The high rate of obesity is having a significant impact on human health. Understanding the underlying biological mechanisms that regulate adipogenesis and adipocyte lipid metabolism is necessary to identify novel approaches that promote weight loss. Conjugated linoleic acid (CLA) is an example of a naturally-derived product reported to exhibit an anti-obesity effect. For this thesis, it was hypothesized that the anti-obesity effects of the t10-c12 CLA isomer is due to lipid droplet dynamics alteration through activation of the Wnt/β-catenin pathway, which leads to weight loss via affecting adipogenesis and/or adipocyte death. Testing of this hypothesis was achieved by examining the effects of the most biologically active CLA isomers, cis-9, trans-11 (c9-t11), trans-10, cis-12 (t10-c12) CLA using in vitro (3T3-L1 cell line) and in vivo (mouse) models. In 3T3-L1 preadipocytes, both c9-t11 and t10-c12 CLA stimulated early stage differentiation, while t10-c12 CLA inhibited late differentiation as indicated by fewer lipid droplets, lower adipokine levels, and decreased levels of perilipin-1 and phospho-perilipin-1 compared to null. The t10-c12 CLA isomer decreased hormone-sensitive lipase (HSL) levels and inhibited lipolysis by activating protein kinase Cα (PKCα). As well, t10-c12-CLA inhibited adipocyte differentiation by stabilizing β-catenin, which sequesters peroxisome proliferator-activated receptor-γ in an inactive complex. Reduced body weight in both db/db and C57B/L6 mice fed t10-c12 CLA was due to less white and brown fat mass without changes in lean body mass or an alteration in feed intake compared to their respective control. t10-c12 CLA did not stimulate cell death in white adipose tissue. Immune cell infiltration was decreased in calorie restricted pair weight control mice, but not with CLA. t10-c12 CLA-induced weight loss did not improve hyperglycemia in db/db mice. In conclusion, the anti-adipogenic effects of t10-c12 CLA in vitro result from stabilization of β-catenin, which alters lipid droplet dynamics through HSL levels and perilipin-1 phosphorylation via the activation of PKCα. In contrast, t10-c12 CLA promotes loss of adipose tissue in vivo, possibly by activating β-catenin, but without influencing either adipogenesis or adipocyte clearance. This study suggests a novel mechanism for the anti-obesity effect of t10-c12 CLA, and highlights the possible side-effects associated with t10-c12 CLA consumption. / February 2016

The effects of conjugated linoleic acid (CLA) on lipogenesis and preadipocyte proliferation in young pigs were evaluated in two separate experiments. The first compared dietary effects of linoleic acid, beef tallow, and CLA on composition, lipogenesis, and DNA synthesis. Eighteen pigs weaned at 17 d of age were allotted randomly to corn-based diets supplemented with 1.5% corn oil, 1.5% tallow, or 1.5% CLA. The second experiment evaluated the effects of CLA included with diets high in polyunsaturated fat or beef tallow. Twenty-four pigs weaned at 17 d of age were allotted randomly to one of four corn-based diets supplemented with: 15% corn oil, 12% corn oil + 3% CLA, 15% tallow, and 12% tallow + 3% CLA. The piglets in both trials were fed a basal diet for 7 d and their respective diet for 35 d. [U-14C]Glucose incorporation into total lipids was (experiment 1): 10.64, 11.04, 13.64; (experiment 2): 21.15, 17.54, 21.34, and 19.52 nmol/(105 cells per h) for subcutaneous (s.c.) adipose tissue from corn oil, tallow, CLA; corn oil, corn oil + CLA, tallow, and tallow + CLA-fed piglets, respectively. Tritiated thymidine incorporation into DNA was not different in s.c. adipocytes across treatment groups, but was 5,581, 2,794, 6,573, and 3,760 dpm/(105 cells per h) in s.c. stromal vascular cells from corn oil, corn oil + CLA, tallow, and tallow + CLA-fed piglets, respectively (CLA main effect p<0.034). Additionally, there was a greater proportion of s.c. adipocytes in the smaller, 180-pL cell fraction from the corn oil + CLA-fed pigs (p<0.0074). CLA in the diet increased the s.c. adipose tissue concentration of 18:0 and decreased 16:1 and 18:1 (p<0.05), suggesting depression of stearoyl-coenzyme A desaturase (SCD) enzyme activity in the CLA-fed pigs. The concentration of CLA isomers was raised only slightly in s.c. adipose tissue with the addition of CLA to the diets even though the CLA oil contained 62% CLA isomers. No effects on the growth of young pigs were observed. However, CLA caused a more saturated fatty acid composition and may suppress preadipocyte proliferation, apparent SCD activity, and lipid filling of smaller cells.

