Systemic loss of CD36 aggravates NAFLD-related HCC through MEK1/2-ERK1/2 signaling pathway.

BACKGROUND & AIMS: CD36, a membrane protein widely present in various tissues, is crucial role in regulating energy metabolism. The rise of HCC as a notable outcome of NAFLD is becoming more apparent. Patients with hereditary CD36 deficiency are at increased risk of NAFLD. However, the impact of CD36 deficiency on NAFLD-HCC remains unclear. METHODS: Global CD36 knockout mice (CD36KO) and wild type mice (WT) were induced to establish NAFLD-HCC model by N-nitrosodiethylamine (DEN) plus high fat diet (HFD). Transcriptomics was employed to examine genes that were expressed differentially. RESULTS: Compared to WT mice, CD36KO mice showed more severe HFD-induced liver issues and increased tumor malignancy. The MEK1/2-ERK1/2 pathway activation was detected in the liver tissues of CD36KO mice using RNA sequencing and Western blot analysis. CONCLUSION: Systemic loss of CD36 leaded to the advancement of NAFLD to HCC by causing lipid disorders and metabolic inflammation, a process that involves the activation of MAPK signaling pathway. We found that CD36 contributes significantly to the maintenance of metabolic homeostasis in NAFLD-HCC.

Loss of Splicing Factor SRSF3 Impairs Lipophagy Through Ubiquitination and Degradation of Syntaxin17 in Hepatocytes.

Lipid accumulation in hepatocytes is the distinctive characteristic of nonalcoholic fatty liver disease. Serine/arginine-rich splicing factor 3 (SRSF3) is highly expressed in the liver and expression decreases in high-fat conditions. However, the role of SRSF3 in hepatic lipid metabolism needs to be clarified. Here, we showed that loss of SRSF3 was associated with lipid accumulation. We determined that SRSF3 regulated lipophagy, the process of selective degradation of lipid droplets by autophagy. Mechanistically, loss of SRSF3 impaired the fusion of the autophagosome and lysosome by promoting the proteasomal degradation of syntaxin 17 (STX17), a key autophagosomal SNARE protein. We found that ubiquitination of STX17 was increased and upregulation of seven in absentia homolog 1 was responsible for the increased posttranslational modification of STX17. Taken together, our data primarily demonstrate that loss of SRSF3 weakens the clearance of fatty acids by impairing lipophagy in the progression of nonalcoholic fatty liver disease, indicating a novel potential therapeutic target for fatty liver disease treatment.

Klotho inhibits renal ox-LDL deposition via IGF-1R/RAC1/OLR1 signaling to ameliorate podocyte injury in diabetic kidney disease.

OBJECTIVE: Diabetic kidney disease (DKD) is characterized by the abnormal deposition of oxidized low-density lipoprotein (ox-LDL), which contributes to podocyte damage. Klotho, an aging suppressor that plays a critical role in protecting podocytes in DKD, is mainly expressed in kidney tubular epithelium and secreted in the blood. However, it has not been established whether Klotho can alleviate podocyte injury by inhibiting renal ox-LDL deposition, and the potential molecular mechanisms require further investigation. METHODS: We conducted a comprehensive analysis of serum and kidney biopsy samples obtained from patients diagnosed with DKD. Additionally, to explore the underlying mechanism of Klotho in the deposition of ox-LDL in the kidneys, we employed a mouse model of DKD with the Klotho genotype induced by streptozotocin (STZ). Furthermore, we conducted meticulous in vitro experiments on podocytes to gain further insights into the specific role of Klotho in the deposition of ox-LDL within the kidney. RESULTS: Our groundbreaking study unveiled the remarkable ability of the soluble form of Klotho to effectively inhibit high glucose-induced ox-LDL deposition in podocytes affected by DKD. Subsequent investigations elucidated that Klotho achieved this inhibition by reducing the expression of the insulin/insulin-like growth factor 1 receptor (IGF-1R), consequently leading to a decrease in the expression of Ras-related C3 botulinum toxin substrate 1 (RAC1) and an enhancement of mitochondrial function. Ultimately, this series of events culminated in a significant reduction in the expression of the oxidized low-density lipoprotein receptor (OLR1), thereby resulting in a notable decrease in renal ox-LDL deposition in DKD. CONCLUSION: Our findings suggested that Klotho had the potential to mitigate podocyte injury and reduced high glucose-induced ox-LDL deposition in glomerulus by modulating the IGF-1R/RAC1/OLR1 signaling. These results provided valuable insights that could inform the development of novel strategies for diagnosing and treating DKD.

