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1.
Diabetes Obes Metab ; 21(11): 2422-2428, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31264758

RESUMO

AIM: To assess the effects of the sodium-glucose co-transporter-2 (SGLT2) inhibitor dapagliflozin on a pre-specified panel of 13 urinary metabolites linked to mitochondrial metabolism in people with type 2 diabetes and elevated urine albumin levels. MATERIALS AND METHODS: Urine and plasma samples were used from a double-blind, randomized, placebo-controlled crossover trial in 31 people with type 2 diabetes, with an albumin:creatinine ratio >100 mg/g, and who were on a stable dose of an angiotensin-converting enzyme inhibitor or an angiotensin receptor blocker. Dapagliflozin or placebo treatment periods each lasted 6 weeks, with a 6-week washout period in between. Urinary and plasma metabolites were quantified by gas-chromatography mass spectrometry, corrected for creatinine level, and then combined into a single-valued urinary metabolite index. Fractional excretion of the metabolites was calculated. RESULTS: All 13 urinary metabolites were detectable. After 6 weeks of dapagliflozin therapy, nine of the 13 metabolites were significantly increased from baseline. The urinary metabolite index increased by 42% (95% confidence interval [CI] 8.5 to 85.6; P = .01) with placebo versus 121% (95% CI 69 to 189; P < .001) with dapaglifozin. The placebo-adjusted effect was 56% (95% CI 11 to 118; P = .012). In plasma, seven of the 13 metabolites were detectable, and none was modified by dapagliflozin. CONCLUSIONS: Dapagliflozin significantly increased a panel of urinary metabolites previously linked to mitochondrial metabolism. These data support the hypothesis that SGLT2 inhibitors improve mitochondrial function, and improvements in mitochondrial function could be a mechanism for kidney protection. Future studies with longer treatment duration and clinical outcomes are needed to confirm the clinical impact of these findings.

2.
Am J Physiol Renal Physiol ; 317(2): F419-F434, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31166707

RESUMO

Na+/H+ exchanger isoform 3 (NHE3) contributes to Na+/bicarbonate reabsorption and ammonium secretion in early proximal tubules. To determine its role in the diabetic kidney, type 1 diabetic Akita mice with tubular NHE3 knockdown [Pax8-Cre; NHE3-knockout (KO) mice] were generated. NHE3-KO mice had higher urine pH, more bicarbonaturia, and compensating increases in renal mRNA expression for genes associated with generation of ammonium, bicarbonate, and glucose (phosphoenolpyruvate carboxykinase) in proximal tubules and H+ and ammonia secretion and glycolysis in distal tubules. This left blood pH and bicarbonate unaffected in nondiabetic and diabetic NHE3-KO versus wild-type mice but was associated with renal upregulation of proinflammatory markers. Higher renal phosphoenolpyruvate carboxykinase expression in NHE3-KO mice was associated with lower Na+-glucose cotransporter (SGLT)2 and higher SGLT1 expression, indicating a downward tubular shift in Na+ and glucose reabsorption. NHE3-KO was associated with lesser kidney weight and glomerular filtration rate (GFR) independent of diabetes and prevented diabetes-associated albuminuria. NHE3-KO, however, did not attenuate hyperglycemia or prevent diabetes from increasing kidney weight and GFR. Higher renal gluconeogenesis may explain similar hyperglycemia despite lower SGLT2 expression and higher glucosuria in diabetic NHE3-KO versus wild-type mice; stronger SGLT1 engagement could have affected kidney weight and GFR responses. Chronic kidney disease in humans is associated with reduced urinary excretion of metabolites of branched-chain amino acids and the tricarboxylic acid cycle, a pattern mimicked in diabetic wild-type mice. This pattern was reversed in nondiabetic NHE3-KO mice, possibly reflecting branched-chain amino acids use for ammoniagenesis and tricarboxylic acid cycle upregulation to support formation of ammonia, bicarbonate, and glucose in proximal tubule. NHE3-KO, however, did not prevent the diabetes-induced urinary downregulation in these metabolites.

