The notion that insulin binds to a specific site on the cell membrane was first proposed many years ago. However, experimental proof of a membrane bound insulin receptor did not come until the early 1970s when biologically active radiolabelled insulin was used in direct binding studies (Cuatrecasas, 1971). Recent advances in understanding the mechanism of insulin action are the result of studies on the structure and function of the insulin receptor. The membrane receptor would appear to have two functions: firstly, it must bind insulin and secondly, it must couple insulin binding to insulin action. Defects in either of these receptor functions will result in an impaired response to insulin, or insulin resistance (Taylor, 1985). Insulin resistance is a common disorder in a number of disease states in man. For example, non-insulin-dependent diabetes mellitus and obesity are associated with mild insulin resistance (Bar et aZ., 1976). There are also a number of relatively rare syndromes of extreme insulin resistance in which there is either impaired receptor function, or an immunological defect resulting in the development of auto-antibodies against the insulin receptor (Taylor et aZ., 1985).Studies on insulin receptor defects associated with these disease states have led to progress in understanding the molecular mechanisms of insulin action. Ideally when investigating these disease states one should study insulin action on classical target cells such as adipocytes, hepatocytes or muscle. However, it is now well established that the kinetics of insulin binding to its membrane receptor is similar in all human tissue whether or not it is a target for insulin action. This has led to a great deal of research on the more accessible human tissues such as monocytes, erythrocytes, cultured fibroblasts and Epstein-Barr virus (EBV) transformed B-Iymphocytes. The most convenient tissue to study is EBV transformed B-Iymphocytes, as these cells can be taken from individual patients and grown in culture in large quantities, which facilitates biochemical studies. Despite these advantages, it is important to establish that this virus-induced receptor is a true insulin receptor and not an artifact of viral transformation. Studies on B-Iymphocyte proliferation have shown that the insulin receptor appears on the cell membrane during the proliferative phase of B-cell activation. However , this is a transient event and once the cell reaches maturation the insulin receptor is no longer evident (Marchalonis & Galbraith, 1987). The insulin receptor has also been demonstrated in a number of cultured human lymphoblastoid cell lines (Gavin et aL, 1983; Maegawa et aL, 1983). It seems, therefore, that the insulin receptor is normally expressed by blast cells. The purpose of this study was to investigate insulin binding characteristics on a human lymphoblastoid cell line with B-cell characteristics which was originally derived from a patient with Burkitt's lymphoma. These cells, which are known as Raji cells, are unusual in that they carry multiple copies of the EBV genome in their DNA. For this reason they provide a useful model system for studying the insulin receptor in EBV transformed lymphocytes. In addition, studies on the mechanism of insulin action in these cells should give some insight into the function of the insulin receptor during B-cell proliferation. In this study four major characteristics of insulin binding to insulin receptors on Raji cells are described. Firstly, on the basis of kinetic studies a model for insulin-receptor interaction was established. Secondly, processing of insulin and the receptor was investigated to determine whether the receptor is functional. A third aspect was elucidation of the receptor structure and the insulin binding site. Finally, the cross-reaction between insulin and type I IGF receptors was studied, and the cellular response mediated by the insulin receptor and growth factor receptor was determined. / Thesis (M.Med.Sc.)-University of Natal, Durban, 1988.

No description available.

