Endocrinol Metab.  2021 Feb;36(1):171-184. 10.3803/EnM.2020.850.

Mechanism of Lipid Accumulation through PAR2 Signaling in Diabetic Male Mice

• Kim DH
• Kim YR
• Bang E
• Ha S
• Noh SG
• Kim BM
• Jeong SH
• Jung HJ
• Lee JY
• Chung HY

• KMID: 2513300
• DOI: http://doi.org/10.3803/EnM.2020.850

Abstract

Background
Protease-activated protein-2 (PAR2) has been reported to regulate hepatic insulin resistance condition in type 2 diabetes mice. However, the mechanism of lipid metabolism through PAR2 in obesity mice have not yet been examined. In liver, Forkhead box O1 (FoxO1) activity induces peroxisome proliferator-activated receptor γ (PPARγ), leading to accumulation of lipids and hyperlipidemia. Hyperlipidemia significantly influence hepatic steatoses, but the mechanisms underlying PAR2 signaling are complex and have not yet been elucidated.
Methods
To examine the modulatory action of FoxO1 and its altered interaction with PPARγ, we utilized db/db mice and PAR2-knockout (KO) mice administered with high-fat diet (HFD).
Results
Here, we demonstrated that PAR2 was overexpressed and regulated downstream gene expressions in db/db but not in db+ mice. The interaction between PAR2/β-arrestin and Akt was also greater in db/db mice. The Akt inhibition increased FoxO1 activity and subsequently PPARγ gene in the livers that led to hepatic lipid accumulation. Our data showed that FoxO1 was negatively controlled by Akt signaling and consequently, the activity of a major lipogenesis-associated transcription factors such as PPARγ increased, leading to hepatic lipid accumulation through the PAR2 pathway under hyperglycemic conditions in mice. Furthermore, the association between PPARγ and FoxO1 was increased in hepatic steatosis condition in db/db mice. However, HFD-fed PAR2-KO mice showed suppressed FoxO1-induced hepatic lipid accumulation compared with HFD-fed control groups.
Conclusion
Collectively, our results provide evidence that the interaction of FoxO1 with PPARγ promotes hepatic steatosis in mice. This might be due to defects in PAR2/β-arrestin-mediated Akt signaling in diabetic and HFD-fed mice.

Keyword

Forkhead box protein O1; PPAR gamma; Receptor, PAR-2; Obesity; Fatty liver; Hyperlipidemia

Figure

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  Fig. 1 Obesity-dependent serum changes in insulin resistance and lipogenesis. (A) Liver weight. (B) Fasting glucose levels. (C) Insulin levels measured in the serum of db/db mice (each n=6). Results represents three independent experiments for each protein. Statistical results of one-factor analysis of variance. WT, wild type. aP<0.01, bP<0.001 vs. WT mice; cP<0.01 vs. 7-week mice.

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  Fig. 2 Obesity increase in Forkhead box O1 (FoxO1)-induced lipid accumulation. (A) Hepatic triglyceride (TG) in male db/db mice. One representative result of three experiments yielding similar outcomes for each protein is listed. Results of one-factor analysis of variance (ANOVA). (B) Western blotting was conducted to find the protein levels of p-FoxO1 and FoxO1 in db/db mice liver. Transcription factor II B (TFIIB) was used as the loading control protein of the nuclear fraction. (C) Western blotting of peroxisome proliferator-activated receptors (PPARs) and sterol regulatory element-binding protein 1c (SREBP-1c) in the nucleus of db/db mouse livers. TFIIB was used as the loading control of the nuclear fraction. (D) Western blotting showed that immunoprecipitated FoxO1 and PPARγ interacted with PPARγ and FoxO1, respectively. (E) Real-time polymerase chain reaction analysis was performed for analyzing the mRNA levels of PPARα, PPARβ, and PPARγ. Results of one-factor ANOVA. (F) Real-time PCR analysis was conducted for analyzing the mRNA expression levels of fatty acid synthase (FASN), acetyl-CoA carboxylase (ACC), and stearoyl-CoA desaturase 1 (SCD1). Results of one-factor ANOVA. (G) Real-time PCR analysis was performed for analyzing the mRNA levels of carnitine palmitoyltransferase I α (CPT1α) and peroxisomal acyl-coenzyme A oxidase 1 (ACOX1). IgG, immunoglobulin G; WT, wild type. aP<0.001 vs. 7 weeks db+ mice; bP<0.001 vs. 10 weeks db+ mice; cP<0.01 vs. 10 weeks db+ mice; dP<0.01 vs. 7 weeks db+ mice; eP<0.05 vs. 10 weeks db+ mice.

