Author response: Mitochondrial fusion supports increased oxidative phosphorylation during cell proliferation

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Proliferating cells often have increased glucose consumption and lactate excretion relative to the same cells in the quiescent state, a phenomenon known as the Warburg effect. Despite an increase in glycolysis, however, here we show that non-transformed mouse fibroblasts also increase oxidative phosphorylation (OXPHOS) by nearly two-fold and mitochondrial coupling efficiency by ~30% during proliferation. Both increases are supported by mitochondrial fusion. Impairing mitochondrial fusion by knocking down mitofusion-2 (Mfn2) was sufficient to attenuate proliferation, while overexpressing Mfn2 increased proliferation. Interestingly, impairing mitochondrial fusion decreased OXPHOS but did not deplete ATP levels. Instead, inhibition caused cells to transition from excreting aspartate to consuming it. Transforming fibroblasts with the Ras oncogene induced mitochondrial biogenesis, which further elevated OXPHOS. Notably, transformed fibroblasts continued to have elongated mitochondria and their proliferation remained sensitive to inhibition of Mfn2. Our results suggest that cell proliferation requires increased OXPHOS as supported by mitochondrial fusion. https://doi.org/10.7554/eLife.41351.001 eLife digest Most cells in the body contain many small compartments called mitochondria. These tiny powerhouses can use oxygen to break down molecules of glucose (a type of sugar) and release the energy that fuels many life processes. Mitochondria can also use oxygen to build certain compounds essential for the cell. Rapidly dividing cells, such as the ones found in tumors, need a lot of energy. Yet, they often ‘choose’ to burn much of their glucose through fermentation, a less efficient process that does not require oxygen or mitochondria. In fact, many theories suggest that cells which divide a lot decrease the quantity of oxygen their mitochondria consume. It is still unclear what role mitochondria have during phases of intense growth, and if they act differently in cancerous and healthy cells. Here, Yao et al. use a cell system where division can be turned on or off, and find that when cells quickly multiply, their mitochondria actually consume more oxygen. Further experiments then reveal that, in both cancerous and healthy cells, the different mitochondria inside a cell merge during periods of intense division. This mechanism allows the compartment to better use oxygen. Yao et al. go on to show that adjusting the fusion process through genetic manipulation helps to control division. When mitochondria cannot combine, cells divide less well; when the compartments can merge more easily, cells multiply faster. If growing cells do not rely on their mitochondria for their energy demands during multiplication, why do these compartments seem to be essential for division? The reason might be that the mitochondria produce aspartate, a molecule that cells use to replicate. The work by Yao et al. suggests that at least certain cancer cells may increase their consumption of oxygen to sustain their mitochondria; armed with this knowledge, it may be possible to design new diagnostic tests and new treatments to identify, and potentially target these oxygen-dependent tumor cells. https://doi.org/10.7554/eLife.41351.002 Introduction Depending on cell type and microenvironment, various adaptations in metabolism have been associated with cellular proliferation. The metabolic adaptation that has received the most attention is a phenomenon known as aerobic glycolysis or the Warburg effect, which is characterized by a high level of glucose consumption and a high rate of glucose fermentation to lactate irrespective of oxygen availability (Liberti and Locasale, 2016; Vander Heiden et al., 2009). Although the Warburg effect is recognized as a typical feature of dividing cancer cells and is the basis for imaging many tumors in the clinic with fluorodeoxyglucose positron emission tomography (Hanahan and Weinberg, 2011; Zhu et al., 2011), it is also found in normal proliferating cells such as