Cluster Differentiating 36 (CD36) Deficiency Attenuates Obesity-Associated Oxidative Stress in the Heart

<div><p>Rationale</p><p>Obesity is often associated with a state of oxidative stress and increased lipid deposition in the heart. More importantly, obesity increases lipid influx into the heart and induces excessive production of reactive oxygen species (ROS) leading to cell toxicity and metabolic dysfunction. Cluster differentiating 36 (CD36) protein is highly expressed in the heart and regulates lipid utilization but its role in obesity-associated oxidative stress is still not clear.</p><p>Objective</p><p>The aim of this study was to determine the impact of CD36 deficiency on cardiac steatosis, oxidative stress and lipotoxicity associated with obesity.</p><p>Methods and Results</p><p>Studies were conducted in control (Lean), obese leptin-deficient (Lep^ob/ob) and leptin-CD36 double null (Lep^ob/obCD36^-/-) mice. Compared to lean mice, cardiac steatosis, and fatty acid (FA) uptake and oxidation were increased in Lep^ob/ob mice, while glucose uptake and oxidation was reduced. Moreover, insulin resistance, oxidative stress markers and NADPH oxidase-dependent ROS production were markedly enhanced. This was associated with the induction of NADPH oxidase expression, and increased membrane-associated p47^phox, p67^phox and protein kinase C. Silencing CD36 in Lep^ob/ob mice prevented cardiac steatosis, increased insulin sensitivity and glucose utilization, but reduced FA uptake and oxidation. Moreover, CD36 deficiency reduced NADPH oxidase activity and decreased NADPH oxidase-dependent ROS production. In isolated cardiomyocytes, CD36 deficiency reduced palmitate-induced ROS production and normalized NADPH oxidase activity.</p><p>Conclusions</p><p>CD36 deficiency prevented obesity-associated cardiac steatosis and insulin resistance, and reduced NADPH oxidase-dependent ROS production. The study demonstrates that CD36 regulates NADPH oxidase activity and mediates FA-induced oxidative stress.</p></div

Effects of CD36 expression on cardiac oxidative stress markers.

<p>Contents of Isoprostanes (A) and lipid peroxides (B) were measured in heart homogenates using gas chromatographic/mass spectrometry and enzymatic kit, respectively. Hydrogen peroxides (C) were evaluated using Amplex Red method. Data are means ± SEM of duplicates from an n = 6 per group. Differences between Lep^ob/ob and Lean mice are indicated with asterisks ** p < 0.01, and differences between Lep^ob/ob CD36^-/- and Lep^ob/ob mice are indicated with alphabetic letters ^a p < 0.01 and ^b p < 0.05.</p

Effects of CD36 deficiency on insulin signaling.

<p>A) Representative blots and B) ratio of phosphorylated (p) to total (t) Akt and IRS1 in hearts 10 minutes after insulin injection. Results are presented as mean ± SEM (n = 5 per group). Statistical differences between Lep^ob/ob and Lean mice are indicated with an asterisk * p < 0.01, and differences between Lep^ob/ob CD36^-/- and Lep^ob/ob mice are indicated with an alphabetic letter with ^a p <0.05.</p

Effects of CD36 deficiency on cardiac lipid contents, fatty uptake and incorporation in lipids.

<p>Cardiac lipid contents (A) were determined enzymatically after lipid extraction as described in the Methods. Fatty acid uptake in indicated organs (B) was assessed using ¹²⁵I-BMIPP. Mice were injected with 5 μCi of ¹²⁵I-BMIPP into the tail vein and tissues were removed 2 h later. Incorporation of BMIPP in heart lipids (C) was examined after extraction and TLC separation. Polar lipids include phospholipids and monoacylglycerides. Results are presented as mean ± SEM (n = 6–7 per group). Differences between Lep^ob/ob and Lean mice are indicated with asterisks ** p < 0.01, and * p < 0.05. Differences between Lep^ob/ob CD36^-/- and Lep^ob/ob mice are indicated with alphabetic letters with ^a p < 0.01 and ^b p < 0.05.</p

Body and organ weights, and plasma parameters for control lean (Lean), leptin null (Lep^ob/ob) and leptin CD36 double null (Lep^ob/obCD36^-/-) mice.

<p>Data are expressed as mean ± SEM with n = 12 per group.</p

Effects of CD36 deficiency on glucose metabolism parameters.

