RESUMO
Animals maintain the ability to survive and reproduce by acclimating to environmental temperatures. We showed here that Caenorhabditis elegans exhibited temperature acclimation plasticity, which was regulated by a head-tail-head neural circuitry coupled with gut fat storage. After experiencing cold, C. elegans individuals memorized the experience and were prepared against subsequent cold stimuli. The cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) regulated temperature acclimation in the ASJ thermosensory neurons and RMG head interneurons, where it modulated ASJ thermosensitivity in response to past cultivation temperature. The PVQ tail interneurons mediated the communication between ASJ and RMG via glutamatergic signaling. Temperature acclimation occurred via gut fat storage regulation by the triglyceride lipase ATGL-1, which was activated by a neuropeptide, FLP-7, downstream of CREB. Thus, a head-tail-head neural circuit coordinated with gut fat influenced experience-dependent temperature acclimation.
Assuntos
Aclimatação , Tecido Adiposo , Caenorhabditis elegans , Temperatura Baixa , Sistema Digestório , Cabeça , Vias Neurais , Cauda , Aclimatação/fisiologia , Tecido Adiposo/metabolismo , Animais , Caenorhabditis elegans/anatomia & histologia , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Sistema Digestório/metabolismo , Ácido Glutâmico/metabolismo , Cabeça/inervação , Interneurônios/metabolismo , Lipase/metabolismo , Neuropeptídeos/metabolismo , Cauda/inervação , Sensação TérmicaRESUMO
RATIONALE: Visceral adipose tissue-derived serine proteinase inhibitor (vaspin) is an adipokine identified from visceral adipose tissues of genetically obese rats. OBJECTIVE: The role of vaspin in the diabetic vascular complications remains elusive, and we investigated the effects of vaspin on the vascular function under the diabetic milieu. METHODS AND RESULTS: Adenovirus carrying the full length of the vaspin gene (Vaspin-Ad) ameliorated intimal proliferation of balloon-injured carotid arteries in diabetic Wistar rats. The expression of Ccl2, Pdgfb, and Pdgfrb genes was significantly reduced by the treatment of Vaspin-Ad. In cuff-injured femoral arteries, the intimal proliferation was ameliorated in vaspin transgenic (Vaspin Tg) mice. The application of recombinant vaspin and Vaspin-Ad promoted the proliferation and inhibited the apoptosis of human aortic endothelial cells. Adenovirus expressing vaspin with calmodulin and streptavidin-binding peptides was applied to human aortic endothelial cells, subjected to tandem tag purification and liquid chromatography-tandem mass spectrometry, and we identified GRP78 (78-kDa glucose-regulated protein) as an interacting molecule. The complex formation of vaspin, GRP78, and voltage-dependent anion channel on the plasma membrane was confirmed by the immunoprecipitation studies using aortas of Vaspin Tg mice. The binding assay using (125)I-vaspin in human aortic endothelial cells revealed high-affinity binding (dissociation constant = 0.565×10(-9) m) by the treatment of 5 µM thapsigargin, which recruited GRP78 from the endoplasmic reticulum to plasma membrane by inducing endoplasmic reticulum stress. In human aortic endothelial cells, vaspin induced phosphorylation of Akt and inhibited the kringle 5-induced Ca(2+) influx and subsequent apoptosis. CONCLUSIONS: Vaspin is a novel ligand for the cell-surface GRP78/voltage-dependent anion channel complex in endothelial cells and promotes proliferation, inhibits apoptosis, and protects vascular injuries in diabetes mellitus.
Assuntos
Adipocinas/metabolismo , Apoptose/fisiologia , Endotélio Vascular/patologia , Proteínas de Choque Térmico/metabolismo , Serpinas/metabolismo , Canais de Ânion Dependentes de Voltagem/metabolismo , Adenoviridae/genética , Adipocinas/genética , Animais , Membrana Celular/metabolismo , Proliferação de Células , Células Cultivadas , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Modelos Animais de Doenças , Chaperona BiP do Retículo Endoplasmático , Humanos , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ratos , Ratos Wistar , Serpinas/genética , Estreptozocina/efeitos adversosRESUMO
An 81-year-old Japanese male with primary Sjögren syndrome (pSS) developed a low-grade fever and productive cough which were refractory to antibiotic therapy. Based on the high level of eosinophils observed in his bronchial alveolar lavage, he was diagnosed with chronic eosinophilic pneumonia (CEP) and successfully treated by oral prednisolone. Interstitial lung diseases associated with pSS (pSS-ILDs) usually present as nonspecific interstitial pneumonia or usual interstitial pneumonia; therefore, the present case is extremely unique in that the patient's condition was complicated with CEP. A diagnosis of advanced gallbladder cancer was made in the patient's clinical course, suggesting the advisability of a whole-body workup in cases of pSS, especially in elderly patients.
