Description
Partial inhibition of the key glycolytic enzyme PFKFB3 in myeloid cells causes increased hepatic steatosis and inflammation Tillie RJHA1, De Bruijn J1, Dobie R2, Van Kuijk K1, Gijbels MJ1,3,4, Carmeliet P5,6, Henderson NC2,7, Wouters K8, Sluimer JC1,9 1. Department of Pathology, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University Medical Center, Maastricht, Netherlands 2. Centre for Inflammation Research, The Queen’s Medical Research Institute, University of Edinburgh, Edinburgh, UK 3. Department of Pathology, GROW-School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht, Netherlands 4. Department of Medical Biochemistry, Experimental Vascular Biology, Amsterdam UMC, University of Amsterdam, Amsterdam, Netherlands 5. Laboratory of Angiogenesis and Vascular Metabolism, Center for Cancer Biology, VIB, Department of Oncology, Leuven Cancer Institute, KU Leuven, Leuven, Belgium 6. State Key Laboratory of Ophthalmology, Zhongshan Opthalmic Center, Sun Yat-Sen University, Guangzhou 510060, Guangdong, P.R. China 7. MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK 8. Department of Internal Medicine, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University Medical Center, Maastricht, Netherlands 9. BHF Centre for Cardiovascular Sciences (CVS), University of Edinburgh, Edinburgh, UK Background Macrophages play a central role in liver steatosis and depend highly on glycolysis for energy metabolism, specifically upon pro-inflammatory stimulation. Blockage of glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) reduces both glycolysis and inflammation, and macrophage survival in vitro. We hypothesised that myeloid PFKFB3 deficiency in vivo reduces glycolysis and subsequent pro-inflammatory activity in macrophages, attenuating liver inflammation. Methods Ldlr-/-LysMCre+/-Pfkfb3wt/wt (n=17, Pfkfb3wt/wt) and Ldlr-/-LysMCre+/-Pfkfb3fl/fl (n=20, Pfkfb3fl/fl) mice were fed a 0.25% cholesterol diet (HCD) for 12 weeks, followed by EdU injection prior to sacrifice to assess proliferation and liver phenotype, or glucose- and insulin tolerance testing to assess glucose metabolism. Circulating immune cells and progenitor cells were analysed by flow cytometry. Liver macrophage- (CD68), lipid- (Oil Red O), and collagen content (Sirius Red) were quantified. Extracellular acidification rate of bone marrow-derived macrophages (BMDMs) was assessed with an Agilent Seahorse XF Analyzer. Glucose and lactate levels were determined in BMDM conditioned medium. BMDM proliferation (EdU incorporation, xCELLigence) and lipid uptake (TopFluor cholesterol) were determined. Results Pfkfb3fl/fl BMDMs showed 50% knockdown of Pfkfb3 mRNA (p
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