Inhibition of stearoyl-CoA desaturase activity by the cis-9,trans-11 isomer and the trans-10,cis-12 isomer of conjugated linoleic acid in MDA-MB-231 and MCF-7 human breast cancer cells

Conjugated linoleic acid (CLA) is a collective term for a group of positional and geometric conjugated dienoic isomers of linoleic acid. CLA has been shown to have strong inhibitory effects on mammary carcinogenesis both in vitro and in vivo. In this study, we investigated the regulation of human stearoyl-CoA desaturase (SCD, EC 1.14.99.5) expression by CLA in human breast cancer cell lines, MDA-MB-231 and MCF-7. Treatment of the cells with the cis-9,trans-11 and trans-10,cis-12 CLA isomers (45 microM) did not repress SCD mRNA in both MDA-MB-231 and MCF-7 cells. However, the cis-9,trans-11 and trans-10,cis-12 CLA isomers significantly decreased SCD protein levels and SCD activity in MDA-MB-231 cells. In MCF-7 cells, both isomers did not affect protein levels, but they inhibited SCD activity. These results suggest that in MDA-MB-231 cells the cis-9,trans-11 and trans-10,cis-12 CLA isomers regulate human SCD by reducing SCD protein levels, while in MCF-7 cells both isomers have a direct inhibitory effect on SCD enzyme activity.     

Leptin-induced matrix metalloproteinase-2 secretion is suppressed by trans-10,cis-12 conjugated linoleic acid

It has long been recognized that leptin, a hormone made by adipocytes, is an important circulating signal for the regulation of body weight. In addition, matrix metalloproteinase (MMP), especially MMP-2, an adipocyte-secreted protein which promotes multi-cellular adipose clusters, is up-regulated in obesity. The present study is designed to evaluate whether trans-10,cis-12 conjugated linoleic acid (t-CLA) can suppress leptin-induced MMP-2 secretion in 3T3-L1 cells. The result showed that expressions of adipocyte marker proteins were significantly reduced by t-CLA-treated cultures, but not by linoleic acid (LA)-treated ones. Interestingly, MMP-2 secretion was significantly increased by leptin-treated cultures, thereby leading to accelerate adipocyte differentiation, indicating that MMP-2 was a necessary mediator of adipogenesis. However, increasing concentration of t-CLA significantly reduced leptin-induced MMP-2 secretion and triglyceride (TG) content. These findings provide support for a role for t-CLA in the regulation of metabolism in leptin-induced adipose tissue development.     

Incorporation of cis-9,trans-11 or trans-10,cis-12 conjugated linoleic acid into plasma and cellular lipids in healthy men

This study investigated the incorporation of cis-9,trans-11 conjugated linoleic acid (c9,t11 CLA) and trans-10,cis-12-CLA (t10,c12 CLA) into plasma and peripheral blood mononuclear cell (PBMC) lipids when consumed as supplements highly enriched in these isomers. Healthy men (n = 49, age 31 +/- 8 years) consumed one, two, and four capsules containing approximately 600 mg of either c9,t11 CLA or t10,c12 CLA per capsule for sequential 8 week periods followed by a 6 week washout before consuming the alternative isomer. Both isomers were incorporated in a dose-dependent manner into plasma phosphatidylcholine (PC) (c9,t11 CLA r = 0.779, t10,c12 CLA r = 0.738; P < 0.0001) and cholesteryl ester (CE) (c9,t11 CLA r = 0.706, t10,c12 CLA r = 0.788; P < 0.0001). Only t10,c12 CLA was enriched in plasma nonesterified fatty acids. Both c9,t11 CLA and t10,c12 CLA were incorporated linearly into PBMC total lipids (r = 0.285 and r = 0.273, respectively; P < 0.0005). The highest concentrations of c9,t11 CLA and t10,c12 CLA in PBMC lipids were 3- to 4-fold lower than those in plasma PC and CE. These data suggest that the level of intake is a major determinant of plasma and PBMC CLA content, although PBMCs appear to incorporate both CLA isomers less readily.     

