Oxidation and apoptosis are induced by CLA-t10c12 similar to omega-3 DHA in 3T3 adipocyte cells
Citation
Meadus WJ, Vahmani P, Duff P, Zantinge JL, Turner TD, and Dugan M.E.R. Oxidation and apoptosis are induced by CLA-t10c12 similar to DHA in 3T3 adipocyte cells. Functional Food in Health and Disease 2017: 7(2): 149-167
Plain language summary
Polyunsaturated fatty acids (PUFA) are toxic to rumen bacteria. To cope, rumen bacteria hydrogenate PUFAs and conjugated linoleic acids (CLAs) can be formed in the process. One type of CLA -t10c12 has anti-obesity effects but the exact mechanism is unknown. Omega-3 fatty acids and t10,c12-CLA may share some common anti-obesity effects and we wanted to determine if these effects are related to oxidative stress. We took immature 3T3 cells and induced them to become mature adipose fat cells. We cultured the adipose cells with fatty acids, t10,c12-CLA, c9,t11-CLA, linoleic acid (18:2n-6) and the omega-3 fatty acids, alpha linolenic acid (18:3n-3) and DHA (22:6n-3). Cellular factors were measured by gene expression at both the mRNA, protein level and through biochemical assays. We found that both the CLA-t10,c12 and DHA caused oxidative stress in fat cells. The oxidative stress was severe enough to induce apoptosis and programmed cell death. CLA-t10-c12 was unique from DHA because it stimulated aldo-keto reductase (AKR1c1) indicating fat oxidation, CCL5 and MCP-1 indicating cell inflammation but not the factor adipoQ which is usually the fat oxidation metabolism factor. We speculate that the unique t10 double bond in t10c12-CLA is responsible for increasing cellular catabolism and anti-obesity effects.
Abstract
Commercial conjugated linoleic acid (CLA) dietary supplements contain an equal mixture of the C18:2 isomers, cis-9,trans-11 and trans-10,cis-12. Predominantly, CLA-c9t11 occurs naturally in meat and dairy products at ~ 0.5% of total fat , whereas the CLA-t10c12 occurs at > 0.1%. Recent studies show that CLA-c9t11 generally promotes lipid accumulation but CLA-t10c12 may inhibit lipid accumulation and may also promote inflammation. The omega-3 fatty acids α-linolenic acid (C18:3n-3) and docosahexaenoic acid (DHA) have also been observed to inhibit lipid accumulation and effect inflammation; therefore we examined the effects of the two main isoforms of CLA and omega -3 fatty acids C18:3n-3 and DHA at the molecular level to determine if they are causing similar oxidative stresses.
Purified CLA-c9t11 and CLA-t10c12 were added to 3T3 cells induced into mature adipocyte cultures at 100uM concentrations and compared with 100uM C18:3(n-3) (α-linolenic acid) and 50uM docosahexaenoic acid (DHA) to observe their effect on growth, gene transcription and general oxidation. The results of multiple separate trials were averaged and compared for significance at levels of p < 0.05, using one way ANOVA and Student’s t-test.
C18:3(n-3), DHA and CLA-t10c12 inhibited 3T3 adipose cell growth and caused a significant increase in lipid hydro peroxide activity. CLA-t10c12 and c9t11 increased AFABP, FAS and ACOX1 mRNA gene expression but DHA and C18:3(n-3) decreased the same mRNAs. CLA-c9t11 but not the t10c12 stimulated adipoQ expression even though; c9t11 had only a slightly greater affinity for PPARγ than CLA- t10c12, according to TR-FRET assays. The expression of the xenobiotic metabolism genes, aldo-keto reductase 1c1 (akr1c1), superoxide dismutase (SOD) and inflammation chemokine secretions of eotaxin (CCL11), Rantes (CCL5), MIG (CCL9) and MCP-1 were increased by DHA, C18:3(n-3) and CLA-t10c12 but not CLA-c9t11. This correlated with an increase in apoptosis factors, caspase 3, Bcl-2 and BAXs. Apoptosis factors were partially reduced by co-treatment with lipophilic anti-oxidant α-tocopherol.
CLA-t10c12 stimulated more reactive oxygen species (ROS) than CLA-c9t11. In response, cascades of genes are activated in detoxification, inflammation or apoptosis, to deal with the potentially damaging effects of ROS including CCL5 and MCP-1. This was similar to treatment with the omega- 3 fatty acids but the fat metabolic enzymes were generally inhibited by C18:3(n-3) and DHA except CLA-t10c12 which did not stimulate adipoQ.