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1.
Mol Metab ; 51: 101239, 2021 09.
Article in English | MEDLINE | ID: mdl-33932586

ABSTRACT

OBJECTIVE: Transport of Ca2+ into pancreatic ß cell mitochondria facilitates nutrient-mediated insulin secretion. However, the underlying mechanism is unclear. Recent establishment of the molecular identity of the mitochondrial Ca2+ uniporter (MCU) and associated proteins allows modification of mitochondrial Ca2+ transport in intact cells. We examined the consequences of deficiency of the accessory protein MICU2 in rat and human insulin-secreting cells and mouse islets. METHODS: siRNA silencing of Micu2 in the INS-1 832/13 and EndoC-ßH1 cell lines was performed; Micu2-/- mice were also studied. Insulin secretion and mechanistic analyses utilizing live confocal imaging to assess mitochondrial function and intracellular Ca2+ dynamics were performed. RESULTS: Silencing of Micu2 abrogated GSIS in the INS-1 832/13 and EndoC-ßH1 cells. The Micu2-/- mice also displayed attenuated GSIS. Mitochondrial Ca2+ uptake declined in MICU2-deficient INS-1 832/13 and EndoC-ßH1 cells in response to high glucose and high K+. MICU2 silencing in INS-1 832/13 cells, presumably through its effects on mitochondrial Ca2+ uptake, perturbed mitochondrial function illustrated by absent mitochondrial membrane hyperpolarization and lowering of the ATP/ADP ratio in response to elevated glucose. Despite the loss of mitochondrial Ca2+ uptake, cytosolic Ca2+ was lower in siMICU2-treated INS-1 832/13 cells in response to high K+. It was hypothesized that Ca2+ accumulated in the submembrane compartment in MICU2-deficient cells, resulting in desensitization of voltage-dependent Ca2+ channels, lowering total cytosolic Ca2+. Upon high K+ stimulation, MICU2-silenced cells showed higher and prolonged increases in submembrane Ca2+ levels. CONCLUSIONS: MICU2 plays a critical role in ß cell mitochondrial Ca2+ uptake. ß cell mitochondria sequestered Ca2+ from the submembrane compartment, preventing desensitization of voltage-dependent Ca2+ channels and facilitating GSIS.


Subject(s)
Calcium Channels , Calcium-Binding Proteins , Calcium , Insulin Secretion , Insulin-Secreting Cells , Animals , Female , Humans , Male , Mice , Rats , Calcium/metabolism , Calcium Channels/genetics , Calcium Channels/metabolism , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Gene Knockdown Techniques , HEK293 Cells , Insulin-Secreting Cells/metabolism , Mice, Knockout , Mitochondria/metabolism , Mitochondrial Membranes/metabolism
2.
Pediatr Obes ; 8(1): 52-61, 2013 Feb.
Article in English | MEDLINE | ID: mdl-22961720

ABSTRACT

UNLABELLED: What is already known about this subject Circulating concentrations of branched-chain amino acids (BCAAs) can affect carbohydrate metabolism in skeletal muscle, and therefore may alter insulin sensitivity. BCAAs are elevated in adults with diet-induced obesity, and are associated with their future risk of type 2 diabetes even after accounting for baseline clinical risk factors. What this study adds Increased concentrations of BCAAs are already present in young obese children and their metabolomic profiles are consistent with increased BCAA catabolism. Elevations in BCAAs in children are positively associated with insulin resistance measured 18 months later, independent of their initial body mass index. BACKGROUND: Branched-chain amino acid (BCAA) concentrations are elevated in response to overnutrition, and can affect both insulin sensitivity and secretion. Alterations in their metabolism may therefore play a role in the early pathogenesis of type 2 diabetes in overweight children. OBJECTIVE: To determine whether paediatric obesity is associated with elevations in fasting circulating concentrations of BCAAs (isoleucine, leucine and valine), and whether these elevations predict future insulin resistance. METHODS: Sixty-nine healthy subjects, ages 8-18 years, were enrolled as a cross-sectional cohort. A subset of subjects who were pre- or early-pubertal, ages 8-13 years, were enrolled in a prospective longitudinal cohort for 18 months (n = 17 with complete data). RESULTS: Elevations in the concentrations of BCAAs were significantly associated with body mass index (BMI) Z-score (Spearman's Rho 0.27, P = 0.03) in the cross-sectional cohort. In the subset of subjects that followed longitudinally, baseline BCAA concentrations were positively associated with homeostasis model assessment for insulin resistance measured 18 months later after controlling for baseline clinical factors including BMI Z-score, sex and pubertal stage (P = 0.046). CONCLUSIONS: Elevations in the concentrations of circulating BCAAs are significantly associated with obesity in children and adolescents, and may independently predict future insulin resistance.


