C9t11 Conjugated Linoleic Acid Research Articles

Conjugated Linoleic Acid Activates AMP-Activated Protein Kinase and Reduces Adiposity More Effectively When Used with Metformin in Mice1–3

Trans-10, cis-12 (t10c12) conjugated linoleic acid (CLA) reduces lipid levels in adipocytes, but the mechanisms involved are still emerging. The hypotheses of this study were that t10c12 CLA treatment activated AMP-activated protein kinase (AMPK) and that the effectiveness of a low dose of t10c12 CLA would be increased when combined with an AMPK activator. We demonstrated t10c12 CLA, directly or indirectly, activated AMPK and increased the amount of phosphorylated acetyl-CoA carboxylase (ACC) in 3T3-L1 adipocytes. Compound C, a potent inhibitor of AMPK, attenuated the phosphorylation of ACC, integrated stress response (ISR), inflammatory response, reduction in key lipogenic transcription factors, and triglyceride (TG) reduction that otherwise occurred in t10c12 CLA-treated adipocytes. Treatment of adipocytes or mice with a low dose of t10c12 CLA in conjunction with the AMPK activator metformin resulted in more TG loss than treatment with the individual chemicals. Additionally, although an inflammatory response was required for robust TG reduction, the combination of t10c12 CLA with AMPK activators had a similar TG loss with a reduced inflammatory response. A microarray analysis of the transcriptional response to either t10c12 CLA, metformin, or the combination, indicated the responses were very similar, with a correlation coefficient of 0.91 or better for genes in the ISR or lipid-related pathways. Altogether, these results support our hypotheses that t10c12 CLA activates AMPK, directly or indirectly, and that metformin increases the effectiveness of t10c12 CLA in reducing TG amounts in adipocytes.

Conjugated Linoleic Acid Isomers, t10c12 and c9t11, are Differentially Incorporated into Adipose Tissue and Skeletal Muscle in Humans

Conjugated linoleic acid (CLA) is a popular supplement believed to enhance immune function, body composition and insulin sensitivity, but results of scientific studies investigating its effects are conflicting. The isomer- and tissue-specific effects of CLA may explain these conflicting results. Therefore, this study quantified the incorporation of the c9t11 and t10c12 CLA isomers into adipose tissue and skeletal muscle in response to supplementation in healthy, regularly-exercising, non-obese persons. The CLA group (n = 14) ingested 3.9 g per day CLA (50:50 t9c11:c10t12) and the placebo group (n = 11) 3.9 g per day high-oleic-acid sunflower oil for 12 weeks. Following supplementation, the t10c12 isomer was incorporated into adipose tissue triacylglycerol (P < 0.001), and the c9t11 isomer tended to increase in skeletal muscle phospholipids (P = 0.056). Therefore, human adipose tissue and skeletal muscle are enriched with CLA in an isomer-specific manner.

Peroxisome proliferator-activated receptor-γ activation and long-chain fatty acids alter lipogenic gene networks in bovine mammary epithelial cells to various extents

Several long-chain fatty acids (LCFA) are natural ligands of nonruminant peroxisome proliferator-activated receptor-γ (PPARG), which, along with its lipogenic target genes, is upregulated in bovine mammary tissue during lactation. Thus, PPARG might represent an important control point of bovine milk fat synthesis. We tested lipogenic gene network expression via quantitative PCR of 19 genes in bovine mammary epithelial cells cultured with 16:0, 18:0, cis-9 18:1, trans-10 18:1, trans-10,cis-12 18:2 [t10c12 conjugated linoleic acid (CLA)], 20:5, ethanol (control), and the PPARG agonist rosiglitazone (ROSI). Triplicate cultures were maintained for 12h with 50μM ROSI or 100μM LCFA. Responses common to 16:0 and 18:0 relative to the control included significantly greater expression of INSIG1 (+298%, +92%), AGPAT6 (+137%, +169%), FABP3 (+755%, +338%), and FABP4 (+171%, 157%). These were coupled with greater intracellular lipid droplet formation and mRNA of ACSS2, LPIN1, SCD, and SREBF2 in response to 16:0, and greater DGAT1 and THRSP with 18:0. Trans-10 18:1 and t10c12 CLA reduced expression of FASN (–60%, –31%), SCD (–100%, –357%), and SREBF1 (–49%, –189%). Furthermore, t10c12 CLA downregulated ACSS2, FABP3, INSIG1, SREBF2, and THRSP expression. Expression of SREBF1 was lower with cis-9 18:1 (–140%) and 20:5 (–125%) compared with the control. This latter LCFA also decreased SCD, SREBF2, and LPL expression. No effects of LCFA or ROSI on PPARG were observed, but ROSI upregulated (+39% to +269%) expression of ACACA, FASN, LPIN1, AGPAT6, DGAT1, SREBF1, SREBF2, and INSIG1. Thus, these genes are putative PPARG target genes in bovine mammary cells. This is the first report showing a direct effect of trans-10 18:1 on bovine mammary cell lipogenic gene expression. The coordinated upregulation of lipogenic gene networks in response to ROSI and saturated LCFA offers support for PPARG activation in regulating bovine milk fat synthesis.

