Interferência do TSPO sobre as vias de ativação de adipócitos da linhagem 3T3-L1 / Interference of TSPO upon the activation pathways of 3T3-L1 adipocytes

A obesidade está associada a um processo inflamatório crônico de baixa intensidade e representa um dos fatores de risco para o desenvolvimento de uma série de comorbidades. A proteína TSPO está envolvida em inúmeras funções celulares, incluindo biossíntese e transporte de esteróides, transporte de porfirinas, apoptose, biossíntese do heme, processos oxidativos e imunomodulação. Ademais, a presença e a função da proteína TSPO no tecido adiposo e na inflamação ainda não estão bem estabelecidas. Deste modo, o objetivo do presente estudo foi validar a expressão e função do TSPO durante a diferenciação de células 3T3-L1, e investigar se o tratamento de adipócitos 3T3-L1 com diazepam, um benzodiazepínico de ação central (GABAA) e periférica (TSPO), é capaz de modular os efeitos inflamatórios induzidos pela incubação das células 3T3-L1 com TNF-α. Nossos resultados evidenciaram que, em nosso estudo, o tratamento de pré-adipócitos com diazepam não modulou a adipogênese. Entretanto, apesar de o diazepam per se não modular o acúmulo de triacilglicerol e a expressão gênica e protéica de PPAR-γ; em células estimuladas pelo TNF-α, o tratamento com diazepam foi capaz de reverter a diminuição da expressão gênica e protéica de PPAR-γ induzida pelo TNF-α. Ademais, o tratamento dos adipócitos com diazepam foi capaz de modular positivamente a expressão protéica de TSPO, efeito este que não observamos em células tratadas pelo clonazepam, um benzodiazepínico de ação exclusivamente central. Em resumo, os dados obtidos neste estudo, pela primeira vez, demonstram a possível relação entre as vias que controlam a sinalização de TSPO, TNF-α e PPAR-γ. Assim, nos é possível inferir que a ativação de TSPO pelo seu ligante diazepam foi capaz de modular a ativação de NF-kB induzida pelo TNF-α, promovendo, com a diminuição da lipólise e aumento da expressão gênica de TSPO e gênica e protéica de PPAR-γ, o reestabelecimento da homeostase celular, o que aumentaria a sobrevida das células, a atividade mitocondrial, e a atividade adipogênica dos adipócitos

Obesity is associated with a chronic low-grade inflammation and these represents one of the risk factors to development of other non-communicable diseases. TSPO 18 kDa is involved in several cellular functions, including biosynthesis and steroids transport, porphyrin transport, apoptosis, heme biosynthesis, oxidative metabolism and immunomodulation. Furthermore, the TSPO expression and function on adipose tissue and in the chronic low-grade inflammation have not been established. Thus, the aim of present study was to validate the TSPO expression and function on the 3T3-L1 differentiation process and to investigate whether diazepam treatment is able to modulate the TNF-α induced inflammatory effects on 3T3-L1 cells. Our results showed that diazepam treatment of preadipocytes was not able to modulate the adipogenesis. However, although diazepam treatment per se does not modulate the triacylglycerol accumulation and gene and protein expression of PPAR-γ; in TNF-α stimulated adipocytes, the treatment with diazepam was able to modulate the decreased of PPAR-γ gene and protein expression induced by TNF-α. In addition, the diazepam treatment of adipocytes was able to positively modulate the TSPO protein expression, an effect that we did not observe in cells treated with clonazepam, a central benzodiazepine ligand. In summary, the data obtained in this study, for the first time, demonstrate the possible relationship between the pathways that control the TSPO, TNF-α and PPAR-γ signaling. Thus, it is possible that the activation of TSPO by diazepam was able to modulate TNF-α-induced activation of NF-kB, promoting the reduction of lipolysis and increased of TSPO gene expression and PPAR-γ gene and protein expression, reestablishment of cellular homeostasis, which would increase cell survival, mitochondrial activity, and adipogenic activity of adipocytes

ANT2 shRNA downregulates miR-19a and miR-96 through the PI3K/Akt pathway and suppresses tumor growth in hepatocellular carcinoma cells

MicroRNAs (miRNAs) are negative regulators of gene expression, and miRNA deregulation is found in various tumors. We previously reported that suppression of adenine nucleotide translocase 2 (ANT2) by short hairpin RNA (shRNA) inhibits hepatocellular carcinoma (HCC) development by rescuing miR-636 expression. However, the tumor-suppressive mechanisms of ANT2 shRNA are still poorly understood in HCC. Here, we hypothesized that miRNAs that are specifically downregulated by ANT2 shRNA might function as oncomiRs, and we investigated the roles of ANT2 shRNA-regulated miRNAs in the pathogenesis of HCC. Our data show that miR-19a and miR-96, whose expression is regulated by ANT2 suppression, were markedly upregulated in HCC cell lines and clinical samples. Ectopic expression of miR-19a and miR-96 dramatically induced the proliferation and colony formation of hepatoma cells in vitro, whereas inhibition of miR-19a and miR-96 reduced these effects. To investigate the in vivo function, we implanted miR-96-overexpressing HepG2 cells in a xenograft model and demonstrated that the increase in miR-96 promoted tumor growth. We also found that miR-19a and miR-96 inhibited expression of tissue inhibitor of metalloproteinase-2. Taken together, our results suggest that ANT2-regulated miR-19a and miR-96 play an important role in promoting the proliferation of human HCC cells, and the knockdown of ANT2 directly downregulates miR-19a and miR-96, ultimately resulting in the suppression of tumor growth.

