title: The role of zinc and the Zip7 transporter in disease processes associated with IR in skeletal muscle
Creators: name: Norouzi, S
Publisher: name: University of Tasmania
Year: 2019

description: Zinc is a critical metal ion that has wide-ranging effects on cellular function. It is required for growth and development, immunity, and metabolism, and is therefore, vital for life. Disturbances in zinc homeostasis lead to various disease states including cancer, neurological disorders and diabetes. Recent studies have highlighted the dynamic role of zinc as an insulin mimetic and a cellular second messenger that controls many processes associated with insulin signalling and other downstream pathways that are amendable to glycaemic control. Therefore, mechanisms that contribute to the decompartmentalization of zinc and dysfunctional zinc transporter mechanisms, including zinc signalling are associated with metabolic disease. As zinc cannot pass through biological membranes unassisted, cellular zinc storage, release and distribution are controlled by a family of proteins: zinc transporters and metallothioneins. Increasing evidence suggests that dysregulation of these proteins might act as key causative or promoting factors in several chronic pathologies such as insulin resistance and type 2 diabetes. Of these, emerging research has highlighted a role for several zinc transporters in the initiation of zinc signalling events in cells that lead to metabolic processes associated with maintaining insulin sensitivity and thus glycaemic homeostasis. One principle zinc transporter, Zip7 is emerging as a ‘gate-keeper’ of zinc flux from intracellular organelles including the Golgi apparatus and endoplasmic reticulum. Recent studies have identified that this transporter facilitates the mobilisation of intracellular zinc flux into the cytosol and the subsequent zinc-mediated activation of cell signalling molecules involved in glucose homeostasis. However, the mechanisms whereby zinc and Zip7 achieve glucose control is not known. The present study evaluated the insulin-like effects of zinc on cell signalling pathways associated with controlling glucose homeostasis. Initially, key molecules involved in controlling glucose metabolism including tyrosine, PRSA40, Akt, ERK1/2, SHP-2, GSK-3β and p38, and the physiological response of glucose oxidation were analysed in the presence of zinc treatment in mouse and human skeletal muscle cells. Insulin and zinc treatment independently led to the phosphorylation of these proteins over a 60-minute time course in both mouse and human skeletal muscle cells and was concomitant with an increase in glucose oxidation. Similarly, utilising a commercially available protein array that contains several key proteins implicated in insulin signalling pathways we identified that zinc could active the phosphorylation of p38, ERK1/2 and GSK-3β in human and ERK1/2 and GSK-3β in mouse skeletal muscle cells. Glucose oxidation assays were performed on skeletal muscle cells treated with insulin, zinc, or a combination of both and resulted in a significant induction of glucose consumption in mouse and human skeletal muscle cells when treated with zinc alone. Insulin, as expected, increased glucose oxidation in mouse and human skeletal muscle cells, however the combination of zinc and insulin did not augment glucose consumption in these cells. Given that we did not observe an additive effect of insulin and zinc treatment together on glucose oxidation, we sought to determine whether a functional insulin receptor is required to facilitate zinc activation of pAkt. We utilised mouse C2C12 skeletal muscle cells treated with an insulin receptor tyrosine kinase inhibitor (HNMPA-(AM)3) in the presence of insulin or zinc. We observed that HNMPA-(AM)3 was sufficient to inhibit insulin-induced pAkt. Similarly, we identified that HNMPA-(AM)3 inhibited zinc-induced pAkt and suggests that zinc potentially acts through the insulin signalling pathway. Previous studies have identified that Zip7 controls cell signalling pathways associated with glycaemic control in skeletal muscle cells. It was suggested that dysfunctional cell signalling processes in disease states such as insulin resistance and type 2 diabetes might be due to aberrant Zip7 expression and/or function. Accordingly, we initially set out to determine the role of Zip7 in an insulin-resistant cell culture model. Initially we tested the ability of glucose to regulate Zip7 protein levels. We found that 25 mM of glucose treatment of C2C12 mouse skeletal muscle cells significantly increased Zip7 protein levels. We also determined that Zip7 (and Glut4) were significantly reduced in insulin resistant skeletal muscle cells through inhibition of insulin receptor signalling and palmitate-induced insulin resistance. These studies suggest that Zip7 plays an important role in maintaining cell signalling processes associated with glucose control. We next focused our studies to determine if the expression of Zip7 changed in a mouse model of obesity. We found that Zip7 and the glucose transporter, Glut4 was reduced in obese mice fed a high fat diet. From the above studies we established a Zip7 overexpression cell culture model to test the ability of this transporter to regulate genes implicated in glucose homeostasis. These studies were based on previous work which identified that a reduction in Zip7 in skeletal muscle cells led to several changes in genes associated with insulin signalling and glycaemic control. Accordingly, utilising an insulin signalling pathway array that contained 84 genes involved in insulin signalling, we identified that overexpression of Zip7 in mouse C2C12 skeletal muscle cells led to significant changes in Akt3, Dok2, Fos, Hras, Kras, Nos2, Pck2, and Pparg. In these studies, we demonstrated that zinc acts as an insulin mimetic, activating key molecules implicated in cell signalling to maintain glucose homeostasis in mouse and human skeletal muscle cells. Similarly, these studies demonstrated that Zip7 is involved in processes associated with insulin signalling and glucose control in skeletal muscle. This study also raises the possibility that zinc transporters could provide novel utility to be targeted experimentally and in a clinical setting to treat patients with insulin resistance and thus introduce a new class of drug target with utility for diabetes pharmacotherapy.