HIF-2α-induced upregulation of CD36 promotes the development of ccRCC.

Clear cell renal cell carcinoma (ccRCC) is characterized by the abundance of lipid droplets and the activation of the hypoxia-inducible factor (HIF) signaling pathway. However, the lipid reprogramming induced by HIF signaling in ccRCC is not fully understood. In this study, we found that the fatty acid receptor CD36 was highly expressed in human ccRCC tissues and ccRCC cell lines. CD36 overexpression increased fatty acid uptake and lipid droplet formation, and enhanced the proliferation and migration of ccRCC cells in a DGAT1-dependent manner. In contrast, the disruption of endogenous CD36 showed the opposite effects. The upregulated expression of CD36 in ccRCC was associated with hypoxia and HIF-2α activation. Furthermore, we identified CD36 as a new target of the transcription factor HIF-2α. The knockdown of CD36 in ccRCC cells reduced lipid accumulation and also blocked the tumor-promoting effects induced by HIF-2α under hypoxia. Our findings suggest that hypoxia-dependent HIF-2α promotes the remodeling of lipid metabolism and the malignant phenotype of ccRCC via CD36, providing a certain theoretical basis for clarifying the mechanism of ccRCC.

CT-based radiomics signature to predict CD8+ tumor infiltrating lymphocytes in non-small-cell lung cancer.

BACKGROUND: An abundance of CD8+ tumor infiltrating lymphocytes (TILs) in the center of solid tumors is a reliable predictive biomarker for patients eligible for immunotherapy. PURPOSE: To develop a computed tomography (CT)-based radiomics signature for a preoperative prediction of an abundance of CD8+ TILs in non-small-cell lung cancer (NSCLC). MATERIAL AND METHODS: In this retrospective study, 117 consecutive patients with pathologically confirmed NSCLC were included and randomly divided into training (n = 77) and test sets (n = 40). A total of 107 radiomics features were extracted from the three-dimensional volumes of interest of each patient. Least absolute shrinkage and selection operator (LASSO) regression was used to select the strongest features for abundance of CD8+ TILs in NSCLC, and the radiomics score was constructed through a linear combination of these selected features. Receiver operating characteristic (ROC) curve analysis was used to evaluate the predictive performance of the radiomics score. RESULTS: The radiomics score was associated with an abundance of CD8+ TILs in NSCLC, which achieved an area under the curve (AUC) of 0.83 (95% CI=0.73-0.92) and 0.68 (95% CI=0.54-0.87) in the training and test sets, respectively. The difference was not statistically significant (P = 0.20). The tumors with high CD8+ TILs tended to have heterogeneous dependences (high value of Dependence Non-Uniformity Normalized) and complicated texture (high value of Informational Measure of Correlation 1). CONCLUSION: CT-based radiomics features have the ability to predict CD8+ TILs expression levels of an abundance of CD8+ TILs in NSCLC, which was shown to be a potential imaging biomarker for stratifying patients who may benefit from immunotherapy.

Novel pathogenic variants in CUBN uncouple proteinuria from renal function.

