Assessing the effects of LXR agonists on cellular cholesterol handling: a stable isotope tracer study

Abstract

The liver X receptors (LXRs) a and b are responsible for the transcriptional regulation of a number of genes involved in cholesterol efflux from cells and therefore may be molecular targets for the treatment of cardiovascular disease. However, the effects of LXR ligands on cholesterol turnover in cells has not been examined comprehensively. In this study, cellular cholesterol handling (e.g., synthesis, catabolism, influx, and efflux) was examined using a stable isotope labeling study and a two-compartment modeling scheme. In HepG2 cells, the incorporation of 13 C into cholesterol from [1-13 C]acetate was analyzed by mass isotopomer distribution analysis in conjunction with nonsteady state, multicompartment kinetic analysis to calculate the cholesterol fluxes. Incubation with synthetic, nonsteroidal LXR agonists (GW3965, T0901317, and SB742881) increased cholesterol synthesis (z10-fold), decreased cellular cholesterol influx (71-82%), and increased cellular cholesterol efflux (1.7-to 1.9-fold) by 96 h. As a consequence of these altered cholesterol fluxes, cellular cholesterol decreased (36-39%) by 96 h. The increased cellular cholesterol turnover was associated with increased expression of the LXR-activated genes ABCA1, ABCG1, FAS, and sterol-regulatory element binding protein 1c.In summary, the mathematical model presented allows time-dependent calculations of cellular cholesterol fluxes. These data demonstrate that all of the cellular cholesterol fluxes were altered by LXR activation and that the increase in cholesterol synthesis did not compensate for the increased cellular cholesterol efflux, resulting in a net cellular cholesterol loss.-Aravindhan, K., C. L. Webb, M. Jaye, A. Ghosh, R. N. Willette, N. J. Di Nardo, and B .M. Jucker. The liver X receptor (LXR) is an oxysterol-sensing nuclear receptor responsible for transcriptional regulation of a number of genes involved in reverse cholesterol transport and therefore may be a molecular target for the treatment of cardiovascular disease (1-3). LXR is a member of the nuclear receptor family that regulates cellular cholesterol efflux (4) and whole body cholesterol excretion (5) and is endogenously activated by various oxysterols (6), including 24(S),25-epoxycholesterol and 22(R)-hydroxycholesterol, an intermediate in steroid hormone production (7, 8). Additionally, synthetic LXR agonists that potently upregulate macrophage ABCA1 expression have been shown to be antiatherogenic in genetic mouse models of atherosclerosis (9, 10).Cellular cholesterol metabolism and handling has been examined in terms of its synthesis by measuring the fraction of newly synthesized cholesterol (11,12), in terms of catabolism by measuring levels of bile acids through biochemical assays or HPLC (13,14), in terms of influx by measuring the radioactivity of absorbed [ 3 H]cholesteryl oleate (15, 16), and in terms of efflux by measuring specific radioactivity in labeled cells (17)(18)(19)(20). However, prior methods address only the unidirectional flow of cholesterol and are not ...

The liver X receptor modulator 22(S)-hydroxycholesterol exerts cell-type specific effects on lipid and glucose metabolism

“…In the present study we observed that T0901317-treatment induced ABCA1 in myotubes, SGBS, HepG2 and CaCo-2 cells, as well as ABCG1 in SGBS and CaCo-2 cells, in accordance with previous reports [7,[40][41][42]. Due to the beneficial effects of LXR activation on cholesterol homeostasis, LXR antagonists could be suspected to exert a negative effect on whole body cholesterol homeostasis.…”

“…Treatment with T0901317 increased apoA1-dependent cholesterol efflux from human myotubes, as well as SGBS and CaCo-2 cells. We did not observe LXR-mediated elevation of apoA1-dependent cholesterol efflux from HepG2 cells, contrary to a previous report [40]. LXR activation has previously been shown to enhance apoA1-dependent cholesterol efflux from murine myotubes [22], HepG2 cells [40], murine 3T3-L1 adipocytes [42] and basolateral cholesterol efflux from CaCo-2 cells [41], but has not, to our knowledge, been studied in human adipocytes or myotubes.…”

Lysosomal Acid Lipase Deficiency Impairs Regulation of ABCA1 Gene and Formation of High Density Lipoproteins in Cholesteryl Ester Storage Disease

“…The absence of low plasma HDL-C in LAL-deficient mice is consistent with these observations, because mouse hepatic ABCA1 is reported to be unresponsive to LXR stimulation (45,46) and would not be expected to show reduced expression in the face of reduced flux of cholesterol out of lysosomes and oxysterol generation with LAL deficiency. Human hepatocyte ABCA1, conversely, is responsive to LXR activation in both primary human hepatocytes (47,48) and HepG2 cells (49,50). Human hepatocyte ABCA1 would therefore be expected to show reduced expression in the face of reduced LAL (and NPC1) activity and the reduced production of oxysterol agonist of LXR, which we have demonstrated here in fibroblasts.…”

Liver X receptor antagonist reduces lipid formation and increases glucose metabolism in myotubes from lean, obese and type 2 diabetic individuals

“…Aravindhan et al [44] recently showed in HepG2 cells that cellular cholesterol fluxes were altered by LXR activation and that the increase in cholesterol synthesis did not compensate for the increased cellular cholesterol efflux, resulting in a net cellular cholesterol loss. We have previously demonstrated that LXR activation increased levels of cholesterol esters from labelled palmitic acid in human myotubes [33].…”