Flux analysis of cholesterol biosynthesis in vivo reveals multiple tissue and cell-type specific pathways

Abstract

Two parallel pathways produce cholesterol: the Bloch and Kandutsch-Russell pathways. Here we used stable isotope labeling and isotopomer analysis to trace sterol flux through the two pathways in mice. Surprisingly, no tissue used the canonical K–R pathway. Rather, a hybrid pathway was identified that we call the modified K–R (MK–R) pathway. Proportional flux through the Bloch pathway varied from 8% in preputial gland to 97% in testes, and the tissue-specificity observed in vivo was retained in cultured cells. The distribution of sterol isotopomers in plasma mirrored that of liver. Sterol depletion in cultured cells increased flux through the Bloch pathway, whereas overexpression of 24-dehydrocholesterol reductase (DHCR24) enhanced usage of the MK–R pathway. Thus, relative use of the Bloch and MK–R pathways is highly variable, tissue-specific, flux dependent, and epigenetically fixed. Maintenance of two interdigitated pathways permits production of diverse bioactive sterols that can be regulated independently of cholesterol.Cholesterol is important for animals, both as an essential component of the membrane that surrounds cells and as a building block to make hormones and other biologically important molecules. However, cells limit how much cholesterol they make because an excess of this fatty molecule can cause serious health problems, including heart disease and stroke.Cholesterol is made via a complex process that involves more than 30 different steps, which can be organized into two biochemical pathways (named the Bloch pathway and the Kandutsch–Russell pathway). The enzymes that carry out the steps in these pathways have been characterized in detail. Less is known about which of the two pathways is actually used in different cells and tissues, or how much cholesterol each pathway produces. This is partly because it is difficult to distinguish between the closely related intermediate molecules that are formed in each pathway.Mitsche et al. have now used mass spectrometry and isotope labeling techniques to analyze the relative contributions of the two cholesterol-making pathways in both cells grown in the laboratory and in mice. The experiments show that many cells use the Bloch pathway. However, no cells were found to use the Kandutsch–Russell pathway as it was originally described. Rather, some of the cells used a hybrid pathway where the production of cholesterol was started using the Bloch pathway and then after a certain number of steps, the process switched to using part of the Kandutsch–Russell pathway. Mitsche et al. referred to this mixed system as the ‘modified Kandutsch–Russell pathway’.Mitsche et al. next examined the flow of molecules through these two pathways in different tissues and observed that the Bloch pathway is exclusively used in the testes and adrenal glands, which produce high levels of cholesterol. In contrast, the skin and brain use the modified Kandutsch–Russell pathway. In some tissues, a fraction of the building blocks that can be used to make cholesterol were instead diverted to make other products. This suggests that animals have maintained the two pathways over the course of evolution to enable them to generate a variety of products, which can be used to carry out different biological processes. One challenge following this work will be to use the newly developed methods to analyze other complex biochemical pathways.