Direct conversion of human myoblasts into brown-like adipocytes by engineered super-active PPARγ

Abstract

Objective: To determine whether super-activation of PPARγ can reprogram human myoblasts into brown-like adipocytes and to establish a new cell model for browning research.

Methods: To enhance the PPARγ signaling, M3, the transactivation domain of MyoD, was fused to PPARγ. PPARγ and M3-PPARγ-lentiviral vectors were used to convert human myoblasts into adipocytes. Brown adipocyte markers of the reprogrammed adipocytes were assessed by qPCR and protein analyses. White adipocytes differentiated from subcutaneous stromal vascular cells and perithyroid brown fat tissues were used as references.

Results: In transient transfections, M3-PPARγ had a stronger constitutive activity than PPARγ by reporter assay. Although the transduction of either PPARγ or M3-PPARγ induced adipogenesis in myoblasts, M3-PPARγ drastically induced the brown adipocyte markers of UCP1, CIDEA, and PRDM16 by 1,050, 2.4, and 5.0 fold, respectively and increased mitochondria contents by 4 fold, compared to PPARγ.

Conclusions: Super-activation of PPARγ can effectively convert human myoblasts into brown-like adipocytes and a new approach to derive brown-like adipocytes.

Figure 1

Fusion of M3 transacting domain to PPARγ enhances its transcriptional activity of PPARγ. (a) Illustration of M3 domain to N-terminus of PPARγ, which is comprised of a transactivation domain (AF1), a DNA-binding domain (DBD), a C-terminal ligand-binding domain (LBD) and a ligand-dependent transactivation domain (AF2). The entire construct is cloned into the lentiviral expression vector pSMPUW. (b) Transcriptional activity of M3-PPARγ on PPRE3x-luciferase promoter. HEK293 cells are transfected with the reporter construct and GFP, PPARγ and M3-PPARγ in the presence or absence of rosiglitazone (1 μM), and assayed for luciferase activities. Data are expressed as mean ± SE (n = 3). **: p < 0.01.

Figure 2

Lentivirus production and experiment scheme. (a) Western blot analysis of PPARγ and M3-PPARγ. Human myoblasts were transduced with lenti-GFP, lenti-PPARγ or lenti-M3-, and protein lysates were collected for Western blot by antibodies against MyoD, PPARγ, and β-actin (as a loading control) 48 h post-transduction. (b) Experimental scheme testing for the differentiation of human myoblasts into brown-like adipocytes. Human myoblasts were transduced with the indicated lentiviruses and cultured in induction medium containing IBMX, rosiglitazone, insulin and dexamethasone for 3 days. Then the cells were cultured in differentiation medium without IBMX and rosiglitazone for 8 days before analysis.

Figure 3

Representative microscopic images of myoblasts before and during adipocyte differentiation. Myoblasts, transduced with lentiviruses of GFP, PPARγ or M3-PPARγ, were examined with phase-contrast microscopy before differentiation and on the indicated days of differentiation. Scale bar: 50 μm for all images.

Figure 4

Gene expression analyses of human myoblasts transduced with lentiviruses of GFP, PPARγ or M3-PPARγ after treatment with the adipose differentiation cocktail, compared to the in vitro differentiated white adipocytes (iWA) and the perithyorid BAT. iWA are adipocytes differentiated from subcutaneous AdSVCs and BAT are biopsies of perithyroid brown adipose tissues. Total RNAs were isolated from cells on day 11 of differentiation or BAT. (a) Selective white and adiposity markers. (b) Selective brown adipocyte selective markers (c) Selective myocyte markers. Data are presented as mean ± SEM (n = 3 for myocytes and BAT; n = 4 for iWA); *p < 0.05, **p < 0.01; ^#p < 0.05, ^##p < 0.01 versus GFP; ^§p < 0.05, ^§§p < 0.01 versus PPARγ; ^†p < 0.05, ^††p < 0.01 versus M3-PPARγ.

Figure 5

UCP1 protein expression analyses of human myoblasts transduced with GFP, PPARγ and M3-PPARγ 11 days after treatment of adipose differentiation cocktail. (a) Representative image of Western blot of UCP1 protein expression in differentiated cells and (b) quantification in reference to β-actin control. Data are presented as mean ± SEM (n=3); *p < 0.05, **p < 0.01.

Figure 6

Induction of mitobiogenesis by M3-PPARγ. (a) Representative image of MitoTracker (red) staining of human myoblasts transduced with GFP, PPARγ and M3-PPARγ 11 days after treatment of adipose differentiation. (b) Quantitative fluorescence signal of MitoTracker staining. Data are presented as mean ± SEM; **p < 0.01 versus PPARγ (n = 10). Scale bar: 10 μm for all images.