High-energy demand and nutrient exhaustion in MTCH2 knockout cells

  1. Sabita Chourasia
  2. Christopher Petucci
  3. Hu Wang
  4. Xianlin Han
  5. Ehud Sivan
  6. Alexander Brandis
  7. Tevie Mehlman
  8. Sergey Malitsky
  9. Maxim Itkin
  10. Ron Rotkopf
  11. Limor Regev
  12. Yehudit Zaltsman
  13. Atan Gross

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Abstract

Mitochondrial carrier homolog 2 (MTCH2) is a regulator of apoptosis, mitochondrial dynamics, and metabolism. Loss of MTCH2 results in mitochondrial fragmentation, an increase in whole-body energy utilization, and protection from diet-induced obesity. We now show using temporal metabolomics that MTCH2 deletion results in a high ATP demand, an oxidized environment, a high lipid/amino acid/carbohydrate metabolism, and in the decrease of many metabolites. Lipidomics analyses show a strategic adaptive decrease in membrane lipids and an increase in storage lipids in MTCH2 knockout cells. Importantly, all the metabolic changes in the MTCH2 knockout cells were rescued by MTCH2 re-expression. Interestingly, this imbalance in energy metabolism and reductive potential triggered by MTCH2-deletion inhibits adipocyte differentiation, an energy consuming reductive biosynthetic process. In summary, loss of MTCH2 results in an increase in energy demand that triggers a catabolic and oxidizing environment, which fails to fuel the anabolic processes during adipocyte differentiation.

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    Reply to the reviewers

    My response to the reviewers appears in the uploaded "Revision Plan" PDF file

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    Referee #3

    Evidence, reproducibility and clarity

    This study by Chourasia et al determined the effects of MTCH2 deletion on the total metabolite content (polar and lipid metabolites) per total protein of HeLa cells, analyzed at different consecutive times after adding fresh and complete media (high glucose, 10%FBS). They also analyzed the effects of nutrient depletion (no FBS In addition, authors assessed the effects of MTCH2 deletion on mitochondrial morphology, lipid droplet number and size in HeLa cells, as well as in the differentiation of mouse fibroblasts to adipocytes. From the metabolite snaphots under htese differents times and conditions, authors conclude that MTCH2 deletion increases mitochondrial oxidative function to induce a catabolic state, which impedes lipid synthesis and, as a result, adipocyte differentiation. The major concerns are that it is unclear whether the metabolic phenotype observed is a consequence of MTCH2 deletion inducing a decrease in proliferation of HeLa, as well as of fibroblasts that need to reach confluence to differentiate. In this regard, it is also unclear whether MTCH2 deletion increases ATP demand and/or promotes a catabolic program, as metabolic flux analyses are missing. A minor concern is the use of computer (processing system), antenna and wifi analogies to describe the role of MTCH2 in mitochondrial function, which is confusing.

    Significance

    This study represents a thorough characterization of the metabolite content in proliferating HeLa cells in the absence of MTCH2 expression. The changes observed in polar and lipidic metabolites are novel, interesting and contribute to our understanding on the role of MTCH2 function in cellular metabolism. The main limitation of the study is that the levels of most metabolites are normalized by protein content, comparing conditions in which cell number and protein synthesis have changed. Thus, it is unclear whether some of the effects observed are a consequence of the reported role of MTCH2 supporting the proliferation of different tumors and cell lines, or whether it is a direct effect of MTCH2 increasing ATP demand and/or being a direct activator of mitochondrial catabolism. Related to this point, it is unclear whether the defect in adipocyte differentiation induced by MTCH2 KO in NIH3T3 fibroblasts might be caused by an inability of MTCH2 KO to reach confluency at day 0, needed for differentiation.Finally, respirometry and mitochondrial ROS content analyses would be needed to confirm that the changes in the metabolite levels induced by MTCH2 are caused by an increase in mitochondrial oxidation leading to nutrient depletion, as authors conclude. For example, an increase in the ADP/ATP ratio could also be caused by an inhibition of mitochondrial ATP synthase in the mitochondria, concurrent to an increase in ROS production, which would decrease NADH and NADPH content.

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    Referee #2

    Evidence, reproducibility and clarity

    Mitochondrial carrier homolog 2 (MTCH2, SLC25A50) loss induces alterations in mitochondrial dynamics and energy utilization. However, the molecular mechanisms underlying these changes are still unknown. The study employs temporal metabolomic and lipidomic analyses, uncovering heightened catabolism, increased lipid storage, and disrupted adipogenesis in MTCH2 KO cells. The manuscript provides a comprehensive metabolic profile, revealing ATP demand increase, oxidized cellular environment, and adaptive changes in MTCH2 KO cells. Notably, in line with the fundamental role of fatty acid biosynthesis and anabolism in adipogenesis, the authors demonstrates that MTCH2 loss inhibits adipocyte differentiation. This work offers novel insights into the broader metabolic consequences of MTCH2 depletion.

