RESUMO
Mitochondria play an important role in both normal heart function and disease etiology. We report analysis of common genetic variations contributing to mitochondrial and heart functions using an integrative proteomics approach in a panel of inbred mouse strains called the Hybrid Mouse Diversity Panel (HMDP). We performed a whole heart proteome study in the HMDP (72 strains, n=2-3 mice) and retrieved 848 mitochondrial proteins (quantified in ≥50 strains). High-resolution association mapping on their relative abundance levels revealed three trans-acting genetic loci on chromosomes (chr) 7, 13 and 17 that regulate distinct classes of mitochondrial proteins as well as cardiac hypertrophy. DAVID enrichment analyses of genes regulated by each of the loci revealed that the chr13 locus was highly enriched for complex-I proteins (24 proteins, P=2.2E-61), the chr17 locus for mitochondrial ribonucleoprotein complex (17 proteins, P=3.1E-25) and the chr7 locus for ubiquinone biosynthesis (3 proteins, P=6.9E-05). Follow-up high resolution regional mapping identified NDUFS4, LRPPRC and COQ7 as the candidate genes for chr13, chr17 and chr7 loci, respectively, and both experimental and statistical analyses supported their causal roles. Furthermore, a large cohort of Diversity Outbred mice was used to corroborate Lrpprc gene as a driver of mitochondrial DNA (mtDNA)-encoded gene regulation, and to show that the chr17 locus is specific to heart. Variations in all three loci were associated with heart mass in at least one of two independent heart stress models, namely, isoproterenol-induced heart failure and diet-induced obesity. These findings suggest that common variations in certain mitochondrial proteins can act in trans to influence tissue-specific mitochondrial functions and contribute to heart hypertrophy, elucidating mechanisms that may underlie genetic susceptibility to heart failure in human populations.
Assuntos
Insuficiência Cardíaca , Proteoma , Animais , Camundongos , Cardiomegalia/genética , DNA Mitocondrial/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/metabolismo , Camundongos Endogâmicos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Proteoma/metabolismoRESUMO
When stable and near-normoglycemic, patients with "A-ß+" ketosis-prone diabetes (KPD) manifest accelerated leucine catabolism and blunted ketone oxidation, which may underlie their proclivity to develop diabetic ketoacidosis (DKA). To understand metabolic derangements in A-ß+ KPD patients during DKA, we compared serum metabolomics profiles of adults during acute hyperglycemic crises, without (n = 21) or with (n = 74) DKA, and healthy control subjects (n = 17). Based on 65 kDa GAD islet autoantibody status, C-peptide, and clinical features, 53 DKA patients were categorized as having KPD and 21 type 1 diabetes (T1D); 21 nonketotic patients were categorized as having type 2 diabetes (T2D). Patients with KPD and patients with T1D had higher counterregulatory hormones and lower insulin-to-glucagon ratio than patients with T2D and control subjects. Compared with patients withT2D and control subjects, patients with KPD and patients with T1D had lower free carnitine and higher long-chain acylcarnitines and acetylcarnitine (C2) but lower palmitoylcarnitine (C16)-to-C2 ratio; a positive relationship between C16 and C2 but negative relationship between carnitine and ß-hydroxybutyrate (BOHB); higher branched-chain amino acids (BCAAs) and their ketoacids but lower ketoisocaproate (KIC)-to-Leu, ketomethylvalerate (KMV)-to-Ile, ketoisovalerate (KIV)-to-Val, isovalerylcarnitine-to-KIC+KMV, propionylcarnitine-to-KIV+KMV, KIC+KMV-to-C2, and KIC-to-BOHB ratios; and lower glutamate and 3-methylhistidine. These data suggest that during DKA, patients with KPD resemble patients with T1D in having impaired BCAA catabolism and accelerated fatty acid flux to ketones-a reversal of their distinctive BCAA metabolic defect when stable. The natural history of A-ß+ KPD is marked by chronic but varying dysregulation of BCAA metabolism.
