β-Adrenergic stimulation of interleukin-1α and interleukin-6 expression in mouse brown adipocytes

AbstractMouse brown adipocytes in primary culture were shown to contain high levels of mRNA for interleukin-1α (IL-1α) which could be further stimulated up to 9-fold by norepinephrine (NE). Even higher stimulation by NE, up to 40-fold, was found in case of interleukin-6 (IL-6). Time-course of activation of both genes was biphasic, but the response of IL-6 gene was slower than of IL-1α gene. IL-1α mRNA level reached the maximum after 1 h and the second, lower increase, occurred after 8 h. IL-6 mRNA level showed first maximum after 2 h, but the highest level was found after 8 h. Similarly to NE, the expression of IL-1α and IL-6 genes was stimulated by selective β-adrenergic agonist isoproterenol, β3-selective agonist CGP-12117, forskoline and db-cAMP. The activation of both genes by CGP-12177 was dose-dependent with the optimum at 100 nM concentration. Stimulation of α-adrenergic receptors by cirazoline and oxymetazoline was without any effect. When the expression of IL-6 was studied at the protein level, the stimulation of IL-6 gene via β3-receptors resulted in secretion of IL-6 up to the concentration 10 ng/ml culture media in 24 h. The results indicate a new type of regulation of expression of IL-1α and IL-6 genes in brown adipocytes by catecholamines acting via β3-adrenergic receptors. The resulting increase in IL-6 production by brown adipocytes could significantly contribute to systemic levels of IL-6

The function and the role of the mitochondrial glycerol-3-phosphate dehydrogenase in mammalian tissues

AbstractMitochondrial glycerol-3-phosphate dehydrogenase (mGPDH) is not included in the traditional textbook schemes of the respiratory chain, reflecting the fact that it is a non-standard, tissue-specific component of mammalian mitochondria. But despite its very simple structure, mGPDH is a very important enzyme of intermediary metabolism and as a component of glycerophosphate shuttle it functions at the crossroads of glycolysis, oxidative phosphorylation and fatty acid metabolism. In this review we summarize the present knowledge on the structure and regulation of mGPDH and discuss its metabolic functions, reactive oxygen species production and tissue and organ specific roles in mammalian mitochondria at physiological and pathological conditions

Mitochondrial diseases and ATPase defects of nuclear origin

AbstractDysfunctions of the F1Fo-ATPase complex cause severe mitochondrial diseases affecting primarily the paediatric population. While in the maternally inherited ATPase defects due to mtDNA mutations in the ATP6 gene the enzyme is structurally and functionally modified, in ATPase defects of nuclear origin mitochondria contain a decreased amount of otherwise normal enzyme. In this case biosynthesis of ATPase is down-regulated due to a block at the early stage of enzyme assembly—formation of the F1 catalytic part. The pathogenetic mechanism implicates dysfunction of Atp12 or other F1-specific assembly factors. For cellular energetics, however, the negative consequences may be quite similar irrespective of whether the ATPase dysfunction is of mitochondrial or nuclear origin

Identification of nucleus-encoded F0I protein of bovine heart mitochondrial H+-ATPase as a functional part of the F0 moiety

AbstractThe F0I protein of apparent Mr 27000, previously characterized [(1988) Eur. J. Biochem. 173, 1–8] as a genuine component of bovine heart F0, has been sequenced and shown to be identical with the nucleus encoded 24668 Da protein characterized earlier [(1987) J. Mol. Biol. 197, 89–100]. It is directly shown by proteolytic cleavage and reconstitution experiments that this protein, denoted here as PVP from the single-letter codes of the last three residues of the N-terminus, is involved in proton conduction by F0 and in its sensitivity to oligomycin

Mitochondrial F0F1 H+-ATP synthase Characterization of F0 components involved in H+ translocation

AbstractThe membrane F0, sector of mitochondrial ATP synthase complex was rapidly isolated by direct extraction with CHAPS from F1-depleted submitochondrial particles. The preparation thus obtained is stable and can be reconstituted in artificial phospholipid membranes to result in oligomycin-sensitive proton conduction, or recombined with purified F1 to give the oligomycin-sensitive F0F1-ATPase complex. The F0 preparation and constituent polypeptides were characterized by SDS-polyacrylamide gel electrophoresis and immunoblot analysis. The functional role of F0 polypeptides was examined by means of trypsin digestion and reconstitution studies. It is shown that, in addition to the 8 kDa DCCD-binding protein, the nuclear encoded protein [(1987) J. Mol. Biol. 197, 89–100], characterized as an intrinsic component of F0, (F0I, PVP protein [(1967) J. Biol. Chem. 242, 2547–2551]) is involved in H+ translocation and the sensitivity of this process to the F0 inhibitors, DCCD and oligomycin

