LRP1 controls intracellular cholesterol storage and fatty acid synthesis through modulation of Wnt signaling - PubMed (original) (raw)

. 2009 Jan 2;284(1):381-388.

doi: 10.1074/jbc.M806538200. Epub 2008 Nov 6.

Váronique Bruban 1, Li Zhou 1, Wanfeng Gong 1, Zeina El Asmar 1, Petra May 1, Kai Zurhove 1, Philipp Haffner 1, Claude Philippe 1, Estelle Woldt 1, Rachel L Matz 1, Cáline Gracia 1, Daniel Metzger 1, Johan Auwerx 1, Joachim Herz 2, Philippe Boucher 3

Affiliations

LRP1 controls intracellular cholesterol storage and fatty acid synthesis through modulation of Wnt signaling

Járome Terrand et al. J Biol Chem. 2009.

Abstract

The low-density lipoprotein receptor-related protein LRP1 is a cell surface receptor with functions in diverse physiological pathways, including lipid metabolism. Here we show that LRP1-deficient fibroblasts accumulate high levels of intracellular cholesterol and cholesteryl-ester when stimulated for adipocyte differentiation. We demonstrate that LRP1 stimulates a canonical Wnt5a signaling pathway that prevents cholesterol accumulation. Moreover, we show that LRP1 is required for lipolysis and stimulates fatty acid synthesis independently of the noradrenergic pathway, through inhibition of GSK3beta and its previously unknown target acetyl-CoA carboxylase (ACC). As a result of ACC inhibition, mature LRP1-deficient adipocytes of adult mice are hypotrophic, and lower uptake of fatty acids into adipose tissue leads to their redistribution to the liver. These results establish LRP1 as a novel integrator of adipogenic differentiation and fat storage signals.

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Figures

FIGURE 1.

FIGURE 1.

LRP1 governs adipocyte differentiation. Cells were treated with the adipogenic mixture containing the PPARγ agonist rosiglitazone for 10 days to stimulate differentiation. A, quantitative RT-PCR analysis of LRP1 in 3T3 preadipocytes, B, plates of LRP1(+/+), LRP1(-/-) MEFs, and 3T3-L1 preadipocytes. The extent of cellular lipid accumulation was determined by Oil Red O staining. C, micrographs of LRP1(+/+) and LRP1(-/-) MEFs. c and d show two different morphologies of lipid droplets that accumulate in LRP1(-/-) MEFs. D, triglyceride (at day 10) and (E) cholesterol and cholesteryl-ester quantifications during differentiation of LRP1(+/+) and LRP1(-/-) MEFs. F, quantitative RT-PCR analysis of HMG CoA reductase in MEFs during differentiation. G, Western blot analysis of LDL receptor in MEFs at day 10. T0, day 0; T10, day 10 of treatment. Scale bar, 50 μm. Results are means ± S.D. *, p < 0.05.

FIGURE 2.

FIGURE 2.

LRP1 controls fat accumulation through a Wnt5a and β-catenin-dependent signaling pathway. MEFs were stimulated with the adipogenic mixture containing the PPARγ agonist rosiglitazone (A), Oil Red O staining of LRP1(+/+) and LRP1(-/-) in the absence (-CM) or presence of conditioned medium from LRP1(-/-) MEFs ((-/-) CM) and LRP1(+/+)(+/+ CM), respectively. B, quantification (top) and immunoblotting (bottom) for nuclear β-catenin accumulation over the course of the differentiation regimen. C, Oil Red O staining of LRP1(+/+) and LRP1(-/-) MEFs differentiated in the absence (-LiCl) or presence (+LiCl) of lithium chloride. D, Wnt5a mRNA and protein levels over the course of the differentiation regimen. E, MEFs were stimulated for differentiation in the absence (-CM) or presence of conditioned medium from L-M(TK-) cells overexpressing (Wnt5a + CM) or not (Wnt5a- CM) Wnt5a.F, lipid accumulation in differentiated LRP1(-/-) MEFs transfected with Wnt5a (-/-, Wnt5a), LRP1(+/+) MEFs (+/+) and mock-transfected LRP1(-/-) MEFs. G, cholesterol and cholesteryl-ester quantifications during the course of differentiation of LRP1(-/-)-transfected with Wnt5a (-/-, Wnt5a) and mock-transfected LRP1(-/-) MEFs. Oil Red O staining of (H) plates and (I) representative micrographs of LRP1(+/+) and LRP1(-/-) MEFs differentiated in the presence of recombinant murine Wnt5a (rWnt5a+) or control medium (rWnt5a-). J, top panel, intracellular cholesteryl-ester quantification and (bottom panel) Western blot analysis with anti-Wnt5a antibodies of supernatant from differentiated control cells (+/+) or LRP1(-/-) MEFs untreated (-/-) or treated with recombinant mouse Wnt5a (rWnt5a+) or control medium (_rWnt5a_-).K, immunoblot analysis of nuclear β-catenin during the course of differentiation of LRP1(-/-) cells transfected with Wnt5a (-/-, Wnt5a) and mock-transfected LRP1(-/-) MEFs. Scale bar, 50 μm. Results are means ± S.D. *, p < 0.05.