non-transformed fibroblasts, lymphocytes, macrophages, thymocytes, endothelial cells, and embryonic stem cells (Brand, 1985; Hedeskov, 1968; Hume et al., 1978; Munyon and Merchant, 1959; Wang et al., 1976). Accordingly, even in non-cancerous contexts, the Warburg effect has been classified as a hallmark of rapid proliferation (Abdel-Haleem et al., 2017). Although there is a general consensus that glycolytic flux increases in proliferating cells, the extent to which oxidative metabolism is altered has been historically complicated (DeBerardinis et al., 2007; Seyfried, 2015). Warburg originally proposed that cancer cells rely on enhanced glycolysis because of defects in mitochondria (Warburg, 1956). Some cancers do have defective mitochondrial enzymes (e.g. succinate dehydrogenase and fumarase), but it is now well established that most proliferating cells (including cancer) have functional mitochondria (Ahn and Metallo, 2015; Vyas et al., 2016). Indeed, functional mitochondria are essential to the proliferation of some cell types. Studies have shown that oxidative phosphorylation (OXPHOS) may provide the majority of ATP during proliferation and function to support the synthesis of important molecular building blocks such as aspartate (Birsoy et al., 2015; Fan et al., 2013; Rodríguez-Enríquez et al., 2010; Sullivan et al., 2015; Zu and Guppy, 2004). Elevated levels of OXPHOS, however, may not necessarily be required to fulfill such functions. To the contrary, many reports have suggested that proliferating cells suppress mitochondrial respiration and statements that glycolysis is preferred over OXPHOS during proliferation are prevalent in the literature (Whitaker-Menezes et al., 2011). Certain cancers of the bladder, breast, and kidney are depleted of mitochondrial DNA and have decreased expression of respiratory genes (Reznik et al., 2016). Some cancer cells exhibit high levels of mitochondrial fission and have associated decreases in respiratory capacity as mediated by an imbalance of dynamin-related protein 1 (DRP1) and mitofusin-2 (Mfn2) (Chen and Chan, 2017; Rehman et al., 2012; Serasinghe et al., 2015; Xie et al., 2015). In other cases, increasing glucose oxidation by inhibiting pyruvate dehydrogenase kinase has been shown to slow the proliferation of transformed cells (Bonnet et al., 2007). A challenge of quantitating changes in OXPHOS as a function of proliferation has been the confounding experimental factors that are often associated with cancer studies. Tumors contain non-proliferating cell types that may shift the average of metabolic measurements from bulk tissues. Additionally, cancer cells from tumors often have restricted access to oxygen (Brahimi-Horn et al., 2007). Although oxygen limitation can similarly lead to an enhanced glycolytic phenotype, this metabolic program is distinct from the Warburg effect. Finally, many studies have focused on the proliferative state of cancer cells without having an appropriately matched non-proliferating comparison with the same genetic background tested under the same conditions (Zu and Guppy, 2004). In this study, to directly compare oxidative metabolism in the same cells of the quiescent and proliferative state, we exploited the cell-density-dependent phenotype of non-transformed fibroblasts. We find that even though proliferating fibroblasts exhibit enhanced glycolysis that is consistent with a Warburg phenotype, they also increase OXPHOS by nearly two-fold and increase their mitochondrial coupling efficiency by ~30%. Interestingly, both increases are supported by mitochondrial fusion. Although transformation with the Ras oncogene further elevated OXPHOS, the additional increase was supported by mitochondrial biogenesis rather than changes in mitochondrial dynamics. Blocking mitochondrial fusion slowed proliferation in both non-transformed and transformed cells. Taken together, our results indicate that proliferation of fibroblasts requires an increase in OXPHOS supported by mitochondrial fusion. Results Proliferation increases oxidative phosphorylation and mitochondrial coupling efficiency Mouse 3T3-L1 fibroblasts are immortalized, non-transformed cells that retain