Assuntos
Aminoácidos de Cadeia Ramificada/sangue , Carnitina/sangue , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 2/sangue , Cetoacidose Diabética/sangue , Adulto , Autoanticorpos , Carnitina/análogos & derivados , Feminino , Humanos , Masculino , Metaboloma , Metabolômica , Pessoa de Meia-IdadeRESUMO
HIV patients develop hepatic steatosis. We investigated hepatic steatosis in transgenic mice expressing the HIV-1 accessory protein Vpr (Vpr-Tg) in liver and adipose tissues, and WT mice infused with synthetic Vpr. Vpr-Tg mice developed increased liver triglyceride content and elevated ALT, bilirubin and alkaline phosphatase due to three hepatic defects: 1.6-fold accelerated de novo lipogenesis (DNL), 45% slower fatty acid ß-oxidation, and 40% decreased VLDL-triglyceride export. Accelerated hepatic DNL was due to coactivation by Vpr of liver X receptor-α (LXRα) with increased expression of its lipogenic targets Srebp1c, Chrebp, Lpk, Dgat, Fasn and Scd1, and intranuclear SREBP1c and ChREBP. Vpr enhanced association of LXRα with Lxrα and Srebp1c promoters, increased LXRE-LXRα binding, and broadly altered hepatic expression of LXRα-regulated lipid metabolic genes. Diminished hepatic fatty acid ß-oxidation was associated with decreased mRNA expression of Pparα and its targets Cpt1, Aox, Lcad, Ehhadh, Hsd10 and Acaa2, and blunted VLDL export with decreased expression of Mttp and its product microsomal triglyceride transfer protein. With our previous findings that Vpr circulates in HIV patients (including those with undetectable plasma HIV-1 RNA), co-regulates the glucocorticoid receptor and PPARγ and transduces hepatocytes, these data indicate a potential role for Vpr in HIV-associated fatty liver disease.
Assuntos
Produtos do Gene vpr/metabolismo , Infecções por HIV/complicações , Infecções por HIV/genética , HIV-1/fisiologia , Receptores X do Fígado/genética , Hepatopatia Gordurosa não Alcoólica/etiologia , PPAR alfa/genética , Animais , Modelos Animais de Doenças , Regulação da Expressão Gênica , Infecções por HIV/virologia , Hepatócitos/metabolismo , Metabolismo dos Lipídeos , Testes de Função Hepática , Receptores X do Fígado/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Hepatopatia Gordurosa não Alcoólica/diagnóstico , Hepatopatia Gordurosa não Alcoólica/metabolismo , PPAR alfa/metabolismoRESUMO
Although histone deacetylase inhibitors (HDACi) are a promising class of anti-cancer drugs, thus far, they have been unsuccessful in early phase clinical trials for pancreatic ductal adenocarcinoma (PDAC). One potential reason for their poor efficacy is the tumor stroma, where cancer-associated fibroblasts (CAFs) are a prominent cell type and a source of resistance to cancer therapies. Here, we demonstrate that stromal fibroblasts contribute to the poor efficacy of HDACi's in PDAC. HDACi-treated fibroblasts show increased biological aggressiveness and are characterized by increased secretion of pro-inflammatory tumor-supportive cytokines and chemokines. We find that HDAC2 binds to the enhancer and promoter regions of pro-inflammatory genes specifically in CAFs and in silico analysis identified AP-1 to be the most frequently associated transcription factor bound in these regions. Pharmacologic inhibition of pathways upstream of AP-1 suppresses the HDACi-induced inflammatory gene expression and tumor-supportive responses in fibroblasts. Our findings demonstrate that the combination of HDACi's with chemical inhibitors of the AP-1 signaling pathway attenuate the inflammatory phenotype of fibroblasts and may improve the efficacy of HDACi in PDAC and, potentially, in other solid tumors rich in stroma.
