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Cited by 211Liver Disease Diagnosis and TreatmentCancer, Lipids, and MetabolismLipid metabolism and biosynthesis

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CD36 promotes de novo lipogenesis in hepatocytes through INSIG2-dependent SREBP1 processing

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Zeng, Han, Qin, Hong, Liao, Meng, Zheng, Enze, Luo, Xiaoqing, Xiao, Anhua, Li, Yiyu, Chen, Lin, Wei, Li, Zhao, Lei, Ruan, Xiong Z., Yang, Ping, Chen, Yaxi·2022·Molecular Metabolism
DOI 10.1016/j.molmet.2021.101428PMC PMC8810570
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Abstract

OBJECTIVE: Enhanced de novo lipogenesis (DNL) in hepatocytes is a major contributor to nonalcoholic fatty liver disease (NAFLD). Fatty acid translocase (FAT/CD36) is involved in the pathogenesis of NAFLD through facilitating free fatty acids uptake. Here, we explored the effects of CD36 on DNL and elucidated the underlying mechanisms. METHODS: We generated hepatocyte-specific CD36 knockout (CD36LKO) mice to study in vivo effects of CD36 on DNL under high-fat diet (HFD). Lipid deposition and DNL were analyzed in primary hepatocytes isolated from CD36LKO mice or HepG2 cells with CD36 overexpression. RNA sequence, co-immunoprecipitation, and proximity ligation assay were carried out to determine its role in regulating DNL. RESULTS: Hepatic CD36 expression was upregulated in NAFLD mice and patients, and CD36LKO mice exhibited attenuated HFD-induced hepatic steatosis and insulin resistance. We identified hepatocyte CD36 as a key regulator for DNL in the liver. Sterol regulatory element-binding protein 1 (SREBP1) and its downstream lipogenic enzymes such as FASN, ACCα, and ACLY were significantly downregulated in the liver of HFD-fed CD36LKO mice, whereas overexpression CD36 stimulated insulin-mediated DNL and lipid droplet formation in vitro. Mechanistically, CD36 was activated by insulin and formed a complex with insulin-induced gene-2 (INSIG2) that disrupts the interaction between SREBP cleavage-activating protein (SCAP) and INSIG2, thereby leading to the translocation of SREBP1 from ER to Golgi for processing. Furthermore, treatment with 25-hydroxycholesterol or betulin molecules shown to enhance SCAP-INSIG interaction, reversed the effects of CD36 on SREBP1 cleavage. CONCLUSIONS: Our findings identify a previously unsuspected role of CD36 in the regulation of hepatic lipogenic program through mediating SREBP1 processing by INSIG2, providing additional evidence for targeting CD36 in NAFLD.

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Figure 1 Hepatic CD36 is regulated by nutritional signals. A–B: C57BL/6J male mice were fed with either the normal chow diet (NCD) or high-fat diet (HFD) for 16 weeks. The hepatic expression of CD36 was analyzed by immunoblotting (A) or real-time quantitative PCR (Q-PCR) (B). n = 3, ∗P < 0.05 vs. NCD group. C: The hepatic CD36 mRNA levels in the nonalcoholic fatty liver disease group (NAFLD, n = 40) and normal control group (control, n = 21) from the GEO database (GSE151158). ∗P < 0.05 vs. control group. D–E: C57BL/6J male mice were overnight-fasted and then fed again 12 h. Livers were collected and the protein level of CD36 (D) and SREBP1 signaling (E) were analyzed by immunoblotting. n = 3, ∗P < 0.05 vs. fasted group. F: C57BL/6J male mice were fasted overnight and injected with PBS or insulin (1U/kg body weight) for 4 h. Livers were collected and the protein level of CD36 was analyzed by immunoblotting. n = 3, ∗P < 0.05 vs. PBS group. G: HepG2 cells were treated with insulin (100 nM) for indicated time points. The protein levels of CD36 were analyzed immunoblotting. n = 3.