Assuntos
Eosinofilia Pulmonar/diagnóstico , Eosinofilia Pulmonar/etiologia , Síndrome de Sjogren/complicações , Administração Oral , Corticosteroides/administração & dosagem , Corticosteroides/uso terapêutico , Idoso de 80 Anos ou mais , Doença Crônica , Humanos , Masculino , Prednisolona/administração & dosagem , Prednisolona/uso terapêutico , Eosinofilia Pulmonar/tratamento farmacológico , Resultado do TratamentoRESUMO
INTRODUCTION: ACE cleaves angiotensin I (Ang I) to angiotensin II (Ang II) inducing vasoconstriction via Ang II type 1 (AT1) receptor, while ACE2 cleaves Ang II to Ang (1-7) causing vasodilatation by acting on the Mas receptor. In diabetic kidney disease (DKD), it is still unclear whether plasma or urine ACE2 levels predict renal outcomes or not. RESEARCH DESIGN AND METHODS: Among 777 participants with diabetes enrolled in the Urinary biomarker for Continuous And Rapid progression of diabetic nEphropathy study, the 296 patients followed up for 9 years were investigated. Plasma and urinary ACE2 levels were measured by the ELISA. The primary end point was a composite of a decrease of estimated glomerular filtration rate (eGFR) by at least 30% from baseline or initiation of hemodialysis or peritoneal dialysis. The secondary end points were a 30% increase or a 30% decrease in albumin-to-creatinine ratio from baseline to 1 year. RESULTS: The cumulative incidence of the renal composite outcome was significantly higher in group 1 with lowest tertile of plasma ACE2 (p=0.040). Group 2 with middle and highest tertile was associated with better renal outcomes in the crude Cox regression model adjusted by age and sex (HR 0.56, 95% CI 0.31 to 0.99, p=0.047). Plasma ACE2 levels demonstrated a significant association with 30% decrease in ACR (OR 1.46, 95% CI 1.044 to 2.035, p=0.027) after adjusting for age, sex, systolic blood pressure, hemoglobin A1c, and eGFR. CONCLUSIONS: Higher baseline plasma ACE2 levels in DKD were protective for development and progression of albuminuria and associated with fewer renal end points, suggesting plasma ACE2 may be used as a prognosis marker of DKD. TRIAL REGISTRATION NUMBER: UMIN000011525.
Assuntos
Enzima de Conversão de Angiotensina 2 , Biomarcadores , Nefropatias Diabéticas , Taxa de Filtração Glomerular , Peptidil Dipeptidase A , Humanos , Masculino , Feminino , Nefropatias Diabéticas/sangue , Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/diagnóstico , Enzima de Conversão de Angiotensina 2/sangue , Biomarcadores/sangue , Pessoa de Meia-Idade , Peptidil Dipeptidase A/sangue , Idoso , Prognóstico , Progressão da Doença , SeguimentosRESUMO
The peroxisome proliferator activated receptor-γ (PPARγ) agonist, pioglitazone (PIO), exerts anti-diabetic properties associated with increased fat mass, whereas the retinoid X receptor (RXR) antagonist HX531 demonstrates anti-obesity and anti-diabetic effects with reduced body weight and fat pad mass. The cell cycle abnormality in adipocytes has not been well-investigated in obesity or during treatment with modulators of nuclear receptors. We therefore investigated cell size and cell cycle distributions of adipocytes in vivo and examined the expression of cell cycle regulators in cultured human visceral preadipocytes. The cell size distribution and cell cycle analyses of in vivo adipocytes derived from OLETF rats demonstrated that HX531 brought about G0/G1 cell cycle arrest associated with the inhibition of cellular hypertrophy, which resulted in the reduction of fat pad mass. In contrast, PIO promoted proliferation activities associated with the increase in M + late M:G0 + G1 ratio and the appearance of both small and hypertrophied adipocytes. In cultured human visceral preadipocytes HX531 up-regulated cell cycle regulators, p53, p21(Cip1), cyclin D1, Fbxw7 and Skp2, which are known contributors towards G0 /G1 cell cycle arrest. The knockdown of p53 with a shRNA lentivirus reversed the HX531-induced up-regulation of p21(Cip1), which is one of the major p53-effector molecules. We conclude that HX531 exerts anti-obesity and anti-diabetes properties by up-regulating the p53-p21(Cip1) pathway, resulting in G0/G1 cell cycle arrest and the inhibition of cellular hypertrophy of adipocytes.
