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.     

Mapping a high oleic acid mutation in winter oilseed rape (Brassica napus L.)

Two winter oilseed rape mutant lines, 7488 and 19661, with a high oleic (HO) acid content in the seed oil were characterized phenotypically. In both mutant lines the HO trait was monogenically inherited. Segregation analysis in an F₂ population derived from a cross between 7488 and 19661 showed the two mutations to be allelic. From a comparison of seed, leaf and root fatty acid composition it was concluded that fad2, the endoplasmic oleic acid desaturase, is affected by the mutation. In a bulked segregant analysis three AFLP markers linked to this mutation were detected and localized on the genetic map of Brassica napus. The markers mapped near the locus of one copy of the fad2 gene in the rapeseed genome. Received: 16 February 2000 / Accepted: 28 March 2000     

Fatty acid double bond orientation alters interaction with L-cell fibroblasts

Relatively little is known of fatty acid specificity in cellular fatty acid uptake. In this study L-cells, a fibroblastic cell line with very low levels of endogenous cytosolic fatty acid binding protein, were used to examine the role of cis and trans unsaturation on fatty acid uptake. The fluorescent fatty acids, trans-parinaric acid and cis-parinaric acid, were used as analogs of straight-chain saturated, and kinked-chain unsaturated fatty acids, respectively, in order to evaluate the fatty acid specificity of the uptake system. Parinaric acid is poorly metabolizable; greater than 97% was unesterified while ³H-oleic acid was almost totally metabolized after 30 min uptake. Cis- and trans-parinaric acid uptake was saturable and dependent on the concentration of fatty acid. However, the initial rate and maximal amount of trans-parinaric acid taken up by the L-cells was greater than for cis-parinaric acid under the same conditions. The affinity of L-cell uptake for trans-parinaric acid (K_m = 0.12 uM) was 35-fold higher than that for cis-parinaric acid (K_m = 4.17 uM) . Based on competition studies with oleic and stearic acids, it was concluded that the cis- and trans-parinaric acid were taken up by the same L-cell fatty acid uptake system. The results suggest that the L-cell fatty acid uptake system has selectivity for straight chain rather than kinked chain unsaturated fatty acids.Abbreviations Cis-parinaric acid 9Z, 11E, 13E, 15Z-octatetraenoic acid - trans-parinaric acid 9E, I IE, 13E, 15E-octatetraenoic acid - EGTA ethylene glycol-bis(beta-amlno-ethyl ether) N,N,N,N-tetratacetic acid - BSA bovine serum albumin - PBS phosphate buffered saline     

Changes in mitochondrial membrane composition and oxidative status during rapid growth,maturation and aging in zebrafish, Danio rerio

Considering membranes and membrane components as possible pacemakers of the main processes taking place inside mitochondria, changes in phospholipids or fatty acids could play a central role linking different mechanisms involved in cumulative damage to cell molecules and dysfunction during periods of high stress, such as rapid growth and aging. Changes affecting either lipid class or fatty acid compositions could affect phospholipid and membrane properties and alter mitochondrial function and cell viability. In the present study, mitochondrial oxidative status and mitochondrial membrane phospholipid compositions were analyzed throughout the life-cycle of zebrafish. TBARS content significantly increased in 18-month-old fish while aconitase activity decreased in 24-month-old fish, which have been related with oxidative damage to molecules. Mitochondria-specific superoxide dismutase decreased in 24-month-old animals although this change was not statistically significant. Age affected both mitochondrial phospholipid content and the peroxidation index of most phospholipid classes suggesting that oxidative damage to mitochondrial lipids was occurring.     

Oleic acid induces intracellular calcium mobilization, MAPK phosphorylation, superoxide production and granule release in bovine neutrophils

Oleic acid (OA) is a nonesterified fatty acid that is released into the blood during lipomobilization at the time of calving in cows, a period where increased risk of infection and acute inflammation is observed. These data suggest potential OA-mediated regulation of innate immune responses. In the present study, we assessed the effects of OA on intracellular calcium release, ERK1/2 phosphorylation, superoxide production, CD11b expression and matrix metalloproteinase-9 (MMP-9) release in bovine neutrophils. Furthermore, the presence of GPR40, an OA receptor, was assessed by RT-PCR, immunoblotting and confocal microscopy. OA induced, in a dose-dependent manner, intracellular calcium mobilization, superoxide production and CD11b expression in bovine neutrophils; these effects were reduced by the intracellular chelating agent BAPTA-AM. OA also induced ERK2 phosphorylation and MMP-9 release. RT-PCR analysis detected mRNA expression of a bovine ortholog of the GPR40 receptor. Using a polyclonal antibody against human GPR40, we detected a protein of 31 kDa by immunoblotting that was localized predominately in the plasma membrane. The selective agonist of GPR40, GW9508, induced intracellular calcium mobilization and ERK2 phosphorylation. In conclusion, OA can modulate bovine neutrophil responses in an intracellular calcium-dependent manner; furthermore, these responses could be induced by GPR40 activation.     