sensitivity to contact inhibition (Green and Kehinde, 1975). They provide a simple, well-controlled model to compare metabolism in the proliferative and quiescent states, as has been demonstrated previously (Yao et al., 2016a). The first step in our analysis was to verify that proliferating fibroblasts exhibit the Warburg effect. Relative to quiescent fibroblasts in the contact-inhibited state, proliferating cells had increased glucose consumption and lactate excretion (Figure 1A). As expected, proliferating cells excreted a greater percentage of glucose as lactate (47%) compared to quiescent cells (32%) (Figure 1—source data 1). Of note, the absolute amount of glucose having a non-lactate fate was also increased by over two-fold in the proliferative state (0.38 pmol/cell/hr) relative to the quiescent state (0.16 pmol/cell/hr) (Figure 1—source data 1). Glucose carbon that is not excreted as lactate is potentially available to support an increased rate of oxidative metabolism, which we next aimed to quantify. Figure 1 with 2 supplements see all Download asset Open asset In addition to increasing glucose consumption and lactate excretion, proliferating fibroblasts also increase mitochondrial respiration and mitochondrial coupling efficiency. (A) Glucose consumption and lactate excretion rates for quiescent and proliferating fibroblasts (n = 4). As expected, proliferating cells exhibit an enhanced glycolytic phenotype that is consistent with the Warburg effect. (B) Mitochondrial stress test of quiescent and proliferating fibroblasts. OCR was normalized to protein amount to take into account differences in cell size. Displayed OCR values were corrected for non-mitochondrial respiration (n = 3). (C) Measured and calculated parameters of mitochondrial respiration (using results from Figure 1B). We note that the coupling efficiency is calculated as the ratio of the OCR required for ATP production relative to the basal OCR in the same sample and therefore is independent of the sample normalization method (n = 3). (D) Glutamine consumption and glutamate excretion rates for quiescent and proliferating fibroblasts (n = 4). (E) Palmitate and oleate consumption rates for quiescent and proliferating fibroblasts (n = 4). (F–H) Isotopologue distribution pattern of citrate after cells were labeled with U-13C glucose (F), U-13C palmitate (G), or U-13C glutamine (H) for 6 hr (n = 3). Data are presented as mean ±SEM. **p<0.01, ***p<0.001, n.s. not statistically significant. OCR, oxygen consumption rate; oligo, oligomycin; rot, rotenone; AA, Antimycin A. https://doi.org/10.7554/eLife.41351.003 Figure 1—source data 1 Total accounting of glucose utilization in quiescent and proliferating cells. Data are presented as mean ±SEM (n = 4). https://doi.org/10.7554/eLife.41351.006 Download elife-41351-fig1-data1-v1.pptx Figure 1—source data 2 Labeling percentages of 13C-enriched precursors for Figure 1. Data are presented as mean ±SEM (n = 3). https://doi.org/10.7554/eLife.41351.007 Download elife-41351-fig1-data2-v1.pptx Figure 1—source data 3 Mass isotopologue distributions for all metabolites analyzed by LC-MS in Figure 1F–H. https://doi.org/10.7554/eLife.41351.008 Download elife-41351-fig1-data3-v1.xlsx Strikingly, on a per cell basis, we found that the basal respiration rate was ~81% higher during proliferation compared to quiescence (Figure 1—figure supplement 1). Given that proliferating cells are larger in size relative to quiescent cells, we also independently normalized the oxygen-consumption data by protein content instead of cell number. Even when normalized by protein content, the respiration rate of proliferating cells was ~59% higher than quiescent cells (Figure 1B–C). Intriguingly, proliferating cells also exhibited a decrease in proton leak and a ~ 112% increase in ATP production (Figure 1B–C). Taken together, the coupling efficiency of proliferating cells was determined to be 34% higher than quiescent cells. We note that the coupling efficiency was calculated as the ratio of the ATP production rate and the basal respiration rate, which is therefore independent of sample normalization method. These data suggest that proliferating