RESUMO
mTORC1 is known to activate sterol regulatory element-binding proteins (SREBPs) including SREBP-2, a master regulator of cholesterol synthesis. Through incompletely understood mechanisms, activated mTORC1 triggers translocation of SREBP-2, an endoplasmic reticulum (ER) resident protein, to the Golgi where SREBP-2 is cleaved to translocate to the nucleus and activate gene expression for cholesterol synthesis. Low ER cholesterol is a well-established trigger for SREBP-2 activation. We thus investigated whether mTORC1 activates SREBP-2 by reducing cholesterol delivery to the ER. We report here that mTORC1 activation is accompanied by low ER cholesterol and an increase of SREBP-2 activation. Conversely, a decrease in mTORC1 activity coincides with a rise in ER cholesterol and a decrease in SERBP-2 activity. This rise in ER cholesterol is of lysosomal origin: blocking the exit of cholesterol from lysosomes by U18666A or NPC1 siRNA prevents ER cholesterol from increasing and, consequently, SREBP-2 is activated without mTORC1 activation. Furthermore, when mTORC1 activity is low, cholesterol is delivered to lysosomes through two membrane trafficking pathways: autophagy and rerouting of endosomes to lysosomes. Indeed, with dual blockade of both pathways by Atg5-/- and dominant-negative rab5, ER cholesterol fails to increase when mTORC1 activity is low, and SREBP-2 is activated. Conversely, overexpressing constitutively active Atg7, which forces autophagy and raises ER cholesterol even when mTORC1 activity is high, suppresses SREBP-2 activation. We conclude that mTORC1 actively suppresses autophagy and maintains endosomal recycling, thereby preventing endosomes and autophagosomes from reaching lysosomes. This results in a reduction of cholesterol in the ER and activation of SREBP-2.
Assuntos
Autofagossomos/fisiologia , Colesterol/metabolismo , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Células HEK293 , HumanosRESUMO
When recombinant human (rh) thyroid-stimulating hormone (TSH) is administered to thyroid cancer survivors, an acute extra-thyroidal effect raises pro-inflammatory cytokines and activates platelets. Thymic stromal lymphopoietin (TSLP) is a cytokine recently implicated in platelet activation. Our aim was to measure platelet microparticle levels after rhTSH stimulation in vivo, and to investigate TSLP expression in TSH-stimulated human adipocytes in culture. Blood samples for total and platelet microparticle analysis were obtained from thyroid cancer survivors before (day 1) and after rhTSH administration (day 5). Adipocytes, differentiated from stromal preadipocytes isolated from adipose tissue from surgical patients, were stimulated with TSH. TSLP mRNA expression, protein expression, and protein release into the adipocyte medium were measured. The level of platelet microparticles in thyroid cancer patients rose 5-fold after rhTSH stimulation. TSH upregulated TSLP mRNA expression in adipocytes in culture through a pathway that was inhibited by 66% by H89, a protein kinase A inhibitor. TSLP protein expression rose in response to TSH, and TSH-stimulated TSLP release into the medium was completely blocked by dexamethasone. In conclusion, TSLP is a novel TSH-responsive adipokine. Future studies will be needed to address the potential role of adipocyte-derived TSLP and whether it is linked to TSH-dependent platelet activation.
Assuntos
Adipócitos/metabolismo , Plaquetas/metabolismo , Citocinas/metabolismo , Ativação Plaquetária , Neoplasias da Glândula Tireoide/metabolismo , Tireotropina/farmacologia , Adipócitos/efeitos dos fármacos , Adipócitos/patologia , Células Cultivadas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias da Glândula Tireoide/tratamento farmacológico , Neoplasias da Glândula Tireoide/patologia , Linfopoietina do Estroma do TimoRESUMO
OBJECTIVES: Thyroid-stimulating hormone (TSH) acts in an extra-thyroidal fashion and induces a pro-inflammatory, pro-coagulant state. Blood monocytes can be activated by vascular stress, but it is not known if this occurs upon TSH administration. Our aim was to determine if recombinant human (rh) TSH, administered acutely to patients being screened for thyroid cancer recurrence, alters blood monocyte gene expression. DESIGN AND SETTING: Patients (14 women, 1 man) had a mean (±SD) age of 48±10 years, a body mass index of 26±6 kg/m2, a history of total thyroidectomy and radioablation for thyroid cancer, and were on L-thyroxine therapy at a university teaching hospital. They received 2 intramuscular doses of rhTSH (0.9 mg), administered on days 1 and 2. Blood samples were obtained at baseline on day1, and on days 3 and 5. RESULTS: Monocyte MCP-1 mRNA (mean±SE) increased significantly by 1.7±0.3 fold on day 5 following rhTSH stimulation (p=0.03, n=15). IL-1ß and CD36 mRNA expression also increased on day 5 (1.9±0.4 fold, p=0.07, n=14) and 2.5±0.4 fold, p=0.1, n=10), respectively, although did not quite reach statistical significance. Significant correlations were detected between the BMI of patients and their TSH-stimulated monocyte mRNA responses at day 5 for CD11a, (r=0.66, n=14, p=0.01); CD14 (r=0.638, n=13, p=0.019), and CD16, r=0.84, n=13, p=0.0003). CONCLUSION: TSH administration increases pro-atherogenic monocyte gene expression.
