Author response: Flux analysis of cholesterol biosynthesis in vivo reveals multiple tissue and cell-type specific pathways (2015)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics 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. https://doi.org/10.7554/eLife.07999.001 eLife digest 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. https://doi.org/10.7554/eLife.07999.002 Introduction Cholesterol is an essential structural component of vertebrate cell membranes (Maxfield and Tabas, 2005) and a precursor of vital end products such as bile acids (Russell, 2009) and steroid hormones (Sih and Whitlock, 1968). First identified as a crystalline component of gallstones more than 200 years ago, cholesterol consists of a rigid, planar tetracyclic nucleus and a flexible, iso-octyl side-chain at carbon 17 (Nes, 2011). The molecule is synthesized entirely from acetate through a complex series of over 30 enzymatic reactions that are clustered into four major processes: condensation of acetate to isoprene, polymerization of isoprene to squalene, cyclization of squalene to lanosterol, and finally the conversion of lanosterol to cholesterol (Figure 1). Figure 1 Download asset Open asset Schematic representation of the Bloch and Kandutsch–Russell (K–R) pathways for the enzymatic conversion of squalene to cholesterol. The Bloch pathway, indicated by solid black arrows, is shown on the left. The Kandutsch–Russell pathway, indicated by red arrows, is shown on the right. Additional potential sites of crossover from the Bloch to K–R pathway are indicated by broken arrows. Sterol intermediates that were not measured using deuterium water labeling are shown in gray. https://doi.org/10.7554/eLife.07999.003 Two intersecting pathways have been described for biosynthesis of cholesterol from lanosterol. The two pathways use the same catalytic steps and are distinguished by the stage at which the double bond at C24 in the side chain is reduced. Bloch et al. (1965) proposed that reduction of the double bond in the side chain (Δ24) is the last reaction in the pathway (Figure 1, black arrows). Thus, in the Bloch pathway, cholesterol synthesis proceeds via a series of side-chain unsaturated intermediates to desmosterol, which is reduced by DHCR24 to cholesterol. Subsequently, Kandutsch and Russell (Kandutsch and Russell, 1960a, 1960b) reported that the preputial glands of mice synthesized dihydrolanosterol and other side-chain saturated intermediates that were different from those in the Bloch pathway. They proposed an alternative pathway, the Kandutsch–Russell (K–R) pathway, in which the Δ24 bond of lanosterol is reduced and the conversion of dihydrolanosterol to cholesterol proceeds via 7-dehydrocholesterol (Figure 1, red arrows) using the same enzymes as the Bloch pathway. Later studies showed the presence of saturated side chain intermediates in brain, skin, and eventually in all tissues examined (Schroepfer, 1982). The catalytic mechanisms and regulation of the enzymes that catalyze the Bloch and K–R pathways have been extensively investigated but much less is known about their relative use and physiological significance. Cholesterol biosynthesis is usually measured using radioisotope methods (Dietschy and Spady, 1984) that are highly sensitive but do not provide information about the turnover of intermediate sterols in the biosynthetic pathway. Flux through the Bloch and K–R pathways has not been systematically studied in cultured cells or in vivo. Therefore, the relative use of the two pathways and their responses to changes in cholesterol availability are not known. The reason why both pathways have been maintained is also not known. The biosynthetic intermediates of the two pathways have powerful, but distinct, effects on cholesterol homeostasis (Yang et al., 2006; Lange et al., 2008), fatty acid synthesis (Spann et al., 2012), and inflammation (Spann et al., 2012). Varying the concentrations of specific cholesterol biosynthetic intermediates by differential use of the Bloch and K–R pathways may thus contribute to regulation of diverse cellular processes. The analytical challenges associated with distinguishing the multiple closely related sterol intermediates in each pathway, and interpreting the complex patterns of isotope enrichment generated by in vivo labeling have been major obstacles to in vivo studies of the Bloch and K–R pathways. Kelleher and Masterson (1992) demonstrated that stable isotope methods and isotopomer spectral analysis (ISA) could be used to measure the flux of cholesterol biosynthetic intermediates such as lathosterol in cultured cells and in living animals using gas chromatography–MS (Lindenthal et al., 2002). More recently, McDonald et al. (2012) developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to