The Role of Zinc Transporters in Modulating Insulin Signalling

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The Role of Zinc Transporters in Modulating

Insulin Signalling PhD Thesis

Alexander Nield 2015

School of Applied and Biomedical Sciences

Faculty of Science and Technology

Federation University Australia, Mt Helen

Victoria, 3350

Declaration of Authorship

I hereby certify that the thesis I am submitting is entirely my own original work except where otherwise indicated. I am aware of the University's regulations concerning plagiarism, including those regulations concerning disciplinary actions that may result from plagiarism.

Any use of the works of any other author, in any form, is properly acknowledged at their point of use.

About This Thesis

This thesis contains 6 chapters in total, with Chapter 1 being a literature overview and

Chapter 2 being a summary of materials and methods. The three results chapters contain all

my own work with the exception of Chapter 4, which includes data that has been published as

a paper containing team-based research on which I was equal first author with my associate

supervisor Dr Stephen Myers. Results chapter contributions are summarised in Table i.

Chapter 6 contains the final discussions and conclusions, linking together the work

undertaken in this thesis.

Table i: Contribution to thesis results chapters. Chapter Title Extent of Candidates Contribution 3 Understanding the Structure and Designed study and carried out all Function of Zip7: A Histidine experiments. Wrote the chapter. Rich Zinc Transporter 85% Implicated in Cellular Signalling 4 The Zinc Transporter Zip7 is Co-designed study and carried out most Implicated in Glycaemic experiments. Co-wrote the chapter. Control in Skeletal Muscle Cells 55% 5 Insulin Induces an Increase in Designed study and carried out all Cytosolic Free Zinc in C2C12 experiments. Wrote the chapter. Skeletal Muscle Cells as a 85% Positive Feedback Mechanism for Insulin Signalling

Each results chapter is presented in a publication ready format, with figures and figure

legends shown at the end of each chapter. This resulted in some content overlap and repetition in introductory concepts and methodologies. Each chapter also has a separate references section.

Thesis Summary

Zinc is a cell impermeable transition metal with a large number of biological functions, and is an essential component of the insulin signalling pathway. Cellular free zinc increases insulin sensitivity though it is also toxic at high levels, making it essential for cells to tightly regulate bioavailable levels. This homeostasis is maintained by three groups of proteins known as

Zips, ZnTs and metallothioneins (MTs). Zips and ZnTs are zinc transporters with Zips increasing cytosolic zinc by pumping it outside the cell or from organelle stores, while the

ZnTs decrease cytosolic zinc. The MTs bind to free zinc in the cytosol, reducing its bioavailability. The rapid release of zinc mediated by these proteins has been implicated as a mechanism of signal pathway activation, through zinc activating and deactivating various signalling proteins. This thesis investigated one zinc transporter in particular, known as Zip7.

Zip7 is a novel Zip transporter localised to the endoplasmic reticulum and has been implicated in cell signalling in breast cancer cells through release of zinc from cellular stores in response to extracellular stimuli. The aim of this thesis was to investigate the potential role of this zinc transporter in modulating the insulin signalling pathway.

The human Zip7 protein sequence was analysed using various bioinformatics tools to identify regions that may contribute to the proposed novel function of this transporter. The loop regions of Zip7 were found to be poorly conserved between species with the exception of histidine rich regions, which showed a high level of conservation when compared to a diverse series of species and so are suspected to have an essential role in modulating the transport function of Zip7 by binding to zinc. These findings implicate histidine residues as an important functional component of Zip7.

In order to identify whether Zip7 expression is essential for a normal insulin response, Zip7 mRNA was reduced via transfection of siRNA in mouse skeletal muscle cells and

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measurement of markers of insulin signalling. When Zip7 expression was reduced there was a

subsequent decrease in the expression of several markers of insulin signalling including Glut4 protein levels, Akt phosphorylation and insulin-mediated glycogen synthesis, indicating that

the cells were insulin resistant compared to the control.

It was hypothesised that given the proposed role for Zip7 in mediating rapid zinc release and

that Zip7 expression is important for normal insulin signalling, Zip7 activation is stimulated

by insulin treatment to temporarily increase cytosolic zinc bioavailability as a positive

feedback mechanism for prolonging pathway activation. To test this, live cell imaging of zinc

flux in cells was performed in cells with reduced Zip7 expression compared to controls.

Insulin was shown to cause an increase in cytosolic zinc in C2C12 cells. However when Zip7

expression was reduced, even though the cells showed signs of insulin resistance, there was

still an increase in zinc levels mediated by insulin. Insulin treatment is known to induce

cellular ROS production and hydrogen peroxide has been suggested to cause a release of zinc

due to oxidation of MTs leading to a release of bound zinc. These findings indicate that

insulin-stimulated zinc flux is the result of MT oxidation rather than Zip7 activation.

Taken together, these results highlight an important role for Zip7 in the insulin signalling

pathway and show a previously undescribed positive feedback loop whereby insulin mediates

a release of zinc to potentially inhibit PTP1B and other phosphatases to prolong insulin

signalling activation. Further work is needed to fully elucidate the role of Zip7 in this

pathway.

Acknowledgments

Firstly I would like to thank my primary and associate supervisors, A/Prof Mark Myers and

Dr Stephen Myers (no relation). Mark was always a font of wisdom and guidance throughout

my candidature and I will be eternally grateful for all his support. Steve provided me with an

incredible level of support during the first two years of my PhD and gave me the skills

needed go on to complete a thesis that I am proud of. I am extremely lucky to have had the opportunity to work with these two world-class researchers and cannot express the level of my gratitude for all of their help.

I would also like to thank all the friends and family who have not seen much of me over the last few years. I promise to make up for lost time with everyone now that I am taking the final steps towards leaving this PhD cave. Thank you especially to my parents for their love and support over the past 27 years.

A huge thank you to all of my work colleagues for all the support and friendship, with a special mention to the zinc research team comprised of Farah Ahmady, Michelle Maier,

Ingrid Wise, Scott Nankervis and Usha Kalluri. Also thank you to Scott Boon for all of the future career conversations to keep the motivation levels up. Thank you to the biomedical science Technical Officer Fahima Ahmady for all of her assistance over the years.

Publications

Peer Reviewed Journal Articles

Published Works

• Myers, S.A., Nield, A., and Myers, M. (2012). Zinc transporters, mechanisms of action and therapeutic utility: implications for type 2 diabetes mellitus. J Nutr Metab 2012, 173712.

• Nield, A*., Myers, S.A*., Chew, G.S., and Myers, M.A. (2013). The zinc transporter, slc39a7 (zip7) is implicated in glycaemic control in skeletal muscle cells. PLoS One 8, e79316.

*Contributed equally

Book Chapter

• Myers, S., Nield, A (2014). Zinc, Zinc Transporters and Type 2 Diabetes. In Endocrine Diseases.

In Preparation

• Nield, A., Myers, SA, Myers, M., Increased cytosolic free zinc in C2C12 skeletal muscle cells as a positive feedback mechanism for insulin signalling.

Conference Contributions

• Poster Presentation – International Society for Zinc Biology 4th biennial meeting, Pacific Grove, CA (2014) – Awarded ISZB poster prize

• Oral presentation - Federation University Postgraduate Research Conference, Victoria, Australia (2014)

• Poster presentation - Federation University Postgraduate Research Conference, Victoria, Australia (2013)

• Poster presentation - 52nd annual ASMR National Scientific Conference, Victoria, Australia (2013)

• Poster presentation - BIT's 3rd Annual World Congress of Endobolism, Xi’an, China (2013)

• Oral presentation - University of Ballarat Postgraduate Research Conference, Victoria, Australia (2012)

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Abbreviations

PTP Protein tyrosine phosphatase ZnT/SLC30A Zinc efflux transporter Zip/SLC39A Zinc influx transporter MT Metallothionein TMD Transmembrane domains ROS Reactive oxygen species MAPK Mitogen-activated kinase ER Endoplasmic reticulum IR Insulin receptor IRS Insulin receptor substrates PI3-K Phosphoinositide 3-kinase

PIP2 Phosphatidylinositol (4, 5)-bisphosphate

PIP3 Phosphatidylinositol (3, 4, 5)-trisphosphate PDK 3-phosphoinositide-dependent protein kinase GSK-3 Glycogen synthase kinase-3 GS Glycogen synthase GLUT Glucose transporter T2DM Type 2 diabetes mellitus

H2O2 Hydrogen peroxide SOD Superoxide dismutase IGF Insulin-like growth factors IGFR IGF Receptor AGE Advanced glycation end-product RAGE Receptors for glycation end-product LIRKO Liver-specific IR knockout mouse MIRKO Muscle-specific IR knockout mouse BIRKO Beta-cell specific knockout mouse FIRKO Fat-specific IR knockout mouse BATIRKO Brown adipose tissue knockout mouse GWAS Genome-wide association study EZS Early zinc signalling

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LZS Late zinc signalling CK-2 Casein kinase-2 MINNOU Membrane protein IdeNtificatioN withOUt explicit use of hydropathy profiles and alignments SLiM Short linear motifs ELM Eukaryotic Linear Motif siRNA Small interfering RNA qPCR Quantitative PCR GOI Gene of interest GAPDH Glyceraldehyde 3-phosphate dehydrogenase EEF2 Eukaryotic elongation factor 2 BCA Bicinchoninic acid BSA Bovine serum albumin HRP Horseradish peroxidase TBHP Tert-butyl hydrogen peroxide TTHB Tetra-thiomolybdate LZT LIV-1 subfamily of Zip zinc Transporters AA Amino acid LXR Liver X receptor PPAR Peroxisome proliferating activated receptor EGF Epidermal growth factor

Table of Contents

Declaration of Authorship ...... I About This Thesis ...... II Thesis Summary ...... III Acknowledgments ...... V Publications ...... VI Peer Reviewed Journal Articles ...... VI Published Works ...... VI Book Chapter ...... VI In Preparation ...... VI Conference Contributions ...... VII Abbreviations ...... VIII Chapter 1: Literature Review ...... 1 Zinc in Biology...... 2 Catalytic Roles ...... 2 Structural Roles ...... 3 Regulatory Roles ...... 3 Zinc in Health ...... 3 Cellular Zinc Homeostasis ...... 4 ZnTs ...... 7 Zips ...... 7 Metallothioneins ...... 8 Cellular Zinc Storage ...... 10 Zinc and Insulin Signalling ...... 10 Insulin Synthesis ...... 11 Insulin Signalling Pathway ...... 11 PTP1B ...... 15 Insulin-stimulated ROS production ...... 15 Blood Sugar Homeostasis ...... 16 Glucagon ...... 16 Incretins ...... 16

Insulin-like Growth Factors ...... 16 Diabetes Mellitus...... 17 Type 2 Diabetes ...... 17 Insulin Resistance ...... 18 Oxidative Stress ...... 18 β-Cell Dysfunction ...... 19 Disease Progression ...... 19 Current Treatments ...... 20 Diabetic Animal Models ...... 20 Zinc in Diabetes ...... 22 Effect of Zinc Supplementation on Glycaemic Control ...... 22 ZnT8 and Insulin Production ...... 22 Zinc Flux and Insulin Signalling ...... 23 GHTD-amide ...... 24 MTs and Zinc Signalling ...... 25 The Emerging Role of Zip7 ...... 25 Conclusion ...... 26 References ...... 27 Chapter 2: Materials and Methods ...... 51 Introduction ...... 52 Protein Sequence Information ...... 52 Protein Sequence alignments and phylogenetic analysis ...... 53 Signal Peptide Prediction...... 53 TMD Prediction ...... 53 Prediction of SLiMs...... 53 C2C12 Skeletal muscle cells ...... 54 Cell Culture...... 54 siRNA Transfection ...... 54 RNA Extraction ...... 55 cDNA synthesis ...... 55 Primer Design ...... 56 Real-time PCR ...... 57

Gene expression analysis ...... 58 Protein Extraction ...... 59 Bicinchoninic acid assay ...... 59 Western Blotting ...... 59 Fluorescent Zinc Imaging ...... 60 Fluorescent ROS Imaging ...... 61 Confocal Microscopy ...... 61 Fluorescence Intensity Analysis ...... 62 References ...... 63 Chapter 3: Understanding the Function of Zip7: A Histidine Rich Zinc Transporter Implicated in Cellular Signalling ...... 65 Abstract ...... 66 Introduction ...... 67 Methods ...... 69 Obtaining Protein Sequence Information ...... 69 Protein Sequence Alignments and Phylogenetic Analysis ...... 70 Signal Peptide Identification ...... 70 Predictive Analysis of Transmembrane Domains ...... 70 Identification of Functional Motifs...... 71 Characterisation of Histidine rich regions in the human Zip family members ...... 71 Results ...... 71 Zip7 secondary structure...... 71 Histidine residues are conserved across a broad range of species...... 72 ELM Database analysis reveals key areas of importance in the activation and cellular localisation of Zip7...... 72 Zip7 is most closely related to Zip13 and has high histidine content in loop regions. .... 73 Zip7 has a unique profile of SLiMs compared to other Zips...... 74 Discussion ...... 77 References ...... 81 Figure Legends ...... 87 Chapter 4: The Zinc Transporter Zip7 is Implicated in Glycaemic Control in Skeletal Muscle Cells ...... 92 Abstract ...... 93

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Introduction ...... 94 Materials and Methods ...... 95 Cell culture ...... 95 RNA Extraction and cDNA Synthesis...... 96 Mouse glucose metabolism and zinc transporter arrays ...... 96 cDNA synthesis of RT2 mouse glucose metabolism and zinc transporter PCR array ..... 96 Quantitative Real-time PCR ...... 97 Quantitative Real-time PCR of RT2 mouse glucose metabolism and zinc transporter PCR array ...... 97 Transient transfections of siRNA molecules ...... 98 Protein Extraction and Western Blot ...... 98 Glycogen synthesis assay ...... 99 Statistics ...... 99 Results ...... 100 The Slc39a (Zip) zinc transporters are differentially expressed in C2C12 skeletal muscle cells and mouse quadriceps...... 100 Slc39a7 (Zip7) is expressed during C2C12 skeletal muscle differentiation...... 100 siRNA-Zip7 represses endogenous Zip7 mRNA in skeletal muscle cells...... 101 Zip7 knockdown resulted in no change in other zinc transporters...... 102 Reduced expression of Zip7 in C2C12 cells is associated with changes in several genes implicated in glucose metabolism...... 103 Reduced Zip7 compromises insulin-induced glycogen synthesis and phosphorylation of Akt in C2C12 skeletal muscle cells...... 104 Discussion ...... 105 References ...... 112 Chapter 5: Insulin Induces an Increase in Cytosolic Free Zinc in C2C12 Skeletal Muscle Cells as a Positive Feedback Mechanism for Insulin Signalling ...... 128 Abstract ...... 129 Introduction ...... 130 Methods ...... 132 Cell culture ...... 132 Transient siRNA transfection ...... 133 RNA extraction and cDNA synthesis ...... 133

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Real-time PCR ...... 134 Determining myoblast insulin responsiveness and the effect of impermeable and permeable zinc on Akt phosphorylation ...... 134 Protein extraction and Western blot ...... 135 Inhibition of insulin signalling using wortmannin ...... 135 Live cell fluorescent probe staining for analysis of zinc flux ...... 136 Confocal microscopy ...... 136 Fluorescence intensity analysis ...... 137 Reactive oxygen species analysis ...... 137 Results ...... 137 Characterising insulin response in C2C12 skeletal muscle myoblasts...... 137 Insulin induces an increase in cytosolic zinc fluorescence...... 138 Insulin mediated cytosolic zinc increase in skeletal muscle cells was inhibited by wortmannin...... 139 Reduction in Zip7 expression results in reduced insulin-mediated Akt phosphorylation...... 139 Transient Zip7-siRNA transfection results in no change in the insulin-stimulated increase in cytosolic free zinc...... 139 MT1 and 2 gene expression is increased when Zip7 expression is reduced...... 140 Insulin treatment of C2C12 myoblasts results in increased cellular ROS production. .. 140 Discussion ...... 140 References ...... 146 Figure Legends ...... 152 Chapter 6: Discussion and Conclusions ...... 166 Overview ...... 167 Zip7 has a high level of evolutionary conservation ...... 167 Zip7 knockdown reduces skeletal muscle insulin sensitivity ...... 169 Insulin treatment stimulates increased cytosolic zinc in skeletal muscle...... 170 Zip7 silencing does not alter insulin-mediated zinc release ...... 171 ROS and zinc signalling ...... 172 Conclusion ...... 175 Future Studies ...... 176 References ...... 178

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Chapter 1: Literature Review

Zinc in Biology

Zinc is an essential trace element required for the normal physiological function of all living

organisms [1], with new biological roles being discovered on a regular basis. The essential

role of zinc in biology was first shown in 1869 when it was discovered to be required for the

growth of the common bread mould Aspergillus niger [2]. The importance of zinc in biology

was slowly pieced together over the next 100 years following this preliminary finding.

Initially zinc proved a difficult ion to study in comparison to similar transition elements such

as iron, as it is found in very low concentrations and researchers lacked simple methods of

detection [3]. As more sensitive detection methods have been developed in recent years, there

has been an explosion of discoveries in the field of zinc biology.

Zinc has a number of interesting properties that make it unique amongst transition metals

such as iron and copper. Zinc rarely undergoes redox reactions due to a filled orbital d shell

[3], making it a relatively stable ion that instead functions as a Lewis acid, accepting two

electrons from reactants [4]. These properties make this metal surprisingly nontoxic

compared to other metals and highly versatile in how it can be used in biological functions.

Numerous roles for zinc are currently known which are separated into three general groups being catalytic, structural and regulatory [5].

Catalytic Roles

Zinc is essential for the function of numerous enzymes, with one of the first major findings of

the involvement of zinc in biology being the discovery of carbonic anhydrase requiring zinc

to function [6]. Zinc has since been found to be required for the activity of over 300 enzymes

[7], with each of the six enzyme classes having zinc-requiring enzymes. The zinc ion generally acts as a cofactor for the function of the enzyme, but also has co-catalytic roles,

increasing the efficacy of other metal cofactors. Zinc may also have a structural role in

shaping the active site of the enzyme [7].