Assuntos
Monócitos/metabolismo , Neoplasias da Glândula Tireoide/diagnóstico , Tireotropina/administração & dosagem , Feminino , Humanos , Radioisótopos do Iodo , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/diagnóstico , Proteínas Recombinantes , TiroxinaRESUMO
PURPOSE: Fetuin-A is a hepatokine that is linked to lipid metabolism, obesity, insulin resistance, type 2 diabetes and cardiovascular disease. Elevated thyroid-stimulating hormone (TSH) levels are associated with metabolic and cardiovascular disturbances. Our aim was to determine if TSH can regulate fetuin-A levels. METHODS: Fetuin-A serum levels were examined in 26 subjects (19 women; previous thyroidectomy and radioactive iodine ablation) undergoing recombinant human TSH (rhTSH) stimulation to screen for thyroid cancer recurrence. Their age was 49±10 years, and body mass index (BMI) was 28±6 (both expressed as mean±SD). The patients received two doses of rhTSH (0.9 mg), administered 24 hours apart on days 1 and 2, without discontinuation of ongoing L-thyroxine therapy. Morning blood samples were obtained on days 1 (prior to the first dose of rhTSH), 3 and 5. RESULTS: The baseline value of fetuin-A (mean±SD) for all participants was 527±186 mg/L. Values of fetuin-A did not change in response to rhTSH administration. The lack of response was not dependent on gender, age, baseline free thyroxine level or BMI. CONCLUSION: Fetuin-A has been implicated in metabolic and inflammatory conditions, but there have been no reports on whether fetuin-A is influenced by TSH. Within the context of rhTSH administration for surveillance of thyroid cancer recurrence, there was no effect on serum levels of fetuin-A.
Assuntos
Recidiva Local de Neoplasia/sangue , Neoplasias da Glândula Tireoide/sangue , Tirotropina Alfa/administração & dosagem , alfa-2-Glicoproteína-HS/metabolismo , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/prevenção & controle , Neoplasias da Glândula Tireoide/terapia , Tiroxina/administração & dosagemRESUMO
Obesity is associated with adipose tissue remodeling, characterized by macrophage accumulation, adipocyte hypertrophy, and apoptosis. We previously reported that macrophage-conditioned medium (MacCM) protects preadipocytes from apoptosis, due to serum withdrawal, in a platelet-derived growth factor (PDGF)-dependent manner. We have now investigated the role of intracellular signaling pathways, activated in response to MacCM versus PDGF, in promoting preadipocyte survival. Exposure of 3T3-L1 preadipocytes to J774A.1-MacCM or PDGF strongly stimulated Akt and ERK1/2 phosphorylation from initially undetectable levels. Inhibition of the upstream regulators of Akt or ERK1/2, i.e. phosphoinositide 3-kinase (PI3K; using wortmannin or LY294002) or MEK1/2 (using UO126 or PD98509), abrogated the respective phosphorylation responses, and significantly impaired pro-survival activity. J774A.1-MacCM increased reactive oxygen species (ROS) levels by 3.4-fold, and diphenyleneiodonium (DPI) or N-acetyl cysteine (NAC) significantly inhibited pro-survival signaling and preadipocyte survival in response to J774A.1-MacCM. Serum withdrawal itself also increased ROS levels (2.1-fold), and the associated cell death was attenuated by DPI or NAC. In summary, J774A.1-MacCM-dependent 3T3-L1 preadipocyte survival requires the Akt and ERK1/2 signaling pathways. Furthermore, ROS generation by J774A.1-MacCM is required for Akt and ERK1/2 signaling to promote 3T3-L1 preadipocyte survival. These data suggest potential mechanisms by which macrophages may alter preadipocyte fate.