3.
Science ; 364(6436)2019 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-30975860

RESUMO

To understand the health impact of long-duration spaceflight, one identical twin astronaut was monitored before, during, and after a 1-year mission onboard the International Space Station; his twin served as a genetically matched ground control. Longitudinal assessments identified spaceflight-specific changes, including decreased body mass, telomere elongation, genome instability, carotid artery distension and increased intima-media thickness, altered ocular structure, transcriptional and metabolic changes, DNA methylation changes in immune and oxidative stress-related pathways, gastrointestinal microbiota alterations, and some cognitive decline postflight. Although average telomere length, global gene expression, and microbiome changes returned to near preflight levels within 6 months after return to Earth, increased numbers of short telomeres were observed and expression of some genes was still disrupted. These multiomic, molecular, physiological, and behavioral datasets provide a valuable roadmap of the putative health risks for future human spaceflight.


Assuntos
Adaptação Fisiológica , Astronautas , Voo Espacial , Imunidade Adaptativa , Peso Corporal , Artérias Carótidas/diagnóstico por imagem , Espessura Intima-Media Carotídea , Dano ao DNA , Metilação de DNA , Microbioma Gastrointestinal , Instabilidade Genômica , Humanos , Masculino , Homeostase do Telômero , Fatores de Tempo , Estados Unidos , United States National Aeronautics and Space Administration
4.
Semin Nephrol ; 38(2): 111-120, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29602394

RESUMO

Diabetic kidney disease (DKD) is the leading cause of morbidity and mortality in diabetic patients. Defining risk factors for DKD using a reductionist approach has proven challenging. Integrative omics-based systems biology tools have shed new insights in our understanding of DKD and have provided several key breakthroughs for identifying novel predictive and diagnostic biomarkers. In this review, we highlight the role of the Warburg effect in DKD and potential regulating factors such as sphingomyelin, fumarate, and pyruvate kinase muscle isozyme M2 in shifting glucose flux from complete oxidation in mitochondria to the glycolytic pathway and its principal branches. With the development of highly sensitive instruments and more advanced automatic bioinformatics tools, we believe that omics analyses and imaging techniques will focus more on singular-cell-level studies, which will allow in-depth understanding of DKD and pave the path for personalized kidney precision medicine.

5.
Metabolomics ; 14(6): 84, 2018 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-30830355

RESUMO

INTRODUCTION: Little is known about the association of urine metabolites with structural lesions in persons with diabetes. OBJECTIVES: We examined the relationship between 12 urine metabolites and kidney structure in American Indians with type 2 diabetes. METHODS: Data were from a 6-year clinical trial that assessed renoprotective efficacy of losartan, and included a kidney biopsy at the end of the treatment period. Metabolites were measured in urine samples collected within a median of 6.5 months before the research biopsy. Associations of the creatinine-adjusted urine metabolites with kidney structural variables were examined by Pearson's correlations and multivariable linear regression after adjustment for age, sex, diabetes duration, hemoglobin A1c, mean arterial pressure, glomerular filtration rate (iothalamate), and losartan treatment. RESULTS: Participants (n = 62, mean age 45 ± 10 years) had mean ± standard deviation glomerular filtration rate of 137 ± 50 ml/min and median (interquartile range) urine albumin:creatinine ratio of 34 (14-85) mg/g near the time of the biopsy. Urine aconitic and glycolic acids correlated positively with glomerular filtration surface density (partial r = 0.29, P = 0.030 and r = 0.50, P < 0.001) and total filtration surface per glomerulus (partial r = 0.32, P = 0.019 and r = 0.43, P = 0.001). 2-ethyl 3-OH propionate correlated positively with the percentage of fenestrated endothelium (partial r = 0.32, P = 0.019). Citric acid correlated negatively with mesangial fractional volume (partial r=-0.36, P = 0.007), and homovanillic acid correlated negatively with podocyte foot process width (partial r=-0.31, P = 0.022). CONCLUSIONS: Alterations of urine metabolites may associate with early glomerular lesions in diabetic kidney disease.


