Lipoxin A4 Attenuates Obesity-Induced Adipose Inflammation and Associated Liver and Kidney Disease

Abstract

The role of inflammation in obesity-related pathologies is well established. We investigated the therapeutic potential of LipoxinA4 (LXA4:5(S),6(R),15(S)-trihydroxy-7E,9E,11Z,13E,-eicosatetraenoic acid) and a synthetic 15(R)-Benzo-LXA4-analog as interventions in a 3-month high-fat diet (HFD; 60% fat)-induced obesity model. Obesity caused distinct pathologies, including impaired glucose tolerance, adipose inflammation, fatty liver, and chronic kidney disease (CKD). Lipoxins (LXs) attenuated obesity-induced CKD, reducing glomerular expansion, mesangial matrix, and urinary H2O2. Furthermore, LXA4 reduced liver weight, serum alanine-aminotransferase, and hepatic triglycerides. LXA4 decreased obesity-induced adipose inflammation, attenuating TNF-α and CD11c(+) M1-macrophages (MΦs), while restoring CD206(+) M2-MΦs and increasing Annexin-A1. LXs did not affect renal or hepatic MΦs, suggesting protection occurred via attenuation of adipose inflammation. LXs restored adipose expression of autophagy markers LC3-II and p62. LX-mediated protection was demonstrable in adiponectin(-/-) mice, suggesting that the mechanism was adiponectin independent. In conclusion, LXs protect against obesity-induced systemic disease, and these data support a novel therapeutic paradigm for treating obesity and associated pathologies.

Figure 1. Lipoxins attenuated adipose inflammation and shift adipose macrophage phenotype towards resolution in vivo

A) Schematic illustration of the protocol: C57BL/6 mice fed a Standard-Fat-Diet (SFD; 10% fat) or a High- Fat-Diet (HFD; 60% fat) for 12 wks, were treated with vehicle, LXA₄ (5ng/g) or benzo-LXA₄ (1.7ng/g) 3 times/wk, between wks 5–12. B) White Adipose Tissue (WAT) macrophage (MΦ) phenotype was analysed by flow cytometry. Single, live cells, pan-leukocytes were identified as inflammatory M1 MΦs (CD11c⁺ of CD45⁺F480^hiCD11b^hi cells) vs. anti-inflammatory M2 MΦs (CD206⁺ of CD45⁺F480^hiCD11b^hi cells). Representative dotplots are shown as well as quantification of both % positive cells and absolute cell numbers, n=4. C) WAT TNF-α and IL-6 expression was analysed by qPCR, n=4. D) WAT adiponectin was analysed by ELISA, n=4. E) WAT Annexin-A1 protein was determined by western blot; a representative blot is shown and densitometry quantification of n=3 experiments. Data is presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001; ANOVA with Bonferroni correction.

Figure 2. Lipoxins attenuated adipose inflammation and shift adipose macrophage phenotype towards resolution in vitro

J774 macrophages (MΦs), constitutively expressing an M1 phenotype, were incubated with vehicle, 1 nM LXA₄ or 10 pM benzo-LXA₄ for 16h. MΦ phenotype was analysed by flow cytometry and supernatants were collected. Serum-starved hypertrophic 3T3-L1 adipocytes were treated with vehicle, 1 nM LXA₄ or 10 pM benzo-LXA₄, or alternatively with MΦ-conditioned supernatants, as indicated. Following 24h incubation, adipocyte supernatants were collected and analysed by ELISA. A) J774 MΦ phenotype was analysed by flow cytometry and representative dotplots are shown of anti-inflammatory M2 (CD206⁺) MΦ and pro-inflammatory M1 (CD11c⁺9 ) MΦ, with respective quantification below, n=3. B) Adipocyte cytokine production was analysed by ELISA, n=3. Data are presented as mean ± SEM; n=3. *P<0.05, **P<0.01, ***P<0.001; ANOVA with Bonferroni correction.

