Abstract
Recent studies have identified the liver X receptors (LXRalpha and LXRbeta) as important regulators of cholesterol and lipid metabolism. Although originally identified as liver-enriched transcription factors, LXRs are also expressed in skeletal muscle, a tissue that accounts for approximately 40% of human total body weight and is the major site of glucose utilization and fatty acid oxidation. Nevertheless, no studies have yet addressed the functional role of LXRs in muscle. In this work we utilize a combination of in vivo and in vitro analysis to demonstrate that LXRs can functionally regulate genes involved in cholesterol metabolism in skeletal muscle. Furthermore we show that treatment of muscle cells in vitro with synthetic agonists of LXR increases the efflux of intracellular cholesterol to extracellular acceptors such as high density lipoprotein, thus identifying this tissue as a potential important regulator of reverse cholesterol transport and high density lipoprotein levels. Additionally we demonstrate that LXRalpha and a subset of LXR target genes are induced during myogenesis, suggesting a role for LXR-dependent signaling in the differentiation process.
Highlights
Recent studies have identified the liver X receptors (LXR␣ and LXR) as important regulators of cholesterol and lipid metabolism
Several studies have demonstrated that the LXRs play a dynamic role in the regulation of genes involved in cholesterol and fatty acid metabolism
Regulation of LXR Target Genes in Muscle—Treatment of rodents with synthetic LXR agonists has been shown to increase expression of genes involved in cholesterol and lipid metabolism in liver, intestine, and adipose tissue [1, 7,8,9,10,11, 21, 23]
Summary
In Vivo Analysis—Homozygous LXR␣-double knockout mice (LXR␣Ϫ/Ϫ) were from a breeding colony established and maintained at X-Ceptor Therapeutics Inc. C2C12 Cell Culture and Transient Transfection Assays—Mouse myogenic C2 cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 20% fetal bovine serum in 5% CO2. Cells were induced to biochemically and morphologically differentiate into multinucleated myotubes by mitogen withdrawal (Dulbecco’s modified Eagle’s medium supplemented with 2% horse serum) as previously described [2]. RNA Isolation and Analysis of Gene Expression by Quantitative Reverse Transcription-PCR—Total RNA from mouse tissues and C2C12 cells was isolated using RNeasy kits (Qiagen Inc.) according to the supplier’s total RNA isolation procedure. Quantitative PCR of reverse transcriptase reactions was carried out with 1.25 units of Taq polymerase (Invitrogen), 1ϫ Taq buffer, 3 mM MgCl2, 200 M dNTPs, 400 nM target-specific forward and reverse primers, and 100 nM target-specific fluorogenic probe. Statistical Analysis—Statistical analysis was carried out using a two-tailed unpaired t test
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