Assuntos
Adipócitos/efeitos dos fármacos , Fármacos Antiobesidade/farmacologia , Benzoatos/farmacologia , Compostos de Bifenilo/farmacologia , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Hipoglicemiantes/farmacologia , Obesidade/tratamento farmacológico , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Receptores X de Retinoides/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo , Adipócitos/metabolismo , Adipócitos/patologia , Animais , Proliferação de Células/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Células Cultivadas , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Modelos Animais de Doenças , Humanos , Hipertrofia , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Masculino , Obesidade/genética , Obesidade/metabolismo , Obesidade/patologia , PPAR gama/efeitos dos fármacos , PPAR gama/metabolismo , Pioglitazona , Interferência de RNA , Ratos , Ratos Endogâmicos OLETF , Receptores X de Retinoides/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tiazolidinedionas/farmacologia , Fatores de Tempo , Transfecção , Proteína Supressora de Tumor p53/genética , Regulação para CimaRESUMO
BACKGROUND: Galectin-9 (Gal-9) induces apoptosis in activated T helper 1 (TH1) cells as a ligand for T cell immunoglobulin mucin-3 (Tim-3). Gal-9 also inhibits the G1 phase cell cycle arrest and hypertrophy in db/db mice, the hallmark of early diabetic nephropathy, by reversing the high glucose-induced up-regulation of cyclin dependent kinase inhibitors such as p27(Kip1) and p21(Cip1). METHODS: We investigated the serum levels of Gal-9 in the patients with type 2 diabetes and various stages of chronic kidney disease (CKD) (n=182). RESULTS: Serum Gal-9 levels in the patients with type 2 diabetes were 131.9 ± 105.4 pg/ml and Log(10)Gal-9 levels significantly and positively correlated with age (r=0.227, p=0.002), creatinine (r=0.175, p=0.018), urea nitrogen (r=0.162, p=0.028) and osmotic pressure (r=0.187, p=0.014) and negatively correlated with estimated glomerular filtration rate (eGFR) (r=-0.188, p=0.011). Log(10)Gal-9 levels increased along with the progression of GFR categories of G1 to G4, and they were statistically significant by Jonckheere-Terpstra test (p=0.012). Log(10)Gal-9 levels remained similar levels in albuminuria stages of A1 to A3. CONCLUSION: The elevation of serum Gal-9 in the patients with type 2 diabetes is closely linked to GFR and they may be related to the alteration of the immune response and inflammation of the patients with type 2 diabetes and CKD.
Assuntos
Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/epidemiologia , Galectinas/sangue , Insuficiência Renal Crônica/sangue , Insuficiência Renal Crônica/epidemiologia , Biomarcadores/sangue , Comorbidade , Diabetes Mellitus Tipo 2/diagnóstico , Feminino , Humanos , Incidência , Japão/epidemiologia , Masculino , Pessoa de Meia-Idade , Insuficiência Renal Crônica/diagnóstico , Reprodutibilidade dos Testes , Medição de Risco , Sensibilidade e EspecificidadeRESUMO
Extracellular vesicles (EVs) have gained considerable attention as vital circulating biomarkers since their structure and composition resemble the originating cells. The investigation of EVs' biochemical and biophysical properties is of great importance to map them to their parental cells and to better understand their functionalities. In this study, a novel frequency-dependent impedance measurement system has been developed to characterize EVs based on their unique dielectric properties. The system is composed of an insulator-based dielectrophoretic (iDEP) device to entrap and immobilize a cluster of vesicles followed by utilizing electrical impedance spectroscopy (EIS) to measure their impedance at a wide frequency spectrum, aiming to analyze both their membrane and cytosolic charge-dependent contents. The EIS was initially utilized to detect nano-size vesicles with different biochemical compositions, including liposomes synthesized with different lipid compositions, as well as EVs and lipoproteins with similar biophysical properties but dissimilar biochemical properties. Moreover, EVs derived from the same parental cells but treated with different culture conditions were characterized to investigate the correlation of impedance changes with biochemical properties and functionality in terms of pro-inflammatory responses. The system also showed the ability to discriminate between EVs derived from different cellular origins as well as among size-sorted EVs harbored from the same cellular origin. This proof-of-concept approach is the first step towards utilizing EIS as a label-free, non-invasive, and rapid sensor for detection and characterization of pathogenic EVs and other nanovesicles in the future.
Assuntos
Espectroscopia Dielétrica , Vesículas Extracelulares , Impedância ElétricaRESUMO
BACKGROUND: Glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1) plays a crucial role in lipolytic processing. Previous studies have shown that GPIHBP1 mutations cause severe hypertriglyceridemia and that serum GPIHBP1 levels are marginally higher in patients with coronary heart disease; however, the role of GPIHBP1 in type 2 diabetes mellitus (T2DM) remains unknown. OBJECTIVE: We investigated the association between circulating GPIHBP1 levels and the prevalence of microvascular complications in T2DM. METHODS: A total of 237 subjects with T2DM and 235 non-diabetic control subjects were enrolled in this study. Their serum GPIHBP1 levels were evaluated using ELISA assays. RESULTS: Circulating GPIHBP1 levels were higher in patients with T2DM (952.7 pg/mL [761.3-1234.6], p < 0.0001) than in non-diabetic subjects (700.6 [570.8-829.6]), but did not differ in T2DM patients with or without hypertriglyceridemia. Serum GPIHBP1 levels were significantly higher in patients with T2DM with diabetic retinopathy (DR), diabetic nephropathy (DN), and microvascular complications than in those without these complications. Multivariable logistic regression and receiver operating characteristic (ROC) curve analyses revealed that the presence of microvascular complications, but not macrovascular complications, was independently associated with serum GPIHBP1 levels, which could predict the presence of diabetic microvascular complications. CONCLUSIONS: Elevated GPIHBP1 levels are associated with microvascular complications in T2DM and may help to predict their progression.
