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1.
J Biol Chem ; 294(13): 5121-5136, 2019 03 29.
Article in English | MEDLINE | ID: mdl-30718282

ABSTRACT

Viperin (also known as radical SAM domain-containing 2 (RSAD2)) is an interferon-inducible and evolutionary conserved protein that participates in the cell's innate immune response against a number of viruses. Viperin mRNA is a substrate for endoribonucleolytic cleavage by RNase mitochondrial RNA processing (MRP) and mutations in the RNase MRP small nucleolar RNA (snoRNA) subunit of the RNase MRP complex cause cartilage-hair hypoplasia (CHH), a human developmental condition characterized by metaphyseal chondrodysplasia and severe dwarfism. It is unknown how CHH-pathogenic mutations in RNase MRP snoRNA interfere with skeletal development, and aberrant processing of RNase MRP substrate RNAs is thought to be involved. We hypothesized that viperin plays a role in chondrogenic differentiation. Using immunohistochemistry, real-time quantitative PCR, immunoblotting, ELISA, siRNA-mediated gene silencing, plasmid-mediated gene overexpression, label-free MS proteomics, and promoter reporter bioluminescence assays, we discovered here that viperin is expressed in differentiating chondrocytic cells and regulates their protein secretion and the outcome of chondrogenic differentiation by influencing transforming growth factor ß (TGF-ß)/SMAD family 2/3 (SMAD2/3) activity via C-X-C motif chemokine ligand 10 (CXCL10). Of note, we observed disturbances in this viperin-CXCL10-TGF-ß/SMAD2/3 axis in CHH chondrocytic cells. Our results indicate that the antiviral protein viperin controls chondrogenic differentiation by influencing secretion of soluble proteins and identify a molecular route that may explain impaired chondrogenic differentiation of cells from individuals with CHH.


Subject(s)
Chemokine CXCL10/metabolism , Chondrogenesis , Proteins/metabolism , Animals , Cell Line , Gene Knockdown Techniques , Humans , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , Mice , Oxidoreductases Acting on CH-CH Group Donors , Proteins/analysis , Proteins/genetics , Signal Transduction , Smad Proteins/metabolism , Transforming Growth Factor beta/metabolism
2.
Obesity (Silver Spring) ; 19(4): 792-9, 2011 Apr.
Article in English | MEDLINE | ID: mdl-20595951

ABSTRACT

Accumulation of triacylglycerols (TAGs) and acylcarnitines in skeletal muscle upon high-fat (HF) feeding is the resultant of fatty acid uptake and oxidation and is associated with insulin resistance. As medium-chain fatty acids (MCFAs) are preferentially ß-oxidized over long-chain fatty acids, we examined the effects of medium-chain TAGs (MCTs) and long-chain TAGs (LCTs) on muscle lipid storage and whole-body glucose tolerance. Rats fed a low-fat (LF), HFLCT, or an isocaloric HFMCT diet displayed a similar body weight gain over 8 weeks of treatment. Only HFLCT increased myocellular TAG (42.3 ± 4.9, 71.9 ± 6.7, and 48.5 ± 6.5 µmol/g for LF, HFLCT, and HFMCT, respectively, P < 0.05) and long-chain acylcarnitine content (P < 0.05). Neither HF diet increased myocellular diacylglycerol (DAG) content. Intraperitoneal (IP) glucose tolerance tests (1.5 g/kg) revealed a significantly decreased glucose tolerance in the HFMCT compared to the HFLCT-fed rats (802 ± 40, 772 ± 18, and 886 ± 18 area under the curve for LF, HFLCT, and HFMCT, respectively, P < 0.05). Finally, no differences in myocellular insulin signaling after bolus insulin injection (10 U/kg) were observed between LF, HFLCT, or HFMCT-fed rats. These results show that accumulation of TAGs and acylcarnitines in skeletal muscle in the absence of body weight gain do not impede myocellular insulin signaling or whole-body glucose intolerance.


Subject(s)
Carnitine/analogs & derivatives , Dietary Fats/administration & dosage , Fatty Acids/pharmacokinetics , Glucose Intolerance/metabolism , Muscle, Skeletal/metabolism , Triglycerides/pharmacokinetics , Analysis of Variance , Animals , Blood Glucose/metabolism , Blotting, Western , Body Weight , Carnitine/metabolism , Dietary Fats/metabolism , Diglycerides/analysis , Energy Metabolism , Glucose Tolerance Test , Insulin/blood , Insulin Resistance , Lipid Peroxidation , Male , Muscle, Skeletal/drug effects , Rats , Rats, Wistar
3.
Biochem Cell Biol ; 84(2): 215-31, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16609703