Assuntos
Fibroblastos Associados a Câncer/efeitos dos fármacos , Fibroblastos Associados a Câncer/patologia , Carcinoma Ductal Pancreático/patologia , Inibidores de Histona Desacetilases/farmacologia , Neoplasias Pancreáticas/patologia , Animais , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Imunoprecipitação da Cromatina , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Fenótipo , Reação em Cadeia da Polimerase em Tempo Real , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
UNLABELLED: Pancreatic ductal adenocarcinoma (PDAC) has a characteristically dense stroma comprised predominantly of cancer-associated fibroblasts (CAF). CAFs promote tumor growth, metastasis, and treatment resistance. This study aimed to investigate the molecular changes and functional consequences associated with chemotherapy treatment of PDAC CAFs. Chemoresistant immortalized CAFs (R-CAF) were generated by continuous incubation in gemcitabine. Gene expression differences between treatment-naïve CAFs (N-CAF) and R-CAFs were compared by array analysis. Functionally, tumor cells (TC) were exposed to N-CAF- or R-CAF-conditioned media and assayed for migration, invasion, and viability in vitro Furthermore, a coinjection (TC and CAF) model was used to compare tumor growth in vivo R-CAFs increased TC viability, migration, and invasion compared with N-CAFs. In vivo, TCs coinjected with R-CAFs grew larger than those accompanied by N-CAFs. Genomic analysis demonstrated that R-CAFs had increased expression of various inflammatory mediators, similar to the previously described senescence-associated secretory phenotype (SASP). In addition, SASP mediators were found to be upregulated in response to short duration treatment with gemcitabine in both immortalized and primary CAFs. Inhibition of stress-associated MAPK signaling (P38 MAPK or JNK) attenuated SASP induction as well as the tumor-supportive functions of chemotherapy-treated CAFs in vitro and in vivo These results identify a negative consequence of chemotherapy on the PDAC microenvironment that could be targeted to improve the efficacy of current therapeutic regimens. IMPLICATIONS: Chemotherapy treatment of pancreatic cancer-associated fibroblasts results in a proinflammatory response driven by stress-associated MAPK signaling that enhances tumor cell growth and invasiveness. Mol Cancer Res; 14(5); 437-47. ©2016 AACR.
Assuntos
Fibroblastos Associados a Câncer/citologia , Carcinoma Ductal Pancreático/patologia , Desoxicitidina/análogos & derivados , Inflamação/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Neoplasias Pancreáticas/patologia , Células Tumorais Cultivadas/citologia , Animais , Fibroblastos Associados a Câncer/efeitos dos fármacos , Fibroblastos Associados a Câncer/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Meios de Cultivo Condicionados , Desoxicitidina/farmacologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Transplante de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Células Tumorais Cultivadas/metabolismo , GencitabinaRESUMO
BACKGROUND: We previously identified a correlation between increased expression of the phosphoinositide 3-kinase (PI3K) regulatory subunit p85α and improved survival in human pancreatic ductal adenocarcinoma (PDAC). The purpose of this study was to investigate the impact of changes in p85α expression on response to chemotherapy and the regulation of p85α by microRNA-21 (miR-21). MATERIALS AND METHODS: PDAC tumor cells overexpressing p85α were generated by viral transduction, and the effect of p85α overexpression on sensitivity to gemcitabine was tested by MTT assay. Primary human PDAC tumors were stained for p85α and miR-21 via immunohistochemistry and in situ hybridization, respectively. Additionally, PDAC cells were treated with miR-21 mimic, and changes in p85α and phospho-AKT were assessed by Western blot. Finally, a luciferase reporter assay system was used to test direct regulation of p85α by miR-21. RESULTS: Higher p85α expression resulted in increased sensitivity to gemcitabine (P < 0.01), which correlated with decreased PI3K-AKT activation. Human tumors demonstrated an inverse correlation between miR-21 and p85α expression levels (r = -0.353, P < 0.001). In vitro, overexpression of miR-21 resulted in decreased levels of p85α and increased phosphorylation of AKT. Luciferase reporter assays confirmed the direct regulation of p85α by miR-21 (P < 0.01). CONCLUSIONS: Our results demonstrate that p85α expression is a determinant of chemosensitivity in PDAC. Additionally, we provide novel evidence that miR-21 can influence PI3K-AKT signaling via its direct regulation of p85α. These data provide insight into potential mechanisms for the known relationship between increased p85α expression and improved survival in PDAC.