Assuntos
Adipócitos/citologia , Sobrevivência Celular , Macrófagos/fisiologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Células 3T3-L1 , Animais , Camundongos , Fosforilação/efeitos dos fármacosRESUMO
Macrophage infiltration into adipose tissue, associated with obesity, is thought to contribute to abnormal adipose tissue remodeling, low-grade inflammation, and insulin resistance. Medium conditioned by macrophages (MacCM) inhibits 3T3-L1 and human adipocyte differentiation, as well as early adipogenic cell cycle events including MCE and retinoblastoma protein (Rb) phosphorylation. Our objective was to determine if the inhibition of Rb phosphorylation was linked to changes in cell cycle-related proteins. We treated 3T3-L1 preadipocytes with adipogenic inducers for 24 h in control medium versus J774A.1-MacCM. The differentiation-induced mRNA and protein expression of cyclin A, an activator of cyclin-dependent kinase (cdk) 2 which phosphorylates Rb, was inhibited by 82% and 73%, respectively, by J774A.1-MacCM; adipogenic expression of Myc, a transcriptional regulator of cyclin A, was also suppressed significantly. Consistent with the reduction in cyclin A levels, the activation of cdk2 by adipogenic inducers was inhibited by 75% by J774A.1-MacCM. J774A.1-MacCM also lowered levels of cyclins D1 and D2. Inhibition studies demonstrated that platelet-derived growth factor, an anti-adipogenic factor found in J774A.1-MacCM, was not responsible for the inhibitory effect on differentiation. The anti-adipogenic effect of J774A.1-MacCM was resistant to proteinase K and heat treatment, and was present in a <3 kDa fraction. Our data indicate that J774A.1-MacCM interferes with the upregulation of cyclin A levels and cdk2 activity that are required for Rb phosphorylation and MCE in 3T3-L1 adipogenesis.
Assuntos
Adipócitos/efeitos dos fármacos , Adipócitos/enzimologia , Adipogenia/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Quinase 2 Dependente de Ciclina/metabolismo , Macrófagos/metabolismo , Células 3T3-L1 , Adipócitos/citologia , Animais , Ciclina A/metabolismo , Ciclina D/metabolismo , Endopeptidase K/metabolismo , Ativação Enzimática/efeitos dos fármacos , Temperatura Alta , Humanos , Macrófagos/efeitos dos fármacos , Camundongos , Fosforilação/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteína do Retinoblastoma/metabolismo , Transdução de Sinais/efeitos dos fármacosAssuntos
Micropartículas Derivadas de Células , Coagulantes/sangue , Tireotropina/sangue , Adenocarcinoma Folicular/sangue , Adenocarcinoma Folicular/radioterapia , Adenocarcinoma Folicular/cirurgia , Adulto , Idoso , Índice de Massa Corporal , Carcinoma/sangue , Carcinoma/radioterapia , Carcinoma/cirurgia , Carcinoma Papilar , Células Endoteliais/citologia , Feminino , Citometria de Fluxo , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Recombinantes/química , Análise de Regressão , Câncer Papilífero da Tireoide , Neoplasias da Glândula Tireoide/sangue , Neoplasias da Glândula Tireoide/radioterapia , Neoplasias da Glândula Tireoide/cirurgiaRESUMO
This study examines the mechanisms underlying the anti-adipogenic effect of macrophage-secreted products. 3T3-L1 preadipocytes were induced to differentiate over 8 days in medium conditioned by murine J774 macrophages (MacCM). The inhibitory effect on lipid accumulation and expression of adipogenic markers was diminished when addition of MacCM was delayed to day 2 of differentiation. Clonal expansion, an early event required for 3T3-L1 adipogenesis, was reduced in the presence of MacCM (89%; n=3; p<0.001), and BrdU incorporation was impaired by 55% (n=3; p<0.01). Activation of ERK1/2 was not affected by MacCM, and neither was the expression of p27(kip1), a cyclin-dependent kinase inhibitor. However, phosphorylation of the retinoblastoma protein (Rb), required for cell cycle progression, was impaired by MacCM (94% inhibition; n=3; p<0.01). Differentiation-dependent expression, nuclear localization, and DNA binding ability of C/EBPbeta were not inhibited by MacCM. Alterations in cell cycle-associated proteins may be important with respect to the anti-adipogenic action of MacCM.