Figure 1 Hepatic CD36 is regulated by nutritional signals. A–B: C57BL/6J male mice were fed with either the normal chow diet (NCD) or high-fat diet (HFD) for 16 weeks. The hepatic expression of CD36 was analyzed by immunoblotting (A) or real-time quantitative PCR (Q-PCR) (B). n = 3, ∗P < 0.05 vs. NCD group. C: The hepatic CD36 mRNA levels in the nonalcoholic fatty liver disease group (NAFLD, n = 40) and normal control group (control, n = 21) from the GEO database (GSE151158). ∗P < 0.05 vs. control group. D–E: C57BL/6J male mice were overnight-fasted and then fed again 12 h. Livers were collected and the protein level of CD36 (D) and SREBP1 signaling (E) were analyzed by immunoblotting. n = 3, ∗P < 0.05 vs. fasted group. F: C57BL/6J male mice were fasted overnight and injected with PBS or insulin (1U/kg body weight) for 4 h. Livers were collected and the protein level of CD36 was analyzed by immunoblotting. n = 3, ∗P < 0.05 vs. PBS group. G: HepG2 cells were treated with insulin (100 nM) for indicated time points. The protein levels of CD36 were analyzed immunoblotting. n = 3.CC BY-NC-ND · PubMed Central
Figure 2 CD36LKO mice were protected from HFD-induced hepatic steatosis. The hepatocyte-specific CD36 knockout (LKO) mice and control mice (fl/fl) were fed a HFD for 16 weeks (n = 12). A: Schematic experimental design to determine the effects of hepatocyte-specific CD36 on NAFLD. B-C: Body weight and daily food intake during the 16-week HFD condition were measured. D: Liver weight of 16-week-HFD-fed was measured. E–I: Overnight-fasted serum glucose levels (E), total triglycerides (TG) levels (F), total cholesterol (TC) levels (G), as well as ALT (H) and AST (I) levels were analyzed (n = 6). J–K: Glucose tolerance tests (GTTs, J) and insulin tolerance tests (ITTs, K) in HFD-fed mice. Quantification of the area under the curve (AUC) was shown in the right (n = 6). L–N: Representative images of hematoxylin & eosin (H&E)-stained livers (L), Sirius red-stained livers (M), and H&E-stained epididymal white adipose tissues (eWAT, N). Scale bar, 100 μm. O-P: Liver contents of TG and TC were determined (n = 6). Q: Representative images of Oil red O-stained livers. Quantification of Oil red O-stained area was shown in the right (n = 4; Scale bar, 100 μm). ∗P < 0.05 vs. fl/fl mice.
Figure 2 CD36LKO mice were protected from HFD-induced hepatic steatosis. The hepatocyte-specific CD36 knockout (LKO) mice and control mice (fl/fl) were fed a HFD for 16 weeks (n = 12). A: Schematic experimental design to determine the effects of hepatocyte-specific CD36 on NAFLD. B-C: Body weight and daily food intake during the 16-week HFD condition were measured. D: Liver weight of 16-week-HFD-fed was measured. E–I: Overnight-fasted serum glucose levels (E), total triglycerides (TG) levels (F), total cholesterol (TC) levels (G), as well as ALT (H) and AST (I) levels were analyzed (n = 6). J–K: Glucose tolerance tests (GTTs, J) and insulin tolerance tests (ITTs, K) in HFD-fed mice. Quantification of the area under the curve (AUC) was shown in the right (n = 6). L–N: Representative images of hematoxylin & eosin (H&E)-stained livers (L), Sirius red-stained livers (M), and H&E-stained epididymal white adipose tissues (eWAT, N). Scale bar, 100 μm. O-P: Liver contents of TG and TC were determined (n = 6). Q: Representative images of Oil red O-stained livers. Quantification of Oil red O-stained area was shown in the right (n = 4; Scale bar, 100 μm). ∗P < 0.05 vs. fl/fl mice.CC BY-NC-ND · PubMed Central
Figure 3 Decreased hepatic lipogenesis in LKO mice. RNA sequence analysis of the livers from fl/fl and LKO mice under 16-week-HFD (n = 3). A: Top 20 biological process gene ontology (GO) terms enriched by differentially expressed genes (DEGs) in fl/fl and LKO mice. The DEGs were selected with a criterion at adjusted P-value < 0.05. B: The protein–protein interactions (PPI) network analysis of genes including in top 20 BP GO terms. The top 15 hub genes were selected and visualized with Cytoscape software. The size and color of map nodes were determined by degree score. C: Heatmap representation of genes involved in fatty acid metabolism. The gradual color ranged from pink to blue represented the mRNA expression changing from up-regulation to down-regulation. D: Volcano plot comparison of genes in fl/fl versus LKO mice, showing the distribution of significance [-log10 (P adjust value)] vs. fold change (FC) [log2 (FC)] for all genes. The red points represented up-regulated genes (log2 (FC) > 1 and P adjust value < 0.05, 222 genes) and green points represented down-regulated genes (log2 (FC) < −1 and P adjust value < 0.05, 222genes), while gray points indicated genes without significant differential expression (|log2 (FC) | < 1 or a P adjust value > 0.05). E–G: The liver mRNA levels of lipogenic genes (E), inflammatory genes (F), and fatty acid β-oxidation genes (G) determined by Q-PCR (n = 3). H: Hepatic expression of the SREBP1 and downstream lipogenic enzymes determined by immunoblotting. Quantitation of protein levels was shown in the right (n = 3). ∗P < 0.05 vs. fl/fl mice.