Differential effects of conjugated linoleic acid isomers in insulin-resistant female C57Bl/6J mice

Obesity is associated with a high risk of developing diabetes and cardiovascular disease. Therefore, management of body weight to prevent obesity remains as an important priority. The present investigation addresses the effects of conjugated linoleic acid (CLA) isomers on body weight and composition of body fat in female C57Bl/6J mice. To investigate the differential effects of individual CLA isomers and their mixture on changes in lean mass, fat mass, glucose and insulin, 6-month-old female C57BL/6J mice were fed with 10% corn oil (CO) as a dietary fat source and either supplemented with purified cis 9, trans 11 (c9t11) CLA (0.5%) or trans 10, cis 12 (t10c12) CLA (0.5%) and/or their mixture (50:50) for 6 months. As a result of 6 months' dietary intervention, both the t10c12-CLA and CLA mix showed increased lean mass and reduced fat mass compared to the CO and c9t11-CLA groups. Insulin resistance was, however, increased in t10c12-CLA and CLA mix-fed groups based on the results of homeostasis model assessment (HOMA), the revised quantitative insulin-sensitivity check index (R-QUICKI) and also with intravenous glucose tolerance test (IVGTT). In conclusion, long-term feeding of the major CLA isomers in 12-month-old C57Bl/6J mice revealed a contrasting effect on fat mass, glucose and insulin metabolism. The t10c12 isomer is found to reduce the fat mass and increase the lean mass but significantly contributed to increase insulin resistance and liver steatosis, whereas c9t11 isomer prevented the insulin resistance.

T10c12 Conjugated Linoleic Acid Suppresses HER2 Protein and Enhances Apoptosis in SKBr3 Breast Cancer Cells: Possible Role of COX2

BackgroundHER2-targeted therapy with the monoclonal antibody trastuzumab (Herceptin®) has improved disease-free survival for women diagnosed with HER2-positive breast cancers; however, treatment resistance and disease progression are not uncommon. Current data suggest that resistance to treatment in HER2 cancers may be a consequence of NF-κB overexpression and increased COX2-derived prostaglandin E2 (PGE2). Conjugated linoleic acid (CLA) has been shown to have anti-tumor properties and to inhibit NF-κB activity and COX2.MethodsIn this study, HER2-overexpressing SKBr3 breast cancer cells were treated with t10c12 CLA. Protein expression of the HER2 receptor, nuclear NF-κB p65, and total and phosphorylated IκB were examined by western blot and immunofluorescence. PGE2 levels were determined by ELISA. Proliferation was measured by metabolism of 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), and apoptosis was measured by FITC-conjugated Annexin V staining and flow cytometry.Results/ConclusionsWe observed a significant decrease in HER2 protein expression on western blot following treatment with 40 and 80 µM t10c12 CLA (p<0.01 and 0.001, respectively) and loss of HER2 protein in cells using immunoflourescence that was most pronounced at 80 µM. Protein levels of nuclear NF-κB p65 were also significantly reduced at the 80 µM dose. This was accompanied by a significant decrease in PGE2 levels (p = 0.05). Pretreatment with t10c12 CLA significantly enhanced TNFα-induced apoptosis and the anti-proliferative action of trastuzumab (p = 0.05 and 0.001, respectively). These data add to previous reports of an anti-tumor effect of t10c12 CLA and suggest an effect on the HER2 oncogene that may be through CLA mediated downregulation of COX2-derived PGE2.

