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Proceedings of the National Academy of... Jun 2003Mitochondria are the primary site of skeletal muscle fuel metabolism and ATP production. Although insulin is a major regulator of fuel metabolism, its effect on...
Mitochondria are the primary site of skeletal muscle fuel metabolism and ATP production. Although insulin is a major regulator of fuel metabolism, its effect on mitochondrial ATP production is not known. Here we report increases in vastus lateralis muscle mitochondrial ATP production capacity (32-42%) in healthy humans (P < 0.01) i.v. infused with insulin (1.5 milliunits/kg of fat-free mass per min) while clamping glucose, amino acids, glucagon, and growth hormone. Increased ATP production occurred in association with increased mRNA levels from both mitochondrial (NADH dehydrogenase subunit IV) and nuclear [cytochrome c oxidase (COX) subunit IV] genes (164-180%) encoding mitochondrial proteins (P < 0.05). In addition, muscle mitochondrial protein synthesis, and COX and citrate synthase enzyme activities were increased by insulin (P < 0.05). Further studies demonstrated no effect of low to high insulin levels on muscle mitochondrial ATP production for people with type 2 diabetes mellitus, whereas matched nondiabetic controls increased 16-26% (P < 0.02) when four different substrate combinations were used. In conclusion, insulin stimulates mitochondrial oxidative phosphorylation in skeletal muscle along with synthesis of gene transcripts and mitochondrial protein in human subjects. Skeletal muscle of type 2 diabetic patients has a reduced capacity to increase ATP production with high insulin levels.
Topics: Adenosine Triphosphate; Adult; Cell Nucleus; Citrate (si)-Synthase; DNA, Complementary; Diabetes Mellitus, Type 2; Dose-Response Relationship, Drug; Electron Transport Complex IV; Female; Glucose Tolerance Test; Humans; Insulin; Male; Mitochondria; Muscle, Skeletal; Oxygen; Protein Biosynthesis; RNA; RNA, Messenger; Reverse Transcriptase Polymerase Chain Reaction; Substrate Specificity; Time Factors
PubMed: 12808136
DOI: 10.1073/pnas.1332551100 -
Proceedings of the National Academy of... Nov 1987The development of a cell culture system that produces erythropoietin (Epo) in a regulated manner has been the focus of much effort. We have screened multiple renal and... (Comparative Study)
Comparative Study
The development of a cell culture system that produces erythropoietin (Epo) in a regulated manner has been the focus of much effort. We have screened multiple renal and hepatic cell lines (including MDCK, LLC-PK1, BHK, WRL 68, CLCL, A704, CRFK, A498, ACHN, TCMK-1, LLC-MK2, CaKi-2, HepG2, and Hep3B) for either constitutive or regulated expression of Epo. Only the human hepatoma cell lines, Hep3B and HepG2, made significant amounts of Epo as measured both by radioimmunoassay and in vitro bioassay (as much as 330 milliunits per 10(6) cells in 24 hr). The constitutive production of Epo increased dramatically as a function of cell density in both cell lines. At cell densities less than 3.3 X 10(5) cells per cm2, there was little constitutive release of Epo in the medium (less than 30 milliunits per 10(6) cells in 24 hr). With Hep3B cells grown at low cell densities, a mean 18-fold increase in Epo expression was seen in response to hypoxia and a 6-fold increase was observed in response to incubation in medium containing 50 microM cobalt(II) chloride. At similar low cell densities, Epo production in HepG2 cells could be enhanced an average of about 3-fold by stimulation with either hypoxia or cobalt(II) chloride. Upon such stimulation, both cell lines demonstrated markedly elevated levels of Epo mRNA. Hence, both Hep3B and HepG2 cell lines provide an excellent in vitro system in which to study the physiological regulation of Epo expression.
Topics: Carcinoma, Hepatocellular; Cell Line; Erythropoietin; Gene Expression Regulation; Humans; Liver Neoplasms; Neoplasm Proteins; RNA, Messenger; Tumor Cells, Cultured
PubMed: 2825172
DOI: 10.1073/pnas.84.22.7972 -
The Journal of Biological Chemistry Mar 1981Perfusion of livers from fed rats with medium containing glucagon (2 x 10(-10) or 1 x 10(-8) M) resulted in both time- and concentration-dependent inactivation of...
