br Discussion Hepatic dysfunction in the metabolic syndrome
Discussion Hepatic dysfunction in the metabolic syndrome is a driver of negative patient outcomes that requires more therapeutic options. The complexity of liver alterations in this disease – elevated de novo synthesis of fatty acids and cholesterol, reduced clearance of circulating atherogenic LDL, and inflammation and fibrosis that is secondary to steatosis – has proven difficult to treat with single agents. The observation here that pharmacological activation of the energy sensor AMPK, first identified for its actions on lipid and cholesterol pathway enzymes ACC and HMGCR, is capable of such multifactorial impact on the liver in this disease setting is a promising advance. These finding are exciting yet we remain cautious given their preliminary nature; due to the lack of extensive fibrosis development in the DIO and many other mouse NAFLD models and because our observations are based on mRNA expression profiles. Activation of AMPK has been reported to lower hepatic TAG in multiple studies, yet the exact mechanism for TAG lowering has been unclear because of a large number of potential mechanisms for liver TAG lowering that exist. Here we definitively show that a direct activator of AMPK, PF-06409577, is capable of significant reductions in liver TAG through actions originating in the hepatocytes, and therefore rule out a significant role for non-autonomous control of hepatic lipids driven by changes in adipose lipolysis or signaling through hepatic enervation in this model. The primary mechanisms that have been proposed whereby AMPK can lower hepatic TAG autonomously are the inhibition of ACC to cause reduced de novo lipogenesis and increased fatty growth hormone secretagogue oxidation and inhibition of SREBP-1 transcriptional activity leading to reductions of lipogenic gene expression. Here we show that activation of AMPK results in an increase in the expression of SREBP target genes, and resulting increase in protein levels suggesting that the phosphorylation of ACC predominates over this pathway; a finding consistent with recent studies targeting ACC (Fullerton et al., 2013; Harriman et al., 2016; Kim et al., 2017). Our studies do not quantitatively evaluate the importance of hepatic fatty acid oxidation and de novo lipogenesis in the reductions of hepatic triglyceride. Additionally, our in vivo evaluation of PF-06409577 in a high fat fed DIO mouse model, where de novo lipogenesis is not as quantitatively important as other models or diabetic humans, suggests that fat oxidation is likely playing a significant role in the hepatic TAG reduction. Our observation of PF-06409577-mediated increases in plasma ketone bodies is consistent with this. PF-06409577 was also capable of robust reductions in liver TAG in the ZSF1 rat, a genetic model that would be expected to have elevated de novo lipogenesis. Taken together, these data suggest that AMPK activation with PF-06409577 is capable of lowering hepatic lipids in diverse models and likely through a combination of both inhibition of de novo lipogenesis and stimulation of fatty acid oxidation. The increase in hepatic SREBP target genes, both lipogenic and those in the cholesterol signaling pathway, is contradictory to a previous work suggesting AMPK phosphorylates and inhibits SREBP-1c (Li et al., 2011). However, this effect is consistent with the AMPK-mediated phosphorylation and inhibition of HMGCR or ACC, reducing hepatic cholesterol and plasma mevalonic acid or lipid species, initiating increased processing of the SREBP transcription factors, and an increase in the mRNA of their target genes. In the case of ACC inhibition this has been shown carefully in recent reports describing hepatic knockout and pharmacological inhibition of ACC (Kim et al., 2017). This cascade of events is also triggered by pharmacological inhibitors of HMGCR, the statin class of cholesterol lowering drugs, and results in lower de novo cholesterol synthesis and increased LDL clearance because of increases in cell surface LDL-receptor (Goldstein and Brown, 2015). It is also observed with inhibition of lipid synthesis involving pharmacological blockage of ATP citrate lyase or genetic removal of ACC (Kim et al., 2017; Pinkosky et al., 2016). AMPK activation with PF-06409577 is capable of this ultimate outcome of reduced plasma LDL in two models – cynomolgus monkeys and a rat model of metabolic syndrome – suggesting this is an effect with potential for clinical translation. Although the precise mechanisms underlying this LDL lowering remain to be confirmed, the similarities between pharmacological inhibition of HMGCR with statin-class drugs and phosphorylation-based inhibition of HMGCR with PF-06409577 are evident.