Assuntos
Biomarcadores/urina , Diabetes Mellitus Tipo 2/complicações , Nefropatias Diabéticas/diagnóstico , Metaboloma , Adulto , Estudos Transversais , Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/urina , Feminino , Taxa de Filtração Glomerular , Humanos , Índios Norte-Americanos , Testes de Função Renal , Masculino , Pessoa de Meia-Idade
6.
EBioMedicine ; 26: 68-77, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29128444

RESUMO

Chronic kidney disease (CKD) is a public health problem with very high prevalence and mortality. Yet, there is a paucity of effective treatment options, partly due to insufficient knowledge of underlying pathophysiology. We combined metabolomics (GCMS) with kidney gene expression studies to identify metabolic pathways that are altered in adults with non-diabetic stage 3-4 CKD versus healthy adults. Urinary excretion rate of 27 metabolites and plasma concentration of 33 metabolites differed significantly in CKD patients versus controls (estimate range-68% to +113%). Pathway analysis revealed that the citric acid cycle was the most significantly affected, with urinary excretion of citrate, cis-aconitate, isocitrate, 2-oxoglutarate and succinate reduced by 40-68%. Reduction of the citric acid cycle metabolites in urine was replicated in an independent cohort. Expression of genes regulating aconitate, isocitrate, 2-oxoglutarate and succinate were significantly reduced in kidney biopsies. We observed increased urine citrate excretion (+74%, p=0.00009) and plasma 2-oxoglutarate concentrations (+12%, p=0.002) in CKD patients during treatment with a vitamin-D receptor agonist in a randomized trial. In conclusion, urinary excretion of citric acid cycle metabolites and renal expression of genes regulating these metabolites were reduced in non-diabetic CKD. This supports the emerging view of CKD as a state of mitochondrial dysfunction.


Assuntos
Metabolômica , Mitocôndrias/metabolismo , Insuficiência Renal Crônica/sangue , Insuficiência Renal Crônica/genética , Ácido Aconítico/metabolismo , Idoso , Biópsia , Ciclo do Ácido Cítrico/genética , Feminino , Regulação da Expressão Gênica/genética , Humanos , Isocitratos/metabolismo , Ácidos Cetoglutáricos/metabolismo , Rim/metabolismo , Rim/patologia , Masculino , Redes e Vias Metabólicas/genética , Pessoa de Meia-Idade , Mitocôndrias/genética , Insuficiência Renal Crônica/patologia , Insuficiência Renal Crônica/urina , Ácido Succínico/metabolismo
7.
J Cell Sci ; 130(19): 3248-3260, 2017 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-28808085

RESUMO

Each mitochondrial compartment contains varying protein compositions that underlie a diversity of localized functions. Insights into the localization of mitochondrial intermembrane space-bridging (MIB) components will have an impact on our understanding of mitochondrial architecture, dynamics and function. By using the novel visualizable genetic tags miniSOG and APEX2 in cultured mouse cardiac and human astrocyte cell lines and performing electron tomography, we have mapped at nanoscale resolution three key MIB components, Mic19, Mic60 and Sam50 (also known as CHCHD3, IMMT and SAMM50, respectively), in the environment of structural landmarks such as cristae and crista junctions (CJs). Tagged Mic19 and Mic60 were located at CJs, distributed in a network pattern along the mitochondrial periphery and also enriched inside cristae. We discovered an association of Mic19 with cytochrome c oxidase subunit IV. It was also found that tagged Sam50 is not uniformly distributed in the outer mitochondrial membrane and appears to incompletely overlap with Mic19- or Mic60-positive domains, most notably at the CJs.


Assuntos
Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Linhagem Celular Transformada , Humanos , Proteínas de Membrana/genética , Mitocôndrias/genética , Proteínas Mitocondriais/genética
8.
Diabetologia ; 60(4): 729-739, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28188334