    Major comments:

    • The key conclusions of the paper align with the conducted experiments, but a few additional experiments are necessary to state some claims and provide more robust conclusions.
    • The paper could benefit from the inclusion of specific experiments, particularly those that address the following aspects:
    1. Validate MTCH2 ablation in HeLa and NIH3T3L1 through sequencing of clonal lines, Western blot analysis to confirm the absence of the protein, and real-time PCR to assess whether the mechanism involves mRNA decay.
    2. Provide a more detailed rationale for their temporal metabolomics approach, elucidating the choice of the media and the timepoints of cell collection. The method involves an initial culture of the cells in DMEM medium, followed by a switch to complete medium (CM) for overnight cell growth, and subsequent refreshment with CM for different timepoints before the metabolomics analyses. Authors should articulate the reasoning for opting for CM. Furthermore, authors should explicitly explain the rationale behind selecting specific timepoints for cell collection after the addition of the fresh medium.
    3. In Figure 1, authors conclude that MTCH2 ablation stimulates oxidative metabolism and ATP production to fulfill increased cellular ATP demands. However, this conclusion is based only on metabolomic analyses of the ADP/ATP ratio. To comprehensively assess the impact on cellular respiration, the authors should monitor the Oxygen Consumption Rate (OCR) and report the Respiratory Control Ratio (RCR).
    4. NAD+/NADH ratio: authors should measure NADH levels in both mitochondria and cytosol. This can be accomplished through NADH autofluorescence (recommended) or commercially available kits. This additional analysis would contribute to a more comprehensive interpretation of the observed changes in oxidative metabolism. They should also include measurement of mitochondrial membrane potential using TMRM. Suggested experiment: measuring NADH autofluorescence. The autofluorescence of mitochondrial NADH can be distinguished from cytosolic NADH by optimizing substrate consumption followed by the complete inhibition of electron feeding to the ETC. The redox state of NADH reflects the equilibrium between mitochondrial ETC activity and the rate of substrate supply. After acquiring basal autofluorescence levels through live imaging, max signal is obtained by stimulating maximal respiration (FCCP), and min signal is obtained by inhibiting respiration (NaCN or Rot+AA). Subsequently, "NADH redox indexes" are generated by expressing the basal NADH levels as a percentage of the difference between the oxidized and reduced signals. Furthermore, by examining the fluorescence signal increase after NaCN addition, the rate of NADH production can be monitored. This rate serves as a proxy of TCA efficiency.
    5. Authors observe a reduction in the levels of various amino acids and TCA cycle intermediates, indicative of an increased flux through the TCA cycle. This proposition could be further supported by measuring the kinetics of NADH autofluorescence. Additionally, a decrease in metabolites associated with the urea cycle, such as citrulline and ornithine, is observed, yet this observation remains uncommented and warrants discussion. Intriguingly, an elevation in Branched-Chain Amino Acids (BCAAs) and unsaturated acyl carnitines is noted, leading to the hypothesis of an increased transport and breakdown of fatty acids in the mitochondria to meet the heightened cellular demand for ATP in MTCH2 KO cells. To substantiate this, and to quantitatively measure mitochondrial fuel utilization in live cells, authors shall perform a Mitofuel Flex Test by measuring the Oxygen Consumption Rate (OCR) in cells treated with inhibitors of each mitochondrial oxidative pathway including etomoxir. This approach would enable the measurement of the dependency, capacity, and flexibility of cells concerning the pathway of interest in meeting ATP demand. It is also recommended to perform MitoStress test in cells supplemented with only one of the carbon sources (such as Glucose, Glutamine, Long chain and Short Chain Fatty acids).
    6. In Fig 3, a reduction in membrane lipids, free fatty acids, and non-esterified fatty acids is observed, while there is an increase in esterified fatty acids, storage lipids like Triacylglycerols (TAG) and Cholesterol Esters (CE), and lipid droplet number and size. Notably, these lipid droplets are positioned closer to mitochondria in MKO cells. The authors propose that MKO results in enhanced transfer and metabolism of lipid moieties at the mitochondria to generate ATP. To provide insights into the molecular mechanisms underlying the observed lipid changes in MTCH2 KO cells, the following experiments are recommended: Employ Western blot and real-time PCR to measure the levels of enzymes crucial in TAG and CE formation and accumulation (e.g., Long-chain acyl-CoA synthetase (Acsl), Stearoyl-CoA desaturase (SCD) or others). Evaluate the enzymatic activity of these identified enzymes to understand their functional role in lipid metabolism in MTCH2 KO cells.
    • The suggested experiments are realistic in terms of time and resources, ensuring practical feasibility.
    • The data and methods are presented in a clear and reproducible manner.
    • The experiments appear adequately replicated, and the statistical analysis seems OK.