Structural Roles

Zinc was shown to be an essential element in protein structure and folding with the discovery

of zinc finger protein structures in 1985 [8]. These zinc fingers were found to contain a

repeating cysteine (C) and histidine (H) C2H2 zinc binding motifs that creates a finger-like structure capable of binding nucleic acid. This discovery revealed that zinc is essential for the formation of transcription factors and hence is required for gene regulation. Through searching the sequenced human genome it is estimated that zinc is required for the structure and folding of a predicted 3000 proteins [9].

Regulatory Roles

In more recent years, zinc has been found to have a regulatory role, acting as a secondary messenger in numerous signalling pathways through the inhibition or activation of various proteins [10]. This regulation is independent of zinc-requiring enzymes, instead being the result of zinc ions directly interacting with proteins to modulate their activity. An example of one such role is the modulation of tyrosine kinase receptors through the inhibition of protein tyrosine phosphatases (PTPs) [11]. PTPs dephosphorylate tyrosine kinase-linked receptors and zinc-mediated inhibition of PTPs increases the duration of receptor activation. Cellular cytosolic zinc concentrations have also been shown to be regulated by extracellular stimuli

[10], supporting the notion that zinc acts as an intracellular messenger in signalling pathways.

Zinc in Health

Zinc is a trace metal of relatively high abundance within humans with only iron having a

higher concentration. The vital importance of zinc in human health was first shown in 1961

when Prasad and associates revealed that zinc deficiency resulted in stunted growth and

impaired sexual development in a group of young males from Shiraz, Iran [12]. These effects

were found to be almost entirely reversed through zinc supplementation [13]. Before this initial discovery, little thought was given regarding zinc as an essential trace element for

growth and development. Zinc deficiency is now known as a major health risk, especially in

developing countries [14]. Deficiencies in zinc can lead to immune system dysfunction [15]

and growth retardation [16] whilst overabundance causes uncontrolled cell growth, as well as

abnormalities in cell migration and organism development [17]. Zinc has also been identified

as being essential for normal nervous system function [18] and bone formation [19] and has

been shown to be a negative regulator of apoptosis [20].

There are numerous diseases associated with aberrant zinc levels such as Alzheimer’s disease

[21], Acrodermatitis enteropathica [22], cardiovascular disease [23] and certain types of cancer [24, 25] (Table 1). Dysfunction of zinc homeostasis is also strongly linked to aberrant

insulin signalling and may potentially have a role in the pathophysiology of diseases such as

diabetes [26, 27].

Cellular Zinc Homeostasis

Due to the numerous biological roles of zinc as well as the inability of free zinc cations to passively diffuse across lipid membranes due to their charge, the management of cellular zinc homeostasis via specific transport proteins is essential for normal cell physiology. A large proportion of recent zinc biology research has been focused on understanding the mechanisms of zinc homeostasis. As different amounts of zinc are necessary for various functions, cells must also be able to manipulate the amount of bioavailable zinc within the cytosol as required. To this end, there are a large number of proteins responsible for the regulation of cellular zinc, all with various patterns of tissue expression and cellular localisation. These proteins are divided into three main families, with the SLC30A (ZnT) and

SLC39A (Zip) solute carrier families being the two primary protein groups responsible for the transport of zinc across membranes [28]. The ZnT family of zinc transporters reduce cytosolic zinc by transporting it into organelles for storage or out of the cell. The Zips have the opposite function to the ZnTs, excreting zinc into the cytosol from intracellular stores or

4 across the plasma membrane. The third family of proteins is a group of heavy metal binding metallothioneins (MTs) that act as a zinc muffler by binding zinc and so reducing its bioavailability. In mammals, there are 10 members of the ZnT family and 14 members of the

Zip family and 4 well characterised MTs [29]. The tissue expression and cellular localisation for the majority of the zinc transporters and MTs has been well studied in recent years (Table

1) however their various functions and mechanisms of action are still yet to be fully understood. Due to zinc being involved in various cellular signalling pathways, manipulation of zinc transporter gene expression and protein activation are potential methods of regulating these pathways.

Table 1: Zinc transporters and metallothioneins; cellular location, tissue-specific expression and disease associations.

Zinc Transporter Cellular Localisation Tissue Expression Disease Refs Associations SLC39A1/Zip1 Plasma membrane Ubiquitously expressed Prostate Cancer [30-32] SLC39A2/Zip2 Plasma membrane Blood, prostate Carotid Artery Disease [33-35] SLC39A3/Zip3 Plasma membrane, Mammary gland, Unknown [31, 36] intracellular compartments prostate SLC39A4/Zip4 Apical membranes Small intestine, Pancreatic Cancer, AE [22, 25, stomach, colon, kidney, 33] brain SLC39A5/Zip5 Basolateral membranes Pancreas, kidney, liver, Unknown [37, 38] spleen, colon, stomach SLC39A6/Zip6 Plasma membrane Ubiquitously expressed Breast Cancer [39] SLC39A7/Zip7 Golgi apparatus, Ubiquitously expressed Breast Cancer [40, 41] endoplasmic reticulum SLC39A8/Zip8 Vesicles Ubiquitously expressed Unknown [42, 43] SLC39A9/Zip9 Trans Golgi network Ubiquitously expressed Unknown [44] SLC39A10/Zip10 Plasma membrane Ubiquitously expressed Breast Cancer [45, 46] SLC39A11/Zip11 Unknown Mammary gland Unknown [47] SLC39A12/Zip12 Unknown Retina, brain, testis, Schizophrenia [48] lung SLC39A13/Zip13 Golgi apparatus Ubiquitously expressed Ehlers-Danlos syndrome [49, 50] SLC39A14/Zip14 Plasma membrane Ubiquitously expressed Asthma [51, 52] SLC30A1/ZnT1 Plasma membrane Ubiquitously expressed Alzheimer's disease, [21, 53, Pancreatic cancer 54] SLC30A2/ZnT2 Vesicles, lysosomes Pancreas, kidney, testis, Unknown [55] epithelial cells, small intestine SLC30A3/ZnT3 Synaptic vesicles Brain, testis Alzheimer's disease [56, 57] SLC30A4/ZnT4 Intracellular compartments Mammary gland, brain, Alzheimer's disease [21, 58] small intestine, placenta, blood, epithelial cells SLC30A5/ZnT5 Secretory vesicles, Golgi Ubiquitously expressed Osteopenia [59, 60] apparatus SLC30A6/ZnT6 Secretory vesicles, Golgi Small intestine, liver, Alzheimer's disease [21, 61] apparatus brain, adipose tissue SLC30A7/ZnT7 Golgi apparatus Retina, small intestine, Prostate Cancer [62, 63] liver, blood, epithelial cells, spleen SLC30A8/ZnT8 Secretory vesicles Pancreatic B-cells Type 1 and 2 diabetes [64-66] mellitus SLC30A9/ZnT9 Cytoplasm, nucleus Ubiquitously expressed Unknown [67] SLC30A10/ZnT10 Unknown Liver, brain Parkinson's disease, [68-70] dystonia, liver disease MT1 Cytoplasm, nucleus, Ubiquitously expressed Unknown [71, 72] mitochondria MT2 Cytoplasm, nucleus, Ubiquitously expressed Unknown [73, 74] mitochondria MT3 Cytoplasm, nucleus Brain, Testis Alzheimer's disease [75] MT4 Cytoplasm, nucleus Squamous epithelia Unknown [76, 77]

ZnTs

The ZnT family of zinc transporters are responsible for decreasing the level of cytosolic zinc

either by transporting it out of the cell or by moving it into organelles for storage [78]. There are 10 members of the mammalian ZnT family, most of which are predicted to have 6 transmembrane domains (TMDs) with the amino and carboxy termini predicted to be located in the cytosol [28]. ZnT5 is an exception to this, with a total of 15 predicted TMD regions

[60]. The first discovered ZnT, known as ZnT1, was initially identified as a protein that protects cells from toxicity associated with high levels of zinc [54]. Mutations of ZnT1 resulted in cells that were more susceptible to zinc toxicity [54]. The majority of the ZnTs are associated with cellular organelles, with ZnT1 being the only currently known plasma membrane associated ZnT [54], indicating that the greater part of excess cellular zinc is stored within organelles rather than excreted.

Zips

The Zip family of zinc transporters was named due to their similarity to the family of Iron

transporters and hence given the name “Zrt-, Irt-like Proteins” or Zips. This family of zinc

transporters is responsible for increasing the concentration of cytosolic zinc by facilitating

influx from outside the cell or via efflux from intracellular compartments [79]. This was shown with the first discovered member of the Zip family known as Zrt1 (Zip1) [80], with overexpression of this zinc transporter causing an increase in cellular zinc uptake, whilst

down-regulation resulted in poor growth when cultured in low zinc conditions [80]. Fourteen

members of the Zip family have thus far been identified in mammals [81], the majority of which are predicted to contain 8 TMDs with the amino and carboxy termini predicted to be extracellular [28]. A number of Zips contain a histidine rich cytosol orientated loop between

TMDs III and IV which is believed to play an important role in their function through the binding of zinc. This region has been predicted to act as a cytosolic zinc sensor, which may

activate or deactivate transport depending on cytosolic zinc availability [82]. Studies with

Zip4 have shown that the binding of zinc to this region results in protein degradation [82],

potentially acting as a negative feedback system for preventing excess build-up of zinc within

the cytosol.

Metallothioneins

The MTs are a family of cysteine rich, low molecular weight proteins that bind a variety of

heavy metals including cadmium, copper and zinc [83]. These proteins are capable of binding

4-12 heavy metal ions per molecule. There are four mammalian MTs, designated MT1 to 4.

MT1 and MT2 were first discovered in 1957 with the isolation of a cadmium associated protein from equine kidney cortex [84]. These two proteins are ubiquitously expressed in most tissues and their gene expression has been shown to be regulated by a number of factors including reactive oxygen species (ROS), cytokines and heavy metal concentrations [85].

MT3 and MT4 were discovered in the 1990s and have restricted tissue expression [83]. MT3 is primarily expressed in the brain [86], while MT4 is expressed in squamous epithelial cells during differentiation [87]. Although the physiological functions of these proteins are not yet well understood, the MTs are believed to play an important protective role within cells by binding free heavy metals within the cytosol, limiting their bioavailability. The metal binding functional regions of MTs are rich with thiol clusters which can be oxidised, leading to a release of bound zinc [88, 89]. This has been proposed as a possible functional mechanism by which zinc levels are regulated by cellular ROS levels.

Figure 1: Cellular localisation of Zip and ZnT zinc transporters. A generalised mammalian cell showing the localisation and predicted domains of the various Zip (red) and ZnT (green) zinc transporters with the arrows indicating the flow of zinc across the plasma membrane and organelles. The figure was produced using Servier Medical Art, www.servier.com.

Cellular Zinc Storage

Zinc is required for the normal physiological function of all living organisms, but is also toxic

in large enough doses [90]. An average 70kg adult will contain approximately 2.3g of zinc in

total [91] though plasma zinc concentrations are kept around 16µM [92]. Significant zinc-

mediated enzyme inhibition occurs above concentrations of 10-8M [93] and hence it has been

estimated that cytosolic bioavailable zinc concentrations are maintained around this level

[17]. The remaining cellular zinc is stored within cellular organelles until required. Cells have

been found to contain vesicles with high zinc concentration [17] called zincosomes, which act

as a site of rapid storage and efflux of zinc as required by cellular processes. There is currently very little known regarding these zinc vesicles, though they have been identified as late endosomes in cultured astrocytes using fluorescent microscopy [94]. These zinc-rich

vesicles were found to rapidly fluoresce when the cells were treated with 10 µM zinc. The

formation of these zinc vesicles has been shown to be inhibited by the presence of ethanol

[95], indicating a negative impact of alcohol consumption on cellular zinc uptake.

Zinc and Insulin Signalling

Zinc is known to be an important component of insulin signalling, an essential pathway for

the homeostasis of blood glucose levels. Zinc is involved in the synthesis, storage and efflux of insulin from the pancreas and is also involved in modulating the signalling pathway initiated by this peptide hormone. The primary role of insulin signalling is to stimulate the

uptake of glucose from the bloodstream (summarised in Figure 2). It is also responsible for

the storage of glucose as glycogen in the liver. The insulin signalling pathway is linked to a number of other important pathways such as mitogen-activated kinase (MAPK) and growth

factor signalling associated with cell growth and survival.

Insulin Synthesis

Insulin is a 51 amino acid peptide hormone produced in the β-cells of the islets of Langerhans within the pancreas and is a key regulator of carbohydrate and lipid metabolism. It is initially synthesised as a precursor known as preproinsulin, containing an endoplasmic reticulum (ER) retention signal peptide. After cleavage of the signal peptide within the ER, this molecule becomes proinsulin. The peptide is then cleaved again into its mature form and packaged by the Golgi apparatus into secretory granules. These vesicles are then ready to be translocated to the plasma membrane to secrete the mature insulin in response to increases in blood glucose concentrations. It was first discovered nearly 100 years ago that zinc is essential for the crystallisation of insulin [96]. It was later discovered that mature insulin is stored as hexamers formed around 2 zinc cations [97]. These hexamers are essential for the normal function of insulin as they increase stability. Insulin is excreted into the blood stream in its hexameric form where it is slowly broken down into monomeric peptides [98]. This increases the duration and reduces the intensity of insulin action.

Studies of insulin release of rat pancreas have revealed two phases of insulin secretion known as rapid and slow release respectively [99]. Rapid insulin release occurs within minutes after glucose stimulation, with a high concentration of insulin being rapidly released to counteract a postprandial spike in blood glucose levels. The slow release insulin secretion involves a steady, low concentration release of insulin that continues for as long as the glucose stimulus is present.

Insulin Signalling Pathway

Insulin signalling is a complex pathway initiated by insulin binding to the extracellular domain of the insulin receptor (IR), which spans the plasma membrane of insulin sensitive cells (illustrated in Figure 2). The IR is made up of two extracellular alpha subunits

connected to two transmembrane beta subunits that contain intracellular tyrosine kinase

domains. The binding of insulin to the IR leads to autophosphorylation of three tyrosine

residues (Tyr1146, Tyr1150 and Tyr1151) on each beta subunit which induces the kinase

activity of this receptor. The activated kinase phosphorylates a group of adaptor molecules

known as insulin receptor substrates (IRS). IRS proteins are scaffold proteins essential for

insulin signal transduction. There are four IRS proteins found in mammals (IRS1 to 4), with

an additional two (IRS5 and 6) found only in humans [100].

When phosphorylated by the insulin receptor, IRS proteins bind to phosphoinositide 3-

kinases (PI3-Ks) leading to its activation. PI3Ks are a group of enzymes that phosphorylate

inositides. These lipids control a number of cellular processes associated with cell growth and

division [101] and play a key role in insulin signalling through the phosphorylation of

phosphatidylinositol (4, 5)-bisphosphate (PIP2) to phosphatidylinositol (3, 4, 5)-trisphosphate

(PIP3). PIP3 in turn binds to Akt at the plasma membrane so allowing its phosphorylation and

activation by 3-phosphoinositide-dependent protein kinase (PDK) [102].

When active, Akt phosphorylates a number of proteins associated with metabolic pathways

such as glycogen synthase kinase-3 (GSK-3). Phosphorylation of GSK-3 causes kinase

inhibition leading to reduced phosphorylation of glycogen synthase (GS). GS is inactive in its

phosphorylated form and hence the inhibition of GSK leads to increased glycogen synthesis

[103].

Activation of PDK1 also leads to a cascade of phosphorylation events culminating in the

translocation of vesicles carrying glucose transporters from the cytosol to the plasma

membrane [104]. There are a number of glucose transporters with different tissue expressions

and glucose affinities. These transporters are separated into three classes, with the four

primary glucose transporters in mammals (Glut1 to 4) comprising Class I. Classes II and III

are made up of a group of recently characterised glucose-like transporters [105]. The two

primary transporters associated with insulin-mediated glucose disposal are Glut2 and Glut4 while Glut3 is responsible for glucose uptake in neurons [106]. Glut2 is a bidirectional, low affinity glucose transporter that is primarily associated with the liver and pancreas [107].

Glut4 is the primary glucose transporter protein responsible for insulin mediated cellular glucose uptake [108]. This transporter is primarily expressed in skeletal and cardiac muscle and adipose tissues. Skeletal muscle, adipose and liver tissues are the three primary sites for insulin activity [104] and skeletal muscle is believed to be the site of the majority of insulin- mediated glucose disposal [109].

Figure 2: The role of zinc in the insulin signalling pathway. Insulin binds to the insulin receptor which activates a cascade of phosphorylation events leading to the translocation of Glut4 to the plasma membrane. PTP1B acts through the dephosphorylation of the insulin receptor which prevents insulin signalling from occurring. The function of PTP1B is inhibited by cellular free zinc which is regulated by the Zip (red) and ZnT (green) zinc transporters. The arrows indicate the flow of zinc through the plasma membrane and cellular organelles. The figure was produced using Servier Medical Art, www.servier.com.

PTP1B

PTPs are a family of enzymes responsible for the dephosphorylation of tyrosine residues. A

member of the PTP family known as PTP1B has been found to be a potent negative regulator

of insulin signalling through dephosphorylation of the IR. This phosphatase is activated by

the phosphorylated IR as a part of a negative feedback system [110]. Knockout studies of

PTP1B in skeletal muscle cells [111] and mice [112] have shown that insulin sensitivity increases in the absence of this phosphatase due to an increased level of IR phosphorylation and subsequent downstream effects. Knockout mice were also found to be resistant to diet- induced obesity [113]. PTP1B deficiency has been shown to improve glucose homeostasis in a polygenic insulin resistant mouse model [114]. This had led to PTP1B becoming an increasingly popular target for research for the treatment of diseases associated with insulin resistance such as Type 2 diabetes mellitus (T2DM) [115]. Furthermore, PTP1B has been shown to be inhibited by zinc [11, 116-118], revealing a potential zinc-modulated mechanism of controlling insulin signalling activity.