Assuntos
Diabetes Mellitus Tipo 2 , Nefropatias Diabéticas , Retinopatia Diabética , Hipertrigliceridemia , Receptores de Lipoproteínas , Estudos Transversais , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/epidemiologia , Nefropatias Diabéticas/complicações , Feminino , Humanos , Hipertrigliceridemia/complicações , Masculino , Receptores de Lipoproteínas/genéticaRESUMO
Argonaute 2 (Ago2) is the main component of the RNA-induced silencing complex. We recently showed that liver-specific Ago2-deficiency in mice (L-Ago2 knockout [KO] mice) enhances mitochondrial oxidation and alleviates obesity-associated pathophysiology. However, the precise mechanisms behind the role of hepatic Ago2 in regulating the mitochondrial oxidation associated with glucose metabolism are still unclear. Here, we show that hepatic Ago2 regulates the function of peroxisome proliferator-activated receptor α (PPARα) for oxidative metabolism. In both genetically and diet-induced severe obese conditions, L-Ago2 KO mice developed obesity and hepatic steatosis but exhibited improved glucose metabolism accompanied by lowered expression levels of pathologic microRNAs (miRNAs), including miR-802, miR-103/107, and miR-152, and enhanced expression of PPARα and its target genes regulating oxidative metabolism in the liver. We then investigated the role of hepatic Ago2 in the outcomes of vertical sleeve gastrectomy (VSG) in which PPARα plays a crucial role in a drastic transcription reprogram associated with improved glycemia post VSG. Whereas VSG reduced body weight and improved fatty liver in wild-type mice, these effects were not observed in hepatic Ago2-deficient mice. Conversely, glucose metabolism was improved in a hepatic Ago2-dependent manner post VSG. Treating Ago2-deficient primary hepatocytes with WY-14643, a PPARα agonist, showed that Ago2-deficiency enhances sensitivity to WY-14643 and increases expression of PPARα target genes and mitochondrial oxidation. Our findings suggest that hepatic Ago2 function is intrinsically associated with PPARα that links Ago2-mediated RNA silencing with mitochondrial functions for oxidation and obesity-associated pathophysiology.
Assuntos
Proteínas Argonautas/deficiência , Fígado/metabolismo , Obesidade/metabolismo , Obesidade/cirurgia , PPAR alfa/metabolismo , Animais , Proteínas Argonautas/genética , Cirurgia Bariátrica , Glucose/metabolismo , Teste de Tolerância a Glucose , Controle Glicêmico , Hepatócitos/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/tratamento farmacológico , Obesidade/genética , Estresse Oxidativo , PPAR alfa/genética , Pirimidinas/administração & dosagemRESUMO
Background: Although various biomarkers predict cardiovascular event (CVE) in patients with diabetes, the relationship of urinary glycan profile with CVE in patients with diabetes remains unclear. Methods: Among 680 patients with type 2 diabetes, we examined the baseline urinary glycan signals binding to 45 lectins with different specificities. Primary outcome was defined as CVE including cardiovascular disease, stroke, and peripheral arterial disease. Results: During approximately a 5-year follow-up period, 62 patients reached the endpoint. Cox proportional hazards analysis revealed that urinary glycan signals binding to two lectins were significantly associated with the outcome after adjustment for known indicators of CVE and for false discovery rate, as well as increased model fitness. Hazard ratios for these lectins (+1 SD for the glycan index) were UDA (recognizing glycan: mixture of Man5 to Man9): 1.78 (95% CI: 1.24-2.55, P = 0.002) and Calsepa [High-Man (Man2-6)]: 1.56 (1.19-2.04, P = 0.001). Common glycan binding to these lectins was high-mannose type of N-glycans. Moreover, adding glycan index for UDA to a model including known confounders improved the outcome prediction [Difference of Harrel's C-index: 0.028 (95% CI: 0.001-0.055, P = 0.044), net reclassification improvement at 5-year risk increased by 0.368 (0.045-0.692, P = 0.026), and the Akaike information criterion and Bayesian information criterion decreased from 725.7 to 716.5, and 761.8 to 757.2, respectively]. Conclusion: The urinary excretion of high-mannose glycan may be a valuable biomarker for improving prediction of CVE in patients with type 2 diabetes, and provides the rationale to explore the mechanism underlying abnormal N-glycosylation occurring in patients with diabetes at higher risk of CVE. Trial Registration: This study was registered with the University Hospital Medical Information Network on June 26, 2012 (Clinical trial number: UMIN000011525, URL: https://upload.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000013482).
