Journal: Genes & Diseases

Article Title: Tirzepatide, a dual GIP/GLP-1 receptor agonist, alleviates metabolic dysfunction-associated steatotic liver disease by reducing the expression of CD36 and OBP2A

doi: 10.1016/j.gendis.2025.101761

Figure Lengend Snippet: Tirzapetide reduces hepatic lipid accumulation by down-regulating the expression of CD36 and OBP2A. (A) Venn analysis of the differentially expressed genes in the livers of mice fed with NCD, HFFC, and HFFC treated with tirzapetide. (B) Clustering analysis was performed on overlapping differentially expressed genes with padjust values less than 0.05. (C) Quantitative PCR was performed to detect the mRNA expression of specific genes (CD36, Cxcl10, Cxcl9, OBP2A, Tgtp1, Ubiquitin D, Aldoc, Dusp1, Socs2) in the livers of mice fed with NCD, HFFC, and HFFC supplemented with tirzapetide. (D) Immunohistochemical staining was performed to detect the protein expression of CD36 and OBP2A in the livers of mice fed with NCD, HFD, HFFC, and HFD or HFFC supplemented with tirzapetide. (E) Western blot analysis of CD36 protein expression in the livers of mice fed with NCD, HFFC, and HFFC supplemented with tirzapetide. (F) siRNAs targeting CD36 or OBP2A (siCD36 or siOBP2A) were transfected into HepG2 cells for 48 h, followed by treatment with 0.2% BSA or 0.2 mM palmitic acid for the last 24 h. Subsequently, the cells were stained with HCS LipidTOX™ Deep Red neutral lipid stain. (G) HepG2 cells were treated for 48 h with 0.2% BSA, 0.2 mM palmitic acid, or palmitic acid combined with 42 nM tirzapetide, with or without transfection of CD36 or OBP2A overexpression plasmids, followed by staining with HCS LipidTOX™ Deep Red neutral lipid stain to assess intracellular lipid accumulation. The average lipid content per cell was quantified using ImageJ software. n = 5 for A–E, n = 3 for F–G. The data are presented as mean ± SD.

Article Snippet: The CD36 overexpression lentivirus, OBP2A overexpression plasmid, and siRNA targeting OBP2A were purchased from OBiO technology (Shanghai, China).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Ubiquitin Proteomics, Immunohistochemical staining, Staining, Western Blot, Transfection, Over Expression, Software

Journal: Genes & Diseases

Article Title: Tirzepatide, a dual GIP/GLP-1 receptor agonist, alleviates metabolic dysfunction-associated steatotic liver disease by reducing the expression of CD36 and OBP2A

doi: 10.1016/j.gendis.2025.101761

Figure Lengend Snippet: Tirzepatide decreased CD36 and OBP2A expression and increased ATGL expression in adipose tissue. (A) HE staining of white adipose tissue from mice fed with NCD, HFD, HFFC, and HFD or HFFC supplemented with tirzapetide. For each group, the areas of at least thirty adipocytes were measured and analyzed statistically. (B) Immunohistochemical staining of CD36, OBP2A, and ATGL in white adipose tissue. (C) Western blotting analysis of CD36 expression in white adipose tissue. (D) ATGL expression in brown adipose tissue was detected via western blotting. (E) CD36 protein expression in skeletal muscle tissue was detected via western blotting. n = 5. The data are presented as mean ± SD.

Article Snippet: The CD36 overexpression lentivirus, OBP2A overexpression plasmid, and siRNA targeting OBP2A were purchased from OBiO technology (Shanghai, China).