Assuntos
Carcinoma Ductal Pancreático/metabolismo , Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , MicroRNAs/metabolismo , Neoplasias Pancreáticas/metabolismo , Antimetabólitos Antineoplásicos/uso terapêutico , Carcinoma Ductal Pancreático/tratamento farmacológico , Linhagem Celular Tumoral , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapêutico , Células HEK293 , Humanos , Neoplasias Pancreáticas/tratamento farmacológico , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , GencitabinaRESUMO
Viral infections, such as HIV, have been linked to obesity, but mechanistic evidence that they cause adipose dysfunction in vivo is lacking. We investigated a pathogenic role for the HIV-1 accessory protein viral protein R (Vpr), which can coactivate the glucocorticoid receptor (GR) and co-repress peroxisome proliferator-activated receptor γ (PPARγ) in vitro, in HIV-associated adipose dysfunction. Vpr circulated in the blood of most HIV-infected patients tested, including those on antiretroviral therapy (ART) with undetectable viral load. Vpr-mediated mechanisms were dissected in vivo using mouse models expressing the Vpr transgene in adipose tissues and liver (Vpr-Tg) or infused with synthetic Vpr. Both models demonstrated accelerated whole-body lipolysis, hyperglycemia and hypertriglyceridemia, and tissue-specific findings. Fat depots in these mice had diminished mass, macrophage infiltration, and blunted PPARγ target gene expression but increased GR target gene expression. In liver, we observed blunted PPARα target gene expression, steatosis with decreased adenosine monophosphate-activated protein kinase activity, and insulin resistance. Similar to human HIV-infected patients, Vpr circulated in the serum of Vpr-Tg mice. Vpr blocked differentiation in preadipocytes through cell cycle arrest, whereas in mature adipocytes, it increased lipolysis with reciprocally altered association of PPARγ and GR with their target promoters. These results delineate a distinct pathogenic sequence: Vpr, released from HIV-1 in tissue reservoirs after ART, can disrupt PPAR/GR co-regulation and cell cycle control to produce adipose dysfunction and hepatosteatosis. Confirmation of these mechanisms in HIV patients could lead to targeted treatment of the metabolic complications with Vpr inhibitors, GR antagonists, or PPARγ/PPARα agonists.
Assuntos
Produtos do Gene vpr/metabolismo , HIV-1/metabolismo , Receptores de Glucocorticoides/metabolismo , Células 3T3-L1 , Animais , Cromatografia em Camada Fina , Ensaio de Imunoadsorção Enzimática , Produtos do Gene vpr/genética , HIV-1/genética , Humanos , Immunoblotting , Masculino , Camundongos , Camundongos Transgênicos , PPAR alfa/agonistas , PPAR alfa/metabolismo , PPAR gama/metabolismo , Receptores de Glucocorticoides/agonistasRESUMO
Aâ»ß⺠ketosis-prone diabetes (KPD) is an emerging syndrome of obesity, unprovoked ketoacidosis, reversible ß-cell dysfunction, and near-normoglycemic remission. We combined metabolomics with targeted kinetic measurements to investigate its pathophysiology. Fasting plasma fatty acids, acylcarnitines, and amino acids were quantified in 20 KPD patients compared with 19 nondiabetic control subjects. Unique signatures in KPD--higher glutamate but lower glutamine and citrulline concentrations, increased ß-hydroxybutyryl-carnitine, decreased isovaleryl-carnitine (a leucine catabolite), and decreased tricarboxylic acid (TCA) cycle intermediates--generated hypotheses that were tested through stable isotope/mass spectrometry protocols in nine new-onset, stable KPD patients compared with seven nondiabetic control subjects. Free fatty acid flux and acetyl CoA flux and oxidation were similar, but KPD had slower acetyl CoA conversion to ß-hydroxybutyrate; higher fasting ß-hydroxybutyrate concentration; slower ß-hydroxybutyrate oxidation; faster leucine oxidative decarboxylation; accelerated glutamine conversion to glutamate without increase in glutamate carbon oxidation; and slower citrulline flux, with diminished glutamine amide-nitrogen transfer to citrulline. The confluence of metabolomic and kinetic data indicate a distinctive pathogenic sequence: impaired ketone oxidation and fatty acid utilization for energy, leading to accelerated leucine catabolism and transamination of α-ketoglutarate to glutamate, with impaired TCA anaplerosis of glutamate carbon. They highlight a novel process of defective energy production and ketosis in Aâ»ß⺠KPD.