Subject(s)
Amino Acids, Branched-Chain/blood , Child Nutrition Disorders/blood , Diabetes Mellitus, Type 2/blood , Insulin Resistance , Insulin/blood , Obesity/blood , Adolescent , Biomarkers/blood , Blood Glucose/metabolism , Body Mass Index , Child , Child Nutrition Disorders/epidemiology , Child Nutrition Disorders/prevention & control , Cross-Sectional Studies , Diabetes Mellitus, Type 2/epidemiology , Diabetes Mellitus, Type 2/prevention & control , Fasting/blood , Female , Humans , Insulin/metabolism , Insulin Secretion , Isoleucine/blood , Leucine/blood , Longitudinal Studies , Male , Massachusetts/epidemiology , Obesity/epidemiology , Obesity/etiology , Obesity/prevention & control , Predictive Value of Tests , Valine/blood
3.
Indian J Chest Dis Allied Sci ; 53(1): 21-4, 2011.
Article in English | MEDLINE | ID: mdl-21446220

ABSTRACT

BACKGROUND AND AIMS: Medical thoracoscopy, also called pleuroscopy, has received renewed interest in the recent past for diagnostic as well as therapeutic uses. In this study, we describe our experience with thoracoscopy for undiagnosed pleural effusions. METHODS: In a retrospective analysis of thoracoscopic procedures we performed between January 2007 and December 2008, yield of thoracoscopic pleural biopsy for achieving a diagnosis in undiagnosed pleural effusions, defined as pleural effusions with adenosine deaminase (ADA) levels less than 70 IU/L and negative pleural fluid cytology for malignancy on three occasions was evaluated. Complications of thoracoscopy were also analysed. RESULTS: Overall diagnostic yield of thoracoscopic pleural biopsy was 74.3% in patients with undiagnosed pleural effusions. Pleural malignancy was diagnosed in 48.6% of patients. There was only one case of mesothelioma and the rest were due to pleural metastasis. Lung cancer and breast cancer were the most common sites of primary malignancy. Tuberculosis was diagnosed with pleural biopsy in 22.8% of patients. We had low complication rate after thoracoscopy. Only two cases of empyema were observed. CONCLUSION: Medical thoracoscopy is a safe procedure and has good diagnostic yield in patients with undiagnosed pleural effusions.


Subject(s)
Pleural Effusion/diagnosis , Pleural Effusion/etiology , Thoracoscopy , Adult , Cohort Studies , Female , Humans , India , Male , Middle Aged , Pleural Effusion/therapy , Reproducibility of Results , Retrospective Studies
4.
Neurology ; 64(3): 539-41, 2005 Feb 08.
Article in English | MEDLINE | ID: mdl-15699391

ABSTRACT

Primary muscle coenzyme Q10 (CoQ10) deficiency is an apparently autosomal recessive condition with heterogeneous clinical presentations. Patients with these disorders improve with CoQ10 supplementation. In a family with ataxia and CoQ10 deficiency, analysis of genome-wide microsatellite markers suggested linkage of the disease to chromosome 9p13 and led to identification of an aprataxin gene (APTX) mutation that causes ataxia oculomotor apraxia (AOA1 [MIM606350]). The authors' observations indicate that CoQ10 deficiency may contribute to the pathogenesis of AOA1.