The effects of conjugated linoleic acid (CLA) on lipogenesis and preadipocyte proliferation in young pigs were evaluated in two separate experiments. The first compared dietary effects of linoleic acid, beef tallow, and CLA on composition, lipogenesis, and DNA synthesis. Eighteen pigs weaned at 17 d of age were allotted randomly to corn-based diets supplemented with 1.5% corn oil, 1.5% tallow, or 1.5% CLA. The second experiment evaluated the effects of CLA included with diets high in polyunsaturated fat or beef tallow. Twenty-four pigs weaned at 17 d of age were allotted randomly to one of four corn-based diets supplemented with: 15% corn oil, 12% corn oil + 3% CLA, 15% tallow, and 12% tallow + 3% CLA. The piglets in both trials were fed a basal diet for 7 d and their respective diet for 35 d. [U-14C]Glucose incorporation into total lipids was (experiment 1): 10.64, 11.04, 13.64; (experiment 2): 21.15, 17.54, 21.34, and 19.52 nmol/(105 cells per h) for subcutaneous (s.c.) adipose tissue from corn oil, tallow, CLA; corn oil, corn oil + CLA, tallow, and tallow + CLA-fed piglets, respectively. Tritiated thymidine incorporation into DNA was not different in s.c. adipocytes across treatment groups, but was 5,581, 2,794, 6,573, and 3,760 dpm/(105 cells per h) in s.c. stromal vascular cells from corn oil, corn oil + CLA, tallow, and tallow + CLA-fed piglets, respectively (CLA main effect p<0.034). Additionally, there was a greater proportion of s.c. adipocytes in the smaller, 180-pL cell fraction from the corn oil + CLA-fed pigs (p<0.0074). CLA in the diet increased the s.c. adipose tissue concentration of 18:0 and decreased 16:1 and 18:1 (p<0.05), suggesting depression of stearoyl-coenzyme A desaturase (SCD) enzyme activity in the CLA-fed pigs. The concentration of CLA isomers was raised only slightly in s.c. adipose tissue with the addition of CLA to the diets even though the CLA oil contained 62% CLA isomers. No effects on the growth of young pigs were observed. However, CLA caused a more saturated fatty acid composition and may suppress preadipocyte proliferation, apparent SCD activity, and lipid filling of smaller cells.

