AICAR attenuated HFD-induced adipose inflammation independent of adiponectin
Wild-type and Adipoq
−/− mice were fed a sucrose-matched SFD (10% fat) or HFD (60% fat) for 12 weeks (ESM Fig. 1a). Because this HFD regime has previously been shown to cause systemic disease after 4 weeks, such as renal impairment , we initiated AICAR treatment at week 4 to test the effect of AICAR as an intervention.
As expected, HFD-fed mice gained significantly more weight than mice fed an SFD (ESM Fig. 1b). AICAR is known to increase metabolism and weight loss , even in sedentary mice . Accordingly, we observed that AICAR attenuated weight gain during the final few weeks of the diet regimen, both in wild-type and Adipoq
-/- mice (ESM Fig. 1b). However, AICAR-treated HFD-fed mice weighed significantly more than controls fed an SFD in both mouse strains throughout the study (ESM Fig.1b).
The total number of F4/80+ macrophages was not affected by HFD in perigonadal WAT from wild-type mice (Fig. 1a), in accordance with our previous studies . However, HFD-fed Adipoq
−/− mice presented with an increased number of F4/80+ macrophages (p < 0.05; Fig. 1a). Adipoq
−/− mice fed either diet exhibited a higher percentage of CD11c+ M1 macrophages compared with their respective wild-type controls (p < 0.001; Fig. 1b).
AICAR attenuated HFD-induced WAT inflammation in both mouse strains; in wild-type mice, AICAR treatment reduced the percentage of CD11c+ M1 macrophages (p < 0.01) and increased levels of anti-inflammatory CD206+ M2 macrophages (p < 0.001; Fig. 1b–d). Similarly, AICAR attenuated HFD-induced CD11c+ M1 macrophages in Adipoq
−/− mice (p < 0.001). Macrophage CD206+ expression was increased in SFD-fed Adipoq
−/− mice compared with SFD-fed wild-type mice, but there were no changes between Adipoq
−/− mice on an SFD or an HFD with or without AICAR treatment (Fig. 1c). AICAR also reduced the percentage of cytotoxic CD8+ T cells in both mouse strains but did not affect levels of CD4+ T cells (Fig. 1e–h). Although we and others have previously shown that HFD reduces p-AMPK/AMPK , surprisingly HFD did not alter AMPK activity in WAT in the present study. This may be explained by the fact that we matched sucrose levels in SFD and HFD regimens in the present study. However, as expected, AICAR increased WAT p-AMPK/AMPK levels (Fig. 1i). Furthermore, AICAR did not restore HFD-mediated attenuation of WAT adiponectin in wild-type mice (Fig. 1j).
WAT comprises a myriad of cells, including adipocytes, epithelial cells and leucocytes. To determine if AICAR could alter the macrophage phenotype via direct or indirect effects, we also investigated AICAR-mediated effects on murine macrophages in vitro, using the J774 cell line. Similar to the in vivo findings, AICAR promoted an M1-to-M2 phenotype switch in cultured macrophages, attenuating CD11c++ expression (p < 0.01), while promoting CD206+ expression (p < 0.05; Fig. 2a,b). This correlated with increased p-AMPK/AMPK in this cell line (p < 0.05; Fig. 2c,d), but AMPK/β-actin remained unaltered (Fig. 2c,e).
AICAR partially restored glucose tolerance in obese mice independent of adiponectin
An IPGTT was performed to assess glucose tolerance (Fig. 3a–c). HFD significantly impaired glucose clearance in both wild-type and Adipoq
−/− mice (p < 0.001). However, HFD-fed Adipoq
−/−mice presented with exaggerated glucose intolerance compared with HFD wild-type controls (p < 0.05) (Fig. 3c).
