The Malate–Aspartate Shuttle supports thermogenic lipid mobilization in brown adipocytes

  1. Michaela Veliova
  2. Caroline M. Ferreira
  3. Katrina Montales
  4. Francisco Villalobos
  5. Alexandra J. Brownstein
  6. Rebeca Acín-Pérez
  7. Gabrielly S. Ferreira
  8. Linsey Stiles
  9. Marc Liesa
  10. Orian S. Shirihai
  11. Marcus F. Oliveira

Reviewed by

Read the full article See related articles

Discuss this preprint

Start a discussion What are Sciety discussions?

Listed in

Log in to save this article

Abstract

Brown adipose tissue (BAT) plays a central role in thermogenesis by coupling fatty acid oxidation to heat production. Efficient BAT thermogenic activity requires enhanced glycolytic flux, which in turn depends on continuous regeneration of cytosolic NAD⁺ to sustain glyceraldehyde-3-phosphate dehydrogenase activity. This regeneration is mediated by three main pathways: lactate dehydrogenase, the glycerol-3-phosphate shuttle, and the malate–aspartate shuttle (MASh). We previously showed that inhibition of the mitochondrial pyruvate carrier increases energy expenditure in brown adipocytes via MASh activation. However, the specific contribution of MASh to BAT energy metabolism remains poorly defined. Here, we show that MASh is functional and directly regulates lipid metabolism in BAT. Enzymatic activities of cytosolic and mitochondrial malate dehydrogenases and glutamic–oxaloacetic transaminases in BAT were comparable to those in the liver. Using a reconstituted system of isolated BAT mitochondria and cytosolic MASh enzymes, we demonstrated that extra-mitochondrial NADH is efficiently reoxidized in a glutamate-dependent manner via MASh. Genetic silencing of the mitochondrial carriers critical to MASh—namely the oxoglutarate carrier (OGC1) and aspartate–glutamate carrier (Aralar1) had no apparent effects on respiratory rates. However, silencing either OGC1 or Aralar1 led to the accumulation of small lipid droplets and impaired norepinephrine-induced lipolysis. Taken together, our data indicate a novel role of MASh in regulating BAT lipid homeostasis with potential implications to body energy expenditure and thermogenesis.

Article activity feed

  1. Review Commons

    Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

    Learn more at Review Commons

    Reply to the reviewers

    __Reviewer #1 __

    Major comments

    1. The manuscript posits that the loss of function of MASh components (Ogc1 and Aralar) decreases adrenergic-stimulated lipolysis by altering the cytosolic NAD⁺/NADH ratio, with AMPK/ACC mentioned as possible mediators. However, this remains speculative. Please provide mechanistic data directly linking MASh-dependent NAD⁺/NADH changes to the regulation of lipolysis in brown adipocytes during adrenergic stimulation. Answer 1) The reviewer raises an important point regarding the direct assessment of cytosolic NAD⁺/NADH redox changes as a mechanistic link for altered lipolysis in brown adipocytes lacking MASh components. To address this point, we added new data to the revised manuscript showing lactate/pyruvate ratio as measured by metabolomics. This is a well-established surrogate marker to monitor changes in redox balance. Notably, under basal (non-stimulated) conditions, the lactate/pyruvate ratio did not display any significant differences between Aralar 1 KD and control cells, suggesting preservation of cytosolic NAD⁺/NADH levels in the absence of functional MASh under these conditions. This finding is consistent with reports showing the robustness of NAD⁺ regeneration via multiple shuttles and the possibility of metabolic compensation when one shuttle is compromised (PMID: 40540398; PMID: 37647199).

    The results have been added as new supplementary Figure 1 as following:

    Our new metabolomics data also revealed substantial reductions in the aspartate/glutamate ratio in Aralar 1 knockdown cells, serving as a metabolomic signature of impaired MASh function and reduced exchange of these amino acids between the cytosol and mitochondria. Given that the MASh is a major mechanism for exporting cytosolic reducing equivalents into the mitochondria under high metabolic demand, its loss would be expected to impact redox homeostasis, particularly under adrenergic stimulation when glycolytic flux and lipolytic activity are elevated (PMID: 40540398).

    Importantly, although our redox surrogate marker did not detect alterations, this may be explained by activation of compensatory pathways, most notably the glycerol phosphate shuttle (GPSh), which is highly expressed and active in brown adipocytes. Indirect support for this compensation comes from data shown in figure 4I showing reduced glycerol release in *Aralar 1 *KD cells upon norepinephrine stimulation and blocked lipolysis. This suggests a redirection of glycolytically derived G3P away from release and toward enhanced cycling within the GPSh, supporting cytosolic NAD⁺ regeneration via mitochondrial FAD-dependent G3PDH and cytosolic NAD⁺-dependent G3PDH activity. This is consistent with studies documenting that the combined action of MASh and GPSh maintains NAD redox homeostasis in brown adipocytes especially during non-thermogenic conditions (PMID: 168075; PMID: 40540398; PMID: 37647199). We have included a discussion about this possibility at page 9, third paragraph as follows:

    *“Previous studies have shown that BAT exhibits high activity of mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase (mG3PDH), which functions as an electron sink to sustain low cytosolic NADH levels essential for continuous glycolytic flux [11]. Accordingly, suppression of the MASh, either genetically or pharmacologically, is likely to induce a compensatory upregulation of the GPSh. This adaptation would enhance G3P turnover, contributing to the maintenance of cytosolic NAD redox balance. Moreover, the increased flux through the GPSh could favor fatty acid esterification and triglyceride synthesis or re-esterification, consistent with our findings in Ogc and/or Aralar 1 KD cells, where (i) triglyceride content rises (Fig. 3), (ii) overall respiratory rates remain largely unaltered (Figs. 2D–G), and (iii) glycerol release declines significantly (Fig. 4I). Notably, the decrease in glycerol release persists even when lipolysis is blocked by ATGlistatin, suggesting that the available G3P pool is rerouted from dephosphorylation and extracellular release toward oxidation to DHAP by mG3PDH to regenerate cytosolic NAD+ under MASh-deficient conditions. We propose that interference with the MASh does not directly impact lipolysis but instead alters the cellular balance between DHAP and G3P owing to enhanced activity of the GPSh. This metabolic shift would favor the esterification of G3P with free fatty acids, thereby promoting triglyceride synthesis. These results support the notion that, even during adrenergic stimulation—when long-chain unsaturated fatty acids and their CoA esters strongly inhibit mG3PDH activity [11]—the residual flux through the glycerophosphate shuttle remains critical for sustaining cytosolic NAD redox equilibrium [11,19,32].” *