BACKGROUND: Proteinuria is an unfavorable clinical condition highly associated with a risk of renal and cardiovascular disease in chronic kidney disease (CKD). However, whether all proteinuria forms are linked to renal impairment are still unclear. Cubilin is an endocytic receptor highly expressed in renal proximal tubules mediating uptake of albumin, transferrin and α1-microglobulin. METHODS: Exome sequencing method initially identified candidate genes. With the application of exome sequencing combined with Sanger sequencing, we further focused on CUBN through bioinformatics analysis. The pathogenic effects of the potentially causative variants were verified utilizing complementary analysis of clinical data and systematic characterization of the variants' expression and function with clinical samples and in vitro experiments in HEK293T cell lines along with in vivo experiments in mice. RESULTS: In this study, we identified four novel variants locating after the vitamin B12 (vitB12)-binding domain of Cubilin (encoded by CUBN, NM_001081.3: c.4397G > A (p.C1466Y), c.6796C > T (p.R2266X), c.6821 + 3A > G and c.5153_5154delCT (p.S1718X)) in two families. Moreover, the variants severely affected the expression and function of Cubilin in renal proximal tubules and caused albuminuria, increasing levels in urine transferrin and α1-microglobulin, but without progressive glomerular filtration barrier (GFB) impairment, vitB12 deficiencies or abnormal blood levels of HDL and albumin. Further mechanistic insights showed that the variants after the vitB12-binding domain of CUBN merely disrupted the association with Amnionless (AMN) that exhibited aberrant localization in cell cytoplasm rather than membrane. CONCLUSIONS: Here, our findings suggested that different mutation types after the vitB12-binding domain of CUBN uncouple proteinuria from glomerular filtration barrier, that may be an unexpectedly common benign condition in humans and may not require any proteinuria-lowering treatment or renal biopsy.

Src-mediated Tyr353 phosphorylation of IP3R1 promotes its stability and causes apoptosis in palmitic acid-treated hepatocytes.

Palmitic acid (PA)-induced hepatocyte apoptosis is critical for the progression of nonalcoholic fatty liver disease (NAFLD). Inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) is an intracellular Ca2+-release channel and is involved in PA-induced hepatocyte apoptosis. While the expression of IP3R1 is elevated in patients with NAFLD and in hepatocytes treated with PA, it remains unclear how PA promotes the expression of IP3R1. In present study, our results showed that PA induced mitochondrial dysfunction and apoptosis, which is accompanied with the increase of the IP3R1 expression in hepatic cells. The inhibition of IP3R1 expression using siRNA ameliorated the PA-induced mitochondrial dysfunction. Furthermore, PA enhanced the stability of the IP3R1 protein instead of an increase in its mRNA levels. PA also promoted the phosphorylation of IP3R1 at the Tyr353 site and increased the phosphorylation of src in hepatic cells. Moreover, an inhibitor of src kinase (SU6656) significantly reduced the Tyr353 phosphorylation of IP3R1 and decreased its stability. In addition, SU6656 improved mitochondrial function and reduced apoptosis in hepatocytes. Conclusion: PA promotes the Tyr353 phosphorylation of IP3R1 by activating the src pathway and increasing the protein stability of IP3R1, which consequently results in mitochondrial Ca2+ overload and mitochondrial dysfunction in hepatic cells. Our results also suggested that inhibition of the src/IP3R1 pathway, such as by SU6656, may be a novel potential therapeutic approach for the treatment of NAFLD.

Loss of CD36 impairs hepatic insulin signaling by enhancing the interaction of PTP1B with IR.

A contradictory role of CD36 in insulin resistance was found to be related to the nutrient state. Here, we examined that the physiological functions of CD36 in insulin signal transduction in mice fed a low-fat diet. CD36 deficiency led to hepatic insulin resistance and decreased insulin-stimulated tyrosine phosphorylation of insulin receptor ß (IRß) in mice fed a low-fat diet. The ability of insulin to bind with IR did not differ between WT and CD36-deficient hepatocytes. CD36 formed a complex with IRß and dissociation of CD36/Fyn complex or inhibition of Fyn only partially reversed the effects of CD36 on hepatic insulin signaling. Furthermore, we found that CD36 deficiency led to abnormally increased hepatic protein-tyrosine phosphatase 1B (PTP1B) expression and enhanced PTP1B and IR interactions, which contributed to the decreased insulin signaling and disordered glucose metabolism. In addition, increased endoplasmic reticulum (ER) stress was found in the livers of the CD36-deficient mice, while inhibited ER stress normalized the PTP1B expression and restored insulin signaling in the CD36-deficient mice. Our findings suggest that the loss of CD36 impairs hepatic insulin signaling by enhancing the PTP1B/IR interaction that is induced by ER stress, indicating a possible critical step in the progression of hepatic insulin resistance.

The usefulness of the combined high-dose dexamethasone suppression test and desmopressin stimulation test in establishing the source of ACTH secretion in ACTH-dependent Cushing's syndrome.