Techniques: Expressing, Staining, Immunohistochemical staining, Western Blot

Journal: Experimental cell research

Article Title: Fatty acid translocase promoted hepatitis B virus replication by upregulating the levels of hepatic cytosolic calcium.

doi: 10.1016/j.yexcr.2017.07.012

Figure Lengend Snippet: Fig. 1. Overexpression of CD36 promoted HBV replication. (A) Western blotting and quantitative analysis of CD36 protein in HepG2.2.15 and HepAD38 cells transfected with human CD36 overexpression lentivirus (CD36OE) or vector (n = 3) (*** p < 0.001). Actin was used as a loading control. (B) Core particles associated with HBV DNA were extracted from CD36OE and vector cells after transfection for 4 days and were identified by southern blot. M, marker; rcDNA: intracellular HBV relax circle; ssDNA: single-stranded DNA. (C) HBV DNA levels were also determined by real-time PCR (n = 4). The secretion levels of HBeAg and HBsAg in culture medium of CD36OE and vector cells in HepG2.2.15 and HepAD38 cells were collected and measured using ELISA. O.D. values were expressed as a percentage relative to the vector (n = 3) (*P < 0.05, **P < 0.01).

Article Snippet: To determine the effect of chemical reagents on the process of CD36 affecting HBV replication, CD36 overexpression cells were treated with calcium chelator BAPTA-AM (Selleck, Houston, TX, USA) at a concentration of 25 μM and Src family kinase inhibitor SU6656 (Selleck, Houston, TX, USA) at a concentration of 0.6 μM, and CD36 knockdown cells were treated with calcium mobilizing agent thapsigargin (SigmaAldrich, St. Louis, MO, USA) at a concentration of 5 nM and 2.5 nM in HepAD38 and HepG2.2.15, respectively.

Techniques: Over Expression, Western Blot, Transfection, Plasmid Preparation, Control, Southern Blot, Marker, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Experimental cell research

Article Title: Fatty acid translocase promoted hepatitis B virus replication by upregulating the levels of hepatic cytosolic calcium.

doi: 10.1016/j.yexcr.2017.07.012

Figure Lengend Snippet: Fig. 2. Gene silencing of CD36 inhibited HBV replication. (A) Western blotting and quantitative analysis of the CD36 protein in HepG2.2.15 and HepAD38 cells transfected with lentiviruses expressing shRNA-targeting CD36 (shCD36) or shRNA-scramble control (shCont) (n = 3) (*** p < 0.001). Actin was used as a loading control. (B) Core particles associated with HBV DNA were extracted from shCD36 and shCont cells after transfection for 4 days and were identified by southern blot. M, marker; rcDNA: intracellular HBV relax circle; ssDNA: single-stranded DNA. (C) HBV DNA levels were also determined by real-time PCR (n = 4). The secretion levels of HBeAg and HBsAg in culture medium of shCD36 and shCont in HepG2.2.15 and HepAD38 cells were collected and measured using ELISA. O.D. values were expressed as a percentage relative to the shCont (n = 5). (*P < 0.05, **P < 0.01 and*** p < 0.001). (D) The levels of HBs Ag and HBe Ag in serum of HBV-infected WT and CD36KO mice by chemiluminescence immunoassay (n = 3). HBV DNA levels in the serum and liver were detected by real-time PCR (n = 4) (*P < 0.05, **P < 0.01). HBV core protein expression in the liver of HBV-infected WT and CD36KO mice was visualized by immunohistochemistry using anti-HBcAg; arrows indicate HBcAg.

Article Snippet: To determine the effect of chemical reagents on the process of CD36 affecting HBV replication, CD36 overexpression cells were treated with calcium chelator BAPTA-AM (Selleck, Houston, TX, USA) at a concentration of 25 μM and Src family kinase inhibitor SU6656 (Selleck, Houston, TX, USA) at a concentration of 0.6 μM, and CD36 knockdown cells were treated with calcium mobilizing agent thapsigargin (SigmaAldrich, St. Louis, MO, USA) at a concentration of 5 nM and 2.5 nM in HepAD38 and HepG2.2.15, respectively.