Dietary trans-10,cis-12 conjugated linoleic acid induces hyperinsulinemia and fatty liver in the mouse

Conjugated linoleic acids (CLA) are a class of positional, geometric, conjugated dienoic isomers of linoleic acid (LA). Dietary CLA supplementation results in a dramatic decrease in body fat mass in mice, but also causes considerable liver steatosis. However, little is known of the molecular mechanisms leading to hepatomegaly. Although c9,t11- and t10,c12-CLA isomers are found in similar proportions in commercial preparations, the respective roles of these two molecules in liver enlargement has not been studied. We show here that mice fed a diet enriched in t10,c12-CLA (0.4% w/w) for 4 weeks developed lipoatrophy, hyperinsulinemia, and fatty liver, whereas diets enriched in c9,t11-CLA and LA had no significant effect. In the liver, dietary t10,c12-CLA triggered the ectopic production of peroxisome proliferator-activated receptor gamma (PPARgamma), adipocyte lipid-binding protein and fatty acid transporter mRNAs and induced expression of the sterol responsive element-binding protein-1a and fatty acid synthase genes. In vitro transactivation assays demonstrated that t10,c12- and c9,t11-CLA were equally efficient at activating PPARalpha, beta/delta, and gamma and inhibiting liver-X-receptor. Thus, the specific effect of t10,c12-CLA is unlikely to result from direct interaction with these nuclear receptors. Instead, t10,c12-CLA-induced hyperinsulinemia may trigger liver steatosis, by inducing both fatty acid uptake and lipogenesis.     

Cryosurvival of bovine blastocysts is enhanced by culture with trans-10 cis-12 conjugated linoleic acid (10t,12c CLA)

An excessive lipid content in embryo cells is a consequence of embryo culture in the presence of serum which is suggested to be responsible for their high susceptibility to cryopreservation. The objective of the present study was to examine the effects of supplementing serum-containing culture media with trans-10 cis-12 conjugated linoleic acid (10t,12c CLA) on embryo lipid accumulation and its subsequent cryopreservation. Abattoir-derived oocytes were matured and fertilized in vitro (IVF=day 0). On day 1, presumptive zygotes (n=3390) were randomly placed in: (I) (MS), granulosa cell monolayer cultured with M199 and 10% serum; (II) (SCLA), granulosa cell monolayer cultured with M199, 10% serum and 100 microM 10t,12c CLA and (III) (SOF), modified synthetic oviduct fluid, where embryo culture proceeded for 8 days. Cleavage rates or D7/D8 embryo quality did not vary among treatments. D7/D8 embryo production rate was significantly (P<0.001) lower in SOF (17.9+/-1.6%) than in groups MS (29.8+/-2.5%) and SCLA (27.8+/-2.0%). After cytoplasmic lipid droplets observation under Nomarski microscopy, classified embryos were the leanest when cultured in SOF, intermediate in SCLA and the fattest in MS (P<0.02). Post-thawing intact blastocyst rates where significantly higher in the SCLA group (84.7+/-4.1%) than in SOCS (50.3+/-4.8%, P=0.0007) or SOF (65.3+/-6.9%, P=0.03) groups. Post-thawing re-expanding rates were significantly lower when embryos were cultured in MS (34.7+/-3.7%) than in SCLA (63.7+/-5.3%, P=0.0006) or SOF (49.0+/-4.6%, P=0.04). Moreover, re-expanding rates were lower (P=0.05) in SOF than in SCLA cultured embryos. These results clearly show that addition of CLA to serum-containing media reduced lipid accumulation during in vitro culture and significantly improved cryopreservation survival.     

Quantitative gas chromatographic method for the analysis of cis-9, trans-11 and trans-10, cis-12 isomers of the conjugated linoleic acid in liver

A quantitative GC method for conjugated linoleic acid (CLA) isomers of physiological significance (cis-9, trans-11 CLA and trans-10, cis-12 CLA) as non-esterified fatty acids (NEFA) or triacylglycerols (TAG) was developed. Furthermore, the effect of the internal standard addition point (sample or fat extract) was studied. Response linearity, recovery and precision assays, detection and quantification limits were determined. Linearity was demonstrated over a range from 0.1 to 10 microg/mL. When CLA isomers were present as NEFA, the recovery significantly decreased (P< or =0.05) from 76% to 27.1% (cis-9, trans-11 CLA) and 28.5% (trans-10, cis-12 CLA) when the standards were added to the fat extract or to the initial tissue, respectively. As an application, liver samples from hamsters fed a diet supplemented with both CLA isomers were analyzed. The CLA isomers in liver samples were detected with reasonable reproducibility.     

