performed electroporation experiments and analyzed the data. NADPH in differentiating 3T3-L1 mouse adipocytes is made by malic enzyme. The associated metabolic cycle is disrupted by hypoxia, which switches the main adipocyte NADPH source to the oxPPP. Thus, 2H-labeled tracers enable dissection of NADPH production routes across cell types and environmental conditions. NADPH is a key cofactor involved in antioxidant defense and reductive biosynthesis1. It can be produced in cells by several enzymes, including glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase in the oxPPP, methylenetetrahydrofolate dehydrogenase (MTHFD) and aldehyde dehydrogenases (ALDHs) in folate metabolism, and isocitrate dehydrogenases (IDHs) and malic enzyme associated with the tricarboxylic acid (TCA) cycle. The oxPPP is localized to the cytosol and is NADPH specific, whereas different isozymes of MTHFD, ALDH, malic enzyme and IDH are found in the cytosol and mitochondria and may generate NADPH or NADH2,3. The importance of the oxPPP in NADPH production is the best established. G6PDH deficiency is the most common human enzyme deficiency and leads to oxidative stress in red blood cells4. 13C-labeled tracers have long been used to follow metabolic activity, but they provide only indirect information on the sources of redox cofactors such as NADPH. They are inadequate when the same carbon transformation can produce NADPH or NADH depending on the isozyme involved. To address this limitation, hydride transfer from [2H]glucose or [2H]serine into NADPH in cells has been tracked directly by mass spectrometry5. Related work has traced compartment-specific NADPH hydride 2H labeling using 2-hydroxyglutarate as a reporter metabolite6. 2-hydroxyglutarate is made by NADPH-driven -ketoglutarate reduction by mutant IDH, with IDH1 localized into the cytosol and IDH2 to mitochondria. Both the direct NADPH 2H labeling measurements A 967079 and the 2-hydroxyglutarate reporter approach revealed that the oxPPP is the largest cytosolic NADPH source in typical transformed cells in culture, with other pathways collectively making a roughly comparable contribution. Whether different enzymes have a predominant role in certain cell types or conditions remains unknown. The most NADPH-demanding biosynthetic activity in mammals is fat synthesis, which consumes a majority of cytosolic NADPH in typical transformed cells in culture5. In intact mammals, fat synthesis is thought to be localized primarily to liver and adipose tissue7. Significant malic enzyme activity was described in adipose tissue more than 50 years ago8,9. During adipocyte differentiation, there is coordinate upregulation of ATP citrate lyase and cytosolic malic enzyme (ME1), which together with A 967079 cytosolic malate dehydrogenase and at the expense of 1 1 ATP molecule, can convert citrate and NADH into acetyl-CoA, NADPH and pyruvate10. Acetyl-CoA and NADPH are the two key substrates for fat synthesis, and the resulting pyruvate can be used to make more citrate. Thus, it is efficient to use malic enzyme to make NADPH in adipose tissue. The quantitative contribution of different NADPH-producing enzymes in adipose, however, remains ill defined. Prior quantitative studies suggest a ~60% contribution from the oxPPP and the remainder from other pathways11C14. Here we employ 2H tracing to quantitatively analyze NADPH metabolism in the common tissue culture model of adipose, 3T3-L1 adipocytes. 2H tracers for the oxPPP and folate metabolism were recently established5,6, but suitable tracers for malic enzyme were lacking. We demonstrate the utility of both [2,2,3,3-2H]dimethyl-succinate and [4-2H]glucose for tracing hydride flux from malate to NADPH and into fat. Combining this approach with 13C labeling studies shows that malic enzyme is the main NADPH source in normoxic 3T3-L1 adipocytes, with total NADPH production more than double that from the oxPPP. Adipocyte differentiation and associated A 967079 fat synthesis continue in hypoxia, but the mode of NADPH production changes dramatically, with malic enzymes contribution becoming minimal and the oxPPPs predominant. RESULTS Quantitative analysis of 3T3-L1 cell NADPH consumption 3T3-L1 preadipocytes cells grow in standard tissue culture medium and can be induced to differentiate into adipocytes by addition of a hormone cocktail15,16. We monitored cell proliferation, cell volume expansion and associated lipid accumulation during the differentiation process (Fig. 1a,b). As we observed the A 967079 fastest lipid accumulation between days 4 Rabbit Polyclonal to ROCK2 and 7 of differentiation, we used adipocytes at day 5 of differentiation in subsequent analyses of metabolic activity. Open in a separate window Figure 1 NADPH consumption during adipogenesis(a) Changes in cell number and packed cell volume (PCV) in normoxic 3T3-L1 cells after initiation of differentiation; values are relative to day 0. (b) Total saponified fatty acids from cells in a. (c) NADPH biosynthetic consumption flux in proliferating and differentiating (day 5) 3T3-L1 cells. Data are mean s.d., = 3 independent culture dishes analyzed in parallel (similar results were obtained on two independent occasions). NADPH drives the synthesis of deoxyribonucleotides, proline and fatty acids. We investigated NADPH biosynthetic consumption flux in proliferating and differentiating 3T3-L1 cells. To determine the.