Korean J Physiol Pharmacol.  2016 Nov;20(6):649-655. 10.4196/kjpp.2016.20.6.649.

TonEBP suppresses adipocyte differentiation via modulation of early signaling in 3T3-L1 cells

• Kim SJ
• Kim T
• Choi HN
• Cho EJ
• Park JB
• Jeon BH
• Lee SD

• KMID: 2364269
• DOI: http://doi.org/10.4196/kjpp.2016.20.6.649

Abstract

TonEBP belongs to the Rel family of transcription factors and plays important roles in inflammation as well as kidney homeostasis. Recent studies suggest that TonEBP expression is also involved in differentiation of several cell types such as myocytes, chondrocytes, and osteocytes. In this study, we investigated the roles of TonEBP during adipocyte differentiation in 3T3-L1 cells. TonEBP mRNA and protein expression was dramatically reduced during adipocyte differentiation. Sustained expression of TonEBP using an adenovirus suppressed the formation of lipid droplets as well as the expression of FABP4, a marker of differentiated adipocytes. TonEBP also inhibited the expression of PPARγ, a known master regulator of adipocytes. RNAi-mediated knock down of TonEBP promoted adipocyte differentiation. However, overexpression of TonEBP did not affect adipogenesis after the initiation of differentiation. Furthermore, TonEBP expression suppressed mitotic clonal expansion and insulin signaling, which are required early for adipocyte differentiation of 3T3-L1 cells. These results suggest that TonEBP may be an important regulatory factor in the early phase of adipocyte differentiation.

Keyword

Adipogenesis; Akt; Insulin; TonEBP

MeSH Terms

3T3-L1 Cells*
Adenoviridae
Adipocytes*
Adipogenesis
Chondrocytes
Homeostasis
Humans
Inflammation
Insulin
Kidney
Lipid Droplets
Muscle Cells
Osteocytes
RNA, Messenger
Transcription Factors
Insulin
RNA, Messenger
Transcription Factors

Figure

• 

  Fig. 1 Expression of TonEBP during adipocyte differentiation.3T3-L1 preadipocytes were induced to differentiate into adipocytes. (A) Total cell lysates were prepared at indicated times and protein expression was analyzed by immunoblotting. (B) TonEBP expression (A) was normalized to that of GAPDH and then presented as a relative value based on D0. (C) Expression of TonEBP mRNA at indicated times was measured by quantitative RT-PCR and presented as a relative value to D0. GAPDH mRNA expression was used as a reference. Values are mean±SEM. *p<0.05, **p<0.01 compared with D0 for each group (n≥3).

• 

  Fig. 2 Suppression of adipocyte differentiation by TonEBP.3T3-L1 cells were infected with recombinant adenovirus expressing β-galactosidase or TonEBP and then induced to differentiate into adipocytes. Expression of TonEBP (A) or adipogenic proteins (D) was analyzed by immunoblotting. (B) The degree of adipocyte differentiation was visualized by staining lipid droplets with Oil Red O at D6. (C) Lipid droplets shown in (B) were extracted with isopropyl alcohol and quantified as a relative value to Adβ-gal. (E) The quantity of FABP4 shown in (D) was normalized to that of GAPDH and then presented as a relative value to D6 of Adβ-gal. Values are mean±SEM. *p<0.05, **p<0.01 compared to Adβ-gal for each group (n=3).

• 

  Fig. 3 Promotion of adipocyte differentiation by TonEBP knockdown.3T3-L1 cells were transfected with scrambled or TonEBP siRNA and then induced to differentiate into adipocytes. Expression of TonEBP (A) or adipogenic proteins (D) were analyzed by immunoblotting. (B) The degree of adipocyte differentiation was visualized by staining lipid droplets with Oil Red O at D6. (C) Lipid droplets shown in (B) were extracted with isopropyl alcohol and quantified as a relative value to scrambled siRNA. (E) The quantity of FABP4 shown in (D) was normalized to that of GAPDH and then presented as a relative value to D6 of scrambled siRNA. Values are mean±SEM. *p<0.05, **p<0.01 compared to scrambled siRNA for each group (n≥3).

• 

  Fig. 4 Limited anti-adipogenic property of TonEBP in the early stages of adipocyte differentiation3T3-L1 cells were treated with differentiation factors at D0 for adipogenesis. Recombinant adenoviruses were infected at indicated times as described in (A). (B) Expression of each protein was analyzed by immunoblotting. (C) The degree of adipocyte differentiation was visualized by staining lipid droplets with Oil Red O at D6. (D) The quantity of FABP4 shown in (B) was normalized to that of GAPDH and then presented as a relative value to D-3 of Adβ-gal. Values are mean±SEM. **p<0.01, ***p<0.001 compared to Adβ-gal for each group (n=6).

• 

  Fig. 5 Suppression of MCE and insulin signaling with TonEBP overexpression.3T3-L1 cells infected with recombinant adenoviruses were treated with differentiation factors (IDI) for the indicated times. (A) Cell number was determined via MTT assay and presented as a relative value to DO. (B) The protein levels of phosphorylated IRS-1 (p-IRS-1), IRS-1, phosphorylated Akt (p-Akt), and Akt in these cells were detected by immunoblotting using specific antibodies. The experiment was repeated three times with similar results.

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