determine steady-state concentrations of most of post squalene cholesterol biosynthetic intermediates of the Bloch and K–R pathways in biological fluids. Here we combined these approaches to examine the flux of substrate though the Bloch and K–R pathways in cultured cells and in vivo in the tissues of mice. Results Applying stable isotopes to measure sterol biosynthesis rates As a first step towards characterizing the flux of precursor sterols through the cholesterol biosynthetic pathway, we used LC-MS/MS and deuterium oxide (D2O) labeling to analyze the turnover of a representative sterol, lanosterol, in immortalized human skin fibroblasts (SV-589 cells). The isotopomer distributions of lanosterol before and 24 hr after addition of 5% D2O to the medium are shown in Figure 2A. In unlabeled cells, the isotopomer spectrum matched the predicted distribution based on a 1.1% natural abundance of 13C and a 0.015% natural abundance of deuterium (Figure 2A, top, left panel). Lanosterol molecules containing exclusively 12C, 16O and 1H isotopes had an m/z peak of 409 Da (M = 0) and comprised 72% of the total. Lanosterol molecules containing a single extra neutron (i.e., one atom of 13C or 2H) had an m/z peak of 410 Da (M = 1) comprised 24% of the total while those containing two extra neutrons (M = 2) comprised 4%. Figure 2 with 1 supplement see all Download asset Open asset Sterol biosynthesis in cultured cells. (A) Deuterated water (D2O) labeling of lanosterol in cultured cells. (Top) Isotopomer spectrum of lanosterol in SV-589 cells grown for 24 hr in the absence (left) or presence (right) of 5% D2O added to the medium. (Bottom) Turnover of lanosterol in SV-589 cells. Cells were grown in NCLPPS (open circles) or in NCLPPS plus 25-hydroxycholesterol (1 μg/ml) (closed circles) to ∼60% confluence. After 16 hr , the medium was supplemented with 5% D2O and cells were harvested at 0, 0.5, 1, 2, 4, 6, 8, 12, and 24 hr (last two points not shown, but used for modeling). Sterols were analyzed by LC-MS/MS and the fraction of lanosterol that was newly synthesized was determined using isotopomer analysis (IA) (see Figure 2—figure supplement 1) and the results were fit to a first-order kinetic model (solid lines) as described in the ‘Materials and methods’. The rates of synthesis of lanosterol in the presence or absence of sterols were calculated by multiplying the first-order rate constant by the concentration of lanosterol. Standard deviations are reported based on four independent replicates. (B) Biosynthetic rates of intermediary sterols in cultured cells. The rate of synthesis of intermediary sterols in the Bloch (left) and K–R (right) pathways was measured using D2O labeling. Cells were plated at a density of 500,000/60 mm dish and grown to ∼60% confluence. D2O was then added to the medium to a final concentration of 5% (vol/vol). Cells were harvested at 0, 0.5, 1, 2, 4, 6, 8, 12, and 24 hr, and lipids were extracted using methanol-dichloromethane. Sterols were analyzed by LC-MS/MS and the fraction of each sterol that was newly synthesized was determined using IA and the results were fit to a first-order kinetic model as described in the ‘Materials and methods’. Y1-BS1 cells, a mouse adrenal cell line (Top) were grown in 15%horseserum. SV-589 cells, an immortalized human skin fibroblast line (bottom), were grown in either 10% FCS. Means and standard deviations are reported based on four independent replicate experiments in each cell line. ***p < 0.001. https://doi.org/10.7554/eLife.07999.004 Cells grown in the presence of D2O incorporate deuterium into newly synthesized sterols, primarily via deuterated NADPH, causing a decrease in the abundance of M0 isotopomers and an increase in the proportion of heavier molecules. After cells were grown in 5% D2O long enough for the lanosterol pool to be replaced (in this case, for 24 hr), only 25% of the lanosterol had not incorporated any deuterium atoms (M = 0). Over 75% of the lanosterol molecules had incorporated at least one heavy atom (M1 + M2 + M3) (Figure 2A, top, right panel). The largest fraction of lanosterol isotopomers (37%) contained a single deuterium or 13C atom (M = 1). The shift in isotopomer distribution over time was used to infer the incorporation of deuterium, which was then used to measure the rate of lanosterol synthesis as described in the ‘Materials and methods’ and reviewed in Figure 2—figure supplement 1. Next, we examined the effect of 25-hydroxycholesterol (25-OH Chol), a potent suppressor of cholesterol biosynthesis (Kandutsch et al., 1977), on lanosterol turnover in cultured fibroblasts (Figure 2A, bottom, left panel). Cells were grown for 16 hr in the presence or absence of 25-OH Chol (1 μg/ml) prior to addition of 5% D20 to the medium. The rate of lanosterol biosynthesis