Insulin-stimulated ROS production

Insulin signalling is known to increase cellular ROS production [119, 120], with hydrogen peroxide (H2O2) being a signalling molecule that positively regulated insulin signalling [119],

even being described as having insulin-mimetic properties [121]. Increased insulin signalling

induced by H2O2 appears to be mediated by H2O2-mediated inhibition of PTP activity [120].

Insulin signalling stimulates increased mitochondrial ROS production in the form of highly

reactive superoxides, which are linked to oxidative stress and cell death [122]. These are then

converted to the less reactive and more easily diffused H2O2 via the activity of a family of

metalloenzymes known as superoxide dismutases (SODs). There are three well characterised

SOD forms in mammals known as SOD1 to 3. SOD1 is cytosolic [123], SOD2 is

mitochondrial [124] and SOD3 is extracellular [125]. SOD has been implicated as being

essential for the production of H2O2, which acts as a signalling molecule in the insulin

signalling pathway [126].

Blood Sugar Homeostasis

Blood sugar homeostasis is an essential mechanism for life that is regulated via a complex

series of pathways. In addition to insulin, there are a number of hormones involved in this complex balance including glucagon, incretins and insulin-like growth factors.

Glucagon

Glucagon is a hormone with the opposite effect on glucose metabolism to that of insulin. This hormone is secreted by the A-cells of the islets of Langerhans within the pancreas. Glucagon acts by stimulating breakdown of hepatic glycogen stores into glucose, resulting in an increase in blood glucose. Glucagon secretion is inhibited by insulin [127] as a negative feedback mechanism for limiting postprandial hepatic glucose secretion.

Incretins

Incretins are a family of proteins that are secreted by the gastrointestinal tract in response to food uptake. These hormones enhance the secretion of insulin from the pancreas. Orally ingested glucose has been shown to stimulate a greater insulin response compared to intravenous glucose uptake [128], highlighting the important role for incretin hormones in managing blood glucose homeostasis.

Insulin-like Growth Factors

The insulin-like growth factors (IGFs) are a family of peptide hormones that regulate a number of growth associated pathways including insulin signalling. This group is made up of two primary peptide hormones; IGF-1 and IGF-2 and a group of six IGF binding proteins

(IGFBPs) that act as carriers [129]. IGF-1 is believed to be the primary functional peptide and

is released from the liver in response to growth hormone. This peptide is capable of binding

16 to, and activating, the insulin receptor, although with poor affinity compared to insulin. IGF function is primarily modulated through IGF Receptor (IGFR) binding. There are two IGF receptors, IGF1R and IGF2R. Both IGF peptides are capable of binding to both receptors.

IGF1R is the response receptor while IGH2R binds and internalises without generating a physiological response, acting as a clearance mechanism [130].

Diabetes Mellitus

Aberrations in blood sugar homeostasis leads to a condition known as diabetes mellitus.

There are two major types of diabetes which share common characteristics, but differ in key ways. Type 1 diabetes mellitus is caused by a loss of glycaemic control due to insufficient insulin production and efflux. This is usually the result of autoimmune destruction of the B- cells of the pancreas, resulting in a permanent loss of insulin production [131]. Patients with type 1 diabetes are fully dependant on insulin injections for survival. The more common form is type 2 diabetes (T2DM).

Type 2 Diabetes

T2DM is an increasingly prevalent metabolic disorder characterised by a chronic loss of blood glucose homeostasis, leading to chronic hyperglycaemia. Uncontrolled blood glucose in T2DM is due to aberrations in the insulin signalling pathway resulting in resistance to insulin in peripheral tissues such as skeletal muscle, adipose and liver as well as decreased insulin production and efflux from the pancreas, leading to relative insulin deficiency [132].

There were an estimated 171 million people diagnosed with diabetes in the year 2000 [133] and this is predicted to increase to 366 million by the year 2030 [134]. This increase in prevalence is primarily attributed to changes in nutrition and lifestyle combined with multiple genetic factors contributing to an increased predisposition to this disease [135, 136]. The growing incidence of T2DM is directly correlated with the increasing rate of obesity [137].

Symptoms of T2DM are a direct result of the chronic hyperglycaemia which causes

17 numerous secondary symptoms including polyuria, fatigue, blurred vision and nausea. The majority of symptoms are caused by the formation of advanced glycation end-products

(AGEs) as a result of high levels of glucose in the bloodstream [138]. These AGEs bind to plasma membrane bound receptors for AGEs (RAGE) [139], causing activation of pro- inflammatory cell pathways [140], resulting in tissue damage. Damage of the microvascular systems of extremities can result in poor wound healing leading to gangrene, potentially requiring amputation. Cardiovascular complications are also a common symptom of diabetes and are the leading cause of diabetes related death [141]. T2DM has a large impact on morbidity and mortality, causing an estimated 3.4 million deaths in 2004 world-wide [142].

Onset of diabetes is the result of a combination of insulin resistance and pancreatic β-cell dysfunction, resulting in a loss of glucose regulation.

Insulin Resistance

Insulin resistance is characterised by the reduced efficacy of insulin in peripheral tissues, resulting in reduced blood glucose clearance. Genetic factors play an important role in insulin resistance susceptibility contributing approximately 50% to disease susceptibility with environmental influences such as over eating and reduced exercise contributing the remainder of the risk [143, 144]. Insulin resistance is commonly associated with obesity although the exact mechanisms are still unknown. This is supported by the fact that increased lipid overabundance has been linked to reduced insulin efficacy [145]. Examination of insulin resistant animal models and T2DM patients has revealed alterations in the insulin signalling pathway that reveals clues to explain how the reduced efficacy of insulin occurs [146-148].

Insulin resistance characteristically worsens over time in cases of T2DM.

Oxidative Stress

Though the mechanisms leading to insulin resistance are not yet fully understood, oxidative stress is strongly implicated as at least partly responsible. Repeated activation of the insulin

signalling pathway leads to the accumulation of ROS. Chronic ROS build-up in cells has

been proposed to lead to compensatory responses in the form of insulin resistance, protecting

the cell from further harm by limiting insulin signalling activation [149].

β-Cell Dysfunction

Progressive insulin resistance results in an increased demand upon the pancreas to produce

insulin in order to maintain glucose homeostasis. This high demand progressively increases

pancreatic β-cell stress, eventually leading to damage if left untreated. This damage is often not reversible, leading to a permanent decrease in insulin production that progressively worsens as more damage accumulates [150].

Disease Progression

Initially insulin resistance causes impaired glucose tolerance, also known as prediabetes [151] although patients are usually asymptomatic at this point. The pancreas attempts to compensate for the insulin resistance by increasing production of insulin. Eventually insulin resistance worsens until the pancreas can no longer keep up with the increasing demand, leading to β-cell dysfunction and reduced insulin efflux [152]. At this point blood glucose levels are not adequately regulated resulting in hyperglycaemia. Early stage T2DM is often treatable through lifestyle changes such as improved diet, weight loss and increased exercise which can all help reduce insulin resistance [137] so reducing the associated hyperglycaemia and preventing, or at least delaying, further pancreatic damage [153]. If the insulin resistance and pancreatic dysfunction are at a point where lifestyle changes alone are insufficient then pharmaceutical intervention is required in order to maintain blood glucose levels within physiologically normal ranges.

Current Treatments

There are a number of commonly prescribed oral anti-hyperglycaemia agents such as biguanides (metformin), insulin secretagogues, α-glucosidase inhibitors and thiazolidinediones, which are generally the first line drugs for the treatment of T2DM [154] and may be administered in combination [155]. These drugs all have numerous side effects which include weight gain, fluid retention, hypoglycaemia and increased risk of congestive heart failure and coronary artery disease [137]. As the insulin resistance and pancreatic dysfunction continue to progress these drugs may begin to lose their effectiveness, leading to the requirement for larger doses with a potential for increased associated side effects.

Eventually the dosages of anti-diabetic drugs become limited by their side effects at which point insulin injections are required for effective control of blood glucose levels [137]. Due to the difficulty of treating T2DM it is important that new efficacious methods of treatment with reduced side effects are found.

Diabetic Animal Models

The primary tissues involved in the development of T2DM are muscle, liver and adipose along with impaired insulin secretion from dysfunctional pancreatic B cells. Mammalian insulin signalling is a highly conserved pathway [100], making the study of insulin signalling in animal studies feasible for downstream human disease research. A number of mouse models have been developed to help further understand the underlying mechanisms within the various tissues involved in this disease.

Mice with complete insulin receptor knockout are born with slight growth retardation, but quickly perish within a week due to a lack of blood glucose regulation during feeding [156].

A number of tissue specific knockouts of the insulin receptor have been created to better

understand the roles of the various tissues involved in this pathway. A liver-specific IR

knockout (LIRKO) mouse model presents with severe insulin resistance and glucose

intolerance and declining hepatic function over time, indicating the importance of the liver in

blood sugar regulation. The muscle-specific IR knockout (MIRKO) mouse model interestingly does not present with glucose intolerance. This is a very interesting finding, considering that skeletal muscle is considered to be the site of the vast majority (70-90%) of human postprandial glucose disposal [157], though early onset skeletal muscle insulin resistance may stimulate compensatory actions through increased liver glucose disposal.

Beta-cell specific knockout (BIRKO) mice lose glucose mediated insulin secretion, resulting

in symptoms of T2DM [158]. A fat-specific knockout (FIRKO) in mice resulted in lower fat

mass with a resistance to obesity and obesity-mediated glucose intolerance [159]. Neuron-

specific knockout (NIRKO) resulted in mild insulin resistance and increased susceptibility to

diet induced obesity [160]. Brown adipose tissue knockout (BATIRKO) mice develop a loss

of insulin secretion, leading to glucose intolerance without insulin resistance [161]. In a

complete IR knockout, restoration of insulin receptor activity in muscle, adipose and brain

failed to restore glucose homeostasis [162]. Restoration of IR function in the liver was able to

partially restore glycaemic control [163], although only in 10% of cases. These findings

highlight the importance of the liver in insulin signalling and potentially show skeletal

muscle as being less important than initially thought, at least in mice, even though this tissue

is still thought to be the primary site of insulin-mediated glucose disposal in humans [109].

Knockout of IRS1 in mice results in severe growth retardation and insulin resistance [164],

although glucose homeostasis could still be maintained due to B-cell compensation. Mouse

IRS2 knockout resulted in liver insulin resistance, leading to development of T2DM after 10

weeks [165].

A mouse muscle specific knockout of Glut4 resulted in severe insulin resistance and glucose

intolerance [166], indicating the importance of glucose transport in muscle, though strangely

contradicting the findings of the IR muscle knockout study as mentioned above. It is

21 uncertain how much can be deduced from these studies as these dramatic physiological changes may have resulted in the development of compensatory mechanisms for overcoming insulin resistance.

Zinc in Diabetes

There is mounting evidence supporting a role for zinc in the modulation of the insulin signalling pathway. Serum zinc levels have been shown to be significantly decreased in patients with T2DM [167-169]. The mechanisms behind this are yet to be fully understood however it has also been shown that zinc is able to inhibit PTP1B [11], allowing the insulin receptor to remain phosphorylated for longer, thereby increasing the amount of insulin- mediated cellular glucose uptake and glycogen synthesis [170] (Figure 2).

Effect of Zinc Supplementation on Glycaemic Control

There have been numerous studies examining the effect of zinc supplementation on glycaemic control, although its efficacy is still being debated. Studies performed using diabetic animal models found a positive effect of zinc on diabetic parameters [171, 172], indicating a potential benefit of zinc supplementation for diabetic patients. Several zinc supplementation clinical trials in diabetic patients have shown a positive effect on glycaemic control [173, 174]. A comprehensive review of zinc supplementation trials published in 2012 by Jayawardena and colleagues found that zinc supplementation improved glycaemic control

[175], although further well designed and controlled trials are still required to make this finding definitive.

ZnT8 and Insulin Production

The majority of recent research on zinc transport in diabetes has been focused on the production and efflux of insulin due to the discovery of ZnT8, which is predominantly expressed within secretory granules of pancreatic β-cells [66]. This zinc transporter has been

22 comprehensively studied in the context of insulin secretion and diabetes [66, 176] and is responsible for the influx of zinc into secretory vesicles for the hexamerisation of insulin before it is excreted [66]. These hexamers are formed by six insulin molecules co-ordinated by two zinc ions [98] and are important for the compact storage of insulin in secretory granules and also for the slow release of injected insulin once it is secreted. Slow release prolongs the effect of a single insulin injection as it is not until the hexamers dissociate into monomers that insulin becomes available to activate the insulin signalling pathway. It is well documented that ZnT8 down regulation in pancreatic β-cells leads to impaired insulin secretion [177-180]. There have also been a number of genome-wide association studies

(GWAS) that have implicated mutations of the ZnT8 gene in genetic susceptibility to T2DM

[181, 182].

There has been minimal research into the regulation of zinc levels via zinc transporters in regards to modulating the insulin signalling pathway in peripheral tissues, however recently it has been shown that knockout of ZnT7, a zinc transporter localised to the Golgi apparatus, increases susceptibility to reduced glucose tolerance and insulin resistance [183]. The overexpression of ZnT7 has been shown to increase insulin production and efflux [184].

Currently, there is currently very little known regarding the role of the Zip family of transporters in the insulin signalling pathway.

Zinc Flux and Insulin Signalling

Changes in zinc concentrations are required for various cellular functions with zinc levels in constant flux throughout a cells lifespan. For example, an acute increase in cytosolic zinc may temporarily be required for the activation of a signalling pathway. A slow, steady increase may also be required for an increased demand for a zinc-associated protein or to compensate for changes in levels of available extracellular zinc. To this end, biological systems have developed mechanisms for delivering zinc via transport proteins as required.

Two patterns of intracellular zinc flux in cells have been described, known as early zinc signalling (EZS) and late zinc signalling (LZS) [185]. EZS describes the rapid release of zinc from cellular stores in response to an extracellular stimulus through the activation, via post-

translational protein modification, of the various zinc transporters [186]. This free zinc is then

quickly sequestered away via the ZnTs and MTs. This form of zinc movement occurs

independently of transcription factor regulation of genes. LZS describes the slow

manipulation of zinc levels through the increased expression of the transporter genes,

resulting in an eventual increase in transporter function so creating a net change in zinc levels

over time [186]. This is the result of transcription factors binding to the promoter regions of

the zinc transporter coding genes. EZS occurs in a matter of minutes while LZS may take

hours. Both forms of zinc movement are necessary for the management of cellular processes

such as regulation of molecular pathways and influencing cell signalling.

GHTD-amide

A tetrapeptide isolated from urine in the 1970s known as GHTD-amide [187] was found to

increase glycogen synthesis in muscle cells and reduce blood glucose levels in rats through

causing an increased sensitivity to insulin [188]. This novel pancreatic β-cell associated peptide was found to increase the dispersal of insulin hexamers through the binding of zinc

[189] allowing biologically active insulin monomers to be more rapidly available after release of hexameric insulin from the pancreas. This was observed in cells treated with insulin alongside the peptide and interestingly was also able to reduce glucose levels in cells treated with no additional insulin. Subsequent studies have also revealed that GHTD-amide stimulates a dramatic increase in cytosolic zinc concentrations when administered to C2C12 skeletal muscle cells [190] although the mechanisms behind this are yet to be determined.

These findings indicate that as well as making insulin more bioavailable, this peptide may

also increase insulin sensitivity through the stimulation of a rapid, acute increase in cytosolic

zinc, inhibiting PTP1B and hence prolonging insulin signalling activity.

MTs and Zinc Signalling

The ROS-mediated release of zinc has been described as a mechanism for the regulation of signalling pathways [191]. It has been proposed that ROS production causes the oxidation of

MTs, leading to release of bound zinc [88, 89]. This zinc is then free to inhibit PTP1B as a positive feedback mechanism. ROS is also able to directly inhibit phosphatases [119, 192], adding another level of ROS regulation of insulin signalling. ROS production is associated with insulin signalling activation, leading to the proposed mechanism by which insulin- stimulated ROS production causes MT oxidation and subsequent zinc release.

The Emerging Role of Zip7

Zip7, a zinc transporter primarily localised to the Golgi apparatus and endoplasmic reticulum within cells [40], has recently been identified as having a unique role in the mediation of signalling pathways [193]. This ubiquitously expressed transporter was shown to be essential for the growth and development of zebrafish [194]and is unique among the Zips in that its gene expression and protein cellular localisation are not regulated by changes in intracellular zinc levels [40], although treatment with high levels of zinc does cause post-translational suppression of Zip7 protein levels [40]. Zip7, like the majority of members of this protein family, is predicted to contain 8 TMDs [195] with a histidine rich cytosolic loop region between TMDs III and IV [195]. In a recent study, Taylor and associates found that casein kinase-2 (CK2)-mediated serine phosphorylation of Zip7 was responsible for modulating a rapid release of zinc from cellular stores into the cytoplasm [193]. When mutated at the CK2 phosphorylation site, Zip7 was inactivated. This activation site was found to be highly conserved across a large variety of different species, indicating that it is essential for Zip7 function [193]. CK2 activity has also been shown to increase in response to insulin treatment

[196], which further implicates Zip7 in having a crucial role in modulating insulin signalling.

These findings indicate a role for this transporter as a gatekeeper for acute zinc release which may potentially have a role in the regulation of insulin signalling.

