• 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2020-03
  • 2020-07
  • 2020-08
  • br Material and methods br Results br Discussion


    Material and methods
    Discussion The discovery of active BAT in adult humans has brought renewed interest in this tissue as a potential therapeutic target for the treatment of obesity and its associated metabolic diseases by means of increasing MIK665 (S-64315) expenditure. The capacity of brown adipocytes to dissipate energy as heat relies on the acquisition during the course of their differentiation of a dense network of mitochondria distinctively equipped with UCP1 [32,33]. The balanced acquisition of all the protein machinery of the respiratory chain is absolutely needed in order to build up the membrane electrochemical potential that will be dissipated by UCP1 to generate heat. The present study demonstrates that MTERF4, a mitochondrial protein of the MTERF family of transcription termination factors, is essential for the full oxidative function of brown adipocytes. Our findings showing that lack of MTERF4 severely impairs the translation of proteins encoded by the mitochondrial genome in brown adipocytes fully supports the current view of MTERF4 as a central factor for mitochondrial protein translation. Indeed, the current action model for MTERF4 proposes that it controls mitochondrial protein translation by participating in mitochondrial ribogenesis [22]. Specifically, MTERF4 would form a complex with NSUN4, another mitochondrial protein with methyltransferase activity, and facilitate its recruitment to the large mitochondrial ribosome subunit. The MTERF4/NSUN4 complex would enable the interaction and subsequent assembly of the large and small mitochondrial ribosomal subunits to form fully functional mitoribosomes [23]. Although our results strongly support a critical role of MTERF4 mitoribogenesis and protein translation, some variable but still detectable translation of mtDNA-encoded genes was detected in BAT of mice devoid of MTERF4. The detection of some basal translation of mtDNA-encoded proteins could be explained by the translational activity in adipocytes in which efficient recombination of the Mterf4 gene has not occurred [31]. Residual translation is also compatible with some MIK665 (S-64315) degree of mitoribogenesis still occurring in the absence of MTERF4 [22], suggesting that other factors may contribute to the assembly of mitochondrial ribosomes to ensure certain basal level of mitochondrial protein translation. Although MTERF4 exclusively controls the translation of mtDNA-encoded proteins, the levels of protein subunits encoded by the nDNA but belonging to OxPhos complexes composed by both nDNA- and mtDNA-encoded proteins were found reduced in BAT of MTERF4-FAT-KO mice. mtDNA-encoded OxPhos subunits are highly hydrophobic proteins residing in the inner mitochondrial membrane and act as seeding cores for the assembly of the rest of the subunits that form the functional OxPhos complex. In this regard, given the defect in their translation in the absence of MTERF4, mtDNA-encoded proteins become limiting for the stoichiometric assembly of the OxPhos complexes. As a result, the unassembled subunits are eventually degraded to avoid their accumulation within the adipocytes of MTERF4-FAT-KO mice [34]. As a consequence, the loss of MTERF4 in BAT leads to a net decrease of approximately 40–60% in the levels of assembled OxPhos complexes I, III and IV that translates into a similar reduction in their enzymatic activity and an overall decreased in the oxidative capacity of brown adipocytes, both under basal conditions and upon adrenergic stimulation. The primary physiological defect that stems from ablation of MTERF4 in adipose tissues is the severe cold intolerance exhibited by MTERF4-FAT-KO mice when acutely exposed to low temperatures. The reduced capacity for cold-induced thermogenesis is neither due to inadequate UCP1 expression -MTERF4-FAT-KO mice exhibit similar levels of UCP1 than their Wt littermates- nor to a reduced capacity to mobilize fatty acids by lipolysis. Although lipolysis is critical for BAT thermogenesis, it has been shown that mice with defective lipolysis specifically in brown adipocytes have their cold-induced thermogenic capacity preserved due to the capacity to use fatty acids released by white adipocytes as thermogenic substrates [30,35]. Therefore, even if the increased triglyceride accumulation observed in BAT of MTERF4-FAT-KO mice would suggest an impaired brown adipocyte lipolytic capacity, the similar response of Wt and MTERF4-FAT-KO mice to isoproterenol in raising the circulating levels of free fatty acids indicates that the availability of lipid substrates for thermogenesis is guaranteed despite the lack of MTERF4. Consequently, our results unequivocally point towards the reduced respiratory chain complex activity as the major cause of the cold intolerance exhibited by MTERF4-FAT-KO mice, which are unable to build up the mitochondrial electrochemical gradient required by UCP1 to carry out thermogenesis.