Mechanisms of capsaicin and zinc in the control of glucose metabolism in skeletal muscle cells

Signalling pathways play a critical role in a range of metabolic processes including glucose metabolism. Growing evidence suggests that the dysregulation of these signalling events acts as key causative or promoting factors in glucose metabolism disorders including insulin resistance (IR) and type 2 diabetes (T2DM). Accordingly, substantial work has been conducted to investigate the role of these signalling pathways and their stimulators on cellular events that lead to glycaemic control; however, more studies are needed to further examine intracellular signalling pathways involved in glucose metabolism aiming to investigate novel therapeutic targets for IR and T2DM. In recent years there has been an increased interest in studying biochemical and clinical aspects of bioactive food components including zinc and capsaicin on glucose metabolism. Growing evidence supports the beneficial impacts of zinc on glycaemic control in human subjects. Capsaicin, the active ingredient of chilli peppers, also has been shown to improve glucose metabolism in humans. Both zinc and capsaicin seem to induce their effects on glucose homeostasis through the activation of signalling pathways involved in glucose uptake independent of insulin signalling. Therefore, they could substantially benefit patients with disorders associated with abnormal glucose metabolism including IR and T2DM through the stimulation of glucose uptake by bypassing the defective insulin signal transduction. Accordingly, many attempts have been made to shed light on the mechanism of action of zinc and capsaicin in glucose homeostasis. Studies demonstrate that zinc ameliorates glycaemic control through the activation of insulin signal transduction in skeletal muscle. This tissue plays a critical role in maintaining glucose homeostasis in the body. Activation of insulin signalling molecules such as protein kinase B (AKT), extracellular signal-regulated kinase 1/2 (ERK1/2), and SH2 containing protein tyrosine phosphatase-2 (SHP2) lead to glucose transporter type 4 (GLUT4) translocation to the cell membrane of the skeletal muscle cells and subsequently maintains glucose homeostasis in these cells. On the other hand, research shows that capsaicin does not affect the stimulation of the insulin signalling pathway and induces glucose uptake by the activation of other key regulatory proteins including calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) and AMP-activated protein kinase (AMPK). Although research on zinc and capsaicin has revealed important information on their mechanism of action in skeletal muscle glucose metabolism, several crucial signalling events have remained unstudied. Therefore, the present thesis, in three main studies, focused on further investigating the cellular processes involved in glucose uptake induced by zinc and capsaicin, aiming to find novel signalling molecules and pathways regulated by these two bioactive food components in mouse skeletal muscle cells. Throughout studies conducted in this thesis, the skeletal muscle cell line, C2C12, was utilised due to the critical role of skeletal muscle in maintaining whole-body glucose metabolism. Molecular biology techniques including western blotting, cDNA synthesis and quantitative polymerase chain reaction (qPCR), cell-based assays, and microscopy techniques were applied to investigate the impact of zinc and capsaicin on signalling events leading to glucose uptake. Firstly, the activation status of insulin signalling proteins and transcription factors involved in glucose metabolism including AKT, ERK1/2, and cAMP-response element- binding (CREB) was examined in the presence and absence of insulin, zinc, and zinc chelator, N,N,N,N-Tetrakis (2-pyridylmethyl)-ethylenediamine (TPEN), in skeletal muscle cells. Insulin and zinc treatment independently led to the phosphorylation and activation of AKT and ERK1/2 over a 30-minute incubation time in these cells. Moreover, we showed that the blocking of zinc diminishes activation of AKT and ERK1/2 which confirms the crucial role of zinc in the activation of these signalling molecules. Following the responsive effect of zinc on the activation of key insulin signalling proteins, a Cignal Reporter Array was utilised to determine the impact of zinc on the expression of transcription factors. Results demonstrated the upregulation of several transcription factors including nuclear respiratory factors-1/2 (Nrf1/Nrf2), activating transcription factor 6 (Atf6), T-cell factor/lymphoid enhancer factor (Tcf/Lef), early growth response 1 (Egr1), signal transducer and activator of transcription 1 (Stat1), Activator protein 1 (Ap1), peroxisome proliferator-activated receptors (Ppar), and Creb in skeletal muscle cells treated with zinc. Similarly, in the second study, the impact of capsaicin on the activation of signalling proteins involved in glucose metabolism was investigated. Western blot analysis showed that, unlike zinc, capsaicin does not activate insulin signalling molecules including AKT, SHP2, and glycogen synthase kinase-3 β (GSK-3β) and decreases activation of ERK1/2 in the cells. Furthermore, results demonstrated that transient receptor potential cation channel subfamily V member 1 (TRPV1) activation by capsaicin activates other key signalling proteins involved in glucose metabolism such as CAMKK2 and AMPK which was concomitant with an increase in glucose uptake and ATP production in skeletal muscle cells. Finally, due to the critical role of calcium signalling in glucose metabolism, the effect of zinc, capsaicin, and their co-treatment on calcium signal transduction was assessed. The results obtained from live-cell imaging showed that zinc, capsaicin, and their co- treatment induce calcium release from both extracellular space and intracellular stores in the skeletal muscle cells. Data from this study revealed a greater calcium flux in the co-treatment group compared with capsaicin-treated cells but less than in the zinc treatment group. Subsequently, activation of calcium signalling proteins including CAMKK2, CREB, and target of rapamycin complex-2 (TORC2) was assessed in the zinc, capsaicin, and co-treated cells which were concomitant with glucose uptake in the treatment groups except for the co-treated cells. Moreover, the reduction of cytosolic calcium levels by calcium chelator (BAPTA-AM) deactivated CAMKK2, CREB, and TORC1 and reduced capsaicin- and zinc-mediated glucose uptake in mouse skeletal muscle cells. This indicates the significant role of calcium in capsaicin- and zinc-induced signalling events involved in glucose metabolism and glucose uptake in skeletal muscle cells. Moreover, results from live-cell imaging and glucose uptake studies suggest that co-treatment of capsaicin and zinc does not have a synergic effect on calcium flux and glucose uptake in skeletal muscle cells. The results from these studies confirm the essential role of calcium signal transduction in maintaining cell signalling processes associated with glycaemic control in skeletal muscle cells. To conclude, results from this thesis raises the possibility that zinc- and capsaicin- activated signalling events involved in glucose metabolism could provide novel utility to be targeted experimentally and in a clinical setting in helping individuals with IR and T2DM. This also could potentially introduce a new class of medication with advantages for diabetes pharmacotherapy