Figure 3 Decreased hepatic lipogenesis in LKO mice. RNA sequence analysis of the livers from fl/fl and LKO mice under 16-week-HFD (n = 3). A: Top 20 biological process gene ontology (GO) terms enriched by differentially expressed genes (DEGs) in fl/fl and LKO mice. The DEGs were selected with a criterion at adjusted P-value < 0.05. B: The protein–protein interactions (PPI) network analysis of genes including in top 20 BP GO terms. The top 15 hub genes were selected and visualized with Cytoscape software. The size and color of map nodes were determined by degree score. C: Heatmap representation of genes involved in fatty acid metabolism. The gradual color ranged from pink to blue represented the mRNA expression changing from up-regulation to down-regulation. D: Volcano plot comparison of genes in fl/fl versus LKO mice, showing the distribution of significance [-log10 (P adjust value)] vs. fold change (FC) [log2 (FC)] for all genes. The red points represented up-regulated genes (log2 (FC) > 1 and P adjust value < 0.05, 222 genes) and green points represented down-regulated genes (log2 (FC) < −1 and P adjust value < 0.05, 222genes), while gray points indicated genes without significant differential expression (|log2 (FC) | < 1 or a P adjust value > 0.05). E–G: The liver mRNA levels of lipogenic genes (E), inflammatory genes (F), and fatty acid β-oxidation genes (G) determined by Q-PCR (n = 3). H: Hepatic expression of the SREBP1 and downstream lipogenic enzymes determined by immunoblotting. Quantitation of protein levels was shown in the right (n = 3). ∗P < 0.05 vs. fl/fl mice.CC BY-NC-ND · PubMed Central
Figure 4 CD36 is involved in insulin-stimulated DNL in vitro. A: Experimental design for insulin-stimulate DNL in vitro. B: HepG2 cells were treated with insulin (100 nM) for indicated time points. The protein levels of SREBP1 and downstream lipogenic enzymes were analyzed by immunoblotting. C–D: BODIPY staining for lipid droplets in HepG2 cells (C) or primary mouse hepatocytes (D) with or without insulin (100 nM) treatment for 12 h. Quantitative of lipid droplets was shown in the right. ∗P < 0.05 vs. control group. E: BODIPY staining for lipid droplets in CD36 overexpression (CD36OE) or NC cells with insulin (100 nM) treatment for 12 h. Quantitative of lipid droplets was shown in the right. ∗P < 0.05 vs. NC or fl/fl group. F: BODIPY staining for lipid droplets in primary hepatocytes isolated from fl/fl or LKO mice with insulin (100 nM) treatment for 12 h. Quantitative of lipid droplets was shown in the right. ∗P < 0.05 vs. fl/fl group. G–H: Protein levels of SREBP1 and downstream lipogenic enzymes in CD36OE cells (G) or primary hepatocytes lacking CD36 (H). Quantitation of protein levels was shown in the bottom. n = 3, ∗P < 0.05 vs. NC, or fl/fl group.