was determined by sampling cells at the indicated time points. At each time point the fraction of newly synthesized lanosterol molecules (termed ‘g’) was determined from the isotopomer spectrum. The relationship between time and g was fitted to a first-order kinetic model to determine the rate constant (k), which was multiplied by the lanosterol concentration to determine the rate of synthesis (ng/hr/μg protein). The addition of 25-OH Chol to the medium decreased the rate of lanosterol biosynthesis by 90% (Figure 2A, bottom). Relative utilization of the Bloch and K–R pathways in cultured adrenal cells and fibroblasts To determine the relative utilization of the Bloch and K–R pathways in various cell types, we measured and compared the rates of deuterium incorporation from D2O into post-squalene cholesterol biosynthetic intermediates in cultured mouse adrenal cells (Y1-BS1 cells) (Watt and Schimmer, 1981) and transformed human fibroblasts (SV-589 cells) (Yamamoto et al., 1984) (Figure 2B). Deuterium was incorporated almost exclusively into Bloch pathway intermediates in Y1BS1 cells, in which the rates of incorporation were similar for lanosterol, ff-MAS, t-MAS, dehydrodesmosterol, and desmosterol (Figure 2B, top left panel). Little turnover of K–R intermediates was detected in these cells (Figure 2B, top right panel). In SV-589 fibroblasts (Figure 2B, bottom panel), lanosterol was quantitatively converted to ff-MAS and t-MAS (Bloch pathway) with almost no detectable incorporation into the corresponding K–R intermediates (dihydro-ff-MAS and dihydro-t-MAS). Incorporation into the downstream Bloch intermediates dehydrodesmosterol and desmosterol was minimal, but robust labeling of 7-dehydrocholesterol was observed, indicating a crossover from the Bloch to the K–R pathway between t-MAS and dehydrodesmosterol (Figure 1). The methylated biosynthetic intermediates between lanosterol and 7-dehydrocholesterol in the K–R pathway (i.e., dihydrolanosterol, dihydro-ff-MAS, and dihydro-t-MAS) did not turnover at comparable rates to either lanosterol or 7-dehydrocholesterol, suggesting that the classical K–R pathway was not used to synthesize cholesterol in these cells. Instead, the cells used a hybrid pathway that we will refer to as the modified Kandutsch–Russell (MK–R) pathway. In this hybrid pathway, intermediates proceed down the Bloch pathway until demethylation of the sterol nucleus is complete, and then they undergo reduction of the double bond at C24 to enter the K–R pathway. The step at which sterol synthesis crosses over from the Bloch to the K–R pathway could not be pinpointed in this experiment since some intermediates (shown in light gray in Figure 1) could not be measured due to either isobaric interference with cholesterol or because the levels were below the detection limits of the assay (see ‘Material and methods’). Our data suggest that in SV-589 cells, sterols are demethylated via the Bloch pathway and then undergo Δ24 reduction upstream of desmosterol. Therefore, in cultured fibroblasts the cross-over to the MK–R pathway occurs at either zymosterol, dehydrolathosterol, or dehydrodesmosterol. These experiments confirmed the conclusion of Kandutsch and Russell that significant differences exist between cells of different types in the pathways utilized for cholesterol synthesis (Kandutsch and Russell, 1960a, 1960b); however, they did not provide evidence that the K–R pathway as originally conceived (Figure 1, red arrows) is utilized, at least in these two cell lines. Pathway utilization and cholesterol biosynthesis To interrogate the factors that determine the relative utilization of the Bloch and MK–R pathway, we examined two cell culture systems in which rates of cholesterol biosynthesis were altered. First, we compared sterol flux in WT Chinese Hamster Ovary (CHO-7 cells) grown in cholesterol-containing medium (10% FCS) or in cholesterol-depleted serum (NCLPPS) to upregulate cholesterol synthesis. Second, cholesterol synthesis was examined in a mutant line of CHO-7 cells (SRD13A cells) that lack SREBP cleavage activating protein (SCAP), a protein required to activate the transcription of cholesterol biosynthesis genes (Rawson et al., 1999). SRD13A cells have no active SREBPs and thus exhibit low rates of cholesterol synthesis (Rawson et al., 1999). In WT CHO-7 cells, the Bloch pathway accounted for ∼90% of cholesterol synthesis (Figure 3A). Deletion of SCAP reduced lanosterol biosynthesis by 80%, with similar reductions in the biosynthesis of other unsaturated side chain Bloch intermediates (Figure 3A). Despite the massive reduction in net sterol synthesis in the SRD13A cells, the biosynthetic rate of 7-dehydrocholesterol was similar to that observed in the parental cell line (Figure 3A, inset). This finding suggests