RESUMO

AIMS/HYPOTHESIS: In this study, we aimed to evaluate the therapeutic potential of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), an activator of AMP-activated protein kinase, for ameliorating high-fat diet (HFD)-induced pathophysiology in mice. We also aimed to determine whether the beneficial effects of AICAR were dependent on adiponectin. Furthermore, human adipose tissue was used to examine the effect of AICAR ex vivo. METHODS: Six-week-old male C57BL/6J wild-type and Adipoq -/- mice were fed a standard-fat diet (10% fat) or an HFD (60% fat) for 12 weeks and given vehicle or AICAR (500 µg/g) three times/week from weeks 4-12. Diet-induced pathophysiology was examined in mice after 11 weeks by IPGTT and after 12 weeks by flow cytometry and western blotting. Human adipose tissue biopsies from obese (BMI 35-50 kg/m2) individuals were incubated with vehicle or AICAR (1 mmol/l) for 6 h at 37°C, after which inflammation was characterised by ELISA (TNF-α) and flow cytometry. RESULTS: AICAR attenuated adipose inflammation in mice fed an HFD, promoting an M1-to-M2 macrophage phenotype switch, while reducing infiltration of CD8+ T cells. AICAR treatment of mice fed an HFD partially restored glucose tolerance and attenuated hepatic steatosis and kidney disease, as evidenced by reduced albuminuria (p < 0.05), urinary H2O2 (p < 0.05) and renal superoxide levels (p < 0.01) in both wild-type and Adipoq -/- mice. AICAR-mediated protection occurred independently of adiponectin, as similar protection was observed in wild-type and Adipoq -/- mice. In addition, AICAR promoted an M1-to-M2 macrophage phenotype switch and reduced TNF-α production in tissue explants from obese human patients. CONCLUSIONS/INTERPRETATION: AICAR may promote metabolic health and protect against obesity-induced systemic diseases in an adiponectin-independent manner. Furthermore, AICAR reduced inflammation in human adipose tissue explants, suggesting by proof-of-principle that the drug may reduce obesity-induced complications in humans. TRIAL REGISTRATION: ClinicalTrials.gov NCT02322073.


Assuntos
Adiponectina/metabolismo , Dieta Hiperlipídica/efeitos adversos , Adiponectina/genética , Animais , Inflamação/imunologia , Inflamação/metabolismo , Nefropatias/imunologia , Nefropatias/metabolismo , Hepatopatias/imunologia , Hepatopatias/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/imunologia , Obesidade/metabolismo
9.
JCI Insight ; 1(15): e86976, 2016 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-27699244

RESUMO

Diabetes is associated with altered cellular metabolism, but how altered metabolism contributes to the development of diabetic complications is unknown. We used the BKS db/db diabetic mouse model to investigate changes in carbohydrate and lipid metabolism in kidney cortex, peripheral nerve, and retina. A systems approach using transcriptomics, metabolomics, and metabolic flux analysis identified tissue-specific differences, with increased glucose and fatty acid metabolism in the kidney, a moderate increase in the retina, and a decrease in the nerve. In the kidney, increased metabolism was associated with enhanced protein acetylation and mitochondrial dysfunction. To confirm these findings in human disease, we analyzed diabetic kidney transcriptomic data and urinary metabolites from a cohort of Southwestern American Indians. The urinary findings were replicated in 2 independent patient cohorts, the Finnish Diabetic Nephropathy and the Family Investigation of Nephropathy and Diabetes studies. Increased concentrations of TCA cycle metabolites in urine, but not in plasma, predicted progression of diabetic kidney disease, and there was an enrichment of pathways involved in glycolysis and fatty acid and amino acid metabolism. Our findings highlight tissue-specific changes in metabolism in complication-prone tissues in diabetes and suggest that urinary TCA cycle intermediates are potential prognostic biomarkers of diabetic kidney disease progression.


Assuntos
Metabolismo dos Carboidratos , Diabetes Mellitus Experimental/complicações , Nefropatias Diabéticas/fisiopatologia , Metabolismo dos Lipídeos , Adulto , Animais , Biomarcadores , Ciclo do Ácido Cítrico , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Nefropatias Diabéticas/metabolismo , Feminino , Humanos , Índios Norte-Americanos , Rim , Masculino , Metabolômica , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Ensaios Clínicos Controlados Aleatórios como Assunto , Transcriptoma
10.
JCI Insight ; 1(17): e87877, 2016 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-27777973