Bilateral inferior petrosal sinus sampling (BIPSS) is the current gold standard test for differentially diagnosing ACTH-dependent Cushing's syndrome (CS). However, BIPSS is an invasive procedure, and its availability is limited. We retrospectively analysed the 24-hour urinary free cortisol (UFC) level during the high-dose dexamethasone suppression test (HDDST) and plasma ACTH/cortisol levels after the desmopressin stimulation test (DDAVP test) in subjects with confirmed Cushing's disease (CD) (n = 92) and ectopic ACTH-dependent CS (EAS) (n = 16), and evaluated the positive predictive value (PPV) of the two combined-tests in the aetiological diagnosis of ACTH-dependent CS. The percent changes in UFC levels after the HDDST and in ACTH/cortisol levels after DDAVP administration relative to the corresponding basal levels and the area under the receiver operating characteristic (ROC) curve (AUC) were analysed. UFC suppression below 62.7% suggested a pituitary origin with a sensitivity (SE) of 80% (95% CI: 70-88) and a specificity (SP) of 80% (95% CI: 52-96). A threshold increase in the ACTH level after DDAVP stimulation of 44.6% identified CD with an SE of 91% (95% CI: 83-97) and an SP of 75% (95% CI: 48-93). The combination of both tests yielded an SE of 95.5% and PPV of 98.4% for CD, and significantly improved the efficiency of the differential diagnosis between CD and EAS. These dual non-invasive endocrine tests may substantially reduce the need for BIPSS in the etiological investigation of ACTH-dependent CS.

Lipid Metabolism and Tumor Antigen Presentation.

Tumors always evade immune surveillance and block T cell activation in a poorly immunogenic and immunosuppressive environment. Cancer cells and immune cells exhibit metabolic reprogramming in the tumor microenvironment (TME), which intimately links immune cell function and edits tumor immunology. In addition to glucose metabolism, amino acid and lipid metabolism also provide the materials for biological processes crucial in cancer biology and pathology. Furthermore, lipid metabolism is synergistically or negatively involved in the interactions between tumors and the microenvironment and contributes to the regulation of immune cells. Antigen processing and presentation as the initiation of adaptive immune response play a critical role in antitumor immunity. Therefore, a relationship exists between antigen-presenting cells and lipid metabolism in TME. This chapter introduces the updated understandings of lipid metabolism of tumor antigen-presenting cells and describes new directions in the manipulation of immune responses for cancer treatment.

[Deletion of CD36 gene ameliorates glucose metabolism abnormality induced by high-fat diet and promotes liver lipid accumulation].

This study aimed to investigate the effects and the underlying mechanism of CD36 gene on glucose and lipid metabolism disorder induced by high-fat diet in mice. Wild type (WT) mice and systemic CD36 knockout (CD36-/-) mice were fed with high-fat diet for 14 weeks (n = 12). Mice were intraperitoneally injected with glucose (1 g/kg) or insulin (5 units/kg) to perform glucose tolerance test (GTT) or insulin tolerance test (ITT). Liver lipid deposition was observed by HE staining, and the contents of total triglyceride (TG), free fatty acid (FFA), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum were determined by automatic biochemical analyzer. Real-time PCR and Western blot were used to detect insulin signaling pathways in liver and muscle tissues of mice. The mRNA levels of genes encoding phosphoenolpyruvate carboxykinase (PEPCK) in primary hepatocytes of mice were detected by real-time PCR, and glucose detection kit was used to detect gluconeogenesis. Co-immunoprecipitation (Co-IP) and ELISA were used to detect insulin receptor ß (IRß) tyrosine phosphorylation in mouse muscle. Real-time PCR and immunofluorescence staining (IF) were used to detect the expression and location of glucose transporter 4 (GLUT4) in muscle of mice. After high-fat diet feeding, serum FFA, TG, AST and ALT levels of CD36-/- mice were significantly higher than WT mice (P < 0.05). The appearance of CD36-/- mouse liver presented fatty degeneration, and HE staining results showed increased lipid accumulation in the liver, suggesting that CD36 knockout promoted the occurrence of fatty liver. However, CD36-/- mice showed decreased fasting glucose levels, increased glucose tolerance, and decreased insulin tolerance compared with WT mice (P < 0.05), suggesting that CD36 knockout protects against the abnormal glucose metabolism induced by high-fat diet. Compared with WT mice, there was no significant difference in insulin signaling pathway in CD36-/- mouse liver, and there were no significant differences in PEPCK expression and gluconeogenesis between the two groups of primary hepatocytes. In muscle tissue, Co-IP and ELISA experiments showed that the phosphorylation level of IRß tyrosine was significantly increased in CD36-/- mice compared with that in WT mice. Besides, the levels of p-AKT in CD36-/- mouse muscle were significantly increased (P < 0.05). At the same time, IF experiment indicated that GLUT4 localization in cell membrane was enhanced in the muscle of CD36-/- mice, indicating that insulin sensitivity and glucose utilization ability were enhanced in CD36-/- mouse muscle. The results suggested that deletion of CD36 gene increased lipid accumulation in liver of mice with high-fat diet, but had no significant effect on liver gluconeogenesis. CD36 deficiency improves the abnormal glucose metabolism in mice with high-fat diet mainly through improving insulin sensitivity of muscle tissue and promoting GLUT4-mediated glucose utilization.