Conjugated linoleic acid (CLA) prevents age associated skeletal muscle loss in mice by maintaining redox balance during aging

Age related skeletal muscle sarcopenia is a major health problem. Currently, there is no safe and effective therapies that selectively increase skeletal muscle mass. Previously, we have shown that CLA maintains higher skeletal muscle mass in mice. This study was designed to elucidate the molecular mechanisms of muscle loss prevention by CLA using 12 mo old C57BL/6 mice fed 10% corn oil (CO) or a diet supplemented with 0.5% c9t11 CLA (CLA 1), t10c12 CLA (CLA 2) or c9t11 CLA+t10c12 CLA (CLA mix) for 6 months. Dual energy X‐ray absorptiometry (DXA) scan was performed at baseline, and end of dietary intervention. CLA 2 and CLA mix groups showed significantly higher muscle mass as compared to CO and CLA 1 groups by DXA and muscle wet weight. Production of muscle mitochondrial ATP, and H2O2 as reactive oxygen species and muscle antioxidant enzymes (catalase and glutathione peroxidase) has been analyzed. Enhanced production of ATP, and antioxidant enzymes accompanied by slight increase in H2O2 level was noted in CLA 2 and CLA mix groups as compared to that of CO and CLA 1 groups. Finally, we have analyzed serum malondialdehyde (MDA) level. The lower level of serum MDA in CLA 2 and CLA mix groups indicate less oxidative stress in these groups. These data suggest that CLA 2 and CLA mix groups may preserve muscle mass by enhancing muscle mitochondrial energy production as well as by maintaining the redox balance during aging. Research support: NIH

Inhibitory Effect of Conjugated α‐Linolenic Acid from Bifidobacteria of Intestinal Origin on SW480 Cancer Cells

In this study, we assessed the ability of six strains of bifidobacteria (previously shown by us to possess the ability to convert linoleic acid to c9, t11-conjugated linoleic acid (CLA) to grow in the presence of alpha-linolenic acid and to generate conjugated isomers of the fatty acid substrate during fermentation for 42 h. The six strains of bifidobacteria were grown in modified MRS (mMRS) containing alpha-linolenic acid for 42 h at 37 degrees C, after which the fatty acid composition of the growth medium was assessed by gas liquid chromatography (GLC). Indeed, following fermentation of one of the strains, namely Bifidobacterium breve NCIMB 702258, in the presence of 0.41 mg/ml alpha-linolenic acid, 79.1% was converted to the conjugated isomer, C18:3 c9, t11, c15 conjugated alpha-linolenic acid (CALA). To examine the inhibitory effect of the fermented oils produced, SW480 colon cancer cells were cultured in the presence of the extracted fermented oil (10-50 microg/ml) for 5 days. The data indicate an inhibitory effect on cell growth (p <or= 0.001) of CALA, with cell numbers reduced by 85% at a concentration of 180 microM, compared with a reduction of only 50% with alpha-linolenic acid (p <or= 0.01).

Abnormal Immune Responses in fa/fa Zucker Rats and Effects of Feeding Conjugated Linoleic Acid

The objective of this study was to characterize immune function in the fa/fa Zucker rat, and to determine the effects of feeding conjugated linoleic acid (CLA) isomers on immune function. Lean and fa/fa Zucker rats were fed for 8 weeks nutritionally complete diets with different CLA isomers (%wt/wt): control (0%), c9t11 (0.4%), t10c12 (0.4%), or MIX (0.4% c9t11 + 0.4% t10c12). Isolated splenocytes were used to determine phospholipid (PL) fatty acid composition and cell phenotypes, or stimulated with mitogen to determine their ability to produce cytokines, immunoglobulins (Ig), and nitric oxide (NO). Splenocyte PL of fa/fa rats had a higher proportion of total monounsaturated fatty acids and n -3 polyunsaturated fatty acids (PUFA), and lower n -6 PUFA and n -6-to-n -3 PUFA ratio (P < 0.05). Feeding CLA increased the content of CLA isomers into PL, but there were lower proportions of each CLA isomer in fa/fa rats. Splenocytes of fa/fa rats produced more amounts of IgA, IgG, and IgM, NO, and interleukin-1beta (IL-1beta), IL-6, and tumor necrosis factor-alpha (TNF-alpha) (P < 0.05). Obese rats fed the t10c12 diet produced less TNF-alpha and IL-1beta (lippopolysaccharide (LPS), P < 0.05). Splenocytes of fa/fa rats produced less concanavalin A (ConA)-stimulated IL-2 (P < 0.0001) than lean rats, except fa/fa rats fed the c9t11 diet (P < 0.05). The c9t11 and t10c12 CLA isomers were incorporated into the membrane PL of the fa/fa Zucker rat, but to a lesser extent than lean rats. Splenocytes of obese rats responded in a proinflammatory manner and had reduced T-cell function and feeding the t10c12 and c9t11 CLA isomers may improve some of these abnormalities by distinct methods.