    At the mechanistic level, adrenergic stimulation in brown adipocytes activates robust lipolysis and thermogenic gene programs, generating high NADH that must be efficiently reoxidized to sustain flux through glycolysis and lipolysis-linked pathways. Our findings are consistent with a model in which the loss of MASh does not prevent cytosolic NAD⁺ regeneration or lipolytic flux during acute adrenergic stimulation, due to compensatory upregulation of the GPSh, as suggested by the glycerol release changes. Thus, while MASh normally acts as a conduit for NADH export and aspartate/glutamate exchange, in its absence, the GPSh maintains cytosolic redox balance, thereby sustaining glycolytic and lipolytic capacity.

    We agree that future studies should employ direct measurements of cytosolic NAD⁺/NADH ratios (e.g., genetically-encoded redox sensors) during adrenergic stimulation and specific pharmacological inhibition of both shuttles to dissect these relationships in greater detail. We sincerely appreciate the reviewer's input, which has prompted us to clarify the indirect but robust evidence supporting a role for compensatory redox shuttle activity in preserving brown adipocyte lipolysis in the setting of MASh impairment.

    We have further added a new paragraph in the discussion section (page 10)::

    *“Mechanistically, the connection between the MASh and lipolysis appears to involve regulation of the cytosolic NAD⁺/NADH redox balance. MASh activity facilitates the regeneration of NAD⁺ from NADH in the cytosol, primarily through the reduction of oxaloacetate to malate by cytosolic malate dehydrogenase (Fig. 1G-H). Despite the theoretical expectation that reductions in MASh activity would disturb redox homeostasis, our metabolomic data show that the lactate/pyruvate ratio remains unchanged under conditions of MASh impairment, indicating that the overall cytosolic NAD⁺/NADH ratio is maintained (Figure S1A-C). While direct measurements of cytosolic NAD⁺/NADH were not performed, the preserved lactate/pyruvate ratio in Aralar 1 KD cells under basal conditions strongly suggests redox stability, likely due to compensatory activity by alternative mitochondrial shuttles or metabolic adaptations that maintain NAD redox homeostasis despite MASh impairment [18,33]. *

    Previous evidence indicates that BAT exhibits high activity of mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase (G3PDH), which acts as an electron sink to sustain low cytosolic NADH levels critical for glycolysis [34]. In this sense, it is conceivable that genetic or pharmacological suppression of MASh triggers compensatory enhancement of the G3P shuttle, increasing G3P availability and facilitating the maintenance of cytosolic NAD redox balance. This adaptation could also promote fatty acid esterification and triglyceride synthesis or re-esterification, aligning with our observations that in Ogc and/or Aralar 1 KD cells: (i) triglyceride levels increase (Fig. 3); (ii) overall respiratory rates are preserved (Figs. 2D–G); and (iii) glycerol release is significantly reduced (Fig 4I).”

    __ The absence of in vivo analysis of lipid-droplet size in MASh loss-of-function models is a major concern. In vitro results could be confounded by differences in differentiation stage between groups. Please document equivalent adipogenesis across groups (e.g., Pparg/Cebpa/Plin1/Fabp4 expression).__

    Answer 2) We thank the reviewer for the thoughtful and constructive comment regarding potential confounding by differences in differentiation stage, and for highlighting the importance of documenting equivalence between experimental groups. We appreciate the opportunity to clarify and provide additional assurance on this point.

    As detailed in our manuscript, we have performed qPCR analysis of multiple well-established markers of brown adipocyte differentiation, including Ucp1, Elovl3, Prdm16, Pparg, Cebpa, Plin1, and Fabp4, in both scramble, aralar1 KD, and Ogc KD cells (see Fig. S1A and accompanying text). Our results show no apparent effect of these genetic interventions on overall differentiation, as the expression levels of these key markers were consistently unaltered across groups. Furthermore, adenoviral-mediated knockdown of Ogc achieved an approximate 80% reduction in Ogc mRNA (see Fig. S1B), yet most differentiation markers remained unaffected. We did observe significant increases in Atgl, Pgc1α, and Tfam mRNA levels, which may indicate a degree of pathway reprogramming without affecting the general differentiation profile. We propose that interference with the MASh does not directly impact lipolysis but instead alters the cellular balance between DHAP and G3P owing to enhanced activity of the GPSh. This metabolic shift would favor the esterification of G3P with free fatty acids, thereby promoting triglyceride synthesis.

    Additional experimental support for equivalent differentiation can be drawn from our respirometry data presented in Figures 2E and 2G. These figures demonstrate that respiratory rates upon norepinephrine stimulation, which is a sensitive indicator of brown adipocyte thermogenic capacity, were essentially identical in scramble, aralar1 KD, and Ogc KD cells. Since norepinephrine-stimulated respiration requires both functional mitochondria and the full differentiation of brown adipocytes, these results strongly support the conclusion that silencing either MASh component does not impair the fundamental ability of cells to undergo brown adipocyte differentiation or achieve functional thermogenic competence.