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  Fig. 3 Changes in insulin signaling in db/db mice. (A)Western blotting was conducted in order to detect the protein expression levels of the factors involved in insulin signaling and Akt signaling. Obesity-related insulin signaling factors phospho-serine-insulin receptor substrate 1 (pSer-IRS1), phospho-tyrosine-insulin receptor substrate 1 (pTyr-IRS1), p-Akt, and total Akt levels. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the loading control protein of the cytoplasm. Results are representative of three independent experiments for each protein. Results of one-factor analysis of variance. (B) Western blot analysis was performed to detect protein levels of phospho-AMP-activated protein kinase (p-AMPK), and AMPK in liver homogenates. GAPDH was used as the loading control protein of the cytoplasm. WT, wild type. aP<0.05, bP<0.001 vs. 7 weeks db+ mice; cP<0.05, dP<0.001 vs. 10 weeks db+ mice.

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  Fig. 4 Interaction of protease-activated protein-2 (PAR2) and β-arrestin in db/db mice. (A) PAR2 and β-arrestin concentrations were analyzed using cytosolic fraction extracted from the liver samples of db/db mice. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the loading control protein of the cytosolic fractions. Results of one-factor analysis of variance. (B) Western blotting showed that immunoprecipitated PAR2 and β-arrestin were physically interacted with PAR2 and β-arrestin, respectively. (C) Western blotting showed that immunoprecipitated β-arrestin and Akt were interact with β-arrestin and Akt, respectively. (D) A potential mechanism underlying the effect of Forkhead box O1 (FoxO1) on PAR2/β-arrestin signaling in obesity. WT, wild type; IP, immunoprecipitation; IB, immunoblotting; IgG, immunoglobulin G; FoxO1, forkhead box O1; PPARγ, peroxisome proliferator-activated receptor γ. aP<0.05, bP<0.01 vs. 7 weeks db+ mice; cP<0.01, dP<0.001 vs. 10 weeks db+ mice.

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  Fig. 5 High-fat diet (HFD)-mediated serum changes in protease-activated protein-2 (PAR2)-knockout (KO) mice. (A) Fasting glucose levels, (B) triglycerides, (C) free fatty acids level in the serum of HFD-feeding PAR2-KO mice (each n=6). One representative result of three experiments yielding similar outcomes for each protein is demonstrated. Results of one-factor analysis of variance. WT, wild type; NEFA, non-esterified fatty acid. aP<0.01 vs. WT chop mice; bP<0.05 vs. HFD-feeding mice.

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  Fig. 6 Forkhead box O1 (FoxO1) induces hepatic steatosis in high-fat diet (HFD)-feeding protease-activated protein-2 (PAR2)-knockout (KO). Western blotting analyses of liver cytosolic fraction (A) FoxO1 and peroxisome proliferator-activated receptor γ (PPARγ) concentrations were analyzed using nuclear proteins extracted from obese mice (n=6 in each group). Transcription factor II B (TFIIB) was the loading control protein of the nuclear fractions. (B) Hepatic triglyceride (TG) in HFD-feeding PAR2-KO mice. One representative result of three experiments yielding similar outcomes for each protein is demonstrated. Results of one-factor analysis of variance (ANOVA). (C) Real-time polymerase chain reaction analyses was conducted for analyzing the mRNA levels of lipogenic genes such as PPARγ, fatty acid synthase (FASN), acetyl-CoA carboxylase (ACC), and stearoyl-CoA desaturase 1 (SCD1). Results of one-factor ANOVA. WT, wild type. aP<0.01 vs. normal control mice; bP<0.05 vs. HFD-feeding mice; cP<0.05, dP<0.01, eP<0.001 vs. WT chow; fP<0.05, gP<0.01, hP<0.001 vs. WT-HFD.

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