Influence of pasture on fatty acid profile of goat milk

The effect of pasture on the fatty acid profile of milk was evaluated using 30 pluriparous goats, which were divided into two homogeneous groups (H and P) of 15 goats each after kidding. Group H (control) goats were housed in a stall and received alfalfa hay, while group P was led to pasture. The goats of both the groups received the same concentrate. Average milk yield did not differ statistically between the groups. The goats of the P group had significantly higher fat content in milk than those of group H (53.7 vs. 61.5 g/day, p < 0.01), while lactose content was significantly higher for group H (66.2 and 61.4 g/day, for groups H and P, respectively, p < 0.05). Among milk fatty acids, pasture significantly affected the percentages of C18:1 cis9, C18:1 trans11, octadecadienoic acid, mono-unsaturated fatty acid and polyunsaturated fatty acid. Pasture highly influenced the c9 t11 conjugated linoleic acid (CLA) (0.778 and 0.513 g/100 g of fat, for group P and H, respectively, p < 0.01), t10 c12 CLA (0.046 vs. 0.029 g/100 g of fat: p < 0.01, for group P and H, respectively, p < 0.01) and SigmaCLA (0.84 vs. 0.56 g/100 g of fat for group P and H, respectively, p < 0.01) concentrations of milk.

T10c12 CLA isomer prevents age associated bone loss by modulating osteoclastogenesis

Osteoporosis is one of the major causes of morbidity in the elderly. Inflammation exerts a significant influence on bone turnover, inducing the chronic form of osteoporosis. Dietary nutrition has the capacity to modulate inflammatory response. Therefore, nutritional strategies and lifestyle changes may prevent age‐related osteoporosis, thereby improving the quality of life of the elderly population. Conjugated linoleic acid (CLA) has been shown to positively influence calcium and bone metabolism. Earlier we have shown that CLA (equal ratio of c9t11 and t10c12 isomers) can protect age associated bone loss by modulating inflammatory markers and osteoclastogenesis. However, c9t11 and t10c12 CLA individual isomers differentially regulate functional parameters and gene expression in different cell types. Therefore, the present study was undertaken to examine the differential effect of c9t11 and t10c12 CLA isomers on bone mineral density (BMD) in middle‐aged (12 mo) C57BL/6 female mice. After 5 months on diet, t10c12 CLA‐isomer fed mice maintained a significantly higher BMD in femoral, tibial and lumbar regions than c9t11 and corn oil (CO)‐fed mice. The increased BMD was accompanied by a decreased activity of osteoclastogenic factor, RANKL and decreased osteoclast activity as measured by TRAP5b in serum. A significant reduction of osteoclasts differentiation was also observed in RANKL stimulated RAW 264.7 cells in t10c12 CLA isomer treated culture as compared to c9t11 CLA isomer or linoleic acid treated culture. In conclusion, these findings suggest that t10c12 isomer is the most potent CLA isomer to prevent the bone loss by modulating osteoclastogenesis. Research support: NIH R21 AG027562.