the flux through the MK–R pathway is constitutive in these cells. Figure 3 Download asset Open asset Modification of cholesterol biosynthesis rate. (A) Biosynthetic rate of intermediary sterols of the Bloch (left; black) and K–R (right; red) pathways of CHO-7 (wt, closed bars) and SRD13A cells (SCAP−/−, open bars). Cells were grown to ∼60% confluence in 10% NCLPPS before measuring sterol biosynthesis rates using D2O. The inset of the right panel rescales the dehydrocholesterol values shown below. (B) Sterol biosynthesis rate in HuH7 cells grown in FCS (open bars) or the cholesterol depleted medium NCLPPS (solid bars). Cells were grown in their respective medium to ∼60% confluence before D2O labeled to measure biosynthetic rates. In panels A and B, means and standard deviations are reported based on four independent replicates of each cell line or condition. (C) Expression of genes encoding enzymes catalyzing the conversion of squalene to cholesterol (shown in the Bloch sequence from left to right) in HuH7 cells grown in either FCS or NCLPPS. Expression is normalized to 36B4. Means and standard deviations are based on six replicates from two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. https://doi.org/10.7554/eLife.07999.006 In cultured human hepatoma cells (HuH-7 cells) grown in cholesterol-replete serum (FCS) (Figure 3B), the pattern of sterol turnover resembled that seen in SV-589 cells (Figure 2B); most of the sterol flux traversed the MK–R biosynthetic pathway (Figure 3B, open bars). When cells were grown for 16 hr in cholesterol-depleted serum (NCLPPS) to stimulate cholesterol biosynthesis, sterol turnover doubled. Essentially all of the increase occurred via the Bloch pathway: flux through the MK–R pathway did not change (Figure 3B, open bars). Therefore, cholesterol depletion shifted the relative utilization of the two pathways such that approximately equal amounts of lanosterol were metabolized via Bloch and MK–R intermediates. Cholesterol depletion also resulted in the incorporation of a small amount of deuterium into dihydrolanosterol, presumably reflecting conversion from lanosterol, but none of the other K–R intermediates prior to 7-dehydrocholesterol were labeled in these cells. To determine if the increase in Bloch pathway utilization associated with upregulation of cholesterol synthesis was coordinated at the transcriptional level, we compared levels of mRNAs encoding cholesterol biosynthetic enzymes in the HuH7 cells grown in FCS or NCLPPS (Figure 3C). Despite a significant increase in Bloch pathway utilization in the cells that were grown in NCLPPS, no corresponding changes were observed in expression of the post-squalene cholesterol biosynthetic enzymes, including DHCR24. Thus, the increase in flux through the Bloch pathway cannot be attributed simply to changes in the expression of SREBP-2 and the coordinated transcriptional upregulation of genes encoding enzymes in the pathway. Next, we examined the relationship between the level of expression of DHCR24, which desaturates the side-chain of the sterol, and the relative use of the Bloch and MK–R pathways. DHCR24 expression and pathway utilization To determine if changing DHCR24 expression altered the relative use of the Bloch and MK–R pathways, we expressed recombinant mouse DHCR24 in human embryonal kidney cells (HEK-293 cells). In HEK-293 cells transfected with vector alone, 78% of cholesterol biosynthesis proceeded through the Bloch pathway (Figure 4A, open bars). Overexpression of DHCR24 resulted in lanosterol being quantitatively converted into the Bloch pathway intermediate zymosterol, but did not increase incorporation of label into the early methyl sterols of the classic K–R pathway. Only about one-third of the labeled was converted to dehydrodesmosterol and desmosterol (Figure 4A, solid however, flux through 7-dehydrocholesterol was increased in these cells, indicating cross-over of intermediates to the MK–R pathway. these data that overexpression of DHCR24 can flux through the MK–R pathway, thus flux through the of the Bloch pathway. evidence was seen of utilization of the classical K–R pathway these Figure Download asset Open asset DHCR24 expression and sterol (A) Sterol turnover in cells DHCR24. HEK-293 cells were cultured in FCS for 2 3 the cells were transfected with vector (open bars) or a encoding DHCR24 (closed bars). After hr, the medium was supplemented with 5% D2O and the incorporation of label into sterols was measured using The rates of synthesis for intermediates in the Bloch (left) and K–R (right) pathways were determined using IA and fitted to a first kinetic DHCR24 was measured by analysis at the hr time point in right panel). Means and standard deviations shown are based on independent experiments. (B) HEK-293 cells were