RESUMO

To derive new insights in diabetic complications, we integrated publicly available human protein-protein interaction (PPI) networks with global metabolic networks using metabolomic data from patients with diabetic nephropathy. We focused on the participating proteins in the network that were computationally predicted to connect the urine metabolites. MDM2 had the highest significant number of PPI connections. As validation, significant downregulation of MDM2 gene expression was found in both glomerular and tubulointerstitial compartments of kidney biopsy tissue from 2 independent cohorts of patients with diabetic nephropathy. In diabetic mice, chemical inhibition of MDM2 with Nutlin-3a led to reduction in the number of podocytes, increased blood urea nitrogen, and increased mortality. Addition of Nutlin-3a decreased WT1+ cells in embryonic kidneys. Both podocyte- and tubule-specific MDM2-knockout mice exhibited severe glomerular and tubular dysfunction, respectively. Interestingly, the only 2 metabolites that were reduced in both podocyte and tubule-specific MDM2-knockout mice were 3-methylcrotonylglycine and uracil, both of which were also reduced in human diabetic kidney disease. Thus, our bioinformatics tool combined with multi-omics studies identified an important functional role for MDM2 in glomeruli and tubules of the diabetic nephropathic kidney and links MDM2 to a reduction in 2 key metabolite biomarkers.


Assuntos
Nefropatias Diabéticas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Biologia de Sistemas , Albuminúria , Animais , Biologia Computacional , Diabetes Mellitus Experimental/metabolismo , Humanos , Glomérulos Renais/fisiopatologia , Túbulos Renais/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Podócitos
11.
Am J Nephrol ; 44(2): 92-103, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27410520

RESUMO

The development of new therapies for chronic diseases, such as diabetic kidney disease (DKD), will continue to be hampered by lack of sufficient biomarkers that will provide insights and will be responsive to treatment interventions. The recent application of metabolomic technologies, such as nuclear magnetic resonance and mass spectroscopy, has allowed large-scale analysis of small molecules to be interrogated in a targeted or untargeted manner. Recent advances from both human and animal studies that have arisen from metabolomic analysis have recognized that mitochondrial function and fatty acid oxidation play key roles in the development and progression of DKD. Although many challenges in the technology for clinical chronic kidney disease (CKD) are yet to be validated, there will very likely be ongoing major contributions of metabolomics to develop new biochemical understanding for diabetic and CKD. The clinical application of metabolomics and accompanying bioinformatic tools will likely be a cornerstone of personalized medicine triumphs for CKD.


Assuntos
Nefropatias Diabéticas/diagnóstico , Nefropatias Diabéticas/metabolismo , Metabolômica/métodos , Insuficiência Renal Crônica/diagnóstico , Insuficiência Renal Crônica/metabolismo , Aminoácidos/metabolismo , Animais , Biomarcadores/metabolismo , Ciclo do Ácido Cítrico , Ácidos Graxos/metabolismo , Humanos , Metabolismo dos Lipídeos , Espectrometria de Massas , Mitocôndrias/metabolismo , Ressonância Magnética Nuclear Biomolecular , Medicina de Precisão/métodos , Transdução de Sinais , Ureia/metabolismo
12.
EBioMedicine ; 7: 121-34, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-27322466

RESUMO

AMP-activated protein kinase (AMPK) is suppressed in diabetes and may be due to a high ATP/AMP ratio, however the quantitation of nucleotides in vivo has been extremely difficult. Via matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to localize renal nucleotides we found that the diabetic kidney had a significant increase in glomerular ATP/AMP ratio. Untargeted MALDI-MSI analysis revealed that a specific sphingomyelin species (SM(d18:1/16:0)) accumulated in the glomeruli of diabetic and high-fat diet-fed mice compared with wild-type controls. In vitro studies in mesangial cells revealed that exogenous addition of SM(d18:1/16:0) significantly elevated ATP via increased glucose consumption and lactate production with a consequent reduction of AMPK and PGC1α. Furthermore, inhibition of sphingomyelin synthases reversed these effects. Our findings suggest that AMPK is reduced in the diabetic kidney due to an increase in the ATP/AMP ratio and that SM(d18:1/16:0) could be responsible for the enhanced ATP production via activation of the glycolytic pathway.