fibroblasts with Warburg metabolism not only have increased OXPHOS, but also that they respire more efficiently. Glutamine is the major carbon source for fueling the TCA cycle and mitochondrial respiration We next aimed to investigate which carbon sources fuel mitochondrial respiration by analyzing the utilization of glucose, glutamine, and fatty acids. In addition to increasing glucose consumption and lactate excretion (Figure 1A), proliferating fibroblasts take up two-fold more glutamine compared to quiescent fibroblasts (Figure 1D). Since the rate of glutamate excretion was unchanged, more glutamine carbon was being used for anaplerosis. We also found that the consumption rates of palmitate and oleate were increased in proliferation by 194% and 98%, respectively (Figure 1E). Since proliferating fibroblasts showed increased utilization of all three nutrients we examined, we next applied stable isotope tracing and metabolomics to access the relative contribution of each carbon source to the TCA cycle. In three separate experiments, cells were fed either uniformly 13C-labeled glucose (U-13C glucose), uniformly 13C-labeled glutamine (U-13C glutamine), or uniformly 13C-labeled palmitate (U-13C palmitate) for 6 hr. The isotope enrichments in citrate, a representative TCA cycle intermediate, were evaluated to infer the relative contribution of each carbon source to the TCA cycle and mitochondrial respiration. Even though proliferating cells consumed more glucose and more fatty acids, citrate labeling from these two carbon sources was significantly decreased in the proliferative state (Figure 1F; Figure 1G). In contrast, labeling of citrate by glutamine was substantial and significantly increased in proliferating cells relative to quiescent cells, suggesting that glutamine is a major energy source to fuel mitochondrial respiration (Figure 1H). This result is consistent with reports from other cells (Fan et al., 2013). Given that previous studies have shown that glutamine dependence is correlated with cystine uptake through the cystine/glutamate antiporter SLC7A11 (Muir et al., 2017), we examined whether these metabolite changes were enabled by transporter expression. Even though the level of SLC7A11 protein was unchanged, we observed more than a two-fold increase in cystine comsumption for proliferating fibroblasts compared to quiescent fibroblasts (Figure 1—figure supplement 2). Increased influx of cystine may drive the export of glutamate, thereby depleting the pool of intracellular glutamate/αKG and promoting glutamine anaplerosis (Muir et al., 2017). Increased oxidative phosphorylation during proliferation is supported by mitochondrial fusion We next sought to understand how fibroblasts support increased OXPHOS during proliferation. We reasoned that one mechanism might be by increasing mitochondrial mass in the proliferative state. We used RT-PCR to determine the relative copy number of mitochondrial DNA (mtDNA) to genomic DNA (gDNA), from which we inferred mitochondrial mass. The mtDNA to gDNA ratio was unchanged between quiescent and proliferating cells (Figure 2A). Consistent with these data, we also observed no change in expression of respiratory enzymes, as determined by immunoblotting of electron transport chain (ETC) subunits (SDHA for complex II, cytochrome c for complex III, COX IV for complex IV, and ATP5A for complex V) (Figure 2B). Our results indicate that proliferating fibroblasts do not support elevated levels of OXPHOS by increasing mitochondrial mass or by increasing the expression of respiratory enzymes. Figure 2 with 5 supplements see all Download asset Open asset Proliferating fibroblasts regulate respiration by mitochondrial fusion. (A) Mitochondrial mass remains the same for quiescent (Q) and proliferating (P) fibroblasts as estimated by the ratio of mtDNA to gDNA (n = 3). (B) Proliferating fibroblasts have similar protein expression levels of ETC subunits as quiescent fibroblasts. (C) Representative EM images of mitochondria in quiescent fibroblasts, proliferating fibroblasts, and proliferating Mfn2 knockdowns show changes in mitochondrial elongation. (D) Statistical analysis of mitochondrial