Resolution of NASH and hepatic fibrosis by the GLP-1R and GCGR dual-agonist cotadutide via modulating mitochondrial function and lipogenesis

Abstract

Non-alcoholic fatty liver disease and steatohepatitis are highly associated with obesity and type 2 diabetes mellitus. Cotadutide, a glucagon-like protein-1 receptor (GLP-1R) and glucagon receptor (GCGR) agonist, was shown to reduce blood glycaemia, body weight and hepatic steatosis in people with type 2 diabetes mellitus. Here, we demonstrate that the effects of cotadutide in reducing body weight and food intake and improving glucose control are predominantly mediated through Glp-1 signalling, whereas its action on the liver to reduce lipid content, drive glycogen flux and improve mitochondrial turnover and function are directly mediated through Gcg signalling. 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, consistent with a unique therapeutic contribution of GCGR agonism by cotadutide in vivo. Notably, cotadutide also alleviated fibrosis to a greater extent than liraglutide or obeticholic acid, despite dose adjustment to achieve similar weight loss in two preclinical mouse models of NASH. Thus, cotadutide, via direct hepatic (GcgR) and extrahepatic (Glp-1R) effects, exerts multifactorial improvement in liver function and is a promising therapeutic option for the treatment of steatohepatitis.

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Fig. 1: Metabolic and hepatic parameters following 2-week treatment of DIO Glp-1R WT or KO mice with cotadutide, liraglutide, g1437 or liraglutide+g1437.
Fig. 2: Temporal changes in metabolic and hepatic parameters following 1-week treatment of DIO C57Bl6/J mice with cotadutide, liraglutide, g1437 or liraglutide+g1437.
Fig. 3: Hepatic glycogen flux following 1-week treatment of DIO C57Bl6/J mice with cotadutide, liraglutide or g1437.
Fig. 4: Cotadutide-induced changes in the hepatic phosphoproteome of carbohydrate-metabolism- and lipid-metabolism-related molecules.
Fig. 5: Cotadutide induces mitochondrial turnover and improves mitochondrial respiration.
Fig. 6: Superior efficacy of cotadutide on NASH end points in C57BL6/J mice fed an AMLN diet for 29 weeks, compared with liraglutide and OCA treatment.
Fig. 7: Superior reductions in liver lipids with cotadutide versus liraglutide in an ob/ob AMLN mouse model of NASH.
Fig. 8: Cotadutide further reduces hepatic fibrosis and inflammation compared with that after liraglutide or diet switch in an ob/ob AMLN mouse model of NASH.
Fig. 9: Summary of cotadutide mechanism of action at target organs.

Data availability

The datasets generated during these studies are available from the corresponding author on reasonable request. These datasets include: in vivo data for the mouse studies and encompass metabolic, biochemical and histological data/images; mass spectrometry data from mouse liver and/or serum, mouse hepatocytes or human hepatocytes; imaging and quantitation data for mitochondrial analyses; qRT–PCR data for select gene expression analyses. RNA-seq datasets are available in the SRA database with accession number PRJNA574649.

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Acknowledgements

The authors wish to thank the Lab Animal Resource staff at MedImmune/AstraZeneca for their assistance with animal husbandry and care. We thank D. Leeming and M. Karsdal (Nordic Biosciences, Denmark) for assistance with circulating collagen fragment detection and interpretation. We thank K. Hightower (Metabolon, Durham, NC) for assistance with metabolomics analyses and interpretation. The authors also wish to thank M. Jain (AstraZeneca, Cambridge, UK) for critical review and comments on the manuscript during preparation. The Villum Center for Bioanalytical Sciences at University of Southern Denmark is acknowledged for access to high-end MS instruments. Vanderbilt MMPC and their NIH funding (DK059637).

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Contributions

M.L.B., R.C.L., A.N., M.F., M.R.L., L.L., O.P.M. and J.L.T. designed experiments; M.L.B., R.C.L., A.N., S.O., B.B.B., H.L., J.C., K.M., J.N., M.F., M.R.L., L.L., O.P.M., C.J.R. and J.L.T. collected and/or analysed and interpreted experimental data; S.G. and S.S.V. provided pathology analysis of mouse NASH studies; M.L.B, R.C.L., J.G., M.R.L, C.J.R. and J.L.T. wrote the paper; C.M.R. and L.J. reviewed and edited the manuscript.

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Correspondence to Christopher J. Rhodes.

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The authors declare competing interests as defined by Nature Research. Employee of AstraZeneca (R.C.L., K.M., S.O., J.C., J.N., J.G., L.J., C.J.R.). Owns stock in AstraZeneca (K.M., S.O., J.C., J.N., J.G., L.J., C.M.R., J.L.T., C.J.R.).

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Extended data

Extended Data Fig. 1 Metabolic and hepatic parameters following six-week treatment of NASH C57Bl6/J mice.

