Uncoupling Lipid Synthesis from Adipocyte Development

Abstract

Obesity results from the expansion of adipose tissue, a versatile tissue regulating energy homeostasis, adipokine secretion, thermogenesis, and inflammation. The primary function of adipocytes is thought to be lipid storage through lipid synthesis, which is presumably intertwined with adipogenesis. However, during prolonged fasting, adipocytes are depleted of lipid droplets yet retain endocrine function and an instant response to nutrients. This observation led us to question whether lipid synthesis and storage can be uncoupled from adipogenesis and adipocyte function. By inhibiting key enzymes in the lipid synthesis pathway during adipocyte development, we demonstrated that a basal level of lipid synthesis is essential for adipogenesis initiation but not for maturation and maintenance of adipocyte identity. Furthermore, inducing dedifferentiation of mature adipocytes abrogated adipocyte identity but not lipid storage. These findings suggest that lipid synthesis and storage are not the defining features of adipocytes and raise the possibility of uncoupling lipid synthesis from adipocyte development to achieve smaller and healthier adipocytes for the treatment of obesity and related disorders.

Keywords: ACC; DGAT; FASN; adipocyte; adipogenesis; fatty acid; lipogenesis; obesity.

Figure 1

Prolonged fasting deprives lipid storage but adipocyte identity in mice. (A): Experimental design. Chow diet-fed C57BL/6 mice were subjected to 72-h fasting with or without 3 h refeeding. (B,C,D,E) staining of iWAT (B) and eWAT (C) from ad libitum fed, fasted mice or fasted-refed mice. (D,E): Confocal immunofluorescence of iWAT (D) and eWAT (E) from fasted mice for indicated proteins. (F): Western blotting analysis of plasma adipokines. (G,H): qPCR analysis of gene expression of adipogenic and lipolytic markers (G) and synthetic lipid genes (H) in iWAT from ad libitum fed, fasted mice, or fasted-refed mice. The fold changes for gene expression in iWAT (Y-axis on the left side of the graph) and liver (Y-axis on the right side of the graph) were normalized to its expression in iWAT from mice on ad libitum condition. n = 5/group. Data were represented as mean ± SEM. Statistical significance was calculated via two-tailed Student’s t-tests (Fasted or refed group versus ad libitum group). N.S.: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 2

Biotin deprivation causes adipogenesis without TG accumulation. (A): Experimental design. C3H10T1/2 pre-adipocytes were differentiated in the presence of 10⁻⁷ M Avidin from Day 0 to Day 7 of differentiation. (B): Oil Red O staining of lipid droplets in C3H10T1/2 cells on Day 7 of differentiation. (C): Quantification of Oil Red O staining (n = 3, 3). (D): qPCR analysis of gene expression of adipogenic markers and lipogenic genes on Day 7 of differentiation with or without Avidin treatment. (n = 3, 4). Data were represented as mean ± SEM. Statistical significance was calculated via two-tailed Student’s t-tests. N.S.: not significant, **** p < 0.0001.

Figure 3

Inhibition of FASN shows a stage-dependent effect on adipogenesis. (A): Experimental design. C3H10T1/2 pre-adipocytes were treated with FASN inhibitor TVB-3664 (200 nM) from the beginning of differentiation Day 0 to Day 6/7 or from Day 6 to Day 9. (B): Oil Red O staining of lipid droplets in C3H10T1/2 cells (fixed on Day 7 or Day 9 with or without TVB-3664 treatment). (C): Quantification of Oil Red O staining. (D): Gene expression of adipogenic markers and lipogenic genes in C3H10T1/2 cells were treated with TVB-3664 from Day 0 to Day 6 during differentiation (n = 4, 4). (E): Gene expression of C3H10T1/2 cells after TVB-3664 treatment from Day 6 to Day 9 (n = 3, 3). N.S.: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001 for control vs. TVB-3664 treatment group by 2-tailed Student’s t-test. Data were represented as mean ± SEM.

Figure 4

PPARγ2 cannot rescue adipogenesis from FASN inhibition. (A): Experimental design. PPARγ2 OE MEF cells were treated with the FASN inhibitor TVB-3664 (200 nM) from differentiation Day 0 to Day 6 or Day 3 to Day 6. (B): Oil Red O staining of lipid droplets of differentiated PPARγ2 OE MEF cells (fixed on Day 6 with or without treatment). (C): Quantification of Oil Red O staining (n = 3, 3). (D,E): Gene expression of PPARγ2 OE MEF cells treated with TVB-3664 at Day 0 to Day 6 (D) or Day 3 to Day 6 (E) during differentiation (n = 3, 3). N.S.: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 for control vs. TVB-3664 treatment group by 2-tailed Student’s t-test. Data were represented as mean ± SEM.

Figure 5

DGAT1/2 inhibition shows normal adipogenesis. (A): Experimental design. C3H10T1/2 pre-adipocytes were differentiated in the presence of DGAT1 inhibitor (PF-04620110, 3 µM) and DGAT2 inhibitor (PF-06427878, 3 µM) from Day 0 to Day 6. (B): Oil Red O staining of lipid droplets in C3H10T1/2 cells on Day 6 of differentiation. (C): Quantification of Oil Red O staining (n = 3, 3). (D): qPCR analysis of gene expression of adipogenic markers and lipogenic genes on Day 6 of differentiation (n = 4, 4). N.S.: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001 for control vs. DGAT1/2 inhibitor treatment group by 2-tailed Student’s t-test. Data were represented as mean ± SEM.

Figure 6

TGF-β induces adipocyte dedifferentiation. (A): Experimental design. Mature C3H10T1/2 cells were treated with TGF-β (5 ng/mL) from differentiation Day 6 to Day 12. (B): Oil Red O staining of lipid droplets in C3H10T1/2 cells on Day 12 of differentiation. (C): Quantification of Oil Red O staining (n = 3, 3). (D): qPCR analysis of gene expression in C3H10T1/2 cells on Day 12 of differentiation (n = 3, 3). ** p < 0.01, *** p < 0.001 for control vs. TGF-β group by 2-tailed Student’s t-test. Data were represented as mean ± SEM.

Figure 7

Lipid synthesis in adipocyte differentiation and functions. (A): The differentiation of mesenchymal stem cells (MSCs) into adipocytes begins with the first stage of fate determination, in which the cell commits to an adipocyte fate. Subsequently, the early stage of differentiation is accompanied by morphological and functional alterations and requires a basal level of lipogenesis. Lipid synthesis is required for the hypertrophic growth of adipocytes and adipocyte functions but is dispensable in the maintenance of adipocyte identity. (B): The lipid synthesis pathway and adipocyte functions. Abbreviations: ACC1/2, acetyl CoA carboxylase1/2; AT, Adipose tissue; C/EBPα, CCAAT/enhancer binding protein α; C/EBPβ, CCAAT/enhancer binding protein β; CD36, a cluster of differentiation 36; Adispin, complement factor D; DGAT1/2, diglyceride acyltransferase 1/2; LCFA, Long-chain fatty acid; FASN, fatty acid synthase; GLUT1/4, glucose transporter type 1/4; IL-6, interleukin 6; PPARγ, peroxisome proliferator-activated receptor γ; TG, triglyceride; TCA, tricarboxylic acid cycle; TNF-α, tumor necrosis factor-α.