Figure 4 CD36 is involved in insulin-stimulated DNL in vitro. A: Experimental design for insulin-stimulate DNL in vitro. B: HepG2 cells were treated with insulin (100 nM) for indicated time points. The protein levels of SREBP1 and downstream lipogenic enzymes were analyzed by immunoblotting. C–D: BODIPY staining for lipid droplets in HepG2 cells (C) or primary mouse hepatocytes (D) with or without insulin (100 nM) treatment for 12 h. Quantitative of lipid droplets was shown in the right. ∗P < 0.05 vs. control group. E: BODIPY staining for lipid droplets in CD36 overexpression (CD36OE) or NC cells with insulin (100 nM) treatment for 12 h. Quantitative of lipid droplets was shown in the right. ∗P < 0.05 vs. NC or fl/fl group. F: BODIPY staining for lipid droplets in primary hepatocytes isolated from fl/fl or LKO mice with insulin (100 nM) treatment for 12 h. Quantitative of lipid droplets was shown in the right. ∗P < 0.05 vs. fl/fl group. G–H: Protein levels of SREBP1 and downstream lipogenic enzymes in CD36OE cells (G) or primary hepatocytes lacking CD36 (H). Quantitation of protein levels was shown in the bottom. n = 3, ∗P < 0.05 vs. NC, or fl/fl group.CC BY-NC-ND · PubMed Central
Figure 5 CD36 regulates ER-to-Golgi transport of SREBP1. A: The protein level of SREBP1 in Golgi and ER of organelle fractions from fl/fl and LKO mice was analyzed by immunoblotting. B: Nuclear location of SREBP1 (red-TRITC) was determined by immunofluorescence staining and confocal analysis in primary hepatocytes from fl/fl and LKO mice. Scale bars, 25 μm. C: Localization of SREBP1 (green-FITC) in the Golgi (red-TRITC) was determined by immunofluorescence staining and confocal analysis in primary hepatocytes from fl/fl and LKO mice. Scale bars, 25 μm. D: The protein levels of SREBP1 of cytoplasmic and nuclear fractions from NC and CD36OE cells were analyzed by immunoblotting. E: Nuclear location of SREBP1 (red-TRITC) was determined by immunofluorescence staining and confocal analysis in NC and CD36OE cells. Scale bars, 25 μm. F: The localization of SREBP1 (green-FITC) in the Golgi (red-TRITC) was determined by immunofluorescence staining and confocal analysis in NC and CD36OE cells. Scale bars, 25 μm. G–H: HepG2 cells were co-transfected with stably expressing HA-tagged full-length SREBP1 (FL-SREBP1) and FLAG-tagged-CD36. The precursor and active nuclear form of SREBP1 were visualized using HA antibody (G) and the SREBP1 downstream targets, including ACCα, FASN, SCD1, and ACLY, were analyzed by immunoblotting (H). Quantitation of protein levels was shown in the bottom. n = 4, ∗P < 0.05 vs. SREBP1OE group without CD36OE.
Figure 5 CD36 regulates ER-to-Golgi transport of SREBP1. A: The protein level of SREBP1 in Golgi and ER of organelle fractions from fl/fl and LKO mice was analyzed by immunoblotting. B: Nuclear location of SREBP1 (red-TRITC) was determined by immunofluorescence staining and confocal analysis in primary hepatocytes from fl/fl and LKO mice. Scale bars, 25 μm. C: Localization of SREBP1 (green-FITC) in the Golgi (red-TRITC) was determined by immunofluorescence staining and confocal analysis in primary hepatocytes from fl/fl and LKO mice. Scale bars, 25 μm. D: The protein levels of SREBP1 of cytoplasmic and nuclear fractions from NC and CD36OE cells were analyzed by immunoblotting. E: Nuclear location of SREBP1 (red-TRITC) was determined by immunofluorescence staining and confocal analysis in NC and CD36OE cells. Scale bars, 25 μm. F: The localization of SREBP1 (green-FITC) in the Golgi (red-TRITC) was determined by immunofluorescence staining and confocal analysis in NC and CD36OE cells. Scale bars, 25 μm. G–H: HepG2 cells were co-transfected with stably expressing HA-tagged full-length SREBP1 (FL-SREBP1) and FLAG-tagged-CD36. The precursor and active nuclear form of SREBP1 were visualized using HA antibody (G) and the SREBP1 downstream targets, including ACCα, FASN, SCD1, and ACLY, were analyzed by immunoblotting (H). Quantitation of protein levels was shown in the bottom. n = 4, ∗P < 0.05 vs. SREBP1OE group without CD36OE.CC BY-NC-ND · PubMed Central
Figure 6 CD36 interacts with INSIG2 but not INSIG1. A–B: The hepatic protein (A) and mRNA (B) levels of INSIG1 and INSIG2 in fl/fl and LKO mice under 16-week-HFD. Quantitation of protein levels was shown in the right. n = 4, ∗P < 0.05 vs. fl/fl mice. C: HepG2 cells expressing FLAG-CD36 were subjected to immunoprecipitation with FLAG (top), INSIG1 (middle), or INSIG2 (bottom) antibodies, and the levels of the co-immunoprecipitated protein were immunoblotted with INSIG1, INSIG2, or CD36 antibodies. D: The co-localization of CD36 (red-TRITC) and INSIG2 (green-FITC) in NC and CD36OE cells was determined by confocal microscopy. Scale bars, 25 μm. E: The physical interactions of CD36 with INSIG1 (top) or INSIG2 (bottom) in CD36OE cells were detected by proximity ligation assay (PLA). Representative PLA images where the PLA signal (red) represented close proximity (<40 nm) between two proteins. Scale bars, 25 μm. F: The physical interactions of INSIG2 with SCAP in NC and CD36OE cells were detected by PLA. Scale bars, 25 μm. G–H: The localization of SCAP (green-FITC) in the Golgi (red-TRITC) was determined by immunofluorescence staining and confocal analysis in NC and CD36OE cells (G) or fl/fl and LKO mouse primary hepatocytes (H). Scale bars, 25 μm.
