Prolonged induction activates Cebpα independent adipogenesis in NIH/3T3 cells

Abstract

Background: 3T3-L1 cells are widely used to study adipogenesis and insulin response. Their adipogenic potential decreases with time in the culture. Expressing exogenous genes in 3T3-L1 cells can be challenging. This work tries to establish and characterize an alternative model of cultured adipocytes that is easier to work with than the 3T3-L1 cells.

Methodology/principal findings: INDUCED CELLS WERE IDENTIFIED AS ADIPOCYTES BASED ON THE FOLLOWING THREE CHARACTERISTICS: (1) Accumulation of triglyceride droplets as demonstrated by oil red O stain. (2) Transport rate of 2-deoxyglucose increased after insulin stimulation. (3) Expression of fat specific genes such as Fabp4 (aP2), Slc2a4 (Glut4) and Pparg (PPARγ). Among the cell lines induced under different conditions in this study, only NIH/3T3 cells differentiated into adipocytes after prolonged incubation in 3T3-L1 induction medium containing 20% instead of 10% fetal bovine serum. Rosiglitazone added to the induction medium shortened the incubation period from 14 to 7 days. The PI3K/AKT pathway showed similar changes upon insulin stimulation in these two adipocytes. C/EBPα mRNA was barely detectable in NIH/3T3 adipocytes. NIH/3T3 adipocytes induced in the presence of rosiglitazone showed higher 2-deoxyglucose transport rate after insulin stimulation, expressed less Agt (angiotensinogen) and more PPARγ. Knockdown of C/EBPα using shRNA blocked 3T3-L1 but not NIH/3T3 cell differentiation. Mouse adipose tissues from various anatomical locations showed comparable levels of C/EBPα mRNA.

Conclusions/significance: NIH/3T3 cells were capable of differentiating into adipocytes without genetic engineering. They were an adipocyte model that did not require the reciprocal activation between C/EBPα and PPARγ to differentiate. Future studies in the C/EBPα independent pathways leading to insulin responsiveness may reveal new targets to diabetes treatment.

Figure 1. NIH/3T3 cells formed insulin responsive adipocytes after prolonged induction.

(A) The whole cell culture dish view of the oil red O stain of NIH/3T3 cells induced with (R7) or without rosiglitazone (R-7) for 7 days and regular 3T3-L1 (3T3-L1) adipocytes. (B) Oil red O stained images of NIH/3T3 cells induced without rosiglitazone for 7 (R-7) or 14 days (R-14) and with rosiglitazone for 7 days (R7). (C) The glucose uptake rate in response to insulin with the standard errors (n = 3) of measurement of cells in panel (B).

Figure 2. NIH/3T3 adipocytes did not express C/EBPα.

The relative mRNA levels and the standard errors (n = 3) of measurement of 3 adipocyte marker genes, (A) Fabp4 (Ap2), (B) Cebpa (C/EBPα) and (C) Pparg (PPARγ) were determined daily by qPCR during adipogenesis in 3T3-L1 and NIH/3T3 (R7) cells. Because the required induction times were different in these 2 cell lines, day 0 on the x-axis indicated the day the induction medium was removed and the cells were incubated in maturation medium. (D) Western blot of labeled genes in 3T3-L1 and NIH/3T3 fibroblasts and adipocytes. HSP70 was used as the internal control. Lanes 1: 3T3-L1fibroblasts, 2: NIH/3T3 fibroblasts, Lanes 3: 3T3-L1adipocytes, 4: NIH/3T3 adipocytes.

Figure 3. C/EBPα was not required for adipogenesis in NIH/3T3 cells.

(A) The whole cell culture dish view in panel (A) and micrographs in panel (B) of the oil red O staining of 3T3-L1 (L1) and NIH/3T3 (NIH) cells induced after the cells were infected with lentiviruses expressing shRNA targeting luciferase (Luc), C/EBPα (C1 and C5) or PPARγ (P3 and P5). (C) The 2-deoxyglucose uptake and the standard errors (n = 3) of measurement of NIH/3T3 adipocytes infected with lentiviruses expressing shRNA targeting luciferase (-Luc) or C/EBPα (-C/EBP) before (f-) or after (a-) differentiation of NIH/3T3 cells.

Figure 4. Temporal expression profiles of the C/EBPs during differentiation of 3T3-L1 and NIH/3T3 cells.

The mRNA levels and the standard errors (n = 3) of measurement of (A) C/EBPα, (B) C/EBPβ, and (C) C/EBPδ were determined by qPCR and normalized by Gapdh signal on day 0 (D0), 2 (D2) and 4(D4) in differentiation medium and day 8 in DMEM +10% FBS medium.

Figure 5. Comparison of PI3K/AKT signaling in NIH/3T3 and 3T3-L1 adipocytes.

(A) Western blotting of phosphorylated-IRS-1(pIRS-1) and phosphorylated-insulin receptor (pIR), insulin receptor (IR), IRS-1, PDK1, PI3K,phospho-AKT (pAKT), total AKT (AKT), and β-actin in NIH/3T3 and 3T3-L1 adipocytes treated with (for 15 minutes) or without insulin. β-actin was the internal loading control. Labels were: L-Ins : 3T3-L1 -insulin, L+Ins: 3T3-L1 +insulin, N-Ins: NIH/3T3 -insulin, N+Ins: NIH/3T3 +insulin. pIRS-1 and pIR were blotted with 4G10 antibody, the rest of the proteins were probed with the antibody against the labeled protein. The blots were representative of 3 independent experiments. (B) The average band intensity normalized to beta-actin and the corresponding standard errors (error bars) were calculated after densitometry determination of each band in 3 independent blots as described in (A). None of the bands showed statistical significant difference in intensity.

Figure 6. NIH/3T3 adipocytes induced without rosiglitazone expressed more Pparg (PPARγ) and Agt (Angiotensiongen).

The mRNA levels and the standard errors (n = 3) of measurement of selected genes involved in blood pressure regulation, glucose transport and lipid metabolism in NIH/3T3 adipocytes induced in the presence (+Rosi) or absence (-Rosi) of rosiglitazone were compared with 3T3-L1 adipocytes. The gene symbols used were those approved by the HUGO gene nomenclature committee (HANG) for the mouse. Asterisks on top of the graph bars indicated the difference between (+Rosi) and (-Rosi) cells was significant after Bonferroni correction for multiple comparison (p <0.0045).

Figure 7. The relative mRNA levels and the standard errors (n = 3) of measurement of Cebpa (C/EBPα), Pparg (PPARγ), Fabp4 (Ap2) genes were determined by qPCR and in adipose tissues taken from 8 weeks old male C57BL/6 mice.

G: epididymal fat, M: mesenteric fat, I: interscapular fat, S: posterior thigh fat.