Three experiments were conducted for this dissertation with the goals to; 1) determine conjugated linoleic acid (CLA) effects on net-energy balance (EBAL) and milk production parameters during periods of nutrient/energy stress, and 2) investigate temporal CLA effects on mammary lipogenic gene expression. Study one was designed to determine if abomasal CLA infusion could reduce milk fat synthesis and partition nutrients towards alternative milk components in feed restricted rotationally grazed dairy cows. Data indicate abomasally-infusing CLA reduced milk fat synthesis in nutrient restricted grazing dairy cows and improved calculated EBAL and milk protein production. Another period of transitory stress experienced by the lactating dairy cow is immediately postpartum and study two objectives were to feed rumen inert-CLA to evoke milk fat depression (MFD) and investigate production and bioenergetic parameters. Data indicated a high CLA dose (3 x greater than needed in established lactation) inhibited milk fat synthesis immediately postpartum and improved calculated EBAL in grazing dairy cows. A curvilinear relationship existed between the severity of CLA-induced MFD and milk yield response. Moderate CLA-induced MFD (<~35%) tended to increase milk yield whereas severe MFD (>~35%) diminished this response. Previous research speculated that extensive MFD might decrease Δ⁹-desaturase (stearoyl CoA desaturase; SCD) and subsequent membrane fluidity to such an extent as to adversely affect cellular functions and inhibit milk secretion, thus decreasing milk yield. However, SCD inhibition was temporally independent in the present study, offering little support for the aforementioned theory. Mammary sensitivity to CLA increased as lactation progressed and previous speculations attributed this to reduced contribution of de novo synthesised fatty acids or increased competition from circulating non-esterified fatty acids immediately postpartum. However, data indicate that de novo fatty acids and milk fat trans-10, cis-12 CLA content don’t appreciably change during early lactation, (even though MFD became more severe) offering little support for either hypothesis. Study three investigated the effects of intravenous CLA infusion on temporal expression of mammary lipogenic genes to determine if trans-10, cis-12 CLA down regulates expression of a key gene (i.e. acetyl CoA carboxylase, ACC, the rate limiting enzyme in de novo fatty acid synthesis) and reduction in other mammary lipid synthesis genes is due to lack of substrate (i.e. malonyl CoA), or an alternative indirect mechanism. Data indicated however, that mammary lipogenic genes (ACC, fatty acid synthetase and SCD) followed a similar temporal pattern, providing more support for a global regulator (i.e. sterol regulatory element binding protein-1, peroxisome proliferator-activated receptor-γ or nuclear factor- κB) rather than a specific key enzyme effect.

The objectives of this study was to examine the efficacy and safety of graded doses of c9, t11, t10, c12 CLA isomers on body composition, energy expenditure, lipid profile and hepatic biomarkers in hamsters. Male Golden Syrian hamsters (n=105) were randomized to seven treatments (control; 1, 2, 3% of c9, t11; 1, 2, 3% of t10, c12) for 28 days. Compared with control, 1% and 3% t10, c12 had lowered food intake with all three doses of t10, c12 lowering (p<0.0001) body fat mass (g). Groups fed with 1, 2, 3% t10, c12 and 3% c9, t11 treatments showed higher lean mass compared to control and other treatment groups. However, neither body weights, nor serum HDL or triglyceride levels differed across treatment groups. The 3% t10, c12 groups exhibited higher (p<0.0001) cholesterol and LDL-C levels compared to control or other treatment groups. The 2% and 3% t10, c12 groups also presented elevated ALT level (p<0.05). The present data suggest that 3% t10, c12 possess potential adverse effects on liver and posing unfavorable change in lipid profile.

The objectives of this study was to examine the efficacy and safety of graded doses of c9, t11, t10, c12 CLA isomers on body composition, energy expenditure, lipid profile and hepatic biomarkers in hamsters. Male Golden Syrian hamsters (n=105) were randomized to seven treatments (control; 1, 2, 3% of c9, t11; 1, 2, 3% of t10, c12) for 28 days. Compared with control, 1% and 3% t10, c12 had lowered food intake with all three doses of t10, c12 lowering (p<0.0001) body fat mass (g). Groups fed with 1, 2, 3% t10, c12 and 3% c9, t11 treatments showed higher lean mass compared to control and other treatment groups. However, neither body weights, nor serum HDL or triglyceride levels differed across treatment groups. The 3% t10, c12 groups exhibited higher (p<0.0001) cholesterol and LDL-C levels compared to control or other treatment groups. The 2% and 3% t10, c12 groups also presented elevated ALT level (p<0.05). The present data suggest that 3% t10, c12 possess potential adverse effects on liver and posing unfavorable change in lipid profile.

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Conjugated linoleic acids (CLA) are geometrical and positional isomers of n-6 octadecadenoic acid (linoleic acid, LA, 18:2n-6), which form part of a family of essential polyunsaturated fatty acids (PUFA). There are 28 identified CLA isomers that mostly found in the meat and milk from ruminant animals. CLA has shown to possess a number of health benefits including; reduction in body fat and increased lean body mass, prevention of atherosclerosis, hypertension, increased immune function and in particular the prevention of cancer. The effects of CLA on cancer cell lines will be evaluated to discover the mechanisms that are employed to achieve this great phenomenon on cell growth. The aim of this study was to determine the effect of CLA on various parameters that are essential in the development of cancer cell phenotype. The objectives were to evaluate the effect of CLA on iron-induced lipid peroxidation of microsomes isolated from rat liver cells and in vitro cytotoxicity, cell proliferation and apoptosis in HepG2 hepatocarcinoma cells. The Fatty acid incorporation in HepG2 cells was also assessed.