Assuntos
Autoanticorpos/análise , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Cetoacidose Diabética/etiologia , Metabolismo Energético , Células Secretoras de Insulina/metabolismo , Adulto , Algoritmos , Índice de Massa Corporal , Ciclo do Ácido Cítrico , Estudos de Coortes , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/fisiopatologia , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/imunologia , Diabetes Mellitus Tipo 2/fisiopatologia , Feminino , Humanos , Resistência à Insulina , Células Secretoras de Insulina/imunologia , Cinética , Estudos Longitudinais , Masculino , Metabolômica/métodos , Pessoa de Meia-Idade , Obesidade/complicaçõesRESUMO
PURPOSE: To carry out an integrative profile of human pancreatic ductal adenocarcinoma (PDAC) to identify prognosis-significant genes and their related pathways. EXPERIMENTAL DESIGN: A concordant survival-based whole genome in silico array analysis of DNA copy number, and mRNA and miRNA expression in 25 early-stage PDAC was carried out. A novel composite score simultaneously integrated gene expression with regulatory mechanisms to identify the signature genes with the most levels of prognosis-significant evidence. The predominant signaling pathways were determined via a pathway-based approach. Independent patient cohorts (n = 148 and 42) were then used as in vitro validation of the array findings. RESULTS: The composite score identified 171 genes in which expressions were able to define two prognosis subgroups (P = 3.8e-5). Eighty-eight percent (151 of 171) of the genes were regulated by prognosis-significant miRNAs. The phosphoinositide 3-kinase/AKT pathway and SRC signaling were densely populated by prognosis-significant genes and driven by genomic amplification of SRC and miRNA regulation of p85α and CBL. On tissue microarray validation (n = 148), p85α protein expression was associated with improved survival for all patients (P = 0.02), and activated P-SRC (Y418) was associated shorter survival for patients with low-grade histology tumors (P = 0.04). Interacting P-SRC and p85α revealed that they define two distinct PDAC patient subgroups (P = 0.0066). Furthering the importance of these pathways, CBL protein expression was associated with improved survival (P = 0.03) on a separate cohort (n = 42). CONCLUSIONS: These pathways and related genes may represent putative clinical biomarkers and possible targets of individualized therapy in the distinct patient subgroups they define.
Assuntos
Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/mortalidade , Perfilação da Expressão Gênica , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/mortalidade , Idoso , Idoso de 80 Anos ou mais , Análise por Conglomerados , Variações do Número de Cópias de DNA , Feminino , Genômica , Humanos , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Fosfatidilinositol 3-Quinases/metabolismo , Prognóstico , RNA Mensageiro/genética , Reprodutibilidade dos Testes , Transdução de Sinais , Análise de Sobrevida , Quinases da Família src/metabolismoRESUMO
Obesity, type 2 diabetes, and HIV-associated lipodystrophy are associated with abnormalities in adipocyte growth and differentiation. In persons with these conditions, adipose depots contain increased numbers of macrophages, but the origins of these cells and their specific effects are uncertain. Peripheral blood mononuclear cells (PBMC)-derived monocytes, but not T cells, cocultured via transwells with primary subcutaneous preadipocytes, increased proliferation (approximately twofold) and reduced differentiation (~50%) of preadipocytes. Gene expression analyses in proliferating preadipocytes (i.e., prior to hormonal induction of terminal differentiation) revealed that monocytes down-regulated mRNA levels of CCAAT/enhancer binding protein, alpha (C/EBPα) and up-regulated mRNA levels of G0/G1 switch 2 (G0S2) message, genes important for the regulation of adipogenesis and the cell cycle. These data indicate that circulating peripheral blood monocytes can disrupt adipogenesis by interfering with a critical step in C/EBPα and G0S2 transcription required for preadipocytes to make the transition from proliferation to differentiation. Interactions between preadipocytes and monocytes also increased the inflammatory cytokines IL-6 and IL-8, as well as a novel chemotactic cytokine, CXCL1. Additionally, the levels of both IL-6 and CXCL1 were highest when preadipocytes and monocytes were cultured together, compared to each cell in culture alone. Such cross-talk amplifies the production of mediators of tissue inflammation.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Síndrome de Lipodistrofia Associada ao HIV/metabolismo , Monócitos/metabolismo , Obesidade/metabolismo , RNA Mensageiro/metabolismo , Adipócitos , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Western Blotting , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Diferenciação Celular , Proliferação de Células , Quimiocina CXCL1/metabolismo , Citocinas/metabolismo , Diabetes Mellitus Tipo 2/genética , Síndrome de Lipodistrofia Associada ao HIV/genética , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Obesidade/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