Effects of trans-10, cis-12 conjugated linoleic acid on body fat and serum lipids in young and adult hamsters

The aim of the present work was to determine whether t-10, c-12 conjugated linoleic acid (CLA) feeding was able to reduce body fat accumulation and improve the serum lipid profile in adult hamsters fed an atherogenic diet, in order to compare these effects with those observed in young growing hamsters. Young and adult hamsters were fed semi-purified atherogenic diets supplemented with 0.5 % linoleic acid or 0.5% t-10, c-12 CLA for 6 weeks. Body weight and food intake were measured every two days. Adipose tissue from different anatomical locations, liver and gastrocnemious muscle were dissected and weighed. Cholesterol, triacylglycerols, non-esterified fatty acids and proteins were determined spectrophotometrically and water content by gravimetry. In young hamsters, no significant differences were found in food intake, final body weight and gastrocnemious muscle weight. White adipose tissue weights were reduced, liver weight was increased and cholesterol and triacyl-glycerols in both serum and liver were reduced. In adult hamsters, CLA feeding decreased food intake and adipose tissue weights. No changes were observed in other parameters. The present study demonstrates that age has an influence in hamster responsiveness to t-10, c-12 CLA because, although when this isomer is added to an atherogenic diet it reduces body fat accumulation in both young and adults hamsters, the lessening of the effects on serum lipids brought about by atherogenic feeding is only observed in young animals. Moreover, it is clear that liver is a target for CLA in young but not in adult hamsters.     

Modulation of lipid droplet size and lipid droplet proteins by trans-10,cis-12 conjugated linoleic acid parallels improvements in hepatic steatosis in obese,insulin-resistant rats

The isomer-specific effects of conjugated linoleic acid (CLA) on hepatic steatosis were assessed in fa/fa Zucker rats, a model for insulin resistance and the metabolic syndrome. Eight weeks of feeding trans-10,cis-12 CLA significantly improved glucose tolerance without changing body weight or visceral adipose mass. The trans-10,cis-12 isomer was also associated with reduced liver lipid content, improved liver function and reduced inflammation; these effects were not observed in rats fed the cis-9,trans-11 CLA isomer. Reduced liver lipid content did not correlate with activation of AMP-activated protein kinase or suppressed activation of sterol-regulatory element binding protein-1, two key regulators of hepatic lipid metabolism. Interestingly, rats fed cis-9,trans-11 CLA had fewer cytoplasmic lipid droplets in hepatocytes compared to rats fed control diet, but these droplets were larger in size. Conversely, fa/fa rats fed the trans-10,cis-12 CLA isomer had greater numbers of hepatic lipid droplets that were smaller in size, resulting in overall lower total lipid within these droplets. Changes in lipid droplets were associated with lower hepatic levels of PERILIPIN-2 (formerly known as adipophilin) in rats fed trans-10,cis-12 CLA, whereas amounts of other members of the PERILIPIN family of lipid droplet proteins were unaffected by dietary CLA. However, CLA isomers differentially affected the subcellular localization of these proteins. Treatment of H4IIE rat hepatoma cells with CLA isomers neither prevented nor reversed, but rather induced cytoplasmic lipid droplet formation, suggesting that the anti-steatotic effects of trans-10,cis-12 CLA are likely indirect and potentially mediated via increased lipid utilization by peripheral tissues.     

Different mechanisms of cis-9,trans-11- and trans-10,cis-12- conjugated linoleic acid affecting lipid metabolism in 3T3-L1 cells

Conjugated linoleic acid (CLA) has been shown to reduce body fat mass in various experimental animals. It is valuable to identify its influence on enzymes involved in energy expenditure, apoptosis, fatty acid oxidation and lipolysis. We investigated isomer-specific effects of high dose, long treatment of CLA (75.4 μmol/L, 8 days) on protein and gene expression of these enzymes in cultured 3T3-L1 cells. Proteomics identified significant up- or down-regulation of 52 proteins by either CLA isomer. Protein and gene expression of uncoupling protein (UCP) 1, UCP3, perilipin and peroxisome proliferator-activated receptor (PPAR) α increased whereas UCP2 reduced for both CLA isomers. And eight-day treatment of trans-10,cis-12 CLA, but not cis-9,trans-11 CLA, significantly up-regulated protein and mRNA levels of PKA (P<.05), CPT-1 and TNF-α (P<.01). Compared to protein expression, both isomers did not significantly influence the mRNA expression of HSL, ATGL, ACO and leptin. In conclusion, high-dose, long treatment of cis-9,trans-11 CLA did not promote apoptosis, fatty acid oxidation and lipolysis in adipocytes, but may induce an increase in energy expenditure. trans-10,cis-12 CLA exhibited greater influence on lipid metabolism, stimulated adipocyte energy expenditure, apoptosis and fatty acid oxidation, but its effect on lipolysis was not obvious.     

