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1.
Cells ; 10(3)2021 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-33668197

RESUMO

The bioactivity of the IGF system is not a function of isolated hormone concentrations in a given biological matrix. Instead, the biological activities of IGFs are regulated by IGFBPs, IGFBP proteases, and inhibitors of IGFBP proteases. Therefore, assays based on IGF-related bioactivity may describe functions of the complete IGF system in a given biological matrix. Of particular interest are the IGF system effects on the AKT/mTOR pathway, as a dominant system for controlling growth, metabolism, and aging. In order to improve the sensitivity of IGF-dependent bioactivity, we made use of the known short-term and enhancing effects of IGFBP2 on the intracellular PI3K pathway. As a specific readout of this pathway, and further as a marker of the mTOR pathway, we assessed the phosphorylation of AKT-Ser473. Preincubation using IGFBP2 enhanced IGF1-dependent AKT-Ser473 phosphorylation in our experimental system. The assay's specificity was demonstrated by inhibition of IGF1 receptors outside or inside the cell, using antiserum or small molecule inhibitors, which reduced AKT phosphorylation in response to exogenous IGF1 (p < 0.05). The maximal response of AKT phosphorylation was recorded 15 to 60 min after the addition of IGF1 to cell monolayers (p < 0.001). In our cellular system, insulin induced AKT phosphorylation only at supra-physiological concentrations (µM). Using this novel assay, we identified the differential biological activity of the IGF system in AKT-Ser473 phosphorylation in serum (mouse, naked mole rat, and human), in cerebrospinal fluid (human), and in colostrum or mature milk samples (dairy cow). We have developed a sensitive and robust bioassay to assess the IGF-related activation of the AKT/mTOR pathway. The assay works efficiently and does not require expensive cell culture systems. By using capillary immuno-electrophoresis, the readout of IGF-related bioactivity is substantially accelerated, requiring a minimum of hands-on time. Importantly, the assay system is useful for studying IGF-related activity in the AKT/mTOR pathway in a broad range of biological matrices.


Assuntos
Bioensaio/métodos , Fator de Crescimento Insulin-Like I/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Técnicas de Cultura de Células , Humanos , Transdução de Sinais
2.
PLoS One ; 7(6): e39711, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22768110

RESUMO

We have investigated molecular mechanisms for muscle mass accretion in a non-inbred mouse model (DU6P mice) characterized by extreme muscle mass. This extreme muscle mass was developed during 138 generations of phenotype selection for high protein content. Due to the repeated trait selection a complex setting of different mechanisms was expected to be enriched during the selection experiment. In muscle from 29-week female DU6P mice we have identified robust increases of protein kinase B activation (AKT, Ser-473, up to 2-fold) if compared to 11- and 54-week DU6P mice or controls. While a number of accepted effectors of AKT activation, including IGF-I, IGF-II, insulin/IGF-receptor, myostatin or integrin-linked kinase (ILK), were not correlated with this increase, phosphatase and tensin homologue deleted on chromosome 10 (PTEN) was down-regulated in 29-week female DU6P mice. In addition, higher levels of PTEN phosphorylation were found identifying a second mechanism of PTEN inhibition. Inhibition of PTEN and activation of AKT correlated with specific activation of p70S6 kinase and ribosomal protein S6, reduced phosphorylation of eukaryotic initiation factor 2α (eIF2α) and higher rates of protein synthesis in 29-week female DU6P mice. On the other hand, AKT activation also translated into specific inactivation of glycogen synthase kinase 3ß (GSK3ß) and an increase of muscular glycogen. In muscles from 29-week female DU6P mice a significant increase of protein/DNA was identified, which was not due to a reduction of protein breakdown or to specific increases of translation initiation. Instead our data support the conclusion that a higher rate of protein translation is contributing to the higher muscle mass in mid-aged female DU6P mice. Our results further reveal coevolution of high protein and high glycogen content during the selection experiment and identify PTEN as gate keeper for muscle mass in mid-aged female DU6P mice.