Conclusion

Zinc has roles in numerous cellular pathways and the tight regulation of zinc levels is vital for the production and efflux of insulin. Zinc has been shown to inhibit PTP1B, a phosphatase that inactivates the insulin signalling pathway via dephosphorylation of the IR. The recent findings that the insulin sensitising peptide, GHTD-amide, stimulates a rapid increase in cytosolic zinc levels supports the theory that zinc is an important regulator in the insulin signalling pathway and may have a role in increasing insulin sensitivity. Zip7 has been shown to be a gatekeeper for rapid CK2-mediated zinc release in breast cancer cells, but this has yet to be explored in other tissues. This thesis will investigate the potential role of this zinc transporter in modulating the insulin signalling pathway, which may have implications for new options for the management of diseases associated with aberrant insulin signalling such as T2DM. This will be investigated based on the following objectives:

1. To use a bioinformatics approach to investigate the human Zip7 protein sequence for

areas of potential functional importance.

2. To reduce the expression of Zip7 in skeletal muscle using siRNA silencing to

determine the involvement of this zinc transporter in the insulin signalling pathway.

3. To investigate whether treatment of skeletal muscle cells with insulin induces a

change in cytosolic zinc levels to understand the mechanisms of insulin-stimulated

zinc flux.

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Chapter 2: Materials and Methods

Introduction

The combined materials and methods used for the completion of this thesis are outlined in detail below. Methods are also included in their relevant chapters to aid both in describing their specific application and in the publishing of thesis chapters.

Protein Sequence Information

Human, Orangutan, Pig, Sheep, Rat, Mouse and Zebrafish Zip7 protein sequences were obtained from the UniProt protein database (http://www.uniprot.org/) (Table 1). Protein sequences for the 14 human Zips were also obtained from this database (Table 2). Swiss-Prot reviewed sequences were used wherever possible.

Table 2: Zip7 cross-species protein sequence accession information. Species Accession Number Orangutan Q5RFD5 Pig Q29175 Sheep C7BDW5 Rat Q6MGB4 Mouse Q31125 Zebrafish Q9PUB8

Table 3: Human Zip transporter accession information. Transporter Accession Number Zip1 Q9NY26 Zip2 Q9NP94 Zip3 Q9BRY0 Zip4 Q6P5W5 Zip5 Q6ZMH5 Zip6 Q13433 Zip7 Q92504 Zip8 Q9C0K1 Zip9 Q9NUM3 Zip10 Q9ULF5 Zip11 Q8N1S5 Zip12 Q504Y0 Zip13 Q96H72 Zip14 Q15043

Protein Sequence alignments and phylogenetic analysis

Sequence alignments were performed with the Clustal Omega algorithm and editing was

performed using JalView v. 2.8 (http://www.jalview.org/). FigTree v. 1.4.0

(http://tree.bio.ed.ac.uk/software/figtree/) was used to generate phylogenetic trees from

alignments using the Neighbour joining method and % identity.

Signal Peptide Prediction

Prediction of signal peptides within protein sequences was performed using SignalP 4.1. A

positive score was based on detection of an early N-terminal alpha-helical region followed by a viable cleavage site [1]. This was performed to eliminate potential false transmembrane

TMDs identified using detection tools as they often have difficulty differentiating between early TMDs and signal peptides due to their similar α-helical secondary structure [2].

TMD Prediction

Analysis of transmembrane domains was performed using MINNOU software

(http://minnou.cchmc.org/). This tool predicts transmembrane domains based on a combination of predicted secondary structure formation, known sequence comparisons and hydropathy profiling. This predictive tool has been shown to have an 80% accuracy rating when tested on protein sequences of a known structure [3].

Prediction of SLiMs

Short linear motifs (SLiMs) were predicted within protein sequences using the Eukaryotic

Linear Motif (ELM) database (http://elm.eu.org/), which compares the sequence against a

database of >2400 experimentally validated motifs [4]. Identified SLiMs were aligned to

other vertebrate species to determine evolutionary conservation of these regions and hence

their potential importance for protein function. ELM analysis was performed in all human

Zips to identify SLiMs that may be shared between them. Listed SLiMs were chosen based

on their predicted function relative to their location within the 2-dimensional Zip structure in

regards to loop or TMD regions.

C2C12 Skeletal muscle cells

Mouse C2C12 skeletal muscle cells were used in all cell culture experiments performed in

this thesis. This cell line was initially isolated from the thigh muscle of a female CH3 mouse

after a crush injury [5]. These myoblasts can be differentiated into mature fully differentiated

myotubes via mitogen withdrawal [6]. C2C12 cells are frequently used in mechanistic studies

involving skeletal muscle [7-10].

Cell Culture

Proliferating mouse C2C12 myoblasts were cultured and maintained in DMEM supplemented

with 100 ml per litre Foetal bovine serum, 10 ml per litre Penicillin/Streptomycin, L-

Glutamate and 20 µM ZnSO4, (Life Technologies, Mulgrave, Australia). Differentiation of

myoblasts into post-mitotic, multi-nucleated myotubes was induced by mitogen withdrawal

(i.e. DMEM supplemented with 20 ml per litre horse serum for three days). Assessment of

the muscle-specific, contractile and metabolic C2C12 muscle phenotype was assessed by

measuring the expression of markers of differentiation and metabolic processes as previously

described. Cells were maintained in a humidified 37°C incubation chamber with 5% CO2.

siRNA Transfection

Small interfering RNA (siRNA) molecules for Zip7 (Catalogue No: AM16708), Zip1

(Catalogue No: AM16708), GAPDH (Catalogue No. 4390771) and a scramble control were mixed with 2.5 µl/ml RNAiMAX Lipofectamine (Life Technologies) to a final working concentration of 10 nM for each siRNA. Solutions were prepared in Opti-MEM Reduced

Serum Media (Life Technologies). Transfection solution was added to 2% HS media for

differentiated cells or 10% FBS media for myoblast transfections. Transfections were performed in 6-well cell culture dishes over 48 hours.

RNA Extraction

Total RNA was extracted from C12C2 cells and C57Bl/6J mouse quadriceps using TRI-

Reagent (Sigma-Aldrich, Castle Hill, Australia) with the phenol/chloroform method according to the manufacturer’s protocol. Cells collected in TRI-reagent and RNA was separated in chloroform via a 10 minute centrifugation at 12000 RCF at 4°C. RNA was precipitated by adding isopropanol and incubation on ice for 10 minutes. Samples were then centrifuged at 12000 RCF at 4°C for 10 minutes. The resulting RNA pellet was washed twice in 75% ethanol before finally resuspending the RNA in 25 µl of RNAase free water. Total

RNA was then treated with 2U of DNase1 for 30 min at 37°C followed by purification of the

RNA through an RNeasy purification column system (Qiagen, Chadstone, Victoria,

Australia). RNA quantity and quality was measured using a Nanodrop spectrophotometer

(Thermo Scientific). All RNA samples used had a 260/280 quality ratio of 1.95-2.00. cDNA synthesis

A High Capacity cDNA Synthesis kit (Life Technologies) was used to synthesise cDNA according to the manufacturer’s instructions. A solution containing reaction buffer, random hexamers, free nucleotides and reverse transcriptase was added to 2 µg of total RNA to make a 20 µl reaction and incubated according to the protocol in Table 3. The completed reaction was diluted to 400 µl in nuclease-free water to reach a final working concentration of 5 ng/µl and stored at -20°C.

Table 4: cDNA synthesis temperature conditions Annealing Extension Inactivation Hold Temperature 25°C 37°C 85°C 4°C Time 10 minutes 120 minutes 5 minutes ∞

Primer Design

Primers were designed using the NCBI Primer Blast Tool

(http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi), with the exception of Irs1

(PrimerBank ID: 29825829a1), Irs2 (PrimerBank ID: 3661525a1) and Insr (PrimerBank ID:

6754360a1) which were obtained from the PrimerBank Database

(http://pga.mgh.harvard.edu/primerbank/index.html). Primers were purchased from

GeneWorks (Thebarton, Australia) with the exception of Mt1 and Mt2 which were purchased

from Bioneer (Kew, Australia). Note: all primers were rigorously analysed by BLAST for

target gene specificity and designed to be genomic resistant, with at least one primer in set

spanning an exon-exon junction. All primers were designed to have a melting temperature of

as close to 60°C as possible to ensure that all PCR reactions could be run with the same annealing temperature.

Table 5: PCR primer sequences. MyoG 5’-CCTTAAAGCAGAGAGCATCC-3’ 5’-GGAATTCGAGGCATAATATGA-3’

Tnni1 5’-GCCTATGCGCACACCTTTG-3’ 5’-CGGGTACCATAAGCCCACACT-3’

Tnni2 5’- AAATGTTCGAGTCTGAGTCCTAACTG-3’ 5’-GCCAAGTACTCCCAGACTGGAT-3’

Srebp-1c 5’-CGTCTGCACGCCCTAGG-3’ 5’-CTGGAGCATGTCTTCAAATGTG-3’

Fabp3 5’-CCCCTCAGCTCAGCACCAT-3’ 5’-CAGAAAAATCCCAACCCAAGAAT-3’

Abca1 5’-GCTCTCAGGTGGGATGCAG-3’ 5’-GGCTCGTCCAGAATGACAAC-3’

Zip7 5’-TCTGGGCCTACGCACTGGGG-3’ 5’-TGGAGCAGAGAGCGGTGCCT-3’

Eef2 5’-ACGATGAGGCCGCCATGGGTAT-3’ 5’-AAGTGGGCCTTTGGGGTCGC-3’

Il-6 5’-TTCCTCTCTGCAAGAGACTTCC-3’ 5’-AGCATCAGTCCCAAGAAGGC-3’

Pygm 5’-AGCTGGAGCCTCACAAGTTC-3 5’-CAATGCGCTCAGCAATGACC-3’

Phkb 5’-GCTAGCATACAGGCGCATTG-3’ 5’-TATCAGCCTGCCGCATGTAG-3’

Pgm2 5’-CATCCCGACCCCAATCTCAC-3’ 5’-ATGTTTCGATCCCCGTCACC-3’

Gbe1 5’-ACTGCTTTGATGGCTTCCGT-3’ 5’-AACCTTGACCCATTCCGTGG-3’

Zip1 5’-TGCATGTGACGCTTCAGTTC-3’ 5’-TAAGCCAGCGTGATCTGCTC-3’

Zip13 5’-GCCAGCTTCCTTGTGAGCAA-3’ 5’-GATAGCAAAGTCACCCACCTCA-3’

Zip14 5’-GGCTGGAGGACTTCAGTGTG-3’ 5’-GGTGAGGCCAAGGCTAATGT-3’

Irs1 5’-CGATGGCTTCTCAGACGTG-3’ 5’-CAGCCCGCTTGTTGATGTTG-3’

Irs2 5’-CTGCGTCCTCTCCCAAAGTG-3’ 5’-GGGGTCATGGGCATGTAGC-3’

Insr 5’-ATGGGCTTCGGGAGAGGAT-3’ 5’-GGATGTCCATACCAGGGCAC-3’

Mt1 5’-CAAGTGCAGGAGCCGCCGGT-3’ 5’-TCTCGCAATGGACCCCAACTGCT-3’

Mt2 5’-TCCACTCGCCATGGACCCCAA-3’ 5’-AGCAGGATCCATCGGAGGCA-3’

Real-time PCR

Quantitative PCR (qPCR) was performed on a RealPlex PCR detection system (Eppendorf,

North Ryde, Australia) in triplicate on at least three independent RNA preparations. Target cDNA levels were analysed in 10 µl reactions with SensiMix SYBR No-ROX (Bioline,

Alexandria, Australia). qPCR was performed using 4 µl of cDNA template (20 ng) and

primers at a concentration of 1 µM according to the parameters presented in Table 4.

Table 6: PCR Cycle Settings. Activation Denaturation Annealing Extension Temperature 95°C 95°C 58°C 72°C Time 5 minutes 15 seconds 15 seconds 20 seconds Cycles 1 40X

Gene expression analysis

Real-time qPCR data was analysed using the relative expression formula. To calculate this,

the gene of interest (GOI) cycle threshold (Ct), the point at which PCR fluorescence is measured, was made relative to a reference (REF) gene, Glyceraldehyde 3-phosphate

dehydrogenase (Gapdh) or eukaryotic elongation factor 2 (Eef2), using the following

equation:

CtGOI – CtREF = ∆Ct

To inverse the values to aid in graph comprehension the ∆Ct values were transformed using the relative expression equation:

2-∆Ct = Relative Expression

In order to test for statistical significance, a Student’s t-test was performed on ∆Ct values in

the control verses the experimental group as defined in each study. The ∆Ct is the most

appropriate value to analyse as this is the least transformed relative data [11]. Depending on

the study hypothesis, either a 1- or 2-tailed t-test was performed. As an example, a 1-tailed

test was performed when testing the gene knockdown efficiency of an siRNA as the change

was expected to occur in one direction only, while measuring compensatory effects in other

genes where expression may be increased or decreased was performed using a 2-tailed test.

Statistical significance threshold was set to 0.05. Error bars on gene expression graphs are

shown as the standard deviation of the relative expression of at least three replicates.

Protein Extraction

Total cellular protein from samples was isolated by scraping cells in RIPA buffer (Thermo scientific) with added protease and phosphatase inhibitors using a rubber policeman [12].

After collection, samples were incubated on ice for 10 minutes and then vigorously vortexed

for 20 seconds and centrifuged at 12,000 RCF for 5 minutes at 4°C to collect cellular debris,

after which the supernatant containing total soluble protein was collected and stored at -80°C.

Bicinchoninic acid assay

Total cellular protein concentrations were measured using a bicinchoninic acid (BCA) assay

kit (Bio-Rad, Gladesville, Australia) against a known standard of bovine serum albumin

(BSA) (Thermo Scientific, Melbourne, Australia) as outlined by the manufacturer’s instructions. Briefly, cell samples were diluted 1 in 5 in a flat bottom 96-well dish along with the known standard at 0, 0.2, 0.4, 0.6, 0.8 and 1 µg/µl concentrations. The BCA solution was added to each well and the plate was lightly shaken before being incubated for 1 hour at

37°C. Colorimetric absorbance was measured using a Multiskan Microplate Photometer

(Thermo Scientific) at 570 nm. Protein samples were diluted 1:5 to ensure concentrations fit within the standard curve of the known BSA.

Western Blotting

Total soluble protein (30 µg per sample) were mixed with β-Mercaptoethanol (Sigma) and denatured at 100°C for 5 minutes. Samples were resolved on a 4-15% SDS-PAGE gradient gel (Bio-Rad) at 80 V for 1.5 hours and transferred to a nitrocellulose membrane at 100 V for one hour at 4°C. A SNAP i.d.® 2.0 vacuum assisted blotting system (Merk Millipore,

Bayswater, Australia) was used to block and probe protein bound membranes according to

the manufacturer’s guidelines. Briefly, membranes were blocked in 0.5% skim milk in TBS

Tween for 10 minutes. Membranes were probed with a primary antibody for 20 minutes

(Antibodies and their concentrations used are listed in Table 7). Following 4 x 20 second washes in TBS Tween the membranes were incubated with 1:2500 horseradish peroxidase

(HRP)-linked anti-Rabbit secondary antibody (Cell Signaling, Catalog No:7074) for 10 minutes. An Enhanced SuperSignal West Pico Substrate kit (Thermo Scientific) was used to generate a chemiluminescent signal from the HRP tag which was visualised using a UVITEC

Alliance digital imaging system (Thermo Scientific).

Table 7: Western blotting antibodies. Antibody Antibody Company Product ID Concentration Target species pAkt Rabbit Cell Signalling 4058 1:2500 Akt Rabbit Cell Signalling 9272 1:2500 Glut4 Mouse Cell Signalling 2213 1:2000 Gapdh Goat Santa Cruz sc-20357 1:2000 Biotechnology

Fluorescent Zinc Imaging

Fluorescent stains were all purchased from Life Technologies. C2C12 cells grown to 60%

confluence were washed twice in PBS and loaded with 5 µM cell permeable Fluozin-3 (Life

Technologies) (Excitation/Emission: 494/516 nm) in 10% FBS DMEM media containing no phenol red indicator and incubated at 37°C for 30 minutes. Cells used for visualisation of cytosolic zinc were again washed twice in PBS and loaded with 1 µM of the nuclear stain

Hoechst 33342 (Excitation/Emission: 350⁄461 nm) and 5 µg/ml of the plasma membrane stain Alexa Fluor® 594 wheat germ agglutinin (Excitation/Emission: 590/617 nm). Cells were then incubated at 37°C for 5 minutes before being washed a final two times and adding fresh media containing 20 µM ZnSO4. Cells were incubated for a final 10 minutes after

removal of stains to ensure complete de-esterification of the FluoZin-3. Cells used for pixel

intensity analysis of fluorescent intensity were incubated with Fluozin-3 for 30 minutes

before washing, adding fresh media and incubating at 37°C for 15 minutes. Cells were then

brought to the 37°C incubation chamber attached to the confocal microscope and left to

equilibrate for a further 15 minutes prior to imaging. 25 mM Hepes buffer was included in

the imaging media to buffer pH fluctuations brought on by the change from 5% CO2 in the

cell culture incubator to the conditions of the microscope incubation chamber.

Fluorescent ROS Imaging

Insulin-mediated ROS production was measured using an Image-iT LIVE Green Reactive

Oxygen Species Detection Kit (Life Technologies) according to the manufacturer’s protocol.

Briefly, Cellular ROS was labelled with 25 µM 5-(and-6)-carboxy-2′,7′- difluorodihydrofluorescein diacetate (carboxy-H2DCFDA), a compound that fluoresces when

oxidised (Excitation/Emission: 492–495nm/517–527nm). Cells were co-treated treated with

0, 10 or 100 nM insulin for 30 minutes. 2 µM of Hoechst 33342 was added in the final 5

minutes of incubation. Cells were then washed twice in warm imaging media before adding a

final 1 ml of media prior to imaging. For a positive control, cells treated with 100 µM tert-

butyl hydrogen peroxide (TBHP), a known inducer of ROS production, for 90 minutes were

stained as described above. To assess the role of superoxide dismutase (SOD) in insulin-

stimulated ROS production, cells were treated with the SOD1 inhibitor tetra-thiomolybdate

(TTHB) (Sigma Aldrich) at a concentration of 20 µM for 24 hours prior to ROS staining.

