测细胞周期;油红O染色法检测细胞内脂质含量;Image-Pro Plus5．0软件测量细胞直径;逆转录PCR检测过氧化物酶体增殖物激活受体-γ（peroxisome proliferator-activatedreceptor-γ PPAR-γ）mRNA表达。结果显示：前脂肪细胞及诱导5d获得的脂肪细胞均有SUR2mRNA表达,且后者明显高于前者;格列本脲抑制前脂肪细胞SUR2mRNA表达,剂量依赖性地促进前脂肪细胞增殖,增加G2／M＋S期细胞百分比,增加细胞脂质含量,使脂肪细胞直径增大,增加PPAR-γ mRNA的表达;二氮嗪在这些方面的作用与格列本脲相反。以上结果提示,ATP敏感钾通道在前脂肪细胞增殖和分化中可能起调节作用,PPAR-γ可能参与这些作用。     

Niacin improves renal lipid metabolism and slows progression in chronic kidney disease

Background

Mounting evidence points to lipid accumulation in the diseased kidney and its contribution to progression of nephropathy. We recently found heavy lipid accumulation and marked dysregulation of lipid metabolism in the remnant kidneys of rats with chronic renal failure (CRF). Present study sought to determine efficacy of niacin supplementation on renal tissue lipid metabolism in CRF.

Methods

Kidney function, lipid content, and expression of molecules involved in cholesterol and fatty acid metabolism were determined in untreated CRF (5/6 nephrectomized), niacin-treated CRF (50 mg/kg/day in drinking water for 12 weeks) and control rats.

Results

CRF resulted in hypertension, proteinuria, renal tissue lipid accumulation, up-regulation of scavenger receptor A1 (SR-A1), acyl-CoA cholesterol acyltransferase-1 (ACAT1), carbohydrate-responsive element binding protein (ChREBP), fatty acid synthase (FAS), acyl-CoA carboxylase (ACC), liver X receptor (LXR), ATP binding cassette (ABC) A-1, ABCG-1, and SR-B1 and down-regulation of sterol responsive element binding protein-1 (SREBP-1), SREBP-2, HMG-CoA reductase, PPAR-α, fatty acid binding protein (L-FABP), and CPT1A. Niacin therapy attenuated hypertension, proteinuria, and tubulo-interstitial injury, reduced renal tissue lipids, CD36, ChREBP, LXR, ABCA-1, ABCG-1, and SR-B1 abundance and raised PPAR-α and L-FABP.

Conclusions and general significance

Niacin administration improves renal tissue lipid metabolism and renal function and structure in experimental CRF.     

Hepatic ATGL mediates PPAR-α signaling and fatty acid channeling through an L-FABP independent mechanism

Adipose TG lipase (ATGL) catalyzes the rate-limiting step in TG hydrolysis in most tissues. We have shown that hepatic ATGL preferentially channels hydrolyzed FAs to β-oxidation and induces PPAR-α signaling. Previous studies have suggested that liver FA binding protein (L-FABP) transports FAs from lipid droplets to the nucleus for ligand delivery and to the mitochondria for β-oxidation. To determine if L-FABP is involved in ATGL-mediated FA channeling, we used adenovirus-mediated suppression or overexpression of hepatic ATGL in either WT or L-FABP KO mice. Hepatic ATGL knockdown increased liver weight and TG content of overnight fasted mice regardless of genotype. L-FABP deletion did not impair the effects of ATGL overexpression on the oxidation of hydrolyzed FAs in primary hepatocyte cultures or on serum β-hydroxybutyrate concentrations in vivo. Moreover, L-FABP deletion did not influence the effects of ATGL knockdown or overexpression on PPAR-α target gene expression. Taken together, we conclude that L-FABP is not required to channel ATGL-hydrolyzed FAs to mitochondria for β-oxidation or the nucleus for PPAR-α regulation.     

Liver fatty acid binding protein gene ablation enhances age-dependent weight gain in male mice

