Figure 1.
SIRT activity affects TG levels in t10c12 CLA treated 3T3-L1 adipocytes.
a–c Triglyceride (TG) levels were measured in differentiating adipocytes after incubation with LA (L) or t10c12 CLA (C), with or without the SIRT1 activator SRT1720 (SRT), or the sirtuin inhibitors sirtinol (SOL), nicotinamide (NAM), or etomoxir (ETO) for 24 h. d Immunoblot analysis of whole cell extracts for the amount of SIRT1 and β-actin proteins after exposure to 80 nmol/L of control siRNA (siCON), or siRNA against SIRT1 (siSirt1), or transfection reagent only. Duplicate loadings of each sample were analyzed and the lanes marked by 1/3 have one third of the indicated samples loaded. e Triglyceride (TG) amounts after treatments as in d. Each bar in panels a–c and e represents the mean + SEM (n = 3), and is representative of three independent experiments. Means not sharing a common letter differ, P0.05. Panel d is a representative blot of three independent experiments.
Figure 2.
SIRT1 affects fatty acid metabolism and inflammatory mRNA levels.
a–c The effects of 100 µmol/L of t10c12 CLA, with or without the sirtuin inhibitor sirtinol (SOL), on the rates of fatty acid metabolism were measured by radioactive tracers (disintegrations per minute: dpm) as follows: a fatty acid biosynthesis by incorporation of [1-14C]-acetate; b lipolysis by the release of lipids previously labeled with [1-14C]-acetate; c fatty acid oxidation by the release of [14C]-CO2 from lipids previously labeled with [1-14C]-oleate. d–e The effects of the SIRT1 activator SRT1720 (SRT), or the sirtuin inhibitors sirtinol (SOL) or nicotinamide (NAM) on RNA levels in LA or t10c12 CLA treated adipocytes were analyzed for MCP-1 and COX2 relative to GAPDH by reverse transcription and quantitative PCR. The relative amounts of MCP-1 and COX2 mRNA are shown as bar graphs. Each bar in panels a–e represents the mean + SEM (n = 3 for a–b or n = 2 for c–e), and is representative of three independent experiments. Means not sharing a common letter differ, P0.05.
Figure 3.
SIRT1 affects ACC and AMPK phosphorylation levels after t10c12 CLA treatment.
Differentiated 3T3-L1 adipocytes were incubated with 100 µmol/L LA (L) or t10c12 CLA (C): a with or without SIRT1 activator SRT1720 (SRT), or sirtuin inhibitors sirtinol (SOL) or nicotinamide (NAM) for 2 or b 12 h; or c with 40 nmol/L of SIRT1 siRNA (siSIRT1) or control siRNA (siCON). Representative western blots indicate the proteins detected in cytosolic extracts with antibodies to p-ACC (p-Ser79), or total ACC, p-AMPK (p-Thr172), or total AMPK. Each bar in panels a–c represents the mean + SEM (n = 3) of the ratio of the phosphorylated to total form of each protein (phospho/total) for three independent experiments. Means for p-AMPK/AMPK (a–e) or p-ACC/ACC (u–x) not sharing a common letter differ, P0.05.
Figure 4.
PPARγ antagonists and agonists affect TG levels and modulate p-AMPK activity levels in t10c12 CLA treated 3T3-L1 adipocytes.
a Differentiated adipocytes were incubated with 50 µmol/L LA (L) or t10c12 CLA (C) with or without PPARγ antagonist GW9662 (9662) for 24 h and TG levels were measured. b Differentiating adipocytes were treated with either control media (0), troglitazone (Tro), ciglitazone (C), rosiglitazone (R), or pioglitazone (P) to determine which PPARγ agonist was most effective for increasing TG levels. c Differentiated 3T3-L1 adipocytes were incubated with 100 µmol/L LA or t10c12 CLA, with or without PPARγ agonist troglitazone (Tro), and TG levels were measured after 24 h. d Adipocytes were treated as in a or c for 2 or 12 h using 100 µmol/L LA or t10c12 CLA, and representative western blots of cytosolic extracts indicate the proteins detected with antibodies to p-ACC (p-Ser79), total ACC, p-AMPK (p-Thr172), or total AMPK. The ratio of the phosphorylated to total form of each protein (phospho/total) is shown in the bar graphs. e–f The amount of phosphorylated or total PPARγ in nuclear extracts was measured after 2 or 12 h of treatment with 100 µmol/L LA or t10c12 CLA, and with or without compound C (Comp. C) at 12 h. The ratio of the amount of phosphorylated to total PPARγ is shown in the bar graphs. Each bar in panels a–f represents the mean + SEM (n = 3), and is representative of three independent experiments (a–c) or is the mean of three independent experiments (d–f). Means within each data type (a–e or u–x) not sharing a common letter differ, P0.05.
Figure 5.
PPARγ agonists or antagonists affect the TG loss caused by AMPK activators without t10c12 CLA being present.
a Differentiated 3T3-L1 adipocytes were incubated with or without 0.1 mmol/L phenformin (Phen), with or without PPARγ agonist troglitazone (Tro), and TG levels were measured after 24 h. b TG levels were measured in differentiated 3T3-L1 adipocytes in media lacking or containing 2 mmol/L metformin (Met), with or without PPARγ antagonist GW9662 (9662) for 24 h. Each bar represents the mean + SEM (n = 3), and is representative of three independent experiments. Means not sharing a common letter differ, P0.05.
Figure 6.
SIRT1, AMPK and PPARγ affect the t10c12 CLA-dependent decrease in the amount of acetylated NF-κB.
The amount of acetylation on the p65 subunit of NF-κB or the total amount of p65 in nuclear extracts was detected by immunoblot analysis with antibodies specific for acetylated p65 or total p65. The ratio of the acetylated to total p65 is shown in the bar graphs (Acetyl-p65/p65). a–c Differentiated 3T3-L1 adipocytes were incubated with 100 µmol/L LA (L) or t10c12 CLA (C) a for 2 h, or b for 12 h, with or without sirtuin inhibitors sirtinol (SOL) or nicotinamide (NAM), or c with or without AMPK inhibitor compound C (Comp.C). d The effects of PPARγ agonist troglitazone (Tro) or PPARγ antagonist GW9662 (9662), in combination with LA or t10c12 CLA, on the acetylation of the p65 subunit of NF-κB was measured. Each bar in panels a–d represents the mean + SEM (n = 3) of three independent experiments. Means not sharing a common letter differ, P0.05.
Figure 7.
Treatment with t10c12 CLA increases the interaction of SIRT1 with PPARγ or NCoR1.
Differentiated 3T3-L1 adipocytes were incubated with 100 µmol/L LA (L) or t10c12 CLA (C) for 12 h, and a portion of the nuclear extracts were immunoprecipitated with antibody to SIRT1 (IP: SIRT1). Representative immunoblots (IB) indicate the proteins detected from the nuclear extracts (5% input) or when the immunoprecipitated proteins were probed with antibodies to SIRT1, PPARγ, or NCoR1. Each bar represents the mean + SEM (n = 3) of three independent experiments. Means within each data type not sharing a common letter differ, P0.05.
Figure 8.
Summary diagram for proposed effects of AMPK, SIRT1, PPARγ, and their activators or inhibitors in 3T3-L1 adipocytes.
AMPK and SIRT1 impair lipid synthesis and PPARγ activity (red lines with stop bars), and stimulate each other (blue arrows). PPARγ impairs AMPK and SIRT1 activity and stimulates lipid synthesis. The activating (blue arrows) or inhibiting (red line with stop bar) effects of the different chemical activators and inhibitors are also shown.