Figure 6 CD36 interacts with INSIG2 but not INSIG1. A–B: The hepatic protein (A) and mRNA (B) levels of INSIG1 and INSIG2 in fl/fl and LKO mice under 16-week-HFD. Quantitation of protein levels was shown in the right. n = 4, ∗P < 0.05 vs. fl/fl mice. C: HepG2 cells expressing FLAG-CD36 were subjected to immunoprecipitation with FLAG (top), INSIG1 (middle), or INSIG2 (bottom) antibodies, and the levels of the co-immunoprecipitated protein were immunoblotted with INSIG1, INSIG2, or CD36 antibodies. D: The co-localization of CD36 (red-TRITC) and INSIG2 (green-FITC) in NC and CD36OE cells was determined by confocal microscopy. Scale bars, 25 μm. E: The physical interactions of CD36 with INSIG1 (top) or INSIG2 (bottom) in CD36OE cells were detected by proximity ligation assay (PLA). Representative PLA images where the PLA signal (red) represented close proximity (<40 nm) between two proteins. Scale bars, 25 μm. F: The physical interactions of INSIG2 with SCAP in NC and CD36OE cells were detected by PLA. Scale bars, 25 μm. G–H: The localization of SCAP (green-FITC) in the Golgi (red-TRITC) was determined by immunofluorescence staining and confocal analysis in NC and CD36OE cells (G) or fl/fl and LKO mouse primary hepatocytes (H). Scale bars, 25 μm.CC BY-NC-ND · PubMed Central
Figure 7 INSIG2 is a key pathway by which CD36 regulates SREBP1 processing. A–C: After a 24 h period of transfection with siCon, siCd36, and/or siInsig2, HepG2 cells were cultured in serum-free medium for 12 h and then treated with 100 nM insulin for 12 h. The expression of SREBP1 and downstream targets were analyzed by Q-PCR (A). n = 4–6. Nuclear localization of SREBP1 was determined by immunofluorescence staining and confocal analysis (B). Scale bars, 25 μm. Quantitative of nuclear SREBP1 density was shown in the right. The expression of SREBP1 and downstream targets were analyzed by immunoblotting (C). Quantification of protein levels was shown in the bottom. n = 3, ∗P < 0.05 vs. siCon, #P < 0.05 vs. siCd36. D–E: CD36OE or NC cells were cultured in serum-free medium overnight and then treated with DMSO, 25-hydroxycholesterol (25-HC, 5 μM), or betulin (10 μM) in the presence of insulin (100 nM) for 24 h. Nuclear localization of SREBP1 was determined by immunofluorescence staining and confocal analysis (D). Quantitative of nuclear SREBP1 density was shown in the bottom. Scale bars, 25 μm. The expression of SREBP1 and downstream targets were analyzed by immunoblotting (E). n = 3. Quantification of protein levels was shown in the right. ∗P < 0.05 vs. NC, #P < 0.05 vs. NC + DMSO, †P < 0.05 vs. CD36OE + DMSO.
