Plac8 is required for white adipocyte differentiation in vitro and cell number control in vivo

Abstract

Plac8 belongs to an evolutionary conserved family of proteins, mostly abundant in plants where they control fruit weight through regulation of cell number. In mice, Plac8 is expressed both in white and brown adipose tissues and we previously showed that Plac8(-/-) mice develop late-onset obesity, with abnormal brown fat differentiation and reduced thermogenic capacity. We also showed that in brown adipocytes, Plac8 is an upstream regulator of C/EBPβ expression. Here, we first assessed the role of Plac8 in white adipogenesis in vitro. We show that Plac8 is induced early after induction of 3T3-L1 adipocytes differentiation, a process that is prevented by Plac8 knockdown; similarly, embryonic fibroblasts obtained from Plac8 knockout mice failed to form adipocytes upon stimulation of differentiation. Knockdown of Plac8 in 3T3-L1 was associated with reduced expression of C/EBPβ, Krox20, and Klf4, early regulators of the white adipogenic program, and we show that Plac8 could transactivate the C/EBPβ promoter. In vivo, we show that absence of Plac8 led to increased white fat mass with enlarged adipocytes but reduced total number of adipocytes. Finally, even though Plac8(-/-) mice showed impaired thermogenesis due to brown fat dysfunction, this was not associated with changes in glycemia or plasma free fatty acid and triglyceride levels. Collectively, these data indicate that Plac8 is an upstream regulator of C/EBPβ required for adipogenesis in vitro. However, in vivo, Plac8 is dispensable for the differentiation of white adipocytes with preserved fat storage capacity but is required for normal fat cell number regulation.

Figure 1. Plac8 knockdown in 3T3-L1 decreases adipogenesis.

A, Time-course (0, 3, and 6 h, 1 to 7 days) of Plac8, C/EBPβ and Krox20 mRNA expression during 3T3-L1 cells differentiation. Three independent experiments are shown. B, Oil red O staining of 3T3-L1 transduced with two different Plac8 (sh1 and sh2 and a control (shLuc) shRNAs at 7 days after induction of differentiation. Upper row: culture dishes; lower row: photomicrographs of the cells (scale bars = 100 µm). C, Western blot analysis of Plac8 protein in shRNA-transduced 3T3-L1 cells at day 0 and 3 and 6 hours after induction of differentiation. Actin served as loading control D, mRNA levels of Plac8 and of the transcriptional regulators C/EBPβ, C/EBPδ, Krox20, Klf4 and PPARγ2 and of the general adipogenic gene aP2 at day 0 and 7 of differentiation. One representative experiment out of three is shown. Values are means ± SD, n = 3.,*p<0.05 vs. shLuc, #p<0.05 vs. shLuc at day 0.

Figure 2. Plac8 overexpression in 3T3-L1 enhances adipogenesis.

A, 3T3-L1 cells were transduced with Plac8 or control retroviruses. Oil red O staining was performed at 7 days after induction of differentiation. Upper row: culture dishes; lower row: photomicrographs of the cells (scale bars = 100 µm). B, Western blot analysis of Plac8 in shRNA transduced 3T3-L1 cells at day 0 and 3 and 6 hours after induction of differentiation. Actin served as loading control C, mRNA levels of Plac8 and of the transcriptional regulators C/EBPβ, C/EBPδ, Krox20 and PPARγ2 and of the general adipogenic gene aP2 at days 0 and 7 of differentiation. One representative experiment out of three is shown. Values are means ± SD, n = 3.,*p<0.05 vs. shLuc, #p<0.05 vs. shLuc at day 0.

Figure 3. Absence of Plac8 prevents adipogenic differentiation of primary mouse embryonic fibroblasts.

A, Oil red O staining of primary mouse embryonic fibroblasts from Plac8^+/+; Plac8^+/− and Plac8^−/− mice 7 days after induction of differentiation. Upper row: culture dishes; lower row: photomicrographs of the cells (scale bars = 50 µm). B, mRNA expression of aP2, PPARγ2 and adiponectin at day 0 and day 7 after induction of differentiation. One representative experiment out of three is shown. Values are means ± SD. *p<0.05 vs. Plac8^+/+ at day 7, #p<0.05 vs. Plac8^+/+ at day 0.

Figure 4. Plac8 increases C/EBPβ-induced adipogenesis of NIH-3T3 fibroblasts.

A, NIH-3T3 cells were transduced with Plac8 and C/EBPβ, or control retroviruses and Oil red O staining was performed 7 days after induction of differentiation. Upper row: culture dishes; lower row: photomicrographs of the cells (scale bars = 50 µm). B, mRNA levels of the Plac8, C/EBPβ and aP2 and of the white fat genes adiponectin and resistin at days 0 and 7 after induction of differentiation. One representative experiment out of three is shown. Values are means ± SD, n = 4. *p<0.05 vs. Plac8^+/+ at day 7, #p<0.05 vs. Plac8^+/+ at day 0, $p<0.05 vs. Plac8^−/− at day 7. C, Plac8 transactivates C/EBPβ and Krox20 promoters in 3T3-L1 preadipocytes. Transcriptional activity of a 3 kb C/EBPβ promoter-luciferase reporter construct (black bars) or empty vector (pGL3) (white bars, close to the zero level and not visible) cotransfected into 3T3-L1 preadipocytes with a Plac8 expression or an empty vector. Luciferase activities were determined 48 hours after transfection. Results are means ± SD (n = 3), one representative experiment is shown. *p<0.05 vs. empty vector.

Figure 5. Adipocytes in visceral and subcutaneous adipose tissues are enlarged in Plac8^−/− mice.

Hematoxylin-eosin staining of VAT (A) and SCAT (B) sections of 29-weeks-old Plac8^+/+, Plac8^+/− and Plac8^−/− mice (scale bars = 50 µm). C, Analysis of VAT and SCAT adipocytes size distribution of Plac8^+/+, Plac8^+/− and Plac8^−/− mice. D, VAT and SCAT weight in Plac8^+/+, Plac8^+/− and Plac8^−/− mice. E, Total DNA content in VAT and SCAT of the same mice. Values are means ± SEM (n = 7–13). *p<0.05 vs. Plac8^+/+.

Figure 6. Adipogenic transcriptional regulators are decreased in white adipose tissues of Plac8^−/− mice.

mRNA levels of the transcriptional regulators C/EBPβ, C/EBPα, PPARγ2, C/EBPδ and of aP2 in VAT (A) and SCAT (B) of 24 weeks-old Plac8^+/+, Plac8^+/− and Plac8^−/− mice. Values are means ± SEM (n = 7–13), *p<0.05 vs. Plac8^+/+.

Figure 7. Expression of lipogenic and lipolytic genes in VAT and SCAT of Plac8^−/− mice.

mRNA levels of the lipogenic genes Acc1, SREBP-1c, Fas and SCD-1, and of the lipolytic genes Atgl and Hsl in VAT (A,B) and (C,D) of 24 weeks-old Plac8^+/+, Plac8^+/− and Plac8^−/− mice. Values are means ± SEM (n = 7–13), *p<0.05 vs. Plac8^+/+.

Figure 8. Normal glycemia and plasma free fatty acids and triglycerides in Plac8^−/− mice.

Blood glucose (A), and plasma free fatty acids (FFA) (B) and triglycerides (C) levels in fed and 16-hours fasted Plac8^+/+ and Plac8^−/− 24-weeks old mice Values are means ± SEM (n = 7–13), *p<0.05 vs. Plac8^+/+ at fed state.