Individual and combined effects of docosahexaenoic acid, stearic acid, trans‐vaccenic acid, and c9,t11‐conjugated linoleic acid on hepatic gene expression

Dietary long-chain fatty acids (LCFA), polyunsaturated fatty acids (PUFA) and their combinations alter metabolism by modulating expression of fatty acid synthase (FAS), carnitine palmitoyltransferase-1a (CPT-1a), and acyl-CoA oxidase (ACO). The effects of LCFA and PUFA on mRNA expression were determined in H4IIE rat hepatoma cells incubated with docosahexaenoic acid (DHA), c9, t11-conjugated linoleic acid (CLA), trans-vaccenic acid (TVA), and stearic acid (SA) or their combinations. Treatments were: 200 μM of individual fatty acids DHA, CLA, SA, or TVA; Replacement of 100 μM DHA with either 100 μM CLA or 100 μM SA; 100 μM CLA replaced by 100 μM TVA (final fatty acid concentration of 200 μM); 200 μM of DHA or CLA supplemented with 200μM of CLA, SA, or TVA. When CLA replaced DHA, FAS mRNA decreased by 53.7% (P< 0.05). Replacement of DHA with SA resulted in a 74.9% decrease (P< .05) in FAS mRNA. When 200 μM CLA was supplemented with 200 μM DHA or with 200 μM TVA, FAS mRNA decreased (P< 0.05) by 62.4% and 48.6% respectively. When CLA was replaced with TVA, CTP-1a mRNA tended (P= 0.05) to decrease by 47.5%. When CLA was supplemented with DHA there was a tendency (P< 0.10) for a 40.2% decrease in CPT-1a mRNA. Fatty acid concentration and mixture had no effect on ACO mRNA. The data suggest that individual and combined LCFA/PUFA exert different potencies on expression of genes for fatty acid metabolism in liver.

Production of trans C18:1 and conjugated linoleic acid in continuous culture fermenters fed diets containing fish oil and sunflower oil with decreasing levels of forage

Previously, feeding fish oil (FO) and sunflower seeds to dairy cows resulted in the greatest increases in the concentrations of vaccenic acid (VA, t11 C18:1) and conjugated linoleic acid (CLA) in milk fat. The objective of this study was to evaluate the effects of forage level in diets containing FO and sunflower oil (SFO) on the production of trans C18:1 and CLA by mixed ruminal microbes. A dual-flow continuous culture system consisting of three fermenters was used in a 3 × 3 Latin-square design. Treatments consisted of (1) 75:25 forage:concentrate (HF); (2) 50:50 forage:concentrate (MF); and (3) 25:75 forage:concentrate (LF). FO and SFO were added to each diet at 1 and 2 g/100 g dry matter (DM), respectively. The forage source was alfalfa pellets. During 10-day incubations, fermenters were fed treatment diets three times daily (140 g/day, divided equally between three feedings) as TMR diet. Effluents from the last 3 days of incubation were collected and composited for analysis. The concentration of trans C18:1 (17.20, 26.60, and 36.08 mg/g DM overflow for HF, MF, and LF treatments, respectively) increased while CLA (2.53, 2.35, and 0.81 mg/g DM overflow) decreased in a linear manner (P < 0.05) as dietary forage level decreased. As dietary forage levels decreased, the concentrations of t10 C18:1 (0.0, 10.5, 33.5 mg/g DM) in effluent increased ( P < 0.05) and t10c12 CLA (0.08, 0.12, 0.35 mg/g DM) tended to increases (P < 0.09) linearly. The concentrations of VA (14.7, 13.9, 0.0 mg/g DM) and c9t11 CLA (1.78, 1.52, 0.03 mg/g DM) in effluent decreased in a linear manner ( P < 0.05) as dietary forage levels decreased. Decreasing dietary forage levels resulted in t10 C18:1 and t10c12 CLA replacing VA and c9t11 CLA, respectively, in fermenters fed FO and SFO.

The effect of pH and polyunsaturated C18 fatty acid source on the production of vaccenic acid and conjugated linoleic acids in ruminal cultures incubated with docosahexaenoic acid