RESUMO
BACKGROUND: Collectrin, a homologue of angiotensin converting enzyme 2, is expressed in pancreatic beta cells and renal proximal tubular and collecting duct cells under the control of hepatocyte nuclear factors-1alpha and -1beta. Because collectrin interacts with the soluble N-ethylmaleiamide-sensitive factor attachment protein receptor (SNARE) complexes, we investigated whether collectrin is involved in sodium handling in hypertension by vesicle trafficking of apical membrane proteins. METHODS AND RESULTS: Collectrin physically interacts with the SNARE complex: snapin, synaptosomal-associated protein 23 kDa, syntaxin-4, and vesicle-associated membrane protein-2 in mIMCD-3 cells. siRNA knockdown of collectrin resulted in a reduction in membrane-associated aquaporin-2, alpha-epithelial Na+ channel, and H+-ATPase. Collectrin and SNARE proteins were abundantly expressed in collecting ducts of Wistar-Kyoto rats. Wistar-Kyoto rats and spontaneously hypertensive rats 7 weeks of age were subjected to normal-salt (1% NaCl) and high-salt (8% NaCl) chow for 10 weeks. High-salt chow prominently elevated blood pressure, oral intake, and urinary excretion of NaCl and water in both groups. Although urinary excretion of aldosterone was significantly suppressed in both groups, collectrin expression was upregulated and associated with the maintenance of aquaporin-2, alpha-epithelial Na+ channel, and H+-ATPase in membrane fractions. Collectrin promoter activities and mRNA and protein expressions were upregulated and ubiquitinated collectrin was reduced by high NaCl (175 to 225 mmol/L) and not altered by 1 micromol/L aldosterone in mIMCD-3 cells. CONCLUSIONS: Upregulation of collectrin by high NaCl independent of aldosterone functionally links to the trafficking of apical membrane proteins via the SNARE complex, and collectrin may be responsible for the sodium retention in salt-sensitive hypertension.
Assuntos
Colectinas/biossíntese , Hipertensão/induzido quimicamente , Complexos Multiproteicos/metabolismo , Proteínas SNARE/metabolismo , Cloreto de Sódio/toxicidade , Regulação para Cima/efeitos dos fármacos , Aldosterona/urina , Animais , Aquaporina 2/metabolismo , Pressão Sanguínea/efeitos dos fármacos , Linhagem Celular , Canais Epiteliais de Sódio , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Fator 1-beta Nuclear de Hepatócito/metabolismo , Hipertensão/metabolismo , Células Secretoras de Insulina/metabolismo , Túbulos Renais Coletores/metabolismo , Túbulos Renais Proximais/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Proteínas Qa-SNARE/metabolismo , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Proteína 2 Associada à Membrana da Vesícula/metabolismoRESUMO
Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for systemic energy homeostasis. Primary hepatocytes isolated from the murine liver constitute a valuable biological tool to understand the functional properties or alterations occurring in the liver. Herein we describe a method for the isolation and culture of primary mouse hepatocytes by performing a two-step collagenase perfusion technique and discuss their utilization for investigating protein metabolism. The liver of an adult mouse is sequentially perfused with ethylene glycol-bis tetraacetic acid (EGTA) and collagenase, followed by the isolation of hepatocytes with the density gradient buffer. These isolated hepatocytes are viable on culture plates and maintain the majority of endowed characteristics of hepatocytes. These hepatocytes can be used for assessments of protein metabolism including nascent protein synthesis with non-radioactive reagents. We show that the isolated hepatocytes are readily controlled and comprise a higher quality and volume stability of protein synthesis linked to energy metabolism by utilizing the chemo-selective ligation reaction with a Tetramethylrhodamine (TAMRA) protein detection method and western blotting analyses. Therefore, this method is valuable for investigating hepatic nascent protein synthesis linked to energy homeostasis. The following protocol outlines the materials and methods for the isolation of high-quality primary mouse hepatocytes and detection of nascent protein synthesis.