Perfusion of livers from fed rats with medium containing glucagon (2 x 10(-10) or 1 x 10(-8) M) resulted in both time- and concentration-dependent inactivation of glycogen synthase phosphatase. Expected changes occurred in cAMP, cAMP-dependent protein kinase, glycogen synthase, and glycogen phosphorylase. The effect of glucagon on synthase phosphatase was partially reversed by simultaneous addition of insulin (4 x 10(-8) M), an effect paralleled by a decrease in cAMP. Addition of arginine vasopressin (10 milliunits/ml) resulted in a similar inactivation of synthase phosphatase and activation of phosphorylase, but independent of any changes in cAMP or its kinase. Phosphorylase phosphatase activity was unaffected by any of these hormones. Synthase phosphatase activity, measured as the ability of a crude homogenate to catalyze the conversion of purified rat liver synthase D to the I form, was no longer inhibited by glucagon or vasopressin when phosphorylase antiserum was added to the phosphatase assay mixture in sufficient quantity to inhibit 90-95% of the phosphorylase a activity. These data support the following conclusions: 1) hepatic glycogen synthase phosphatase activity is acutely modulated by hormones, 2) hepatic glycogen synthase phosphatase and phosphorylase phosphatase are regulated differently, 3) the hormone-mediated changes in synthase phosphatase cannot be explained by an alteration of the synthase D molecule affecting its behavior as a substrate, and 4) glycogen synthase phosphatase activity is at least partially controlled by the level of phosphorylase a.
Topics: Animals; Arginine Vasopressin; Cyclic AMP; Glucagon; Glycogen Synthase; Glycogen-Synthase-D Phosphatase; Insulin; Kinetics; Liver; Male; Perfusion; Phosphoprotein Phosphatases; Phosphorylase a; Protein Kinases; Rats
PubMed: 6259145
DOI: No ID Found -
The Journal of Biological Chemistry Oct 2001The genes coding for two different proteins with homologies to glutaredoxins have been identified in the genome of the malarial parasite Plasmodium falciparum. Both...
The genes coding for two different proteins with homologies to glutaredoxins have been identified in the genome of the malarial parasite Plasmodium falciparum. Both genes were amplified from a gametocytic cDNA and overexpressed in Escherichia coli. The smaller protein (named PfGrx-1) with 12.4 kDa in size exhibits the typical glutaredoxin active site motif "CPYC," shows glutathione-dependent glutaredoxin activity in the beta-hydroxyethyl disulfide (HEDS) assay, and reduces Trypanosoma brucei ribonucleotide reductase. Glutathione:HEDS transhydrogenase activity (approximately 60 milliunits/mg of protein) was clearly detectable in trophozoite extracts from eight different P. falciparum strains and did not differ between chloroquine-resistant and -sensitive parasites. Five different antimalarial drugs at 100 microm did not significantly influence isolated PfGrx-1 activity. In contrast, the second protein (deduced mass 19.9 kDa) with homology to glutaredoxins (31% identity to Schizosaccharomyces pombe in a 140-amino acid overlap) was not active in the HEDS assay; however, its general dithiol reducing activity was demonstrated in the insulin assay in the presence of dithiothreitol. Interestingly, the sequence contains a PICOT (for protein kinase C-interacting cousin of thioredoxin) homology domain, which might suggest regulatory functions of the protein. We named this protein PfGLP-1, for P. falciparum 1-Cys-glutaredoxin-like protein-1. In contrast to glutaredoxins, PfGLP-1 could not be reduced by glutathione. This is the first report on glutaredoxin-like proteins in the family of Plasmodia.
Topics: Amino Acid Sequence; Animals; Base Sequence; Carrier Proteins; Cloning, Molecular; DNA, Protozoan; Glutaredoxins; Insulin; Molecular Sequence Data; Oxidoreductases; Plasmodium falciparum; Protein Structure, Tertiary; Proteins; Protozoan Proteins; Recombinant Proteins; Ribonucleotide Reductases; Sequence Homology, Amino Acid
PubMed: 11479312
DOI: 10.1074/jbc.M105524200 -
The Journal of Biological Chemistry Aug 1991The 34-kDa subunit of rat liver phosphoribosylpyrophosphate synthetase is a mixture of the two highly homologous isoforms, PRS I and PRS II. Heretofore, it was not...
