PMC

Trans-10,cis-12 conjugated linoleic acid (CLA) decreases lipid accumulation in differentiating human preadipocytes. Cultures were continuously treated with either a bovine serum albumin (BSA) vehicle, or 30 μM linoleic acid (LA), cis-9,trans-11 (9,11-CLA), or trans-10,cis-12 (10,12-CLA) for 12 days during differentiation. On Day 12, cultures were stained with oil red O and phase-contrast photomicrographs were taken using an Olympus inverted microscope with a 10× objective.

Isomer-specific regulation of stearoyl-CoA desaturase-1 (SCD-1) by CLA. The effects of BSA (B), 30 μM cis-9,trans-11 CLA (), or 30 μM trans-10,cis-12 CLA () on SCD-1 gene expression over the differentiation time course (Days 0, 1, 3, 6, 9, and 12 postinduction) were examined using multi-plex semi-quantitative RT-PCR. 18S rRNA was used as an internal control. Results are representative of two separate experiments from independent human subjects.

Acute effect of CLA isomers on PPARγ activity in 3T3-L1 adipocytes. 3T3-L1 adipocytes (Day 4 after induction of differentiation) were transiently transfected with (A) the ACBP (−392/+979)-luc constructs containing the rat ACBP proximal promoter and intron 1 or (B) the rACBP(−392/+979)-Δ-peroxisome proliferator response element (PPRE)-luc reporter where the intronic PPRE has been mutated. Cells were transfected using LipofectAMINE PLUS, and 3 h later media was changed and fatty acids and/or BRL49653 were added as indicated. LA, cis-9,trans-11 CLA, or trans-10,cis-12 CLA were added to final concentrations of 0.1 μM, 0.3 μM, 1 μM, or 30 μM. Cells were harvested after 24 h and luciferase activity was measured and normalized to β-galactosidase activity.

Isomer-specific down-regulation of adipocyte fatty acid binding protein (aP2) and perilipin protein levels by CLA are not rescued by LA supplementation. Western blot analyses of perilipin A and aP2 proteins were conducted in differentiating human preadipocytes under the following treatment conditions: A: Cultures were continuously treated with either a BSA vehicle control or 30 μM LA, cis-9,trans-11 CLA (, ), or trans-10,cis-12 CLA (, ), and total cellular protein was harvested on Day 8 (d8) and Day 16 (d16) of differentiation. B: Cultures were treated continuously with either a vehicle control (BSA), 30 μM trans-10,cis-12 CLA alone (CLA), or 30 μM trans-10,cis-12 CLA plus increasing levels (10 μM, 30 μM, or 100 μM) of LA (CLA + 10 LA, CLA + 30 LA, CLA + 100 LA), or increasing levels of LA alone (10 μM LA, 30 μM LA, 100 μM LA), and total cellular protein was harvested on Day 12 of differentiation. The abundant protein signal transducer and activator of transcription six was used as a loading control.

Isomer-specific regulation of human adipocyte glucose metabolism by CLA. Cultures of differentiating human preadipocytes were continuously treated with increasing concentrations (0, 3, 10, or 30 μM) of either LA (diamond), cis-9, trans-11 CLA (square), or trans-10, cis-12 CLA (triangle) for 12 days during differentiation. A: Uptake of 4 nmol [³H]2-deoxy-glucose (specific activity = 25–50 Ci/mmol) was measured following a 90 min incubation in the presence of 100 nm insulin; control rate was 146 pmol/[h · mg protein]. B: [¹⁴C]CO₂ production from [¹⁴C]glucose was measured following a 5 h incubation with 2.2 nmol [¹⁴C]glucose (specific activity = 231 mCi/mmol) in the presence of 100 nm insulin; control rate was 15 pmol/[h · mg protein]. For A and B, data are expressed as a percentage of vehicle control (BSA) levels. Means (±SEM; n = 6) not sharing a common superscript differ, P < 0.05. C: In addition, the effects of BSA (B), 30 μM cis-9,trans-11 CLA () or 30 μM trans-10,cis-12 CLA () on insulin-dependent glucose transporter 4 (GLUT4) gene expression over the differentiation time course (Days 0, 1, 3, 6, 9, and 12 postinduction) was conducted using multiplex semi-quantitative RT-PCR. 18S rRNA was used as an internal control. Results for GLUT4 expression are representative of two separate experiments from independent human subjects.

Time course and CLA-induced alterations of human adipocyte gene expression. A: For the time course data, confluent cultures of human preadipocytes were induced to differentiate under standard adipogenic conditions, and total RNA was harvested throughout adipose conversion (Day 0, 8 h, 24 h, 72 h, and 216 h postinduction). Total RNA was used for first-strand cDNA synthesis, and real-time quantitative RT-PCR analyses were performed to analyze the expression of acyl-CoA binding protein (ACBP), adipocyte fatty acid binding protein (aP2), CAAT/enhancer binding protein α (C/EBPα), glycerol dehydrogenase (GPDH), hormone-sensitive lipase (HSL), perilipin, peroxisome proliferator-activated receptors γ1 and γ2 (PPARγ1 and PPARγ2), leptin, and acetyl-CoA carboxylase (ACC). The relative expression level of a given gene was calculated after normalization to TATA-binding protein expression, and was expressed relative to Day 0 (confluent, noninduced) controls. B: To examine the effects of fatty acid treatment on gene expression, cultures were continuously treated with either a BSA vehicle control, 30 μM cis-9,trans-11 CLA (, ), or 30 μM trans-10,cis-12 CLA (, ) for either 0 h, 8 h, 24 h, 72 h, or 216 h during differentiation. Real-time quantitative RT-PCR analyses were performed as stated above to analyze gene expression. Results shown are only for the 216 h treatments and are representative of three separate experiments from independent human subjects.

Isomer-specific regulation of human adipocyte lipid metabolism by CLA. Cultures of differentiating human preadipocytes were continuously treated with increasing concentrations (0, 3, 10, or 30 μM) of either LA (diamond), cis-9,trans-11 CLA (square), or trans-10,cis-12 CLA (triangle) for 6 days during differentiation. On Day 6, [¹⁴C]oleic acid metabolism was measured by determining the incorporation of the radionuclide into distinct cellular and gaseous fractions. A: [¹⁴C]oleic acid (6.2 nmol) uptake after 20 min incubation; control rate was 38.1 nmol/(h · mg protein). B: [¹⁴C]oleic acid (12.5 nmol) incorporation into the lipid-soluble cellular fraction after 2 h incubation; control rate was 5.3 nmol/(h · mg protein). C: [¹⁴C]oleic acid (12.5 nmol) incorporation into the water-soluble cellular fraction after 2 h incubation; control rate was 53.4 pmol/(h · mg protein). D: [¹⁴C]CO₂ production from [¹⁴C]oleic acid following 90 min incubation with 12.5 nmol [¹⁴C]oleic acid; control rate was 100 pmol/(h · mg protein). For A–D, data are expressed as a percentage of vehicle control (BSA) levels. Means (±SEM; n = 6) not sharing a common superscript differ, P < 0.05. In addition, the effects of BSA (B), 30 μM cis-9,trans-11 CLA (), or 30 μM trans-10,cis-12 CLA () on lipoprotein lipase (LPL) (E), adipocyte fatty acid binding protein (aP2) (F), and fatty acid translocase (CD-36) (G) gene expression over the differentiation time course (Days 0, 1, 3, 6, 9, and 12 postinduction) were examined using multiplex semi-quantitative RT-PCR. 18S rRNA was used as an internal control. Results for LPL, aP2, and cd36 are representative of two separate experiments from independent human subjects.