Thymidine Kinase 2 Deficiency-Induced Mitochondrial DNA Depletion Causes Abnormal Development of Adipose Tissues and Adipokine Levels in Mice

Mammal adipose tissues require mitochondrial activity for proper development and differentiation. The components of the mitochondrial respiratory chain/oxidative phosphorylation system (OXPHOS) are encoded by both mitochondrial and nuclear genomes. The maintenance of mitochondrial DNA (mtDNA) is a key element for a functional mitochondrial oxidative activity in mammalian cells. To ascertain the role of mtDNA levels in adipose tissue, we have analyzed the alterations in white (WAT) and brown (BAT) adipose tissues in thymidine kinase 2 (Tk2) H126N knockin mice, a model of TK2 deficiency-induced mtDNA depletion. We observed respectively severe and moderate mtDNA depletion in TK2-deficient BAT and WAT, showing both tissues moderate hypotrophy and reduced fat accumulation. Electron microscopy revealed altered mitochondrial morphology in brown but not in white adipocytes from TK2-deficient mice. Although significant reduction in mtDNA-encoded transcripts was observed both in WAT and BAT, protein levels from distinct OXPHOS complexes were significantly reduced only in TK2-deficient BAT. Accordingly, the activity of cytochrome c oxidase was significantly lowered only in BAT from TK2-deficient mice. The analysis of transcripts encoding up to fourteen components of specific adipose tissue functions revealed that, in both TK2-deficient WAT and BAT, there was a consistent reduction of thermogenesis related gene expression and a severe reduction in leptin mRNA. Reduced levels of resistin mRNA were found in BAT from TK2-deficient mice. Analysis of serum indicated a dramatic reduction in circulating levels of leptin and resistin. In summary, our present study establishes that mtDNA depletion leads to a moderate impairment in mitochondrial respiratory function, especially in BAT, causes substantial alterations in WAT and BAT development, and has a profound impact in the endocrine properties of adipose tissues

Mitochondria Express α7 Nicotinic Acetylcholine Receptors to Regulate Ca2+ Accumulation and Cytochrome c Release: Study on Isolated Mitochondria

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that mediate synaptic transmission in the muscle and autonomic ganglia and regulate transmitter release in the brain. The nAChRs composed of α7 subunits are also expressed in non-excitable cells to regulate cell survival and proliferation. Up to now, functional α7 nAChRs were found exclusively on the cell plasma membrane. Here we show that they are expressed in mitochondria and regulate early pro-apoptotic events like cytochrome c release. The binding of α7-specific antibody with mouse liver mitochondria was revealed by electron microscopy. Outer membranes of mitochondria from the wild-type and β2−/− but not α7−/− mice bound α7 nAChR-specific antibody and toxins: FITC-labeled α-cobratoxin or Alexa 555-labeled α-bungarotoxin. α7 nAChR agonists (1 µM acetylcholine, 10 µM choline or 30 nM PNU-282987) impaired intramitochondrial Ca2+ accumulation and significantly decreased cytochrome c release stimulated with either 90 µM CaCl2 or 0.5 mM H2O2. α7-specific antagonist methyllicaconitine (50 nM) did not affect Ca2+ accumulation in mitochondria but attenuated the effects of agonists on cytochrome c release. Inhibitor of voltage-dependent anion channel (VDAC) 4,4′-diisothio-cyano-2,2′-stilbene disulfonic acid (0.5 µM) decreased cytochrome c release stimulated with apoptogens similarly to α7 nAChR agonists, and VDAC was co-captured with the α7 nAChR from mitochondria outer membrane preparation in both direct and reverse sandwich ELISA. It is concluded that α7 nAChRs are expressed in mitochondria outer membrane to regulate the VDAC-mediated Ca2+ transport and mitochondrial permeability transition

Sex differences of nicotinate-induced hyperbilirubinemia in Gilbert's syndrome. Implication of bilitranslocase function.

Intravenous administration of nicotinic acid (NA) is followed by an increase in serum unconjugated bilirubin level. This effect is higher in Gilbert's syndrome (GS) and this test has been used in the diagnosis of the syndrome. After administration of 5.9 mumol NA/kg body weight, the maximal increment of serum unconjugated bilirubin and the area under the bilirubin concentration time curve (AUC) were significantly higher (P less than 0.01) in GS males than in GS females. The half-life of the first fast slope of plasma disappearance curve of the drug was also significantly prolonged in GS males as compared to GS females (15.91 +/- 1.12 vs 9.13 +/- 1.25 min, mean +/- SEM, P less than 0.005). The maximal bilirubin increment and AUC were linearly correlated (P less than 0.01) with NA plasma half-life. Purified preparations of bilitranslocase, a liver plasma-membrane protein involved in bilirubin and sulfobromophthalein (BSP) transport, specifically bound NA and the drug competitively inhibited BSP uptake in rat liver plasma membrane vesicles (Ki = 50 nM). These data suggest that, in addition to the hemolytic effect of the drug, NA-induced hyperbilirubinaemia could be also due to a competition between the two anions at the sinusoidal plasma membrane level. A possible implication of bilitranslocase in GS is considered