Previously, combining docosahexaenoic acid (DHA) with linoleic acid in rumen cultures enhanced vaccenic acid (VA) and conjugated linoleic acid (CLA) accumulations. The objective of this experiment was to examine the effect of two pH levels and two polyunsaturated C18 fatty acid (FA) sources on VA and CLA accumulations in rumen cultures incubated with DHA. High pH culture treatments consisted of 10 mg DHA + 20 mg linoleic acid (LOH), or 10 mg DHA + 20 mg linolenic acid (LNH). Low pH culture treatments consisted of 10 mg DHA + 20 mg linoleic acid (LOL), or 10 mg DHA + 20 mg linolenic acid (LNL). Treatments were incubated in triplicate in 125 ml flasks containing 500 mg finely ground TMR, 10 ml strained ruminal fluid, 40 ml media, and 2 ml reducing solution. Ruminal fluid was collected from fermenters fed high forage (pH 6.7) or high grain diets (pH 5.4). A 5-ml sample of culture contents was taken at 0 and 24 h for fatty acid analysis by gas liquid chromatography. After 24 h of incubation, VA was the predominant trans C18:1 FA isomer in the high pH cultures and its concentration was greater with LOH (20.9 mg/culture) than with LNH (9.3 mg/culture). Similarly, t10 C18:1 was the predominant trans C18:1 FA isomer in the low pH cultures and its concentration was greater with LOL (12.8 mg/culture) than with LNL (4.5 mg/culture). The c9 t11 CLA (0.74 mg/culture) and tt CLA (0.67 mg/culture) were the predominant CLA isomers in the LOH cultures, while t10 c12 CLA (1.47 mg/culture) and tt CLA (1.27 mg/culture) were the predominant CLA isomers in the LOL cultures. The t11 t13 CLA increased in linolenic acid cultures, particularly when linolenic acid incubated under low pH condition (0.95 mg/culture). Additions of linolenic acid to rumen cultures caused a dramatic increase in the concentration of t11 c15 C18:2 (9.84 and 8.45 mg/culture, for treatments LNH and LNL, respectively). The concentrations of VA and c9 t11 CLA in rumen cultures were greatest when DHA was incubated with linoleic acid at high pH. In contrast, t10 C18:1 replaced VA and c9 t11 CLA disappeared when linoleic and linolenic acids were incubated under the low pH condition. This study shows that the best approach to increase ruminal VA production, and potentially c9 t11 CLA levels in ruminant food products, is by combining a DHA source (such as fish oil or algae) and linoleic acid source under high ruminal pH condition.

Isomer-Specific Effect of Conjugated Linoleic Acid on Inflammatory Adipokines Associated with Fat Accumulation in 3T3-L1 Adipocytes

The purpose of this study was to examine the isomer-specific effect of conjugated linoleic acid (CLA) on inflammatory markers associated with fat accumulation in cultures of differentiating 3T3-L1 adipocytes. trans-10,cis-12 CLA (t10c12 CLA) reduced leptin secretion and fat accumulation. Linoleic acid (LA) and cis-9,trans-11 CLA (c9t11 CLA) increased them, but not significantly. t10c12 CLA and LA showed similar effects on mRNA expression of inflammatory markers. t10c12 CLA and LA tended to up-regulate the mRNA levels of inflammatory cytokines such as interleukin (IL)-6 (not significantly), tumor necrosis factor (TNF)-alpha, and C-reactive protein (CRP) with no significant change in the secretion of adiponectin, an anti-inflammatory adipokine. However, c9t11 CLA induced no significant change in the mRNA expression of IL-6, TNF-alpha, or CRP, but significantly increased adiponectin secretion. In conclusion, CLA exerted isomer-specific effects on fat accumulation and mRNA expression of inflammatory markers in 3T3-L1 adipocytes. t10c12 CLA up-regulated inflammatory markers in spite of the decreased fat accumulation, and TNF-alpha might be one of the causal factors.

Trans-10,cis-12 Conjugated Linoleic Acid Inhibits the G1–S Cell Cycle Progression in DU145 Human Prostate Carcinoma Cells

Conjugated linoleic acid (CLA) is a group of positional and geometric isomers of linoleic acid, and has evidenced anti-cancer activities in experimental animal cancer models and in vitro studies. The two predominant isomers of CLA are cis-9,trans-11 CLA (c9t11) and trans-10,cis-12 CLA (t10c12). The present study was performed to study the effect of the individual CLA isomers on DU145 cell growth. The cells were incubated in serum-free medium with different concentrations of the fatty acids. Treatment of cells with t10c12 (at 2.5-10 micromol/L) resulted in a dose-dependent reduction in the numbers of viable cells, whereas c9t11 CLA at a concentration of 5 micromol/L slightly increased viable cell numbers at 3 days (P < .05). DNA flow cytometric analysis revealed that the treatment of DU145 cells with t10c12 for 24 hours induced a small but significant increase in the number of cells in the G1 phase, accompanied by a complementary decrease in cells in the S phase. c9t, however, had no effect on cell cycle progression. To determine the molecular mechanisms underlying t10c12-induced G1 arrest, the levels of cell cycle regulatory proteins were estimated by western blot analyses. t10c12 induced a marked increase in p21(CIP1/WAF1) protein levels in a dose-dependent manner. p27(KIP1) was not affected by t10c12. t10c12 moderately decreased cyclin A and cyclin D1 protein levels (P > .05). However, t10c12 did not affect the expression of cyclin-dependent kinase (CDK) 2, CDK4, or cyclin E. t10c12 increased p21(CIP1/WAF1) bound to CDK2 and attenuated CDK2 activity. These results indicate that t10c12-induced p21(CIP1/WAF1) binds to CDK, and inhibits the activity of this enzyme, which results in the observed decrease in the G1-S progression in DU145 cells.

