Unique mode of lipogenic activation in rat preputial sebocytes

Abstract

Lipoprotein delivery of fatty acids and cholesterol is linked with peroxisome proliferator-activated receptor (PPAR) activation in adipocytes and macrophages. We postulated that similar interactions exist in sebaceous epithelial cells (sebocytes) in which PPAR activation induces differentiation. High-density lipoprotein (HDL) and very low-density lipoprotein (VLDL) markedly enhanced sebocyte differentiation above that found with PPAR agonists and were more potent than explicable by their lipid content. The PPARγ antagonist GW5393 reduced sebocyte differentiation to all PPAR isoform agonists, HDL and VLDL, suggesting that the lipoprotein effect on differentiation occurs partially through activation of PPARγ. Furthermore, we found that sebocytes expressed a unique pattern of lipogenic genes. Our results demonstrate that HDL and VLDL are the most potent inducers of sebocyte differentiation tested to date, and these actions are partially inhibited by PPAR antagonists. This suggests that substrates provided by lipoproteins are targeted to sebocytes and affect their own disposition via PPAR activation.

Figure 1

Comparison of sebocyte differentiation induced by PPAR agonists and lipoproteins before and after pretreatment with two PPAR antagonists. Maximally effective doses of the selective PPAR agonists troglitazone (TRO), carbaprostacyclin (cPGI2), and linoleic acid (LIN) were used as indicated. HDL and LDL were used at 100 μg protein/mL. The specific PPARδ (GW742) and PPARγ (GW845) agonists were used at the doses indicated. The PPARγ antagonist GW5393 (a) and the PPAR binding pocket antagonist GW9662 (b) were added to the cells at a dose of 1 μg 2 hours prior to treatment with the PPAR agonists or lipoproteins on day 7 of primary culture. LFC determination was made on day 9 of culture after fixing and staining the cells with Oil Red O (ORO). Striped bars indicate colonies with 6–50 ORO-stained cells, and solid bars those colonies with >50 ORO-stained cells. Means +/− SEMs are presented.

Figure 2

Triglyceride and cholesterol content of cultured sebocytes following treatment with HDL and VLDL. HDL induced significantly greater concentrations of both triglyceride and cholesterol, whereas VLDL induced a high level of triglyceride accumulation, but had no significant effect on cholesterol accumulation. Triglyceride (n = 5) and cholesterol (n = 6) were assayed per 3 wells. Means +/− SEMs are shown.

Figure 3

Comparison of protein versus lipid content of HDL, VLDL, and LIN on induction of sebocyte differentiation. VLDL was more potent than HDL in the induction of sebocyte differentiation in terms of protein content but less potent when comparing lipid content (n = 4). Means +/− SEMs are shown.

Figure 4

Comparison of the expression of lipogenic regulatory genes in freshly dispersed (Fr) and cultured (Cx) ± dihydrotestosterone (DHT) preputial sebocytes and epidermal cells, using RT-PCR. Total RNA was isolated from adult rat preputial sebocytes, epidermal cells and homogenized epididymal fat pad. Primers specific for the listed genes were used (see Table 1). Adipsin, aP2, CD36, and MC5-R were only detected in sebocytes, not in fresh or cultured epidermal cells. Leptin was detected in cultured sebocytes (less mature) to a greater extent than in freshly dispersed sebocytes (more mature) or epidermal cells. MC5-R was detected in freshly dispersed sebocytes to a greater extent than cultured sebocytes.

Figure 5

Feed-forward model of sebocyte differentiation. We postulate that lipoproteins target lipid substrates within skin to sebocytes through interactions with specific receptors. The substrates in turn stimulate sebocyte differentiation by acting through cell-specific PPARγ-mediated lipogenic pathways.