Assuntos
Alanina/análogos & derivados , Hepatócitos/metabolismo , Proteínas/metabolismo , Alanina/metabolismo , Animais , Células Cultivadas , Hepatócitos/citologia , CamundongosRESUMO
RNA silencing inhibits mRNA translation. While mRNA translation accounts for the majority of cellular energy expenditure, it is unclear if RNA silencing regulates energy homeostasis. Here, we report that hepatic Argonaute 2 (Ago2)-mediated RNA silencing regulates both intrinsic energy production and consumption and disturbs energy metabolism in the pathogenesis of obesity. Ago2 regulates expression of specific miRNAs including miR-802, miR-103/107, and miR-148a/152, causing metabolic disruption, while simultaneously suppressing the expression of genes regulating glucose and lipid metabolism, including Hnf1ß, Cav1, and Ampka1. Liver-specific Ago2-deletion enhances mitochondrial oxidation and ATP consumption associated with mRNA translation, which results in AMPK activation, and improves obesity-associated pathophysiology. Notably, hepatic Ago2-deficiency improves glucose metabolism in conditions of insulin receptor antagonist treatment, high-fat diet challenge, and hepatic AMPKα1-deletion. The regulation of energy metabolism by Ago2 provides a novel paradigm in which RNA silencing plays an integral role in determining basal metabolic activity in obesity-associated sequelae.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Argonautas/metabolismo , Obesidade/enzimologia , Interferência de RNA , Animais , Dieta Hiperlipídica , Fatores de Iniciação em Eucariotos/metabolismo , Deleção de Genes , Genótipo , Glucose/metabolismo , Teste de Tolerância a Glucose , Glicólise , Humanos , Hiperglicemia/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/metabolismo , Mitocôndrias/metabolismo , Oxigênio/metabolismo , Ácido Pirúvico/metabolismoRESUMO
Coxsackie virus and adenovirus receptor-like membrane protein (CLMP) was identified as the tight junction-associated transmembrane protein of epithelial cells with homophilic binding activities. CLMP is also recognized as adipocyte adhesion molecule (ACAM), and it is upregulated in mature adipocytes in rodents and humans with obesity. Here, we present that aP2 promoter-driven ACAM transgenic mice are protected from obesity and diabetes with the prominent reduction of adipose tissue mass and smaller size of adipocytes. ACAM is abundantly expressed on plasma membrane of mature adipocytes and associated with formation of phalloidin-positive polymerized form of cortical actin (F-actin). By electron microscopy, the structure of zonula adherens with an intercellular space of â¼10-20 nm was observed with strict parallelism of the adjoining cell membranes over distances of 1-20 µm, where ACAM and γ-actin are abundantly expressed. The formation of zonula adherens may increase the mechanical strength, inhibit the adipocyte hypertrophy, and improve the insulin sensitivity.
Assuntos
Citoesqueleto de Actina/metabolismo , Junções Aderentes/metabolismo , Adipócitos Brancos/metabolismo , Adiposidade , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus/metabolismo , Obesidade/prevenção & controle , Regulação para Cima , Células 3T3-L1 , Citoesqueleto de Actina/patologia , Citoesqueleto de Actina/ultraestrutura , Junções Aderentes/patologia , Junções Aderentes/ultraestrutura , Adipócitos Brancos/citologia , Adipócitos Brancos/patologia , Adipócitos Brancos/ultraestrutura , Animais , Adesão Celular , Tamanho Celular , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus/genética , Diabetes Mellitus/etiologia , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Diabetes Mellitus/prevenção & controle , Dieta Hiperlipídica/efeitos adversos , Sacarose Alimentar/efeitos adversos , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Microscopia Imunoeletrônica , Obesidade/etiologia , Obesidade/metabolismo , Obesidade/patologia , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Although obesity is undoubtedly major risk for non-alcoholic steatohepatitis (NASH), the presence of lean NASH patients with normal body mass index has been recognized. Here, we report that the insufficiency of phosphatidylethanolamine N-methyltransferase (PEMT) is a risk for the lean NASH. The Pemt-/- mice fed high fat-high sucrose (HFHS) diet were protected from diet-induced obesity and diabetes, while they demonstrated prominent steatohepatitis and developed multiple liver tumors. Pemt exerted inhibitory effects on p53-driven transcription by forming the complex with clathrin heavy chain and p53, and Pemt-/- mice fed HFHS diet demonstrated prominent apoptosis of hepatocytes. Furthermore, hypermethylation and suppressed mRNA expression of F-box protein 31 and hepatocyte nuclear factor 4α resulted in the prominent activation of cyclin D1. PEMT mRNA expression in liver tissues of NASH patients was significantly lower than those with simple steatosis and we postulated the distinct clinical entity of lean NASH with insufficiency of PEMT activities.