Assuntos
Adipócitos/metabolismo , Diferenciação Celular/fisiologia , Macrófagos/metabolismo , Proteína do Retinoblastoma/metabolismo , Células 3T3-L1 , Adipócitos/citologia , Adipogenia , Animais , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Ciclo Celular , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Núcleo Celular/metabolismo , Meios de Cultivo Condicionados , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Metabolismo dos Lipídeos , Camundongos , FosforilaçãoRESUMO
Inadequate proliferation and/or differentiation of preadipocytes may lead to adipose tissue dysfunction characterized by hypertrophied, insulin-resistant adipocytes. Platelet-derived growth factor (PDGF) may alter adipose tissue function by promoting proliferation of preadipocytes. Two principal signaling pathways that regulate proliferation are PI3K/PI(3,4,5)P3/Akt and Shc/Ras/ERK1/2. SH2 domain-containing inositol 5-phosphatase 2 (SHIP2) dephosphorylates PI(3,4,5)P3, and also binds to Shc. Our goal was to determine how SHIP2 affects these PDGF signaling routes. To assess the role of the 5-phosphatase domain, we expressed wild-type or catalytically inactive dominant-negative SHIP2 (P686A-D690A-R691A; PDR/AAA) in 3T3-L1 preadipocytes. Surprisingly, SHIP2 PDR/AAA inhibited proliferation more potently than wild-type SHIP2. After three days of proliferation, phospho-Akt, phospho-ERK1/2, and PDGF receptor (PDGFR) levels were reduced in PDR/AAA-expressing preadipocytes. SHIP2 PDR/AAA interference with PDGFR signaling was demonstrated using imatinib, an inhibitor of PDGFR tyrosine kinase. The anti-proliferative effect of imatinib observed in control preadipocytes was not significant in SHIP2 PDR/AAA-expressing preadipocytes, indicating a pre-existing impairment of PDGFR-dependent mitogenesis in these cells. The inhibition of PDGF-activated mitogenic pathways by SHIP2 PDR/AAA was consistent with a decrease in PDGFR phosphorylation caused by a drop in receptor levels in SHIP2 PDR/AAA-expressing cells. SHIP2 PDR/AAA promoted ubiquitination of the PDGFR and its degradation via the lysosomal pathway independently of the association between the E3 ubiquitin ligase c-Cbl and PDGFR. Overall, our findings indicate that SHIP2 PDR/AAA reduces preadipocyte proliferation by attenuating PDGFR signaling.
Assuntos
Adipócitos/citologia , Adipócitos/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Substituição de Aminoácidos , Animais , Sequência de Bases , Benzamidas , Diferenciação Celular , Proliferação de Células/efeitos dos fármacos , Primers do DNA/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Mesilato de Imatinib , Inositol Polifosfato 5-Fosfatases , Camundongos , Mutagênese Sítio-Dirigida , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Monoéster Fosfórico Hidrolases/genética , Fosforilação , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Pirimidinas/farmacologia , Receptores do Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , UbiquitinaçãoRESUMO
OBJECTIVES: Obesity and type 2 diabetes often coexist. The effect of hyperglycemia on adipose tissue is, therefore, of interest. Although studies have shown that high glucose (HG) concentrations do not inhibit adipocyte differentiation, the resulting adipocyte phenotype has not been investigated. In particular, the levels of the glucose-responsive transcription factor carbohydrate-responsive response element binding protein (ChREBP) isoforms have not been assessed. METHODS: Human preadipocytes were differentiated into adipocytes in either normal glucose (NG) or HG conditions. RNA and protein analyses were used to measure the expression of ChREBP isoforms, thioredoxin interacting protein (TXNIP) and lipogenic genes. Insulin-stimulated glucose uptake was measured. RESULTS: HG- vs. NG-differentiated adipocytes expressed more ChREBPß and more TXNIP at the mRNA and protein levels. There was no change in lipogenic gene expression. HG- vs. NG-differentiated adipocytes displayed an inhibition of insulin-stimulated glucose uptake. CONCLUSIONS: HG-differentiated human adipocytes have distinct molecular differences and are insulin resistant. More studies are warranted to investigate potential mechanisms linking changes in ChREBPß and TXNIP to insulin responsiveness.
