Trans-10, cis-12, but not cis-9, trans-11 CLA isomer,inhibits brown adipocyte thermogenic capacity

Conjugated linoleic acid (CLA) is reported to have health benefits, including reduction of body fat. Previous studies have shown that brown adipose tissue (BAT) is particularly sensitive to CLA-supplemented diet feeding. Most of them use mixtures containing several CLA isomers, mainly cis-9, trans-11 and trans-10, cis-12 in equal concentration. Our aim was to characterize the separate effects of both CLA isomers on thermogenic capacity in cultured brown adipocytes. The CLA isomers showed opposite effects. Hence, on the one hand, trans-10, cis-12 inhibited uncoupling protein (UCP) 1 induction by norepinephrine (NE) and produced a decrease in leptin mRNA levels. These effects were associated with a blockage of CCAAT-enhancer-binding protein-alpha and peroxisome proliferator-activated receptor-gamma(2) mRNA expression. On the other hand, cis-9, trans-11 enhanced the UCP1 elicited by NE, an effect reported earlier for polyunsaturated fatty acids and also observed here for linoleic acid. These findings could explain, at least in part, the effects observed in vivo when feeding a CLA mixture supplemented diet as a result of the combined action of CLA isomers (reduction of adipogenesis and defective BAT thermogenesis that could be through trans-10, cis-12 and enhanced UCP1 thermogenic capacity through cis-9, trans-11).     

Conjugated linoleic acid induces human adipocyte delipidation: autocrine/paracrine regulation of MEK/ERK signaling by adipocytokines

Dietary conjugated linoleic acid (CLA) reduces body fat in animals and some humans. Here we show that trans-10, cis-12 CLA, but not cis-9, trans-11 CLA, when added to cultures of stromal vascular cells containing newly differentiated human adipocytes, caused a time-dependent decrease in triglyceride content, insulin-stimulated glucose and fatty acid uptake, incorporation into lipid, and oxidation compared with controls. In parallel, gene expression of peroxisome proliferator-activated receptor-gamma and many of its downstream targets were diminished by trans-10, cis-12 CLA, whereas leptin gene expression was increased. Prior to changes in gene expression and metabolism, trans-10, cis-12 CLA caused a robust and sustained activation of mitogen-activated protein kinase kinase/extracellular signal-related kinase (MEK/ERK) signaling. Furthermore, the trans-10, cis-12 CLA-mediated activation of MEK/ERK could be attenuated by pretreatment with U0126 and pertussis toxin. In parallel, pretreatment with U0126 blocked the ability of trans-10, cis-12 CLA to alter gene expression and attenuate glucose and fatty acid uptake of the cultures. Intriguingly, the induction by CLA of MEK/ERK signaling was linked to hypersecretion of adipocytokines interleukin-6 and interleukin-8. Collectively, these data demonstrate for the first time that trans-10, cis-12 CLA decreases the triglyceride content of newly differentiated human adipocytes by inducing MEK/ERK signaling through the autocrine/paracrine actions of interleukins-6 and 8.     

The biologically active isomers of conjugated linoleic acid.

Numerous physiological effects are attributed to conjugated linoleic acid (CLA). The purpose of this presentation is to consider these effects with respect to the cis-9,trans-11 and trans-10,cis-12 CLA isomers. We review previously published data and present new findings that relate to underlying biochemical mechanisms of action. Both isomers are natural products. The cis-9,trans-11 isomer is the principal dietary form of CLA, but the concentrations of this isomer and the trans-10,cis-12 isomer in dairy products or beef vary depending on the diet fed to cows or steers, respectively. The trans-10,cis-12 CLA isomer exerts specific effects on adipocytes, in particular reducing the uptake of lipid by inhibiting the activities of lipoprotein lipase and stearoyl-CoA desaturase. The trans-10,cis-12 CLA isomer also affects lipid metabolism in cultured Hep-G2 human liver cells, whereas both the cis-9,trans-11 and trans-10,cis-12 CLA isomers appear to be active in inhibiting carcinogenesis in animal models. We present new findings indicating that the cis-9,trans-11 CLA isomer enhances growth and probably feed efficiency in young rodents. Accordingly, the effects of CLA on body composition (induced by trans-10,cis-12 CLA) and growth/feed efficiency (induced by cis-9,trans-11 CLA) appear to be due to separate biochemical mechanisms. We also show that a 19-carbon CLA cognate (conjugated nonadecadienoic acid, CNA) inhibits lipoprotein lipase activity as effectively as CLA in cultured 3T3-L1 adipocytes. Presumably, CNA is metabolized differently than the 18-carbon CLA isomers, so this finding indicates direct activity of the administered compound as opposed to acting via a metabolite.     

