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

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 Electronic supplementary material The online version of this article (doi:10.1007/s00125-017-4211-9) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

established after 4 weeks HFD-feeding [1]. Thus, as an interventional therapeutic, AICAR (500 μg/g) was given 3 times weekly via intraperitoneal (i.p.) injections, between weeks 4-12 (ESM Fig. 1a). Animals tolerated the treatment well, but displayed signs of hypothermia and cages were thus supplemented with heat 24 h post-injections. Animals were housed in a temperature/humidity controlled room on a 12 h light/12 h dark cycle and were allowed to acclimatize for a minimum of one week prior to experiments. All animal procedures were approved by the institutional animal care and use committee (IACUC), as detailed in the study approval section of the manuscript.
One week prior to sacrifice, a 24 h urine sample was collected through metabolic cages, where animals were housed in duplicate to minimise stress. Albumin, creatinine and albumin-tocreatinine ratio were analysed using DCA Vantage™ Analyzer (Siemens), to assess microalbuminuria. Furthermore, a glucose-tolerance test (GTT) was performed the week prior to sacrifice. Briefly, mice were fasted for 4 h and D-glucose (1 mg glucose per gram body weight) was given intraperitoneally, after which tail-vein blood was drawn to measure plasma glucose levels using a Bayer glucometer at 0, 15, 30, 60 and 120 min post glucose challenge.
At time of harvest, organs were collected under isoflurane sedation to minimise hypoxia. Briefly, 500 mg perigonadal white adipose tissue (WAT) and one half of the right kidney (sectioned vertically) was immediately placed into PBS for flow cytometry analysis. The remaining tissues were either frozen in liquid nitrogen for isolation of protein or RNA, or fixed for a minimum of 24 h in 4% paraformaldehyde (PFA) and embedded in paraffin for immunohistochemistry analysis.

Liver function analysis
Liver tissue (100 mg) suspended in 3 mol/l KOH (in 65% EtOH) was incubated at 70°C for 1 h, to activate digestion, and diluted 1:3 with 2 mol/l Tris-HCl pH 7.5. Subsequently, total free cholesterol and triglycerides were determined in the liver extracts using standardised kits (WAKO Richmond, VA, Cat# 439-17501; Pointe Scientific, Canton, MI, Inc. Cat# T7532-500). Finally, paraformaldehyde fixed and paraffin embedded liver samples were stained by haematoxylin and eosin to visualise liver morphology and vacuolisation.

HPLC plasma creatinine analysis
Plasma (25 μl) was diluted 1:10 with ice-cold acidified acetonitrile (ACN), vortexed and placed on ice for 15 min. Following centrifugation (15 min, 15000 RPM, 4ºC), supernatants were collected and evaporated to dryness to remove the ACN, prior to reconstitution in 50 μl HPLC mobile phase (20 mmol/l sodium acetate pH = 4.2 ± 0.1). Samples were loaded onto a Perkin-Elmer HPLC autosampler tray and run at an isocratic load and elution at a flow rate of 0.3 ml/min over a Zorbax SCX Column (Agilent, Wilmington, DE: 150 mm x 2.1 mm, 5µ particle size) at 50 ± 0.5ºC and UV detector set at 225 nm, resulting in an elude time of approximately 3.5 min. Plasma concentrations were determined by comparing eluted peaks to known creatinine standards (Cayman Biochemicals).

Renal superoxide measurements
Renal superoxide production was measured in a subgroup of mice (n=4 animals/group). These mice received intraperitoneal dihydroethidium (DHE) injections 16 h prior to harvest. Specifically, DHE (total 50 mg/kg) was administered via two injections of 25 mg/kg, given 30 min apart, in order to avoid the DMSO toxicity. At harvest, animals underwent cardiac perfusion for 1 min with saline, followed by 2 min perfusion with 4% paraformaldehyde (PFA) infusion. The kidneys were subsequently harvested and fixed for an additional 48 h in 4% PFA. Organs were sectioned using a vibratome into 55 μm slides and allowed to rest in PBS for 24 h before being mounted onto slides, in order to remove excess PFA and reduce nonspecific florescence. DHE oxidation in postmortem slices were detected using confocal imaging, as a measurement of superoxide levels.

Human adipose explant culture
Adipose biopsies were isolated from the omentum of obese (BMI 35-50 kg/m 2 ), non-diabetic bariatric surgery patients (n=4), undergoing laparoscopic antecolic-antegastric Roux-en-Y gastric bypass construction. Patients received pre-surgical antibiotics, but other medications (e.g. NSAIDs and anti-thrombotic drugs) were given after the collection of tissue biopsies. Larger microvasculature was dissected away and adipose explants were incubated ex vivo with vehicle or AICAR (1 mmol/l) for 6 h at 37˚C. 1 g tissue was used per 2 ml DMEM media. Supernatants were collected and cleared of debris by centrifugation (500g, 5 min, 4˚C) and TNF-α levels were determined using ELISA. Tissues were homogenised and leukocytes were isolated and characterised as described above.

Western blot analysis
Protein lysate in RIPA buffer was obtained from homogenised adipose tissue and serum-starved J774 MΦs, which had been incubated with vehicle or AICAR (1 mmol/l) for 24 h. 40 μg protein was loaded onto a 16% SDS-PAGE gel and was transferred onto PVDF membranes (    Wild-type and Adipoq -/mice fed a 12-week standard-fat diet (SFD; 10% fat) or high-fat diet (HFD; 60% fat) received vehicle or AICAR (500 μg/g) between weeks 4-12. Organ hypertrophy was recorded and normalised to the length of the tibia, as indicated, n=7-10. Data are presented as mean±SEM and were analysed by ANOVA with Bonferroni correction, *p<0.05, ***p<0.001.