BACKGROUND: Aging is associated with oxidative stress, but underlying mechanisms remain poorly understood. OBJECTIVE: We tested whether glutathione deficiency occurs because of diminished synthesis and contributes to oxidative stress in aging and whether stimulating glutathione synthesis with its precursors cysteine and glycine could alleviate oxidative stress. DESIGN: Eight elderly and 8 younger subjects received stable-isotope infusions of [2H(2)]glycine, after which red blood cell (RBC) glutathione synthesis and concentrations, plasma oxidative stress, and markers of oxidant damage (eg, F(2)-isoprostanes) were measured. Elderly subjects were restudied after 2 wk of glutathione precursor supplementation. RESULTS: Compared with younger control subjects, elderly subjects had markedly lower RBC concentrations of glycine (486.7 ± 28.3 compared with 218.0 ± 23.7 µmol/L; P < 0.01), cysteine (26.2 ± 1.4 compared with 19.8 ± 1.3 µmol/L; P < 0.05), and glutathione (2.08 ± 0.12 compared with 1.12 ± 0.18 mmol/L RBCs; P < 0.05); lower glutathione fractional (83.14 ± 6.43% compared with 45.80 ± 5.69%/d; P < 0.01) and absolute (1.73 ± 0.16 compared with 0.55 ± 0.12 mmol/L RBCs per day; P < 0.01) synthesis rates; and higher plasma oxidative stress (304 ± 16 compared with 346 ± 20 Carratelli units; P < 0.05) and plasma F(2)-isoprostanes (97.7 ± 8.3 compared with 136.3 ± 11.3 pg/mL; P < 0.05). Precursor supplementation in elderly subjects led to a 94.6% higher glutathione concentration, a 78.8% higher fractional synthesis rate, a 230.9% higher absolute synthesis rate, and significantly lower plasma oxidative stress and F(2)-isoprostanes. No differences in these measures were observed between younger subjects and supplemented elderly subjects. CONCLUSIONS: Glutathione deficiency in elderly humans occurs because of a marked reduction in synthesis. Dietary supplementation with the glutathione precursors cysteine and glycine fully restores glutathione synthesis and concentrations and lowers levels of oxidative stress and oxidant damages. These findings suggest a practical and effective approach to decreasing oxidative stress in aging.
Assuntos
Envelhecimento/fisiologia , Cisteína/uso terapêutico , Suplementos Nutricionais , Glutationa/biossíntese , Glicina/uso terapêutico , Doenças Metabólicas/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , Adulto , Fatores Etários , Idoso , Cisteína/sangue , Cisteína/farmacologia , Eritrócitos/metabolismo , F2-Isoprostanos/sangue , Glutationa/sangue , Glutationa/deficiência , Glicina/sangue , Glicina/farmacologia , Humanos , Isótopos , Doenças Metabólicas/sangue , Coloração e RotulagemRESUMO
HIV-associated dyslipemic lipodystrophy (HADL) is a heterogeneous syndrome of fat redistribution, hypertriglyceridemia, and insulin resistance, associated with markedly accelerated rates of lipolysis, intraadipocyte and intrahepatic reesterification, and very low-density lipoprotein-triglyceride synthesis and release. The objective of the study was to determine if rosiglitazone can ameliorate these lipid kinetic defects in patients with HADL. Infusions of [(13)C(1)]palmitate and [(2)H(5)]glycerol were used to measure total and net lipolysis, adipocyte and hepatic reesterification, and plasma free fatty acid (FFA) oxidation in 9 men with HADL, before and after 3 months of treatment with rosiglitazone (8 mg/d). Rosiglitazone treatment significantly increased both total lipolysis (R(a) FFA(total) from 3.37 ± 0.40 to 4.57 ± 0.68 mmol FFA per kilogram fat per hour, P < .05) and adipocyte reesterification (1.25 ± 0.35 to 2.43 ± 0.65 mmol FFA per kilogram fat per hour, P < .05). However, there was no change in net lipolysis (R(a) FFA(net) 2.47 ± 0.43 to 2.42 ± 0.37 mmol FFA per kilogram fat per hour), plasma FFA oxidation (0.30 ± 0.046 to 0.32 ± 0.04 mmol FFA per kilogram lean body mass per hour), or FFA flux available for hepatic reesterification (0.59 ± 0.07 to 0.56 ± 0.10 mmol FFA per kilogram fat per hour). There were significant decreases in fasting plasma insulin concentrations and insulin resistance, but not in fasting plasma lipid or glucose concentrations. There was a significant decrease in waist to hip ratio (0.98 ± 0.02 to 0.95 ± 0.02, P < .05) consistent with a significant increase in hip circumference (0.93 ± 0.02 to 0.95 ± 0.02 m, P < .05), without change in waist circumference. Rosiglitazone significantly increased adipocyte reesterification and improved insulin sensitivity, but the potential benefit of these changes was compromised by increase in total lipolysis. Combining rosiglitazone with agents designed to blunt lipolysis could expand depleted peripheral adipose depots in patients with HIV lipodystrophy.
