Enhanced ANGPTL2 expression in adipose tissues and its association with insulin resistance in obese women

Figures

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  Figure 1. ANGPTL2 mRNA expression in human adipose tissues. ANGPTL2 mRNA in the VAT and SAT of the normal-weight control (n = 32), non-diabetic obesity (n = 31), and obesity with type 2 diabetes (n = 14) groups. β-actin was used as the reference gene; ANGPTL2 mRNA levels are presented in relative units (RU). Data are log-transformed for statistical analysis and shown as the mean ± SE on the original (back-transformed) scale. *p < 0.05 vs. the corresponding fat depot in the control group by ANOVA with Tukey’s test.

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  Figure 2. ANGPTL2 and MMP9 mRNA levels in adipocyte and the SVF obtained from adipose tissues. (a,b) VAT of normal-weight women (n = 8) who received benign gynecological surgery and non-diabetic obese women (n = 8) and obese women with type 2 diabetes (n = 4) who received RYGB surgery, were removed during surgery and digested with collagenase to separate floating adipocytes and non-floating SVF. β-actin was used as the reference gene. *p < 0.05 vs. control group by ANOVA with Tukey’s test, #p < 0.01 vs. adipocyte by paired t-test.

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  Figure 3. Serum ANGPTL2 concentrations and ANGPTL2 mRNA expression in human. (a) Serum ANGPTL2 concentrations in patients with obesity (n = 31), obesity and type 2 diabetes (n = 14), and normal-weight control patients (n = 32). Data are log-transformed for statistical analysis and shown as the mean ± SE on the original (back-transformed) scale. *p < 0.01 vs. control, #p < 0.01 vs. obesity group according to ANOVA and the Tukey’s test. (b) Significant reduction in serum ANGPTL2 concentration following RYGB surgery in obese patients. Serum ANGPTL2 concentration and metabolic parameters were measured before and 5–9 months (average 7.3 months) after surgery in a subgroup of obese patients (n = 23). *p < 0.01 vs. before surgery according to paired t-test. (c) Close association between percentage reduction in serum ANGPTL2 concentrations and percentage reduction in serum IL-34 following the surgery. Percent reduction in serum ANGPTL2 was calculated as [(serum ANGPTL2 before surgery – serum ANGPTL2 after surgery) ÷ serum ANGPTL2 before surgery] × 100. The correlation coefficient (r) between changes in measurements was calculated using Spearman’s correlation.

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  Figure 4. Induction of ANGPTL2 in human adipocytes. (a,b) Up-regulation of ANGPTL2 by ER stressors and proinflammatory cytokines. Fully differentiated human adipocytes (n = 3) were incubated in serum-free media for 24 hours with or without (control) various stressors such as thapsigargin (500 nM), tunicamycin (2 μg/mL), homocysteine (4 mM), TNF-α (10 ng/mL), and IL-1β (10 ng/mL). Effect of chemical chaperon was examined by adding 4-PBA (500 μM) to the culture media 2 hours prior to the treatment with stressors. ANGPTL2 mRNA level was measured by qPCR using 36B4 as the reference gene (a) and secreted ANGPTL2 protein was assessed by measuring its concentration in the culture media by ELISA (b). *p < 0.05 vs. control by ANOVA with Tukey test, #p < 0.05 vs. without 4-PBA by paired t-test. Dotted line represents ANGPTL2 mRNA or protein level at control state without stimulation. (c) Correlation between ANGPTL2 mRNA expression and ANGPTL2 protein secretion in differentiated human adipocytes. The correlation coefficient (r) between the measurements in experiments described above in A & B was calculated using Spearman’s correlation. (d,e) Effects of hypoxia or glucose on ANGPTL2 mRNA expression.…

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  Figure 5. Induction of ANGPTL2 and MMP9 mRNA expression by co-culture of adipocytes and macrophages and reduction of MMP9 mRNA expression by ANGPTL2 blockade. (a,b) Differentiated 3T3-L1 adipocytes were co-cultured for 24 hours with RAW264.7 macrophages using transwell system. Measurement of ANGPTL2 mRNA expression in differentiated adipocytes (a) and MMP9 mRNA expression in macrophages (b) was performed by qPCR (n = 3). Incubation with tunicamycin (2 μg/mL) was conserved in both of cells for 16 hours. *p < 0.05 vs. each of cells alone, #p < 0.05 vs. co-cultured cells. The data were analyzed by ANOVA with Tukey test. (c) Induction of MMP9 mRNA by adipocyte-conditioned media (CM) and partially reduction of MMP9 mRNA expression by anti-ANGPTL2 antibody (4 μg/mL). RAW264.7 macrophages were incubated for 16 hours in the presence of CM, CM+ anti-ANGPTL2 Ab, or CD+ non-specific IgG (n = 3). *p < 0.05 vs. control, #p < 0.05 vs. CM by ANOVA with Tukey test.