    This is consistent with published findings showing that norepinephrine triggers robust respiration and thermogenic activation only in fully differentiated and functional brown adipocytes, making such measurements a widely accepted proxy for differentiation status and mitochondrial integrity. Thus, the equivalent respiratory responses observed in all groups further validate that differentiation was not compromised by the genetic interventions.

    We hope this clarifies that equivalent adipogenesis was carefully documented and that any observed phenotypes are unlikely to be attributable to differences in differentiation stages. Thank you again for your rigorous assessment and for helping to ensure the robustness of our study.

    __ Please include rescue experiments (add-back OGC1 and Aralar) to rule out siRNA/shRNA off-target effects and verify that the phenotype stems from MASh loss of function.__

    Answer 3) We thank the reviewer for this important suggestion regarding the inclusion of rescue experiments with add-back of Ogc and Aralar to definitively exclude off-target effects of the siRNA/shRNA-mediated knockdowns.

    We would like to kindly point out that although we did not perform add-back rescue experiments directly, the consistency of phenotypes observed across two independent genetic interventions—aralar 1 KD and Ogc KD—strongly argues against off-target effects being responsible for the observed metabolic and functional alterations. Specifically, both knockdowns yielded remarkably similar phenotypes in multiple assays, including respirometry analyses, mitochondrial morphology, lipid droplet homeostasis, and lipid metabolism, supporting the conclusion that these effects stem from MASh loss of function rather than nonspecific silencing.

    Furthermore, our new supplementary data (new Supplementary Figure 1A-F) reveals a significant reduction in the aspartate/glutamate ratio in *Aralar 1 *KD cells, a compelling functional readout for MASh impairment. This molecular evidence corroborates that our genetic interventions effectively disrupted MASh activity as intended.

    We sincerely appreciate the reviewer’s thorough evaluation and understand the importance of rescue experiments. While recognizing their value, we believe the convergent genetic, metabolic, and functional evidence presented across two different MASh components provides strong and consistent support that the phenotypes observed are due to specific loss of MASh function.

    __ Please expand on physiological significance: What is the importance of MASh regulation of BAT lipolysis in long-term adaptive thermogenesis?__

    Answer 4) This is a very interesting aspect, and we have included a new paragraph in the discussion section (page 14) to address it as follows:

    “Our results, supported by recent literature, strongly indicate that the malate–aspartate shuttle (MASh) plays a key role in facilitating fatty acid–dependent thermogenesis in brown adipocytes. Specifically, BAT-targeted overexpression of GOT1 has been shown to enhance β-oxidation and support acute cold-induced thermogenesis (PMID: 40540398). Interestingly, genetic ablation of GOT1—and thus MASh inhibition—preserves cold-induced thermogenesis by promoting a metabolic shift from fatty acid to glucose oxidation. Our findings corroborate and extend these observations by demonstrating that MASh impairment sustains overall respiratory activity in norepinephrine-stimulated brown adipocytes (Figures 2D–2G), while concurrently impairing lipolysis and resulting in an accumulation of small lipid droplets (Figures 3 and 4). Collectively, these data suggest that MASh not only modulates substrate preference towards fatty acid oxidation but also facilitates lipolysis, an essential upstream step that enables lipid oxidation and supports thermogenic heat production.”

    Minor comments

    1. __ Fig. 4 legend/title contains a typo ("lypolysis" → lipolysis).__ Answer 1) Corrected

    __ In Fig. 2 legend line: "Adevirus-mediated" → Adenovirus-mediated; "OCAR" → OCR.__

    Answer 2) Corrected

    __ For lipolysis imaging, you already show Forskolin/Atglistatin/Etomoxir controls; add a vehicle-only time course overlay in the main figure (currently in text/legend) to aid visual comparison.__

    Answer 3) We thank the reviewer for pointing this out. To improve clarity, we have updated the labeling in Figures 3 and 4: “basal” now clearly refers to the unstimulated/untreated condition, and the previously labeled “UT” condition has been clarified as “untransduced.” These changes make the figure legends and data presentation more consistent and easier to interpret.

    __ Ensure consistent gene symbols (Atgl/Pnpla2), and protein capitalization.__

    Answer 4) Corrected.

    __Reviewer #2 __

    Major points:

    1. __ In the current manuscript, mitochondrial morphology (area, aspect ratio, and roundness) was analyzed in OGC1 KD cells using TMRE, whereas MitoTracker Deep Red (MTDR) was used in Aralar1 KD cells. Notably, TMRE is a ΔΨm-dependent probe. The signal intensity can change, or the distribution may reflect alterations in membrane potential rather than true morphological changes. Therefore, the observed differences in OGC1 KD cells based on TMRE staining may be confounded by the dye's functional dependence, potentially biasing the conclusions. It is recommended to evaluate mitochondrial morphology with consistent trackers across conditions. In addition, in the subsequent OCR analysis, mitochondrial area was used for normalization. Please clarify which staining method was employed, and provide justification for its suitability.__ Answer 1) We thank the reviewer for this insightful comment. Indeed, TMRE is a membrane potential-sensitive dye and could therefore potentially affect measurements of mitochondria.

    We would like to point out that mitochondrial morphology was quantified based on mitochondrial area rather than fluorescence intensity. To create an accurate binary map of mitochondria, we used a low threshold, which allowed us to include even weakly stained mitochondria and thereby detect them independently of their membrane potential. In all imaged cells, TMRE signal was sufficient to reliably identify mitochondrial pixels. Moreover, these images were acquired using a confocal microscope, where the risk of pixel expansion due to higher fluorescence intensity is minimized. Lastly, given that overall mitochondrial oxygen consumption in these cells remains largely intact, we do not expect a substantial loss of membrane potential, although minor effects cannot be entirely excluded.