<p>A) Glucose tolerance test (GTT) in overnight fasted control lean (Lean), leptin null (Lep^ob/ob) and leptin and CD36 double null (Lep^ob/obCD36^-/-) mice. B) Insulin tolerance test (ITT) in 4-h fasted mice. C) Uptake of ¹⁸F-2-FDG in organs of overnight fasted mice. Mice were injected with 5 μCi of ¹⁸F-2-FDG in a lateral tail vein and glucose uptake in indicated organ was determined as described in the Methods. Results are presented as mean ± SEM (n = 5–7 per group). For GTT and ITT, statistical differences between initial time (0 min) and subsequent time points (after glucose and insulin injection) were performed by repeated measurement ANOVA test. Differences between groups Tukey’s and student t tests. Statistical significance between Lep^ob/ob and Lean mice are indicated with asterisks ** p < 0.01, and * p < 0.05. Significance between Lep^ob/ob CD36^-/- and Lep^ob/ob mice are indicated with alphabetic letters with ^a p < 0.001, ^b p < 0.01 and ^b p < 0.05. Statistical significance between Lep^ob/ob CD36^-/- and Lean mice are indicated with a capital alphabetic letter ^A p < 0.01.</p

Effects of CD36 deficiency on protein expression.

<p>Representative blots (A) and mRNA abundance (B) of CD36, FATP1, H-FABP, PPARα in hearts of mice (n = 6 per group). Protein levels were examined by Western blotting and mRNA abundance was investigated with qPCR as described in the Methods. Differences between Lep^ob/ob and Lean mice are indicated with an asterisk * p < 0.05, and differences between Lep^ob/ob CD36^-/- and Lep^ob/ob mice are indicated with an alphabetic letter ^a p <0.05.</p

Effects of CD36 expression on NADPH-dependent superoxide production and expression of NADPH oxidase.

<p>A) NADPH-dependent and SOD-inhibitable superoxide was measured in heart homogenates by the lucigenin chemiluminescent method. Measurements were performed in preparations without inhibitors (basal) or in presence of superoxide dismutase (SOD) used to determine the specific of measurements, L-NAME (nitric oxide synthase inhibitor), Oxypurinol (oxidase inhibitor), Rotenone (complex I mitochondrial electron chain inhibitor), Apocynin (NADPH oxidase) and DPI (flavoprotein inhibitor). Measurements were conducted in triplicates from an n = 5 per group. B) Representative blots and C) ratio of p22^phox, NADPH oxidase 2 (Nox2) and 4 (Nox4) to β-actin. Results are presented as Mean ± SEM and differences between Lep^ob/ob and Lean mice are indicated with asterisks with ** p < 0.01 and * p < 0.05. Differences between Lep^ob/obCD36^-/- and Lep^ob/ob mice are indicated with alphabetic letters ^a p < 0.01 and ^b p < 0.05. Differences between Lep^ob/ob CD36^-/- and Lean mice are indicated with A capital alphabetic letter ^A p < 0.05.</p

Effects of CD36 expression on the distribution of NADPH oxidase (Nox) and PKC in membrane and cytosol fractions.

<p>Representative blots (A and C) and means of membrane-to-cytosol ratios of optic density (B and D) of p47^phox, p67^phox, PKCα and PKCδ proteins. The procedures of separation of membrane and cytosol fractions and western blotting are described in the Methods. Differences between Lep^ob/ob and Lean mice are indicated with asterisks with ** p < 0.01 and * p < 0.05, and differences between Lep^ob/ob CD36^-/- and Lep^ob/ob mice are indicated with alphabetic letters ^a p < 0.01 and ^b p < 0.05.</p

DataSheet1_The zhuyu pill relieves rat cholestasis by regulating the mRNA expression of lipid and bile metabolism associated genes.DOCX

Background: The Zhuyu pill (ZYP), composed of Coptis chinensis Franch. and Tetradium ruticarpum (A. Jussieu) T. G. Hartley, is an effective traditional Chinese medicine with potential anti-cholestatic effects. However, the underlying mechanisms of ZYP remain unknown.Objective: To investigate the mechanism underlying the interventional effect of ZYP on mRNA-seq analysis in cholestasis rat models.Materials and methods: This study tested the effects of a low-dose (0.6 g/kg) and high-dose (1.2 g/kg) of ZYP on a cholestasis rat model induced by α-naphthyl-isothiocyanate (ANIT, 50 mg/kg). Serum biochemistry and histopathology results were used to evaluate the therapeutic effect of ZYP, and mRNA-Seq analysis was performed and verified using real-time fluorescence quantitative PCR (qRT-PCR). GO, KEGG, and GSEA analyses were integrated to identify the mechanism by which ZYP impacted cholestatic rats.Results: ZYP was shown to significantly improve abnormal changes in the biochemical blood indexes and liver histopathology of cholestasis rats and regulate pathways related to bile and lipid metabolism, including fatty acid metabolism, retinol metabolism, and steroid hormone biosynthesis, to alleviate inflammation, cholestasis, and lipid metabolism disorders. Relative expression of the essential genes Cyp2a1, Ephx2, Acox2, Cyp1a2, Cyp2c11, and Sult2a1 was verified by qRT-PCR and showed the same trend as mRNA-seq analysis.Conclusion: ZYP has a significant anti-cholestatic effect by regulating bile metabolism and lipid metabolism related pathways. These findings indicate that ZYP is a novel and promising prospect for treating cholestasis.</p