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  • An unbiased evaluation of hepatic mRNA was

    2024-10-01

    An unbiased evaluation of hepatic mRNA was performed to assess the impact of hepatocyte AMPK activation on the transcriptome. The expression of 913 genes were altered in control mice after chronic PF-06409577 treatment, 188 genes were affected by knocking out AMPK in hepatocytes, and expression of 29 genes were altered as a result of PF-06409577 treatment in the AMPK HepKO mice (Fig. 4A). To understand the cellular pathways affected by PF-06409577, we used the Ingenuity pathway analysis (IPA) software, which determined hepatic fibrosis/hepatic stellate cell activation as the main pathway affected by PF-06409577 treatment (Fig. 4B). Within this pathway, PF-06409577 reduced the expression of genes in the collagen family in control but not in AMPK HepKO livers, an effect that was confirmed by qPCR measurements of mRNA levels (Fig. 4C-D). In addition to markers of fibrosis, transcriptional targets of the SREBP transcription factors and mitochondrial proteins exhibited elevated mRNA in the livers of control mice treated chronically with PF-06409577 (Fig. 5A-B). Changes in SREBP-target genes were not accompanied by changes in SREBP-1 protein or mRNA levels, and we did not observe significant changes in the nuclear form of SREBP-1 or SREBP-2 (Supplemental Fig. 3). Given these paradoxical findings which were in contrast to reports of AMPK inhibition of SREBPs and the reduction in hepatic lipids we had observed, we performed an unbiased evaluation of proteomic changes induced by PF-06409577 in the HepG2 hepatoma cell line. Overnight PF-06409577 treatment caused a number of changes to the proteome (Fig. 5C-E), with a clear induction of SREBP target genes involved in fat and cholesterol biosynthesis (Fig. 5C), consistent with the observed effects in livers of mice treated with PF-06409577 chronically. We also observed a cluster of ribosomal proteins related to mTOR signaling with reduced protein expression (Fig. 5D), and a cluster of mitochondrial proteins (Fig. 5E) that were significantly elevated in HepG2 Z-DEVD-AFC receptor treated with PF-06409577. These data suggested that AMPK activation was eliciting a robust elevation in SREBP signaling, a result that may be expected given the reduction in hepatic cholesterol biosynthesis secondary to HMGCR inhibition (Goldstein and Brown, 2015). Circulating cholesterol levels were modestly elevated following PF-06409577 treatment, in both AMPK HepKO and control mice (Fig. 3J), suggesting potential for a non-hepatic effect of the compound in this model. However, the normal HFD-fed mouse has a systemic cholesterol profile that is dissimilar to humans, with very low LDL and high HDL cholesterol, making this model's relevance to humans unclear. Because of the complexity of interpreting the circulating cholesterol profile of the HFD-fed mouse we sought a rodent model with a more human-like cholesterol profile. The ZSF1 rat, a cross between a leptin receptor-deficient and hypertensive-heart failure (SHHF) rats, is Z-DEVD-AFC receptor a cardio-renal model that exhibits hypercholesterolemia but low HDL, similar to humans with hypercholesterolemia (Dower et al., 2017). In chronic studies of PF-06409577 dosed orally at 100, 30, or 10mg/kg in this model there was a dose-dependent increase in liver AMPK activation (Fig. 6A), with no impact on glycemic measures (Salatto et al., 2017). Chronic treatment with PF-06409577 caused a dose dependent reduction in circulating total cholesterol and non-HDL cholesterol in the plasma, and a dose dependent increase in plasma HDL cholesterol (Fig. 6B-C). Similar to the effects observed in DIO mice, treatment of ZSF1 rats with PF-006409577 was capable of reducing the amount of liver triglycerides following chronic treatment (Fig. 6D). The lower AMPK β1 subunit expression in the livers and hepatocytes of cynomolgus monkeys and humans as compared to rodents, and requirement for higher drug concentrations to elicit cellular effects, compelled the in vivo evaluation of PF-06409577 in monkeys to confirm the compound could elicit pharmacology in primates and give confidence in potential for clinically viable efficacy. We evaluated the impact of PF-06409577 on plasma levels of cholesterol, LDL, and HDL during 6weeks of dosing in cynomolgus monkeys. Cynomolgus monkeys showed a reduction in circulating plasma cholesterol and LDL after 2weeks of dosing that persisted over the course of the 6week study (Fig. 6E-F). There was no change in the HDL levels as compared to vehicle treated animals (Fig. 6G).