Assuntos
Adipócitos/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Proteínas de Transporte/metabolismo , Glucose/metabolismo , Resistência à Insulina , Adipócitos/citologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Glicemia , Proteínas de Transporte/genética , Diferenciação Celular , Células Cultivadas , Humanos , Fenótipo , Regulação para CimaRESUMO
Our objective was to identify the signaling pathway activated by TSH that induces IL-6 secretion from human abdominal sc differentiated adipocytes. Human abdominal sc preadipocytes in culture were differentiated into adipocytes. IL-6 release stimulated by TSH was inhibited by 35% (P < 0.05) with SN50, an inhibitor of nuclear factor-kappaB (NF-kappaB) nuclear translocation, and 60% (P < 0.01) with sc-514, an inhibitor of inhibitory-kappaB (IkappaB) kinase (IKK)-beta. Phosphorylation of IKKbeta increased upon TSH treatment (10.3-fold, P < 0.01), and IkappaBalpha levels were reduced by 78% (P < 0.01). TSH activated NF-kappaB (23-fold, P < 0.001), a process that was inhibited (60%, P < 0.01) by SN50. Inhibition of protein kinase A by H89 did not affect TSH-stimulated IKKbeta phosphorylation or IkappaBalpha degradation. TSH-mediated NF-kappaB activation and IL-6 induction also specifically occurred in Chinese hamster ovarian cells expressing the human TSH receptor, resulting in a 5.9-fold (P < 0.001) increase in IKKbeta phosphorylation and a 9.5-fold increase in IL-6 mRNA expression. Our data demonstrate that the IKKbeta/NF-kappaB pathway is a novel TSH target that is required for TSH-induced IL-6 release from human adipocytes.
Assuntos
Adipócitos/metabolismo , Interleucina-6/metabolismo , NF-kappa B/metabolismo , Tireotropina/farmacologia , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Animais , Células CHO , Técnicas de Cultura de Células , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Feminino , Humanos , Cinética , Reação em Cadeia da PolimeraseRESUMO
The proatherogenic state of obesity is associated with hypertrophied adipocytes that may arise because of deficient adipogenesis. Macrophages infiltrate adipose tissue as a function of obesity and may release factors that attenuate adipogenesis. Macrophage-conditioned medium inhibits human and 3T3-L1 adipocyte differentiation in culture, but underlying molecular mechanisms have yet to be defined. Exposure of 3T3-L1 cells throughout the 8-day period of differentiation to medium conditioned by THP-1 macrophages (THP-1-MacCM) blocked adipogenesis. Triacylglycerol (TG) accumulation and induction of peroxisome proliferator-activated receptor gamma and fatty acid synthase protein levels were inhibited by 59% (n = 4, P < .001), 29% (n = 4, P < .01), and 47% (n = 4, P < .01), respectively. THP-1-MacCM had no effect when added after the first 2 days of differentiation, indicating that early exposure of its targets must be needed to inhibit 3T3-L1 adipogenesis. Cell enumeration revealed a 44% decrease in clonal expansion compared with standard differentiation (n = 3, P < .01). Addition of THP-1-MacCM to 3T3-L1 preadipocytes increased ERK1/2 phosphorylation by 6.5-fold (n = 3, P < .01). PD98059 (an inhibitor of the ERK1/2 pathway) impaired the negative effect of THP-1-MacCM on TG accumulation, indicated by an inhibition of 25% vs 69% (n = 3, P < .001), without altering fatty acid synthase or peroxisome proliferator-activated receptor gamma levels. Our data implicate ERK1/2 as an important signaling mediator for the inhibitory effect of THP-1-MacCM on TG accumulation during 3T3-L1 adipogenesis.
Assuntos
Adipogenia , Macrófagos/fisiologia , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Células 3T3-L1 , Adipócitos/citologia , Animais , Diferenciação Celular , Meios de Cultivo Condicionados , Ativação Enzimática , Humanos , Camundongos , Fosforilação , Triglicerídeos/metabolismoRESUMO
Adipogenesis is an important component of adipose tissue development and growth. Alterations in adipogenesis may promote adipose tissue insulin resistance and inflammation. The ability of preadipocytes to differentiate into mature adipocytes depends on the activation of phosphoinositide 3-kinase (PI3K). This chapter describes the methodology used to measure the cellular accumulation of phosphoinositide products of PI3K. This approach involves labeling the cells with myo-[2-3H]-inositol, extraction and deacylation of the phosphoinositides, and HPLC separation of the deacylated derivatives. The assay of PI3K activity itself is also described in detail. The ability to analyze PI3K and its phosphoinositide products is a useful tool for ongoing endeavours to understand adipogenesis and adipose tissue dysfunction.
Assuntos
Adipócitos/metabolismo , Adipogenia/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/fisiologia , Adipócitos/citologia , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Animais , Humanos , Lipídeos/química , Camundongos , Fosfatidilinositóis/metabolismo , Ensaio Radioligante/métodosRESUMO
Surgical resection of a gastrointestinal stromal cell tumor by double enterectomy and partial pancreatectomy on a 13-year-old mixed breed dog. A 13 year-old, male, mixed breed dog, has been presented for an abdominal mass. The exam showed the presence of an ileo-caeco-colic mass adhered to the distal portion of the pancreas and the mid duodenum. A double enterectomy and a partial pancreatectomy were carried out and a diagnosis of gastrointestinal stromal cell tumor has been established.