The 34-kDa subunit of rat liver phosphoribosylpyrophosphate synthetase is a mixture of the two highly homologous isoforms, PRS I and PRS II. Heretofore, it was not possible to separate the two. We now describe isolation and characterization of the recombinant isoforms, named rPRS I and rPRS II. The respective rat cDNAs were inserted into vectors constructed from pKK233-2 by replacing its replication origin with that of pGEM-1 and expressed in Escherichia coli. The rPRS I and rPRS II were purified to apparent homogeneity with specific activities of 33,400 and 46,200 milliunits/mg, respectively; these values were at least 2.5-fold higher than the highest value for the mammalian enzyme so far reported. Both isoforms showed a similar dependency on Pi as an absolute activator. Sulfate partially substituted for Pi. The maximal activities of rPRS I and rPRS II with sulfate were 43 and 7%, respectively, of those seen with Pi. The two isoforms differed in sensitivity to inhibition by ADP and GDP. Inhibition of rPRS I and rPRS II by 0.3 mM ADP was 87 and 54%, respectively, and inhibition by 1 mM GDP was 93 and 24%, respectively. rPRS II was 180-fold more sensitive than rPRS I to heat inactivation at 49 degrees C.
Topics: Animals; Cloning, Molecular; DNA; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Genes, Bacterial; Isoenzymes; Liver; Plasmids; Rats; Recombinant Proteins; Ribose-Phosphate Pyrophosphokinase; Sulfuric Acids
PubMed: 1651917
DOI: No ID Found -
The Journal of Biological Chemistry Mar 1997To investigate the role in catalysis and/or substrate binding of the Walker motif residues of rat testis fructose 6-phosphate, 2-kinase:fructose-2,6-bisphosphatase (Fru...
To investigate the role in catalysis and/or substrate binding of the Walker motif residues of rat testis fructose 6-phosphate, 2-kinase:fructose-2,6-bisphosphatase (Fru 6-P,2-kinase:Fru-2,6-Pase), we have constructed and characterized mutant enzymes of Asp-128, Thr-52, Asn-73, Thr-130, and Tyr-197. Replacement of Asp-128 by Ala, Asn, and Ser resulted in a small decrease in Vmax and a significant increase in Km values for both substrates. These mutants exhibited similar pH activity profiles as that of the wild type enzyme. Mutation of Thr-52 to Ala resulted in an enzyme with an infinitely high Km for both substrates and an 800-fold decreased Vmax. Substitution of Asn-73 with Ala or Asp caused a 100- and 600-fold increase, respectively in KFru 6-P with only a small increase in KATP and small changes in Vmax. Mutation of Thr-130 caused small changes in the kinetic properties. Replacement of Tyr-197 with Ser resulted in an enzyme with severely decreased binding of Fru 6-P with 3-fold decreased Vmax. A fluorescent analog of ATP, 2'(3')-O-(N-methylanthraniloyl)ATP (mant-ATP) served as a substrate with Km = 0.64 microM, and Vmax = 25 milliunits/mg and was a competitive inhibitor with respect to ATP. When mant-ATP bound to the enzyme, fluorescence intensity at 440 nm increased. mant-ATP binding of the wild type and the mutant enzymes were compared using the fluorometric method. The Kd values of the T52A and D128N enzymes were infinitely high and could not be measured, while those of the other mutant enzymes increased slightly. These results provide evidence that those amino acids are involved in substrate binding, and they are consistent with the crystallographic data. The results also suggest that Asp-128 does not serve as a nucleophile in catalysis, and since there are no other potential nucleophiles in the active site, we hypothesize that the Fru 6-P,2-kinase reaction is mediated via a transition state stabilization mechanism.
Topics: Amino Acids; Animals; Binding Sites; Hydrogen-Ion Concentration; Kinetics; Male; Muscles; Mutagenesis, Site-Directed; Phosphofructokinase-2; Phosphoric Monoester Hydrolases; Phosphotransferases (Alcohol Group Acceptor); Rabbits; Rats; Testis
PubMed: 9065453
DOI: 10.1074/jbc.272.12.7867 -
The Journal of Biological Chemistry Jan 1992Clinical and experimental data indicate that activated oxygen species interfere with vascular endothelial cell function. Here, the impact of extracellular oxidant injury...