Loss of COX4I1 leads to combined respiratory chain deficiency and impaired mitochondrial protein synthesis

The oxidative phosphorylation (OXPHOS) system localized in the inner mitochondrial membrane secures production of the majority of ATP in mammalian organisms. Individual OXPHOS complexes form supramolecular assemblies termed supercomplexes. The complexes are linked not only by their function but also by interdependency of individual complex biogenesis or maintenance. For instance, cytochrome c oxidase (cIV) or cytochrome bc1 complex (cIII) deficiencies affect the level of fully assembled NADH dehydrogenase (cI) in monomeric as well as supercomplex forms. It was hypothesized that cI is affected at the level of enzyme assembly as well as at the level of cI stability and maintenance. However, the true nature of interdependency between cI and cIV is not fully understood yet. We used a HEK293 cellular model where the COX4 subunit was completely knocked out, serving as an ideal system to study interdependency of cI and cIV, as early phases of cIV assembly process were disrupted. Total absence of cIV was accompanied by profound deficiency of cI, documented by decrease in the levels of cI subunits and significantly reduced amount of assembled cI. Supercomplexes assembled from cI, cIII, and cIV were missing in COX4I1 knock-out (KO) due to loss of cIV and decrease in cI amount. Pulse-chase metabolic labeling of mitochondrial DNA (mtDNA)-encoded proteins uncovered a decrease in the translation of cIV and cI subunits. Moreover, partial impairment of mitochondrial protein synthesis correlated with decreased content of mitochondrial ribosomal proteins. In addition, complexome profiling revealed accumulation of cI assembly intermediates, indicating that cI biogenesis, rather than stability, was affected. We propose that attenuation of mitochondrial protein synthesis caused by cIV deficiency represents one of the mechanisms, which may impair biogenesis of cI

Loss of COX4I1 Leads to Combined Respiratory Chain Deficiency and Impaired Mitochondrial Protein Synthesis.

Funder: Akademie Věd České Republiky; Grant(s): RVO:67985823The oxidative phosphorylation (OXPHOS) system localized in the inner mitochondrial membrane secures production of the majority of ATP in mammalian organisms. Individual OXPHOS complexes form supramolecular assemblies termed supercomplexes. The complexes are linked not only by their function but also by interdependency of individual complex biogenesis or maintenance. For instance, cytochrome c oxidase (cIV) or cytochrome bc1 complex (cIII) deficiencies affect the level of fully assembled NADH dehydrogenase (cI) in monomeric as well as supercomplex forms. It was hypothesized that cI is affected at the level of enzyme assembly as well as at the level of cI stability and maintenance. However, the true nature of interdependency between cI and cIV is not fully understood yet. We used a HEK293 cellular model where the COX4 subunit was completely knocked out, serving as an ideal system to study interdependency of cI and cIV, as early phases of cIV assembly process were disrupted. Total absence of cIV was accompanied by profound deficiency of cI, documented by decrease in the levels of cI subunits and significantly reduced amount of assembled cI. Supercomplexes assembled from cI, cIII, and cIV were missing in COX4I1 knock-out (KO) due to loss of cIV and decrease in cI amount. Pulse-chase metabolic labeling of mitochondrial DNA (mtDNA)-encoded proteins uncovered a decrease in the translation of cIV and cI subunits. Moreover, partial impairment of mitochondrial protein synthesis correlated with decreased content of mitochondrial ribosomal proteins. In addition, complexome profiling revealed accumulation of cI assembly intermediates, indicating that cI biogenesis, rather than stability, was affected. We propose that attenuation of mitochondrial protein synthesis caused by cIV deficiency represents one of the mechanisms, which may impair biogenesis of cI

Wars2 is a determinant of angiogenesis.

Coronary flow (CF) measured ex vivo is largely determined by capillary density that reflects angiogenic vessel formation in the heart in vivo. Here we exploit this relationship and show that CF in the rat is influenced by a locus on rat chromosome 2 that is also associated with cardiac capillary density. Mitochondrial tryptophanyl-tRNA synthetase (Wars2), encoding an L53F protein variant within the ATP-binding motif, is prioritized as the candidate at the locus by integrating genomic data sets. WARS2(L53F) has low enzyme activity and inhibition of WARS2 in endothelial cells reduces angiogenesis. In the zebrafish, inhibition of wars2 results in trunk vessel deficiencies, disordered endocardial-myocardial contact and impaired heart function. Inhibition of Wars2 in the rat causes cardiac angiogenesis defects and diminished cardiac capillary density. Our data demonstrate a pro-angiogenic function for Wars2 both within and outside the heart that may have translational relevance given the association of WARS2 with common human diseases