Conjugated linoleic acid and obesity control: efficacy and mechanisms.

Obesity is associated with high blood cholesterol and high risk for developing diabetes and cardiovascular disease. Therefore, management of body weight and obesity are increasingly considered as an important approach to maintaining healthy cholesterol profiles and reducing cardiovascular risk. The present review addresses the effects of conjugated linoleic acid (CLA) on fat deposition, body weight and composition, safety, as well as mechanisms involved in animals and humans. Animal studies have shown promising effects of CLA on body weight and fat deposition. The majority of the animal studies have been conducted using CLA mixtures that contained approximately equal amounts of trans-10, cis-12 (t10c12) and cis-9, trans-11 (c9t11) isomers. Results of a few studies in mice fed CLA mixtures with different ratios of c9t11 and t10c12 isomers have indicated that the t10c12 isomer CLA may be the active form of CLA affecting weight gain and fat deposition. Inductions of leptin reduction and insulin resistance are the adverse effects of CLA observed in only mice. In pigs, the effects of CLA on weight gain and fat deposition are inconsistent, and no adverse effects of CLA have been reported. A number of human studies suggest that CLA supplementation has no effect on body weight and insulin sensitivity. Although it is suggested that the t10c12 CLA is the antiadipogenic isomer of CLA in humans, the effects of CLA on fat deposition are marginal and more equivocal as compared to results observed in animal studies. Mechanisms through which CLA reduces body weight and fat deposition remain to be fully understood. Proposed antiobesity mechanisms of CLA include decreased energy/food intake and increased energy expenditure, decreased preadipocyte differentiation and proliferation, decreased lipogenesis, and increased lipolysis and fat oxidation. In summary, CLA reduces weight gain and fat deposition in rodents, while produces less significant and inconsistent effects on body weight and composition in pigs and humans. New studies are required to examine isomer-specific effects and mechanisms of CLA in animals and humans using purified individual CLA isomers.

Evidence that the anti-obesity effect of conjugated linoleic acid is independent of effects on stearoyl-CoA desaturase1 expression and enzyme activity

The trans-10, cis-12 isomer of conjugated linoleic acid (CLA) reduces body fat gain in animals and inhibits stearoyl-CoA desaturase (SCD) activity in 3T3-L1 adipocytes. To test whether CLA’s body fat reduction is mediated by SCD1, wild-type and SCD1-null mice were fed diet supplemented with 0.2% trans-10, cis-12 (t10c12) CLA for 4 weeks. The t10c12 CLA-supplemented diet significantly reduced body fat mass in both wild type and SCD1-null mice. Similarly, t10c12 CLA diet decreased blood triglyceride and free fatty acid levels regardless of SCD1 genotypes. Mice fed t10c12 CLA exhibited increased mRNA expression of fatty acid synthase and uncoupling protein 2 in both genotypes. Taken together, the effects of t10c12 CLA on reduction of body fat gain, blood parameters, and mRNA expression in both SCD1-null mice and wild-type mice were similar, indicating that the anti-obesity effect of t10c12 CLA may be independent of the effects of this CLA isomer on SCD1 gene expression and enzyme activity.

Trans-10, cis-12 conjugated linoleic acid reduces insulin-like growth factor-II secretion in HT-29 human colon cancer cells.

