Diss. (sammanfattning) Linköping : Linköpings universitet, 2004. / Härtill 4 uppsatser.

The mechanism by which insulin and other polypeptide growth factors alter cellular metabolism is not fully understood. In the case of insulin, it is thought that phosphorylation/dephosphorylation mechanisms may play a central role in the signalling pathway. This is based on evidence which includes demonstration that the receptor for insulin is a tyrosine-specific protein kinase which is activated in response to insulin binding. Ultimately, insulin binding to its receptor on the surface of intact fat cells leads to altered levels of serine phosphorylation of several soluble proteins, including the phosphorylation of ATP-citrate lyase and acetyi-CoA carboxylase. Recently, studies involving site-specific mutagenesis have shown that the tyrosine kinase function of the insulin receptor is essential for insulin signalling. The studies described in this thesis have addressed the problem of how activation of the insulin receptor/tyrosine kinase results in the altered serine phosphorylation observed in intact cells in response to insulin. To gain further understanding of the cellular components required for insulin signalling, reconstitution experiments have been carried out mixing isolated cellular fractions with preparations of insulin receptors. The effects of insulin on altering protein-serine and protein-tyrosine phosphorylation have been determined in this reconstituted system. Results show that in a high-speed (100,000 x g) supernatant fraction prepared from rat adipose tissue endogenous protein-serine kinases are sensitive to conditions which are commonly employed for assaying insulin receptor/kinase activity. This includes inhibition by micromolar concentrations of MnCI₂, by 40 mM NaF, and by low reaction temperature (0°C). When the insulin receptor, present in a WGA-Sepharose-purified preparation of detergent-solublized rat liver membranes, was assayed in the complete absence of both MnCI₂ and NaF, receptor/tyrosine kinase activity was only slightly reduced with little or no decrease in the responsiveness to insulin. Furthermore, when the WGA-Sepharose-purified membrane fraction was incubated at 37°C in the presence of [ɣ -³²P]ATP several endogenous proteins were observed to be phosphorylated in addition to the β-subunit of the insulin receptor. These membrane proteins appear to be phosphorylated on tyrosine as indicated by their resistance to alkali hydrolysis. Upon reconstitution of the adipose tissue high-speed supernatant fraction with the WGA-Sepharose-purified preparation of insulin receptors the most striking effects observed were the phosphorylation of a 40 kd protein subunit (pp40) and the dephosphorylation of a 25 kd protein subunit (pp25) present in adipose tissue. The phosphorylation of pp40 occurs on tyrosine and is insulin-responsive, whereas the dephosphorylation of pp25 occurs following reconstitution with either untreated control, or insulin-activated insulin receptors. To assess the effect that reconstituted insulin receptors may have on the phosphorylation of endogenous ATP-citrate lyase in adipose tissue high-speed supernatant, it was found that a more pure preparation of insulin receptors was required. Further purification of the insulin receptor to homogeneity was therefore attempted using insulin-agarose affinity chromatography. However, difficulties including low yield and instability of the receptor through purification have prevented progress with these studies at present. In a separate study, highly purified acetyl-CoA carboxylase was reconstituted with a crude fraction consisting of total Triton-solublized membrane proteins. In this reconstituted system phosphorylation of acetyl-CoA carboxylase was enhanced to an extent greater than 6-fold after incubation with [ɣ -³²P]ATP. Following chromatography of the crude Triton-solublized extract over WGA-Sepharose this acetyl-CoA carboxylase kinase activity was found to be present in the flow-through void fraction and not in the N-acetylglucosamine eluted fraction. The acetyl-CoA carboxylase kinase, at present, does not appear to be insulin-responsive, but further studies are needed to confirm this observation. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Insulin and insulin-like growth factor 1 (IGF-1) and 2 (IGF-2) are related protein hormones with different but overlapping biological functions. All the hormones interact with a receptor within the insulin-IGF system (insulin receptor A and B, IGF-1 receptor), however with different affinity. The different interaction with individual receptors is just one of the main tools for regulation of the system that is essential for the proper functioning of the organism. Although the residues directly interacting with receptors are mainly located in A and B domains, the C and D domains probably play a role in receptor specificity. Here, we firstly focused on the impact of D domains of IGF-1 and 2 (D1 and D2 domains) and C domain of IGF- 2 (C2 domain). To probe the impact of C and D domains, we prepared insulin analogues containing a part of or an entire domain following a pattern seen in IGFs. The receptor-binding affinities of these analogues and their receptor autophosphorylation potentials were characterised. Our results revealed that the initial part of D1 domain has a detrimental effect on IR affinity that is only slightly enhanced by the rest of the D1 domain. D2 domain has rather neutral effect on IR affinity. We further showed that the addition of amino acids derived from the C2 domain to the...