Figure 7 INSIG2 is a key pathway by which CD36 regulates SREBP1 processing. A–C: After a 24 h period of transfection with siCon, siCd36, and/or siInsig2, HepG2 cells were cultured in serum-free medium for 12 h and then treated with 100 nM insulin for 12 h. The expression of SREBP1 and downstream targets were analyzed by Q-PCR (A). n = 4–6. Nuclear localization of SREBP1 was determined by immunofluorescence staining and confocal analysis (B). Scale bars, 25 μm. Quantitative of nuclear SREBP1 density was shown in the right. The expression of SREBP1 and downstream targets were analyzed by immunoblotting (C). Quantification of protein levels was shown in the bottom. n = 3, ∗P < 0.05 vs. siCon, #P < 0.05 vs. siCd36. D–E: CD36OE or NC cells were cultured in serum-free medium overnight and then treated with DMSO, 25-hydroxycholesterol (25-HC, 5 μM), or betulin (10 μM) in the presence of insulin (100 nM) for 24 h. Nuclear localization of SREBP1 was determined by immunofluorescence staining and confocal analysis (D). Quantitative of nuclear SREBP1 density was shown in the bottom. Scale bars, 25 μm. The expression of SREBP1 and downstream targets were analyzed by immunoblotting (E). n = 3. Quantification of protein levels was shown in the right. ∗P < 0.05 vs. NC, #P < 0.05 vs. NC + DMSO, †P < 0.05 vs. CD36OE + DMSO.CC BY-NC-ND · PubMed Central
Figure S1 Phenotype of WT mice fed with HFD. C57BL/6 mice were fed with NCD or HFD for 16 weeks (n = 3–5). A-B: Body weight (A) and liver weight (B) were measured. C-H: Overnight-fasted serum ALT levels (C), AST levels (D), TC levels (E), TG levels (F), glucose levels (G), and insulin levels (H) were measured. I-J: Representative images of H&E-stained (I) and Sirius red stained (J) livers. Scale bar, 100 μm. K: Representative images of Oil red O-stained livers. Quantification of Oil red O-stained area was shown in the right. Scale bar, 100 μm ∗P < 0.05 vs. NCD group.
Figure S1 Phenotype of WT mice fed with HFD. C57BL/6 mice were fed with NCD or HFD for 16 weeks (n = 3–5). A-B: Body weight (A) and liver weight (B) were measured. C-H: Overnight-fasted serum ALT levels (C), AST levels (D), TC levels (E), TG levels (F), glucose levels (G), and insulin levels (H) were measured. I-J: Representative images of H&E-stained (I) and Sirius red stained (J) livers. Scale bar, 100 μm. K: Representative images of Oil red O-stained livers. Quantification of Oil red O-stained area was shown in the right. Scale bar, 100 μm ∗P < 0.05 vs. NCD group.CC BY-NC-ND · PubMed Central
Figure S2 Phenotype of LKO mice fed with NCD. A: Schematic diagram showing the targeting strategy for generating LKO mice. B-C: The expression of hepatic CD36 was analyzed by Q-PCR (B) and immunoblotting (C). D: The expression of CD36 in heart, kidney, and adipose tissues was analyzed by immunoblotting. E: Quantitation of protein levels in (B) and (C). n=3, ∗P < 0.05 vs. fl/fl mice. F-P: The fl/fl and LKO mice were fed with NCD for 16 weeks (n=6-12). Body weight (F), daily intake (G), and liver weight (H) were measured. Overnight-fasted serum glucose levels (I), TG levels (J), TC levels (K), as well as ALT (L) and AST levels (M) were measured. Representative images of H&E-stained livers (N). Scale bar, 100 μm. GTTs (O) and ITTs (P) in NCD-fed mice, respectively. Quantification of AUC was shown in the right.
Figure S2 Phenotype of LKO mice fed with NCD. A: Schematic diagram showing the targeting strategy for generating LKO mice. B-C: The expression of hepatic CD36 was analyzed by Q-PCR (B) and immunoblotting (C). D: The expression of CD36 in heart, kidney, and adipose tissues was analyzed by immunoblotting. E: Quantitation of protein levels in (B) and (C). n=3, ∗P < 0.05 vs. fl/fl mice. F-P: The fl/fl and LKO mice were fed with NCD for 16 weeks (n=6-12). Body weight (F), daily intake (G), and liver weight (H) were measured. Overnight-fasted serum glucose levels (I), TG levels (J), TC levels (K), as well as ALT (L) and AST levels (M) were measured. Representative images of H&E-stained livers (N). Scale bar, 100 μm. GTTs (O) and ITTs (P) in NCD-fed mice, respectively. Quantification of AUC was shown in the right.CC BY-NC-ND · PubMed Central
Figure S3 Effects of CD36 on liver SREBP2 activation. A: The mRNA levels of Srebp2 and downstream targets Hmgcr and Ldlr in HFD-fed fl/fl and LKO mice were analyzed by Q-PCR (B). n=8, ∗P < 0.05 vs. fl/fl mice. B: The hepatic expression of SREBP2 and downstream target HMGCR in HFD-fed fl/fl and LKO mice were analyzed by immunoblotting. Quantitation of protein levels was shown in the right. n=3, ∗P < 0.05 vs. fl/fl mice. C: The expression of SREBP2 and downstream target HMGCR in primary hepatocytes from fl/fl and LKO mice were analyzed by immunoblotting. Quantitation of protein levels was shown in the right. n=3, ∗P < 0.05 vs. fl/fl mice. E: The protein levels of SREBP2 of cytoplasmic and nuclear fractions from NC and CD36OE cells were analyzed by immunoblotting.