Clinical and experimental data indicate that activated oxygen species interfere with vascular endothelial cell function. Here, the impact of extracellular oxidant injury on the fibrinolytic response of cultured human umbilical vein endothelial (HUVE) cells was investigated at the protein and mRNA levels. Xanthine (50 microM) and xanthine oxidase (100 milliunits), which produces the superoxide anion radical (O2-) and hydrogen peroxide (H2O2), was used to sublethally injure HUVE cells. Following a 15-min exposure, washed cells were incubated for up to 24 h in serum-free culture medium. Tissue-type plasminogen activator (t-PA) antigen, plasminogen activator inhibitor-1 (PAI-1) antigen, and PAI-1 activity were determined in 1.25 ml of conditioned medium and t-PA and PAI-1 mRNA in the cell extracts of 2 x 10(6) HUVE cells. Control cells secreted 3.9 +/- 1.3 ng/ml (mean +/- S.D., n = 12) within 24 h. Treatment with xanthine/xanthine oxidase for 15 min induced a 2.8 +/- 0.4-fold increase (n = 12, p less than 0.05) of t-PA antigen secretion after 24 h. The t-PA antigen was recovered predominantly in complex with PAI-1. The oxidant injury caused a 3.0 +/- 0.8-fold increase (n = 9, p less than 0.05) in t-PA mRNA within 2 h. Total protein synthesis was unaltered by xanthine/xanthine oxidase. The oxidant scavengers superoxide dismutase and catalase, in combination, abolished the effect of xanthine/xanthine oxidase on t-PA secretion and t-PA mRNA synthesis. Xanthine/xanthine oxidase treatment of HUVE cells did not affect the PAI-1 secretion in conditioned medium nor the PAI-1 mRNA levels in cell extracts. Thus extracellular oxidant injury induces t-PA but not PAI-1 synthesis in HUVE cells.
Topics: Antigens; Blotting, Northern; Blotting, Western; Cells, Cultured; Endothelium, Vascular; Fibrinolysis; Free Radicals; Humans; Hydrogen Peroxide; Oxygen; Plasminogen Inactivators; Precipitin Tests; RNA, Messenger; Tissue Plasminogen Activator; Umbilical Veins; Xanthine; Xanthine Oxidase; Xanthines
PubMed: 1730619
DOI: No ID Found -
The Journal of Biological Chemistry Aug 1986Hind leg muscles of female rats (85-99 g) were unloaded by tail cast suspension for 6 days. In the fresh-frozen unloaded soleus, the significantly greater concentration...
Hind leg muscles of female rats (85-99 g) were unloaded by tail cast suspension for 6 days. In the fresh-frozen unloaded soleus, the significantly greater concentration of glycogen correlated with a lower activity ratio of glycogen phosphorylase (p less than 0.02). The activity ratio of glycogen synthase also was lower (p less than 0.001), possibly due to the higher concentration of glycogen. In isolated unloaded soleus, insulin (0.1 milliunit/ml) increased the oxidation of D-[U-14C]glucose, release of lactate and pyruvate, incorporation of D-[U-14C]glucose into glycogen, and the concentration of glucose 6-phosphate more (p less than 0.05) than in the weight-bearing soleus. At physiological doses of insulin, the percent of maximal uptake of 2-deoxy-D-[1,2-3H]glucose/muscle also was greater in the unloaded soleus. Unloading of the soleus increased by 50% the concentration of insulin receptors, due to no decrease in total receptor number during muscle atrophy. This increase may account for the greater response of glucose metabolism to insulin in this muscle. The extensor digitorum longus, which generally shows little response to unloading, displayed no differential response of glucose metabolism to insulin.
Topics: Animals; Carbon Radioisotopes; Female; Glucose; Glycogen Synthase; Insulin; Kinetics; Muscles; Phosphorylases; Physical Exertion; Rats; Rats, Inbred Strains
PubMed: 3090039
DOI: No ID Found -
The Journal of Biological Chemistry Jun 2002We have investigated the ability of a receptor-mediated gene transfer strategy (cross-correction) to restore ganglioside metabolism in fibroblasts from Tay-Sachs (TS)...