FIGURE 3.

FIGURE 3.

Impaired adipogenic program and inhibition of fatty acid synthesis in LRP1(-/-) fibroblasts. A, Western blot analysis of PPARγ in 3T3-L1 preadipocytes and MEFs LRP1(+/+) and LRP1(-/-). B, quantitative RT-PCR analysis of the indicated genes in MEFs LRP1(+/+) and LRP1(-/-) during the course of adipogenesis. C, Western blot analysis of the indicated genes in MEFs LRP1(+/+) and LRP1(-/-) during the course of adipogenesis. D, Western blot analysis of p-ACC, p-AMPK, AMPK, LRP1, and loading control in LRP1(+/+) and LRP1(-/-) MEFs. E, ACC activity in MEFs upon differentiation. F, Western blot analysis of p-ACC in LRP1(+/+) and LRP1(-/-) MEFs after 10 days of lithium chloride treatment.G, Western blot analysis of p-ACC in LRP1(+/+) and LRP1(-/-) MEFs after 10 days of treatment with GSK3α (Aloisine A) or GSK3β (inhibitor VIII) inhibitor. Results are means ± S.D. *,p < 0.05 day 0 versus day 10. §, p < 0.05 LRP1(+/+) versus LRP1(-/-).

FIGURE 4.

FIGURE 4.

Decreased lipolysis in LRP1(-/-) fibroblasts. A, lipase activity in MEFs upon differentiation. B, relative mRNA levels of HSL and LPL determined by quantitative RT-PCR analysis during adipocyte differentiation. C, LPL mRNA levels analyzed by RT-PCR during the course of differentiation of LRP1(-/-) transfected with Wnt5a (-/-, Wnt5a) and mock-transfected LRP1(-/-) MEFs. D, mobilization of stored lipids; MEFs were exposed to adipogenic differentiation mixture (Diff) (Day 10), followed by starvation in medium containing only 0.5% serum for 4 days (Day 14). Quantitative RT-PCR analysis of HSL and LPL at 0, 10, and 14 days and (E), plates of cells. Results are means ± S.D. **,p = 0.05 day 0 versus day 10. *, p < 0.05 day 0 versus day 10 or day 14. §, p < 0.05 LRP1(+/+) versus LRP1(-/-).

FIGURE 5.

FIGURE 5.

Adipocyte-selective LRP1 knock-out mice exhibit decreased epididymal fat and hepatosteatosis. A, aspect (arrows) and (B), quantification of the epididymal fat and body weight in 20-week-old tamoxifen-treated aP2-CreERT2; LRP1flox/flox;LDLr(-/-) mice (adLRP1(-/-)Tam).C, H&E staining of epididymal WAT and BAT sections from 12-week-old tamoxifen-treated aP2-CreERT2;LRP1flox/flox;LDLr(-/-) mice (adLRP1(-/-)Tam) and tamoxifen-treated controls (aP2CreERT2;LRP1flox/flox;LDLr(-/-)) mice (adLRP1+/+Tam) fed 5 weeks with a high fat diet. D, immunoblot analysis of p-AMPK, AMPK, p-ACC, and loading control in epididymal WAT and BAT, and of LRP1 in BAT and purified epididymal WAT adipocytes from adLRP1+/+Tam (lanes 1, 3) and adLRP1(-/-)Tam (lanes 2, 4) mice. E, H&E (left) and Oil Red O (middle and right) staining of liver sections from adLRP1(+/+)Tam and adLRP1(-/-)Tam mice fed 5 weeks with a high fat diet (HFD) or a regular chow diet (CD).F, Oil Red O staining of liver sections from aP2Cre; LRP1flox/flox;LDLr(+/+) mice. Panels show adLRP1+/+ (aP2Cre-; LRP1flox/flox; LDLr(+/+)) and adLRP1(-/-) (aP2Cre+; LRP1flox/flox;LDLr(+/+)) mice that had been fed a high fat diet (HFD) for 4 weeks. Results are means ± S.D. *,p < 0.05. Scale bar, 50 μm.

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