Peroxisome Proliferator-activated Receptor γ Stimulation of Adipocyte ApoE Gene Transcription Mediated by the Liver Receptor X Pathway

Lili Yue,Theodore Mazzone

doi:10.1074/jbc.m808482200

Abstract

Peroxisome proliferator-activated receptor (PPARgamma) agonists increase insulin sensitivity in humans and are useful for treating human diabetes. Treatment with these agonists leads to increased apoE expression and triglyceride accumulation in adipocytes. The importance of apoE for adipocyte triglyceride accumulation is demonstrated by observations that triglyceride accumulation is impaired in apoE knockout adipocytes treated with PPARgamma agonists. The current studies investigate the molecular mechanism for PPARgamma stimulation of the adipocyte apoE gene and demonstrate that the liver receptor X (LXR) response element within an apoE gene downstream enhancer is required for the apoE response to PPARgamma agonists. The response of the apoE gene to treatment with PPARgamma agonists was delayed beyond 12 h suggesting the involvement of an intermediary pathway. The combined addition of PPARgamma and LXR agonists did not increase apoE response beyond that observed with addition of either alone. Deletion or mutation of the LXR response element completely eliminated the adipocyte apoE gene response to a PPARgamma agonist. Chromatin immunoprecipitation analyses performed using isolated adipocytes, or adipose tissue from mice treated with PPARgamma agonists, showed increased LXR binding to the apoE gene after PPARgamma agonist treatment. Knockdown of LXR expression completely eliminated the increase in apoE message, protein, and triglyceride in response to PPARgamma stimulation. The LXR response element has been previously shown to mediate sterol responsiveness of the apoE gene, and apoE expression plays an important role in adipocyte triglyceride balance. The current observations suggest that the PPARgamma-LXR-apoE regulatory cascade could be an important molecular link for cross-talk between adipocyte triglyceride and cholesterol homeostasis.

Highlights

The above results established that treatment with PPAR␥ on our previous observations showing that the response of the agonists increased LXR binding to the apoE gene enhancer in apoE gene to PPAR␥ agonists required the presence of a downvitro and in vivo, and that the LXR response element in the apoE stream apoE gene enhancer, the presence of an LXRE in this gene enhancer was required for increased apoE gene expression enhancer, and the absence of a clearly identifiable PPRE within after PPAR␥ stimulation
We performed experiments to this enhancer, we hypothesized that the LXR pathway mediated further confirm that an intact adipocyte LXR pathway was the apoE gene response to PPAR␥ agonists [4]
Consistent with required for the response of the endogenous adipocyte apoE this hypothesis, we show that increased expression of an gene to PPAR␥ stimulation

Summary

EXPERIMENTAL PROCEDURES

Materials—Cell culture media, supplements, and antibiotics were purchased from Invitrogen. Real-time RT-PCR and Immunoblot Analysis—Total RNA was extracted from cells or tissues after the treatments described in figure legends. Following the treatments described in the figure legends, cells were washed twice in cold phosphate-buffered saline and lysed in 120 ␮l of cell culture lysis reagent provided in the luciferase assay kit. Chromatin immunoprecipitation was carried out using the ChIP-IT Express kit (Active Motif, Carlsbad, CA) according to the manufacturer’s instructions as previously described [7]. The DNA was purified with DNA mini columns provided with the kit, and real-time PCR was performed with primers (A 5Ј-AAC TGA CAC GTG GGT TGC AGG GGC AAC A-3Ј) and the reverse primer (5Ј-CAA CAT CCT TTC TCT CAG GCT AGA GTC C-3Ј), designed to amplify a 152-bp portion of the apoE enhancer containing the LXRE. Statistical differences were evaluated using analysis of variance (SPSS, Chicago, IL). p Ͻ 0.05 was considered significant

RESULTS

LXR also completely prevented the

DISCUSSION