α-Conotoxin PIA Recent work has detailed the impact of

Recent work has detailed the impact of genetic α-Conotoxin PIA of AMPK in the liver, revealing only minor phenotypes upon removal in mice (Boudaba et al., 2018). However, these studies very elegantly showed the potential for small molecule-mediated activation of AMPK to elicit large changes in lipid metabolism in hepatocytes. This included increased hepatic fat oxidation and reductions in hepatic lipid and cholesterol synthesis, corroborating other work showing AMPK activation to be a robust inhibitor of anabolic pathways in the liver. These authors also showed that in animals that had elevated de novo lipogenesis because of SREBP1 overexpression AMPK activation using a small molecule activator resulted in a striking reduction in hepatic lipids (Boudaba et al., 2018). In the current study, we use a small molecule activator biased towards AMPK β1 subunits, PF-06409577, in combination with a rodent model that lacks the ability to phosphorylate and inhibit ACC in the liver, to demonstrate the potential for therapeutic benefit in NAFLD and hyperlipidemia. Using both rodent and primate studies, we show that AMPK activation with PF-06409577 is capable of impacting lipid and cholesterol pathways beneficially, suggesting potential for a general improvement in liver health. Our work extends the findings of Boudaba et al. (2018) by studying the impact of AMPK activation in dietary NAFLD models in mice and in primates using a clinically viable small molecule, increasing the expectation of human translation of this line of study.
Materials and Methods
Results The small molecule AMPK activator PF-06409577 potently activates AMPK heterotrimeric complexes that contain the AMPK β1 subunit (Supplemental Figs. 1A-B) (Cameron et al., 2016). The AMPK β1 subunit is the predominant β subunit isoform in mouse and rat livers, unlike the livers of cynomolgus monkeys and humans which express the AMPK β1 isoform but have relatively higher expression of the AMPK β2 isoform (Supplemental Fig. 1C) (Cokorinos et al., 2017; Stephenne et al., 2011; Wu et al., 2013). We sought to evaluate whether there were sufficient AMPK β1 containing heterotrimers in rodent and primate hepatocytes to derive pharmacological benefit from a biased AMPK β1 activator such as PF-06409577. Despite the varying amounts of AMPK β1, treatment of rat, monkey, or human primary hepatocytes with PF-06409577 resulted in a dose-dependent increase in the phosphorylation of ACC at serine 79, a known AMPK phosphorylation site, with an EC50 of 69, 875, or 255nM for rat, monkey, and human primary hepatocytes (Fig. 1A). Consistent with this increase in ACC phosphorylation, PF-06409577 dose dependently lowered acetate incorporation into lipid (a combined measure of de novo lipogenesis and cholesterol synthesis) with an IC50 of 49, 444, or 128nM for rat, monkey, or human primary hepatocytes (Fig. 1B). These data suggested that PF-06409577 would be capable of eliciting pharmacology in a clinical setting in human liver; however, it does suggest that species with lower abundance of AMPK β1 in liver (monkeys and humans) may require higher concentrations of PF-06409577 to elicit the same response as observed in rodents. Hepatocytes isolated from mice lacking AMPK α subunits were studied to assess the requirement of AMPK for the effects of PF-06409577 on lipogenesis. AMPK α1/α2 mice infected with either AAV-TBG-GFP (WT) or AAV-TBG-Cre (HepKO) were isolated and showed a dramatic reduction in AMPK α subunits, ACC phosphorylation at Serine 79, and stimulation of AMPK or ACC phosphorylation following treatment with 10μM PF-06409577 (Fig. 2A). In studies of de novo lipogenesis PF-06409577 delivered at a concentration of 10μM was capable of lowering acetate incorporation into the saponifiable lipid fraction in hepatocytes from wild type but not AMPK HepKO mice (Fig. 2B). When acetate incorporation was measured in the non-saponifiable fraction, indicative of sterol synthesis, we observed a reduction of synthesis in hepatocytes from wild-type mice that was only partially blunted in the hepatocytes from AMPK HepKO mice (Fig. 2C). To examine the specificity of PF-06409577 to inhibit fatty acid synthesis through AMPK inhibition of ACC, hepatocytes were isolated from mice that lacked the key inhibitory AMPK phosphorylation sites on ACC1 (Ser79Ala) and ACC2 (Ser212Ala) (ACC-DKI mice). In hepatocytes from ACC-DKI mice, the effect of PF-06409577 to acutely suppress fatty acid (Fig. 2D) was blunted relative to wild-type controls.