Figure S3 Effects of CD36 on liver SREBP2 activation. A: The mRNA levels of Srebp2 and downstream targets Hmgcr and Ldlr in HFD-fed fl/fl and LKO mice were analyzed by Q-PCR (B). n=8, ∗P < 0.05 vs. fl/fl mice. B: The hepatic expression of SREBP2 and downstream target HMGCR in HFD-fed fl/fl and LKO mice were analyzed by immunoblotting. Quantitation of protein levels was shown in the right. n=3, ∗P < 0.05 vs. fl/fl mice. C: The expression of SREBP2 and downstream target HMGCR in primary hepatocytes from fl/fl and LKO mice were analyzed by immunoblotting. Quantitation of protein levels was shown in the right. n=3, ∗P < 0.05 vs. fl/fl mice. E: The protein levels of SREBP2 of cytoplasmic and nuclear fractions from NC and CD36OE cells were analyzed by immunoblotting.CC BY-NC-ND · PubMed Central

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  1. Zhang, 2020, The CC genotype of insulin-induced gene 2 rs7566605 is a protective factor of hypercholesteremia susceptible to mild cognitive impairment, especially to the executive function of patients with type 2 diabetes mellitus, BioMed Research International, 2020

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Authors
Zeng, H., Qin, H., Liao, M., Zheng, E., Luo, X., Xiao, A., Li, Y., Chen, L., Wei, L., Zhao, L., Ruan, X. Z., Yang, P., & Chen, Y.
Affiliations
  • Second Affiliated Hospital of Chongqing Medical University (CN)
  • The Royal Free Hospital (GB)
Corresponding author
  • Yang, Ping
  • Chen, Yaxi
ORCID
Venue
Molecular Metabolism
Publisher
Elsevier BV
Published
2022-03
Volume
57
Pages
101428
ISSN
2212-8778 (ISSN-L)
DOI
10.1016/j.molmet.2021.101428
PMC
PMC8810570
OpenAlex
W4200530196
Semantic Scholar
9eab22cddc8a889e5bc861e52f4c9cca8b021238
Cited by
211
References
56
Citations by year
2026: 35 · 2025: 68 · 2024: 61 · 2023: 35 · 2022: 12
FWCI
19.45
Type
journal article
Language
en
Open access
Licence
Locations
  • Molecular Metabolism (publishedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • National Natural Science Foundation of China (20190120, 2018YFC1312700, 81873569, 32030054, cstc2020jcyj, D20028)
  • Chongqing Medical University (D20028)
  • Chongqing Research Program of Basic Research and Frontier Technology (cstc2020jcyj-msxmX0205)
  • Higher Education Discipline Innovation Project (D20028)
Topics
  • Health Sciences › Medicine › Hepatology › Liver Disease Diagnosis and Treatment
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Cancer, Lipids, and Metabolism
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biochemistry › Lipid metabolism and biosynthesis
Keywords
CD36, de novo lipogenesis, hepatocytes, non-alcoholic fatty liver disease, INSIG2, SREBP1, SCAP, insulin signaling, hepatic steatosis, insulin resistance, lipid droplet formation, 25-hydroxycholesterol, betulin, free fatty acid uptake, high-fat diet
MeSH terms
Animals, Humans, Membrane Proteins, CD36 Antigens, Hepatocytes, Intracellular Signaling Peptides and Proteins, Lipogenesis, Mice, Sterol Regulatory Element Binding Protein 1, Non-alcoholic Fatty Liver Disease

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Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

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