Confocal Microscopy

Live cell imaging was performed using a Nikon Eclipse Ti-E confocal microscope system with an attached 37°C chamber. Cells were imaged every 5 minutes using a 60X oil emersion objective. In order to capture total cell fluorescence, images were taken over a 15 µm z-range with an image captured every 1 µm. Cells were imaged for 15 minutes prior to treatment to establish baseline fluorescence. Cells were then treated with either 200 µl of fresh culture

media or 200 µl of media containing either insulin of the zinc ionophore NaP, at

concentrations required to achieve a final concentration of 10 nM for insulin or 20 µM for

NaP and imaged for a further 30 minutes.

Fluorescence Intensity Analysis

Pixel density analysis of zinc fluorescence obtained from the confocal microscopy

experiments was performed using ImageJ software (http://imagej.nih.gov/ij/index.html).

Multi-image cell z-stacks were collapsed into a single image using Z-projection. Pixel intensity of the Fluozin-3 fluorescence of each individual cell was measured over the course of the experiment with the pixel threshold adjusted to remove background. Data collected was normalised, with the time point of the initial treatment at 15 minutes set as 0.

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12. Jensen, W.B. (2008). The Origin of the Rubber Policeman. Journal of Chemical

Education 85, 776.

Chapter 3: Understanding the Structure of Zip7: A Histidine Rich Zinc Transporter Implicated in Cellular Signalling

Abstract

Zinc is essential for life and its bioavailability must be tightly controlled within cells. Zinc homeostasis is controlled by two families of transporters known as ZnTs and Zips. ZnTs reduce cellular cytosolic zinc by pumping it out of the cell or into organelles for storage while the Zips have the opposite effect and increase cytosolic zinc. There are 14 Zips in mammals with diverse tissue expression and cellular localisation. There is substantial evidence for histidine having an essential role in Zip function through the binding of zinc to initiate a functional change in the transporter. This study examined Zip7, a distinct Zip transporter which is localised to the endoplasmic reticulum and modulates a rapid increase in cytosolic zinc in response to extracellular stimuli in breast cancer cells. The Zip7 protein sequence was analysed using various bioinformatics tools to predict its 2-dimensional structure. There were

8 predicted transmembrane domains and three distinct histidine rich regions located in the N- terminus and loop regions. Cross-species comparisons showed that the transmembrane domains were highly conserved and essential for the ion transport function of this transporter.

The loop regions of Zip7 were found to be poorly conserved between the species examined, with the exception of the histidine rich regions, which showed a high level of conservation and so are suspected to have an essential role in modulating the transport function of Zip7 by binding to zinc. These findings implicate histidine residues as an important functional component of Zip7. Further analysis through mutagenic studies is needed to fully elucidate the functional effect of the histidine-rich regions of Zip7.

Introduction

Zinc is a non-cell permeable metal that is essential for life. Zinc has numerous biological functions and is required for the formation and function of approximately 3000 metalloproteins [1] including catalytic and co-catalytic functions in numerous biological reactions required for the activity of over 300 enzymes [2]. More recently zinc has been shown to act as a secondary messenger in cellular signaling pathways [3]. An example of one such pathway is insulin signalling, where zinc has been shown to positively regulate the insulin signalling pathway [4] which is believed to occur through the zinc-induced inhibition of PTP1B, a phosphatase that deactivates the insulin receptor [5].

The diversity of functions for zinc ions requires strict control of zinc levels in cells but the mechanisms controlling zinc homeostasis are not yet fully understood. Zinc is one of the most abundant trace elements in biology, second only to iron. Although total levels of zinc are around 30mg/kg, actual concentrations of bio-available cytosolic zinc are kept relatively low [6], due to toxicity when at higher concentrations. Tightly regulated transport mechanisms are required to ensure zinc is kept at nontoxic levels, but remains readily available as required for cellular functions. This is achieved through the collective action of

24 characterised zinc transporters divided in to two families defined by sequence similarities, the ZnTs and Zips [7]. The ZnTs reduce cytosolic zinc concentrations by transport out of the cell or into cellular compartments. The Zip family of zinc transporters have the opposite effect, increasing cytosolic zinc by transporting it into the cytosol from outside the cell or out of cellular compartments. Individual transporters have different tissue expression and cellular localisation and some having direct disease associations (reviewed in [8]). Another group of proteins involved in cellular zinc homeostasis are the metallothioneins, a group of heavy metal binding proteins, that aid in buffering cytosolic metal concentrations within the cytosol

[9].

Zinc movement within cells occurs via two different phases described as early and late zinc

signalling. Late zinc signaling (LZS) describes slow, subtle changes in cytosolic zinc levels

caused by transcriptional regulation of the zinc transporter genes whereas early zinc signaling

(EZS) describes rapid fluctuations in zinc concentrations caused by post-translational changes of zinc transport proteins [10]. These two forms of zinc movement work together to tightly control levels of cytosolic zinc.

Zinc transporters and their kinetics have been well studied in recent years, but there are still many unknowns associated with their control mechanisms. New novel roles for specific zinc transporters are still being discovered. Zip7 has been shown to have an interesting and potentially novel role in modulating rapid zinc flux from the endoplasmic reticulum in response to extracellular stimulus. Taylor and associates found that EGF treatment of cells expressing Zip7 as the most abundant zinc transporter resulted in CK2 mediated phosphorylation, which led to a rapid increase in cytosolic zinc concentrations [11]. Co- treatment with zinc was also required to initiate this response, suggesting a mechanism of zinc induced zinc release. Other studies have shown zinc transporter function being influenced by zinc concentrations [12], with histidine implicated as a necessary component.

Histidine is an essential amino acid with an imidazole side-chain that, along with the sulfhydryl group of cysteine residues and carboxyl containing side chains, is well known for transition metal co-ordination within proteins [13-16]. Within Zips there are three highly conserved histidine residues within TMDs 4 and 5 that are considered essential for transmembrane pore formation [13]. TMD histidines have also been shown to control the selectivity of ZnT transporters for zinc, as other similarly structured heavy metals such as cadmium are highly toxic [15]. Alanine replacement of these residues in the plant Zip transporter TjZNT1 resulted in a loss of zinc selectivity [14].

As well as being required for zinc transporter pore formation, histidine has also been shown

to have important regulatory roles within loop regions of transporter proteins. Sagar and

associates reported that a region of histidine repeats located in a cytoplasmic loop region of

Zip4 played a key role in transporter function [17]. These regions were found to bind

transition metals which resulted in increased degradation of Zip4, which was suggested to be

a mechanism to switch off zinc transport once cytosolic zinc concentration was sufficiently

increased [18]. Alanine replacement of these histidine residues resulted in reduced Zip4

degradation, causing increased cell susceptibility to zinc toxicity. Similar alanine replacement

studies of cytosolic loop histidines in Zip1 resulted in reduced zinc transport [19].

Due to the novel role reported for Zip7 in mediating a rapid zinc release from intracellular

stores and the high histidine content in both cytosolic and extracellular loop regions, further

analysis of potential functional sites is warranted. In this study, the human Zip7 protein

sequence was examined using various bioinformatics tools, sequence alignments and

prediction software to investigate this distinctive transporter. The results highlight histidine

rich regions that may be important in regulating Zip function, particularly in those

transporters that have been implicated in cell signalling pathways.

Methods

Obtaining Protein Sequence Information

Human, Orangutan, Pig, Sheep, Rat, Mouse and Zebrafish Zip7 protein sequences were

obtained from the UniProt protein database (http://www.uniprot.org/), with only Swiss-Prot

reviewed sequences being chosen to ensure sequence quality (Thesis Chapter 2, Table 1).

This study used a diverse range of mammalian sequences as well as the much more divergent

zebrafish sequence to observe areas of evolutionary conservation. Protein sequences for the

14 known human Zips were also obtained from this database (Thesis Chapter 2, Table 2).

Protein Sequence Alignments and Phylogenetic Analysis

Sequence alignments were performed with the Clustal Omega algorithm and visualisation

and editing was performed using JalView v. 2.8 (http://www.jalview.org/). Cross-species

alignments of the Zip7 protein sequences were performed to determine evolutionary

conservation of various functional regions. The human Zip7 sequence was also compared to

the other 13 members of the Zip family to investigate the evolutionary diversification of this family of zinc transporters. FigTree v. 1.4.0 (http://tree.bio.ed.ac.uk/software/figtree/) was

used to generate a phylogenetic tree from alignments using the Neighbour joining method

and percent identity. The tree was rooted to Zip11 as this is considered to be the most

dissimilar Zip member, being the sole member of a subfamily known as gufA [20].

Signal Peptide Identification

The human Zip7 protein sequence was analysed for the presence of a signal peptide using

SignalP 4.1. Analysis was based on detection of N-terminal alpha-helical regions combined with a viable cleavage site [21]. This was performed to eliminate potential false TMDs as prediction tools often have difficulty differentiating between TMDs and signal peptides [22].

Predictive Analysis of Transmembrane Domains

To understand the various functional sites detected within the Zip7 sequence, the 2- dimensional structure was first predicted. Analysis of transmembrane domains was performed using MINNOU software (http://minnou.cchmc.org/). This tool predicts transmembrane domains based on a combination of predicted secondary structure formation, known sequence comparisons and hydropathy profiling. This predictive tool has been tested to have an 80% accuracy rating when tested on protein sequences of a known structure [23].

Identification of Functional Motifs

SLiMs are important regions within a protein sequence for predicting function [24]. SLiMs were predicted within protein sequences using the ELM database (http://elm.eu.org/), which compares the protein sequence against a database of >2400 experimentally validated motifs

[25]. Identified motifs were aligned to other vertebrate species to determine evolutionary conservation and hence potential importance for the function of this protein. ELM analysis was performed in all human Zips to identify SLiMs that may be shared between them. Listed

SLiMs were selected based on their predicted function relative to their location within the 2- dimensional Zip structure. Cytosolic loop regions were given high priority in the context of

SLiM accessibility.

Characterisation of Histidine rich regions in the human Zip family members

To investigate whether there is any link between transporter histidines and their location within the protein sequence, the histidine residues within each Zip transporter were grouped depending on their location within TMDs or loop regions. Histidine residues within predicted signal peptides were excluded from the analysis.

Results

Zip7 secondary structure.

To determine the presence of a signal peptide within the N-terminus of the Zip7 protein, the sequence was analysed using SignalP prediction software. This analysis revealed the presence of a signal peptide with a cleavage site between residues 28 and 29 (Figure 1 A). Prediction of secondary structure for Zip7 highlighted a total of nine areas as potential transmembrane domains (Figure 1 B), with the first being a part of the identified signal peptide sequence, which was therefore excluded.

Zip7 protein sequence alignments against other vertebrate species revealed a high degree of

cross-species sequence conservation within the predicted TMD regions (Figure 2). A very high level of conservation was observed throughout the eight TMD regions in all species, including the distantly related zebrafish sequence which showed a high level of similarity to the mammalian sequences.

Histidine residues are conserved across a broad range of species.

Among the N- and C-termini and intracellular and extracellular loop regions of the Zip7 sequence there was generally a low level of conservation relative to what was observed in the

TMD regions. Three non-TMD regions identified within the human Zip7 protein sequence have high histidine content. When these areas are aligned with other species, the majority of the loop region is poorly conserved. However, the histidine residues within these regions appear to be the only loop region and termini feature that remains highly conserved (Figure

3).

ELM Database analysis reveals key areas of importance in the activation and cellular localisation of Zip7.

Analysis of the Zip7 protein sequence for short linear motifs revealed many areas that may potentially play important roles in the function of this transporter (Table 1). Cross-species sequence alignments revealed that the majority of these motifs are highly evolutionarily

conserved in the examined species.In regards to cellular localisation the sequences contains a

di-arginine motif (RXR) that usually acts as a protein anchor in the endoplasmic reticulum

and a lysosomal-endosomal-melanosomal targeting motif (DFAILV). The Zip7 sequence also

contains a number of potential phosphorylation sites for glycogen synthase kinase-3 (GSK-3),

NIMA-related kinase 2 (NEK2), proline-directed kinase (MAPK), multiple casein kinase 1

(CK1) sites and one CK2 site.

Table 1: Human Zip7 ELM predicted motifs cross-species conservation.

Motif Name Matched Positions Species Sequence (aa) Conservation1 O P S R M Z CK1 phosphorylation site SHESLYH 97-103      X SHHTLEQ 197-203 X      SGCSKKQ 373-379      X CK2 phosphorylation site KQSSEEE 272-278       Glycosaminoglycan attachment site ESGA 119-122 X     X VSGY 317-320       QSGC 372-375 X      GSK3 phosphorylation recognition HGHSHRHS 43-50       site HGHSHGYS 90-97  X X   X NEK2 phosphorylation motif. LRVSGY 315-320      X LVQSGC 370-375  X     Secondary preference for PKA-type HRHSHED 47-53      X AGC kinase phosphorylation. TRGSHGH 257-263 X X X X X X ERSTKEK 267-273 X X    X LRVSGYL 315-321       Proline-Directed Kinase (e.g. MAPK) ESNSPRH 159-165      X phosphorylation site in higher eukaryotes. Tyrosine-based sorting signal YLNL 320-323       responsible for the interaction with mu subunit of AP (Adaptor Protein) complex Di-Arginine ER retention motif. PRHR 163-166      X 1 O = Orangutan, P = Pig, S = Sheep, R = Rat, M = Mouse, Z = Zebrafish

Zip7 is most closely related to Zip13 and has high histidine content in loop regions.

The 14 human Zip proteins were aligned and a phylogenetic tree was generated displaying

the evolutionary distance between the various transporters. Zip7 was shown to be most

closely related to Zip13 (Figure 4). After determining the protein sequence of the N- and C- termini and extracellular and intracellular loop regions of the 14 human Zips (Table 2), the histidine residues within these regions as well as TMD regions were counted. Zip7 was shown to have one of the highest concentrations of histidine, with only Zip6 and Zip10

containing more. Zip13, the transporter found to be most similar to Zip7, contains only a single loop region histidine and none within loop regions (Table 3).

Table 2: Zip transporter loop regions (amino acids).

Zip N- Loop 2 Loop 3 Loop 4 Loop 5 Loop 6 Loop 7 Loop 8 C- Term (C) (L/E) (C) (L/E) (C) (L/E) (C) Term (L/E) (L/E) Zip1 1-26 55-64 92-108 135-177 203-208 231-236 264-270 296-302 323-324 Zip2 1-4 33-41 68-101 128-162 188-193 216-221 249-256 282-288 309-309 Zip3 1-4 31-39 67-83 110-167 193-198 221-226 580-260 286-292 313-323 Zip4 1-322 351-356 384-398 425-494 520-527 550-555 467-584 610-621 642-647 Zip5 1-208 237-241 269-283 310-381 407-414 437-442 467-472 498-509 530-540 Zip6 1-320 349-353 381-419 446-593 619-626 649-654 679-683 710-725 746-755 Zip7 1-130 159-164 192-215 242-316 342-349 372-377 387-413 440-448 469-469 Zip8 1-124 153-157 185-194 221-301 327-334 357-362 387-390 416-433 454-460 Zip9 - 24-30 58-104 133-142 168-176 199-204 236-240 267-284 305-307 Zip10 1-402 431-435 463-493 520-667 693-700 723-728 753-757 784-801 822-831 Zip11 1-3 31-35 63-76 103-192 217-231 254-259 284-288 315-321 341-342 Zip12 1-362 391-396 424-443 470-538 564-571 594-599 624-628 654-665 686-691 Zip13 1-63 92-101 129-147 174-215 241-248 271-276 304-314 341-349 370-371 Zip14 1-149 178-182 210-219 246-334 360-367 390-395 420-423 449-465 486-492 L/E= Lumenal/Extracellular, C= Cytosolic

Table 3: Human Zip family signal peptide and histidine residues.

Transporter Predicted SP Cleavage TMD1 Loop Total Signal Site (aa) Histidines Histidines Histidines Peptide (SP) Zip1 No - 3 5 8 Zip2 No - 4 7 11 Zip3 No - 2 5 7 Zip4 Yes 22-23 6 19 25 Zip5 Yes 24-25 6 15 21 Zip6 Yes 20-21 7 61 68 Zip7 Yes 22-23 6 51 57 Zip8 Yes 22-23 2 11 13 Zip9 No - 6 15 21 Zip10 Yes 25-26 7 77 84 Zip11 No - 1 2 3 Zip12 Yes 23-24 7 16 23 Zip13 Yes 32-33 4 1 5 Zip14 Yes 30-31 2 14 16 1Transmembrane domain

Zip7 has a unique profile of SLiMs compared to other Zips.

The SLiM profile for each transporter was assessed and each motif located within a loop region, and hence likely to be accessible for modification, was tabulated (Table 4). Various phosphorylation sites were found in each transporter in both cytosolic and extracellular loops,

with only Zip9 having no phosphorylation sites in cytosolic loop regions. In regards to

potential localisation motifs, Zips 4, 5, 7, 11, 12 and 13 all had ER retention motifs. With the

exception of Zip7 and Zip13, all of the Zips with ER retention motifs also contained an

endosomal/lysosome sorting signal (SORT). Of the Zips containing a large amount of loop region histidine (Zips 6, 7 and 10), Zips 6 and 10 both contained a Breast cancer gene

BRCA1 interaction motif which was not present in Zip7.

Table 4: Zip transporter predicted loop functional motifs.

