Flux analysis of cholesterol biosynthesis in vivo reveals multiple tissue 2 and cell - type specific pathways 3 4 5 Matthew

Key Words: 14 15 Abbreviations: 16 BCA, bicinchoninic acid assay 17 DHCR24, 24 dehydrocholesterol reductase 18 DCM, dichloromethane 19 DMEM, Dulbecco's modified Eagle's medium 20 D 2 O, deuterium oxide 21 FCS, fetal calf serum 22 HS, horse serum 23 IA: isotopomer analysis 24 ISA, isotopomer spectral analysis 25 K-R, Kandutsch-Russell 26 LC, liquid chromatography 27 MeOH, methanol 28 MIDA, mass isotopomer distribution analysis 29 MS, mass spectrometry 30 NCLPPS, newborn calf lipoprotein-deficient serum 31 PBS, phosphate buffered saline 32 SREBP, the sterol response element-binding protein 33 SREBP cleavage activating protein (SCAP) 34 2 Abstract 35 36 Two parallel pathways produce cholesterol: the Bloch and Kandutsch-Russell pathways. Here we used 37 stable isotope labeling and isotopomer analysis to trace sterol flux through the two pathways in mice. 38 Surprisingly, no tissue used the canonical K-R pathway. Rather, a hybrid pathway was identified that we 39 call the modified K-R (MK-R) pathway. Proportional flux through the Bloch pathway varied from 8% in 40 preputial gland to 97% in testes, and the tissue-specificity observed in vivo was retained in cultured cells. 41 The distribution of sterol isotopomers in plasma mirrored that of liver. Sterol depletion in cultured cells 42 increased flux through the Bloch pathway, whereas overexpression of 24-dehydrocholesterol reductase 43 (DHCR24) enhanced usage of the MK-R pathway. Thus, relative use of the Bloch and MK-R pathways is 44 highly variable, tissue-specific, flux dependent, and epigenetically fixed. Maintenance of two 45 interdigitated pathways permits production of diverse bioactive sterols that can be regulated 46 independently of cholesterol. 47 48 3