Subject(s)
DNA-Binding Proteins/genetics , Hypoalbuminemia/genetics , Nuclear Proteins/genetics , Spinocerebellar Degenerations/genetics , Ubiquinone/deficiency , Amino Acid Substitution , Child, Preschool , Chromosomes, Human, Pair 9/genetics , DNA Mutational Analysis , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/physiology , Exons/genetics , Female , Genes, Recessive , Humans , Hyperlipoproteinemia Type II/genetics , Infant , Lod Score , Male , Muscle Weakness/genetics , Muscle, Skeletal/chemistry , Muscle, Skeletal/pathology , Mutation, Missense , Nuclear Proteins/deficiency , Nuclear Proteins/physiology , Phenotype , Point Mutation , Spinocerebellar Degenerations/drug therapy , Ubiquinone/therapeutic use
5.
Neurology ; 62(11): 2119-21, 2004 Jun 08.
Article in English | MEDLINE | ID: mdl-15184630

ABSTRACT

A woman with typical features of myoclonic epilepsy with ragged red fibers (MERRF) had a novel heteroplasmic mutation (G611A) in the mitochondrial DNA tRNA phenylalanine gene. The mutation was heteroplasmic (91%) in muscle but undetectable in accessible tissues from the patient and her maternal relatives. Single-fiber PCR analysis showed that the proportion of mutant genomes was higher in cytochrome c oxidase (COX)-negative ragged red fibers (RRFs) than in COX-positive non-RRFs. This report shows that typical MERRF syndrome is not always associated with tRNA lysine mutations.


Subject(s)
DNA, Mitochondrial/genetics , MERRF Syndrome/genetics , RNA, Transfer, Phe/genetics , Adolescent , Adult , Amino Acid Substitution , Electron Transport Complex IV/analysis , Genetic Heterogeneity , Humans , MERRF Syndrome/pathology , Mitochondria/chemistry , Muscles/chemistry , Muscles/ultrastructure , Mutation, Missense , Organ Specificity , Point Mutation , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length
6.
Mol Cell ; 8(5): 971-82, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11741533

ABSTRACT

Cachexia is a chronic state of negative energy balance and muscle wasting that is a severe complication of cancer and chronic infection. While cytokines such as IL-1alpha, IL-1beta, and TNFalpha can mediate cachectic states, how these molecules affect energy expenditure is unknown. We show here that many cytokines activate the transcriptional PPAR gamma coactivator-1 (PGC-1) through phosphorylation by p38 kinase, resulting in stabilization and activation of PGC-1 protein. Cytokine or lipopolysaccharide (LPS)-induced activation of PGC-1 in cultured muscle cells or muscle in vivo causes increased respiration and expression of genes linked to mitochondrial uncoupling and energy expenditure. These data illustrate a direct thermogenic action of cytokines and p38 MAP kinase through the transcriptional coactivator PGC-1.


Subject(s)
Cachexia/physiopathology , Cytokines/pharmacology , Energy Metabolism , Enzyme Activation/physiology , Mitogen-Activated Protein Kinases/metabolism , Transcription Factors/metabolism , Animals , Calcium-Calmodulin-Dependent Protein Kinases/genetics , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cell Line , Cell Respiration/physiology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Genes, Reporter , Humans , Lipopolysaccharides/pharmacology , Mice , Mice, Transgenic , Mitochondria/genetics , Mitochondria/metabolism , Models, Biological , Muscle, Skeletal/cytology , Muscle, Skeletal/drug effects , Muscle, Skeletal/metabolism , Nuclear Respiratory Factors , Oxygen/metabolism , Phosphorylation , Trans-Activators/genetics , Trans-Activators/metabolism , Transcription Factors/genetics , Transcription, Genetic , p38 Mitogen-Activated Protein Kinases
7.
EMBO J ; 20(4): 661-71, 2001 Feb 15.
Article in English | MEDLINE | ID: mdl-11179211