Zip Cell Predicted SLiMs Localisation Cytosolic loops Extracellular/lumenal loops Zip1 Plasma membrane CK11, CK2, NEK2, PLK, MAPK, - [26] SORT Zip2 Plasma membrane CDK, NEK2, MAPK CK2, GSK3, CRM1 [27] Zip3 Plasma membrane CDK, CK1, CK2, GSK3, MAPK - [26] Zip4 Plasma membrane NR, CK1, CK2, GSK3, MAPK, CK1, CK2, GSK3, NEK2, PLK, [17] ER, SORT SORT, CRM1 Zip5 Basolateral NEK2, ER, SORT NR, CK1, GSK3, NEK2 membranes [28] Zip6 Plasma membrane CK1, CK2, GSK3, NEK2, SORT BRCA, CK1, CK2, GSK3, [29] NEK2, SORT Zip7 Golgi apparatus CK2, NEK2, ER CK1, GSK3 [30], endoplasmic reticulum [11] Zip8 Plasma membrane, CASP, CK1, CK2, GSK3, NR, CK1, GSK3, NEK2, SORT, vesicles [31] CRM1 Zip9 Trans Golgi network - GSK3, PLK, SORT [32] Zip10 Plasma membrane CASP, CDK, CK1, CK2, GSK3, BRCA, NR, CK1, CK2, GSK3, [33] NEK2 NEK2 Zip11 Golgi apparatus [34] CK1, CK2, NEK2, ER BRCA, SORT Zip12 Plasma membrane CK1, CK2, GSK3, NEK2, SORT NR, CK1, CK2, GSK3, NEK2, [35] ER, SORT Zip13 Golgi apparatus [36] GSK3 CK1, NEK2, ER Zip14 Plasma membrane CASP, CK1, GSK3, NEK2, SORT BRCA, NR, CK1, GSK3 [37]

1CK1 – Casein kinase 1 phosphorylation site

CK2 - Casein kinase 2 phosphorylation site

GSK3 – Glycogen synthase kinase 3 phosphorylation site

NEK2 - NIMA-related kinase 2 phosphorylation site

MAPK – Mitogen-activated protein kinase phosphorylation site

PLK – Polo-like kinase phosphorylation site

ER – Endoplasmic reticulum retention signal

SORT – Endosome/Lysosome sorting signal

NR – Nuclear receptor binding motif

CRM1 – Exportin-1 interaction site

CDK - Cyclin-dependent kinase phosphorylation site

CASP – Caspase 3-7 cleavage site

BRCA – Breast cancer gene BRCA1 interaction motif

Discussion

Zip7 is a zinc transporter of particular interest due to its potential role as the gatekeeper of

zinc release from the endoplasmic reticulum in response to extracellular stimuli as a means of

regulating cell signalling pathways, as reported in breast cancer cells [38]. This zinc

transporter is a member of the LIV-1 subfamily of Zip zinc Transporters (LZT), which are

characterised by the presence of a highly conserved metalloprotease associated motif

(HEXPHEXGD) in the fifth TMD [39].

The predicted TMD regions of Zip7 are highly conserved when compared to evolutionary

diverse species, which is expected for TMDs as they are essential for ion channel formation.

The highly conserved nature of all eight TMDs indicates that even small changes in TMD

structure are enough to impact channel formation. Two metal binding motifs (HN and

HEXXH) found in Zip7 in TMDs 4 and 5 respectively are believed to be essential for zinc

channel function [40]. The HEXXH motif found in TMD 5 is also a part of the

metalloprotease associated sequence that defines the LIV-1 subfamily of transporters [41].

TMDs 3 and 4 both have a predicted TMD of about half of the predicted α-helical structure though subsequent cross-species analysis showed that the entire region was highly conserved, indicating these were a part of the TMDs.

The loop and terminus regions of the human Zip7 sequence show a greatly reduced level of cross-species conservation compared to the TMD regions, indicating reduced selective pressure for areas outside of the transmembrane channel, though certain areas had a high level of conservation, indicating functional importance. Of particular interest were distinct histidine rich regions. Histidine residues within loop regions have previously been shown to be essential for the function of Zip family members and other zinc transporters, which is likely due to the metal binding affinity of the imidazole side-chain of histidine [17, 18].

Removal of the histidine rich region of a cytosolic loop of the zinc antiporter AtMTP from

Arabidopsis thaliana greatly increased the transport function of this protein [42]. These findings suggest that the transporter regions rich in histidine are predicted to aid in transporter activation through binding zinc ions thereby changing protein conformation to either activate or inactivate the transporter.

The importance of histidine in zinc transporter protein function raised the question of the variability in histidine content between different Zips. Zip6 and Zip10 are both similar to

Zip7 in having high levels of histidine residues in loop regions and the N-terminus. Zip6, initially dubbed LIV-1, is a plasma membrane associated transporter [41]. Increased expression of this transporter has been linked to breast cancer, potentially through causing an overabundance of cytosolic zinc, stimulating uncontrolled activation of cellular growth signalling pathways. Zip10 has been shown to be essential for pancreatic β-cell development through the JAK-STAT growth signalling pathway [43]. These findings support the theory that Zip family members with a large number of histidines in loop regions are associated with

EZS for the activation of cellular pathways.

The human Zip7 protein sequence has an overall low level of similarity to the other thirteen members of the Zip family, with only the regions identified as TMDs showing levels of similarity. From an evolutionary perspective, Zip7 was found to be most closely related to

Zip13 (27.94% similar), a transporter with no loop histidine residues. These two transporters have a high level of similarity between their TMD regions, however the loop regions are highly divergent. The Zip13 sequence lacks the CK2 phosphorylation site that was confirmed in Zip7 by Taylor and associates [11]. Zip13 is associated with Ehlers-Danlos syndrome [40], a disease caused by defects in the processing of collagen and proteins that interact with this structural protein. Decreases in zinc have been shown to affect collagen levels [44], which may indicate that Zip13 is a continuously active transporter associated with protein formation

rather than the rapid efflux associated with modulation of signalling pathways. This further

supports the hypothesis that Zips lacking histidine rich loop motifs are more associated with

protein formation than stimulation of signalling pathways.

The loop region histidine concentrations of the various Zips may indicate whether or not

these transporters are active in EZS or LZS. Zinc transporters with a low number of histidine

residues within loop regions may be constantly active, slowly transporting zinc. Transcription

factor regulation of these transporters may be used as a slow and subtle method of adjusting

zinc levels based on zinc intake. Histidine rich Zips may have a more rapid signalling role,

activated as required via post-translational modification. Zinc binding to the loop histidine

regions has been shown to inactivate and initiate ubiquitin-mediated degradation of Zip4, a protein that is activated via glycosylation [18]. This may indicate that protein stability in addition to transcriptional control is a method for regulating transporters involved in EZS.

Zip7 and Zip13 have both been shown to be associated with the Golgi apparatus and ER and were both found to have a corresponding ER retention signal. Other Zips with this motif also contained a SORT signal, indicating that the ER retention motif in the absence of a SORT signal keeps the protein anchored to the ER. The BRCA1 interaction motif found in the two

Zips with histidine rich regions, but not in Zip7, is likely to be important regarding differentiating the roles in breast cancer of the histidine rich Zips. Both Zip6 and Zip10 have been associated with breast cancer invasion and metastasis [33, 41]. Zip7 has also been implicated in breast cancer progress, although as a gatekeeper of rapid zinc flux, for the activation of cellular growth signalling pathways, in response to an extracellular stimulus rather than being a constitutively active transporter [11].

Zip7 has been shown to be an important regulator of rapid zinc flux. This is important in the context of cell signalling pathways such as insulin signalling, where zinc has been shown to

increase insulin sensitivity. This study highlights areas within the Zip7 protein sequence for future functional analyses, with histidine regions being linked to the function of Zips associated with signalling pathways. The regions highlighted here should be followed up with mutagenesis studies to investigate their contribution towards the function of Zip7.

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28. Wang, F., Kim, B.E., Petris, M.J., and Eide, D.J. (2004). The mammalian Zip5 protein

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33. Kagara, N., Tanaka, N., Noguchi, S., and Hirano, T. (2007). Zinc and its transporter

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36. Fukada, T., Civic, N., Furuichi, T., Shimoda, S., Mishima, K., Higashiyama, H.,

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explants. J Dentist 39, 148-153.

Figure Legends

Figure 1: Signal peptide and TMD prediction for Zip7. A signal peptide was predicted

within the Zip7 protein sequence (A) with a cleavage site at 28aa. C-score represents the likelihood of a cleavage site while S-score indicates the presence of a signal peptide. Y-score is a combination of C and S-scores. The threshold for signal peptide prediction is set at 0.450 as indicated by the magenta line. TMD analysis of Zip7 reveals 8 transmembrane domains, determined by the yellow highlighted areas, when the signal peptide is accounted for (B).

Figure 2: Cross-species conservation of predicted TMDs. All 8 of the TMDs predicted

with MINNOU were found to be highly conserved across all species investigated with >95%

identity between all mammalian sequences. The Zebrafish was shown to have the most

diversity, but still had a very high level of similarity with the mammalian sequences, with the

most poorly conserved (TMD) having a 70% identity to the human sequence.

Figure 3: Loop Region histidine conservation alignment. All loop regions were found to be poorly conserved across the analysed species. There were however a high number of histidine residues (highlighted in green) that were found to be highly conserved in the three histidine rich loop region sections (A) 31-108aa, (B) 190-215aa, (C) 230-277aa. The Zebra fish sequence showed the greatest divergence, but still had a number of common histidine residues with the other sequences analysed.

Figure 4: Phylogenetic tree of the human Zip family. Human Zip protein sequences were aligned and a phylogenetic tree was generated using neighbour joining with % identity. Zip7 was found to be mostly closely related to Zip13, a Zip with many similarities such as ER localisation, but entirely lacking the histidine rich motifs found in Zip7. Node ages are given as percentages showing the level of similarity at each separation.

Chapter 4: The Zinc Transporter Zip7 is Implicated in Glycaemic Control in Skeletal Muscle Cells

Abstract

Zinc improves the glucose lowering activity of insulin and defective zinc homeostasis has

been linked to reduced insulin response, however the cellular mechanisms behind these

effects are not known. Zip7, a zinc transporter localised to sub-cellular compartments can

mediate a rapid increases in cytosolic zinc levels in response to an external stimuli. In this

study we have investigated the role of this distinctive zinc transporter in the modulation of

insulin activity in skeletal muscle. Zip7 gene was reduced in mouse skeletal muscle cells via siRNA transfection technology. The reduced gene expression of Zip7 was concomitant with the reduction of several genes associated with glucose metabolism including; Glycogen branching enzyme, Insulin receptor and the Insulin receptor substrates 1 and 2 and the primary insulin-mediated glucose transporter, Glut4. There was also a significant reduction in the phosphorylation of AKT and subsequent cellular glycogen synthesis in the Zip7 knockdown cells. These studies identify a potential mechanism by which Zip7 modulates insulin activity in skeletal muscle, identifying a potential new target for developing novel treatments for diseases associated with aberrant insulin signalling.

Introduction

Cellular zinc storage, release and distribution are controlled by zinc transporter proteins and metallothioneins. Two families of zinc transporters exist in mammals: the zinc efflux ZnT and the zinc influx Zip proteins [1]. ZnT proteins transport zinc out of the cell or into subcellular compartments for storage. In contrast, Zip proteins transport zinc into the cell or out of subcellular compartments when cytosolic zinc is low or as required for cellular functions [2].

There is increasing interest in the importance of zinc transporters in diseases associated with dysfunctional cellular signalling. In particular, a novel role for these transporters in maintaining essential glucose and lipid metabolism has been identified. For example, in myocytes isolated from the femoral muscle of ZnT7 knockout mice, a reduction in insulin signalling activity was observed [3]. ZnT7 knockout mice were shown to be susceptible to diet-induced glucose intolerance and insulin resistance which was associated with decreased expression of the insulin receptor, insulin receptor substrate 2 and Akt1 [3]. ZnT3, ZnT5 and

ZnT8 gene expression are differentially regulated by glucose in INS-IE cells, and streptozotocin-treated ZnT3 null mice have decreased insulin gene expression and insulin secretion that resulted in hyperglycaemia [4]. Moreover, ZnT8, a transporter localised to secretory granules in the pancreas, plays a critical role in the synthesis, hexamerisation and secretion of insulin and therefore represents a pharmacological target for treating disorders of insulin secretion including diabetes [5].

Zinc mediates its effects through two mechanisms; EZS and LZS [6]. LZS occurs several hours after an extracellular signalling event and depends on changes in the expression of zinc-related molecules such as zinc transporters and metallothioneins, leading to increased protein production [6, 7]. In contrast, EZS occurs minutes after an extracellular stimulus and

does not involve transcriptional-dependent changes [6, 7]. EZS signalling mechanisms are

involved in rapidly increasing intracellular zinc concentrations known as the ‘zinc wave’ [8].

In this situation zinc acts as a second messenger that activates pathways associated with cellular signalling. In fact, zinc has been categorised as an insulin-mimetic with several

groups confirming a role in glucose [9-13] and lipid [13, 14] metabolism. In this context Zip7 has been identified as a key zinc transporter implicated in the “zinc wave” and is suggested to be a “gatekeeper” of cytosolic zinc release from the ER [8]. Endogenous Zip7 is

predominately localised to the Golgi apparatus [15], the ER [16], or both [17] and has been

implicated in breast cancer progression [8, 17, 18]. Studies in tamoxifen-resistant MCF-7

breast cancer cells identified that Zip7 was responsible for activation of tyrosine kinases that

are implicated in the aggressive phenotype of tamoxifen-resistant breast cancer [8, 19, 20].

Recent evidence in MCF7 cells suggests that Zip7 is phosphorylated by CK2 and is associated with the regulated release of zinc from intracellular stores to phosphorylate kinases implicated in cell proliferation and migration [8].

Given the role of Zip7 in modulating zinc flux, and the role of zinc as an insulin mimetic in cellular processes, this transporter may also be implicated in metabolic processes associated with glycaemic control. This study investigated whether Zip7 is required for the modulation of glycaemic control in mouse skeletal muscle cells.

Materials and Methods

Cell culture

Proliferating mouse C2C12 myoblasts were cultured in DMEM supplemented with 10%

Foetal bovine serum and physiological zinc concentrations (20 µM ZnSO4) [8], (Life

Technologies, Mulgrave, Victoria, Australia). Myoblasts were differentiated into post-

mitotic, multi-nucleated myotubes via mitogen withdrawal with DMEM supplemented with

2% horse serum. Assessment of the muscle-specific, contractile and metabolic C2C12 muscle

phenotype was assessed by measuring the expression of markers of differentiation and

metabolic processes as previously described [21].

RNA Extraction and cDNA Synthesis

Mouse quadricep muscle samples were a kind gift from Dr. Paul Lewandowski, Deakin

University, Australia, extracted with approval from the Deakin University Animal Welfare

Committee (A37/2007). Total RNA was extracted from C12C2 cells and C57Bl/6J mouse

quadriceps using TRI-Reagent (Sigma-Aldrich, Castle Hill, NSW, Australia) with the

phenol/chloroform method according to the manufacturer’s protocol. Total RNA was then

treated with 2U of DNase1 for 30 min at 37°C followed by purification of the RNA through

an RNeasy purification column system (Qiagen, Chadstone, Victoria, Australia). RNA

quantity and quality was measured using a Nanodrop spectrophotometer (Thermo Scientific,

Scoresby, Victoria, Australia). RNA was synthesised into cDNA using A High Capacity

cDNA Synthesis kit according to the manufacturer’s instructions (Life Technologies).

Mouse glucose metabolism and zinc transporter arrays

The Mouse Glucose Metabolism RT2 Profiler PCR Array (Qiagen) contained primers for

analysing expression of 84 genes involved in the regulation and enzymatic pathways of

glucose and glycogen metabolism. The Zinc Transporter RT2 Profiler Custom PCR Array

(Qiagen) contained primers targeting the genes for the two zinc transporter families (Slc30a1-

10 and Slc39a1-14) and the metallothioneins (Mt1-4). cDNA synthesis of RT2 mouse glucose metabolism and zinc transporter PCR array

cDNA synthesis using the RT2 First Strand Kit was performed as described by the

manufacturer (Qiagen). Briefly, potential genomic DNA was eliminated by digestion with

DNAase from 500 ng of total RNA which was then reverse transcribed before dilution to a

working concentration and storage at -20°C.

Quantitative Real-time PCR

qPCR was performed on a RealPlex PCR detection system (Eppendorf, North Ryde, New

South Wales, Australia) in triplicate on at least three independent RNA preparations. Target

cDNA levels were analysed in 10 µl reactions with SensiMix SYBR No-ROX (Bioline,

Alexandria, New South Wales, Australia). Primers (GeneWorks, South Australia, Australia) for markers of skeletal muscle cell differentiation and metabolism, Myogenin, Tnni1, Tnni2,

Abca1, Fabp3 and Srebp-1c (Refer to Chapter 2, Table 5) have been previously described

[21-23]. Other primers for the amplification of target gene sequences were designed using the

NCBI Primer Blast Tool http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi, with the

exception of Irs1 (PrimerBank ID: 29825829a1), Irs2 (PrimerBank ID: 3661525a1) and Insr

(PrimerBank ID: 6754360a1) which were obtained from the PrimerBank Database

http://pga.mgh.harvard.edu/primerbank/index.html [24-26]. The relative level of target gene

expression was normalised to Gapdh, or Eef2 as described in the results section and

associated errors were calculated using the guidelines described by Bookout and Mangelsdorf

[27].

Quantitative Real-time PCR of RT2 mouse glucose metabolism and zinc transporter PCR array

The qPCR for the RT2 glucose metabolism and zinc transporter arrays were performed as

outlined in the guidelines supplied by the manufacturers (Qiagen). Briefly, a master mix

containing PCR reaction ingredients and cDNA was prepared and added to each well of the

glucose metabolism array 96-well plate. Refer to chapter 2 for a detailed outline of the PCR

parameters used.

Transient transfections of siRNA molecules

The transient transfection of siRNA molecules Zip7 (Catalogue No: AM16708), Zip1, Gapdh

and the scramble control (Life Technologies) were performed using lipofectamine

RNAiMAX reagent as instructed by the manufacturers (Life Technologies). Briefly, C2C12

cells were transfected in 6-well dishes with 10 nM of siRNA molecule or the scramble

control in RNAiMAX reagent. The cells were subsequently maintained in 2% horse serum

and differentiated over three days and collected in 1 ml of TRI-Reagent per three wells for

RNA extraction or 1 ml of RIPA Buffer (Thermo Scientific) containing 10 µl Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific) for protein analysis.