length in quiescent fibroblasts, proliferating fibroblasts, and proliferating Mfn2 knockdowns. For each condition, 100 random mitochondria were measured from EM images. Data are presented as mean ±SD. (E) Mitochondrial stress test of scrambled siRNA control (SSC) cells and Mfn2 knockdown cells (Mfn2KD), both in the proliferating state (n = 3). Data are presented as mean ±SEM. (F) Measured and calculated parameters of mitochondrial respiration (using results from Figure 2E) (n = 3). Data are presented as mean ±SEM. *p<0.05, **p<0.01, ***p<0.001, n.s. not statistically significant. OCR, oxygen consumption rate; oligo, oligomycin; rot, rotenone; AA, Antimycin A. https://doi.org/10.7554/eLife.41351.009 As an alternative, we then considered the possibility that fibroblasts regulate OXPHOS during proliferation by mitochondrial dynamics. Previous studies have shown that mitochondrial fusion is associated with an increased respiration rate in addition to an improved coupling efficiency (Legros et al., 2002; Westermann, 2012). To assess mitochondrial morphology, we applied electron microscopy (EM) imaging (Figure 2C) and fluorescence imaging (Figure 2—figure supplement 1). Both techniques showed that proliferating cells have elongated mitochondria, while mitochondria in quiescent cells were relatively short and fragmented. Quantitative analysis of 100 random mitochondria in each condition showed a statistically significant increase in the mitochondrial length of proliferating cells compared to quiescent cells (Figure 2D). In addition, we found that mitochondria in proliferating cells had a significantly higher level of mitochondrial fusion proteins (Mfn1, Mfn2, and OPA1) compared to mitochondria in quiescent cells (Figure 2—figure supplement 2). When we inhibited mitochondrial fusion by knocking down Mfn2, a protein required for the fusion of the outer mitochondrial membrane, the elongated mitochondrial phenotype in proliferating fibroblasts was suppressed (Figure 2C–D, Figure 2—figure supplement 3). To determine whether mitochondrial fusion is required for increased OXPHOS during proliferation, we compared the oxygen consumption rates of proliferating scrambled siRNA controls (SSC) to Mfn2 knockdowns (Mfn2KD). We point out that these comparisons were conducted when cells were in the proliferating exponential growth phase. Notably, Mfn2 knockdowns had a statistically significant decrease in respiration rate, ATP production, and mitochondrial coupling efficiency (Figure 2E–F). Given that mitochondria in quiescent fibroblasts are already largely fragmented, as expected, the effect of Mfn2 knockdown on basal mitochondrial respiration was minimal in quiescent cells (Figure 2—figure supplement 4). Independent of contact inhibition, cellular quiescence can also be achieved by serum starvation (Yao, 2014). By using serum starvation, we sought to extend our comparison of the proliferative and quiescent states to other cells. Consistent with our contact-inhibition results, we found that serum starved 3T3-L1 and HCT116 cells had fragmented mitochondria relative to the same cells in the non-starved state (Figure 2—figure supplement 5). Mitochondrial elongation occurred as soon as 3 hr after reintroducing serum and continued as cells exited the quiescent state (Figure 2—figure supplement 5B). Inhibiting mitochondrial fusion decreases proliferation by limiting aspartate synthesis Having established that mitochondrial fusion increased in dividing cells, we wished to consider its effects on proliferation. Upon Mfn2 knockdown, we observed a ~ 30% decrease in proliferation rate compared to scrambled siRNA controls (Figure 3A). Re-expressing siRNA-resistant Mfn2 (Mfn2res) in Mfn2 knockdowns restored Mfn2 protein level, mitochondrial respiration, and cellular proliferation (Figure 3—figure supplement 1). When we overexpressed Mfn2 in wildtype 3T3-L1 fibroblasts, we observed a significant increase in both mitochondrial respiration and proliferation (Figure 3—figure supplement 2). These data suggest that promoting mitochondrial fusion is sufficient to drive proliferation. We also observed considerable changes in nutrient utilization between