Conjugated linoleic acid promotes human adipocyte insulin resistance through NFkappaB-dependent cytokine production

We previously demonstrated that trans-10, cis-12 conjugated linoleic acid (CLA) reduced the triglyceride content of human adipocytes by activating mitogen-activated protein kinase kinase/extracellular signal-related kinase (MEK/ERK) signaling via interleukins (IL) 6 and 8. However, the upstream mechanism is unknown. Here we show that CLA increased (>or=6 h) the secretion of IL-6 and IL-8 in cultures containing both differentiated adipocytes and stromal vascular (SV) cells, non-differentiated SV cells, and adipose tissue explants. CLA isomer-specific induction of IL-6 and tumor necrosis factor-alpha was associated with the activation of nuclear factor kappaB (NFkappaB) as evidenced by 1) phosphorylation of IkappaBalpha, IkappaBalpha kinase, and NFkappaB p65, 2) IkappaBalpha degradation, and 3) nuclear translocation of NFkappaB. Pretreatment with selective NFkappaB inhibitors and the MEK/ERK inhibitor U0126 blocked CLA-mediated IL-6 gene expression. Trans-10, cis-12 CLA suppression of insulin-stimulated glucose uptake at 24 h was associated with decreased total and plasma membrane glucose transporter 4 proteins. Inhibition of NFkappaB activation or depletion of NFkappaB by RNA interference using small interfering NFkappaB p65 attenuated CLA suppression of glucose transporter 4 and peroxisome proliferator-activated receptor gamma proteins and glucose uptake. Collectively, these data demonstrate for the first time that trans-10, cis-12 CLA promotes NFkappaB activation and subsequent induction of IL-6, which are at least in part responsible for trans-10, cis-12 CLA-mediated suppression of peroxisome proliferator-activated receptor gamma target gene expression and insulin sensitivity in mature human adipocytes.     

Conjugated linoleic acids promote human fat cell apoptosis.

Conjugated linoleic acids (CLAs) are conjugated dienoic isomers of linoleic acid. Some isomers have been shown to reduce fat mass in animal and cell culture models. However, controversial results were obtained in studies of supplementation of CLAs in human subjects. In order to get more insights into the direct effects of CLAs on human fat cells, we have studied the influence of cis-9, trans-11 CLA and trans-10, cis-12 CLA on the biology of human SGBS preadipocytes and adipocytes. Both CLA isomers equally inhibited the proliferation of preadipocytes in a dose-dependent manner. Continuous treatment with 1-10 microM trans-10, cis-12 CLA, and to a weaker extent cis-9, trans-11 CLA, inhibited accumulation of lipids during adipogenic differentiation. Treatment with higher doses of CLA induced apoptosis in preadipocytes, in differentiating cells, and adipocytes. The trans-10, cis-12 isomer had a higher apoptotic potency in adipocytes than cis-9, trans-11 CLA. Taken together, the treatment of human preadipocytes and adipocytes with physiological relevant concentrations of CLAs resulted in an impairment of proliferation and differentiation and induction of apoptosis. The trans-10, cis-12 isomer was more potent than the cis-9, trans-11 isomer. Further clinical studies are needed to evaluate the effects of CLAs on human fat mass and metabolism in vivo.     

共轭亚油酸对脂肪代谢相关基因表达的影响

本文利用实时定量PCR的测定方法,分析了两种共轭亚油酸(CLA)异构体对3T3-L1小鼠前脂肪细胞脂肪代谢相关基因表达的影响。本研究CLA异构体的处理浓度和时间为75.4μmol/L,8 d,测定了与能量代谢、细胞凋亡、脂肪酸氧化作用和脂解作用相关的多种基因的mRNA水平。结果显示:两种异构体均能够显著提高UCP1、UCP3、Perilipin和PPARα的mRNA水平,而抑制UCP2的表达水平(P<0.01)。与cis-9t,rans-11CLA相比t,rans-10c,is-12 CLA显著提高PKA(P<0.05)、CPT-1和TNF-α(P<0.01)的mRNA水平。与对照组相比,两种CLA异构体处理组均对HSL、ATGL、ACO和Leptin的基因表达无显著影响。     

Linoleic acid partially restores the triglyceride content of conjugated linoleic acid-treated cultures of 3T3-L1 preadipocytes