[Lipopolysaccharide inhibits lipophagy in HepG2 cells via activating mTOR pathway].

This study aimed to investigate the effect of lipopolysaccharide (LPS) on lipophagy in hepatocytes and the underlying mechanism. Human hepatoma cell line HepG2 was cultured in vitro, treated with 0.1 mmol/L palmitic acid (PA), and then divided into control group (0 µg/mL LPS), LPS group (10 µg/mL LPS), LPS+DMSO group and LPS+RAPA (rapamycin, 10 µmol/L) group. Lipid accumulation in hepatocytes was observed by oil red O staining. The autophagic flux of the cells was assessed using confocal laser scanning microscope after being transfected with autophagy double-labeled adenovirus (mRFP-GFP-LC3). The level of intracellular lipophagy was visualized by the colocalization of lipid droplets (BODIPY 493/503 staining) and lysosomes (lysosome marker, lysosomal associated membrane protein 1, LAMP1). The expression levels of mammalian target of rapamycin (mTOR), phosphorylated mTOR (p-mTOR), ribosome protein subunit 6 kinase 1 (S6K1), p-S6K1, LC3II/I and P62 protein were examined by Western blot. The results showed that the number of red lipid droplets stained with oil red O was significantly increased in LPS group compared with that in control group (P < 0.001). Moreover, in LPS group, the number of autophagosomes was increased, while the number of autophagolysosomes and the colocalization rate of LAMP1 and BODIPY were significantly decreased (P < 0.05). Meanwhile, the ratios of p-mTOR/mTOR and p-S6K1/S6K1, the ratio of LC3II/LC3I and the protein expression of P62 were significantly increased (P < 0.05) in LPS group. Furthermore, compared with LPS+DMSO group, RAPA treatment obviously reduced the number of lipid droplets and autophagosomes, and raised the number of autophagolysosomes and the colocalization rate of LAMP1 and BODIPY (P < 0.05). In conclusion, the results demonstrate that LPS inhibits lipophagy in HepG2 cells via activating mTOR signaling pathway, thereby aggravating intracellular lipid accumulation.

SCAP knockdown in vascular smooth muscle cells alleviates atherosclerosis plaque formation via up-regulating autophagy in ApoE-/- mice.

Sterol regulatory element binding protein (SREBP) cleavage-activating protein (SCAP) is a cholesterol sensor that plays a critical role in regulating intracellular cholesterol levels, but the association between SCAP and foam cell formation in vascular smooth muscle cells (VSMCs) is poorly understood. Using tissue-specific SCAP knockdown in apolipoprotein E (ApoE)-/- mice, we sought to search the mechanism through which SCAP signaling affects VSMC foam cell development. VSMC-specific SCAP knockdown mice were generated by Cre/LoxP-mediated gene targeting in ApoE-/- mice. Breeding SCAPflox/flox mice with SM22α-Cre mice resulted in no viable offspring with the homozygote SM22-Cre: SCAPflox/flox genotype due to embryonic lethality. We found that the heterozygote SM22α-Cre:SCAPflox/+:ApoE-/- mice fed a Western diet for 12 wk had significantly fewer atherosclerotic plaques in their aortas than the control mice due to reduced cholesterol uptake and synthesis. Furthermore, we found that autophagy in VSMCs was increased in SM22α-Cre:SCAPflox/+:ApoE-/- mice. Similarly, in vitro, SCAP knockdown in human coronary artery VSMCs by RNA interference reduced lipid accumulation and increased autophagy under LDL cholesterol loading. SCAP knockdown in VSMCs reduced oxidative stress and increased AMPK phosphorylation, which contributed to the up-regulation of autophagy in vivo and in vitro. VSMC-specific SCAP knockdown decreased the lipid accumulation and intracellular oxidative stress, increased excessive lipid clearance by enhancing lipid autophagy mediated by the reactive oxygen species/AMPK pathway in VSMCs, and consequently alleviated atherosclerosis plaque formation.-Li, D., Chen, A., Lan, T., Zou, Y., Zhao, L., Yang, P., Qu, H., Wei, L., Varghese, Z., Moorhead, J. F., Chen, Y., Ruan, X. Z. SCAP knockdown in vascular smooth muscle cells alleviates atherosclerosis plaque formation via up-regulating autophagy in ApoE-/- mice.