Protein Extraction and Western Blot

Total cellular protein from the scramble control and the siRNA-Zip7 transfected C2C12 cells was isolated by scraping cells with RIPA buffer (contains protease and phosphatase inhibitor cocktail) then the samples were placed on ice for 1 hr with vigorous mixing every 10 minutes. The cells were then sonicated with 5 second pulses for 30 seconds at 50% duty followed by boiling for 10 min. The protein samples were centrifuged at 12,000 RCF for 5 minutes and the supernatant was collected. Total protein concentration was measured using a

BCA kit (BIORAD, Gladesville, New South Wales) against a BSA standard of known concentration (Thermo Scientific) as outlined by the manufacturer’s instructions.

Total soluble protein (30 µg) from the scramble control and siRNA-Zip7 transfected C2C12 cell lines was resolved on a 4-15% SDS-PAGE gradient gel (BIORAD) and transferred to a nitrocellulose membrane. The membranes were blocked overnight in 5% skim milk in TBS-

Tween 20 followed by an overnight incubation with Glut4, Gapdh (Santa Cruz

Biotechnology, Santa Cruz, CA), Akt (Cell Signalling) or phosphorylated Akt (Cell

Signalling) antibodies. Following 4 x 15 minute washes the membrane was incubated with either anti-mouse HRP (Cell Signalling, Catalog No: 7076) for Glut4; anti-Rabbit HRP (Cell

Signalling, Catalog No:7074) for Akt and pAkt, and anti-goat HRP (Santa Cruz; Catalog No: sc-2020) for GAPDH (1:5000) for 1 hr at RT. Immunoreactive signals were detected using

enhanced chemiluminescence SuperSignal West Pico Substrate (Pierce) and visualised by

autoradiography or a UVITEC Alliance digital imaging system (Thermo Scientific, Victoria

Australia). To assess protein loading consistency, the membranes were stripped with Restore

Plus Western Blot Stripping Buffer (Thermo Scientific) and membranes were subsequently

washed in TBS-Tween and blocked with 5% skim milk before adding the primary antibody

for a loading control (Gapdh or Akt).

Glycogen synthesis assay

Glycogen synthesis levels were measured using a Glycogen Colorimetric Assay Kit

(BioVision, Scoresby, Australia) as described by the manufacturer. Briefly, C2C12 cells were transfected with siRNA-Zip7 or the scramble control as described above. Following differentiation, C2C12 cells were treated with 10 nM insulin for 60 minutes. Cell lysates were collected and homogenates were boiled for 5 min to inactivate enzymes. Samples were then centrifuged and the supernatant was collected. Samples were prepared by performing hydrolysis of glycogen to glucose and then mixed with OxiRed probe to generate colour.

Statistics

Data obtained from individual qPCR was assessed by a Student’s unpaired t-test on at least three independent biological replicates. One tailed t-tests were used for testing the significance of gene knockdowns and two tailed tests were used to analyse all subsequent changes. Statistical significance was denoted as the average ± standard deviation of the mean.

Changes were considered statistically significant when the P value was ≤ 0.05. *P < 0.05;

**P < 0.01 and ***P < 0.001. The analysis of the gene arrays was performed with the RT2

Profiler PCR Array Data Analysis Software v3.5 (SA Biosciences).

Results

The Slc39a (Zip) zinc transporters are differentially expressed in C2C12 skeletal muscle cells

and mouse quadriceps.

To determine the expression levels of the Slc39a zinc transporter family in mouse C2C12

skeletal muscle cells and mouse quadriceps a custom zinc gene array (SABiosciences,

Qiagen) with primer sequences that are specific for the Slc39a (Zip) mouse zinc transporter genes (i.e. Slc39a1-14) was utilised. Quantitative real-time PCR (qPCR) was performed and the expression of each zinc transporter transcript was measured relative to Gapdh.

The zinc transporters Slc39a1 and Slc39a7 were highly expressed in C2C12 skeletal muscle cells (Figure 1A). Lower levels of expression were observed for Slc39a3, 6, 9, 10, 11, 13 and

14. Minimal or no expression was observed in Slc39a2, 4, 5, 8, and 12 (Figure 1A). In mouse quadriceps, high levels of expression for all of the Slc39a transporters with the exception of

Slc39a5 (Figure 1B) were observed.

Slc39a7 (Zip7) is expressed during C2C12 skeletal muscle differentiation.

Slc39a7 (Zip7) was the focus of this study as this transporter is predominately localised to the

ER and the Golgi apparatus and is suggested to be involved in modulating rapid zinc release for modulating signalling pathways [8]. To elucidate the role of Zip7 in skeletal muscle the expression profile of this zinc transporter relative to Eef2 in the mouse C2C12 myoblast cell line was investigated. Proliferating myoblasts can be induced to biochemically and morphologically differentiate into post-mitotic multinucleated myotubes by mitogen withdrawal. This transition from a non-muscle phenotype to a contractile phenotype is associated with the activation and repression of a structurally diverse group of genes responsible for contraction and the extreme metabolic demands placed on this tissue. During this period of differentiation, it was observed that Zip7 mRNA was highly expressed in

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proliferating myoblasts and consistently expressed at a lower level during skeletal muscle cell

differentiation when normalised to Eef2 (Figure 2A). In order to assess the differentiation

status of the C2C12 cells and to demonstrate that they had acquired a differentiated,

contractile and metabolic muscle phenotype, qPCR was performed on the marker genes

myogenin (MyoG), a gene that encodes the hierarchical basic helix loop regulator and is

specifically required for differentiation [28], the slow twitch (type I) and the fast twitch (type

II) isoforms of the contractile protein troponin I (Tnni1 and TnniII), and the metabolic genes

Abca1 (ATP-binding cassette proteins), Fabp3 (fatty acid binding protein 3) and Srebp1c

(sterol regulatory binding element protein). Expression of both MyoG and the contractile protein genes (type I and II, Tnni1 and Tnni2, respectively) were dramatically increased and confirmed the differentiation of the myoblast C2C12 skeletal muscle cell line to the myotube phenotype (Figure 2B-D). Additionally, genes involved in lipid metabolism (Abca1 and

Srebp1c) (Figure 2E and 2G) were also induced while Fabp3 was downregulated during

muscle differentiation (Figure 2F) which is consistent with previous studies [22, 29-31] and

confirms that the muscle cells had acquired the appropriate contractile and metabolic

phenotype.

siRNA-Zip7 represses endogenous Zip7 mRNA in skeletal muscle cells.

To elucidate the biological role of Zip7 in the context of glucose metabolism, the expression

of this transporter in C2C12 skeletal muscle cells was selectively ablated utilising a siRNA-

Zip7 molecule. A siRNA targeting mouse Gapdh and a scramble sequence that contains no

known homology to the mouse, rat or human genome were utilised as controls. The siRNA-

Gapdh was used to determine the robustness of the transfection and the ability to successfully

reduce the expression of a specific target gene that is constitutively active. Accordingly,

C2C12 cells were transfected with the scramble control, siRNA-Gapdh or the siRNA-Zip7 and subsequently differentiated for three days.

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Initially, the specificity and robustness of the siRNA transfection was validated in C2C12

cells by transfecting a siRNA-Gapdh to determine transfection efficacy and siRNA

specificity. A significant reduction in Gapdh mRNA (4-fold, p=0.0023) in the siRNA-Gapdh transfected cells compared to the scramble control (Figure 3A) was seen. C2C12 skeletal muscle cells were then transfected with a siRNA targeting Zip7 mRNA. Quantitative PCR was then performed to measure the expression levels of endogenous Zip7 relative to Eef2 in

RNA isolated from the scramble control and Zip7 transfected cell lines. A significant reduction in the mRNA levels of Zip7 (4.6-fold, p=0.0006) when compared to the scramble control (Figure 3B) was observed.

To determine that the reduction in Zip7 expression was not due to differential Eef2 mRNA

expression, qPCR was also performed on Eef2 normalised to Gapdh. No change in the level of Eef2 in the siRNA-Zip7 cell lines were observed when normalised to Gapdh mRNA

(Figure 3C). The relative expression of Zip7 in siRNA-Gapdh C2C12 cells was also measured and no change was seen (Figure 4D).

Since Zip1 was also highly expressed in C2C12 skeletal muscle cells (Figure 1), the expression of this transporter was reduced with a siRNA-Zip1 to determine if there were any compensatory changes in Zip7 expression. C2C12 cells were transfected with the scramble control and siRNA-Zip1 and endogenous Zip1 and Zip7 mRNA was measured. Endogenous levels of Zip1 mRNA were successfully reduced (approximately 3-fold, p<0.003) in the

C2C12 cell lines (Figure 3E). No change in endogenous expression of Zip7 mRNA (p=0.1) was observed in the siRNA-Zip1 cell lines (Figure 3F).

Zip7 knockdown resulted in no change in other zinc transporters.

To determine the expression status of the other zinc transporter family members in the presence of the Zip7 reduced C2C12 cell lines, a custom gene array that contains the primer

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sequences for the Slc30a/ZnT (1-10) and Slc39a/Zip (1-14) family members was utilised. cDNA from the scramble control and the siRNA-Zip7 C2C12 cells were assayed for

compensatory changes in the other family members due to reduced Zip7 mRNA. The reduction of Zip7 was shown to have no effect on the expression of the Slc30a/ZnT family members (Figure 4A). In the Slc39a/Zip arrays, reduced expression of Zip7 resulted in a significant reduction in Zip7 mRNA as expected. A small, but significant increase in Zip3

expression and reduction in the expression of Zip13 and Zip14 mRNA (Figure 4B) was also observed.

To further assess the reduced expression of Zip13 and Zip14 in the Zip7 reduced C2C12 cells, primer pairs specific for Zip13 and Zip14 were designed to independently test the validity of this observation. qPCR was performed on Zip13 and Zip14 expression in the scramble control and siRNA-Zip7 C2C12 cells and no significant changes in expression levels for these zinc transporters were observed (Figure 4C).

Reduced expression of Zip7 in C2C12 cells is associated with changes in several genes implicated in glucose metabolism.

A Mouse Glucose Metabolism RT2 Profiler PCR Array (SABiosciences, Qiagen) that contains profiles for the expression of 84 key genes implicated in the regulation of enzymatic pathways of glucose and glycogen metabolism was utilised to assess potential pathways that are modulated by Zip7. The reduced expression of Zip7 in C2C12 skeletal muscle cells resulted in changes in several genes implicated in glucose metabolism. These include Agl

(Amylo-1,6-glucosidase, 4 alpha-glucanotransferase, p=0.003), Dlst (Dihydrolipoamide S- acetyltransferase, p=0.036), Galm (Galactose mutarotase, p=0.0017), Gbe1 (Glucan-1,4- alpha branching enzyme 1, p=0.003), Idh3g (Isocitrate dehydrogenase 3 NAD+ gamma, p=0.015), Pck2 (Phosphoenolpyruvate carboxykinase 2, p=0.002), Pgam2 (Phosphoglycerate mutase 2, p=0.03), Pgm2 (Phosphoglucomutase 2, p=0.03), Phkb (Phosphorylase kinase beta,

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p=0.03), Pygm (Muscle glycogen phosphorylase, p=0.004), Tpi1 (Triosephosphate isomerase

1, p=0.02) and Gusb (Glucuronidase beta, p=0.01) (Table 1).

Several of these genes were further validated, with a focus on glycogen metabolism (Pgm2,

Phkb, Pygm and Gbe1) by designing new primer pairs and performing qPCR on the scramble control versus the siRNA-Zip7 cDNA. A significant downregulation in these genes was observed in concordance with the PCR array data (Figure 5A-D).

It was speculated that given genes implicated in glycogen metabolism were affected by reduced Zip7 mRNA levels, that the glucose transporter, Glut4 might be downregulated in the siRNA-Zip7 cells. Glut4 predominately transports glucose across the plasma membrane which is further processed by oxidative (glycolysis) or non-oxidative (glycogen synthesis) pathways [32]. Accordingly, qPCR was performed for Glut4 mRNA expression in the scramble control and the siRNA-Zip7 C2C12 cells. There was a significant downregulation of Glut4 in the siRNA-Zip7 cells (p=0.01) (Figure 6A). Glut4 immunoreactive protein in the scramble control and siRNA-Zip7 C2C12 cells was also tested. GAPDH was used as a protein loading control and showed that similar amounts of total soluble protein were resolved (Figure 6B). There was a significant reduction in immunoreactive Glut4 in the siRNA-Zip7 C2C12 cells compared to the scramble control (Figure 6B).

Reduced Zip7 compromises insulin-induced glycogen synthesis and phosphorylation of Akt in

C2C12 skeletal muscle cells.

Given that Zip7 modulates core genes implicated in glucose metabolism, glycogen synthesis was investigated in the Zip7 knockdown skeletal muscle cells. Scramble and siRNA-Zip7

C2C12 cells were treated with 10nM insulin over 60 minutes and glycogen synthesis was measured. There was a significant reduction in glycogen synthesis in the siRNA-Zip7 when compared to the scramble control (Figure 6C). As expected, a significant induction of

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glycogen synthesis was found on exposure to insulin in the scramble control cells, however

this was blunted in the siRNA-Zip7 C2C12 cells (Figure 6C).

Glucose taken up through Glut4 is used for glycogen synthesis in muscle [33] and with

increasing plasma insulin concentration, glycogen synthase is activated by insulin and the

rate of glycogen synthesis increases [34]. Moreover, a core component of glycogen synthesis

is the insulin-induced phosphorylation of Akt in a process that leads to the activation of

glycogen synthase [34]. To determine a potential mechanism for reduced glycogen synthesis

in the presence of reduced Zip7 mRNA, qPCR was performed on the insulin receptor (Insr)

and the isoforms of the insulin receptor substrate (Irs) molecules that are predominantly

expressed in skeletal muscle, insulin receptor substrate 1 (Irs1), and insulin receptor substrate

2 (Irs2) [32]. Irs proteins serve as docking molecules for several SH2-containing proteins and

the subsequent activation of downstream signalling molecules that result in the activation of

Akt which mediates many of insulin’s metabolic effects including modulation of gluconeogenesis, protein synthesis and glycogen synthesis [33]. Accordingly, the reduced expression of Zip7 in the C2C12 skeletal muscle cells resulted in a significant reduction in the expression of the Insr, Irs1 and Irs2 (Figure 7A-C). In order to confirm that the reduction of these key genes was associated with a reduction in signalling, an immunoblot analysis on pAkt was performed. A significant reduction in pAkt in the siRNA-Zip7 compared to the

scramble control was observed (Figure 7D).

Discussion

Intracellular zinc levels are largely regulated by two families of zinc transporters (ZnTs and

Zips) that traffic zinc across biological membranes [35, 36]. Dysregulation of zinc

homeostasis may contribute to a number of disease states including cancer [19, 37],

autoimmune disease [38, 39], cardiovascular disease [40, 41] and diabetes [42-45]. Zip7 has

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been implicated in maintaining cellular zinc homeostasis through its ability to initiate the

‘zinc wave’ and provide cytosolic zinc ions that are involved in cellular signalling processes.

Studies in breast cancer cells have elucidated a role for this transporter in cell signalling

events [8, 20] however, the role of Zip7 with respect to the control of the genetic programs associated with carbohydrate metabolism in skeletal muscle has not been addressed. This study provides the first evidence for a metabolic role for Zip7 in modulating glycaemic control in skeletal muscle and justifies further studies in processes associated with insulin resistance in this tissue.

Zip7 mRNA is highly expressed in differentiated C2C12 cells and mouse quadriceps.

Although Slc39a1 was also highly expressed in C2C12 skeletal muscle cells, homozygous knockout of Slc39a1 in mice produces no phenotype when dietary zinc intake is normal [46] suggesting compensatory actions from other Zip family members. To explore compensatory mechanisms from other zinc transporters upon knockdown of Zip7 expression, all of Zip and

ZnT family members were investigated via qPCR. No major changes in expression of zinc transporters were observed in either the scramble control or the siRNA-Zip7 C2C12 skeletal muscle cells, suggesting that reduced Zip7 expression has minimal effect on these genes. Of the zinc transporters, it should be emphasised that, in addition to Zip13, [47] Zip7 is the only

other Zip localised to the Golgi apparatus and not the plasma membrane [15] hence

compensation in expression of transporters on the plasma membrane or other cellular

compartments would be less likely. Given that Zip7 is localised exclusively on the Golgi

apparatus or the ER [17], and the fact that no compensatory changes in the other transporters

were observed in the siRNA-Zip7 C2C12 cells suggests that this transporter may be unique in

its specialised function in transporting zinc from the ER or Golgi into the cytosol in these

cells [19].

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In contrast to the Zip expression profile in C2C12 cells, a moderate expression level for all of

the Zip transporters (except for Zip5) in mouse quadriceps was also observed. Zip7 mRNA

was more highly expressed in C2C12 cells (approximately 15-fold) when compared to the expression found in quadriceps. It should be noted that quadriceps contain a mix of muscle fibre-types (oxidative type 1 and glycolytic type II) [48] as well as blood vessels, nerves and connective tissue. Similar studies on other muscle fibre types; soleus (type I), plantaris (type

II) and anterior tibialis (type II) also demonstrated differences in the level of expression for the orphan nuclear receptor, Coup-tfII in these tissues in comparison to C2C12 cells [49].

Moreover, studies on protein arginine methyltransferase 3, 4 and 5 (PRMT3-5) in mouse

skeletal muscle tissue and C2C12 cells found high expression of PRMT3-5 in gastrocnemius

in comparison to only high expression of PRMT4 in C2C12 with no or minimal expression of

PRMT3 and 5 respectively [50]. Although these relative expression discrepancies exist

between in vitro and in vivo model systems, the C2C12 cell culture model is a well-

established and validated system to study the effects of skeletal muscle metabolic processes

[21, 51, 52]. For example, data derived from this in vitro model with liver X receptor (LXR)

and peroxisome proliferating activated receptor (PPAR) agonists and their role in metabolism

(e.g. energy expenditure, running endurance, lipid metabolism and cholesterol efflux) has

been validated and reproduced in mice [51-55].

