Endocrinol Metab.  2015 Jun;30(2):177-184. 10.3803/EnM.2015.30.2.177.

Exendin-4 Inhibits the Expression of SEPP1 and Fetuin-A via Improvement of Palmitic Acid-Induced Endoplasmic Reticulum Stress by AMPK

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

• KMID: 2282008
• DOI: http://doi.org/10.3803/EnM.2015.30.2.177

Abstract

BACKGROUND
Selenoprotein P (SEPP1) and fetuin-A, both circulating liver-derived glycoproteins, are novel biomarkers for insulin resistance and nonalcoholic fatty liver disease. However, the effect of exendin-4 (Ex-4), a glucagon-like peptide-1 receptor agonist, on the expression of hepatokines, SEPP1, and fetuin-A, is unknown.
METHODS
The human hepatoma cell line HepG2 was treated with palmitic acid (PA; 0.4 mM) and tunicamycin (tuni; 2ug/ml) with or without exendin-4 (100 nM) for 24 hours. The change in expression of PA-induced SEPP1, fetuin-A, and endoplasmic reticulum (ER) stress markers by exendin-4 treatment were evaluated using quantitative real-time reverse transcription polymerase chain reaction and Western blotting. Transfection of cells with AMP-activated protein kinase (AMPK) small interfering RNA (siRNA) was performed to establish the effect of exendin-4-mediated AMPK in the regulation of SEPP1 and fetuin-A expression.
RESULTS
Exendin-4 reduced the expression of SEPP1, fetuin-A, and ER stress markers including PKR-like ER kinase, inositol-requiring kinase 1alpha, activating transcription factor 6, and C/EBP homologous protein in HepG2 cells. Exendin-4 also reduced the expression of SEPP1 and fetuin-A in cells treated with tunicamycin, an ER stress inducer. In cells treated with the AMPK activator 5-aminoidazole-4-carboxamide ribonucleotide (AICAR), the expression of hepatic SEPP1 and fetuin-A were negatively related by AMPK, which is the target of exendin-4. In addition, exendin-4 treatment did not decrease SEPP1 and fetuin-A expression in cells transfected with AMPK siRNA.
CONCLUSION
These data suggest that exendin-4 can attenuate the expression of hepatic SEPP1 and fetuin-A via improvement of PA-induced ER stress by AMPK.

Keyword

Exendin-4; Palmitic acid; Endoplasmic reticulum stress; AMP-activated protein kinases; Selenoprotein P; Fetuin-A; Hepatokine

MeSH Terms

Activating Transcription Factor 6
alpha-2-HS-Glycoprotein*
AMP-Activated Protein Kinases*
Blotting, Western
Carcinoma, Hepatocellular
Cell Line
Endoplasmic Reticulum
Endoplasmic Reticulum Stress*
Fatty Liver
Glucagon-Like Peptide 1
Glycoproteins
Hep G2 Cells
Humans
Insulin Resistance
Palmitic Acid
Phosphotransferases
Polymerase Chain Reaction
Reverse Transcription
RNA, Small Interfering
Selenoprotein P
Transfection
Tunicamycin
Biomarkers
Glucagon-Like Peptide-1 Receptor
AMP-Activated Protein Kinases
Activating Transcription Factor 6
Glucagon-Like Peptide 1
Glycoproteins
Palmitic Acid
Phosphotransferases
RNA, Small Interfering
Selenoprotein P
Tunicamycin
alpha-2-HS-Glycoprotein

Figure

• 

  Fig. 1 Exendin-4 (Ex-4) reduced the expression of selenoprotein P (SEPP1) and fetuin-A in HepG2 cells treated with palmitic acid (PA). HepG2 cells were incubated in the presence or absence of PA-containing medium, and treated with or without 100 nM Ex-4 for 24 hours. (A, B) The expression of SEPP1 and fetuin-A was analyzed using quantitative real-time reverse transcription polymerase chain reaction and Western blotting, and the data were normalized based on the β-actin. Con, control; mRNA, messenger RNA. aP<0.05; bP<0.01.

• 

  Fig. 2 Exendin-4 (Ex-4) reduced the expression of palmitic acid (PA)-induced endoplasmic reticulum stress markers. HepG2 cells were incubated in the presence or absence of PA-containing medium, and treated with or without 100 nM exendin-4 for 24 hours. Protein expression of inositol-requiring enzyme-1α (IRE1α), PKR-like endoplasmic reticulum kinase (PERK), activating transcription factor 6 (ATF6), and CCAAT/enhancer binding homologous protein (CHOP) were analyzed by Western blotting. P-IRE1α, phosphor-IRE1α; P-PERK, phosphor-PERK.

• 

  Fig. 3 Expression of selenoprotein P (SEPP1) and fetuin-A increased by endoplasmic reticulum (ER) stress was reversed by exendin-4 (Ex-4). HepG2 cells were treated with tunicamycin (Tuni), an ER stress inducer, for 24 hours, after which tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor, or Ex-4 was added for 24 hours. The gene expression levels of X-box binding protein 1 (XBP-1), SEPP1, and fetuin-A were analyzed using quantitative real-time reverse transcription polymerase chain reaction, and the data were normalized based on the β-actin. Con, control; mRNA, messenger RNA. aP<0.05; bP<0.01.

• 

  Fig. 4 Expression of selenoprotein P (SEPP1) and fetuin-A in cells treated with exendin-4 (Ex-4) was regulated by AMP-activated protein kinase (AMPK). (A) HepG2 cells were treated with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), an AMPK activator, for 24 hours. (B-D) Cells were transfected with the specific small interfering RNA (siRNA) for AMPK or scrambled siRNA (Scr) for 24 hours, and then added to a container with or without 100 nM Ex-4 for 24 hours. The expression of AMPK, SEPP1, and fetuin-A messenger RNA (mRNA) was measured using quantitative real-time reverse transcription polymerase chain reaction. Con, control. aP<0.05; bP<0.01.

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