Assuntos
Doenças do Cão/cirurgia , Neoplasias Gastrointestinais/veterinária , Leiomiossarcoma/veterinária , Pancreaticoduodenectomia/veterinária , Animais , Procedimentos Cirúrgicos do Sistema Digestório/métodos , Procedimentos Cirúrgicos do Sistema Digestório/veterinária , Cães , Neoplasias Duodenais/cirurgia , Neoplasias Duodenais/veterinária , Neoplasias Gastrointestinais/cirurgia , Leiomiossarcoma/cirurgia , Masculino , Neoplasias Pancreáticas/cirurgia , Neoplasias Pancreáticas/veterinária , Pancreaticoduodenectomia/métodos , Células Estromais , Resultado do TratamentoRESUMO
OBJECTIVES: Adipose tissue expands via differentiation of preadipocytes into adipocytes (adipogenesis) and/or hypertrophy of existing adipocytes. A low adipogenic capacity promotes adipocyte hypertrophy, causing inflammatory macrophage accumulation and insulin resistance. Macrophage-conditioned medium (MacCM) inhibits adipogenesis and promotes adipocyte inflammation, but it is unknown whether these effects are altered by high glucose (HG) versus normal glucose (NG) concentrations. Our aim was to compare the effect of HG-MacCM versus NG-MacCM on human adipogenesis and adipocyte inflammation. METHODS: Human monocyte-derived macrophages (MDMs) were placed in 5 mmol/L (NG) or 25 mmol/L (HG) glucose for 24 hours. MacCM was collected and its effect on differentiation of human subcutaneous abdominal preadipocytes and adipocyte inflammation was evaluated. RESULTS: HG-MacCM, but not NG-MacCM, inhibited triacylglycerol (TG) accumulation and protein expression of peroxisome proliferator-activated receptor γ (PPARgamma) during human adipogenesis. Preadipocytes differentiated in HG-MacCM displayed a more pro-inflammatory phenotype, as assessed by increased interleukin-6 and monocyte chemotactic protein-1 (MCP-1), as well as reduced adiponectin mRNA expression. In MDMs, HG increased phosphorylation of inhibitor of kappaB kinase (IKK)-beta and decreased protein expression of inhibitor of kappaB alpha. HG also reduced protein expression of PPARgamma in MDMs. However, no MDM changes in mRNA expression of MCP-1, interleukin-1beta or tumor necrosis factor-alpha were detected. The stimulatory effect of HG-MacCM on MCP-1 expression in adipocytes was partially inhibited when MDMs were treated with sc-514 (IKKbeta inhibitor). CONCLUSIONS: High glucose concentration accentuates the anti-adipogenic and pro-inflammatory effects of MacCM on human preadipocytes.
Assuntos
Adipócitos/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Glucose/farmacologia , Adipócitos/citologia , Adipócitos/metabolismo , Adipogenia/imunologia , Adulto , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultivo Condicionados , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Humanos , Macrófagos/fisiologia , Masculino , Pessoa de Meia-IdadeRESUMO
OBJECTIVE: To determine the effect of (1) an oral fat load and (2) pro-protein convertase subtilisin/kexin type (PCSK) 9 loss-of-function (LOF) variant status on the ability of peripheral blood mononuclear cells (PBMC) to inhibit human adipogenesis. METHODS: PBMC from subjects with one or more PCSK9 LOF variants versus non-variant controls were compared in the fasting state and after an oral fat load. RESULTS: Fasting triglyceride (TG) levels were lower in the LOF variant versus non-variant group but rose to the same level after the oral fat load. Conditioned medium from PBMC was obtained in fasting (PBMC-CM-F) and 4-h postprandial (PBMC-CM-PP) states. PBMC-CM-PP from non-variant controls inhibited adipogenesis of human preadipocytes more than did PBMC-CM-F. In contrast, PBMC-CM-F or -PP from PCSK9 LOF variant subjects had no effect on adipogenesis. After the oral fat load, PBMC from PCSK9 LOF variant subjects showed significant increases in mRNA levels of interleukin-1ß, tumor necrosis factor-α, sterol regulatory element binding protein-1c, CD36, and monocyte chemoattractant protein-1 (MCP-1), only MCP-1 mRNA levels increased in PBMC from non-variant controls. CONCLUSIONS: The absence of anti-adipogenic action of PBMC from PCSK9 LOF variant subjects points to a novel role for PCSK9 in PBMC-adipose cell interactions.