ABSTRACT

Multiple apoptotic pathways release cytochrome c from the mitochondrial intermembrane space, resulting in the activation of downstream caspases. In vivo activation of Fas (CD95) resulted in increased permeability of the mitochondrial outer membrane and depletion of cytochrome c stores. Serial measurements of oxygen consumption, NADH redox state and membrane potential revealed a loss of respiratory state transitions. This tBID-induced respiratory failure did not require any caspase activity. At early time points, re-addition of exogenous cytochrome c markedly restored respiratory functions. Over time, however, mitochondria showed increasing irreversible respiratory dysfunction as well as diminished calcium buffering. Electron microscopy and tomographic reconstruction revealed asymmetric mitochondria with blebs of herniated matrix, distended inner membrane and partial loss of cristae structure. Thus, apoptogenic redistribution of cytochrome c is responsible for a distinct program of mitochondrial respiratory dysfunction, in addition to the activation of downstream caspases.


Subject(s)
Apoptosis , Cytochrome c Group/pharmacology , Mitochondria/drug effects , Animals , Calcium/metabolism , Female , Mice , Mice, Inbred C57BL , Mitochondria/enzymology , Mitochondria/metabolism , Signal Transduction , fas Receptor/metabolism
8.
Genes Dev ; 14(16): 2060-71, 2000 Aug 15.
Article in English | MEDLINE | ID: mdl-10950869

ABSTRACT

TNFR1/Fas engagement results in the cleavage of cytosolic BID to truncated tBID, which translocates to mitochondria. Immunodepletion and gene disruption indicate BID is required for cytochrome c release. Surprisingly, the three-dimensional structure of this BH3 domain-only molecule revealed two hydrophobic alpha-helices suggesting tBID itself might be a pore-forming protein. Instead, we demonstrate that tBID functions as a membrane-targeted death ligand in which an intact BH3 domain is required for cytochrome c release, but not for targeting. Bak-deficient mitochondria and blocking antibodies reveal tBID binds to its mitochondrial partner BAK to release cytochrome c, a process independent of permeability transition. Activated tBID results in an allosteric activation of BAK, inducing its intramembranous oligomerization into a proposed pore for cytochrome c efflux, integrating the pathway from death receptors to cell demise.


Subject(s)
Carrier Proteins/metabolism , Cytochrome c Group/metabolism , Membrane Proteins/metabolism , Allosteric Regulation , Animals , BH3 Interacting Domain Death Agonist Protein , Biopolymers , Cell Membrane/metabolism , Mice , bcl-2 Homologous Antagonist-Killer Protein
10.
Am J Physiol Cell Physiol ; 278(2): C423-35, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10666039

ABSTRACT

Ca(2+) has been postulated as a cytosolic second messenger in the regulation of cardiac oxidative phosphorylation. This hypothesis draws support from the well-known effects of Ca(2+) on muscle activity, which is stimulated in parallel with the Ca(2+)-sensitive dehydrogenases (CaDH). The effects of Ca(2+) on oxidative phosphorylation were further investigated in isolated porcine heart mitochondria at the level of metabolic driving force (NADH or Deltapsi) and ATP production rates (flow). The resulting force-flow (F-F) relationships permitted the analysis of Ca(2+) effects on several putative control points within oxidative phosphorylation, simultaneously. The F-F relationships resulting from additions of carbon substrates alone provided a model of pure CaDH activation. Comparing this curve with variable Ca(2+) concentration ([Ca(2+)]) effects revealed an approximate twofold higher ATP production rate than could be explained by a simple increase in NADH or Deltapsi via CaDH activation. The half-maximal effect of Ca(2+ )at state 3 was 157 nM and was completely inhibited by ruthenium red (1 microM), indicating matrix dependence of the Ca(2+) effect. Arsenate was used as a probe to differentiate between F(0)/F(1)-ATPase and adenylate translocase activity by a futile recycling of ADP-arsenate within the matrix, catalyzed by the F(0)/F(1)-ATPase. Ca(2+) increased the ADP arsenylation rate more than twofold, suggesting a direct effect on the F(0)/F(1)-ATPase. These results suggest that Ca(2+) activates cardiac aerobic respiration at the level of both the CaDH and F(0)/F(1)-ATPase. This type of parallel control of both intermediary metabolism and ATP synthesis may provide a mechanism of altering ATP production rates with minimal changes in the high-energy intermediates as observed in vivo.


