Endocrinol Metab.  2014 Dec;29(4):553-560. 10.3803/EnM.2014.29.4.553.

Activation of AMP-Activated Protein Kinase Attenuates Tumor Necrosis Factor-alpha-Induced Lipolysis via Protection of Perilipin in 3T3-L1 Adipocytes

• Hong SW
• Lee J
• Park SE
• Rhee EJ
• Park CY
• Oh KW
• Park SW
• Lee WY

• KMID: 2384254
• DOI: http://doi.org/10.3803/EnM.2014.29.4.553

Abstract

BACKGROUND
Tumor necrosis factor (TNF)-alpha and AMP-activated protein kinase (AMPK) are known to stimulate and repress lipolysis in adipocytes, respectively; however, the mechanisms regulating these processes have not been completely elucidated.
METHODS
The key factors and mechanism of action of TNF-alpha and AMPK in lipolysis were investigated by evaluating perilipin expression and activity of protein kinase RNA-like endoplasmic reticulum kinase (PERK)/eukaryotic initiation factor 2 alpha (eIF2alpha) by Western blot and an immunofluorescence assay in 24-hour TNF-alpha-treated 3T3-L1 adipocytes with artificial manipulation of AMPK activation.
RESULTS
Enhancement of AMPK activity by the addition of activator minoimidazole carboxamide ribonucleotide (AICAR) suppressed TNF-alpha-induced lipolysis, whereas the addition of compound C, an inhibitor of AMPK phosphorylation, enhanced lipolysis. Perilipin, a lipid droplet-associated protein, was decreased by TNF-alpha and recovered following treatment with AICAR, showing a correlation with the antilipolytic effect of AICAR. Significant activation of PERK/eIF2alpha, a component of the unfolded protein response signaling pathway, was observed in TNF-alpha or vesicle-treated 3T3-L1 adipocytes. The antilipolytic effect and recovery of perilipin expression by AICAR in TNF-alpha-treated 3T3-L1 adipocytes were significantly diminished by treatment with 2-aminopurine, a specific inhibitor of eIF2alpha.
CONCLUSION
These data indicated that AICAR-induced AMPK activation attenuates TNF-alpha-induced lipolysis via preservation of perilipin in 3T3-L1 adipocytes. In addition, PERK/eIF2alpha activity is a novel mechanism of the anti-lipolytic effect of AICAR.

Keyword

AMP-activated protein kinases; Lipolysis; Perilipin; PERK/eIF2alpha; Adipocytes

MeSH Terms

2-Aminopurine
Adipocytes*
AMP-Activated Protein Kinases*
Blotting, Western
Endoplasmic Reticulum
Fluorescent Antibody Technique
Lipolysis*
Necrosis*
Phosphorylation
Phosphotransferases
Prokaryotic Initiation Factor-2
Protein Kinases
Tumor Necrosis Factor-alpha
Unfolded Protein Response
2-Aminopurine
AMP-Activated Protein Kinases
Phosphotransferases
Prokaryotic Initiation Factor-2
Protein Kinases
Tumor Necrosis Factor-alpha

Figure

• 

  Fig. 1 Chronic 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. 2 AMP-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. 3 AMP-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. 4 Antilipolytic 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.

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