We have previously demonstrated that a crude mixture of commercially available conjugated linoleic acid (CLA) isomers suppressed triglyceride (TG) content and induced apoptosis in post-confluent cultures of murine 3T3-L1 preadipocytes. Furthermore, we found that 100 μM of trans-10, cis-12 isomer of CLA had a greater TG-lowering and apoptotic effect than the crude mixture of CLA isomers. Therefore, the purpose of this study was to: 1) compare the potencies of the two main isomers found in the crude mixture of CLA isomers, e.g. cis-9, trans-11 (41%) and trans-10, cis-12 (44%); and 2) determine if the TG-reducing actions of CLA could be attenuated by the addition of increasing levels of linoleic acid to the cultures. Preadipocyte differentiation was assessed on day 7 of the differentiation protocol by measuring TG content (per 10⁶ cells), cell size, and lipid staining. In experiment 1, post-confluent cultures of 3T3-L1 preadipocytes treated for the first 6 d of differentiation with 100 μM of a crude mixture of CLA isomers or 44 μM of trans-10, cis-12 CLA had less TG content than all other cultures. In contrast, cultures supplemented with 41 μM of the cis-9, trans-11 CLA isomer had the same amount of TG as the BSA controls. In experiment 2, post-confluent cultures of 3T3-L1 preadipocytes treated for the first 6 d of differentiation with 50 μM trans-10, cis-12 CLA had less TG content and a greater number of smaller cells (10–12.5 microns) compared to all other treatments. CLA-treated cultures supplemented with increasing levels of linoleic acid (50–200 μM) had greater TG contents and greater numbers of larger cells (15–20 microns) than cultures treated with 50 μM of the trans-10, cis-12 CLA isomer alone. These data demonstrate that: 1) the TG-lowering effects of the crude mixture of CLA isomers is due almost exclusively to the trans-10, cis-12 isomer; and 2) linoleic acid partially reverses CLA’s attenuation of TG content, suggesting that these unsaturated fatty acids may compete for incorporation into TG or phospholipid-derived eicosanoids that regulate preadipocyte differentiation.     

Isomers of conjugated linoleic acid induce insulin resistance through a mechanism involving activation of protein kinase Cε in liver cells

Conjugated linoleic acid (CLA) constitutes a group of isomers derived from linoleic acid. Diverse studies have suggested that these unsaturated fatty acids have beneficial effects on human health. However, it has also been reported that their consumption can generate alterations in hepatic tissue. Thus, in the present study, we evaluated the effect of two of the major isomers of CLA, cis-9, trans-11-CLA and trans-10, cis-12-CLA, in the regulation of insulin signaling in a hepatic cell model, clone 9 (C9). We found that the two isomers decrease insulin-stimulated phosphorylation of the main proteins involved in insulin signaling, such as Akt at Ser⁴⁷³ and Thr³⁰⁸, the insulin receptor at Tyr¹¹⁵⁸, IRS-1 at Tyr⁶³², and GSK-3 at Ser^9/21. Protein expression, however, was unaffected. Interestingly, both isomers of CLA promoted phosphorylation and activation of PKCε. Inhibition of PKCε activity by a dominant-negative form or knockdown of endogenous PKCε prevented the adverse effects of CLA isomers on insulin-induced Akt phosphorylation. Additionally, we also found that both isomers of CLA increase phosphorylation of IRS-1 at Ser⁶¹², a mechanism that probably underlies the inhibition of IRS-1 signaling by PKCε. Using confocal microscopy, we found that both isomers of CLA induced lipid accumulation in C9 cells with the presence of spherical cytosolic vesicles, suggesting their identity as neutral lipid droplets. These findings indicate that cis-9, trans-11-CLA and trans-10, cis-12-CLA isomers could have a significant role in the development of insulin resistance in hepatic C9 cells through IRS-1 serine phosphorylation, PKCε activation, and hepatic lipid accumulation.     

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.     

9 cis,11 trans conjugated linoleic acid (CLA) is synthesised and desaturated into conjugated 18:3 in bovine adipose tissues