Figure 3. Lipoxins modulate obesity-induced adipose autophagy

C57BL/6 mice, fed a Standard-Fat-Diet (SFD; 10% fat) or a High-Fat-Diet (HFD; 60% fat) for 12 wks, were treated with vehicle, LXA₄ (5ng/g) and benzo-LXA₄ analogue (1.7ng/g) 3 times/wk, between wks 5–12. A) Representative western blots are shown of adipose autophagy markers (p62 and LC3-II protein, normalized to β-Actin). Quantification are expressed as a fold ratio to control (right panels, n=4). B) Representative immunofluorescence staining of WAT LC3-II (green) and p62 (red) is shown (n=3). Data are presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001; ANOVA with Bonferroni correction.

Figure 4. Lipoxins attenuate obesity-induced liver injury

C57BL/6 mice, fed a Standard-Fat-Diet (SFD; 10% fat) or a High-Fat-Diet (HFD; 60% fat) for 12 wks, were treated with vehicle, LXA₄ (5ng/g) or benzo-LXA₄ (1.7ng/g) 3 times/wk, between wks 5–12. A) Liver weight was analysed at harvest, n=10. To further assess liver injury we analysed B) serum alanine aminotransferase (ALT), and C) hepatic and serum triglyceride content; n=4. D–E) Representative images of hepatic F4/80⁺ macrophages and H&E staining is shown; n=3. Data are presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001; ANOVA with Bonferroni correction.

Figure 5. Lipoxins attenuate obesity-induced chronic kidney disease

C57BL/6 mice, fed a Standard-Fat-Diet (SFD; 10% fat) or a High-Fat-Diet (HFD; 60% fat) for 12 wks, were treated with vehicle, LXA₄ (5ng/g) or benzo-LXA₄ (1.7ng/g) 3 times/wk, between wks 5–12. One wk prior to harvest, 24 h urine samples were collected. Parameters of renal injury, including A) albuminuria and B) urine hydrogen peroxide (H₂O₂)/creatinine and C) renal hypertrophy were assessed, n=10. D) Glomerular expansion and matrix deposition were assessed by Periodic acid-Schiff and E) tubulointerstitial collagen by Sirius Red, n=5. Data are presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001; ANOVA with Bonferroni correction.

Figure 6. Lipoxin-mediated protection of obesity-induced pathology is independent of adiponectin

C57BL/6 or adiponectin^−/− mice, fed a Standard-Fat-Diet (SFD; 10% fat) or a High-Fat-Diet (HFD; 60% fat) for 12 wks, were treated with vehicle, LXA₄ (5ng/g) or benzo-LXA₄ (1.7ng/g) 3 times/wk, between wks 5– 12. Parameters of renal injury, including A) albuminuria and B) urine hydrogen peroxide (H₂O₂)/creatinine were analysed, n=10. In figure C) serum alanine aminotransferase (ALT) was analysed, n=4. In figure D) glucose tolerance was assessed over 120 min one wk prior to harvest. The area under curve (AUC) was calculated for respective group and used for statistical analysis, as well as basal fasting glucose, n=10. E) Hypertrophic adipocytes were incubated with vehicle, LXA₄ (1nM) or benzo-LXA₄ (10 pM) for 24h. Alternatively, adipocytes were incubated for 24h with supernatants from MΦ pretreated with vehicle, LXA₄ (1nM) or benzo-LXA₄ (10 pM). Following respective treatment, the adipocyte supernatants were collected and cytokine production was analysed by ELISA, n=3. In Figure F) the endogenous adipose LXA₄ production was assessed by LC-MS-MS in vehicle-treated SFD and HFD Wild Type (WT) and adiponectin^−/− mice (n=7). In Figure G), the HFD-induced fold increase of endogenous LXA₄ production was calculated, excluding the outlier identified in Figure F. Data are presented as mean ± SEM. *P<0.05, **P<0.01, ***P< 0.001. Statistically significant differences between respective condition in the WT versus Adiponectin^−/− strain is indicated as # P<0.05, ##P<0.01, ### P<0.001; ANOVA with Bonferroni correction.