Evaluation of porin interaction with adenine nucleotide translocase and cyclophilin-D proteins after brain ischemia and reperfusion

Porin is a mitochondrial outer membrane channel, which usually functions as the pathway for the movement of various substances in and out of the mitochondria and is considered to be a component of the permeability transition [PT] pore complex that plays a role in the PT. We addressed the hypothesis that porin interacts with other mitochondrial proteins after ischemic injury. For this purpose, we used in vivo 4-vessel occlusion model of rat brain and porin purification method by hydroxyapatite column. After SDS gel electrophoresis and silver nitrate staining, Western blotting was done for porin, adenine nucleotide translocase and cyclophilin-D proteins. Porin was purified from mitochondrial mixture in ischemic brain and control groups. Investigation of interaction of adenine nucleotide transposes [ANT] and cyclophilin-D with porin by Western blotting showed no proteins co-purified with porin from injured tissues. The present study implies that there may not be interaction between porin, and ANT or cyclophilin-D, and if there is any, it is not maintained during the purification procedure

Expressão de genes relacionados à função adrenocortical no estado de caquexia neoplásica / Expression of genes related to the adrenocortical function in the neoplastic cachexia process

A glândula adrenal tem papel fundamental na resposta neuroendócrina, especialmente em situações em que há comprometimento da homeostasia. No processo de caquexia neoplásica, há prejuízo da homeostasia por alterações nutricionais e metabólicas do câncer em estágio avançado, envolvendo a resposta do eixo hipotálamo-hipófise-adrenal. Neste trabalho, foi utilizado um modelo animal de caquexia induzida pelo tumor de Walker-256 em ratos Wistar. Os animais (n=4) foram sacrificados dez dias após a inoculação de células tumorais e a glândula adrenal foi removida. O RNA foi extraído para o estudo da expressão de genes relacionados ao controle da esteroidogênese por RT-PCR semiquantitativa. A análise dos dados demonstrou expressão significativamente reduzida dos genes MC2R (receptor tipo 2 para melacortina), 3ßHSD I (3β-hidroxiesteroidedesidrogenas e tipo I) e TSPO (proteína translocadora) em animais com caquexia neoplásica(valores de P=0,037; 0,0097 e 0,052, respectivamente), revelando falência do córtex da adrenal.

The adrenal gland plays a crucial role in the neuroendocrine response, especially in situations where homeostasis is disturbed. In the neoplastic cachexiaprocess, there is homeostasis impairment by nutritional and metabolic alterations of advanced-stage cancer, involving hypothalamus-pituitary-adrenal axis response. In this assignment, an experimental model of cachexia induced by Walker-256 tumor was performed in Wistar rats. Animals (n=4) were sacrificed 10 days after inoculation of tumor cells, and the adrenal glands were excised. The RNA was isolated for the study of geneexpression related to the steroidogenesis control by semi-quantitative RT-PCR. Dataanalysis showed a significant reduced expression of MC2R (melancortin type 2 receptor), 3ßHSD I (3-beta-hydroxysteroid dehydrogenase type I) and TSPO (translocator protein)genes in animals with neoplastic cachexia (P=0.037, 0.0097 and 0.052, respectively), revealing adrenal cortex failure.

Mitochondrial Permeability Transition Pore and Cardioprotection Against Ischemia-reperfusion Injury

Opening of mitochondrial permeability transition pore (mPTP) was found to have a critical role in cell death from ischemia/reperfusion (I/R) injury experimentally in the late 1980's. Thereafter, tremendous efforts have been made to define the molecular composition of mPTP and underlying mechanisms of its opening. mPTP opening, so far, has been demonstrated with the conformational changes of the mitochondrial protein components including cyclophilin-D, adenine nucleotide translocase, and voltage-dependent anion channel, which were induced by the modification of the levels of Ca2+, phosphate, mitochondrial membrane potential, intracellular pH and adenine nucleotide. At present, genetic modulation of the expression of protein components are being used in the investigation of its properties, presenting novel mechanisms of mPTP opening, including phosphate carrier. For therapeutic intervention, cyclosporin A and its analogues were first to be demonstrated to inhibit the opening of mPTP, affecting cyclophilin-D. There are numerous pharmacological substances that have direct or indirect effects on mPTP opening, including bongkrekic acid, reactive oxygen species scavengers, calcium channel blockers, and Na+/H+ exchanger-1 inhibitors, but only cyclosporin A was clinically tried to limit the myocardial infarction. Conditioning interventions, ischemic or anesthetic, have also been shown to be effective in limiting the detrimental effects of I/R injury. These interventions are commonly related to specific receptors on cell membrane and then signal transduction pathway consisting of many protein kinases, which eventually lead to mitochondria. And being presented are experimental evidences that inhibition of mPTP opening is a primary mechanism of these conditioning interventions. In conclusion, mPTP opening is now presented as primary mechanism and therapeutic target of I/R injury, but precise mechanism and standardized treatment method are needed to be clarified.