Assuntos
Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Diabetes Mellitus/metabolismo , Glomérulos Renais/química , Obesidade/metabolismo , Esfingomielinas/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Linhagem Celular , Glucose/metabolismo , Humanos , Glomérulos Renais/metabolismo , Ácido Láctico/metabolismo , Células Mesangiais/química , Células Mesangiais/citologia , Células Mesangiais/efeitos dos fármacos , Camundongos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Transdução de Sinais/efeitos dos fármacos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Esfingomielinas/farmacologia
13.
Proc Natl Acad Sci U S A ; 112(41): 12681-6, 2015 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-26417071

RESUMO

Cyclic AMP/protein kinase A (cAMP/PKA) and glucocorticoids promote the death of many cell types, including cells of hematopoietic origin. In wild-type (WT) S49 T-lymphoma cells, signaling by cAMP and glucocorticoids converges on the induction of the proapoptotic B-cell lymphoma-family protein Bim to produce mitochondria-dependent apoptosis. Kin(-), a clonal variant of WT S49 cells, lacks PKA catalytic (PKA-Cα) activity and is resistant to cAMP-mediated apoptosis. Using sorbitol density gradient fractionation, we show here that in kin(-) S49 cells PKA-Cα is not only depleted but the residual PKA-Cα mislocalizes to heavier cell fractions and is not phosphorylated at two conserved residues (Ser(338) or Thr(197)). In WT S49 cells, PKA-regulatory subunit I (RI) and Bim coimmunoprecipitate upon treatment with cAMP analogs and forskolin (which increases endogenous cAMP concentrations). By contrast, in kin(-) cells, expression of PKA-RIα and Bim is prominently decreased, and increases in cAMP do not increase Bim expression. Even so, kin(-) cells undergo apoptosis in response to treatment with the glucocorticoid dexamethasone (Dex). In WT cells, glucorticoid-mediated apoptosis involves an increase in Bim, but in kin(-) cells, Dex-promoted cell death appears to occur by a caspase 3-independent apoptosis-inducing factor pathway. Thus, although cAMP/PKA-Cα and PKA-R1α/Bim mediate apoptotic cell death in WT S49 cells, kin(-) cells resist this response because of lower levels of PKA-Cα and PKA-RIα subunits as well as Bim. The findings for Dex-promoted apoptosis imply that these lymphoma cells have adapted to selective pressure that promotes cell death by altering canonical signaling pathways.


Assuntos
Apoptose/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Dexametasona/farmacologia , Linfoma/tratamento farmacológico , Modelos Biológicos , Animais , Apoptose/genética , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2 , Células COS , Linhagem Celular Tumoral , Cercopithecus aethiops , Colforsina/farmacologia , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/genética , Humanos , Linfoma/enzimologia , Linfoma/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo
14.
J Cell Biol ; 204(7): 1083-6, 2014 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-24687277

RESUMO

The mitochondrial inner membrane contains a large protein complex that functions in inner membrane organization and formation of membrane contact sites. The complex was variably named the mitochondrial contact site complex, mitochondrial inner membrane organizing system, mitochondrial organizing structure, or Mitofilin/Fcj1 complex. To facilitate future studies, we propose to unify the nomenclature and term the complex "mitochondrial contact site and cristae organizing system" and its subunits Mic10 to Mic60.


Assuntos
Membranas Mitocondriais/química , Proteínas Mitocondriais/química , Subunidades Proteicas/química , Animais , Humanos , Modelos Moleculares , Complexos Multiproteicos/química , Terminologia como Assunto
15.
Mol Cell Proteomics ; 12(12): 3744-58, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24030101

RESUMO

Insulin resistance plays a major role in the development of type 2 diabetes and obesity and affects a number of biological processes such as mitochondrial biogenesis. Though mitochondrial dysfunction has been linked to the development of insulin resistance and pathogenesis of type 2 diabetes, the precise mechanism linking the two is not well understood. We used high fat diet (HFD)-induced obesity dependent diabetes mouse models to gain insight into the potential pathways altered with metabolic disease, and carried out quantitative proteomic analysis of liver mitochondria. As previously reported, proteins involved in fatty acid oxidation, branched chain amino acid degradation, tricarboxylic acid cycle, and oxidative phosphorylation were uniformly up-regulated in the liver of HFD fed mice compared with that of normal diet. Further, our studies revealed that retinol metabolism is distinctly down-regulated and the mitochondrial structural proteins-components of mitochondrial inter-membrane space bridging (MIB) complex (Mitofilin, Sam50, and ChChd3), and Tim proteins-essential for protein import, are significantly up-regulated in HFD fed mice. Structural and functional studies on HFD and normal diet liver mitochondria revealed remodeling of HFD mitochondria to a more condensed form with increased respiratory capacity and higher ATP levels compared with normal diet mitochondria. Thus, it is likely that the structural remodeling is essential to accommodate the increased protein content in presence of HFD: the mechanism could be through the MIB complex promoting contact site and crista junction formation and in turn facilitating the lipid and protein uptake.