Techniques: Western Blot, Transfection, Expressing, shRNA, Control, Southern Blot, Marker, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Infection, Chemiluminescence Immunoassay, Immunohistochemistry

Journal: Experimental cell research

Article Title: Fatty acid translocase promoted hepatitis B virus replication by upregulating the levels of hepatic cytosolic calcium.

doi: 10.1016/j.yexcr.2017.07.012

Figure Lengend Snippet: Fig. 3. Effects of CD36 expression on cytosolic calcium levels. (A) Differentially expressed genes from RNA sequencing analysis were involved in ion homeostasis in HepG2.2.15-CD36OE and HepG2.2.15-vector cells. (B) Overexpression of CD36 in HepG2.2.15 and HepAD38 cells increases cytosolic Ca2+. HepG2.2.15-CD36OE, HepAD38-CD36OE and vector cells were loaded with the fluorescent Ca2+ indicator Fluo-4/AM after culture for 48 h and measured using flow cytometry. Ca2+ concentrations are expressed as relative fluorescence (n = 3) (*p < 0.05, **P < 0.01). (C) Knockdown of CD36 in HepG2.2.15 and HepAD38 cells decreases cytosolic Ca2+. HepG2.2.15 and HepAD38 cells were transfected with CD36-shRNA-lentivirus (shCD36) and scramble control-lentivirus (shCont), treated with 4 μM Fluo-4/AM after 48 h posttransfection, and measured using flow cytometry. Ca2+

Article Snippet: To determine the effect of chemical reagents on the process of CD36 affecting HBV replication, CD36 overexpression cells were treated with calcium chelator BAPTA-AM (Selleck, Houston, TX, USA) at a concentration of 25 μM and Src family kinase inhibitor SU6656 (Selleck, Houston, TX, USA) at a concentration of 0.6 μM, and CD36 knockdown cells were treated with calcium mobilizing agent thapsigargin (SigmaAldrich, St. Louis, MO, USA) at a concentration of 5 nM and 2.5 nM in HepAD38 and HepG2.2.15, respectively.

Techniques: Expressing, RNA Sequencing, Plasmid Preparation, Over Expression, Cytometry, Knockdown, Transfection, shRNA, Control

Journal: Experimental cell research

Article Title: Fatty acid translocase promoted hepatitis B virus replication by upregulating the levels of hepatic cytosolic calcium.

doi: 10.1016/j.yexcr.2017.07.012

Figure Lengend Snippet: Fig. 4. CD36 regulation of HBV replication is associated with cytosolic calcium signals. (A) Calcium chelator (BAPTA-AM) reduces HBV replication in CD36OE of HepG2.2.15 and HepAD38 cells. BAPTA-AM or vehicle controls (DMSO) were added to the CD36OE of HepG2.2.15 and HepAD38 cells for 96 h, and HBV DNA were extracted and detected by Real-time PCR (n = 4). Cytosolic Ca2+ was measured after treatment for 48 h using flow cytometry (n = 3). (B) Calcium activator (thapsigargin) rescued HBV replication in CD36 knockdown HepG2.2.15 and HepAD38 cells. Thapsigargin (TG) or vehicle controls (DMSO) were added to the CD36 knockdown HepG2.2.15 and HepAD38 cells for 96 h, and HBV DNA were extracted and detected by real-time PCR (n = 4). Cytosolic Ca2+ was measured after being treated for 48 h using flow cytometry (n = 3). (**P < 0.01 and*** p < 0.001).

Article Snippet: To determine the effect of chemical reagents on the process of CD36 affecting HBV replication, CD36 overexpression cells were treated with calcium chelator BAPTA-AM (Selleck, Houston, TX, USA) at a concentration of 25 μM and Src family kinase inhibitor SU6656 (Selleck, Houston, TX, USA) at a concentration of 0.6 μM, and CD36 knockdown cells were treated with calcium mobilizing agent thapsigargin (SigmaAldrich, St. Louis, MO, USA) at a concentration of 5 nM and 2.5 nM in HepAD38 and HepG2.2.15, respectively.