CD36 plays a negative role in the regulation of lipophagy in hepatocytes through an AMPK-dependent pathway.

Fatty acid translocase cluster of differentiation (CD36) is a multifunctional membrane protein that facilitates the uptake of long-chain fatty acids. Lipophagy is autophagic degradation of lipid droplets. Accumulating evidence suggests that CD36 is involved in the regulation of intracellular signal transduction that modulates fatty acid storage or usage. However, little is known about the relationship between CD36 and lipophagy. In this study, we found that increased CD36 expression was coupled with decreased autophagy in the livers of mice treated with a high-fat diet. Overexpressing CD36 in HepG2 and Huh7 cells inhibited autophagy, while knocking down CD36 expression induced autophagy due to the increased autophagosome formation in autophagic flux. Meanwhile, knockout of CD36 in mice increased autophagy, while the reconstruction of CD36 expression in CD36-knockout mice reduced autophagy. CD36 knockdown in HepG2 cells increased lipophagy and ß-oxidation, which contributed to improving lipid accumulation. In addition, CD36 expression regulated autophagy through the AMPK pathway, with phosphorylation of ULK1/Beclin1 also involved in the process. These findings suggest that CD36 is a negative regulator of autophagy, and the induction of lipophagy by ameliorating CD36 expression can be a potential therapeutic strategy for the treatment of fatty liver diseases through attenuating lipid overaccumulation.

High olive oil diets enhance cervical tumour growth in mice: transcriptome analysis for potential candidate genes and pathways.

BACKGROUND: Numerous epidemiologic studies have found a close association between obesity and cancer. Dietary fat is a fundamental contributor to obesity and is a risk factor for cancer. Thus far, the impact of dietary olive oil on cancer development remains inconclusive, and little is known about its underlying mechanisms. METHODS: Nude mouse xenograft models were used to examine the effects of high olive oil diet feeding on cervical cancer (CC) development and progression. Cell proliferation, migration and invasion were observed by the methods of EdU incorporation, Wound healing and Transwell assay, separately. RNA-sequencing technology and comprehensive bioinformatics analyses were used to elucidate the molecular processes regulated by dietary fat. Differentially expressed genes (DEGs) were identified and were functionally analyzed by Gene Ontology (GO), Kyoto Enrichment of Genes and Genomes (KEGG). Then, protein-protein interaction (PPI) network and sub-PPI network analyses were conducted using the STRING database and Cytoscape software. RESULTS: A high olive oil diet aggravated tumourigenesis in an experimental xenograft model of CC. Oleic acid, the main ingredient of olive oil, promoted cell growth and migration in vitro. Transcriptome sequencing analysis of xenograft tumour tissues was then performed to elucidate the regulation of molecular events regulated by dietary fat. Dietary olive oil induced 648 DEGs, comprising 155 up-regulated DEGs and 493 down-regulated DEGs. GO and pathway enrichment analysis revealed that some of the DEGs including EGR1 and FOXN2 were involved in the transcription regulation and others, including TGFB2 and COL4A3 in cell proliferation. The 15 most strongly associated DEGs were selected from the PPI network and hub genes including JUN, TIMP3, OAS1, OASL and EGR1 were confirmed by real-time quantitative PCR analysis. CONCLUSIONS: Our study suggests that a high olive oil diet aggravates CC progression in vivo and in vitro. We provide clues to build a potential link between dietary fat and cancerogenesis and identify areas requiring further investigation.