    We opted to use TMRE for imaging Ogc KD cells because the scramble control for these shRNA viruses carries an mKate fluorescent tag, which overlaps with the MTDR signal. Since accurate assessment of transduction efficiency relied on detecting mKate, MTDR could not be used in these experiments. Importantly, we only compare mitochondrial morphology within the same staining condition and do not draw conclusions across cells stained with different dyes.

    To ensure transparency, we have added a new section at the discussion (page 17, 2nd paragraph) highlighting the potential influence of ΔΨm-dependent dyes on morphological measurements as follows:

    “It is also important to note that mitochondrial morphology was quantified using MTDR in Aralar 1 KD cells and TMRE in Ogc KD cells due to experimental constraints (see Methods). TMRE is a membrane potential–dependent dye, which could potentially influence morphology measurements. To minimize this risk, we used confocal microscopy, which reduces the likelihood of pixel expansion due to higher fluorescence intensity, and set thresholds to detect even weakly stained mitochondria. Nonetheless, we cannot fully exclude the possibility that the differences in morphology observed between Aralar 1 and Ogc KD are influenced by the use of different dyes; however, statistical comparisons were never performed across samples stained with different dyes.”

    Also, we have expanded the Methods section (page 22, 2nd paragraph) to include a rationale for using these dyes and describe the analysis protocol as following:

    “TMRE was used for Ogc KD cells because the scramble control for the shRNA viruses carries an mKate fluorescent tag, which overlaps with MTDR fluorescence, preventing its use. MTDR was used for Aralar KD cells. Image Analysis was performed in FIJI (ImageJ, NIH). For the quantification of mitochondrial morphology and area, images stained with TMRE or MTDR were analyzed. Thresholds were adjusted to ensure that even weakly stained mitochondria were detected and included in the analysis. Only the mitochondrial area was evaluated, independent of fluorescence intensity.”

    Minor points:

    1. __ In the introduction, the authors state that "LDH activity increases in the context of BAT activation". This point is important for the logic of the manuscript, reference [10] cited here is not sufficient to support this claim. It is recommended to provide appropriate references to support this statement.__ Answer 1) We have substantially changed this paragraph in the revised manuscript to better explain why LDH would not act as a major player in contributing to NAD redox balance in the context of BAT thermogenesis, as follows:

    “In mammalian cells, cytosolic NAD⁺ is regenerated through lactate dehydrogenase (LDH), the glycerol-3-phosphate shuttle (GPSh), or the malate-aspartate shuttle (MASh). In BAT, however, lactate production rises only slightly with adrenergic activation and most lactate is oxidized via the TCA cycle, suggesting that LDH primarily consumes NAD⁺ rather than regenerating it [PMID: 30456392; PMID: 37337122; PMID: 30456392; PMID: 37802078; PMID: 40982723]. Consequently, mitochondrial redox shuttles become critical for sustaining cytosolic NAD⁺ supply”.

    We have also provided additional references to support this new section at the introduction.

    __ In Fig. 1A and B-D, there are inconsistencies and duplications in the abbreviation labels. Please check and revise accordingly. __

    Answer 2) We thank the reviewer for this comment. We would like to clarify that Figure 1A is a schematic overview of the system, while Figures 1B–D show protein expression in specific contexts: whole BAT (B), whole liver (C), and BAT mitochondria (D). In Figures 1B and 1C, all components are shown because both cytosolic (MDH1 and GOT1) and mitochondrial proteins (MDH2, GOT2, Aralar 1 and 2 and OGC) are present. In contrast, Figure 1D shows only mitochondrial components (OGC, Aralar1, MDH2, and GOT2). Although Aralar2 is a mitochondrial protein, it was not detected in this study (Forner et al., 2009). Similarly, cytosolic components such as MDH1 and GOT1 are not shown in Figure 1D because they are absent in the mitochondrial fraction. We have revised the figure legend to make these distinctions clearer.

    __ In Fig. S1, the number of n indicated does not match the number of data points shown. Please clarify whether these represent technical replicates or biological replicates, and provide a detailed description of the statistical methods used throughout the manuscript.__

    Answer 3) We thank the reviewer for catching this and allowing us to correct our mistakes. In the revised version, we have corrected the figure legend of Supplementary Figure 1 so that the number of n matches the data points shown.

    __ Please provide details on the normalization strategy used in the BODIPY-C12/BODIPY-493 staining analysis, such as whether fluorescence intensity was quantified as mean or integrated values, and whether the analysis was normalized to lipid droplet area, cell number, or baseline. Since lipolytic stimulation can reduce droplet size and increase droplet number, these factors may bias the results. __

    Answer 4) We thank the reviewer for this important comment and apologize for the lack of detail regarding this analysis. The analysis of BODIPY-C12 and BODIPY-493 was performed by quantifying the mean fluorescence intensity of BODIPY-C12 detected within a mask generated from the BODIPY-493 signal. This approach allowed us to define all lipid droplets and measure the release of previously esterified C12. To account for variability across samples, the data were normalized to each sample’s individual baseline at time point 0 and expressed as fold change relative to this baseline. In the revised manuscript we have included this description in the Methods section (page 18, last paragraph) for clarity and reproducibility, as following:

    “Lipid Droplet area was defined based on Bodipy 493/503 signal, which was used to generate a mask identifying all lipid droplets. Within this mask, the mean fluorescence intensity of BODIPY C12 was quantified over time to monitor the release of previously esterified C12. To account for variability between samples, data were normalized to each sample’s individual baseline at time point 0 and expressed as fold change relative to this baseline.”