We previously demonstrated that a mixture of conjugated linoleic acid (CLA) isomers decreases colon cancer incidence in rats treated with 1,2-dimethylhydrazine. Our in vitro studies have also shown that CLA inhibits the growth of HT-29 cells, a human colon cancer cell line. When we compared the individual potencies of the two main isomers found in the mixture of CLA isomers (e.g., cis-9, trans-11 [c9t11] and trans-10, cis-12 [t10c12]), t10c12 CLA decreased viable cell numbers in a dose-dependent manner. By contrast, c9t11 CLA had no effect. Therefore, the present study examined whether the decreased cell growth is related to changes in secretion of insulin-like growth factor (IGF)-II and/or IGF-binding proteins (IGFBPs) that have been shown to regulate HT-29 cell proliferation. Cells were incubated in serum-free medium with various concentrations of the individual CLA isomers, and immunoblot analysis of 24-hour, serum-free, conditioned media using a monoclonal anti-IGF-II antibody was performed. HT-29 cells secreted both mature 7,500 apparent molecular weight (M(r)) and higher-M(r) forms of IGF-II. t10c12 CLA decreased the levels of the higher-M(r) and the mature form of IGF-II in a dose-dependent manner, whereas c9t11 CLA had no effect. Ligand blot analysis of conditioned medium using (125)I-IGF-II revealed that the production of IGFBP-2 and IGFBP-4 was also decreased by t10c12 CLA, whereas c9t11 CLA had no effect. Exogenous IGF-II abrogated the growth inhibition induced by t10c12 CLA. These results indicate that inhibition of HT-29 cell growth by t10c12 CLA may be mediated by decreasing IGF-II secretion in these cells.

Trans-10, cis-12 CLA inhibits differentiation of 3T3-L1 adipocytes and decreases PPARγ expression

The trans-10, cis-12 isomer of conjugated linoleic acid (CLA) has been shown to reduce body fat gain in mice. However, the underlying molecular mechanism is not well characterized. Here we report evidence that trans-10, cis-12 (t10c12) CLA inhibits preadipocyte differentiation. Treating differentiating 3T3-L1 preadipocytes with t10c12 CLA and conjugated nonadecadienoic acid (CNA, a 19-carbon CLA cognate) resulted in decreased intracellular triglyceride accumulation and mRNA levels of the adipogenic gene fatty acid synthase and adipocyte lipid binding protein. T10c12 CLA and CNA also reduced protein levels of adipocyte transcription factors, peroxisome proliferator-activated receptor γ and CCAAT/enhancer binding protein α. Similarly, CLA reduced body fat gain and significantly inhibited the expression of PPARγ and its downstream target lipoprotein lipase in mouse adipose tissue. These observations indicate that CLA decreases body fat gain in part by inhibiting the differentiation of preadipocytes.

Trans-10,cis-12-conjugated linoleic acid inhibits Caco-2 colon cancer cell growth.

A commercially available mixture of conjugated linoleic acid (CLA) isomers decreases colon cancer cell growth. We compared the individual potencies of the two main isomers in this mixture [cis-9,trans-11 (c9t11) and trans-10,cis-12 (t10c12)] and assessed whether decreased cell growth is related to changes in secretion of insulin-like growth factor II (IGF-II) and/or IGF-binding proteins (IGFBPs), which regulate Caco-2 cell proliferation. Cells were incubated in serum-free medium with different concentrations of the individual CLA isomers. t10c12 CLA dose dependently decreased viable cell number (55 +/- 3% reduction 96 h after adding 5 microM t10c12 CLA). t10c12 CLA induced apoptosis and decreased DNA synthesis, whereas c9t11 CLA had no effect. Immunoblot analysis of 24-h serum-free conditioned medium using a monoclonal anti-IGF-II antibody revealed that Caco-2 cells secreted both a mature 7,500 molecular weight (M(r)) IGF-II and higher M(r) forms of IGF-II. The levels of the higher M(r) and the mature form of IGF-II were decreased 50 +/- 3% and 22 +/- 2%, respectively, by 5 microM t10c12 CLA. c9t11 CLA had no effect. Ligand blot analysis of conditioned medium using 125I-labeled IGF-II revealed that t10c12 CLA slightly decreased IGFBP-2 production; c9t11 CLA had no effect. Exogenous IGF-II reversed t10c12 CLA-induced growth inhibition and apoptosis. These results indicate that CLA-inhibited Caco-2 cell growth is caused by t10c12 CLA and may be mediated by decreasing IGF-II secretion in Caco-2 cells.