CD36 palmitoylation disrupts free fatty acid metabolism and promotes tissue inflammation in non-alcoholic steatohepatitis.

BACKGROUND AND AIMS: Fatty acid translocase CD36 (CD36) is a membrane protein with multiple immuno-metabolic functions. Palmitoylation has been suggested to regulate the distribution and functions of CD36, but little is known about its significance in non-alcoholic steatohepatitis (NASH). METHODS: Human liver tissue samples were obtained from patients undergoing liver biopsy for diagnostic purposes. CD36 knockout mice were injected with lentiviral vectors expressing wild-type CD36 or CD36 with mutated palmitoylation sites. Liver histology, immunofluorescence, mRNA expression profile, subcellular distributions and functions of CD36 protein were assessed. RESULTS: The localization of CD36 on the plasma membrane of hepatocytes was markedly increased in patients with NASH compared to patients with normal liver and those with simple steatosis. Increased CD36 palmitoylation and increased localization of CD36 on the plasma membrane of hepatocytes were also observed in livers of mice with NASH. Furthermore, inhibition of CD36 palmitoylation protected mice from developing NASH. The absence of palmitoylation decreased CD36 protein hydrophobicity reducing its localization on the plasma membrane as well as in lipid raft of hepatocytes. Consequently, a lack of palmitoylation decreased fatty acid uptake and CD36/Fyn/Lyn complex in HepG2 cells. Inhibition of CD36 palmitoylation not only ameliorated intracellular lipid accumulation via activation of the AMPK pathway, but also inhibited the inflammatory response through the inhibition of the JNK signaling pathway. CONCLUSIONS: Our findings demonstrate the key role of palmitoylation in regulating CD36 distributions and its functions in NASH. Inhibition of CD36 palmitoylation may represent an effective therapeutic strategy in patients with NASH. LAY SUMMARY: Fatty acid translocase CD36 (CD36) is a multifunctional membrane protein which contributes to the development of liver steatosis. In the present study, we demonstrated that the localization of CD36 on the plasma membrane of hepatocytes is increased in patients with non-alcoholic steatohepatitis. Blocking the palmitoylation of CD36 reduces CD36 distribution in hepatocyte plasma membranes and protects mice from non-alcoholic steatohepatitis. The inhibition of CD36 palmitoylation not only improved fatty acid metabolic disorders but also reduced the inflammatory response in vitro and in vivo. The present study suggests that CD36 palmitoylation is important for non-alcoholic steatohepatitis development and inhibition of CD36 palmitoylation could be used to cure non-alcoholic steatohepatitis.

Knocking down Stard3 decreases adipogenesis with decreased mitochondrial ROS in 3T3-L1 cells.

Start domain-containing protein 3 (Stard3) plays roles in intracellular cholesterol distribution, however, the role of Stard3 in the adipogenesis of 3T3-L1 preadipocytes remains unclear. We demonstrated that Stard3 expression was significantly increased during the adipogenesis of 3T3-L1 preadipocytes, accompanied by an increase of mitochondrial Reactive oxygen species (ROS). Stard3 knocking-down inhibited 3T3-L1 preadipocyte adipogenesis with decreased mitochondrial ROS levels, while ROS inducer rescued the stard3 silencing 3T3 cells with increased ROS. Moreover, Stard3 silencing reduced the expression of peroxisome proliferator-activated receptor-γ (PPARγ) and CCAAT/enhancer binding protein (C/EBP)α in 3T3- L1 cells. In conclusion, Stard3 enhanced the adipogenesis of preadipocytes by enhancement of cholesterol redistribution to the mitochondrial, increasing mitochondrial ROS production. These results suggest that Stard3 is an essential factor for the 3T3-L1 cells' differentiation.

CD36 and lipid metabolism in the evolution of atherosclerosis.