    __ The manuscript notes that the unexpected result in Fig. 3K-M in parallel with increased Atgl mRNA expression might be because it does not reflect protein levels or enzymatic activity. To strengthen this point, it is recommended to include data on ATGL and phosphorylation ATGL. __

    Answer 5) We thank the reviewer for this constructive comment. We have clarified these aspects in the revised Results and Discussion sections to reflect this interpretation more accurately as follows:

    “Notably, Atgl mRNA measurement in our study was primarily used as a marker of brown adipocyte differentiation, rather than as a direct indicator of ATGL protein abundance or enzymatic activity. We detected increased Atgl expression only in Ogc KD cells (Fig. S1H), but not in Aralar 1 KD cells (Fig. S1G). This likely does not reflect a major difference in differentiation status, as other brown adipocyte markers and norepinephrine-stimulated respiration were comparable between scramble and knockdown cells (Fig. 2D-G and 2N-O and S1G-H). Although lipolysis was not evaluated in Ogc KD cells, in Aralar 1 KD cells basal lipolysis remained unchanged (Fig. 4D-E and 4G-I), whereas norepinephrine-stimulated lipolysis was delayed or partially inhibited. Notably, the enhanced fatty acid esterification observed in Ogc KD cells despite elevated Atgl expression is not contradictory, since in brown adipocytes lipolysis and re-esterification occur concurrently to sustain high lipid turnover [34].

    __ Red-on-black is not a great color code for IMFs, how about black-and-white? __

    Answer 6) We have changed color text for white on figures 2H and K as suggested.

    __Reviewer #3 __

    Major points;

    1. __ Although in the manuscript Veliova and coworkers demonstrated that MAS is functional in brown adipocytes showing kinetic parameters equivalent to that previously described in other tissues, surprisingly, when its components are downregulated, no effect, or very little, on mitochondrial respiration is found (figure 2). This is an intriguing result since MAS disruption has been widely reported to impair respiration in different cell types and tissues. However, since no direct evidence of MAS dysfunction is provided, it is possible that MAS may still remain partially or fully functional under the conditions used by the authors, and therefore this point needs to be clarified to validate these results.__ Answer 1) We thank the reviewer for the insightful comment and the opportunity to clarify these important points regarding MASh dysfunction validation in our study. We acknowledge the reviewer’s observation that mitochondrial respiration was largely unaffected by MASh component knockdown, which is indeed intriguing. Importantly, as already indicated in our responses to Reviewer 1, we have provided new data showing direct molecular evidence of MASh impairment through substantial reductions in the aspartate/glutamate ratio in *Aralar 1 *KD cells (new Supplementary Figure S1F). This ratio is a well-established functional readout reflecting MASh activity and amino acid exchange between cytosol and mitochondria, as demonstrated in original experimental studies of MASh function in multiple tissues including brown adipocytes (PMID: 4436323). The reduction in the aspartate/glutamate ratio directly confirms loss of MASh functionality even though respiratory rates remained unchanged, likely due to metabolic compensation by robust glycerol phosphate shuttle (GPSh) activity, as further supported by our data showing reduced glycerol release upon norepinephrine stimulation in *Aralar 1 *KD cells cells (Figure 4I). This metabolic rerouting maintains cytosolic NAD⁺ regeneration and partially preserves respiration and energy metabolism under these experimental conditions (PMID: 168075; PMID: 40540398; PMID: 37647199). Thus, the combination of metabolomic, respirometry, and functional lipid data strongly indicates that MASh activity was disrupted specifically and effectively by our genetic interventions. This molecular evidence was already signposted in our original manuscript and responses, underscoring that MASh loss of function—and not residual or compensatory MASh activity—is responsible for the phenotypes reported. We greatly appreciate the reviewer’s insightful attention to this critical mechanistic issue and hope this provides clear reassurance that MASh impairment was indeed achieved and functionally validated within our study framework.

    Furthermore, strategies used to downregulate MAS components produce only a partial reduction in mRNA levels, about 70 %, but its outcome on protein levels has not been determined. and the remaining protein level could be sufficient to maintain shuttle activity. Therefore, the effect of silencing at protein level should be analyzed, because as authors also point out on page 16; "mRNA levels may not reflect actual protein levels or activity".

    Answer 2) We thank the reviewer for this important point. Our knockdowns resulted in ~70–80% reduction in mRNA levels. While not complete, this represents a substantial decrease and is sufficient to produce strong functional effects. At the time the experiments were performed, we did not have access to suitable antibodies, and the available antibodies did not provide reliable signals in our samples, which is why we used qPCR to estimate knockdown efficiency. Importantly, we observed clear phenotypic changes in both knockdowns (Aralar and OGC), and both showed very similar phenotypes. This suggests that the level of knockdown was sufficient to significantly impair MAS activity. In the revised version we added new data which further validated the functional impact of Aralar KD (given that this protein has an alternative isoform, as pointed out by the reviewer). We performed metabolomics experiments measuring aspartate and glutamate levels. Our new data shows that the aspartate-to-glutamate ratio is significantly reduced in Aralar KD cells. This ratio serves as a proxy for glutamate catabolism, and the observed decrease suggests reduced glutamate catabolism, likely due to impaired MAS activity. Therefore, the reduced whole-cell aspartate/glutamate ratio serves as a metabolic signature of MAS impairment, consistent with Aralar KD. These data indicate that Aralar is sufficiently downregulated to produce a functional effect, supporting our conclusion that MAS activity is impaired. The results have been added as new supplementary Figure 1 as follows:

    __ In the case of aspartate/glutamate carriers (AGCs) the role of citrin/slc25a13, the second AGC paralog, should also be analyzed. This AGC isoform is discarded based on proteomic data from brown adipose tissue, but, as it is shown in figure 1B, its levels are similar those of Aralar/slc25a12, the only AGC silenced. Besides, primary brown adipocytes differentiated for 7 days are used here, and it is possible that factors such as culture conditions or differentiation itself could alter AGC levels. Therefore, it is necessary to determine the protein levels of citrin/AGC2, and, if necessary, downregulate it together with the Aralar/AGC1 isoform. citrin/AGC2 activity may be responsible for the observed difference between the OGC and Aralar/AGC1 KD adipocytes.__

    Answer 3) We thank the reviewer for this important point. We chose Aralar1 because it is the isoform predominantly expressed in brown adipose tissue (PMID: 23436904). We acknowledge, however, that compensatory increases in Citrin/AGC2 upon Aralar1 knockdown are possible. To address this, we have included new metabolomics data in the revised manuscript (added as Supplementary Figure 1), which provides additional support that downregulation of Aralar1, even if not complete, is sufficient to cause a metabolic change reflected by a reduced aspartate/glutamate ratio in these cells. This functional change supports that the knockdown of Aralar1 alone is sufficient to study its role in brown adipocytes, although minor compensation by Citrin/AGC2 cannot be entirely excluded.