    Minor comments:

    • There are no specific experimental issues that require addressing.
    • Prior studies are appropriately referenced
    • In general, both the text and figures are clear and accurate. The significant alteration of metabolites found in their metabolomic dataset should be plotted using the online tool MetaboAnalyst to analyze metabolic pathways and generate better visualizations.
    • Overall, the presentation is satisfactory with only minor language adjustments recommended. A minor suggestion for improvement involves refining the language used in the text. Instead of consistently using the term "produce energy," please use "conversion of energy".

    Significance

    General Assessment: The study, through the integration of metabolomic and lipidomic data in MTCH2 KO cells, provide a comprehensive overview of the metabolic rewiring of these cells. This metabolic change is particularly interesting in the context of adipogenesis, offering valuable insights into the interconnectedness of a mitochondrial solute carrier, cellular metabolism and adipogenesis.

    Comparison and Advance: The current study significantly advances our understanding of the mitochondrial carrier homolog 2 (MTCH2) by uncovering its intricate roles in metabolism, and adipogenesis. While prior research identified MTCH2 as a regulator of apoptosis and mitochondrial dynamics, the present study expands our knowledge by elucidating its involvement in cellular metabolism and adipocyte differentiation. The major advance lies in the detailed exploration of MTCH2's impact on cellular metabolism through temporal metabolomic and lipidomic analyses. The study reveals that MTCH2 deletion leads to heightened ATP demand, an oxidized cellular environment, and alterations in lipid, amino acid, and carbohydrate metabolism. Additionally, the adaptive response in MTCH2 knockout cells involves a strategic decrease in membrane lipids and an increase in storage lipids. Furthermore, the study unveils a novel connection between the imbalance in energy metabolism triggered by MTCH2 deletion and the inhibition of adipocyte differentiation-a process that demands substantial energy and reductive biosynthetic activities. This mechanistic insight provides a conceptual advance, indicating how MTCH2, beyond its known role in apoptosis and mitochondrial dynamics, plays a pivotal role in orchestrating cellular metabolism and adipogenesis. Importantly, this work aligns with prior observations that hinted at MTCH2's involvement in fatty acid synthesis, storage, and use through his identified interactome. In summary, the study advances our knowledge of MTCH2 by providing a more comprehensive understanding of its roles in cellular metabolism and adipocyte differentiation, shedding new light on its multifaceted functions beyond its originally identified roles.

    Audience: This research will appeal to a broad audience, ranging from specialists in cellular metabolism to those with a general interest in mitochondrial dynamics and biochemistry.

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    Referee #1

    Evidence, reproducibility and clarity

    In the present study, Chourasia et al. describe the effects of MTCH2 deficiency on various metabolic parameters. Using temporal metabolomics in HeLa cells, they show an increase in ATP demand in cells lacking MTCH2. They also show altered lipid metabolism in NIH3T3L1 preadipocytes lacking MTCH2, associated with impaired maturation.

    This study is mainly descriptive, containing large amount of information that would be of interest to the understanding of the role of MTCH2 in cell metabolism. The manuscript would benefit from thorough editing to make it more focused and accurate. This is challenging since there is a very large amount of data. Below are some suggestions, along with a couple of experiments that I suggest to add in order to clarify the mechanism through which MTCH2 acts.

    Major Comments:

    1. The ratio ADP/ATP as well as AMP/ATP as well as the decrease in TCA metabolites are indications of ATP demand. Nevertheless, these are not fluxes. It would therefore be worthy to complement these results with respirometry.
    2. The dramatic increase in AMP/ATP ratio suggests an increase in AMPK activity. Testing it (by measuring AMPK phosphorylation or ACC phosphorylation) could further strengthen the results but it is not a must-have.
    3. The cause of the increase in AMP in the NIH3T3L1 is not addressed. The involvement of acyl-CoA synthetase is worth discussing or investigating.
    4. Fig. 2F shows MKO and WT, but it doesn't show MKO-R. This is an important control since lactate doesn't seem to be affected in the MKO-R.