AICAR did not significantly alter the HFD-induced increase in fasting blood glucose in either mouse strain (Fig. 3a,b). However, AICAR partially restored HFD-induced impairment of glucose clearance in wild-type mice (p < 0.05). This AICAR-mediated beneficial effect on glucose clearance was sustained in the Adipoq
−/− mice (p < 0.05; Fig. 3c) and significantly lower levels of blood glucose were observed in AICAR-treated vs untreated HFD Adipoq
−/−at 60 min and 120 min post-glucose injection (Fig. 3b).
AICAR attenuated HFD-induced hepatic steatosis independent of adiponectin
AICAR reduced HFD-induced hepatic steatosis, as evidenced by reduced hepatic vacuolisation (Fig. 4a) and triacylglycerol content (Fig. 4b). The drug also attenuated HFD-induced elevations in hepatic cholesterol levels in Adipoq
−/− mice (p < 0.05; Fig. 4c). Furthermore, AICAR-mediated attenuation of hepatic vacuolisation was more pronounced in Adipoq
−/− mice (Fig. 4a). Liver weight/hypertrophy was not altered by AICAR treatment (ESM Fig. 2).
AICAR attenuated HFD-induced kidney disease independent of adiponectin
Wild-type and Adipoq
−/− mice developed significant renal dysfunction following a 3 month HFD regimen, as evidenced by increased albuminuria, urine H2O2 and renal superoxide production compared with SFD (Fig. 5a,b,d), without changes in plasma creatinine (ESM Fig. 3). Furthermore, renal hypertrophy was increased in HFD-fed Adipoq
−/− mice compared with SFD (Fig. 5c). The total number of renal F4/80+ pan-macrophages was not altered by HFD in either mouse strain (Fig. 5e), but HFD significantly increased renal CD11c+ M1 macrophages in wild-type mice (Fig. 5f,g). Renal CD11c+ M1 macrophage levels were higher in Adipoq
−/− mice compared with wild-type mice fed an SFD, but no additional increase was observed in HFD-fed Adipoq
−/− mice (Fig. 5f,h).
AICAR significantly attenuated HFD-induced albuminuria (Fig. 5a), urinary H2O2 (Fig. 5b) and renal superoxide (Fig. 5d) in both wild-type and Adipoq
−/− mice. Furthermore, AICAR attenuated HFD-induced renal hypertrophy in Adipoq
−/− mice (Fig. 5c). Finally, AICAR completely attenuated the HFD-induced increase in renal CD11c+ M1 macrophages in the wild-type strain (Fig. 5f).
AICAR reduced adipose inflammation in tissue explants obtained from obese human patients
WAT inflammation is a key driver of obesity-related pathophysiology [26,27,28,29]. As a proof-of-principle for translating our rodent data to human pathophysiology, we investigated whether AICAR could reduce inflammation and manipulate leucocyte phenotypes in omental WAT explants taken from obese patients undergoing gastric bypass surgery.
The antigens used to phenotype human macrophages varied slightly from the panel used for mice. Thus, we characterised human macrophages using the following activation markers: CD11c+ (M1), CD86+ (M1/M2b), CD206+ (M2a) or CD163+ (M2a/M2c). This classification is based on the Martinez et al scheme, whereby M1 macrophages display potent inflammatory activities, whereas M2a and M2c macrophages downregulate proinflammatory cytokines and promote resolution. Meanwhile, M2b macrophages produce IL-12 and IL-10, and are not anti-inflammatory per se, but rather activate the adaptive B cell responses and regulate B cell and T cell trafficking .
Similar to our findings in mice, compared to vehicle AICAR promoted a shift towards inflammatory resolution in human WAT by increasing the percentage of anti-inflammatory CD206+ macrophages (p < 0.05), while reducing the percentage of proinflammatory CD86+ macrophages (p < 0.05). However, AICAR did not affect human CD11c+ or CD163+ macrophage expression (Fig. 6a,b), or the CD8+ or CD4+ T cell populations (Fig. 6c,d) in this 6 h ex vivo experiment. However, AICAR did reduce TNF-α secretion compared to vehicle (p < 0.001; Fig. 6e).