Background: CD36 is a multi-functional class B scavenger receptor, which acts as an important modulator of lipid homeostasis and immune responses. Sources of data: This review uses academic articles. Areas of agreement: CD36 is closely related to the development and progression of atherosclerosis. Areas of controversy: Both persistent up-regulation of CD36 and deficiency of CD36 increase the risk for atherosclerosis. Abnormally up-regulated CD36 promotes inflammation, foam cell formation, endothelial apoptosis, macrophage trapping and thrombosis. However, CD36 deficiency also causes dyslipidemia, subclinical inflammation and metabolic disorders, which are established risk factors for atherosclerosis. Growing points: There may be an 'optimal protective window' of CD36 expression. Areas timely for developing research: In addition to traditionally modulating protein functions using gene overexpression or deficiency, the modulation of CD36 function at post-translational levels has recently been suggested to be a potential therapeutic strategy.

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

Hepatitis B virus (HBV) is designated a "metabolovirus" due to the intimate connection between the virus and host metabolism. The nutrition state of the host plays a relevant role in the severity of HBV infection. Metabolic syndrome (MS) is prone to increasing HBV DNA loads and accelerating the progression of liver disease in patients with chronic hepatitis B (CHB). Cluster of differentiation 36 (CD36), also named fatty acid translocase, is known to facilitate long-chain fatty acid uptake and contribute to the development of MS. We recently found that CD36 overexpression enhanced HBV replication. In this study, we further explored the mechanism by which CD36 overexpression promotes HBV replication. Our data showed that CD36 overexpression increased HBV replication, and CD36 knockdown inhibited HBV replication. RNA sequencing found some of the differentially expressed genes were involved in calcium ion homeostasis. CD36 overexpression elevated the cytosolic calcium level, and CD36 knockdown decreased the cytosolic calcium level. Calcium chelator BAPTA-AM could override the HBV replication increased by CD36 overexpression, and the calcium activator thapsigargin could improve the HBV replication reduced by CD36 knockdown. We further found that CD36 overexpression activated Src kinase, which plays an important role in the regulation of the store-operated Ca2+ channel. An inhibitor of Src kinase (SU6656) significantly reduced the CD36-induced HBV replication. We identified a novel link between CD36 and HBV replication, which is associated with cytosolic calcium and the Src kinase pathway. CD36 may represent a potential therapeutic target for the treatment of CHB patients with MS.

Long-chain fatty acid activates hepatocytes through CD36 mediated oxidative stress.

BACKGROUND: Accumulating evidence suggests that activated hepatocytes are involved in the deposition of the excess extracellular matrix during liver fibrosis via the epithelial to mesenchymal transition. Lipid accumulation in hepatocytes are implicated in the pathogenesis of chronic liver injury. CD36 is known to mediate long-chain fatty acid (LCFA) uptake and lipid metabolism. However, it is unclear whether LCFA directly promotes hepatocyte activation and the involved mechanisms have not been fully clarified. METHODS: Mice were fed with a high fat diet (HFD) and normal hepatocyte cells (Chang liver cells) were treated with palmitic acid (PA) in vivo and in vitro. Real-time polymerase chain reaction (RT-PCR) and western blotting were used to examine the gene and protein expression of molecules involved in hepatic fibrogenesis and hepatocyte activation. CD36 was knocked down by transfecting CD36 siRNA into hepatocyte cells. Hydrogen peroxide (H2O2) and reactive oxygen species (ROS) levels were detected using commercial kits. RESULTS: HFD induced a profibrogenic response and up-regulated CD36 expression in vivo. Analogously, PA increased lipid accumulation and induced human hepatocyte activation in vitro, which was also accompanied by increased CD36 expression. Interestingly, knockdown of CD36 resulted in a reduction of hepatocyte lipid deposition and decreased expression of Acta2 (34% decrease), Vimentin (29% decrease), Desmin (60% decrease), and TGF-ß signaling pathway related genes. In addition, HFD and PA increased the production of H2O2 in vivo (48% increase) and in vitro (385% increase), and the antioxidant, NAC, ameliorated PA-induced hepatocyte activation. Furthermore, silencing of CD36 in vitro markedly attenuated PA-induced oxidative stress (H2O2: 41% decrease; ROS: 39% decrease), and the anti-activation effects of CD36 knockdown could be abolished by pretreatment with H2O2. CONCLUSIONS: Our study demonstrated that LCFA facilitates hepatocyte activation by up-regulating oxidative stress through CD36, which could be an important mechanism in the development of hepatic fibrosis.