Adiposity and serum parameters in hamsters fed energy restricted diets supplemented or not with trans-10,cis-12 conjugated linoleic acid

Numerous studies have demonstrated that conjugated linoleic acid (CLA) modulates body composition, reducing body fat accumulation in various mammalian species. However, very few studies have been carried out to assess the effect of CLA on previously stored body fat. The aim of the present work was to analyse the effectiveness of trans-10,cis-12 CLA in improving alterations produced by high-fat feeding in body fat and serum parameters when it was included in an energy-restricted diet. For this purpose male Syrian Golden hamsters were fed on high-fat diet for 7 weeks in order to increase their body fat content, and a further 25% energy-restricted diet supplemented or not with 0.5% trans-10,cis-12 CLA for 3 weeks. Adipose tissues, liver and gastrocnemious muscles were dissected and weighed. Adipocyte diameter and number were assessed in epididymal adipose tissue. Total cholesterol, triacylglycerols, non-esterified fatty acids and glucose were measured in serum. Three weeks of energy restriction resulted in a reduction in body weight and white adipose tissue size in all anatomical locations, without changes in liver and gastrocnemious muscle weights. Epididymal adipocyte size was reduced, but total adipocyte number remained unchanged. Serum cholesterol, triacylglycerols and glucose were significantly reduced. No differences were observed between the restricted groups (control and CLA supplemented). In conclusion, under our experimental conditions, the addition of trans-10,cis-12 CLA to the diet does not increase the benefits produced by energy restriction.     

Acute exposure of L6 myotubes to cis-9, trans-11 and trans-10, cis-12 conjugated linoleic acid isomers stimulates glucose uptake by modulating Ca2+/calmodulin-dependent protein kinase II

Conjugated linoleic acid (CLA), a dietary fat, has been considered beneficial in metabolic syndrome. Despite several findings indicating that CLA improves glucose clearance, little information is available regarding the cellular dynamics of CLA on skeletal muscle. We sought to investigate the role of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in cis-9, trans-11(c9,t11) and trans-10, cis-12 (t10,c12) CLA isomer-mediated glucose transport by L6 myotubes. t10,c12-CLA stimulated both intracellular Ca(2+) release (Ca(i)(2+)) and CaMKII phosphorylation, whereas c9,t11-CLA showed only modest effects on both. Sequestering Ca(i)(2+) with BAPTA/AM abrogated the effect of both CLA isomers on Akt substrate-160 kDa (AS160) phosphorylation and glucose uptake by myotubes. Exposing myotubes to KN-93 or autocamtide 2-related inhibitory peptide to block CaMKII activity prevented both CLA isomers from inducing AS160 phosphorylation and glucose transport. Likewise, genetic knockdown of CaMKII in myotubes using siRNA completely abolished CLA isomer-mediated glucose uptake. These results indicate that CLA isomers require Ca(i)(2+)-CaMKII to mediate glucose uptake. Evidence that CaMKII blockers inhibit t10,c12-CLA-mediated AMP-activated protein kinase (AMPK) activation indicated that CaMKII acts upstream of AMPK in response to t10,c12-CLA. Lastly, CLA isomers stimulated the formation of reactive oxygen species but had no effect on stress-activated protein kinase/c-jun NH(2)-terminal kinase. These data establish that t10,c12-CLA acts via Ca(i)(2+)-CaMKII-AMPK-AS160 to stimulate skeletal muscle glucose transport, whereas the mechanism of c9,t11-CLA remains unclear. Given that impairments in muscle glucose utilisation are apparent in metabolic syndrome, delineating the molecular mechanisms by which CLA isomers mediate muscle glucose uptake may identify new approaches to manage this condition.     

Inflammation and insulin resistance induced by trans-10, cis-12 conjugated linoleic acid depend on intracellular calcium levels in primary cultures of human adipocytes