Mfn2 knockdowns and scrambled siRNA controls that were consistent with decreased proliferation and reduced OXPHOS (Figure 3B). Mfn2 knockdowns decreased their glucose uptake by 15% and decreased their lactate excretion by 10%. More notably, knocking down Mfn2 caused cells to decrease glutamine consumption by 40%. Given the reduced rate of OXPHOS in Mfn2 knockdowns, these data are consistent with glutamine serving as a major carbon source for the TCA cycle. Tracing experiments confirmed a significant decrease in labeling of TCA cycle intermediates from U-13C glutamine in Mfn2 knockdowns (Figure 3—figure supplement 3A–C). Consistent with decreased glutamine anaplerosis, Mfn2 knockdowns had a 40% decrease in cystine consumption (Figure 3—figure supplement 3D). Figure 3 with 3 supplements see all Download asset Open asset Inhibition of mitochondrial fusion by Mfn2 knockdown slows proliferation by limiting aspartate synthesis. (A) Proliferation was assessed by using a CyQUANT assay after cells were treated with scrambled siRNA control (SSC) or Mfn2 siRNA for 72 hr (n = 5). (B) Mfn2 knockdown alters nutrient utilization (n = 4). Glutamine consumption decreases in Mfn2 knockdown cells, which is consistent with a decreased demand for glutamine to fuel a reduced level of OXPHOS. (C) Intracellular ATP levels in scrambled siRNA controls (SSC) were lower relative to Mfn2 knockdowns. ATP luminescence signals were normalized to protein amount (n = 5). (D) The intracellular pool of aspartate remained unchanged upon Mfn2 knockdown (n = 3). Pool sizes were normalized by dry cell mass and internal standard. (E) Scrambled siRNA controls excrete aspartate into the media, while Mfn2 knockdowns uptake aspartate from the media (n = 4). (F) Supplementing the media with 1 mM aspartate partially rescued the proliferation of Mfn2 knockdowns (n = 6). Data are presented as mean ±SEM. *p<0.05, **p<0.01, ***p<0.001, n.s. not statistically significant. https://doi.org/10.7554/eLife.41351.015 Figure 3—source data 1 Sequences for dicer-substrate short interfering RNA (DsiRNA) and siRNA-resistant Mfn2res. https://doi.org/10.7554/eLife.41351.019 Download elife-41351-fig3-data1-v1.docx Our results show that mitochondrial fusion supports a high level of OXPHOS, which is needed to sustain rapid cellular proliferation. We speculated that the decrease in proliferation rate upon Mfn2 knockdown might be due to a shortage of energy from the decreased rate of OXPHOS. Surprisingly, however, we found that intracellular levels of ATP actually increased slightly in Mfn2 knockdowns relative to scrambled siRNA control cells (Figure 3C). This result suggested that cells could compensate for a reduced energy yield from OXPHOS, but that OXPHOS may serve another indispensable function in our knockdowns. Previous studies have shown that an essential role of OXPHOS in proliferating cells is to regenerate reducing equivalents in support of aspartate synthesis (Birsoy et al., 2015; Sullivan et al., 2015). Through reactions in the malate-aspartate shuttle, increased oxygen consumption may support a higher rate of aspartate generation. Although we did not observe a difference in the intracellular pool of aspartate between scrambled siRNA controls and Mfn2 knockdowns (Figure 3D), we did find a striking change in aspartate uptake. While the control cells excreted aspartate, the Mfn2 knockdown cells consumed aspartate from the media (Figure 3E). Moreover, the proliferation of Mfn2 knockdowns could be partially rescued by supplementing cell-culture media with aspartate (Figure 3F). H-Ras transformed fibroblasts exhibit higher mitochondrial respiration Although 3T3-L1 fibroblasts are immortalized, unlike transformed cells, they remain sensitive to contact inhibition and retain the ability to differentiate. To evaluate whether transformed cells similarly rely on OXPHOS and mitochondrial fusion, we generated stable transfected 3T3-L1 cells expressing the oncogene H-Ras (G12V), a constitutively active mutant (Figure 4A). H-Ras transfected fibroblasts assumed a transformed morphology and their growth was no longer contact inhibited, with high-density cultures forming multiple layers