Therapeutic Agents from Herbal Source to Address Diabetes Type 2 Problems

Dey, Debleena (2006) Therapeutic Agents from Herbal Source to Address Diabetes Type 2 Problems. PhD thesis, Jadavpur University.

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                                          Supervisors

                                          SupervisorsEmail
                                          Roy, Sib Sankar

                                          Abstract

                                          To understand the molecular mechanism involved in insulin resistance and type 2 diabetes is basically necessary to identify the precise target molecules from plants having antidiabetic property. To investigate the precise target in respect of FFA induced impairment leading to insulin resistance and type 2 diabetes, further investigation was carried out taking IR as the major target. Elevated plasma free fatty acids (FFAs) are known to be associated with insulin resistance and type 2 diabetes mellitus. How FFAs oppose insulin action is still not very clear but a few reports suggest their interference in the insulin signaling pathways. FFA also impairs a number of PKC isoforms, which ultimately reduce insulin-signaling leading to reduced glucose uptake. 2-Deoxyglucose uptake by 3T3L1 adipocytes and L6 skeletal muscle cells incubated with different free fatty acids showed that palmitic acid among other free fatty acids is the key player in inhibiting insulin signaling pathway. Insulin augmented signals starting from the activation of insulin receptor (IR) to Glut 4 translocation was inhibited by palmitate. Insulin stimulated IR phosphorylation was significantly reduced by palmitate treatment. Insulin signaling proteins downstream to IR, which get consequently phosphorylated following IR tyrosine kinase phosphorylation on insulin stimulation, also showed significant inhibition due to palmitate treatment. Inhibition of IR, IRS1, PI3 kinase and Akt/PKB phosphorylation, which are markers of insulin resistance, was observed in palmitate fed rats. Therefore, we could develop a rat model for nutritionally induced insulin resistance. Rats fed with palmitate showed inhibition in phosphorylation of IR, IRS1, PI3 kinase and Akt/PKB, all of which were considered as important markers for insulin resistance, indicating that palmitate-fed rats can be used as a model for nutritionally induced insulin resistance and type 2 diabetes. In insulin signal transduction pathway, PDK1 is the upstream kinase that directly phosphorylates downstream substrates such as Akt/PKB and PKC isoforms. Insulin stimulated phosphorylation of Akt/PKB was inhibited by palmitate along with PDK1 phosphorylation. Since phosphorylation of PKC is also dependent on PDK1 it was expected to be inhibited. On the contrary PKC show PDK1 independent phosphorylation due to FFA. This was really surprising, as in vitro incubation of skeletal muscle cells with palmitate increased PKC phosphorylation while PDK1 phosphorylation was decreased. Inhibition of PDK1 by palmitate was reflected on Akt phosphorylation whose phosphorylation was inhibited in palmitate treated cells, Data demonstrated a significant decrease in IR mRNA in palmitate treated muscle cells. Western blot analysis also showed a decrease in IR protein level as a consequence of decreased IR gene expression in palmitate incubated cells as compared to the control. These results suggested that fatty acid-induced insulin resistance is related to reduced IR gene expression. HMGA1 plays a critical regulatory role in IR gene transcription. Phosphorylation of HMGA1 restricts its movement towards IR promoter leading to inhibition of IR gene transcription, and palmitate activates the PKC phosphorylation, which in turn phosphorylates HMGA1 leading to downregulation of IR gene expression. Our observations show the involvement of one of the possible pathways involved in fatty acid induced insulin resistance. Free fatty acid (i.e. Palmitate) induced PDK1 independent PKC phosphorylation; pPKC was then translocated to the nucleus and phosphorylated HMGA1 that restricts its movement to IR gene promoter. Experimental evidences with V1 supported this contention. Phosphorylated HMGA1 interfered with the reactivation of IR promoter causing downregulation of IR gene expression and reduced IR copies on the cell surface thus reducing insulin sensitivity. After availing all these experimental results in relation to FFA induced insulin resistance, the process of searching for antidiabetic activity of medicinal plants of Indian origin begun. A specific fraction or molecules were tested to find whether there was any intervention in the molecular pathway of insulin insensitivity or not. Extract from two plants, i) Pueraria tuberosa and ii) Aloe vera were found to improve FFA induced impairment when insulin activity was determined in terms of 3H-2 deoxyglucose uptake by 3T3L1 adipocytes. However, Aloe vera activity was far less as compared to P. tuberosa. Therefore, only P tuberosa was studied further. HPLC purified closely related structures from Pueraria tuberosa were identified by 2D-NMR and Mass spectrometry and these are LPA4, LPA5, Soyasaponin1 and Kudzusaponin1.LPA4 is different from the others in having -L-rhamnopyranosyloxy moiety and therefore its improvement in terms of insulin activity is due to the presence of this chemical character.The crude extract from Aloe vera was also examined for antidiabetic property. 2-deoxyglucose uptake showed that both crude extract and sub fractions increases the insulin sensitivity in comparison to palmitate treated cells. Our results demonstrated that palmitate-induced reduction of insulin augmented 3H-2-DOG uptake by adipocyte could be prevented by LPA4. The inhibitory effect of palmitate on insulin-stimulated IRβ tyrosine kinase phosphorylation and Akt phosphorylation was waived by LPA4. The inhibition of IR gene expression by palmitate was also reduced by LPA4.

                                          Item Type: Thesis (PhD)
                                          URI: http://www.eprints.iicb.res.in/id/eprint/1598
                                          Subjects: Cell Biology & Physiology
                                          Divisions: Indian Institute of Chemical Biology
                                          Depositing User: Mr Santanu Sadhukhan
                                          Date Deposited: 23 Aug 2012 11:45
                                          Last Modified: 23 Aug 2012 11:45
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