Diss. (sammanfattning) Linköping : Linköpings universitet, 2007. / Härtill 4 uppsatser.

Made available in DSpace on 2014-06-11T19:27:46Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-03-08Bitstream added on 2014-06-13T19:56:34Z : No. of bitstreams: 1 moraes_kac_me_araca.pdf: 306630 bytes, checksum: d5a66918144c7975dabc631c6c9b84ec (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Nos últimos anos tem ocorrido uma redução acentuada nos índices de cárie dentária em diversas regiões do planeta, fato que tem se atribuído ao consumo de produtos fluoretados. Entretanto, o flúor, quando ingerido em excesso, causa intoxicação crônica ou aguda, como a fluorose dentária e distúrbios na homeostase da glicose. As crianças se tornam foco de preocupação, principalmente às portadoras de diabetes mellitus (DM), pois geralmente ingerem grandes quantidades de dentifrício fluoretado durante a escovação, ultrapassando a dose preconizada como limite de ingestão diária de flúor de 0,05 a 0,07mg/F/kg de peso corpóreo. Este trabalho, que foi dividido em duas partes, pretende realizar uma breve revisão de literatura sobre os efeitos decorrentes da ingestão de NaF no metabolismo de carboidratos e avaliar os efeitos da ingestão do fluoreto na sensibilidade à insulina e na transdução do sinal insulínico. A primeira parte, baseada em artigos científicos publicados, procura discorrer sobre os efeitos da ingestão de flúor no metabolismo de carboidratos, na tolerância à glicose e no sinal insulínico, e algumas considerações sobre o diabetes mellitus e sobre as possíveis complicações que a ingestão de NaF pode ocasionar às crianças portadoras desta doença. Estes trabalhos demonstraram que o tratamento agudo ou prolongado com altas doses de fluoreto de sódio interfere na homeostase da glicose. Convém salientar que esta alteração é similar à observada em casos de diabetes mellitus. Além do mais, o flúor quando ingerido em excesso, também ocasiona diminuição da secreção de insulina, inibição da glicólise e depleção de glicogênio. Muitas dessas respostas sugerem que o NaF pode promover resistência à insulina. Portanto, a ingestão em excesso de NaF pode prejudicar a saúde, principalmente de crianças portadora de DM. / Over the last few years there has been a significant reduction in the incidence of dental caries in several regions of the world. This has been attributed to the consumption of fluoridated products. However, excess of fluoride intake can cause chronic or acute intoxication, such as dental fluorosis and impaired glucose homeostasis. Concern is focused on children, especially those with diabetes mellitus, because children usually swallow large amounts of fluoridated dentifrice during tooth brushing, in excess of the maximum recommended daily fluoride dose of 0.05 to 0.07 mg/F/kg of body weight. This report, divided into two parts, intends to make a brief literature review about effects of NAF intake on glucose metabolism, and to determine the effects of this intake on insulin sensitivity and insulin signal transduction. The first part, based on published scientific articles, endeavors to describe the effects of NaF intake on glucose metabolism, glucose tolerance and insulin signal, and put forward considerations concerning diabetes mellitus (DM), and the possible complications that NaF intake could cause in children with DM. These reports demonstrated that the acute or chronic treatment with high sodium fluoride dose interferes in glucose homeostasis, resulting in conditions such as hyperglycemia. This alteration is similar to that observed in DM. Furthermore, NaF ingestion in high doses can produce abnormalities in insulin secretion, glycolysis inhibition, and glycogen depletion. Many of these evidences suggest that NaF can induce insulin resistance. Thus, excessive fluoride consumption could worsen health, particularly of diabetic children. Based on that fluoride can interfere in the glucose metabolism, it is important for the second part of this report to determine the acute effect of fluoride on insulin sensitivity and pp185 (IRS-1/IRS-2) phosphorylation in insulin sensitive tissues.

