Mitochondrial fatty acid synthesis coordinates oxidative metabolism in mammalian mitochondria
Authors: Jared Rutter
Ashley Solmonson
Ralph J DeBerardinis
Jeffrey T Morgan
Joao A Paulo
Mi-Young Jeong
James E Cox
Sarah Fogarty
J Alan Maschek
Katsuhiko Funai
Scott F Rusin
Steven P Gygi
Yeyun Ouyang
Jordan A Berg
Dennis R Winge
Claire L Bensard
Sara M Nowinski
Sandra Lettlova
Bradley C Naylor
Journal: eLife (2020)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: TCA cycle; biochemistry; chemical biology; electron transport chain; fatty acid synthesis
Affiliations: Department of Biochemistry, Salt Lake City, United States, Children’s Medical Center Research Institute, University of Texas Southwestern Medical Center, Dallas, United States, Department of Cell Biology, Harvard University School of Medicine, Boston, United States, Diabetes & Metabolism Research Center, Salt Lake City, United States, Department of Nutrition and Integrative Physiology, Salt Lake City, United States, Metabolomics, Proteomics and Mass Spectrometry Core Research Facilities University of Utah, Salt Lake City, United States, Howard Hughes Medical Institute, Salt Lake City, United States, Department of Internal Medicine, Salt Lake City, United States

Abstract:  Cells harbor two systems for fatty acid synthesis, one in the cytoplasm (catalyzed by fatty acid synthase, FASN) and one in the mitochondria (mtFAS). In contrast to FASN, mtFAS is poorly characterized, especially in higher eukaryotes, with the major product(s), metabolic roles, and cellular function(s) being essentially unknown. Here we show that hypomorphic mtFAS mutant mouse skeletal myoblast cell lines display a severe loss of electron transport chain (ETC) complexes and exhibit compensatory metabolic activities including reductive carboxylation. This effect on ETC complexes appears to be independent of protein lipoylation, the best characterized function of mtFAS, as mutants lacking lipoylation have an intact ETC. Finally, mtFAS impairment blocks the differentiation of skeletal myoblasts in vitro. Together, these data suggest that ETC activity in mammals is profoundly controlled by mtFAS function, thereby connecting anabolic fatty acid synthesis with the oxidation of carbon fuels. 

Retracted: No

PubPeer comments:
 The authors have genetically inhibited the mitochondrial fatty acid synthesis pathway (mtFAS) in C2C12 myoblasts by ablating the genes encoding enzymes (MCAT, OXSM and MCER) of this pathway. They also inhibited enzymes (Lipt1) the enzyme that catalyzes protein lipoylation. Genetic inhibition of mtFAS reduces mitochondrial activity and authors claim that this is due to inhibition of protein lipoylation, the ones they demonstrate are PDH (DLAT) and KDH (DLST). It is unfortunate that the authors have demonstrated the effects of blocking mtFAS that we already know from previous studies and did not made any effort to determine any novel role of this cellular biosynthetic pathway such as the formation of any unknown fatty acids and their role in the regulation of mitochondrial functions. The following are our concerns. T7E1 assay is used only for screening and not for confirming the specific targeting by site-specific nucleases. Authors should have performed NGS and PCR to determine the specificity and efficiency of targeting by CRISPR Cas9 complexes. They studied the mitochondrial activity in mtFAS knockout cells (Fig 2A) by performing Seahorse XF mitochondrial stress test. The knockout cells shows small reduction in basal response and a greater inhibition of FCCP-stimulated response. During the Seahorse assays, the tested cells (control and test) should be at their optimal density (cell numbers) when OCR ECAR measured to accurately record their responses to drugs used for modulating the mitochondrial activity. If the cell density is at suboptimal levels as in case of the mtFAS KO cells that proliferate slowly, recorded data would be incorrect. The authors have tried to correct variations due to difference in cell numbers by post-assay data normalization to total protein values, but that however would not correct the erroneous responses caused by cells at suboptimal numbers. This issue can be clearly seen in the data shown Fig. 5B where 10,000 cells were used, but both control and mutant cells show very low OCR values, close to background values. The authors measured the mitochondrial membrane potential by staining cells with mitotracker green and mitotracker red dyes. They should have known that both of these stains are used for assessing mitochondrial content measurements since they remain covalently bound to mitochondrial proteins even if there is fall in mitochondrial membrane potential. And dyes like TMRM, TMRE and Rhodamine 123 are used for studying mitochondrial membrane potential. From the micrographs shown for mitotracker stained control and mtFAS KO cells, there is no reduction in total mitochondrial content in KO cells compared to controls. But the fluorescence intensity is clearly low in KO cells. So the question would be whether it is due to poor stain incorporation in the KO mitochondria, incorrect microscopic setting (off focused, one will know from metadata on image acquisition setting) or cells were incubated with the fluorescent dye at a suboptimal concentration. Authors show several immunblots (Fig 2D and F) for mitochondrial respiratory complex subunits without accompanying mitochondrial loading control (Citrate synthase, VDAC1, HSP60) immunoblots and claim reduction in several mitochondrial complex subunit expression in the mtFAS KO cells. To determine whether similar amount of mitochondria exist in control and KO crude mitochondrial preparation, they needed blot for loading controls. This was also necessary because the KO cells grow at a slow rate. Fig 1, 2 and 5: To accurately determine the lipoylated DLAT and DLST, they should have stripped and reprobed the immunoblot with total DLAT and total DLST antibodies, and should have shown the Lipoylated total DLAT and Lipoylated total DLST. Also, they should have shown the uncropped lipoylated protein immunoblot with the molecular weight markers, which would have helped us in confirming the molecular weight of these proteins. Fig. 2F: Why Oxsm re-expression in Oxsm KO cells did not completely revert the ETC complex protein expression back to expression levels seen in GFP control? Supplemental file 1. Table: If PDH (DLAT) lipoylation is inhibited and glycolysis was increased in mtFAS KO cells, why lactate levels unchanged? In the metabolomics data shown in Fig 4, succinate formation from endogenous (m+0, from isocitrate - alpha-KG - succinyl-CoA - Succinate) precursors was unaffected and its generation from exogenous precursor (glutamine) actually increased (m+4, alpha-KG - succinyl-CoA - Succinate). This means loss of DLST lipoylation did not block KDH activity. Since majority of the metabolites formed from Glutamine are increased (refer Supplemental file 1. Table) and that include products of KDH activity, we think that blocking mtFAS did not block KDH activity and these cells could be preferentially using the glutamine. Additionally, M+0 is the major peak for all the TCA metabolites, which was increased in mtFAS KO cells for all the TCA cycle metabolites (citrate, fumarate, malate)when compared with controls. And succinate is unchanged when compared with control (Fig. 4 G-L), This result indicates that mtFAS KO mitochondria is capable of supporting oxidative energy metabolism at similar levels or better as compared to controls. ,