We have investigated the ability of a receptor-mediated gene transfer strategy (cross-correction) to restore ganglioside metabolism in fibroblasts from Tay-Sachs (TS) patients in vitro. TS disease is a GM2 gangliosidosis attributed to the deficiency of the lysosomal enzyme beta-hexosaminidase A (HexA) (beta-N-acetylhexosaminidase, EC ). The hypothesis is that transduced cells overexpressing and secreting large amounts of the enzyme would lead to a measurable activity in defective cells via a secretion-recapture mechanism. We transduced NIH3T3 murine fibroblasts with the LalphaHexTN retroviral vector carrying the cDNA encoding for the human Hex alpha-subunit. The Hex activity in the medium from transduced cells was approximately 10-fold higher (up to 75 milliunits) than observed in non-transduced cells. TS cells were cultured for 72 h in the presence of the cell medium derived from the transduced NIH3T3 cells, and they were analyzed for the presence and catalytic activity of the enzyme. Although TS cells were able to efficiently uptake a large amount of the soluble enzyme, the enzyme failed to reach the lysosomes in a sufficient quantity to hydrolyze the GM2 ganglioside to GM3 ganglioside. Thus, our results showed that delivery of the therapeutic HexA was not sufficient to correct the phenotype of TS cells.
Topics: 3T3 Cells; Animals; Cells, Cultured; DNA, Complementary; G(M2) Ganglioside; Gene Transfer Techniques; Genetic Vectors; Humans; Hydrolysis; Mice; Retroviridae; Tay-Sachs Disease
PubMed: 11923278
DOI: 10.1074/jbc.M106164200 -
The Journal of Biological Chemistry Jul 1998Overexpression of the human GLUT1 glucose transporter protein in skeletal muscle of transgenic mice results in large increases in basal glucose transport and metabolism,...
Overexpression of the human GLUT1 glucose transporter protein in skeletal muscle of transgenic mice results in large increases in basal glucose transport and metabolism, but impaired stimulation of glucose transport by insulin, contractions, or hypoxia (Gulve, E. A., Ren, J.-M., Marshall, B. A., Gao, J., Hansen, P. A., Holloszy, J. O. , and Mueckler, M. (1994) J. Biol. Chem. 269, 18366-18370). This study examined the relationship between glucose transport and cell-surface glucose transporter content in isolated skeletal muscle from wild-type and GLUT1-overexpressing mice using 2-deoxyglucose, 3-O-methylglucose, and the 2-N-[4-(1-azi-2,2, 2-trifluoroethyl)benzoyl]-1,3-bis(D-mannos-4-yloxy)-2-propyl amine exofacial photolabeling technique. Insulin (2 milliunits/ml) stimulated a 3-fold increase in 2-deoxyglucose uptake in extensor digitorum longus muscles of control mice (0.47 +/- 0.07 micromol/ml/20 min in basal muscle versus 1.44 micromol/ml/20 min in insulin-stimulated muscle; mean +/- S.E.). Insulin failed to increase 2-deoxyglucose uptake above basal rates in muscles overexpressing GLUT1 (4.00 +/- 0.40 micromol/ml/20 min in basal muscle versus 3.96 +/- 0.37 micromol/ml/20 min in insulin-stimulated muscle). A similar lack of insulin stimulation in muscles overexpressing GLUT1 was observed using 3-O-methylglucose. However, the magnitude of the insulin-stimulated increase in cell-surface GLUT4 photolabeling was nearly identical (approximately 3-fold) in wild-type and GLUT1-overexpressing muscles. This apparently normal insulin-stimulated translocation of GLUT4 in GLUT1-overexpressing muscle was confirmed by immunoelectron microscopy. Our findings suggest that GLUT4 activity at the plasma membrane can be dissociated from the plasma membrane content of GLUT4 molecules and thus suggest that the intrinsic activity of GLUT4 is subject to regulation.
Topics: 3-O-Methylglucose; Animals; Biological Transport; Glucose; Glucose Transporter Type 1; Glucose Transporter Type 4; Humans; Insulin; Mice; Mice, Transgenic; Monosaccharide Transport Proteins; Muscle Proteins; Muscle, Skeletal
PubMed: 9660777
DOI: 10.1074/jbc.273.29.18173