Subject(s)
Calcium/metabolism , Mitochondria/enzymology , Myocardium/enzymology , Proton-Translocating ATPases/metabolism , Adenosine Triphosphate/biosynthesis , Animals , Arsenates/pharmacology , Calcium/pharmacology , Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone/pharmacology , Dose-Response Relationship, Drug , Membrane Potentials/drug effects , Membrane Potentials/physiology , NAD/biosynthesis , Oxidative Phosphorylation , Oxidoreductases/metabolism , Oxygen Consumption/drug effects , Oxygen Consumption/physiology , Regression Analysis , Substrate Specificity , Swine , Uncoupling Agents/pharmacology
11.
FEBS Lett ; 449(2-3): 129-34, 1999 Apr 23.
Article in English | MEDLINE | ID: mdl-10338118

ABSTRACT

Uncoupling protein 3L, uncoupling protein 1 and the mitochondrial oxoglutarate carrier were expressed in Saccharomyces cerevisae. Effects on different parameters related to the energy expenditure were studied. Both uncoupling protein 3L and uncoupling protein 1 reduced the growth rate by 49% and 32% and increased the whole yeast O2 consumption by 31% and 19%, respectively. In isolated mitochondria, uncoupling protein 1 increased the state 4 respiration by 1.8-fold, while uncoupling protein 3L increased the state 4 respiration by 1.2-fold. Interestingly, mutant uncoupling protein 1 carrying the H145Q and H147N mutations, previously shown to markedly decrease the H+ transport activity of uncoupling protein 1 when assessed using a proteoliposome system (Bienengraeber et al. (1998) Biochem. 37, 3-8), uncoupled the mitochondrial respiration to almost the same degree as wild-type uncoupling protein 1. Thus, absence of this histidine pair in uncoupling protein 2 and uncoupling protein 3 does not by itself rule out the possibility that these carriers have an uncoupling function. The oxoglutarate carrier had no effect on any of the studied parameters. In summary, a discordance exists between the magnitude of effects of uncoupling protein 3L and uncoupling protein 1 in whole yeast versus isolated mitochondria, with uncoupling protein 3L having greater effects in whole yeast and a smaller effect on the state 4 respiration in isolated mitochondria. These findings suggest that uncoupling protein 3L, like uncoupling protein 1, has an uncoupling activity. However, the mechanism of action and/or regulation of the activity of uncoupling protein 3L is likely to be different.


Subject(s)
Carrier Proteins/metabolism , Membrane Transport Proteins , Mitochondria/metabolism , Animals , Carrier Proteins/genetics , Gene Expression , Genetic Vectors , Humans , Intracellular Membranes/metabolism , Ion Channels , Membrane Potentials , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mitochondrial Proteins , Oxygen Consumption , Rats , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/growth & development , Uncoupling Protein 1 , Uncoupling Protein 3
12.
Am J Physiol ; 272(2 Pt 2): H769-75, 1997 Feb.
Article in English | MEDLINE | ID: mdl-9124437

