Increased adiposity in annexin A1-deficient mice

Abstract

Production of Annexin A1 (ANXA1), a protein that mediates the anti-inflammatory action of glucocorticoids, is altered in obesity, but its role in modulation of adiposity has not yet been investigated. The objective of this study was to investigate modulation of ANXA1 in adipose tissue in murine models of obesity and to study the involvement of ANXA1 in diet-induced obesity in mice. Significant induction of ANXA1 mRNA was observed in adipose tissue of both C57BL6 and Balb/c mice with high fat diet (HFD)-induced obesity versus mice on chow diet. Upregulation of ANXA1 mRNA was independent of leptin or IL-6, as demonstrated by use of leptin-deficient ob/ob mice and IL-6 KO mice. Compared to WT mice, female Balb/c ANXA1 KO mice on HFD had increased adiposity, as indicated by significantly elevated body weight, fat mass, leptin levels, and adipocyte size. Whereas Balb/c WT mice upregulated expression of enzymes involved in the lipolytic pathway in response to HFD, this response was absent in ANXA1 KO mice. A significant increase in fasting glucose and insulin levels as well as development of insulin resistance was observed in ANXA1 KO mice on HFD compared to WT mice. Elevated plasma corticosterone levels and blunted downregulation of 11-beta hydroxysteroid dehydrogenase type 1 in adipose tissue was observed in ANXA1 KO mice compared to diet-matched WT mice. However, no differences between WT and KO mice on either chow or HFD were observed in expression of markers of adipose tissue inflammation. These data indicate that ANXA1 is an important modulator of adiposity in mice, with female ANXA1 KO mice on Balb/c background being more susceptible to weight gain and diet-induced insulin resistance compared to WT mice, without significant changes in inflammation.

Figure 1. Modulation of ANXA1 expression in adipose tissue.

Panel A: Time course of ANXA1 mRNA expression in VAT and SAT of male C57BL6 mice fed chow or HFD for 5, 9 or 13 weeks. Data are expressed as fold difference vs. the respective tissue in chow mice at 5 weeks. ***p<0.001 vs. respective tissue in chow mice. Panel B: Expression of ANXA1 mRNA in VAT SVF and adipocytes of male C57BL6 mice fed chow or HFD for 13 weeks (n = 9). *p<0.05, ***p<0.001 vs. respective fraction in chow mice. Panels C and D: Number (Panel C) and Mean Fluorescence Intensity (Panel D) of ANXA1⁺ cells per mg of tissue in the F4/80^- and F4/80⁺ populations from VAT of male C57BL6 mice fed chow or HFD for 13 weeks evaluated by flow cytometry (n = 5). ***p<0.001 vs. respective cell population in chow mice. Panel E: Expression of ANXA1 mRNA at eight weeks of age in VAT of male WT and ob/ob mice fed chow diet (n = 3). Panel F: Expression of ANXA1 mRNA in VAT of male WT and IL-6 KO mice fed chow or HFD for 13 weeks (n = 3 – 5). **p<.005 vs. WT-HFD, p<.05 vs. WT-Chow. Data are mean ± SEM.

Figure 2. Deficiency of ANXA1 modulates adiposity in female Balb/c mice.

Panel A: Body weight in grams, (n = 14 – 20). Panel B: Fat mass in grams measured by DXA. Panel C: Percentage of fat mass to BW (n = 14 – 19). Panel D: Plasma leptin levels (n = 13 – 17). Panel E: Leptin mRNA expression in VAT (n = 4 – 5). Panel F: Plasma adiponectin levels (n = 13 – 17). Panel G: Adiponectin mRNA expression in VAT (n = 5). Panel H: PPARγ mRNA expression in VAT (n = 4 – 5). Panel I: Lean mass in grams measured by DXA. Panel J: Median adipocyte size in VAT and SAT (n = 4 – 5), H&E-stained slides for VAT and SAT magnified to 10X. *p<.05 vs. respective diet-matched WT group, p<.05 vs. WT-Chow, #p<.05 vs. KO-Chow. Data are mean ± SEM.

Figure 3. Expression of markers of lipolysis in VAT.

Panel A: Expression of ATGL, HSL, and Galectin-12 mRNA in fasted mice and FAS, ACC, and SREBF1 mRNA in fed mice in VAT (n = 5). Panel B: Ratio of pHSL to HSL protein in fasted mice in VAT by western blot (n = 3). *p<.05 vs. respective diet-matched WT group, p<.05 vs. WT-Chow, #p<.05 vs. KO-Chow. Data are mean ± SEM.

Figure 4. Dysregulated glucose metabolism in ANXA1 KO mice.

Panel A: Fasting blood glucose (n = 14 – 20 per group). Panel B: Fasting plasma insulin (n = 9 – 15 per group). Panel C: Glucose tolerance test (GTT): change in blood glucose and area under the curve (AUC) (n = 3 – 5). Panel D: Insulin tolerance test (ITT): change in blood glucose as percentage to baseline and AUC (n = 3 – 5). Panel E: PEPCK and G6Pase mRNA expression in liver (n = 5). *p<.05 vs. respective diet-matched WT group, #p<.05 vs. KO-Chow. Data are mean ± SEM.

Figure 5. Corticosterone and 11βHSD1 levels in WT and ANXA1 KO mice.

Panel A: Plasma corticosterone levels in fed mice (n = 5 – 13). Panel B: Expression of 11βHSD1 mRNA in VAT of fed mice (n = 4 – 5). *p<.05 vs. respective diet-matched WT group, p<.05 vs. WT-Chow, #p<.05 vs. KO-Chow. Data are mean ± SEM.

Figure 6. Markers of VAT inflammation in WT and ANXA1 KO mice.

Expression of CD68, IL-6, IL-1β, CCL2, and IL-10 mRNA in VAT (n = 8 – 10). p<.05 vs. WT-Chow, #p<.05 vs. KO-Chow. Data are mean ± SEM.