This work deals with the optimization of the method for determination of conjugated linoleic acid (CLA) using gas chromatography. The summary about formation and occurrence of CLA in animal materials, its biological effects in human organism and methods suitable for its determination is introduced in the theoretical part of this study. The experimental part verify, if the gas chromatography is applicable method for assesment of CLA in selected biological matrices. The chosen method was introduced and verified at FCH of Brno university of technology. At the end the applicability of this method to CLA determination is discussed here.

An experiment was conducted to evaluate the effects of pasture finishing versus high-concentrate finishing, over time, on fatty acid metabolism in Angus crossbred (n = 24) beef steers. Ruminal fluid, serum, and adipose tissue biposies were obtained on d 0, 28, 84, and 140. Pasture forages and diet ingredient samples were obtained at 14 d intervals to determine nutritive value and fatty acid composition. The high-concentrate diet consisted of corn silage, cracked corn, soybean meal, and a vitamin and mineral supplement. The pasture-finished steers grazed sequentially on triticale (Triticale hexaploide)/annual ryegrass (Lolium multiflorum), alfalfa (Medicago sativa)/orchardgrass (Dactylis glomerata), and a cool-season grass/legume mixture. The high-concentrate diet consisted of 57 % linoleic acid and 7 % linolenic acid (of total fatty acids). The pasture forages contained an average 9 % linoleic acid and 66 % linolenic acid (of total fatty acids). Adipose tissue concentrations of 18:2 cis-9, trans-11 CLA were higher (P < 0.05) in the pasture-finished steers than high-concentrate finished steers. Concentrations of 18:2 cis-9, trans-11 CLA declined in the high-concentrate finished steers (P < 0.05) from d 0 to 28 and d 28 to 84. In the pasture-finished steers concentrations peaked (P < 0.10) on d 28, and remained high throughout the duration of the study. Concentrations of linolenic acid were higher (P < 0.05) in adipose tissue, ruminal fluid, and serum of the pasture-finished steers, compared to the high-concentrate finished steers. In the pasture-finished steers linolenic acid concentrations peaked (P < 0.05) on d 28, and remained high throughout the study. Concentrations of linolenic acid gradually decreased (P < 0.05) over time within the high-concentrate finished steers. Thus, it appears that only a short time is needed to alter the omega-3 and CLA composition of adipose tissue in cattle finished on pasture. / Ph. D.

Dairy products are a primary source of c9, t11-CLA, an anti-carcinogenic agent, in the diet of humans. The t10, c12-CLA isomer, typically in trace amounts in bovine milk fat, also may benefit human health. Four cows received abomasal infusions of c9, t11-CLA or t10, c12-CLA to obtain milk fat used to prepare butter and yogurt with enhanced c9, t11-CLA or t10, c12-CLA content. Human subjects (3 males, 3 females, ages 22 to 29) received CLA-enhanced butter and yogurt (14% of total kcal) in a crossover study with 2-wk periods. Prior to the study (2 wk) and during a 2-wk washout period between the experimental periods, subjects received butter and yogurt without enhanced CLA content. Blood samples were obtained at 0, 2, 4, 6, and 8 wk relative to the start of the first experimental period. The t10, c12- CLA isomer was detected in plasma (1.32 ug/mL) only when dairy products with enhanced t10, c12-CLA was consumed. Baseline c9, t11-CLA was 6.94 ug/mL plasma during control periods, but increased to 8.95 ug/mL when dairy products with enhanced c9, t11-CLA content were consumed. Results indicated concentrations of CLA isomers in human plasma respond to small changes in daily intake of the isomers in dietary sources. / Master of Science