Assuntos
Evolução Molecular , Glicogênio/metabolismo , Músculos/anatomia & histologia , Músculos/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Animais , Peso Corporal , Ativação Enzimática , Feminino , Imuno-Histoquímica , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like II/metabolismo , Camundongos , Modelos Biológicos , Tamanho do Órgão , Fenótipo , Biossíntese de Proteínas , Proteólise , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Especificidade por Substrato , Extratos de Tecidos
3.
Endocrinology ; 152(5): 1948-60, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21427215

RESUMO

GH is a well established regulator of growth, lipid, and glucose metabolism and therefore important for fuel utilization. However, little is known about the effects of macronutrients on the GH/IGF system. We used low-carbohydrate/high-fat diets (LC-HFD) as a model to study the impact of fat, protein, and carbohydrates on the GH/IGF-axis; 12-wk-old Wistar rats were fed either regular chow, a moderate, protein-matched LC-HFD, or a ketogenic LC-HFD (percentage of fat/protein/carbohydrates: chow, 16.7/19/64.3; LC-HF-1, 78.7/19.1/2.2; LC-HF-2, 92.8/5.5/1.7). After 4 wk, body and tibia length, lean body mass, and fat pad weights were measured. Furthermore, we investigated the effects of LC-HFD on 1) secretion of GH and GH-dependent factors, 2) expression and signaling of components of the GH/IGF system in liver and muscle, and 3) hypothalamic and pituitary regulation of GH release. Serum concentrations of IGF-I, IGF binding protein-1, and IGF binding protein-3 were lower with LC-HF-1 and LC-HF-2 (P < 0.01). Both LC-HFD-reduced hepatic GH receptor mRNA and protein expression, decreased basal levels of total and phosphorylated Janus kinase/signal transducers and activators of transcription signaling proteins and reduced hepatic IGF-I gene expression. Hypothalamic somatostatin expression was reduced only with LC-HF-1, leading to increased pituitary GH secretion, higher IGF-I gene expression, and activation of IGF-dependent signaling pathways in skeletal muscle. In contrast, despite severely reduced IGF-I concentrations, GH secretion did not increase with LC-HF-2 diet. In conclusion, lack of carbohydrates in LC-HFD induces hepatic GH resistance. Furthermore, central feedback mechanisms of the GH/IGF system are impaired with extreme, ketogenic LC-HFD.


Assuntos
Dieta com Restrição de Carboidratos , Carboidratos da Dieta/farmacologia , Hormônio do Crescimento/metabolismo , Fígado/efeitos dos fármacos , Animais , Western Blotting , Composição Corporal/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Carboidratos da Dieta/administração & dosagem , Gorduras na Dieta/administração & dosagem , Gorduras na Dieta/farmacologia , Proteínas Alimentares/administração & dosagem , Proteínas Alimentares/farmacologia , Expressão Gênica/efeitos dos fármacos , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Hormônio do Crescimento/sangue , Hormônio do Crescimento/genética , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/sangue , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/genética , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/metabolismo , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Fígado/metabolismo , Masculino , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Wistar , Receptores da Somatotropina/genética , Receptores da Somatotropina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/metabolismo
4.
BMC Cancer ; 10: 560, 2010 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-20950443

RESUMO

BACKGROUND: Targeted therapy approaches have been successfully introduced into the treatment of several cancers. The multikinase inhibitor Sorafenib has antitumor activity in solid tumors and its effects on acute lymphoblastic leukemia (ALL) cells are still unclear. METHODS: ALL cell lines (SEM, RS4;11 and Jurkat) were treated with Sorafenib alone or in combination with cytarabine, doxorubicin or RAD001. Cell count, apoptosis and necrosis rates, cell cycle distribution, protein phosphorylation and metabolic activity were determined. RESULTS: Sorafenib inhibited the proliferation of ALL cells by cell cycle arrest accompanied by down-regulation of CyclinD3 and CDK4. Furthermore, Sorafenib initiated apoptosis by cleavage of caspases 3, 7 and PARP. Apoptosis and necrosis rates increased significantly with most pronounced effects after 96 h. Antiproliferative effects of Sorafenib were associated with a decreased phosphorylation of Akt (Ser473 and Thr308), FoxO3A (Thr32) and 4EBP-1 (Ser65 and Thr70) as early as 0.5 h after treatment. Synergistic effects were seen when Sorafenib was combined with other cytotoxic drugs or a mTOR inhibitor emphasizing the Sorafenib effect. CONCLUSION: Sorafenib displays significant antileukemic activity in vitro by inducing cell cycle arrest and apoptosis. Furthermore, it influences PI3K/Akt/mTOR signaling in ALL cells.


