AICAR ameliorates high-fat diet-associated pathophysiology in mouse and ex vivo models, independent of adiponectin

Abstract

Aims/hypothesis: In this study, we aimed to evaluate the therapeutic potential of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), an activator of AMP-activated protein kinase, for ameliorating high-fat diet (HFD)-induced pathophysiology in mice. We also aimed to determine whether the beneficial effects of AICAR were dependent on adiponectin. Furthermore, human adipose tissue was used to examine the effect of AICAR ex vivo.

Methods: Six-week-old male C57BL/6J wild-type and Adipoq -/- mice were fed a standard-fat diet (10% fat) or an HFD (60% fat) for 12 weeks and given vehicle or AICAR (500 μg/g) three times/week from weeks 4-12. Diet-induced pathophysiology was examined in mice after 11 weeks by IPGTT and after 12 weeks by flow cytometry and western blotting. Human adipose tissue biopsies from obese (BMI 35-50 kg/m2) individuals were incubated with vehicle or AICAR (1 mmol/l) for 6 h at 37°C, after which inflammation was characterised by ELISA (TNF-α) and flow cytometry.

Results: AICAR attenuated adipose inflammation in mice fed an HFD, promoting an M1-to-M2 macrophage phenotype switch, while reducing infiltration of CD8+ T cells. AICAR treatment of mice fed an HFD partially restored glucose tolerance and attenuated hepatic steatosis and kidney disease, as evidenced by reduced albuminuria (p < 0.05), urinary H2O2 (p < 0.05) and renal superoxide levels (p < 0.01) in both wild-type and Adipoq -/- mice. AICAR-mediated protection occurred independently of adiponectin, as similar protection was observed in wild-type and Adipoq -/- mice. In addition, AICAR promoted an M1-to-M2 macrophage phenotype switch and reduced TNF-α production in tissue explants from obese human patients.

Conclusions/interpretation: AICAR may promote metabolic health and protect against obesity-induced systemic diseases in an adiponectin-independent manner. Furthermore, AICAR reduced inflammation in human adipose tissue explants, suggesting by proof-of-principle that the drug may reduce obesity-induced complications in humans.

Trial registration: ClinicalTrials.gov NCT02322073.

Keywords: AICAR; Adiponectin; Inflammation; Kidney disease; Liver disease; Macrophages; Obesity.

Conflict of interest statement

Data availability

Data supporting the results reported in the article can be found in the Sharma laboratory (kumarsharma@ucsd.edu).

Duality of interest

The authors declare that there is no duality of interest associated with this manuscript.

Contribution statement

EB, KS, and CG conceived and designed the study; EB, JLR, GHS, MD, VW, MRE, PB and CB contributed to data acquisition; EB, VW, LF and MQ-J analysed the data; all authors interpreted the data, drafted the article, revised it critically for important intellectual content and approved the final version to be published. KS is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Figures

Fig. 1

AICAR attenuates adipose inflammation independent of adiponectin in obese mice. Wild-type and Adipoq−/− mice fed a 12 week SFD (10% fat) or HFD (60% fat) received vehicle or AICAR (500 μg/g) during weeks 4–12. WAT leucocytes were characterised by flow cytometry; (a)–(d) Macrophages were characterised as pan-macrophages (F4/80+), proinflammatory M1-macrophages (CD11c+) or anti-inflammatory M2-macrophages (CD206+) (n = 5). (e)–(h) Lymphocytes were characterised as T killer (CD8+) vs T helper (CD4+) cells (n = 5). (i), (j) AMPK activation and adiponectin levels were analysed by western blot. AMPK blots were cut as indicated (full blots and details of cutting are presented in ESM Fig. 4) (n = 3). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001; ANOVA with Bonferroni correction; †p < 0.05, ††p < 0.01, †††p < 0.001, Adipoq−/− vs wild-type mice for respective conditions. N/A, not applicable