Although endogenous synthesis of conjugated linoleic acid (CLA) in the mammary gland of lactating cows has been already well documented, no study has determined so far as to which tissue and/or organ is involved in CLA synthesis in the growing ruminant except one study showing that CLA synthesis does not occur in ruminant liver. In this context, adipose tissue appears to be a good candidate for endogenous synthesis of CLA in the growing ruminant. The aim of this study was to compare the respective metabolisms of 11trans 18:1 (vaccenic acid, VA) and 9cis,11trans 18:2 (rumenic acid) to that of stearic acid (the preferential substrate of Δ9 desaturase) in adipose tissues (subcutaneous, SC and intermuscular, IM) of six Charolais steers by using the in vitromethod of incubated tissue slices. Samples of SC and IM adipose tissues were incubated at 37°C for 16 h under an atmosphere of 95% O2/5% CO2 in a medium supplemented with 0.75 mM of fatty acid (FA) mixture (representative of circulating non-esterified FA) and 186 μM [1-14C]-18:0 or 58.6 μM [1-14C]-VA or 56 μM [1-14C]-9cis,11trans CLA. Viability of explants was verified by measuring metabolic functions (glucose uptake and glucose-6-phosphate dehydrogenase activity). After 16 h of incubation, FA uptake was similar for all FA (18:0, VA and 9cis,11trans 18:2) in both SC and IM adipose tissues (around 40%). Once in adipose tissue, all FA were preferentially esterified (>80% of cell FA) favouring neutral lipid synthesis (around 90% of esterified FA). Stearic acid was highly (27%) desaturated into oleic acid in SC adipose tissue whereas this desaturation was much lower (6.8%) in IM adipose tissue (P < 0.0001). VA was desaturated into 9cis,11trans CLA at a low extent of about 2.5% to 4.4% in both adipose tissues probably because of a limited affinity of Δ9 desaturase for VA. 9cis,11trans CLA was itself converted by desaturation into 6cis, 9cis,11trans 18:3 at the intensity of 10.8% and 14.5% of cell 9cis,11trans CLA in SC and IM adipose tissues, respectively. In conclusion, bovine adipose tissues of the growing ruminant were especially involved in the endogenous synthesis of CLA from VA and in its desaturation into conjugated derivative, mainly 6cis, 9cis,11trans 18:3, of which biological properties need to be elucidated.     

Fatty acid composition of liver, adipose tissue, spleen, and heart of mice fed diets containing t10, c12-, and c9, t11-conjugated linoleic acid

Conjugated linoleic acid (CLA) isomers have unique effects on tissue lipids. Here we investigated the influence of individual CLA isomers on the lipid weight and fatty acid composition of lipid metabolizing (i.e. liver and retroperitoneal adipose) and lipid sensitive (i.e. spleen and heart) tissues. Female mice (8 week old; n=6/group) were fed either a control or one of the two CLA isomer supplemented (0.5%) diets for 8 weeks. The cis-9, trans-11-CLA diet reduced the 18:1n-9 wt% by 20-50% in liver, adipose tissue, and spleen, reduced the spleen n-3 polyunsaturated fatty acid (PUFA) by 90%, and increased the n-6 PUFA wt% by 20-50% in all tissues except heart. The trans-10, cis-12-CLA reduced both the n-6 and n-3 PUFA wt% in liver (>50%), reduced the heart n-3 PUFA wt% by 25%, and increased the wt% of spleen n-3 PUFA by 700%. The functional consequences of such changes in tissue fatty acid composition need to be investigated.     

Body fat-lowering effect of conjugated linoleic acid is not due to increased lipolysis

The ability of conjugated linoleic acid (CLA) to reduce adiposity may be due to changes in energy expenditure and/or direct effects on adipocyte lipid metabolism. The aim of the present work was to analyse if CLA supplementation modifies lipolytic activity in adipose tissue from hamsters fed on high-fat diet. Hamsters were divided into two groups and fed on diets supplemented with either 0.5% linoleic acid (control) or 0.5% trans-10,cis-12 CLA. After 6 weeks, animals were fasted overnight and adipose tissues were dissected and weighed. Adipocytes were isolated by collagenase digestion and incubated in Krebs-Ringer bicarbonate buffer with or without several agents acting at different levels of the lipolytic cascade. Adipocyte diameters were measured by microscopy. Adipose tissue DNA content was assessed by spectrophotometry. Animals fed on CLA diet showed significantly reduced adipose tissue mass. No differences between both groups was found for basal lipolysis, lipolytic effects of isoproterenol, forskolin, dibutyryl-cAMP and isobutylmethylxanthine, and pD2 for isoproterenol. A similar total DNA amount was found in adipose tissue of both groups, showing that CLA diet had no effect on total cell number per fat pad. Although DNA content per gram tissue, an indirect reverse index of cell size, was significantly increased in CLA fed hamsters, microscopy did not reveal differences in medium mature adipocyte diameter, nor in cell size distribution between both groups. These results suggest that adipose tissue size reduction induced by trans-10,cis-12 CLA intake is not due to changes in lipolysis. Reduced preadipocyte differentiation into mature adipocytes may account for this fat-lowering effect.     