We previously demonstrated that trans-10, cis-12 (10,12) conjugated linoleic acid (CLA) induced inflammation and insulin resistance in primary human adipocytes by activating nuclear factor κB (NFκB) and extracellular signal-related kinase (ERK) signaling. In this study, we demonstrated that the initial increase in intracellular calcium ([Ca²⁺]_i) mediated by 10,12 CLA was attenuated by TMB-8, an inhibitor of calcium release from the endoplasmic reticulum (ER), by BAPTA, an intracellular calcium chelator, and by D609, a phospholipase C (PLC) inhibitor. Moreover, BAPTA, TMB-8, and D609 attenuated 10,12 CLA–mediated production of reactive oxygen species (ROS), activation of ERK1/2 and cJun-NH₂-terminal kinase (JNK), and induction of inflammatory genes. 10,12 CLA–mediated binding of NFκB to the promoters of interleukin (IL)-8 and cyclooxygenase (COX)-2 and induction of calcium-calmodulin kinase II (CaMKII) β were attenuated by TMB-8. KN-62, a CaMKII inhibitor, also suppressed 10,12 CLA–mediated ROS production and ERK1/2 and JNK activation. Additionally, KN-62 attenuated 10,12 CLA induction of inflammatory and integrated stress response genes, increase in prostaglandin F_2α, and suppression of peroxisome proliferator activated receptor γ protein levels and insulin-stimulated glucose uptake. These data suggest that 10,12 CLA increases inflammation and insulin resistance in human adipocytes, in part by increasing [Ca²⁺]_i levels, particularly calcium from the ER.     

Fat area and lipid droplet morphology of porcine oocytes during in vitro maturation with trans-10, cis-12 conjugated linoleic acid and forskolin

Lipid droplets (LD) in porcine oocytes form a dark mass reaching almost all cytoplasm. Herein we investigated changes in fat areas, cytoplasmic tone and LD morphology during in vitro maturation (IVM) of porcine oocytes cultured with 100 μM trans-10, cis-12 conjugated linoleic acid (t10,c12 CLA) or 10 μM forskolin at different time periods. Four groups were constituted: control, excipient, t10,c12 CLA and forskolin, with drugs being supplemented during 44 to 48 h and the initial 22 to 24 h in Experiments 1 and 2, respectively. In Experiment 3, forskolin was supplemented for the first 2 h. Matured oocytes were inseminated with frozen-thawed boar semen and cleavage rate recorded. Before and during IVM, samples of oocytes were evaluated for LD, total and fat areas and fat gray value or for meiotic progression. Results showed that forskolin supplementation during 44 to 48 h or 22 to 24 h inhibits oocyte maturation (exp. 1: forskolin = 5.1 ± 8.0%, control = 72.6 ± 5.0%; exp. 2: forskolin = 24.3 ± 7.4%, control = 71.6 ± 5.6%) and cleavage (exp. 1: forskolin = 0.0 ± 0.0%, control = 55.4 ± 4.1%; exp. 2: forskolin = 8.3 ± 3.3%, control = 54.5 ± 3.0%). Forskolin also reduced oocyte and fat areas. In Experiment 3, forskolin negative effect on oocyte maturation and cleavage disappeared, although minor (P ⩽ 0.03) LD and oocyte fat areas were identified at 22 to 24 h of IVM. Oocytes supplemented with t10,c12 CLA during 44 to 48 h presented a lighter (P ⩽ 0.04) colour tone cytoplasm than those of control and forskolin. In conclusion, t10,c12 CLA and forskolin were capable of modifying the distribution and morphology of cytoplasmic LD during porcine oocyte maturation, thus reducing its lipid content in a time-dependent manner.     

Production of lean beef containing a high content of trans-10, cis-12 conjugated linoleic acid by feeding a high-temperature-micro-time-treated diet with extruded soybean

The effects were observed of feeding a high-temperature-micro-time (HTMT)-treated diet of high-quality extruded soybean (ESB) on trans-10, cis-12 conjugated linoleic acid (CLA) incorporation in intra-muscular fat and on the lipid metabolism of twelve Holstein steers. The animals were randomly allocated to three groups, the control group being fed on a normal diet, and the other two groups being fed on a diet containing high-quality ESB with or without the HTMT treatment. The experimental period was four months. The content of total CLA in both the HTMT and non-HTMT-treated diets was higher than that in the control diet (P<0.05). The concentration of trans-10, cis-12 CLA was significantly higher in the HTMT-treated diet than in the non-treated diets at P<0.05, and the HTMT-treatment decreased the intra-muscular fat content (P<0.05). The expression of fatty acid synthase (FAS) and the plasma insulin-like growth factor-1 (IGF-1) level in the HTMT-treated group were lower than those in the control group (P<0.05). Lipoprotein lipase (LPL) mRNA level in the adipose tissue was lower in the non-HTMT-treated group than in the control (P<0.05). These results show that the HTMT treatment increased the level of trans-10, cis-12 CLA in the intra-muscular fat. This increased level of trans-10, cis-12 CLA may inhibit lipogenesis in the intra-muscular fat of Holstein steers.