Assuntos
Adipogenia/genética , Variação Genética , Leucócitos Mononucleares/metabolismo , Pró-Proteína Convertase 9/genética , Idoso , Antígenos CD36/metabolismo , Estudos de Casos e Controles , Quimiocina CCL2/metabolismo , Meios de Cultivo Condicionados , Jejum/metabolismo , Feminino , Humanos , Interleucina-1beta/metabolismo , Masculino , Pessoa de Meia-Idade , Período Pós-Prandial/genética , RNA Mensageiro/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Triglicerídeos/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The mammalian target of rapamycin (mTOR) pathway has recently emerged as a chronic modulator of insulin-mediated glucose metabolism. In this study, we evaluated the involvement of this pathway in the acute regulation of insulin action in both 3T3-L1 and human adipocytes. Insulin rapidly (t(1/2) = 5 min) stimulated the mTOR pathway, as reflected by a 10-fold stimulation of 70-kDa ribosomal S6 kinase 1 (S6K1) activity in 3T3-L1 adipocytes. Inhibition of mTOR/S6K1 by rapamycin increased insulin-stimulated glucose transport by as much as 45% in 3T3-L1 adipocytes. Activation of mTOR/S6K1 by insulin was associated with a rapamycin-sensitive increase in Ser636/639 phosphorylation of insulin receptor substrate (IRS)-1 but, surprisingly, did not result in impaired IRS-1-associated phosphatidylinositol (PI) 3-kinase activity. However, insulin-induced activation of Akt was increased by rapamycin. Insulin also activated S6K1 and increased phosphorylation of IRS-1 on Ser636/639 in human adipocytes. As in murine cells, rapamycin treatment of human adipocytes inhibited S6K1, blunted Ser636/639 phosphorylation of IRS-1, leading to increased Akt activation and glucose uptake by insulin. Further studies in 3T3-L1 adipocytes revealed that rapamycin prevented the relocalization of IRS-1 from the low-density membranes to the cytosol in response to insulin. Furthermore, inhibition of mTOR markedly potentiated the ability of insulin to increase PI 3,4,5-triphosphate levels concomitantly with an increased phosphorylation of Akt at the plasma membrane, low-density membranes, and cytosol. However, neither GLUT4 nor GLUT1 translocation induced by insulin were increased by rapamycin treatment. Taken together, these results indicate that the mTOR pathway is an important modulator of the signals involved in the acute regulation of insulin-stimulated glucose transport in 3T3-L1 and human adipocytes.
Assuntos
Células 3T3-L1/metabolismo , Adipócitos/citologia , Glucose/metabolismo , Insulina/metabolismo , Proteínas Quinases/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , 3-O-Metilglucose/metabolismo , Adipócitos/metabolismo , Animais , Diferenciação Celular , Membrana Celular/metabolismo , Desoxiglucose/metabolismo , Regulação da Expressão Gênica , Transportador de Glucose Tipo 1 , Transportador de Glucose Tipo 4 , Humanos , Immunoblotting , Imunoprecipitação , Proteínas Substratos do Receptor de Insulina , Cinética , Camundongos , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas Musculares/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Proteínas Quinases/metabolismo , Transporte Proteico , Proteínas Proto-Oncogênicas c-akt , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Serina/química , Transdução de Sinais , Sirolimo/farmacologia , Frações Subcelulares , Serina-Treonina Quinases TOR , Fatores de TempoRESUMO
Aortic carboxypeptidase-like protein (ACLP) is a 175-kDa protein that is expressed in vascular smooth muscle cells and contains a signal peptide sequence, a lysine- and proline-rich repeating motif, a discoidin-like domain with 35% identity to discoidin I, and a carboxypeptidase-like domain that is 39% identical with carboxypeptidase E. It is secreted into the extracellular matrix and may play a role in abdominal wall development and dermal wound healing. ACLP is also expressed in adipose tissue, but at lower levels. In this study we demonstrate that ACLP protein and mRNA are severely down-regulated in the early phase of 3T3-L1 preadipocyte differentiation induced by insulin, dexamethasone, and isobutylmethylxanthine. Neither dexamethasone, isobutylmethylxanthine, nor insulin treatment alone reduced the level of ACLP protein, suggesting that ACLP down-regulation is a differentiation-associated event. ACLP down-regulation coincided with the onset of the postconfluent mitotic clonal expansion phase of adipogenesis. In contrast, subconfluent 3T3-L1 cell proliferation did not alter ACLP expression, suggesting a specific linkage between ACLP and differentiation-induced clonal expansion. Stable overexpression of ACLP had no effect on preadipocyte differentiation assessed by triacylglycerol accumulation and peroxisome proliferator-activated receptor-gamma levels. The role of ACLP and its marked reduction during adipogenesis merit further study.