Adipocyte cellularity in different adipose depots in bulls of seven Spanish breeds slaughtered at two body weights

The influence of body weight (BW) at slaughter and genotype on adipocyte size and number in the omental (OM), perirenal (PR), subcutaneous (SC) and intermuscular (IM) adipose tissues was studied in 168 bulls of Spain's local Asturiana, Avileña, Morucha, Parda Alpina, Pirenaica, Retinta, and Rubia Gallega cattle breeds. The young bulls were slaughtered at two BWs, 320 and 540 kg. The results obtained showed the higher amounts of lipids that accumulated between 320 and 540 kg BW (P < 0.001) to be ascribable primarily to adipose cell hypertrophy, i.e. larger adipocyte size, in the OM and PR depots (P < 0.001). In addition to hypertrophy, there was also an increase (P < 0.001) in the number of adipose cells, i.e. hyperplasia, in the SC and IM adipose depots. Significant differences were observed when comparing the different genotypes, with the Morucha, Retinta and Avileña breeds having the highest amount of adipose tissue and the largest adipocytes. The Asturiana and Rubia Gallega breeds had the lowest amount of adipose tissue and the smallest adipocytes. The Pirenaica and Parda Alpina breeds had intermediate values in between the two groups identified above. In short, the results were indicative of different lipid deposition patterns in the different breeds depending on the individual growth and maturation rates in each. Similar findings were made when comparing the different adipose tissue depots, with adipocyte hypertrophy being the main factor responsible for lipid accumulation in the OM and PR depots, as opposed to adipocyte hyperplasia in the SC and IM depots.     

Administration of a murine diet supplemented with conjugated linoleic acid increases the expression and activity of hepatic uncoupling proteins

Daily intake of conjugated linoleic acid (CLA) has been shown to reduce body fat accumulation and to increase body metabolism; this latter effect has been often associated with the up-regulation of uncoupling proteins (UCPs). Here we addressed the effects of a CLA-supplemented murine diet (~2?% CLA mixture, cis-9, trans-10 and trans-10, cis-12 isomers; 45?% of each isomer on alternating days) on mitochondrial energetics, UCP2 expression/activity in the liver and other associated morphological and functional parameters, in C57BL/6 mice. Diet supplementation with CLA reduced both lipid accumulation in adipose tissues and triacylglycerol plasma levels, but did not augment hepatic lipid storage. Livers of mice fed a diet supplemented with CLA showed high UCP2 mRNA levels and the isolated hepatic mitochondria showed indications of UCP activity: in the presence of guanosine diphosphate, the higher stimulation of respiration promoted by linoleic acid in mitochondria from the CLA mice was almost completely reduced to the level of the stimulation from the control mice. Despite the increased generation of reactive oxygen species through oxi-reduction reactions involving NAD(+)/NADH in the Krebs cycle, no oxidative stress was observed in the liver. In addition, in the absence of free fatty acids, basal respiration rates and the phosphorylating efficiency of mitochondria were preserved. These results indicate a beneficial and secure dose of CLA for diet supplementation in mice, which induces UCP2 overexpression and UCP activity in mitochondria while preserving the lipid composition and redox state of the liver.     

Conjugated linoleic acid improves blood pressure by increasing adiponectin and endothelial nitric oxide synthase activity

Conjugated linoleic acid (CLA) has been reported to reduce blood pressure in obese insulin-resistant rats, but its mechanism of action has not been identified. The objective of this study was to determine whether CLA isomers can reduce obesity-related hypertension in the fa/fa Zucker rat in relation to adiponectin production and endothelial nitric oxide synthase (eNOS) activation. Obese fa/fa Zucker rats were randomly assigned to one of four groups: (1) cis-9,trans-11-CLA, (2) trans-10,cis-12 (t10,c12)-CLA, (3) control or (4) captopril. After 8 weeks, systolic blood pressure increased 30% in control obese rats. This increase was attenuated 11%-13% in the t10,c12-CLA isomer and captopril groups, respectively. The t10,c12-CLA isomer concurrently elevated adiponectin levels in both plasma and adipose tissue and increased phosphorylated eNOS in adipose tissue as well as the aorta. Although a direct effect of CLA was not observed in cultured endothelial cells, direct adiponectin treatment increased phosphorylation of eNOS. Endothelial nitric oxide synthase phosphorylation was also increased in adipose of fa/fa Zucker rats infused with adiponectin in parallel with improvements in blood pressure. Our results suggest that the t10,c12-CLA isomer attenuates development of obesity-related hypertension, at least in part, by stimulating adiponectin production, which subsequently activates vascular eNOS.