Orientador: Mario Jose Abdalla Saad / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-06T23:49:19Z (GMT). No. of bitstreams: 1 Lemos_ChristineMarinhode_M.pdf: 1925163 bytes, checksum: 3618c226fce7bf7ab354e89c68dcfa14 (MD5) Previous issue date: 2006 / Resumo: Estudos recentes demonstraram que tratamentos prolongados com drogas inibidoras da atividade tirosina quinase (TKI) poderiam agir favoravelmente não só controlando crescimento e replicação celular, como também funções fisiológicas responsáveis por manter a homeostase da glicose. Porém, os efeitos diretos de PD 153035, um TKI, na regulação das etapas iniciais da ação da insulina não são conhecidos. A insulina, ao se ligar à subunidade a de seu receptor heterotetramérico, dá início a uma série de ações imediatas e tardias, metabólicas e promotoras de crescimento .Tais eventos ocorrem através da estimulação da subunidade ß transmembrana do receptor, que autofosforila e ativa a fosforilação de substratos endógenos intracelulares, conhecidos como substratos do receptor de insulina ou IRSs. Os principais substratos do receptor de insulina são o IRS-1 e IRS-2, que quando fosforilados em tirosina se ligam e ativam proteínas com porção SH2, como a PI 3-quinase. A ativação destas proteínas desencadeia a ativação de suas serinas-quinases importantes que são a AKT e as ERKs (1/2), que são essenciais, respectivamente, para os efeitos metabólicos e de controle gênico do homônio. No presente estudo investigamos o efeito do tratamento com PD 153035, por 7 dias, na sensibilidade e sinalização da insulina em fígado, musculo e tecido adiposo de ratos Wistar. Foi investigado o grau de fosforilação em tirosina do receptor de insulina, dos substratos 1 e 2 do receptor ( IRS-1 e IRS-2 ), a associação deles com a enzima PI 3-quinase, o grau de fosforilação em serina/treonina da AKT, a fosforilação das ERKs (1/2), da p70s6k, da AMPK, da JNK e I?Ba nos três tecidos. Nenhuma diferença nos níveis glicêmicos foi observado durante o GTT entre os grupos tratado com PD 153035 e controle. As taxas de desaparecimento de glicose plasmática estavam altas nos animais tratados. No fígado de ratos tratados com PD 153035, observamos um aumento da fosforilação em tirosina do IR, IRS-1 e do IRS-2 e um aumento da associação desses substratos com a PI 3-quinase. Porém uma diminuição significativa da fosforilação da AKT e da p70s6k foi observada, sem alteração no grau de fosforilação das ERKS (1/2). Não foi observado diferença significativa no grau de fosforilação da JNK, porém os animais tratados apresentaram uma redução significativa nos níveis de I?Ba . O grau de fosforilação da AMPK mostrou-se aumentado nos animais tratados. Quando estudamos o tecido muscular, após estímulo agudo com insulina observamos uma diminuição significativa no grau de fosforilação do receptor de insulina nos animais tratados com PD 153035. Entretanto, nesses animais, pudemos observar após estímulo agudo com insulina, que a fosforilção em tirosina do IRS-1 aumentou significativamente. O aumento da fosforilação do IRS-1 foi acompanhada pelo aumento na associação IRS-1/ PI 3-quinase e pelo aumento no grau de fosforilação da AKT e da p70s6k. a. O grau de fosforilação da AMPK mostrou-se aumentado nos animais tratados. Não foi observada alteração no grau de fosforilação das ERKs (1/2) neste tecido. Foi observado uma redução do grau de fosforilação das serinas quinases JNK e I??a no músculo dos animais tratados. Os animais que receberam tratamento crônico com PD 153035 por 7 dias apresentaram uma redução da adiposidade visceral, bem como uma perda de peso em relação ao grupo controle. Observamos nesses animais uma redução significativa no grau de fosforilação do IR e do IRS-1 no tecido adiposo. A associação IRS-1/PI 3-quinase mostrou uma redução significativa. A fosforilação da AKT e da P70s6k mostrou-se significativamente reduzida nos animais tratados. Entretanto observamos um aumento significativo da fosforilação do IRS-2 após estímulo insulínico agudo, porém acompanhado pela redução significativa na associação IRS-2/PI 3-quinase. Não observamos diferença nos