Fig. 1Chronic incubation with tumor necrosis factor (TNF)-α and AMP-activated protein kinase (AMPK) activation regulates lipolysis in cultured 3T3-L1 adipocytes. Adipocytes were incubated with or without TNF-α (10 ng/mL) for 24 hours in the presence or absence of activator minoimidazole carboxamide ribonucleotide (AICAR; 1 mM) or compound C (CC; 20 µM). (A) Total and phosphorylated AMPK protein levels were examined by Western blot assay. (B) Lipolysis was quantified by determination of glycerol release into the media. Aliquots of the culture medium were collected at 24 hours, and the amount of released glycerol was measured. The results represent the mean±SE from at least three independent batches of 3T3-L1 adipocytes. The released glycerol level in control adipocytes was designated as 100%. aP<0.001 compared with the control group; bP<0.01 compared with the TNF-α group.
Fig. 2AMP-activated protein kinase (AMPK) attenuates the tumor necrosis factor (TNF)-α-induced decrease in perilipin. 3T3-L1 adipocytes were incubated with TNF-α (10 ng/mL) for 24 hours in the presence or absence of activator minoimidazole carboxamide ribonucleotide (AICAR; 1 mM) or compound C (CC; 20 µM). (A) Perilipin and hormone sensitive lipase (HSL) protein levels were examined by Western blot assay. (B) Quantification of blot shown in (A). (C) At the end of the incubation, cells were fixed, permeabilized, and then incubated with a specific antibody against perilipin (green fluorescence). Nuclei were visualized by DAPI staining (blue fluorescence). Bars=10 µm. aP<0.001 compared with control group; bP<0.01 compared with TNF-α group.
Fig. 3AMP-activated protein kinase (AMPK) stimulates phosphorylation of protein kinase RNA-like endoplasmic reticulum kinase (PERK)/eukaryotic initiation factor 2 α (eIF2α). 3T3-L1 adipocytes were incubated with tumor necrosis factor (TNF)-α (10 ng/mL) for 24 hours in the presence or absence of activator minoimidazole carboxamide ribonucleotide (AICAR; 1 mM). (A) Total and phosphorylated form of eIF2α, and PERK protein were detected by Western blot assay. (B) Quantification of blot shown in (A). aP<0.01; and bP<0.001 compared with control group; cP<0.01; and dP<0.001 compared with TNF-α group.
Fig. 4Antilipolytic effect of AMP-activated protein kinase (AMPK) is abrogated by protein kinase RNA-like endoplasmic reticulum kinase (PERK)/eukaryotic initiation factor 2 α (eIF2α) inhibition. 3T3-L1 adipocytes were incubated with tumor necrosis factor (TNF)-α (10 ng/mL) for 24 hours in the presence or absence of activator minoimidazole carboxamide ribonucleotide (AICAR; 1 mM) and/or 2-aminopurine (2-AP; 5 mM). (A) Total and phosphorylated form of PERK/eIF2α and perilipin protein were detected by Western blot assay. (B) Quantification of perilipin protein from the blot shown in (A). (C) At the end of the incubation, the amount of released glycerol in the culture medium was measured. The released glycerol level in control adipocytes was set at 100%. aP<0.05 compared with control group; bP<0.05 compared with TNF-α group; cP<0.01 compared with TNF-α and AICAR group; dP<0.01 compared with the control group; eP<0.001 compared with the TNF-α group; fP<0.001 compared with TNF-α and AICAR groups.