Fig. 2

AICAR promotes AMPK activation and an M1-to-M2 phenotype switch in cultured macrophages. J774 macrophages were incubated with vehicle or AICAR (1 mmol/l) for 16 h (n = 3). Cells were characterised as (a) proinflammatory M1 (CD11c++) or (b) anti-inflammatory M2 (CD206+) by flow cytometry and (c–e) p-AMPK/AMPK was assessed by western blot. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, paired Student’s t test

Fig. 3

AICAR partially restores glucose tolerance independent of adiponectin in obese mice. Wild-type and Adipoq−/− mice fed a 12 week SFD (10% fat) or HFD (60% fat) received vehicle or AICAR (500 μg/g) during weeks 4–12 of feeding. Glucose tolerance was tested at week 11 by an IPGTT in (a) wild-type (n = 4 for all groups) and (b) Adipoq−/− (n = 3 for SFD and HFD, n = 7 for HFD + AICAR) mice. Circles, SFD; squares, HFD; triangles, HFD + AICAR. (c) Graph of the AUC of IPGTT curves. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ANOVA with Bonferroni correction; †p < 0.05, ††p < 0.01, Adipoq−/− vs wild-type mice for respective conditions

Fig. 4

AICAR attenuates hepatic steatosis independent of adiponectin in obese mice. Wild-type and Adipoq−/− mice fed a 12 week SFD (10% fat) or HFD (60% fat) received vehicle or AICAR (500 μg/g) during weeks 4–12. (a) Representative images of hepatic haematoxylin and eosin staining (magnification × 40). Hepatic (b) triacylglycerol and (c) cholesterol (n = 3). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ANOVA with Bonferroni correction

Fig. 5

AICAR attenuates kidney disease independent of adiponectin in obese mice. Wild-type and Adipoq−/− mice fed a 12 week SFD (10% fat) or HFD (60% fat) received vehicle or AICAR (500 μg/g) during weeks 4–12. (a) Micro-albuminuria, (b) urine H2O2 and (c) renal hypertrophy were assessed (n = 7). (d) DHE was injected 16 h prior to killing the mice and renal DHE oxidation was quantified as a measurement of superoxide production (magnification × 100; n = 4). (e) Renal pan-macrophages (F4/80+) and (f) proinflammatory M1 macrophages (CD11c+) were characterised and quantified by flow cytometry (n = 5). (g–h) Flow cytometry histogram showing number of CD11c+ positive cells in (g) wild-type and (h) Adipoq−/− mice, incubated with vehicle (dotted line), fed an HFD without AICAR (solid line), or fed an HFD with AICAR (dashed line). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ANOVA with Bonferroni correction; †p < 0.05, ††p < 0.01, †††p < 0.001, Adipoq−/− vs wild-type mice for respective conditions

Fig. 6

AICAR reduces inflammation in adipose explants from obese individuals. Omental WAT explants from obese individuals (BMI 35–50 kg/m2; n = 4 individuals) were incubated with vehicle or AICAR (1 mmol/l) for 6 h. (a) Tissue macrophages were characterised as M1 (CD11c+), M1/M2b (CD86+), M2a (CD206+) or M2a/M2c (CD163+) and (b) quantified. (c) T cells were characterised as CD4+ and CD8+ and (d) quantified. (e) Levels of TNF-α in the supernatant fraction were determined by ELISA. Data are presented as mean ± SEM. *p < 0.05, ***p < 0.001, Student’s t tests

Fig. 7

Schematic illustration of proposed AICAR-mediated effects in obesity. (a) In obese mice, AICAR treatment attenuates HFD-induced adipose inflammation, promoting an M1-to-M2 macrophage phenotype switch by reducing CD8+ T cell infiltration, while increasing p-AMPK levels. This results in reduced liver and kidney disease and enhanced glucose tolerance. All of these effects are independent of adiponectin. (b) AICAR mediates a similar M1-to-M2 macrophage phenotype switch in adipose explants isolated from obese individuals undergoing bariatric surgery. MΦ, macrophage