Assuntos
Antineoplásicos/farmacologia , Apoptose , Linfócitos B/patologia , Benzenossulfonatos/farmacologia , Caspase 3/metabolismo , Caspase 7/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Piridinas/farmacologia , Linfócitos T/patologia , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Células Jurkat , Niacinamida/análogos & derivados , Compostos de Fenilureia , Fosforilação , Sorafenibe
5.
Glia ; 58(5): 559-71, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19941340

RESUMO

Hepatocyte growth factor (HGF) is a pleiotropic cytokine able to evoke a wide array of cellular responses including proliferation, migration, and survival through activation of its receptor c-met. Various types of leukocytes have been described to express c-met suggesting that HGF/c-met signaling may directly influence leukocyte responses in inflammation. We have investigated the HGF/c-met pathway in experimental autoimmune encephalomyelitis (EAE), a common mouse model of multiple sclerosis (MS), in which macrophages play a dual role, contributing directly to CNS damage at disease onset but promoting recovery during remission by removing myelin debris. Here we show that during EAE both HGF and c-met are expressed in the CNS and that c-met is activated. We subsequently demonstrate that c-met is primarily expressed in inflammatory lesions by macrophages and a small number of dendritic cells (DCs) and oligodendrocyte progenitor cells (OPCs) but not by microglia or T cells. Complementary in vitro experiments show that only LPS and TNFalpha, but not IL-6, IL-10, or IL-13, are able to induce c-met expression in macrophages. In addition, using TNF signaling deficient macrophages we demonstrate that LPS and TNFalpha induce c-met through distinct pathways. Furthermore, TNFalpha- and LPS-induced c-met is functional because treatment of macrophages with recombinant HGF results in rapid phosphorylation of c-met. Interestingly, HGF/c-met signaling does not modulate cytokine expression, phagocytosis, or antigen presentation but promotes proliferation of activated macrophages. Taken together, our data indicate a pro-inflammatory role for the HGF/c-met pathway in EAE rather than a role in the initiation of repair mechanisms.


Assuntos
Sistema Nervoso Central/patologia , Encefalomielite Autoimune Experimental/patologia , Regulação da Expressão Gênica/fisiologia , Macrófagos/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Animais , Antígenos CD/metabolismo , Células da Medula Óssea , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/induzido quimicamente , Feminino , Citometria de Fluxo , Regulação da Expressão Gênica/efeitos dos fármacos , Glicoproteínas , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/metabolismo , Fator de Crescimento de Hepatócito/farmacologia , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/fisiologia , Glicoproteína Mielina-Oligodendrócito , Nitratos/metabolismo , Oligodendroglia/fisiologia , Fragmentos de Peptídeos , Fagocitose/fisiologia , Proteínas Proto-Oncogênicas c-met/genética , Células-Tronco , Fatores de Tempo , Fator de Necrose Tumoral alfa/deficiência
6.
Exp Biol Med (Maywood) ; 234(8): 1002-9, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19491373

RESUMO

Acromegaly is a disease characterized by chronic growth hormone (GH) excess. Since hypertension is a common finding in patients with acromegaly, interactions between GH and the renin-angiotensin-aldosterone system (RAAS) are under controversial debate. We examined GH, IGF-I, aldosterone, and renin in a well-defined group of acromegalic patients before and after cure by surgery. In addition, we analyzed the impact of chronic GH excess on the RAAS in mouse models over-expressing GH alone (G) or in combination with insulin-like growth factor-binding protein-2 (IGFBP-2; GB). Normalization of GH secretion after cure by surgery was accompanied by significant decreases of serum aldosterone in acromegalic patients (pre-op: 96.5 +/- 37.1 pg/mL, post-op: 41.3 +/- 28.2 pg/ mL; P < 0.001; n = 13), but renin concentrations were unaffected. In addition, aldosterone concentrations were positively correlated to GH levels (Spearman r = 0.39; P = 0.025; n = 26). To further study this association, we analysed two transgenic mouse models and found a similar relationship between GH and aldosterone in G mice, which showed about 3-fold elevated serum aldosterone levels in comparison to non-transgenic controls (males: 442 +/- 331 pg/mL vs. 151 +/- 84 pg/mL; P = 0.002; n > or = 12; females: 488 +/- 161 pg/mL vs. 108 +/- 125 pg/mL; P = 0.05; n > or = 4). Expression of aldosterone synthase was similar in adrenal glands of C and G mice. Aldosterone levels in G and GB mice of both genders were not different, indicating that the elevated aldosterone was due to GH excess and not caused by elevated IGF-I, which is known to be blocked by IGFBP-2 overexpression. Also in the mouse models, changes in aldosterone were independent from renin. In summary, we show that chronic GH excess is associated with increased aldosterone in humans and mice. GH-induced increases of aldosterone potentially contribute to the increased cardiovascular risk in acromegalic patients. The underlying mechanism is likely to be independent of renin, excess IGF-I, or adrenal aldosterone synthase expression.


Assuntos
Acromegalia/sangue , Aldosterona/sangue , Hormônio do Crescimento Humano/sangue , Acromegalia/cirurgia , Glândulas Suprarrenais/enzimologia , Glândulas Suprarrenais/patologia , Animais , Bovinos , Citocromo P-450 CYP11B2 , Feminino , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Tamanho do Órgão
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