of cells (Figure 4B–C). It was confirmed that the transformed cells did not undergo oncogene-induced senescence (Figure 4—figure supplement 1). Figure 4 with 4 supplements see all Download asset Open asset H-Ras transformed fibroblasts (Ras) have elongated mitochondria and increased OXPHOS that is supported by mitochondrial biogenesis. (A) Immunoblotting of whole-cell lysates shows H-Ras expression in transformed fibroblasts, but not in empty vector (EV) controls. (B) Growth curve shows the loss of contact inhibition in H-Ras transformed fibroblasts. The proliferation of EV controls remains cell-density dependent (n = 4). (C) Ras cells exhibit a morphological change and gain the ability to grow on top of each other to form multiple cell layers. (D) Mitochondrial stress test of EV controls and Ras cells (n = 3). The respiration of Ras cells is statistically increased compared to EV controls. (E) Measured and calculated parameters of mitochondrial respiration (using results from Figure 4D) (n = 3). (F) Statistical analysis of mitochondrial length in EV controls and Ras cells. In each condition, 100 random mitochondria were measured from EM images. Data are presented as mean ±SD. (G) RT-PCR shows that Ras cells have over a 20-fold increase in mRNA levels of PGC1α (n = 3). (H) Ras cells have increased mitochondrial mass, as indicated by an increased mtDNA/gDNA compared to EV controls (n = 3). (I) Immunoblotting of whole-cell lysates shows that Ras cells have higher expression levels of ETC subunits compared to EV controls. (J) Mfn2 knockdown decreases cellular proliferation in both EV controls and Ras cells. Proliferation was assessed by manual counting after cells were treated with scrambled siRNA control (SSC) or Mfn2 siRNA (Mfn2KD) for 72 hr. Relative proliferation was normalized to either an EV SSC or a RAS SSC for each condition (n = 4). (K) Mfn2 knockdown decreases cellular proliferation in H460 (lung cancer cells), HeLa (cervical cancer cells), BT549 (breast cancer cells), and MCF7 (breast cancer cells). Proliferation was assessed by using a CyQUANT proliferation assay after cells were treated with scrambled siRNA control (SSC) or Mfn2 siRNA (Mfn2KD) for 72 hr (n = 5). Unless specified, data are presented as mean ±SEM. *p<0.05, **p<0.01, ***p<0.001, n.s. not statistically significant. OCR, oxygen consumption rate; oligo, oligomycin; rot, rotenone; AA, Antimycin A. https://doi.org/10.7554/eLife.41351.020 To study the effect of H-Ras transformation on mitochondrial metabolism, we compared the oxygen consumption rates of proliferating empty vector (EV) control cells in the exponential growth phase to H-Ras transformed (Ras) cells. Surprisingly, we found that Ras cells had a ~ 73% increase in basal respiration and a ~ 72% increase in ATP production compared to EV cells (Figure 4D–E). It is interesting to note that we found no difference in the mitochondrial coupling efficiencies between conditions (Figure 4E). This result is consistent with the observation that mitochondria are not further fused in Ras cells relative to proliferating EV controls (Figure 4F). Given that Ras cell mitochondria remain elongated to the same extent as EV controls, we next evaluated increased mitochondrial mass as a possible mechanism to support elevated levels of OXPHOS. For Ras cells, we observed a > 20-fold increase in the mRNA level of peroxisome proliferator activated receptor coactivator (PGC1α), a master regulator for mitochondrial biogenesis (LeBleu et al., 2014; Scarpulla, 2011) (Figure 4G). Ras cells had more mitochondrial mass and increased protein expression levels of ETC subunits (Figure 4H–I), suggesting that increased OXPHOS in Ras cells is driven by mitochondrial biogenesis. Activation of mitochondrial biogenesis upon Ras transformation did not change protein levels of components in the ERK/AMPK pathway or the KSR1 pathway, as has been previously suggested for other cells (Figure 4—figure supplement 2) (Dard et al., 2018; Weinberg et al., 2010). We wish to emphasize that even though Ras cell mitochondria are not further elongated, they remain fused to the same level as proliferating non-transformed fibroblasts. Therefore,