Remodeling of phosphatidylglycerol in Synechocystis PCC6803

The phosphatidylglycerol deficient ΔpgsA mutant of Synechocystis PCC6803 provided a unique experimental system for investigating in vivo retailoring of exogenously added dioleoylphosphatidylglycerol in phosphatidylglycerol-depleted cells. Gas chromatographic analysis of fatty acid composition suggested that diacyl-phosphatidylglycerols were synthesized from the artificial synthetic precursor. The formation of new, retailored lipid species was confirmed by negative-ion electrospray ionization–Fourier-transform ion cyclotron resonance and ion trap tandem mass spectrometry. Various isomeric diacyl-phosphatidylglycerols were identified indicating transesterification of the exogenously added dioleoylphosphatidyl-glycerol at the sn-1 or sn-2 positions. Polyunsaturated fatty acids were incorporated selectively into the sn-1 position. Our experiments with Synechocystis PCC6803/ΔpgsA mutant cells demonstrated lipid remodeling in a prokaryotic photosynthetic bacterium. Our data suggest that the remodeling of diacylphosphatidylglycerol likely involves reactions catalyzed by phospholipase A₁ and A₂ or acyl-hydrolase, lysophosphatidylglycerol acyltransferase and acyl-lipid desaturases.     

红白花斑种皮高油酸花生种质材料的创制

本研究利用粉色种皮高油酸花生G63与紫白花斑种皮普通油酸花生VG-01配置杂交组合。利用等位基因特异性扩增(AS-PCR,allele-specific PCR)鉴定出16个F_1真杂种。在F_2中筛选到_ol1_ol_2单株,并对64个F_2:3家系间的种皮色泽分离进行χ~2检测。结果表明,纯色花生∶花斑花生在0.05水平上符合9∶7,花生种皮花斑性状由2对互补基因控制,当2对基因同时处于显性时,种皮表现为纯色;当2对基因至少有1对为隐性纯合时,种皮表现为花斑。利用AS-PCR技术对334株花斑种皮F_3单株进行基因型鉴定,筛选到29株ol_1ol_1ol_2ol_2基因型的单株。利用气相色谱测定结果表明,F4种子油酸GC值介于70.77%~82.87%,油酸/亚油酸介于8.15~26.30。F4群体植株主茎高12.4~87.3 cm,平均57.2 cm,较对照冀花2号增高34.97%;第1对侧枝长91.6~196.3 cm,平均134.1 cm,较对照冀花2号增长34.28%。F5新种质的单株果重、单株果数、单株仁重、单株仁数分别为(27.16±9.51)g、(22.11±10.26)个、(20.10±7.17)g和(31.94±15.16)个。新种质9-7、14-2、14-3、17-3和17-7均为红白花斑种皮,在单株果重方面较对照冀花2号增加230.02%、165.80%、210.66%、200.65%和160.26%,在单株果数方面增加280.00%、340.00%、450.00%、210.00%和150.00%,其油酸GC值分别为80.54%、81.75%、80.63%、81.3%和81.56%。该批种质材料不仅油酸含量高,而且具有医疗保健价值,对丰富我国高油酸花生遗传多样性具有重要的现实意义。     

诺卡氏菌对油酸的羟基化作用

诺卡氏菌(Nocardia sp．N13)休止细胞对油酸(顾-9-十八碳烯酸)进行羟基化作用。用休止细胞转化时最适温度为30℃,最适pH为6．5,当菌体(干菌)与底物的最适比例为20 ：1对,对油酸的转化率为83．4％,不同的表面活性剂影响转化的效果。N13休止细胞在磷酸缓冲液中重复转化3次仍可保持50％左右的转化率。红外光谱、质谱、棱磁共振鉴定产物为10－羟基硬脂酸。     

Fatty acids decrease mitochondrial generation of reactive oxygen species at the reverse electron transport but increase it at the forward transport

Long-chain nonesterified (“free”) fatty acids (FFA) can affect the mitochondrial generation of reactive oxygen species (ROS) in two ways: (i) by depolarisation of the inner membrane due to the uncoupling effect and (ii) by partly blocking the respiratory chain. In the present work this dual effect was investigated in rat heart and liver mitochondria under conditions of forward and reverse electron transport. Under conditions of the forward electron transport, i.e. with pyruvate plus malate and with succinate (plus rotenone) as respiratory substrates, polyunsaturated fatty acid, arachidonic, and branched-chain saturated fatty acid, phytanic, increased ROS production in parallel with a partial inhibition of the electron transport in the respiratory chain, most likely at the level of complexes I and III. A linear correlation between stimulation of ROS production and inhibition of complex III was found for rat heart mitochondria. This effect on ROS production was further increased in glutathione-depleted mitochondria. Under conditions of the reverse electron transport, i.e. with succinate (without rotenone), unsaturated fatty acids, arachidonic and oleic, straight-chain saturated palmitic acid and branched-chain saturated phytanic acid strongly inhibited ROS production. This inhibition was partly abolished by the blocker of ATP/ADP transfer, carboxyatractyloside, thus indicating that this effect was related to uncoupling (protonophoric) action of fatty acids. It is concluded that in isolated rat heart and liver mitochondria functioning in the forward electron transport mode, unsaturated fatty acids and phytanic acid increase ROS generation by partly inhibiting the electron transport and, most likely, by changing membrane fluidity. Only under conditions of reverse electron transport, fatty acids decrease ROS generation due to their uncoupling action.