Simultaneous induction of glycolysis and oxidative phosphorylation during activation of hepatic stellate cells reveals novel targets to treat liver fibrosis

Background: Upon liver injury, Hepatic Stellate Cells (HSC) transdifferentiate to migratory and proliferative myofibroblasts that produce excessive extracellular matrix proteins causing liver fibrosis. HSC activation... [ view full abstract ]

Background: Upon liver injury, Hepatic Stellate Cells (HSC) transdifferentiate to migratory and proliferative myofibroblasts that produce excessive extracellular matrix proteins causing liver fibrosis. HSC activation supposedly depends on glycolysis, however, the actual shift in glycolytic and/or mitochondrial oxidative phosphorylation (OXPHOS) of quiescent-to-activated HSC has not been established yet. 

Methods: Freshly-isolated quiescent rat HSC (q-rHSC) were culture-activated to a-rHSC. Primary human HSC were isolated from “healthy” tissue from tumor resections and culture-activated (a-hHSC). Pharmacological compounds were used to inhibit glycolysis (2-DG) or OXPHOS (rotenone, antimycin A). We monitored q-HSC and a-HSC by measuring i) mitochondrial respiration (oxygen consumption rate [OCR]) and glycolysis (extracellular acidification rate [ECAR]) by Agilent Seahorse XF, ii) Activity of the glycolytic enzymes, iii) mitochondrial fusion/fission by immunofluorescence microscopy; iv) proliferation and migration with xCELLigence and IncuCyte and v) HSC activation and mitochondrial dynamics by qPCR and Western blotting.

Results: Both glycolysis and mitochondrial oxidative phosphorylation were strongly increased in a-rHSC compared to q-rHSC (Figure A). Extensive mitochondrial fusion was observed in a-rHSC (Figure B), concomitant with enhanced expression of mitochondrial fusion and fission markers, independent of mitochondrial copy number. Inhibition of glycolysis and OXPHOS suppressed Collagen1A1 and α-Sma expression to variable levels in a-hHSC. Glycolytic inhibition decreased a-hHSC migration and proliferation, while OXPHOS inhibition decreased proliferation. Compound-specific effects on a-hHSC morphology were observed using real-time imaging.

Conclusion: Both glycolysis and mitochondrial metabolism are strongly enhanced in activated HSC. Blocking either of these metabolic pathways suppresses HSC activation and are potential targets to treat liver fibrosis.

Keywords: Liver fibrosis, Hepatic Stellate Cells, Metabolism, Mitochondria, Glycolysis

Acknowledgements: Becas Chile, GUIDE, RuG