níveis de fosforilação das ERKs. Observamos que o grau de fosforilação da proteína AMPK aumentou significativamente no ratos que receberam tratamento com PD 153035. Foi observado uma redução significativa do grau de fosforilação da JNK e I??a no tecido adiposo dos animais tratados. Sumariamente, o tratamento com PD 153035 por 7 dias aumentou a fosforilação em tirosina do IR, IRS-1 e IRS-2 no fígado, apesar da fosforilação da AKT apresentar-se reduzida neste tecido. No músculo dos animais tratados com PD 153035 observamos que a droga melhora a sinalização da insulina, provavelmente pela redução da atividade das serinas quinases JNK, IKKß e mTOR. No tecido adiposo a droga induziu resistência à insulina, acompanhada de redução no ganho de peso e redução da adiposidade visceral, possivelmente pelo aumento da secreção de adiponectina pelos adipócitos. Em conclusão, os resultados do nosso estudo demonstram que o tratamento com PD 153035 aumentou a sensibilidade à insulina, por aumento da adiponectina, aumento da AMPK em fígado, músculo e adiposo, e aumentada via IRS/PI3K/AKT em músculo / Abstract: It has been recently demonstrated that long-term treatment with some of tyrosine kinase inhibitor (TKI) drugs, might favorably act at steps in controlling not only cell growth and replication, but also physiological functions responsible for maintaining glucose homeostasis. However, the direct effects of PD 153035, a TKI, in the regulation of the early steps of insulin action are not known. Insulin initiates its growth and metabolic promoting effects by biding to its receptor at the plasma membrane, which has tyrosine-kinase activity, and is able to autophosphorylates and phosphorylates cytoplasmatic proteins called insulin receptor substrates (IRSs). The main substrates of insulin receptor are IRS-1 and IRS-2, which when phosphorylated in tyrosine bind and activate several proteins, including phosphatidylinositol (PI) 3-kinase. These initial steps lead to the activation of two serine/threonine kinases ¿ AKT and ERK family (1/2) of MAPK. In the present study, we investigated the effect of treatment with PD 153035, for 7 days, on insulin sensitivity and insulin signaling in liver, muscle and adipose tissue of Wistar rats. It was investigated the tyrosine phosphorylation of IR, IRS-1 and IRS-2, their association with PI 3-kinase, and Akt serine/threonie phosphorylation , ERKs (1/2) phosphorylation, p70s6k, AMPK, JNK and ???a phosphorylation, in the three tissues. No differences in fasting plasma glucose levels were observed in animals treated with PD 153035. Plasma glucose disappearance rates were higher in treated animals In the liver of rats treated with PD 153035, we observed an increased IR, IRS-1 and IRS-2 tyrosine phosphorilation and an increased association of these substracs with PI 3-quinase. However a significant decrease of AKT and of the p70s6k phosphorylation was observed too, without alteration in ERKs phosphorylation levels (1/2). No significant difference was observed in JNk phosphorylation levels, however ??? showed a reduced phosphorylation in the liver of these animals. AMPK showed a significant increased phosphorylation in treated animals. When we studied muscle, insulin-induced IR tyrosine phosphorylation showed significantly reduced in these animals, however treating rats with PD153035 significantly increased the insulin-induced IRS-1 phosphorylation in the muscle. The increased phosphorylation of IRS-1 was accompanied by increase in IRS-1/PI3-kinase association and Akt and p70s6k phosphorylation were higher in treated animals after insulin stimulation. AMPK showed a significant increased phosphorylation in treated animals. There was no significant changes in ERKs (/2) phosphorylation in this tissue. Reduced phosphorylation of serine-kinases as c-jun N terminal (Jnk) and I?kß was observed. The animals that received chronic treatment with PD 153035 for 7 days had presented a reduction in visceral fat mass, as well as a loss of weight, regarding the control group We observed in these animals a significant decreased IR and IRS-1 tyrosine phosphorylation