    To address this explicitly, we have added a paragraph to the discussion (page 13, 2nd paragraph) highlighting the potential for partial compensation by Citrin/AGC2 and explaining why the observed metabolic effects are still attributable to Aralar 1 knockdown, as follows:

    “Phenotypes observed in Aralar 1 KD cells closely resemble those in Ogc KD cells, particularly in terms of lipid metabolism alterations and energy expenditure. The main difference lies in mitochondrial morphology, which is altered in Ogc KD cells but remains unchanged in Aralar 1-silenced cells (Fig. 2J,M). Unlike Ogc, which lacks an alternative isoform, Aralar 1 has a paralog Aralar 2 (Citrin, or SLC25A13) that may partially compensate for its loss. This potential compensation might explain the preservation of mitochondrial morphology in Aralar 1 KD cells. Nonetheless, our metabolomics data demonstrate that downregulation of Aralar 1 alone significantly reduces the aspartate/glutamate ratio (Fig. S1D-F). Since this ratio reflects glutamate catabolism, its decrease indicates impaired malate-aspartate shuttle activity and reduced glutamate catabolism. Therefore, although compensation by Aralar 2 cannot be entirely excluded, Aralar 1 KD alone suffices to cause substantial impairment of malate-aspartate shuttle function”.

    __ OGC and Aralar/AGC1 silencing is associated with the accumulation of smaller lipid droplets and impaired norepinephrine-induced lipolysis, but no mechanistical evidence is provided. The authors discuss a role for AMPK signaling associated with the redox unbalance generated by MAS disfunction but neither of them is proven.__

    Answer 4) We thank the reviewer for this insightful question, which was also raised by Reviewer 1 (see Reviewer 1, Question 1 above). Here, we aim to clarify the mechanistic basis by which MASh may regulate lipolysis in BAT in a complementary and refined manner.

    Our new data directly addresses this issue by examining cytosolic redox status through the lactate/pyruvate ratio, a well-established indicator of NAD⁺/NADH balance. Under basal conditions, *Aralar 1 *KD cells showed no change in this ratio compared to controls, indicating preserved cytosolic NAD⁺ regeneration despite reduced MASh activity. This observation is consistent with previous studies demonstrating the resilience of cellular redox homeostasis through overlapping NAD⁺-regenerating systems (PMID: 40540398; PMID: 37647199). The new results are shown in Supplementary Figure 1.

    At the same time, we detected a marked decrease in the aspartate/glutamate ratio in *Aralar 1 *KD cells, confirming impaired MASh function and reduced amino acid exchange between cytosol and mitochondria. The lack of redox imbalance likely reflects compensatory mechanisms, most notably the GPSh, which is highly active in brown adipocytes. Supporting this view, *Aralar 1 *KD cells displayed significantly reduced glycerol release upon norepinephrine stimulation (Fig. 4I), suggesting enhanced metabolic cycling of G3P through mitochondrial and cytosolic G3PDH, thereby sustaining NAD⁺ regeneration and redox equilibrium.

    We therefore propose that, although MASh normally facilitates NADH export and aspartate/glutamate exchange, its loss activates GPSh-mediated compensation that preserves cytosolic NAD⁺/NADH balance and maintains lipolytic flux during adrenergic stimulation. These findings refine our mechanistic understanding of how redox shuttle interplay supports glycolytic and lipolytic processes in BAT. Future studies employing NAD⁺/NADH sensors and simultaneous blockade of both shuttles will be essential to dissect these compensatory mechanisms in greater detail.

    Minor points;

    1. __ Is pyruvate present in respiration medium? If so, no effect on respiration is expected as pyruvate reverses the respiratory defects caused by MAS inactivation. __ Answer 1) Thanks for this important insight. In fact, as indicated in the methods section (page 17, last paragraph) all respirometry experiments were carried out in the absence of pyruvate in the media. Therefore, preserved overall respiratory rates in *Aralar 1 *and *Ogc *KD cannot be explained by compensatory pyruvate oxidation present in the media.

    __ In figure 4, only data from Aralar KD cells in relation to norepinephrine-stimulated lipolysis are shown. What happens when OGC is silenced? __

    Answer 2) This is a very interesting and relevant question. We did not perform the norepinephrine-stimulated lipolysis experiments in Ogc-silenced cells, since in most of the other experiments presented in the manuscript *Ogc *and Aralar 1 silencing converged to very similar, if not identical, phenotypes. Based on these consistent overlaps, we anticipate that *Ogc *KD would likely lead to comparable effects on lipolysis as observed in Aralar 1 KD cells. Nonetheless, we fully agree that direct assessment of lipolysis upon Ogc KD would strengthen this conclusion, and we consider this an important aspect for future studies.

    __ Nomenclature used for mitochondrial carriers is confusing. Please do not use OGC1 as there is only one isoform. Furthermore, different names for OGC are used in the manuscript; oxoglutarate carrier, malate-ketoglutarate carrier or OGC1/SLC25A11. In the case of citrin/AGC2, Aralar2 is used and is a uncommon designation.__

    Answer 3) We corrected all OGC naming in the revised manuscript. We also changed “aralar 2” for “citrin” since this was more commonly used in the literature.