    Minor comments:

    In the intro there are a few instances that could benefit from some more accuracy:

    1. In the abstract there is no mention of the cells that are used.
    2. Line 56: "...(OXPHOS) converts nutrients into adenosine triphosphate (ATP)". It would be more accurate to write that OXPHOS converts the chemical energy that is stored in nutrients into ATP.
    3. Line 58: "The mitochondrial NAD+/NADH pool are substrates for OXPHOS". It would be more accurate to write that NADH is the substrate. (NAD+ is after all the product of oxidation)
    4. Line 59-60: "Along with ADP, NAD+ also plays an important role in the regulation of the Krebs cycle". Instead of "regulation" I think it is more specific to write "stimulates" (otherwise add ATP and NADH which are also involved in regulation. 5. Line 65: "...changing metabolic states. In addition, mitochondria..." A link between the two sentences seems to be missing.
    5. Figure 1 is a heavy figure. Some results are significant and some show only a tendency. The description (Lines 119-125) is too general. It addresses only the "trends". A bit more specificity as to the metabolites or ratios of metabolites and the time points that are significant would be in place.
    6. Lines 136-139 "The metabolomics analyses revealed additional important changes in many more nutrient substrates, which included a decrease in most amino acids (Fig. 2A and Fig. S2A). Notably, the most significant change was seen in glutamine (Fig. 2A, left top graph), one of the major amino acid-nutrient sources" Glutamine is indeed an important amino acid and the effect is strong, but in this case it's increased. From the first sentence one would think it's decreased. Again, be more specific in your sentencing.
    7. Lines 157-159: "Thus, the acyl carnitine profile suggests that 1- to 12-hrs post media change the MKO cells use BCAAs as a nutrient source, and later shift to unsaturated acyl carnitines, specifically to the C16:1 and C18:1 forms". This conclusion does not derive from the description by the result - address the difference between the different carnitines that occurs at different times.
    8. Lines 167-168: "These results suggest that there is higher metabolism of acetyl CoA in the MKO cells leading to a bell-shape dynamics (low-high-low levels)." The interpretation is unclear. I understand that you mean that there is fluctuation within the group, however the acetyl-CoA levels remain lower MKO than in the control at all time points. This further suggests a decrease in TCA cycle.
    9. Lines 172-173 addresses the bell shape of lactate, yet the most prominent result is the 3-fold increase in lactate levels compared to WT which is not mentioned. Unfortunately, the MKO-R shows a similar increase. Still, this should be addressed in the text.
    10. 182-184: "Taken together, the results presented above are consistent with the idea that the increased amino acid/lipid/carbohydrate metabolism and substantial decrease of many metabolites in MKO cells is most likely due to their increased utilization to meet themincreased cellular energy demand." I'm not sure how this conclusion is reached. From metabolite levels alone, it's difficult to conclude about fluxes. Also, in a previous conclusion the authors wrote that the TCA cycle is probably reduced; this contradicts the above conclusion. If the authors mean that the FFAs and amino acids are used for anabolism and glucose for meeting energy demand, they should state so more clearly.
    11. Figure 3A can be split into 2 or 3 subfigures. I think it would make it more comprehensible.
    12. Line 210-211: "Notably, we also found that MTCH2 knockout cells showed accelerated mitochondria elongation (Fig. S3D, top panels), which was further pronounced when cells were grown in HBSS". The increase in mitochondrial elongation comes after fragmentation in MTCH2 KO. Although this is a known phenotype, it is good to address it shortly in the text.
    13. Line 208-209: "LDs from dispersed to a highly clustered distribution that was often observed in close proximity to mitochondria..." The proximity of mitochondria to LD suggests the possibility that there is an increase in peridroplet-mitochondria, which have been shown to be involved in biogenesis LD. It might be interesting to investigate this path as an explanation to the observed phenotype.
    14. Lines 244-245: "These results suggest that the MTCH2 knockout preadipocytes face a cellular energy crisis that is similar to the one seen in the MTCH2 knockout HeLa cells presented earlier" It's true that NAD+ and AMP (as well as AMP/ATP ratio) are increased but in view of the high ATP and NADH, it's difficult reach the conclusion that there's an energy crisis.
    15. In the discussion- Lines 267-269: "Thus, MTCH2 might act like a "relay station" by sensing and connecting between metabolic intermediates/pathways and dynamic changes in mitochondria morphology/energy production by receiving and sending Wi-Fi signals." It's difficult to raise such specific hypothesis from the results. Use milder terms.

    Significance

    Great study

  5. Version published to 10.1101/2023.12.15.571941v1 on bioRxiv