Although studies performed in vitro and with transfected cells in culture suggest a role for liver fatty acid binding protein (L-FABP) in regulating fatty acid oxidation and fat deposition, the physiological significance of this possibility is not completely clear. To begin to address this question, the effect of L-FABP gene ablation on phenotype of standard rodent chow-fed male mice was examined with increasing age up to 18 months. While young (2–3 months old) L-FABP null mice displayed no visually obvious phenotype, with increasing age >9 months the L-FABP null mice were visibly larger, exhibiting increased body weight due to increased fat and lean tissue mass. Liver lipid concentrations were unaffected by L-FABP gene ablation with the exception of triacylglycerol, which was decreased by 74% in the livers of 3-month-old mice. Likewise, serum lipid levels were not altered in L-FABP null mice with the exception of triacylglycerol, which was increased in the serum of 18-month-old mice. Increased body weight, fat tissue mass, and lean tissue mass in 18-month-old L-FABP null mice were accompanied by increased hepatic levels of low-density lipoprotein (LDL) receptor, peroxisome proliferator-activated receptor (PPAR) α, and PPARα-regulated proteins such as fatty acid transport protein (FATP), fatty acid translocase (FAT/CD36), carnitine palmitoyl transferase I (CPT I), and lipoprotein lipase (LPL). A key enzyme in cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl Coenzyme A (HMG-CoA) reductase, was down-regulated in L-FABP null mice. These findings were consistent with a proposed role for L-FABP as an important physiological regulator of PPARα.     

Comparative toxicity of oleic and linoleic acid on human lymphocytes

Commercially available lipid emulsions for parenteral nutrition are mainly composed by long chain triacylglycerol containing a high proportion of linoleic acid (LA) or oleic acid (OA). The immunological impact of such therapy is particularly important because parenteral diets are often administered to critically ill patients as a mechanism to supply adequate nutrition during catabolic stress conditions. The comparative toxicity of OA and LA on human lymphocytes and the type of cell death induced by these fatty acids were determined in vitro. Parameters of cell death were investigated by flow cytometry-cell viability, DNA fragmentation, phosphatidylserine externalization, mitochondrial depolarization, neutral lipid accumulation and production of reactive oxygen species-and by fluorescence microscopy-chromatin condensation. Additionally a spectrofluorometric assay was employed to determine the activities of caspase--3, 6 and 8. Evidence is presented herein that OA is less toxic to human lymphocytes than LA. However, both fatty acids promoted apoptosis and necrosis of these cells. The mechanism of cell death induced by OA involved activation of caspase 3 while the mechanism of death induced by LA involved mitochondrial depolarization and ROS production. Importantly, neutral lipid accumulation may be a mechanism to protect lymphocytes against the toxicity induced by OA. OA may offer an immunological less problematic alternative to LA with respect to fatty acid composition of parenteral nutritional emulsions.     

High levels of arachidonic acid and peroxisome proliferator-activated receptor-alpha in breast cancer tissues are associated with promoting cancer cell proliferation

Fatty acids are endogenous ligands of peroxisome proliferator-activated receptor-alpha (PPARα), which is linked to the regulation of fatty acid uptake, lipid metabolism and breast cancer cell growth. This study was designed to screen candidate fatty acids from breast cancer tissue and to investigate the effects of these candidate fatty acids on PPARα expression, cell growth and cell cycle progression in breast cancer cell lines. One breast cancer tissue and one reference tissue were each taken from 30 individual breasts to examine for fatty acid composition and PPARα expression. The cancer cell lines MDA-MB-231 (ER–), MCF-7 (ER++++) and BT-474 (ER++) were used to explore the mechanisms regulating cell proliferation. We found that arachidonic acid (AA) and PPARα were highly expressed in the breast cancer tissues. AA stimulated the growth of all three breast cancer cells in a time- and dose-dependent manner. The growth stimulatory effect of AA was associated with PPARα activation, and the most potent effect was found in MCF-7 cells. The stimulation of cell proliferation by AA was accompanied by the increased expression of cyclin E, a reduced population of G1 phase cells, and a faster G1/S phase transition. In contrast, AA had no effects on the levels of CDK2, CDK4, cyclin D1, p27, Bcl-2 and Bax. Our results demonstrate that high levels of AA and PPARα expression in human breast cancer tissues are associated with ER-overexpressed breast cancer cell proliferation, which is involved in activating PPARα, stimulating cyclin E expression, and promoting faster G1/S transition.     

Ursolic acid is a PPAR-α agonist that regulates hepatic lipid metabolism

In this study, we confirmed that ursolic acid, a plant triterpenoid, activates peroxisome proliferator-activated receptor (PPAR)-α in vitro. Surface plasmon resonance and time-resolved fluorescence resonance energy transfer analyses do not show direct binding of ursolic acid to the ligand-binding domain of PPAR-α; however, ursolic acid enhances the binding of PPAR-α to the peroxisome proliferator response element in PPAR-α-responsive genes, alters the expression of key genes in lipid metabolism, significantly reducing intracellular triglyceride and cholesterol concentrations in hepatocytes. Thus, ursolic acid is a PPAR-α agonist that regulates the expression of lipid metabolism genes, but it is not a direct ligand of PPAR-α.