Activation of the mTOR pathway enhances PPARγ/SREBP-mediated lipid synthesis in human meibomian gland epithelial cells

Abstract

The involvement of the mechanistic targets of rapamycin (mTOR) pathway in lipid metabolism has been recently elucidated. However, its specific role in the Meibomian gland, where lipid metabolism is significant, remains not fully understood. We investigated the role of mTOR signaling system in the lipogenesis and differentiation of human meibomian gland epithelial cells (HMGECs). Treatment of HMGECs with rapamycin resulted in a reduction in lipid synthesis and the expression of PPARγ and SREBP-1, the major regulators of lipid synthesis. The phosphorylation of p70S6kinase and AKT, which are downstream signals of mTOR complexes 1 and 2, respectively, decreased following rapamycin treatment. In addition, when both mTOR complex 1 and 2 were suppressed using siRNA, there was a significant reduction in the expression of PPARγ and SREBP-1, along with a decrease in lipid synthesis in HMGECs. Our findings suggest that inhibiting the mTOR pathway diminishes the differentiation and adipogenesis of meibomian gland epithelial cells, and both mTOR complexes 1 and 2 appear to play a role in this activity.

Fig. 1

Rapamycin impedes lipogenesis in differentiated HMGECs. (a, b) HMGECs were treated with rapamycin (10 nM, added on days 0, 2, 4, and 6) and lipid accumulation was assessed by Oil-red O staining on day 8 (a). The results of three experiments are summarized (b). (c) Lipid synthesis of HMGECs with or without rapamycin was measured every 2 days by the AdipoRed assay. (d) Lipid accumulation was assessed by the LipidTOX assay on day 8 and the results of experiments are summarized. Data are presented as mean ± standard error of the mean. D, days after differentiation; *P < 0.05; **P < 0.01. ACC acetyl-CoA carboxylase, ADBR adrenergic receptor beta, FAS fatty acid synthase, HMGEC human meibomian gland epithelial cell.

Fig. 2

Rapamycin inhibits PPARγ, SREBP-1, and SREBP-1-dependent lipidogenesis related enzymes. HMGECs were cultured in the presence of rapamycin (10 nM, added on days 0, 2, 4, and 6). Transcription and protein expression of PPARγ, SREBP-1, and SREBP-1-dependent lipidogenesis related enzymes were analyzed. The mRNA (a) and protein (b) of PPARγ and SREBP-1 with or without rapamycin were assessed at 2-day intervals until day 8 after the start of differentiation. mRNA (c) and protein (d) levels of acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) were assessed every two days until day eight after the start of differentiation. The results of experiments are summarized. Data are presented as mean ± standard error of the mean. D, days after differentiation; *P < 0.05; **P < 0.01; ***P < 0.001. HMGEC human meibomian gland epithelial cell, PPAR peroxisome proliferator activator receptor, SREBP sterol regulatory element-binding protein.

Fig. 3

mTOR complex 1 and 2 signaling was inhibited by rapamycin in HMGECs. HMGECs were cultured with rapamycin (10 nM, added on days 0, 2, 4, and 6), and the phosphorylation of mTOR (Ser2448) and p70S6kinase (Thr389) for mTORC1 (a) and AKT (Ser473) for mTORC2 (b) were assessed by immunoblotting. The results of experiments are summarized. Data are presented as mean ± standard error of the mean. D, days after differentiation; *P < 0.05; **P < 0.01; ***P < 0.001. HMGEC human meibomian gland epithelial cell, mTOR mechanistic targets of rapamycin.

Fig. 4

Double knockdown of Raptor and Rictor impeded the expression of PPARγ and SREBP-1. HMGECs were transfected with 60 nM Raptor- or Rictor-specific siRNAs. Control groups were transfected with 60 nM non-specific control. siRNA Raptor groups were transfected with 30 nM raptor siRNA and 30 nM control RNA. si-Rictor groups were transfected with 30 nM siRNA for Rictor and 30 nM control RNA. Double-knockdown groups were transfected with 30 nM Raptor and 30 nM Rictor siRNAs. Transfections were repeated every two days. (a–d) HMGEC lysates were used to evaluate silencing efficiency (b) and to determine the phosphorylation of p70S6kinase and AKT (c) and the expression of PPARγ and SREBP-1 (d) by immunoblotting. The results of experiments are summarized. Data are presented as mean ± standard error of the mean. D, days after differentiation; *P < 0.05; **P < 0.01; ***P < 0.001. HMGEC human meibomian gland epithelial cell, PPAR peroxisome proliferator activator receptor, SREBP sterol regulatory element-binding protein.

Fig. 5

Double knockdown of Raptor and Rictor inhibited the lipid accumulation of HMGECs. HMGECs were transfected with 60 nM siRNA oligonucleotides for Raptor, Rictor, Raptor and Rictor, or non-specific controls. Lipid accumulation was assessed using the LipidTOX (a,b) and AdipoRed assays (c) on day 6. The results of experiments are summarized. Data are presented as mean ± standard error of the mean. D, days after differentiation; * P < 0.05. HMGEC human meibomian gland epithelial cell.