Assuntos
Hepatócitos/citologia , Hepatopatia Gordurosa não Alcoólica/patologia , Fosfatidiletanolamina N-Metiltransferase/genética , Fosfatidiletanolamina N-Metiltransferase/metabolismo , Animais , Apoptose , Células Cultivadas , Cadeias Pesadas de Clatrina/metabolismo , Dieta Hiperlipídica , Modelos Animais de Doenças , Regulação para Baixo , Técnicas de Silenciamento de Genes , Predisposição Genética para Doença , Humanos , Camundongos , Hepatopatia Gordurosa não Alcoólica/enzimologia , Hepatopatia Gordurosa não Alcoólica/genética , Obesidade/prevenção & controle , Proteína Supressora de Tumor p53/metabolismoRESUMO
We report a 55-year-old woman with ectopic adrenocorticotropin (ACTH) secretion caused by extra-adrenal pheochromocytoma. The patient presented with a 6-month history of hypertension and diabetes mellitus. Her serum and urinary cortisol levels were extremely high and dexamethasone failed to suppress the cortisol secretion. Her plasma ACTH levels were also elevated (>300 pg/mL) and irresponsive to corticotropin-releasing hormone (CRH) or metyrapone administration. Gel filtration analysis of the patient's plasma detected the existence of large molecular weight ACTH being eluted with a major peak of authentic 1-39 ACTH. Abdominal computed tomographic scan and magnetic resonance imaging revealed a 5-cm paraganglioma located underneath the left kidney, in which (123)I-MIBG tracer specifically accumulated. Bilateral adrenal glands were diffusely enlarged. After surgical removal of the paraganglioma, the patient's clinical symptoms improved and biochemistry normalized including plasma ACTH, urinary free cortisol, and urinary catecholamines. Subsequent histologic evaluation of the transected paranglioma tissue revealed ACTH, synaptin, and chromogranin-A histologically immunostaining. Culture of primary cells collected from the resected paraganglioma demonstrated of in vitro production of ACTH, noradrenaline, and adrenaline. This is the first report of ectopic ACTH syndrome induced by an extra-adrenal abdominal paraganglioma.
Assuntos
Síndrome de ACTH Ectópico/etiologia , Neoplasias das Glândulas Suprarrenais/complicações , Feocromocitoma/complicações , Síndrome de ACTH Ectópico/diagnóstico , Neoplasias das Glândulas Suprarrenais/diagnóstico , Hormônio Adrenocorticotrópico/sangue , Catecolaminas/urina , Feminino , Humanos , Hidrocortisona/sangue , Hidrocortisona/urina , Pessoa de Meia-Idade , Paraganglioma , Feocromocitoma/diagnóstico , Radiografia Abdominal , Tomografia Computadorizada por Raios XRESUMO
PURPOSE: The unique circulating microRNAs (miRNAs) observed in patients with type 2 diabetes (T2D) are candidates as new biomarkers and therapeutic targets. In order to identify circulating miRNAs relevant to the disease process in case of type 2 diabetes, we performed the Illumina sequencing of miRNAs derived from the serum, liver and epididymal white adipose tissue (WAT) of diet-induced obese male C57BL/6J mice. BASIC PROCEDURES: We selected four miRNAs, miR-101, miR-335, miR-375, and miR-802, which are increased in the sera and tissues of obese mice, and measured the serum levels of miRNAs in T2D and subjects with normal glucose tolerance (NGT). MAIN FINDINGS: The serum concentrations of miRNAs, log(10)miR-101, log(10)miR-375, and log(10)miR-802, were significantly increased in the T2D patients compared with NGT subjects (1.41±2.01 v.s. -0.57±1.05 (P=1.36×10(-5)), 0.20±0.58 v.s. 0.038±1.00 (P=3.06×10(-6)), and 2.45±1.27 v.s. 0.97±0.98 (P=0.014), respectively). The log(10)miR-335 values did not demonstrate any significant differences between the T2D and NGT groups (-1.08±1.35 v.s. -0.38±1.21 (P=0.25)). According to the stepwise regression analysis, the HbA1c was an independent predictor of miR-101. Regarding the serum miR-802 levels, eGFR, HbA1c and HDL-C values were identified as significant determinants. PRINCIPAL CONCLUSIONS: The present findings demonstrated that the circulating miR-101, miR-375 and miR-802 levels are significantly increased in T2D patients versus NGT subjects and they may become the new biomarkers for type 2 diabetes.
Assuntos
Biomarcadores/sangue , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/diagnóstico , MicroRNAs/sangue , Adulto , Idoso , Animais , Glicemia/metabolismo , Estudos Transversais , Feminino , Teste de Tolerância a Glucose , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-IdadeRESUMO
OBJECTIVE: In obesity and type 2 diabetes, the impairment of mitochondrial function in white adipose tissue (WAT) is linked to a reduction in whole body insulin sensitivity. Timm44 is upregulated in the kidneys of streptozotocin-induced diabetic mice. In the inner mitochondrial membrane, Timm44 anchors mitochondrial heat-shock protein 70 (mtHsp70) to the translocase of inner mitochondrial membrane 23 (TIM23) complex and facilitates the import of mitochondria-targeted preproteins into the mitochondrial matrix dependent on the inner membrane potential and ATP hydrolysis on ATPase domain of mtHsp70. METHODS: We generated the aP2-promoter driven Timm44 transgenic (Tg) mouse model and investigated whether Timm44 Tg mice fed high-fat/high-sucrose (HFHS) chow are protected from type 2 diabetes and obesity. RESULTS: The body weight of aP2-promoter driven Timm44 Tg mice was lower than that of wild type mice, and insulin sensitivity was greater in Timm44 Tg mice than in wild type mice. Although WAT weight was not altered in Timm44 Tg mice fed HFHS chow, adipocyte size was reduced, and mitochondrial fusion associated with decreased expression of fission genes, such as Dnm1l and Fis1, was observed. In addition, when fed standard (STD) chow, the expressions of the fusion genes Opa1, Mfn1 and Mfn2, and Mfn1 were significantly increased in Timm44 Tg mice compared to wild type mice, and fused mitochondria were also observed in Timm44 Tg mice fed STD chow. CONCLUSIONS: The Timm44 gene may be a new target for the treatment of type 2 diabetes.