    __ Some panels of figures 3 and 4 should be improved. Panels 3J, 3L and 4G are difficult to see. In panel 3J please clarify UT line from untreated/NE, are they not transduced? No equivalents conditions are assayed in Aralar KD and OGC KO cells.__

    Answer 4) We thank the reviewer for giving us the opportunity to improve this figure and apologize for the confusing labeling. In the revised version, we have clarified the labels in panels 3J, 3L, and 4G to improve visibility, and we have added descriptions of all abbreviations to the figure legends, accordingly.

  2. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #3

    Evidence, reproducibility and clarity

    In this manuscript, Veliova and coworkers explore the contribution of the malate-aspartate NADH shuttle (MAS) to energy metabolism in brown adipose tissue. This work done by a group expert in mitochondrial metabolism, continues an interesting previous one (Veliova, 2020) where it was shown that the inhibition of the mitochondrial pyruvate carrier caused an increase in energy expenditure mediated by the activation of MAS in BAT. Here, the authors have explored the consequences of the lack of MAS activity on BAT metabolism by the silencing of the metabolite transporters that are part of MAS in cultured primary brown adipocytes. Using this loss-of-function approach, the role for MAS in the regulation of lipid homeostasis in BAT is analyzed. The results could be interesting, but in my opinion, they are not sufficiently proven. Much more evidence should be provided to confirm MAS deficiency and the mechanisms involved in the alteration of lipid homeostasis.

    Major points

    1. Although in the manuscript Veliova and coworkers demonstrated that MAS is functional in brown adipocytes showing kinetic parameters equivalent to that previously described in other tissues, surprisingly, when its components are downregulated, no effect, or very little, on mitochondrial respiration is found (figure 2). This is an intriguing result since MAS disruption has been widely reported to impair respiration in different cell types and tissues. However, since no direct evidence of MAS dysfunction is provided, it is possible that MAS may still remain partially or fully functional under the conditions used by the authors, and therefore this point needs to be clarified to validate these results. Furthermore, strategies used to downregulate MAS components produce only a partial reduction in mRNA levels, about 70 %, but its outcome on protein levels has not been determined. and the remaining protein level could be sufficient to maintain shuttle activity. Therefore, the effect of silencing at protein level should be analyzed, because as authors also point out on page 16; "mRNA levels may not reflect actual protein levels or activity".
    2. In the case of aspartate/glutamate carriers (AGCs) the role of citrin/slc25a13, the second AGC paralog, should also be analyzed. This AGC isoform is discarded based on proteomic data from brown adipose tissue, but, as it is shown in figure 1B, its levels are similar those of Aralar/slc25a12, the only AGC silenced. Besides, primary brown adipocytes differentiated for 7 days are used here, and it is possible that factors such as culture conditions or differentiation itself could alter AGC levels. Therefore, it is necessary to determine the protein levels of citrin/AGC2, and, if necessary, downregulate it together with the Aralar/AGC1 isoform. citrin/AGC2 activity may be responsible for the observed difference between the OGC and Aralar/AGC1 KD adipocytes.
    3. OGC and Aralar/AGC1 silencing is associated with the accumulation of smaller lipid droplets and impaired norepinephrine-induced lipolysis, but no mechanistical evidence is provided. The authors discuss a role for AMPK signaling associated with the redox unbalance generated by MAS disfunction but neither of them is proven.

    Minor points

    1. Is pyruvate present in respiration medium? If so, no effect on respiration is expected as pyruvate reverses the respiratory defects caused by MAS inactivation.
    2. In figure 4, only data from Aralar KD cells in relation to norepinephrine-stimulated lipolysis are shown. What happens when OGC is silenced?
    3. Nomenclature used for mitochondrial carriers is confusing. Please do not use OGC1 as there is only one isoform. Furthermore, different names for OGC are used in the manuscript; oxoglutarate carrier, malate-ketoglutarate carrier or OGC1/SLC25A11. In the case of citrin/AGC2, Aralar2 is used and is a uncommon designation.
    4. Some panels of figures 3 and 4 should be improved. Panels 3J, 3L and 4G are difficult to see. In panel 3J please clarify UT line from untreated/NE, are they not transduced? No equivalents conditions are assayed in Aralar KD and OGC KO cells.

    Significance

    General assessment: The robust part of this study is its analysis of some aspects related to lipid metabolism in cultured primary cells derived from brown adipose tissue. The participating teams are well-versed in this topic and the approaches used are correct. However, no data in animal models supporting these results are provided and this fact rests interest.

    Advance: This manuscript is the "logical" continuation of a previous study, Veliova et al., (2020) EMBO Rep, more relevant in my opinion. Also, recently, it has been also proposed using animal models, either by overexpression or using deficient mice for GOT1 a cytosolic protein component of MAS, a role for MAS in BAT thermogenesis (Park et al., Cell Rep. 2025). The novelty in this manuscript is the analysis of deficient cells in the metabolite transporter that regulate the direction of NADH shuttling. However, since no evidence is provided its effect on NAD+/NADH ratio, the conclusions related to the role of MAS, or the mitochondrial carriers silenced, in the regulation of lipolysis in BAT and its involvement in thermogenesis are not convinced.

    Audience: These results could be of interest to the audience interested in basic research, but could also be useful in the translational/clinical area because they address metabolic aspects in adipose tissue.

    My expertise is focus on mitochondrial metabolism, specifically in the function of a subtype of mitochondrial carriers regulated by cytosolic calcium and how they participate in the control of different mitochondrial functions, such as respiration, calcium buffering, cell proliferation. Some of these transporters are components of MAS such as Aralar/AGC1 or citrin/AGC2.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    This manuscript presents novel findings on the role of the malate-aspartate shuttle (MASh) in brown adipose tissue (BAT). Building on the recent advances in elucidating the contribution of MASh to BAT metabolism, the present study provides new evidence by offering direct biochemical validation using a reconstituted BAT mitochondrial system and by introducing genetic data on the mitochondrial carriers OGC1 and Aralar1, thereby adding significant new insight. However, the following points require further clarification.