Assuntos
Tecido Adiposo/crescimento & desenvolvimento , Regulação para Baixo/genética , Biossíntese de Proteínas , 1-Metil-3-Isobutilxantina/farmacologia , Células 3T3 , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Adipócitos/fisiologia , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Northern Blotting , Carboxipeptidases , Contagem de Células , Diferenciação Celular/fisiologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Dexametasona/farmacologia , Regulação para Baixo/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Camundongos , Mitose/efeitos dos fármacos , Proteínas/genética , Proteínas Repressoras , Retroviridae/genética , Células-Tronco/fisiologia , Triglicerídeos/metabolismoRESUMO
Thyroid-associated ophthalmopathy, a process in which the orbital tissues become inflamed and are remodeled, occurs with a variable presentation. In some patients, eye muscle enlargement predominates. In others, the connective/adipose tissue enlargement appears the more significant problem. Orbital fibroblasts exhibit heterogeneous phenotypes in culture. Here we report that fibroblasts derived from the connective/adipose tissue depot are distinct from those investing the extraocular muscles. Connective tissue fibroblasts represent a bimodal population of cells with regard to the surface display of the glycoprotein, Thy-1. Perimysial fibroblasts in contrast express Thy-1 uniformly. In that regard, they resemble those from the skin. When subjected to a newly defined set of culture conditions, adipocyte differentiation occurs in up to 43% of the cells. All adipocytes examined failed to display Thy-1. Fibroblasts derived from perimysium and dermis uniformly do not differentiate into adipocytes when incubated under identical culture conditions. Both Thy-1(+) and Thy-1(-) connective tissue fibroblasts express the adipogenic trigger, peroxisome proliferator activator gamma, suggesting that differences in the potential for differentiation may reside with phenotypic attributes downstream from this receptor/adipogenic transcription factor. These observations enhance our understanding of orbital adipogenesis and define previously unrecognized differences between fibroblasts from the extraocular muscle and connective tissue.
Assuntos
Doença de Graves/patologia , Órbita/patologia , Tiazolidinedionas , Adipócitos/metabolismo , Adipócitos/patologia , Diferenciação Celular , Células Cultivadas , Tecido Conjuntivo/metabolismo , Tecido Conjuntivo/patologia , Fibroblastos/imunologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Fenótipo , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Citoplasmáticos e Nucleares/análise , Receptores Citoplasmáticos e Nucleares/biossíntese , Rosiglitazona , Tiazóis/farmacologia , Antígenos Thy-1/análise , Fatores de Transcrição/agonistas , Fatores de Transcrição/análise , Fatores de Transcrição/biossínteseRESUMO
Differentiation of murine 3T3-L1 preadipocytes into adipocytes is associated with the acquisition of apoptotic resistance accompanied by the upregulation of cell survival genes. We have now examined the effect of adipogenesis on apoptotic susceptibility of human abdominal preadipocytes in primary culture. To induce apoptosis, human preadipocytes, or their differentiated counterparts, were serum-deprived for 24 or 48 hours. When indicated, ceramide was also used as an apoptotic trigger. Cell death was assessed by enumeration of adherent viable cells, and its apoptotic nature was verified by Hoechst staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). After 48 hours of serum withdrawal, cell death was 26% +/- 4% in preadipocytes and was increased to 41% +/- 4% in differentiated adipocytes (mean +/- SE; n = 7 patients; P <.002). Under serum-free conditions for 24 hours, ceramide-induced cell death was 40% +/- 6% in preadipocytes and increased to 68% +/- 8% in adipocytes (mean +/- SE; P <.01; n = 8 patients). Neuronal apoptosis inhibitor protein (NAIP), an antiapoptotic protein cell survival that increases upon 3T3-L1 adipogenesis, was reduced in human preadipocytes undergoing differentiation (n = 6 patients). Preadipocytes derived from omental versus subcutaneous abdominal fat were more susceptible to apoptosis induced by serum deprivation, 16% +/- 4% versus 31% +/- 3% cell death, respectively (mean +/- SE; P <.02; n = 7 patients). Although the murine 3T3-L1 preadipocyte cell line is a useful model that approximates primary preadipocyte cell biology, our data derived from human preadipocyte studies suggest important differences with respect to the regulation of apoptosis.