Characteristics of adenine nucleotide translocator in mitochondria of rat cerebral cortex during hypobaric hypoxia exposure / 生理学报

The purpose of the present study was to explore the effects of hypoxic exposure on mitochondrial adenine nucleotide translocator (ANT) activity and its characteristics. Male Wistar rats were exposed to hypoxia in a hypobaric chamber simulating high altitude at 5 000 m for 1, 5, 15 and 30 d. Control rats were fed outside the hypobaric chamber. Rats were sacrificed by decapitation and mitochondria from the cerebral cortex were isolated by differential centrifugation at each time point. The ANT activity was detected by the atractyloside (ATR)-inhibitor stop technique. Mitochondria was initiated by addition of (3)H-ADP and terminated after 12 s by quick addition of ATR. The radioactivity was measured in a liquid scintillation counter. Nonspecific binding of (3)H-ADP to mitochondria was estimated by incubation of mitochondrial samples with ATR prior to the addition of (3)H-ADP. This blank was substracted from the measured radioactivities. The activity of ANT was expressed as nanomoles (3)H-ADP per minute per milligram protein. The ANT density was determined by titrating the rate of state 3 respiration with increasing concentrations of carboxyatractyloside (CAT). Mitochondria were pre-incubated with CAT in a respiratory medium before ADP addition to initiate state 3 respiration. Plots of O2 consumption versus CAT appeared biphasic with an increasing inhibitory segment followed by a steady respiration, indicating that state 3 respiration was completely inhibited. The density of ANT was determined by the amount of CAT required to completely inhibit state 3 respiration, assuming a 1:1 binding stoichiometry, which was expressed as ANT density per milligram mitochondria protein. (ATP+ADP) in mitochondria was measured by high performance liquid chromatography (HPLC). The results showed that there was an obvious decrease in the ANT activity during hypoxic exposure. The lowest ANT activity was seen in 5 d group. Partial recovery of ANT activity was observed in 15 and 30 d groups, but ANT activity was still lower than that in the control group (P<0.01). Compared with that in normoxic control group, no change of ANT density in mitochondria was observed in hypoxia group (P>0.05). The turnover number of ANT in control, 1, 5, 15, 30 d groups were 16.67, 1.90, 0.40, 1.81, 4.73 pmol ADP/(min.pmol ANT), respectively. However, (ATP+ADP) in mitochondria in 1, 5, 15, 30 d groups were 63.37%, 48.44%, 52.73%, 60.16% of control group respectively. Therefore, the turnover number of energy production and expenditure were reduced. These observations suggest that the change of ANT activity may be one of the mechanisms of cellular oxidative phosphorylation dysfunction during hypoxic exposure.

Possible association of HLA-DRB1 gene with the autoantibody against myocardial mitochondria ADP/ATP carrier in dilated cardiomyopathy / 华中科技大学学报（医学）（英德文版）

To probe the genetic background and immunopathogenesis of dilated cardiomyopathy (DCM) 77 patients with DCM, HLA-DRB1 gene polymorphism were analyzed by using the polymerase chain reaction/sequence specific primer (PCR/SSP) technique and autoantibody against myocardial mitochondria ADP/ATP carrier were examined by using the Immunoblot analysis. The frequency of HLA-DRB1*0901 allele was significantly higher in DCM patients in which autoantibody against ADP/ATP carrier of myocardial mitochondria is positive in contrast with those in which the autoantibody is negative (25.46% vs 3.45%, P < 0.05), the relative risk (RR) being 9.56. The other frequencies of HLA-DRB1 alleles have no significant difference in the antibody positive group and negative group. It is possible that a subset of DCM patients may exist in which autoimmunity is associated with genetic factors.

Regulation of histamine secretion in a mast cell line

La interacción del alergeno con su lg-E específica anclada en la superficie externa de la membrana plasmática del mastocito desencadena la liberación de histamina de dicha célula. Este proceso requiere la presencia de calcio extracelular y de energía bajo la forma de ATP proveniente fundamentalmente de glicólisis donde el lactato es el producto final. La heterogeneidad de los mastocitos ha sido previamente descrita en diversos tejidos de diferentes especies animales. Así, esta línea celular tumoral en ratones LAF1 mostró propiedades similares a las descritas para los mastocitos presentes en la cavidad peritoneal de la rata. Las respuestas metabólicas relacionadas a la secreción de histamina también fueron estudiadas en esta línea de mastocinoma, que exhibieron un comportamiento diferente dependiendo del secretagogo utilizado. En este sentido, una fuerte estimulación de la glicólisis fue observada la presencia de Concanavalina-A en comparación con la estimulación inducida por el A-23187 y el compuesto 48/80. En este modelo experimental se logró establecer relaciones entre la liberación de histamina con el consumo de glucosa, la produción de lactato y la carga adenílica bajo la forma de ATP