Mice were treated with cotadutide, liraglutide, g1437 or liraglutide+g1437 at equimolar dosing (10 nmol/kg, SC, QD for 42 days) compared to vehicle. Animals were given ad libitum access to food for the entirety of the study except on day 14 mice were fasted for 6 h prior to ipPTT. a, Reduction in body weight throughout the 42-day dosing period shown as % change. b, Blood glucose profile during ipPTT (pyruvate given at dose of 2g/kg) and (c) area under the ipPTT curve. d, Fasting plasma insulin, (e) fasting blood glucose and (f) plasma ALT levels at the end of the study. g, Terminal liver glycogen content, (h) triglycerides and (i) cholesterol. Chow vehicle (n=10); NASH vehicle (n=11); Cotadutide (n=12); Liraglutide (n=12); g1437 (n=12); Liraglutide+g1437 (n=12). Data shown as the mean ± SEM. (a and b) Two-way ANOVA, Tukey’s multiple comparisons post-hoc test. In (a and b) colored lines and p-values indicated differences for the corresponding treatment group compared with NASH vehicle at each time point. c-i, Two-sided student’s t-test for chow controls vs. NASH vehicle to determine effect of NASH diet; One-way ANOVA, Tukey’s multiple comparisons post-hoc test, chow group excluded.

Extended Data Fig. 2 Table of phosphopeptides.

Description of hepatic phosphopeptides detected in primary mouse and human hepatocytes following treatment with g1437.

Extended Data Fig. 3 Table of phosphopeptides.

Description of hepatic phosphopeptides detected in primary mouse and human hepatocytes following treatment with g1437.

Extended Data Fig. 4 Cotadutide improves mitochondrial respiratory function in primary mouse hepatocytes through Gcg signaling mechanisms.

a, Mitochondrial oxygen consumption rate (OCR) during mitochondrial stress test of healthy primary murine hepatocytes treated ex vivo for 4h with 100 nM cotadutide, g1437 or liraglutide compared to vehicle control hepatocytes (n=3 biologically independent samples/group). b, Basal respiration and (c) maximal respiration measured following injection of uncoupler FCCP. d, Oxygen consumption of primary mouse hepatocytes shown as the percentage of total respiration that is driven by the oxidation of indicated substrates (n=2 biologically independent samples/group). e-i, Oxygen consumption of mouse primary hepatocytes, during mitochondrial stress test, treated with cotadutide +/- Ampk inhibitor, Compound C (e; 20 uM), p38Mapk inhibitor, SB203580 (f; 20 um), mTor inhibitor, Rapamycin (g; 1 uM), Pi3 inhibitor, LY294 (h; 10 uM) or Mapkk inhibitor, PD98 (i; 20 uM) compared with control treated hepatocytes. n=6 replicates from 1 biological samples for all except for Cotadutide; Cotadutide+SB203580; Rapamycin; Cotadutide+Rapamycin; PD98; Cotadutide+PD98 in which 5 replicates were performed. The experiment was performed on 3 separate occasions with similar results. All data shown as the mean ± SEM. b-d, One-way ANOVA with Dunnett’s multiple comparisons post-hoc test.

Extended Data Fig. 5 Cotadutide reduces hepatic fibrosis and inflammation corresponding to animals shown in Figs. 7 and 8.

a, representative αSma stained liver sections, quantification is provided in Fig. 8d. Scale bar = 100 µm. b, representative PSR stained liver sections, quantification is provided in Fig. 8e. Scale bar = 100 µm. These experiments were performed in ob/ob mice with LFD (n=8); AMLN vehicle (n=11); Cotadutide (n=7); Liraglutide (n= 10); AMLN-to-LFD (n=10) mice/group.

Extended Data Fig. 6 Grades of histopathological NASH features corresponding to animals in Figs. 7 and 8.

a, steatosis grade, (b) lobular inflammation, (c) biliary hyperplasia and (d) hepatocyte ballooning. LFD (n=8); Vehicle (n=11); Cotadutide (n=7); Liraglutide (n=10); AMLN-to-LFD (n=10). Data represented as the mean ± SEM. Two-sided student’s t-test for LFD vs. vehicle to determine effect of NASH diet; one-way ANOVA, Tukey’s multiple comparison post-hoc test, LFD group excluded.

Extended Data Fig. 7 CD68, a marker of immune cell infiltration, is reduced by Cotadutide in animals corresponding to Figs. 7 and 8.

a, Representative images of CD68 IHC staining of mouse liver and the (b) pathologist graded scoring. LFD control slides are set to baseline of 1 to account for resident Kupffer cells (dark stained spots disperse equally between hepatocytes). Infiltration of immune cells identified by accumulation around vacuoles we scored relative to the baseline. Scale bar = 200 µm. LFD (n=8); Vehicle (n=11); Cotadutide (n=7); Liraglutide (n=10); AMLN-to-LFD (n=10). Data represented as the mean ± SEM. Two-sided student’s t-test for LFD vs. vehicle to determine effect of NASH diet; One-way ANOVA, Tukey’s multiple comparisons post-hoc test, LFD group excluded.

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Boland, M.L., Laker, R.C., Mather, K. et al. Resolution of NASH and hepatic fibrosis by the GLP-1R and GCGR dual-agonist cotadutide via modulating mitochondrial function and lipogenesis. Nat Metab 2, 413–431 (2020). https://doi.org/10.1038/s42255-020-0209-6

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