transfected with DHCR24 or an vector After hr, of or to was added to the medium. After hr, cells were harvested and labeled sterols were measured by of labeled cholesterol are shown on the right The inset of the bottom right panel level of lathosterol and The of the labeled sterols were normalized to total protein and to an Means and standard deviations are based on 3 replicates. results were observed in an independent *p < 0.05, **p < 0.01, ***p < 0.001. the point of crossover between the Bloch and MK–R pathways As the flux of some sterol intermediates in the pathway could not be using deuterated which to determine the point of cross-over from the Bloch to the MK–R pathway (Figure 1). To this we HEK-293 cells with isotopomers of lanosterol and or that have distributions not found in natural the conversion of these isotopomers into other sterols we were to all of the stable biosynthetic intermediate sterols in their labeled In this experiment we were also to examine cholesterol biosynthesis from desmosterol and from 7-dehydrocholesterol (Figure bars). The of these experiments from those shown Here the labeled sterol was added to the medium and the distribution of label the sterols of the two pathways was determined at a single time point In cells vector alone, the label in unsaturated side chain sterols of the Bloch pathway, with to no label in the MK–R intermediates (Figure open in panel). In cells DHCR24, the label was detected in lanosterol, ff-MAS, t-MAS, and zymosterol, but not in the downstream Bloch intermediates (Figure closed in panel). Instead, the label in the demethylated saturated side chain sterols the MK–R pathway and These data suggest that is the crossover point between the Bloch and MK–R pathways in these cells these and that the methyl sterols upstream of are for do not have DHCR24. both the of the labeled sterol as Overexpression of DHCR24 did not increase the of or of (Figure panel), suggesting the DHCR24 is not for the conversion of lanosterol to cholesterol. To the crossover point between the Bloch and K–R pathway, HEK-293 cells were with In cells the vector alone, label in zymosterol, dehydrolathosterol, dehydrodesmosterol, and desmosterol (Figure open in panel). A small amount of label was measured in lathosterol and 7-dehydrocholesterol, but not in other K–R intermediates. In cells transfected with DHCR24, only a small fraction of the label was detected in intermediates of either pathway, presumably because they are converted to cholesterol. found no label incorporated into or desmosterol. This may conversion of to or conversion of desmosterol to cholesterol. was detected in the downstream MK–R pathway intermediates and 7-dehydrocholesterol (Figure closed in panel). These are with those using labeled lanosterol and the that is the first substrate for DHCR24 in the MK–R pathway used by these cells. The amount of from was HEK-293 cells were transfected with DHCR24 relative to an vector (Figure suggesting that DHCR24 is for the conversion of to cholesterol in these cells. corresponding to or labeled sterols was seen in cells with Cholesterol biosynthesis in the mouse To examine the pathways of post-squalene cholesterol biosynthesis in we rates of sterol synthesis in different mouse were labeled to approximately 5% D2O by an of D2O via and by their water to D2O. of sterol synthesis were calculated as described in the ‘Materials and methods’ and Figure 2—figure supplement 1. The and sterol concentrations are in 1. In testes, deuterium label was only detected in Bloch pathway intermediates (Figure The rates of synthesis of the methyl sterols ff-MAS and in the Bloch pathway were than those of the demethylated intermediates dehydrolathosterol, dehydrodesmosterol and in the pathway. Thus, a fraction of the t-MAS synthesized was diverted from the cholesterol biosynthetic pathway to produce as sterols et al., The in flux the Bloch pathway between t-MAS and was not observed in any other Figure Download asset Open asset Sterol biosynthesis in mice. (A) Sterol biosynthesis in mouse were to D2O by of and the water to D2O. were at 0, 1, 2, 4, 6, 8, 12, and hr after animals time and analyzed by LC-MS/MS as described in the ‘Materials and methods’. of synthesis of the Bloch (left) and K–R (right) intermediates were calculated using IA and first-order The experiment was in an independent of animals with similar (B) utilization of the Bloch and K–R pathways for post-squalene cholesterol The of cholesterol from the Bloch pathway was determined by the desmosterol synthesis rate by the of the desmosterol and 7-dehydrocholesterol synthesis rates. results were the desmosterol synthesis rate was by the lanosterol synthesis in the testes (C) The utilization of the Bloch pathway using the data shown in compared to the natural of