Assuntos
Proteínas de Transporte/metabolismo , Diabetes Mellitus Tipo 2/enzimologia , Proteínas de Membrana/metabolismo , Membranas Mitocondriais/enzimologia , Proteínas Mitocondriais/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Adipócitos , Animais , Proteínas de Transporte/genética , Diferenciação Celular , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/prevenção & controle , Diabetes Mellitus Tipo 2/genética , Fibroblastos/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Resistência à Insulina , Potencial da Membrana Mitocondrial , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas de Transporte da Membrana Mitocondrial , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Mitocondriais/genéticaRESUMO
Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease worldwide. Gpnmb is classified as a type 1 membrane protein and its soluble form is secreted by ADAM10-mediated cleavage. Gpnmb mRNA was found in the Kupffer cells and white adipose tissues (WATs) and its upregulation in obesity was recently found. Here, we generated aP2 promoter-driven Gpnmb transgenic (Tg) mice and the overexpression of Gpnmb ameliorated the fat accumulation and fibrosis of the liver in diet-induced obesity model. Soluble form of Gpnmb in sera was elevated in Gpnmb Tg mice and Gpnmb concentrated in hepatic macrophages and stellate cells interacted with calnexin, which resulted in the reduction of oxidative stress. In the patients with non-alcoholic steatohepatitis, serum soluble GPNMB concentrations were higher compared with the patients with simple steatosis. The GPNMB is a promising biomarker and therapeutic target for the development and progression of NAFLD in obesity.
Assuntos
Proteínas do Olho/metabolismo , Glicoproteínas de Membrana/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Células 3T3-L1 , Adipócitos/metabolismo , Adipócitos/patologia , Tecido Adiposo Branco/metabolismo , Animais , Biomarcadores/metabolismo , Calnexina/metabolismo , Proteínas do Olho/sangue , Proteínas do Olho/genética , Feminino , Regulação da Expressão Gênica , Células Estreladas do Fígado/metabolismo , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/complicações , Cirrose Hepática/patologia , Modelos Logísticos , Macrófagos/metabolismo , Masculino , Glicoproteínas de Membrana/sangue , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Análise Multivariada , Obesidade/complicações , Obesidade/patologia , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Endogâmicos OLETF , Fatores de RiscoRESUMO
Phosphatidylethanolamine N-methyltransferase (Pemt) catalyzes the methylation of phosphatidylethanolamine (PE) to phosphatidylcholine (PC) mainly in the liver. Under an obese state, the upregulation of Pemt induces endoplasmic reticulum (ER) stress by increasing the PC/PE ratio in the liver. We targeted the Pemt gene in mice to explore the therapeutic impact of Pemt on the progression of diabetic nephropathy and diabetes, which was induced by the injection of streptozotocin (STZ). Although the blood glucose levels were similar in STZ-induced diabetic Pemt+/+ and Pemt-/-mice, the glomerular hypertrophy and albuminuria in Pemt-/- mice were significantly reduced. Pemt deficiency reduced the intraglomerular F4/80-positive macrophages, hydroethidine fluorescence, tubulointerstitial fibrosis and tubular atrophy. The expression of glucose-regulated protein-78 (GRP78) was enriched in the renal tubular cells in STZ-induced diabetic mice, and this was ameliorated by Pemt deficiency. In mProx24 renal proximal tubular cells, the treatment with ER-stress inducers, tunicamycin and thapsigargin, increased the expression of GRP78, which was reduced by transfection of a shRNA lentivirus for Pemt (shRNA-Pemt). The number of apoptotic cells in the renal tubules was significantly reduced in Pemt-/- diabetic mice, and shRNA-Pemt upregulated the phosphorylation of Akt and decreased the cleavage of caspase 3 and 7 in mProx24 cells. Taken together, these findings indicate that the inhibition of Pemt activity ameliorates the ER stress associated with diabetic nephropathy in a model of type 1 diabetes and corrects the functions of the three major pathways downstream of ER stress, i.e. oxidative stress, inflammation and apoptosis.