    Major points:

    1. In the current manuscript, mitochondrial morphology (area, aspect ratio, and roundness) was analyzed in OGC1 KD cells using TMRE, whereas MitoTracker Deep Red (MTDR) was used in Aralar1 KD cells. Notably, TMRE is a ΔΨm-dependent probe. The signal intensity can change, or the distribution may reflect alterations in membrane potential rather than true morphological changes. Therefore, the observed differences in OGC1 KD cells based on TMRE staining may be confounded by the dye's functional dependence, potentially biasing the conclusions. It is recommended to evaluate mitochondrial morphology with consistent trackers across conditions. In addition, in the subsequent OCR analysis, mitochondrial area was used for normalization. Please clarify which staining method was employed, and provide justification for its suitability.

    Minor points:

    1. In the introduction, the authors state that "LDH activity increases in the context of BAT activation". This point is important for the logic of the manuscript, reference [10] cited here is not sufficient to support this claim. It is recommended to provide appropriate references to support this statement.
    2. In Fig. 1A and B-D, there are inconsistencies and duplications in the abbreviation labels. Please check and revise accordingly.
    3. In Fig. S1, the number of n indicated does not match the number of data points shown. Please clarify whether these represent technical replicates or biological replicates, and provide a detailed description of the statistical methods used throughout the manuscript.
    4. Please provide details on the normalization strategy used in the BODIPY-C12/BODIPY-493 staining analysis, such as whether fluorescence intensity was quantified as mean or integrated values, and whether the analysis was normalized to lipid droplet area, cell number, or baseline. Since lipolytic stimulation can reduce droplet size and increase droplet number, these factors may bias the results.
    5. The manuscript notes that the unexpected result in Fig. 3K-M in parallel with increased Atgl mRNA expression might be because it does not reflect protein levels or enzymatic activity. To strengthen this point, it is recommended to include data on ATGL and phosphorylation ATGL.
    6. Red-on-black is not a great color code for IMFs, how about black-and-white?

    Referees cross-commenting

    To my opinion, all three reviewers have provided constructive criticism of the work.

    Significance

    The work dives deeper into mitochondrial function and metabolism of brown adipocytes and, thus, advances our understanding of thermogenesis in an incremental fashion. The work will be relevant to brown adipose tissue researchers and mitochondrial biologist.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    The paper makes a clear, well-supported case that the malate-aspartate shuttle (MAS) is active in brown adipocytes and supports adrenergically stimulated lipolysis. The combination of a functional MAS assay, targeted carrier knockdowns, and multi-modal lipolysis measurements is a strong package. The reconstituted mitochondrial assay paired with live-cell lipolysis imaging is technically elegant and broadly reusable. The main gap is the limited in-vitro scope relative to in-vivo cold adaptation.

    Major comments

    1. The manuscript posits that the loss of function of MASh components (Ogc1 and Aralar) decreases adrenergic-stimulated lipolysis by altering the cytosolic NAD⁺/NADH ratio, with AMPK/ACC mentioned as possible mediators. However, this remains speculative. Please provide mechanistic data directly linking MASh-dependent NAD⁺/NADH changes to the regulation of lipolysis in brown adipocytes during adrenergic stimulation.
    2. The absence of in vivo analysis of lipid-droplet size in MASh loss-of-function models is a major concern. In vitro results could be confounded by differences in differentiation stage between groups. Please document equivalent adipogenesis across groups (e.g., Pparg/Cebpa/Plin1/Fabp4 expression)
    3. Please include rescue experiments (add-back OGC1 and Aralar) to rule out siRNA/shRNA off-target effects and verify that the phenotype stems from MASh loss of function.
    4. Please expand on physiological significance: What is the importance of MASh regulation of BAT lipolysis in long-term adaptive thermogenesis?

    Minor comments

    1. Fig. 4 legend/title contains a typo ("lypolysis" → lipolysis).
    2. In Fig. 2 legend line: "Adevirus-mediated" → Adenovirus-mediated; "OCAR" → OCR.
    3. For lipolysis imaging, you already show Forskolin/Atglistatin/Etomoxir controls; add a vehicle-only time course overlay in the main figure (currently in text/legend) to aid visual comparison.
    4. Ensure consistent gene symbols (Atgl/Pnpla2), and protein capitalization.

    Referees cross-commenting

    In my view, the feedback offered by all three reviewers has been highly constructive, as each of them has contributed thoughtful and meaningful criticism that can help improve the quality, clarity, and overall impact of the work.

    Significance

    Advance - how it fits the literature and what kind of advance.

    Relative to prior work linking MASh (often via GOT1) to fuel preference and redox during thermogenesis, this study fills a mechanistic gap by showing that carrier-level MASh disruption (Aralar1/OGC1) specifically impairs adrenergic lipid mobilization upstream of β-oxidation, while respiration per cell can be buffered by compensatory mitochondrial biogenesis (lower OCR per mitochondrion). Conceptual/fundamental advance: it sharpens the redox - lipolysis axis in BAT and clarifies why changes in fuel availability (lipolysis) may limit thermogenesis even when bulk OCR looks preserved.

    Audience - who will be interested/influenced.

    Specialized but cross-cutting: adipose biology & thermogenesis, mitochondrial/redox metabolism, lipid-droplet and lipolysis communities, and metabolic-disease researchers exploring strategies to modulate BAT fuel handling.

    Reviewer expertise

    Adipose tissue and systemic energy metabolism; mitochondrial bioenergetics; thermogenic mechanisms in BAT/beige fat; transcriptional and metabolic control of lipid mobilization. Not a specialist in membrane-carrier biophysics.

  5. Version published to 10.1101/2025.08.04.667739 on bioRxiv