Techniques: Real-time Polymerase Chain Reaction, Cytometry, Knockdown

Journal: Experimental cell research

Article Title: Fatty acid translocase promoted hepatitis B virus replication by upregulating the levels of hepatic cytosolic calcium.

doi: 10.1016/j.yexcr.2017.07.012

Figure Lengend Snippet: Fig. 5. CD36 overexpression activated Src kinase, and Src inhibitors override the higher HBV replication induced by CD36 overexpression. (A) CD36 overexpression activated Src kinase in HepG2.2.15 and HepAD38 cells. The cell protein was collected from CD36-overexpressing HepG2.2.15 and HepAD38 cells after culture for 72 h. Total Src and the phosphorylation levels were detected using western blotting (n = 3) (**P < 0.01 and*** p < 0.001). (B) IP3 levels measured by ELISA in CD36OE and vector control of HepG2.2.15 and HepAD38 cells (n = 3) (*P < 0.05). (C) Src kinase inhibitor (SU6656) decreases cytosolic Ca2+ in CD36OE of HepG2.2.15 and HepAD38 cells. SU6656 or vehicle controls (DMSO) were added to the CD36OE of HepG2.2.15 and HepAD38 cells for 24 h, Cytosolic Ca2+ was measured after treatment using flow cytometry (n = 3) (*** p < 0.001). (D) Src kinase inhibitor (SU6656) overrides the higher HBV replication induced by CD36 overexpression. SU6656 or vehicle controls (DMSO) were added to the CD36 overexpression of HepG2.2.15 and HepAD38 cells for 96 h. HBV DNA were extracted and detected by real-time PCR (n = 4). (**P < 0.01 and*** p < 0.001).

Article Snippet: To determine the effect of chemical reagents on the process of CD36 affecting HBV replication, CD36 overexpression cells were treated with calcium chelator BAPTA-AM (Selleck, Houston, TX, USA) at a concentration of 25 μM and Src family kinase inhibitor SU6656 (Selleck, Houston, TX, USA) at a concentration of 0.6 μM, and CD36 knockdown cells were treated with calcium mobilizing agent thapsigargin (SigmaAldrich, St. Louis, MO, USA) at a concentration of 5 nM and 2.5 nM in HepAD38 and HepG2.2.15, respectively.

Techniques: Over Expression, Phospho-proteomics, Western Blot, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Control, Cytometry, Real-time Polymerase Chain Reaction

Journal: Nature communications

Article Title: Endothelial cell CD36 regulates membrane ceramide formation, exosome fatty acid transfer and circulating fatty acid levels.

doi: 10.1038/s41467-023-39752-3

Figure Lengend Snippet: Fig. 10 | Schematic of the EC FA export pathway. A The polarized ECs control tissue FA uptake by transcytosis of circulating FAs and their export in small extracellular vesicles (sEVs) to parenchymal cells. FA binding to CD36 recruits Src to phosphorylate Cav-1 at tyrosine14, which disrupts the caveolae coat. Associated activation of nSMase2 generates the destabilizing ceramides in the caveolae. Both events initiate caveolae fission from the membrane. The caveolae internalize into small ceramide-rich intracellular vesicles (IVs) thatare then sorted into the lumen of multivesicular bodies (MVBs). The MVBs fuse with the basolateral membrane to release exosome-like small sEVs in the sub-endothelial space. The sEVs export FA and other cargo to parenchymal cells. B Possible alternate fate of endocytosed vesicles in parenchymal cells. The IVs generated in response to FA are likely to be heterogenous and a subset possibly less enriched in specific ceramides might during traffic or sorting associate with proteins such as FABPs that help target the IVs to cellular organelles instead of for secretion as sEVs. This latter fate might predominate in myocytes, cardiomyocytes, and adipocytes where most FAs are kept for local use or storage.

Article Snippet: For CD36 overexpression, mEmerald-CD36 (Addgene, #54030) and mCherry-Cav-1 (Addgene, #55008) were transfected with Lipofectamaine LTX (ThermoFisher Scientific).

Techniques: Control, Binding Assay, Activation Assay, Membrane, Generated