ABSTRACT

Glutamine synthetase (GS) is the only enzyme that can synthesize glutamine, but it also functions to detoxify glutamate and ammonia. Organs with high cellular concentrations of GS appear to function primarily to remove glutamate or ammonia, whereas those with a low cellular concentration appear to primarily produce glutamine. To validate this apparent dichotomy and to clarify its regulation, we determined the GS concentrations in 18 organs of the mouse. There was a >100-fold difference in GS mRNA, protein, and enzyme-activity levels among organs, whereas there was only a 20-fold difference in the GS protein:mRNA ratio, suggesting extensive transcriptional and posttranscriptional regulation. In contrast, only small differences in the GS enzyme activity : protein ratio were found, indicating that posttranslational regulation is of minor importance. The cellular concentration of GS was determined by relating the relative differences in cellular GS concentration, detected using image analysis of immunohistochemically stained tissue sections, to the biochemical data. There was a >1000-fold difference in cellular concentrations of GS between GS-positive cells in different organs, and cellular concentrations were up to 20x higher in subpopulations of cells within organs than in whole organs. GS activity was highest in pericentral hepatocytes (approximately 485 micromol.g(-1).min-(1), followed in descending order by epithelial cells in the epididymal head, Leydig cells in the testicular interstitium, epithelial cells of the uterine tube, acid-producing parietal cells in the stomach, epithelial cells of the S3 segment of the proximal convoluted tubule of the kidney, astrocytes of the central nervous tissue, and adipose tissue. GS activity in muscle amounted to only 0.4 micromol.g(-1).min(-1). Our findings confirmed the postulated dichotomy between cellular concentration and GS function.


Subject(s)
Glutamate-Ammonia Ligase/metabolism , Adipose Tissue/enzymology , Animals , Base Sequence , Epididymis/enzymology , Female , Glutamate-Ammonia Ligase/genetics , Immunohistochemistry , Intestines/enzymology , Kidney/enzymology , Liver/enzymology , Male , Mice , Organ Specificity , RNA Processing, Post-Transcriptional , RNA, Messenger/genetics , RNA, Messenger/metabolism , Stomach/enzymology , Testis/enzymology , Transcription, Genetic , Uterus/enzymology
4.
Am J Clin Nutr ; 76(1): 113-9, 2002 Jul.
Article in English | MEDLINE | ID: mdl-12081824

ABSTRACT

BACKGROUND: Although the loss of peripheral muscle mass has been shown convincingly in chronic obstructive pulmonary disease (COPD), the underlying pathogenesis remains unclear. OBJECTIVE: The aim of the present study was to determine the relations between skeletal muscle fiber types, fiber cross-sectional area (CSA), enzyme activities, and fat-free mass (FFM) in patients with COPD and in control subjects. DESIGN: In 15 patients with COPD and 15 healthy, age-matched control subjects, FFM was determined by dual-energy X-ray absorptiometry and bioelectrical impedance analysis. In biopsy specimens from the vastus lateralis fiber types, fiber CSA and activities of cytochrome oxidase (EC 1.9.3.1), succinate dehydrogenase (EC 1.3.99.1), and glycogen phosphorylase (EC 2.4.1.1) were examined immunohistochemically and histochemically. RESULTS: Compared with control subjects, patients with COPD had less FFM (49 compared with 59 kg, P = 0.030) and lower mean fiber CSA (3839 compared with 4647 microm(2), P = 0.037). A strong correlation (r = 0.87, P < 0.001) was observed between the FFM measured by bioelectrical impedance analysis and mean fiber CSA in patients with COPD. Within fiber-type categories the mean CSA of only the IIA/IIX and IIX fiber types was lower in patients than in control subjects [3358 compared with 4428 microm(2) (P = 0.022) and 2566 compared with 4248 microm(2) (P = 0.003), respectively]. In COPD, 20% of the type IIX fibers lacked stainable activities of cytochrome oxidase, succinate dehydrogenase, and glycogen phosphorylase, and this proportion correlated negatively with type IIX fiber CSA (r = -0.65, P = 0.012). CONCLUSIONS: Muscle fiber atrophy occurs in the vastus lateralis in patients with COPD and contributes to the loss of muscle mass in COPD. Atrophy is specific to fiber types IIA/IIX and IIX and is associated with a disturbed metabolic capacity.


Subject(s)
Muscle Fibers, Fast-Twitch/pathology , Muscular Atrophy/complications , Pulmonary Disease, Chronic Obstructive/complications , Aged , Body Composition , Electron Transport Complex IV/metabolism , Female , Glycogen Phosphorylase/metabolism , Humans , Male , Middle Aged , Muscle Fibers, Fast-Twitch/enzymology , Muscle Fibers, Fast-Twitch/metabolism , Muscular Atrophy/pathology , Pulmonary Disease, Chronic Obstructive/pathology , Succinate Dehydrogenase/metabolism , Weight Loss
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