Assuntos
Diabetes Mellitus Experimental/metabolismo , Mitocôndrias Hepáticas/metabolismo , Proteínas Mitocondriais/metabolismo , Obesidade/metabolismo , Proteoma/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Ciclo do Ácido Cítrico/genética , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/genética , Dieta Hiperlipídica , Gorduras na Dieta/administração & dosagem , Regulação da Expressão Gênica , Resistência à Insulina , Metabolismo dos Lipídeos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Hepáticas/genética , Mitocôndrias Hepáticas/ultraestrutura , Proteínas Mitocondriais/genética , Anotação de Sequência Molecular , Obesidade/induzido quimicamente , Obesidade/genética , Fosforilação Oxidativa , Mapeamento de Interação de Proteínas , Proteoma/genética , Transdução de Sinais , Espectrometria de Massas em Tandem , Vitamina A/metabolismo
16.
Proc Natl Acad Sci U S A ; 110(5): E387-96, 2013 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-23319652

RESUMO

cAMP-dependent protein kinase (PKA) regulates a myriad of functions in the heart, including cardiac contractility, myocardial metabolism,and gene expression. However, a molecular integrator of the PKA response in the heart is unknown. Here, we show that the PKA adaptor A-kinase interacting protein 1 (AKIP1) is up-regulated in cardiac myocytes in response to oxidant stress. Mice with cardiac gene transfer of AKIP1 have enhanced protection to ischemic stress. We hypothesized that this adaptation to stress was mitochondrial dependent. AKIP1 interacted with the mitochondrial localized apoptosis inducing factor (AIF) under both normal and oxidant stress. When cardiac myocytes or whole hearts are exposed to oxidant and ischemic stress, levels of both AKIP1 and AIF were enhanced. AKIP1 is preferentially localized to interfibrillary mitochondria and up-regulated in this cardiac mitochondrial subpopulation on ischemic injury. Mitochondria isolated from AKIP1 gene transferred hearts showed increased mitochondrial localization of AKIP1, decreased reactive oxygen species generation, enhanced calcium tolerance, decreased mitochondrial cytochrome C release,and enhance phosphorylation of mitochondrial PKA substrates on ischemic stress. These observations highlight AKIP1 as a critical molecular regulator and a therapeutic control point for stress adaptation in the heart.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Coração/fisiologia , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Fator de Indução de Apoptose/metabolismo , Western Blotting , Linhagem Celular Tumoral , Células Cultivadas , Células HEK293 , Células HeLa , Coração/fisiopatologia , Humanos , Peróxido de Hidrogênio/farmacologia , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Mitocôndrias Cardíacas/metabolismo , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miocárdio/citologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Proteínas Nucleares/genética , Oxidantes/farmacologia , Ligação Proteica , Ratos , Ratos Sprague-Dawley
17.
J Biol Chem ; 287(47): 39480-91, 2012 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-23019327

RESUMO

Coiled-coil helix coiled-coil helix domain-containing protein 3 (ChChd3) is a mitochondrial inner membrane (IM) protein facing toward the intermembrane space (IMS). In the IMS, ChChd3 complexes with multiple proteins at the crista junctions and contact sites and plays a key role in maintaining crista integrity. ChChd3 is myristoylated at the N terminus and has a CHCH domain with twin CX(9)C motifs at its C terminus. The CHCH domain proteins are traditionally imported and trapped in the IMS by using a disulfide relay system mediated by Mia40 and Erv1. In this study, we systematically analyzed the role of the myristoylation and the CHCH domain in the import and mitochondrial localization of ChChd3. Based on our results, we predict that myristoylation promotes binding of ChChd3 to the outer membrane and that the CHCH domain translocates the protein across the outer membrane. By analysis of the CHCH domain cysteine mutants, we further show that they have distinct roles in binding to Mia40 in the IMS and proper folding of the protein. The transient disulfide-bonded intermediate with Mia40 is formed preferentially between the second cysteine in helix 1, Cys(193), and the active site cysteine in Mia40, Cys(55). Although each of the four cysteines is essential for folding of the protein and binding to mitofilin and Sam50, they are not involved in import. Together our results indicate that both the myristoylation and the CHCH domain are essential for the import and mitochondrial localization of ChChd3. Once imported, ChChd3 binds to Mia40 for further folding and assembly into macromolecular complexes.