in the adipose tissue. The association IRS-1/PI3-kinase showed a significant reduction. AKT and P70s6K phosphorylation showed significantly decreased in treated animals. However we observe a significant increased phosphorylarion of insulin-induced IRS-2 phosphorylation, but the association IRS-2/PI 3-kinase showed a significant reduction. We did not observe any difference in phosphorylation levels of ERKs (1/ 2). AMPK phosphorylarion increased significantly in animals that received treatment with PD 153035. Reduced phosphorylation of JNK and Ikkßa was observed in adipose tissue of treated animals. In summary, our results demonstrated that in 7 days of treatment with PD 153035 increased tyrosine phosphorylation of IR/IRS-1/IRS-2 in the liver were observed, in spite of AKT phosphorylation had decreased in this tissue. In muscle of animals treated with PD 153035 we observed that the drug had improved the insulin signalling, probably by reduction of the serine kinase activity JNK, ???ß and mTOR. In the adipose tissue, the drug induced insulin resistance, accompanied of visceral fat mass reduction as well as a loss of weight, probably due to an increased adiponectin secretion by fat cels. In conclusion, the results of our study demonstrate that the treatment with PD 153035 increased the insulin sensibility, by increased levels of adiponectin, increased AMPK in liver, muscle and adipose tissue, and increased IRS/PI3K/AKT pathway in muscle / Mestrado / Ciencias Basicas / Mestre em Clinica Medica

Diabetes mellitus is considered as one of world's most common metabolic diseases. Complicated treatment and increasing number of newly diagnosed patients, suffering from diabetes every year, shows the importance and necessity of research in this area. Some of the major aims of this research are the development of new therapeutically utilized drugs and defining the problems of insulin acting in human body. Insulin is a peptide hormone whose main physiological function is to regulate blood glucose level in organism connected with large impact on whole metabolism. Insulin acts through binding of its monomeric form to the insulin receptor. Upon binding to the receptor molecule of insulin undergoes specific structural changes, which put the hormone into an active state. As of now, the structure of the insulin's active monomeric form is still unknown. By testing binding affinities of many modified insulin analogues there was discovered strong evidence between structural conformation of the C-terminus of the B-chain and binding affinity to the receptor. The most crucial data, necessary for this work, were observed from the structure of highly active insulin analogues that possessed unique B26 turn, recently prepared and described by team of Dr. J. Jiráček, IOCB AS CR. The aim of this work was synthesis of...

Recent population studies provide evidence that individuals with high circulating insulin levels have a poor prognosis and/or increased risk of cancer development; however, laboratory studies concerning the role of insulin in breast cancer biology are sparse. We compared the growth of 4T1 murine breast cancer allografts in control mice, alloxan-induced hypoinsulinemic mice, and mice treated with the insulin/insulin-like growth factor-1 receptor tyrosine kinase inhibitor BMS-536924. Both interventions significantly decreased tumor growth versus control and decreased pathway activation downstream of the insulin receptor as reflected by Aktser473 phosphorylation status in the neoplastic tissue. Alloxan-treated mice exhibited signs of insulin deficiency, while BMS-536924-treated animals showed only minor metabolic derangements. Skeletal muscle displayed reduced pAktser473 in alloxan-treated mice. In contrast, BMS-536924 treatment increased pAktser473 in muscle. This raises the possibility that the relative lack of metabolic toxicity of BMS-536924 involves varying tissue levels of the drug. These results support the view that host insulin physiology is a potentially modifiable determinant of breast cancer behaviour.

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