Assuntos
Síndrome de Lipodistrofia Associada ao HIV/sangue , Hipoglicemiantes/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Tiazolidinedionas/farmacologia , Terapia Antirretroviral de Alta Atividade , Glicemia/metabolismo , Composição Corporal/efeitos dos fármacos , Calorimetria Indireta , Ácidos Graxos não Esterificados/sangue , Glicerol/sangue , Síndrome de Lipodistrofia Associada ao HIV/tratamento farmacológico , Humanos , Hipolipemiantes/uso terapêutico , Cinética , Lipídeos/sangue , Lipólise/efeitos dos fármacos , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Palmitatos/metabolismo , Rosiglitazona , Relação Cintura-QuadrilRESUMO
OBJECTIVE: Sustained hyperglycemia is associated with low cellular levels of the antioxidant glutathione (GSH), which leads to tissue damage attributed to oxidative stress. We tested the hypothesis that diminished GSH in adult patients with uncontrolled type 2 diabetes is attributed to decreased synthesis and measured the effect of dietary supplementation with its precursors cysteine and glycine on GSH synthesis rate and oxidative stress. RESEARCH DESIGN AND METHODS: We infused 12 diabetic patients and 12 nondiabetic control subjects with [²H2]-glycine to measure GSH synthesis. We also measured intracellular GSH concentrations, reactive oxygen metabolites, and lipid peroxides. Diabetic patients were restudied after 2 weeks of dietary supplementation with the GSH precursors cysteine and glycine. RESULTS: Compared with control subjects, diabetic subjects had significantly higher fasting glucose (5.0 ± 0.1 vs. 10.7 ± 0.5 mmol/l; P < 0.001), lower erythrocyte concentrations of glycine (514.7 ± 33.1 vs. 403.2 ± 18.2 µmol/l; P < 0.01), and cysteine (25.2 ± 1.5 vs. 17.8 ± 1.5 µmol/l; P < 0.01); lower concentrations of GSH (6.75 ± 0.47 vs. 1.65 ± 0.16 µmol/g Hb; P < 0.001); diminished fractional (79.21 ± 5.75 vs. 44.86 ± 2.87%/day; P < 0.001) and absolute (5.26 ± 0.61 vs. 0.74 ± 0.10 µmol/g Hb/day; P < 0.001) GSH synthesis rates; and higher reactive oxygen metabolites (286 ± 10 vs. 403 ± 11 Carratelli units [UCarr]; P < 0.001) and lipid peroxides (2.6 ± 0.4 vs. 10.8 ± 1.2 pg/ml; P < 0.001). Following dietary supplementation in diabetic subjects, GSH synthesis and concentrations increased significantly and plasma oxidative stress and lipid peroxides decreased significantly. CONCLUSIONS: Patients with uncontrolled type 2 diabetes have severely deficient synthesis of glutathione attributed to limited precursor availability. Dietary supplementation with GSH precursor amino acids can restore GSH synthesis and lower oxidative stress and oxidant damage in the face of persistent hyperglycemia.
