Clusterin Impairs Hepatic Insulin Sensitivity and Adipocyte Clusterin Associates With Cardiometabolic Risk

Abstract

Objective: Components of the adipose tissue (AT) extracellular matrix (ECM) are recently discovered contributors to obesity-related cardiometabolic disease. We identified increased adipocyte expression of ECM-related clusterin (apolipoprotein J) in obese versus lean women by microarray. Our objective was to determine 1) whether subcutaneous AT adipocyte (SAd) clusterin and serum clusterin are associated with insulin resistance (IR) and known markers of cardiometabolic risk and 2) how clusterin may contribute to increased risk.

Research design and methods: We validated increased clusterin expression in adipocytes from a separate group of 18 lean and 54 obese individuals. The relationship of clusterin gene expression and plasma clusterin with IR, cardiovascular biomarkers, and risk of cardiovascular disease (CVD) was then determined. Further investigations in human cultured cells and in aged LDLR^-/- mice prone to development of obesity-associated complications were performed.

Results: SAd clusterin correlated with IR, multiple CVD biomarkers, and CVD risk, independent of traditional risk factors. Circulating human clusterin exhibited similar associations. In human adipocytes, palmitate enhanced clusterin secretion, and in human hepatocytes, clusterin attenuated insulin signaling and APOA1 expression and stimulated hepatic gluconeogenesis. LRP2 (megalin), a clusterin receptor, highly expressed in liver, mediated these effects, which were inhibited by LRP2 siRNA. In response to Western diet feeding, an increase in adipocyte clusterin expression was associated with a progressive increase in liver fat, steatohepatitis, and fibrosis in aged LDLR^-/- mice.

Conclusions: Adipocyte-derived clusterin is a novel ECM-related protein linking cardiometabolic disease and obesity through its actions in the liver.

Figure 1

A: SAd and SATM gene expression of CLU in lean and obese subjects. B: Clusterin protein expression by Western blotting in human lean and obese subjects. C: Serum clusterin concentrations in lean and obese subjects by Student t test. D: SAd fold change in CLU in patients with HOMA-IR <3 or ≥3 by Student t test. *P < 0.05. Multiple linear regression analyses between adipocyte gene expression of CLU by qRT-PCR vs. adjusted HOMA-IR (E) and fasting insulin (F). G: Multiple linear regression analyses between serum clusterin concentrations (pg/mL) vs. adjusted HOMA-IR. Multiple linear regression analyses between SAd gene expression and serum clusterin by qRT-PCR vs. adjusted TGs (H and K), the ratio of Tchol to HDL cholesterol (I and L), and HDL cholesterol levels (J and M). Covariates in regression model included adipocyte CLU expression or serum clusterin and age, sex, BMI, and smoking status. Values on the y-axis are adjusted for dependent variables from the regression model. Patients on HMG-CoA reductase inhibitors (i.e., statins) or other antihyperlipidemic medications were excluded from analyses. Correlation analyses (Spearman) between adipocyte gene expression of CLU by qRT-PCR vs. SBP (N), DBP (O), and 10-year risk of cardiovascular event (P). Patients on antihypertensive medications at the time of measurement were excluded from analyses in G and H, and patients with known CVD were excluded from analyses in I. Ten-year risk score calculated according to the Framingham Risk Score algorithm (25). BP, blood pressure; yr, year.

Figure 2

Clusterin protein in human subcutaneous (SQ) SAd treated with palmitate (PA) (200 μmol/L) and adiponectin (APN) (20 μg/mL) assessed by Western blotting of cell lysates (A) and ELISA (B) to determine the clusterin concentrations in conditioned media of the samples in A and the supernatant of the preadipocytes and mature (differentiated) adipocyte cultures without any treatment. C: Effect of recombinant clusterin (0.5 and 5.0 μg/mL) on insulin (INS)-stimulated protein kinase B (AKT) phosphorylation by Western blotting in HepG2 cells using two different phosphorylated AKT (p-Akt) antibodies. D: Effect of recombinant clusterin (0.1, 1.0, and 5.0 μg/mL) on insulin-stimulated expression by qRT-PCR of genes regulating gluconeogenesis (GCK [glucokinase], PKLR [pyruvate kinase isozyme R], and SREBP1) and APOA1 (apolipoprotein A1) in HepG2 cells. *P < 0.05 vs. insulin samples by one-sample t test. All experiments were completed in triplicate. E: Relative gene expression of clusterin receptor LDL receptor–related protein 2 (LRP2) (megalin) in HepG2, cultured skeletal muscle cells (SKM), and VAd and SAd. F: LRP2 expression in HepG2 cells treated with LRP2 scRNA vs. siRNA and clusterin (2 ng/mL). G: Effect of recombinant clusterin on insulin-stimulated AKT phosphorylation (pAkt) by Western blotting in HepG2 cells transfected with control nontargeting scRNA and LRP2-targeted siRNA. Experiments were performed in duplicate; therefore, statistical analysis was not performed. tAkt, total AKT. H: Effect of recombinant clusterin on insulin-stimulated adipocyte gene expression of LRP2, APOA1, GCK, PCK1, and SREBP1 by qRT-PCR in HepG2 cells transfected with nontargeted scRNA and LRP2-targeted siRNA. *P < 0.05, **P < 0.01, and ***P < 0.001. Veh, vehicle.

Figure 3

Weight (kg) (A), percent body fat (B), percent liver fat by MRI (C), serum clusterin (D), VAd CLU gene expression (E), percent liver fat by histology (H), and individual components of the NAS score (ballooning degeneration, lobular inflammation, and fibrosis) and total NAS score (I) in NASH-prone LDLR^−/− mice fed high-fat WD vs. normal chow (n = 4) for 6 (n = 7), 8 (n = 5), 10 (n = 5), and 11 (n = 6) weeks (wk). Correlation analyses (Spearman) between adipocyte gene expression of CLU by qRT-PCR vs. percent body fat (F) and percent liver fat (G) by MRI. J: Representative pathologic liver specimens in LDLR^−/− mice fed WD vs. normal chow for 6, 8, 10, and 11 weeks. *P < 0.05 vs. chow.