ABSTRACT

It is difficult to estimate the maximum in vivo aerobic ATP production rate of the intact heart independent of limitations imposed by blood flow, oxygen delivery, and maximum mechanical power. This value is critical for establishing the kinetic parameters that control oxidative phosphorylation, as well as for providing insights into the limits of myocardial performance. In this study, the maximum ADP-P(i)-driven heart mitochondrial respiratory rate (MV(O2 mito)) was determined with saturating levels of oxygen, substrates, and cofactors at 37 degrees C. These rates were normalized to cytochrome alpha1 alpha3 (cytochrome oxidase; Cyt a) content. To extrapolate this rate to the intact heart, the Cyt a content of the myocardium (nmol Cyt a/g wet wt myocardium) was determined in the same hearts. The maximum ADP-P(i)-driven mitochondrial respiratory rates were 676 +/- 31 and 665 +/- 65 nmol O2 x min(-1) x nmol Cyt a(-1) in the dog and pig, respectively. The Cyt a content in the two species was 43.6 +/- 2.4 and 36.6 +/- 3.1 nmol Cyt a/g wet wt, respectively. With these values, the MV(O2 mito) was calculated to be 29.5 (dog) and 24.3 (pig) micromol O2 x min(-1) x g wet wt myocardium(-1). Comparison with in vivo studies shows that the exercising heart can utilize 80-90% of its maximum oxidative capacity, implying there is little aerobic ATP production reserve in the mammalian heart.


Subject(s)
Myocardium/metabolism , Oxidative Phosphorylation , Adenosine Diphosphate/pharmacology , Animals , Dogs , Electron Transport Complex IV/metabolism , In Vitro Techniques , Mitochondria, Heart/drug effects , Mitochondria, Heart/metabolism , Oxygen Consumption , Phosphates/pharmacology , Swine
13.
Anal Biochem ; 237(2): 274-8, 1996 Jun 01.
Article in English | MEDLINE | ID: mdl-8660576

ABSTRACT

A simple spectroscopic method for determining the cytochrome c oxidase, cytochrome a, a3, content in tissue and mitochondria samples independent of myoglobin or blood contamination is described. Using tissue homogenates solubilized in Triton X-100, this assay relies on the selective reduction of mitochondrial cytochromes by the action of potassium cyanide. Monitoring the optical absorbance of these samples at 605 nm provided a quantitative determination of cytochrome c oxidase content in the presence of myoglobin or blood. The cytochrome c oxidase content of porcine heart mitochondria and whole tissue was determined to be 0.85 nmol/mg protein and 30.5 nmol/g wet wt, respectively.


Subject(s)
Electron Transport Complex IV/analysis , Hemoglobins/analysis , Myoglobin/analysis , Spectrophotometry/methods , Animals , Detergents , Evaluation Studies as Topic , Mitochondria, Heart/chemistry , Myocardium/chemistry , Octoxynol , Potassium Cyanide , Swine
14.
Anal Biochem ; 236(2): 327-30, 1996 May 01.
Article in English | MEDLINE | ID: mdl-8660512

ABSTRACT

Ca(2+)-selective microelectrodes with Simon's neutral carrier ETH 1001 are commercially available and have been widely used for the measurement of both extra- and intracellular calcium. The electrodes demonstrate high selectivity against other cations such as magnesium, sodium, and potassium. We report, however, that the ETH 1001-based microelectrode is a superior tetraphenylphosphonium (TPP+)-sensitive electrode. The electrode exhibits a Nernstian response for [Ca2+] > 10(-5) M but for [TTP+] > 10(-7) M. Using two different methods, we found that log kTTPCa (selectivity coefficient for TPP+ with respect to Ca2+) is in the range of -3.0 to -5.3. We argue that the ETH 1001 microelectrode can be used as a commercially available TPP+ electrode. We illustrate this application by making membrane potential recordings in respiring mitochondria. The results are identical to those obtained using conventional ion-exchange TPP+ electrodes.


Subject(s)
Calcium/analysis , Diamines , Microelectrodes , Onium Compounds/analysis , Organophosphorus Compounds/analysis , Animals , Male , Membrane Potentials , Molecular Probes , Oxygen Consumption/physiology , Rats , Rats, Sprague-Dawley , Sensitivity and Specificity
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