Assuntos
Cisteína/uso terapêutico , Diabetes Mellitus Tipo 2/metabolismo , Glutationa/biossíntese , Glicina/uso terapêutico , Diabetes Mellitus Tipo 2/dietoterapia , Suplementos Nutricionais , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Ketosis-prone diabetes (KPD) is heterogeneous. Longitudinal follow-up revealed that patients with "A-ß+" KPD (absent autoantibodies and preserved ß-cell function) segregated into 2 subgroups with distinct evolution of ß-cell function and glycemic control. Generalized linear analysis demonstrated that the variable that most significantly differentiated them was presence of a clinically evident precipitating event for the index diabetic ketoacidosis (DKA). Hence, we performed a comprehensive analysis of A-ß+ KPD patients presenting with "provoked" compared with "unprovoked" DKA. Clinical, biochemical, and ß-cell functional characteristics were compared between provoked and unprovoked A-ß+ KPD patients followed prospectively for 1 to 8 years. Human leukocyte antigen class II allele frequencies were compared between these 2 groups and population controls. Unprovoked A-ß+ KPD patients (n = 83) had greater body mass index, male preponderance, higher frequency of women with oligo-/anovulation, more frequent African American ethnicity, and less frequent family history of diabetes than provoked A-ß+ KPD patients (n = 64). The provoked group had higher frequencies of the human leukocyte antigen class II type 1 diabetes mellitus susceptibility alleles DQB1*0302 (than the unprovoked group or population controls) and DRB1*04 (than the unprovoked group), whereas the unprovoked group had a higher frequency of the protective allele DQB1*0602. ß-Cell secretory reserve and glycemic control improved progressively in the unprovoked group but declined in the provoked group. The differences persisted in comparisons restricted to patients with new-onset diabetes. "Unprovoked" A-ß+ KPD is a distinct syndrome characterized by reversible ß-cell dysfunction with male predominance and increased frequency of DQB1*0602, whereas "provoked" A-ß+ KPD is characterized by progressive loss of ß-cell reserve and increased frequency of DQB1*0302 and DRB1*04. Unprovoked DKA predicts long-term ß-cell functional reserve, insulin independence, and glycemic control in KPD.
Assuntos
Autoanticorpos/sangue , Diabetes Mellitus Tipo 1/classificação , Diabetes Mellitus Tipo 1/complicações , Cetoacidose Diabética/etiologia , Antígenos de Histocompatibilidade Classe II/genética , Células Secretoras de Insulina/fisiologia , Adulto , Alelos , Autoanticorpos/fisiologia , Estudos de Casos e Controles , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/fisiopatologia , Cetoacidose Diabética/sangue , Cetoacidose Diabética/imunologia , Cetoacidose Diabética/fisiopatologia , Suscetibilidade a Doenças/imunologia , Suscetibilidade a Doenças/fisiopatologia , Etnicidade , Feminino , Frequência do Gene , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Fatores Sexuais , Síndrome , Adulto JovemRESUMO
BACKGROUND: We previously demonstrated that somatostatin receptor type 5 (SSTR5) gene ablation results in alterations in insulin secretion and glucose metabolism, accompanied by morphologic alterations in the islets of Langerhans. The underlying mechanism(s) by which SSTR5 exerts its cellular functions remain(s) unknown. We hypothesized that SSTR5 mediates the inhibitory effect of somatostatin (SST) on insulin secretion and islet proliferation by regulating a specific set of pancreatic genes. METHODS: To identify SSTR5-regulated pancreatic genes, gene expression microarray analysis was performed on the whole pancreas of 1- and 3-month-old wild-type (WT) and SSTR5 knockout (SSTR5-/-) male mice. Real-time RT-PCR and immunofluorescence were performed to validate selected differentially expressed genes. RESULTS: A set of 143 probes were identified to be differentially expressed in the pancreas of 1-month-old SSTR5-/- mice, 72 of which were downregulated and 71 upregulated. At 3 months of age, SSTR5 gene ablation resulted in downregulation of a set of 30 probes and upregulation of a set of 37 probes. Among these differentially expressed genes, there were 15 and 5 genes that were upregulated and downregulated, respectively, in mice at both 1 and 3 months of age. Three genes, PAP/INGAP, ANG, and TDE1, were selected to be validated by real-time RT-PCR and immunofluorescence. CONCLUSIONS: A specific set of genes linked to a wide range of cellular functions such as islet proliferation, apoptosis, angiogenesis, and tumorigenesis were either upregulated or downregulated in SSTR5-deficient male mice compared with their expression in wild-type mice. Therefore, these genes are potential SSTR5-regulated genes during normal pancreatic development and functional maintenance.