Molecular-targeting therapeutics directed towards growth factor receptors have become promising interventions in cancer. They include the family of mammalian receptor tyrosine kinases such as epidermal growth factor, TrkA and insulin. In particular, the insulin receptor (IR) is one of the most well-known members of the RTK family of receptors playing a role in cancer. IRs are covalently-linked heterodimers of αβ subunits on the cell membrane in the absence of insulin. The IR signaling pathways are initially triggered by insulin binding to the α subunits followed by the interaction of β subunits and ATP. The parameter(s) controlling IR activation remains unknown. Here, we report a membrane receptor signaling platform initiated by insulin binding to its receptor to induce Neu1 in live HTC-IR and MiaPaCa-2 cell lines. Microscopy colocalization and co-immunoprecipitation analyses reveal that Neu1 and MMP9 form a complex with naïve and insulin-treated receptors. Tamiflu (neuraminidase inhibitor), galardin and piperazine (broad range MMP inhibitors), MMP9 specific inhibitor and anti-Neu1 antibody blocked Neu1 activity associated with insulin stimulated live cells. Moreover, Tamiflu, anti-Neu1 antibody, and MMP9 specific inhibitor blocked insulin induced insulin receptor substrate-1 phosphorylation (p-IRS1). The previous findings reveal a molecular organizational signaling platform of Neu1 and MMP-9 crosstalk in alliance with insulin receptors. It proposes that insulin binding to the receptor induces MMP9 to activate Neu1, which hydrolyzes α-2,3 sialic acid in removing steric hindrance to generate a functional receptor. The results predict a prerequisite desialylation process by activated Neu1. A complete understanding of IR activation and the role of sialic acids in the iii signaling pathways may provide a therapeutic strategy in the prevention of different diseases such as diabetes mellitus and cancer. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2013-02-20 11:27:44.861

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Recent studies have confirmed the presence of insulin receptors in the rat brain although their function has still not been well defined. The present study explores the possibility that insulin receptors in the brain can alter or contribute to central neurotransmission. Insulin caused a dose-dependent inhibition of hippocampal pyramidal neurons. The pattern of inhibition mirrored the binding kinetics of insulin in the hippocampus. Two related peptides, proinsulin and desoctapeptide insulin, had neuronal effects consistent with their binding to insulin receptors in the brain. Proinsulin was effective in doses 30-fold greater than insulin, whereas desoctapeptide insulin had little or no effect. These observations indicate that the inhibitory effect of insulin in this tissue may be insulin receptor-mediated and support a previously suggested functional role of insulin in the central nervous system.

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

The mechanism of action of insulin and epidermal growth factor was studied by genetic and biochemical means. Particular emphasis was placed on the ability of these factors to induce DNA synthesis and the relationship of endocytosis to that ability. Insulin was crosslinked to the active fragment A of diphtheria toxin. This conjugate specifically killed cultured mouse cells through an insulin receptor-mediated process. The conjugate was used to select genetic variants resistant to its cytotoxic effect. Six resistant variants were isolated, 2 of which retained very low insulin receptor activity. When these two variants were further analyzed both displayed altered cell shape and growth properties. The CI-3 variant also was shown to have a deficient lysosomal system and failed to efficiently degrade epidermal growth factor. This variant was, however, fully responsive to the mitogenic action of EGF. This suggested that lysosomal processing is unimportant in the production of a mitogenic stimulus by EGF. EGF was found to be endocytosed by fibroblasts through 2 separate pathways. One pathway involves an unidentified organelle and correlated with increased degradation of the ligand. The other pathway involves a Golgi-like component and is correlated with a lack of degradation and uptake into a dense, non-lysosomal organelle. Uptake of EGF into this non-lysosomal component, which we named mitosomes, correlated with the ability of EGF to induce DNA synthesis. From these results, a model was constructed for the coupling of endocytosis, uptake into mitosomes and the stimulation of DNA synthesis.

Grb10 is a member of the Grb7 family of adaptor proteins that also includes Grb7 and Grb14. These three members contain multiple protein binding domains and lack enzymatic activity. Extensive two-hybrid studies have demonstrated binding of Grb10 to numerous activated tyrosine kinase receptors including the insulin receptor (IR) and insulin-like growth factor-I receptor (IGF-IR), as well as many non-receptor molecules such as MEK1, Raf-1, and Nedd4. Grb10 has been implicated in IGF-I anti-apoptotic signaling regulation through interactions with Raf-1 and the mitochondrial membrane. / In this report the pattern of transient Grb10 translocation following IGF-I cellular stimulation was studied. This report also demonstrates the implication of a short variable amino-terminal region of Grb10 in mitochondrial membrane association. Finally, assays were developed with the goal of identifying new Grb10 binding partners.

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

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