Assuntos
Lipoilação/fisiologia , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Dobramento de Proteína , Substituição de Aminoácidos , Redutases do Citocromo/genética , Redutases do Citocromo/metabolismo , Células HeLa , Humanos , Mitocôndrias/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas Mitocondriais/genética , Mutação de Sentido Incorreto , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico/fisiologia
18.
J Biol Chem ; 286(4): 2918-32, 2011 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-21081504

RESUMO

The mitochondrial inner membrane (IM) serves as the site for ATP production by hosting the oxidative phosphorylation complex machinery most notably on the crista membranes. Disruption of the crista structure has been implicated in a variety of cardiovascular and neurodegenerative diseases. Here, we characterize ChChd3, a previously identified PKA substrate of unknown function (Schauble, S., King, C. C., Darshi, M., Koller, A., Shah, K., and Taylor, S. S. (2007) J. Biol. Chem. 282, 14952-14959), and show that it is essential for maintaining crista integrity and mitochondrial function. In the mitochondria, ChChd3 is a peripheral protein of the IM facing the intermembrane space. RNAi knockdown of ChChd3 in HeLa cells resulted in fragmented mitochondria, reduced OPA1 protein levels and impaired fusion, and clustering of the mitochondria around the nucleus along with reduced growth rate. Both the oxygen consumption and glycolytic rates were severely restricted. Ultrastructural analysis of these cells revealed aberrant mitochondrial IM structures with fragmented and tubular cristae or loss of cristae, and reduced crista membrane. Additionally, the crista junction opening diameter was reduced to 50% suggesting remodeling of cristae in the absence of ChChd3. Analysis of the ChChd3-binding proteins revealed that ChChd3 interacts with the IM proteins mitofilin and OPA1, which regulate crista morphology, and the outer membrane protein Sam50, which regulates import and assembly of ß-barrel proteins on the outer membrane. Knockdown of ChChd3 led to almost complete loss of both mitofilin and Sam50 proteins and alterations in several mitochondrial proteins, suggesting that ChChd3 is a scaffolding protein that stabilizes protein complexes involved in maintaining crista architecture and protein import and is thus essential for maintaining mitochondrial structure and function.


Assuntos
Mitocôndrias Hepáticas/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Animais , Células HeLa , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Mitocôndrias Hepáticas/genética , Proteínas Mitocondriais/genética , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo
20.
J Biol Chem ; 282(20): 14952-9, 2007 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-17242405

RESUMO

Due to the numerous kinases in the cell, many with overlapping substrates, it is difficult to find novel substrates for a specific kinase. To identify novel substrates of cAMP-dependent protein kinase (PKA), the PKA catalytic subunit was engineered to accept bulky N(6)-substituted ATP analogs, using a chemical genetics approach initially pioneered with v-Src (1). Methionine 120 was mutated to glycine in the ATP-binding pocket of the catalytic subunit. To express the stable mutant C-subunit in Escherichia coli required co-expression with PDK1. This mutant protein was active and fully phosphorylated on Thr(197) and Ser(338). Based on its kinetic properties, the engineered C-subunit preferred N(6)(benzyl)-ATP and N(6)(phenethyl)-ATP over other ATP analogs, but still retained a 30 microm K(m) for ATP. This mutant recombinant C-subunit was used to identify three novel PKA substrates. One protein, a novel mitochondrial ChChd protein, ChChd3, was identified, suggesting that PKA may regulate mitochondria proteins.


Assuntos
Proteínas Mitocondriais/metabolismo , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Animais , Sítios de Ligação/genética , Domínio Catalítico/genética , Proteínas Quinases Dependentes de AMP Cíclico , Células HeLa , Humanos , Cinética , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Mutação de Sentido Incorreto , Fosforilação , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
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