Non-alcoholic fatty liver disease and steatohepatitis are highly associated with obesity and type 2 diabetes mellitus. Cotadutide, a GLP-1R/GcgR agonist, was shown to reduce blood glycemia, body weight and hepatic steatosis in patients with T2DM. Here, we demonstrate that the effects of Cotadutide to reduce body weight, food intake and improve glucose control are predominantly mediated through the GLP-1 signaling, while, its action on the liver to reduce lipid content, drive glycogen flux and improve mitochondrial turnover and function are directly mediated through Gcg signaling. This was confirmed by the identification of phosphorylation sites on key lipogenic and glucose metabolism enzymes in liver of mice treated with Cotadutide. Complementary metabolomic and transcriptomic analyses implicated lipogenic, fibrotic and inflammatory pathways, which are consistent with a unique therapeutic contribution of GcgR agonism by Cotadutide in vivo . Significantly, Cotadutide also alleviated fibrosis to a greater extent than Liraglutide or Obeticholic acid (OCA), despite adjusting dose to achieve similar weight loss in 2 preclinical mouse models of NASH. Thus Cotadutide, via direct hepatic (GcgR) and extra-hepatic (GLP-1R) effects, exerts multi-factorial improvement in liver function and is a promising therapeutic option for the treatment of steatohepatitis.

MEDI0382 is an oxyntomodulin-like peptide with targeted GLP-1/glucagon receptor activity that is in clinical development for type 2 diabetes (T2DM), NASH, and obesity and provides glycemic control and weight loss as well as marked improvements of liver outcomes in humans and mice. In mice, reductions in hepatic lipid were independent of weight loss and resulted from improved mitochondrial respiration and turnover and reduced de novo lipogenesis. As hepatocytes lack the GLP-1 receptor, this effect of MEDI0382 is predominantly modulated via glucagon receptors. After treatment with MEDI0382 or g1437, a glucagon analog, isolated hepatocytes showed increased basal and maximal oxygen consumption, increased mitophagosome number, and a ∼6-fold induction in expression of mitochondrial biogenesis regulator PGC-1α (P ≤ 0.0001). We used phosphoproteomics to identify potential mechanisms of action of MEDI0382 and g1437 on lipogenesis inhibition. After g1437 treatment, primary murine hepatocytes exhibited increased phosphorylation on AMPK, GSK3-α, PHK-β, ChREBP, and HSL—all known PKA substrates. We also identified new glucagon-induced phosphorylations on PHK-α2, PYGL, and ChREBP that were not necessarily PKA consensus motifs, implicating involvement of kinases downstream of PKA. In obese mice, MEDI0382 given for 7 days showed time-dependent phosphorylation of the same sites on AMPK, ChREBP, ACC2, ACLY, and DGAT1. These phosphorylation events are predicted to be inhibitory for lipogenesis. To understand translational potential, we treated primary human hepatocytes with g1437 and found 12 homologous phospho sites, including those on AMPK, ACLY, and DGAT1. Thus, MEDI0382 provides unique antisteatotic benefits for obesity/T2DM comorbidities of NASH/NAFLD via enhanced mitochondrial function and novel glucagon-mediated inhibition of lipogenesis. Disclosure R.C. Laker: Employee; Self; MedImmune. M. Boland: Employee; Self; Gubra, MedImmune. A. Nawrocki: None. K.M. Mather: None. L. Jermutus: Employee; Self; AstraZeneca. Stock/Shareholder; Self; AstraZeneca. M.R. Larsen: None. J. Trevaskis: Employee; Self; Gilead Sciences, Inc., MedImmune. Stock/Shareholder; Self; AstraZeneca. C.J. Rhodes: Employee; Self; AstraZeneca, MedImmune. Funding AstraZeneca

Nonalcoholic fatty liver disease (NAFLD), is associated with metabolic disease and estimated to affect 25% of adults. NAFLD can progress to nonalcoholic steatohepatitis (NASH) which may lead to cirrhosis and hepatocellular carcinoma. There is no current treatment or cure for NASH. AMPK (5’AMP-activated protein kinase) activators have shown potential for treating NAFLD/NASH due to their effects on fatty acid inhibition and cholesterol synthesis. To evaluate this potential, an AMPK activator (herein known as C455) was selected for a preclinical NASH study. C455 demonstrated potent and selective activation of AMPK (cellular EC50= 85 nM). X-ray crystallography showed the compound binds at the ADaM site, and pharmacokinetics determined a bioavailability of 45% and half-life of 3h. Mice (male ob/ob on Amylin-NASH diet for 14-weeks or low-fat control) were dosed orally with vehicle or C455 at 3 or 30mg/kg daily. After 6 weeks of treatment, plasma ALT and terminal liver weight were decreased 32% and 22% respectively in mice treated with 30mg/kg C455 vs. vehicle. This dose also decreased liver lipid from 33% to 23%. Fibrosis via collagen staining did not improve with treatment, but significant decreases in mRNA transcripts for Col1a1(62%), Col1a2(59%), Col4a1(56%), and Timp1(55%) were seen with the high dose. Histology showed dose dependent increases in heart weight relative to body weight, and increased expression of genes associated with cardiac hypertrophy. Mice given 30mg/kg C455 also had increased cardiac glycogen storage compared to vehicle treated or LFD control mice. Our study results show that systemic activation of AMPK in a diet-induced mouse model of NASH reduces plasma ALT, decreases liver lipid, and suppressed hepatic collagen gene expression but shows no histological improvement in fibrosis. Coupled with increased heart weight and glycogen storage, the data suggests that current small molecule activators of AMPK may not be viable therapies for the treatment of NASH-related fibrotic disease. Disclosure K.M. Mather: None. M.L. Boland: None. E. Rivers: None. A. Srivastava: None. M. Schimpl: Employee; Self; AstraZeneca. Stock/Shareholder; Self; AstraZeneca. J. Robinson: None. P.T.C. Wan: None. J.L. Hansen: None. J.A. Read: Employee; Self; AstraZeneca. Stock/Shareholder; Self; AstraZeneca. J.L. Trevaskis: Employee; Self; Gilead Sciences, Inc. D.M. Smith: Employee; Self; AstraZeneca.