Molecular mechanism of a novel hypoglycaemic pancreatic peptide

Abstract

Type 2 diabetes is often associated with defects in metabolic homeostasis such as glucose homeostasis, fat homeostasis and zinc homeostasis. The treatment initially recommended for patients with type 2 diabetes is the change in lifestyle for optimal diet and exercise regimen. However, drug intervention is often needed in addition to lifestyle changes to control blood glucose levels. In our laboratories, a novel pancreatic peptide, GHTD-amide has been identified. It was initially isolated from human urine and showed to have hypoglycaemia activity in experiment with diabetic animal models. An analogue, ISF402 (VHTD-amide), is being developed to improve current anti-diabetic drug therapy as it has insulin-potentiating properties. In addition, due to the fact that GHTD-amide is a naturally-occurring peptide, it has advantage to be used for the treatment of Type 2 diabetes with few or no side effects. This Ph.D. project was therefore aim to gain more insights into the natural biological function of GHTD-amide to aid the development of GHTD-amide and ISF402 into an anti-diabetic drug. The initial approach of this project began with searching for the gene that encodes GHTD-amide (Chapter 2). The characterization of the gene and prohormone processing mechanism of GHTD-amide was anticipated so to bring us a step forward into gene manipulation and functional studies of this novel tetra-peptide. All candidate precursors of GHTD-amide found in the current vertebrate genome database have been analysed using bioinformatics. The biosynthetic route of GHTD-amide is still uncertain because searches in genome and proteome databases have failed to disclose the gene encoding for GHTD-amide precursor protein(s). A de novo biosynthetic route for the synthesis of GHTD-amide is possible. The second aim of this project was to investigate the molecular mechanism of GHTD-amide at cellular level, particularly in skeletal muscle cells. Previous studies have shown GHTD-amide and ISF402 are able to alter cellular activities, such as increase in glycogen synthesis, increase in phosphorylation of Akt and increase in cytosolic free zinc, with the most prominent effect observed in skeletal muscle cells. Chapter 3 comprises of a study that monitors the changes in cytosolic free zinc with live cell imaging in cultured muscle cells treated with GHTD-amide and its counterparts, HTD-amide and ISF402. The toxic effect of the peptides was also examined. The phenomenon of this increase in cytosolic free zinc induced by GHTD-amide is believed to be beneficial to meet cellular metabolic demand due to the fact that the zinc ions are originated from intracellular zinc source and its does not cause cytotoxicity. Subsequent follow-on investigations were to explore the molecular mechanism of GHTD-amide to understand how it exerts its effect in altering zinc homeostasis. The association of GHTD-amide and zinc metabolism has drawn our attention to see this peptide has an important role in regulating zinc homeostasis, which often abnormal in diabetes subjects. In Chapter 4, global analysis of the transcriptional alterations of all zinc homeostasis regulators have been performed in cultured skeletal muscle treated with the peptides. The peptides were found to mobilize zinc in muscle cells by increasing zinc transport molecules. Cells surface receptor inhibitors were also used to test which signalling pathways GHTD-amide is likely to use for signal transduction. This thesis has provided more insights into the effect of GHTD-amide in increasing cytosolic free zinc. This effect was proven to be not caused by oxidative or toxic effect after administration of the peptides to cells. In fact, the increase in cytosolic fee zinc releasing from intracellular zinc store often has beneficial effect for cellular activity, for example, increase in glycogen synthesis may be positively regulated by zinc ions. The conceptual significance of the data in this thesis substantiates the notion that GHTD-amide alters intracellular zinc level by regulating zinc transpoters and metallothionein at transcriptional level. The transcriptional regulation of these zinc homeostasis regulators requires the activity of several signalling pathways to mediate the effect of GHTD-amide. This finding is a breakthrough in our understanding of the molecular mechanism of this peptide. This property of GHTD-amide in regulating cellular zinc metabolism described in this thesis suggests a new potential opportunity for this peptide to be used in therapeutic intervention for diabetes because diabetes subjects often have unknown cause of abnormal zinc metabolism condition. The administration of GHTD-amide in combination with insulin is known to potential insulin activity, hence lower blood glucose. The data presented in thesis further suggests a combinatory therapy of both GHTD-amide and insulin, which has greater potential to increase the cells’ ability to counteract long term low plasma zinc level in diabetes subjects. One functional role of GHTD-amide is hypothesized to modulate the optimal intracellular zinc level at particularly time depending on cellular demand. This brought renewed hope and vitality for us to understand more on zinc dysregulation in diabetes subjects and more importantly, GHTD-amide may lead to new therapeutic intervention for this cause.