This study revealed that subsets of genes involved in glucose metabolism (Agl, Dlst, Galm,

Gbe1, Idh3g, Pck2, Pgam2, Pgm2, Phkb, Pygm, Tpi1, Gusb and Glut4) are altered when Zip7

expression was reduced. This is further highlighted by the fact that related genes in similar

and other pathways (see Table 1) were refractory to the reduced Zip7 expression. These

findings are noteworthy for several reasons. In skeletal muscle, Glut4 predominately

transports glucose across the plasma membrane for further processing by oxidative

(glycolysis) or non-oxidative (glycogenesis) pathways [32]. Thus, the decline in Glut4

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protein in the siRNA-Zip7 C2C12 cells would suggest a reduction in glucose transport and subsequent genes associated with oxidative and non-oxidative pathways. Similarly, this is supported by the observation that several genes involved in glycolysis (Galm, Gusb, Pgam2,

Pgm2 and Tpi1) and glycogen synthesis (Gbe1, Agl, Pgm2, Pygm and Phkg2) were reduced in the Zip7 knockdown cells. In skeletal muscle, these genes play critical roles in the oxidative and non-oxidative pathways, respectively. This is further supported by the reduction of basal and insulin-mediated glycogen storage in C2C12 myotubes when Zip7 expression was reduced (Figure 6).

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Table 1: Glucose metabolism gene array results summary.

PATHWAY: GLUCOSE METABOLISM GLYCOLYSIS T-TEST Fold Up- or Down-Regulation Gene Symbol Description Gene Name p value* siRNA-Zip7 /Scramble NM_176963 Galm Galactose mutarotase 0.00171 -1.71

NM_010368 Gusb Glucuronidase, beta 0.01363 -1.15

NM_018870 Pgam2 Phosphoglycerate mutase 2 0.03151 -1.6

NM_028132 Pgm2 Phosphoglucomutase 2 0.02798 -1.36

NM_009415 Tpi1 Triosephosphate isomerase 1 0.02108 -1.24

GLUCONEOGENESIS T-TEST Fold Up- or Down-Regulation Gene Symbol Description Gene Name p value* siRNA-Zip7 /Scramble NM_028994 Pck2 Phosphoenolpyruvate carboxykinase 2 0.00219 1.82 (mitochondrial) TCA CYCLE T-TEST Fold Up- or Down-Regulation Gene Symbol Description Gene Name p value* siRNA-Zip7 /Scramble NM_030225 Dlst Dihydrolipoamide S-succinyltransferase 0.03589 -1.14

NM_008323 Idh3g Isocitrate dehydrogenase 3 (NAD+), gamma 0.01532 -1.31

NM_028994 Pck2 Phosphoenolpyruvate carboxykinase 2 0.00219 1.82 (mitochondrial) PATHWAY: GLYCOGEN METABOLISM SYNTHESIS T-TEST Fold Up- or Down-Regulation Gene Symbol Description Gene Name p value* siRNA-Zip7 /Scramble NM_028803 Gbe1 Glucan (1,4-alpha-), branching enzyme 1 0.00322 -1.98

DEGRADATION T-TEST Fold Up- or Down-Regulation Gene Symbol Description Gene Name p value* siRNA-Zip7 /Scramble NM_0010813 Agl Amylo-1,6-glucosidase, 4-alpha- 0.00299 -1.41 26 glucanotransferase NM_011224 Pygm Muscle glycogen phosphorylase 0.00409 -1.75

REGULATION T-TEST Fold Up- or Down-Regulation Gene Symbol Description Gene Name p value* siRNA-Zip7 /Scramble NM_199446 Phkb Phosphorylase kinase beta 0.03224 -1.43

* P values <0.05

Skeletal muscle is particularly important in maintaining glucose homeostasis as approximately 70-80% of whole body insulin-mediated glucose uptake occurs in muscle where it is incorporated into glycogen for storage [56]. Moreover, in insulin-resistant states, insulin-induced glucose uptake and glycogen synthesis is markedly reduced in skeletal muscle [32, 57]. Accordingly, in association with a reduction in genes involved in glycogen

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metabolism and the fact that there was a reduction in glycogen synthesis, there was also a

significant decrease in the phosphorylation status of Akt in the siRNA-Zip7 C2C12 cells.

This is consistent with recent studies where a siRNA targeting Zip7 significantly decreased

pAkt after zinc treatment in MCF-7 tamoxifen-resistant breast cancer cells [8]. Similarly, given the role of Zip7 in facilitating zinc flux into the cytosol [8], and the fact that previous studies have shown that zinc can activate pAkt [8, 58], this study suggests a role for Zip7 in

mediating zinc flux and signalling events that lead to phosphorylation of Akt and the

mobilisation of glucose transporters in skeletal muscle. This is also further supported by the

observed reduction in the mRNA expression of Insr, Irs1 and Isr2 in the reduced Zip7 C2C12

skeletal muscle cells.

Zinc can inhibit PTPs [59] with a reported inhibition constant in the nanomolar range [60].

Zinc inhibits PTP1B, a cytoplasmic phosphatase that interacts with the insulin receptor and

catalyses its dephosphorylation resulting in the inactivation of insulin signalling [61]. Based

on these results, and the fact that the insulin signalling pathway depends on the status of

tyrosine phosphatases and the release of zinc into the cytosol, it was hypothesised that

reduced expression of Zip7 would lead to a reduction in the cytosolic zinc pool that is available for cellular signalling. For example, in the testes of diabetic mice treated with the zinc chelator, TPEN, a significant down-regulation of Akt-mediated glucose metabolism was

observed that was associated with reduced phosphorylation of Akt and GSK-3β [62].

Moreover, treatment of 3T3-L1 adipocytes with ZnCl2 increased tyrosine phosphorylation of

the insulin receptor beta subunit and enhanced the transport of glucose in the absence of

insulin through the PI3-kinase/Akt pathway [53] and in myocytes isolated from the femoral

muscle of mice with a ZnT7 knock-out, which display low zinc status, there was reduced

insulin signalling pathway activity and insulin resistance. This was also consistent with the observed reduction in the expression of Insr, Irs2 and Akt [3].

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Based on the observations that Zip7 plays a crucial role in facilitating cytosolic zinc flux [8], and given the role of zinc as a second messenger that activates pathways associated with cellular signalling, these studies suggest a new role for Zip7 in regulating the critical gene programs involved in glucose uptake and glycogen storage in skeletal muscle. In particular, the down-regulation of Insr, Irs1 and Irs2, in association with reduced phosphorylation of

Akt and reduced Glut4 expression, suggests that Zip7 manipulation may have therapeutic

utility as a novel approach for the treatment of insulin resistance in skeletal muscle. Although

this study implicates Zip7 as being essential for carbohydrate metabolism in skeletal muscle,

the function and method of activation of Zip7 in this pathway requires further investigation.

Gene overexpression experiments would be a beneficial next step in exploring the role of this

transporter in the modulation of skeletal muscle glucose metabolism.

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Figure Legends

Figure 1: mRNA expression of the zinc transporters Slc39a (Zip) relative to the

housekeeping gene, Gapdh in mouse C2C12 skeletal muscle cells and mouse quadriceps.

Slc39a1-14 mRNA expression in C2C12 cells and mouse quadriceps, respectively (A-B).

Error bars indicate ± SD from three independent biological samples.

Figure 2: Relative expression of Slc39a7 (Zip7) and markers of skeletal muscle

differentiation in C2C12 cell lines. Slc39a7 (Zip7) expression relative to Eef2 (A). Markers of skeletal muscle differentiation: myogenin (MyoG) and the troponins 1 and 2 (Tnni1 and

Tnni2), respectively (B-D). Markers of metabolism: ATP-binding cassette transporter protein

1 (Abca1), fatty-acid binding protein 3 (Fabp3) and sterol regulatory element binding protein

1c (Srebp-1c), respectively. PMB = proliferating myoblasts; D1-3 = day 1 to day 3 of differentiation of myotubes, respectively (E-G). Error bars indicated the ± SD from three independent biological samples.

Figure 3: Zip7 mRNA is reduced by siRNA-Zip7. Relative expression of Gapdh, Zip7,

Zip1 and Eef2 in the scramble control and corresponding siRNA cells, respectively. Gapdh relative to Eef2 in siRNA-Gapdh cells (A). Zip7 relative to Eef2 in siRNA-Zip7 cells (B).

Eef2 relative to Gapdh in siRNA-Zip7 cells (C). Zip7 relative to Eef2 in siRNA-Gapdh cells

(D). Zip1 relative to Eef2 in siRNA-Zip1 cells (E), and Zip7 relative to Eef2 in siRNA-Zip1 cells (F). Error bars indicated the ± SD from three independent biological samples. **P<0.01,

***P<0.001.

Figure 4: Reduced Zip7 expression has minimal influence on the expression of other zinc transporter genes.. Relative expression of the ZnTs (Slc30a1-10) to Gapdh in the scramble control versus the siRNA-Zip7 (A). Relative expression of the Zips (Slc39a1-14) to

Gapdh in the scramble control versus the siRNA-Zip7 (B). Relative expression of Zip13 and

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Zip14, respectively in the siRNA-Zip7 cells (C and D). Error bars indicated the ± SD from three independent biological samples. *P<0.05, ***P<0.001.

Figure 5: Reduced Zip7 expression alters gene expression of key glucose metabolic genes. Relative expression of Pgm2, Phkb, Pygm and Geb1 mRNA to Eef2 in the scramble control and the siRNA-Zip7 respectively (A-E). Error bars indicated the ± SD from three independent biological samples. *P<0.05, **P<0.01.

Figure 6: Reduced Zip7 expression reduces the expression of Glut4 and decreases glycogen synthesis.. Relative expression of Glut4 mRNA to Eef2 in the scramble control and siRNA-Zip7 C2C12 cells (A). Western blot for immunoreactive Glut4 and Gapdh in protein lysates from the scramble control and the siRNA-Zip7 C2C12 cells (B). Assay for glycogen synthesis in scramble and siRNA-Zip7 cells treated with 10 nM insulin for 1 hour (C). Error bars indicate the ± SD from three independent cell collections for Glut4 mRNA, and six independent transient transfections of the siRNA-Zip7 for the glycogen synthesis. **P<0.01,

***P<0.001.

Figure 7: Reduced Zip7 expression reduces the mRNA expression of the insulin receptor

(Insr), insulin receptor substrate 1 (Irs1), insulin receptor substrate 2 (Irs2) and the phosphorylation of Akt. . Expression of Insr, Isr1 and Isr2 mRNA relative to Eef2 in the scramble control and the siRNA-Zip7 C2C12 skeletal muscle cells (A-C). Western blot for immunoreactive pAkt and Akt in protein lysates isolated from scramble control and siRNA-

Zip7 transfected C2C12 skeletal muscle cells (D). Error bars indicate the ± SD from three independent biological samples for the mRNA analysis of Inrs, Irs1 and Irs2. **P<0.01,

***P<0.001. Western blot analysis for pAkt and Akt was performed three times on six independent and pooled transient transfections of the scramble control and siRNA-Zip7.

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Chapter 5: Insulin Induces an Increase in Cytosolic Free Zinc in C2C12 Skeletal Muscle Cells as a Positive Feedback Mechanism for Insulin Signalling

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Abstract

The insulin-like activity of zinc is well established but the role of fluctuations in the

concentration of intracellular zinc ions in insulin signalling remains unclear. PTP1B, which

dephosphorylates and so inactivates the insulin receptor, is inhibited by both zinc ions and

oxidation and this has been proposed to prolong insulin signalling activation by preventing

rapid receptor dephosphorylation. Zip7, a novel endoplasmic reticulum associated transporter has been implicated in the regulation of signal transduction through the release of zinc from intracellular stores in response to extracellular stimuli. Reduced Zip7 expression has been

shown to reduce glycogen synthesis in muscle cells leading to the hypothesis that insulin

treatment would stimulate zinc release through activation of Zip7. Here we investigated

whether insulin stimulates zinc release in skeletal muscle and the mechanisms involved.

Treatment of C2C12 mouse skeletal muscle cells with 10 nM of insulin resulted in increased

concentrations of free zinc in the cytosol within 15 minutes of treatment and this was ablated

by the PI3-kinase inhibitor wortmannin. Transient siRNA transfection to reduce the

expression of Zip7 also reduced insulin-stimulated Akt phosphorylation but caused no change

in insulin-mediated zinc increase. Oxidation of thiols in metallothioneins leads to release of

zinc ions which may be an alternative mechanism for insulin-mediated cytosolic zinc release.

To test whether this mechanism was operative ROS production was measured and found to be transiently increased by insulin treatment of C2C12 cells. These results show for the first time that insulin signalling increases cytosolic zinc release downstream of PI3-kinase activation. Zinc release was not influenced by reduced Zip7 expression but instead corresponded with increased ROS production. In conclusion, insulin stimulates mobilisation of zinc ions in the cytosol of muscle cells and this is likely to occur through ROS-mediated zinc release from metallothioneins. The combined effect of zinc and oxidative inhibition of

PTPs may serve as a positive feedback loop to prolong insulin signalling in muscle.

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Introduction

The essential role of zinc in the normal physiological function of insulin was first reported nearly 100 years ago with the discovery of the zinc-mediated crystallisation of insulin [1].

Early investigations into zinc biology revealed that zinc was essential in pancreatic β-cells for the production and secretion of insulin [2]. More recent studies have implicated zinc as a signalling molecule for regulating the response to insulin in peripheral tissues [3-5]. The role of zinc in the production and secretion of insulin is well understood [4, 6], while the mechanisms underlying zinc’s role in modulation of insulin signalling remain largely unclear.

Due to the increasing prevalence of insulin resistance and T2DM, the role of zinc in insulin signalling has become an important area for further investigation.

Many signalling molecules have been identified that are regulated by zinc [7, 8]. In the context of insulin signalling, one well characterised protein inhibited by zinc is PTP1B, which inactivates the insulin receptor via the removal of phosphate groups [9]. PTP1B is activated by the insulin receptor when it complexes with insulin [10], and acts as a negative feedback mechanism for modulating insulin activity. Bioavailable zinc is a potent inhibitor of

PTP1B, with only picomolar concentrations required to reduce activity [11], indicating a potential positive feedback role for zinc in this pathway. Although the effects of zinc on insulin activity are well characterised, the cellular mechanisms for controlling zinc availability in the context of insulin signalling remain poorly understood.

Zinc ions are naturally impermeable to biological membranes, requiring a group of transport proteins to control cellular homeostasis. There are two primary families of zinc transporters, the Zips and the ZnTs, which are responsible for increasing and decreasing cytosolic zinc levels respectively [12]. There are a total of 24 zinc transporters in mammals with 14 Zips

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and 10 ZnTs [12], many of which have varying tissue expression and cellular localisation

[13].

Another group of proteins involved in zinc homeostasis are the heavy metal binding MTs, a family of cysteine rich proteins of low molecular weight that function via binding cytosolic heavy metals to reduce their bioavailability. There are four well characterised mammalian

MTs [14] with MT 1 and 2 being ubiquitously expressed and MTs 3 and 4 having more specific expression profiles, mostly in the central nervous system [14]. The expression of Mts

1 and 2 are regulated by cellular heavy metal concentrations [15]. The metal binding characteristics of the MTs are due to a sulphur rich functional site that attracts positively charged ions to form thiolate clusters [16]. The metal bound thiolate clusters of these proteins have been shown to undergo oxidation leading to the release of bound metals [17, 18]. MTs have been implicated in the progression of diabetes due to their role in zinc homeostasis and have been linked to glucose metabolism. Glucose concentrations have been shown to upregulate MT gene expression in cultured pancreatic β-cells [19].

Free Zinc concentrations change within cells with two phases having been recognised known as EZS) and LZS [20]. EZS is a rapid temporary change in cytosolic zinc levels while LZS describes the gradual change in cytosolic zinc levels caused by transcriptional regulation leading to a change in transporter protein production.

Zip7, an efflux transporter localised to the endoplasmic reticulum and Golgi apparatus [21], has been implicated in modulating a rapid increase in cytosolic zinc in response to an extracellular stimulus [22]. This zinc transporter has previously been shown to be a gatekeeper for zinc flux in breast cancer cells [23]. Zip7 is phosphorylated by CK2 in response to a combination of epidermal growth factor (EGF), cell permeable zinc and calcium [22]. Taylor and associates identified that Zip7 can be activated, however it has yet

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to be shown if modulation of cytosolic zinc concentrations by Zip7 is a general phenomenon

that occurs through other extracellular stimuli.

As shown in Chapter 3 of this thesis, gene silencing of Zip7 via siRNA transfection in C2C12

mouse skeletal muscle cells resulted in the decreased expression of genes associated with

glucose metabolism, reduced glycogen synthesis and decreased insulin-mediated phosphorylation of Akt. This indicates an essential role for Zip7 in the response of skeletal muscle to insulin. CK2 is activated within the insulin signalling pathway [24] with CK2 phosphorylation being dependent on insulin receptor substrates [25]. Although this potentially identifies Zip7 as essential for insulin responsiveness in skeletal muscle, the actual zinc homeostasis mechanisms in the context of skeletal muscle insulin signalling are still speculative.

Insulin signalling is a complex pathway requiring precise control to maintain normal blood glucose levels, with numerous feedback systems in place to regulate signal strength and duration. The aim of this study was to identify a previously unreported mechanism by which insulin treatment of skeletal muscle cells stimulates an increase in cytosolic zinc. Evidence is presented that this increase in zinc may occur through the oxidation of MTs leading to the release of bound zinc rather than through Zip7-mediated transport.

Methods

Cell culture

C2C12 mouse skeletal muscle myoblasts were cultured in DMEM supplemented with 10%

foetal bovine serum, 1% Penicillin/streptomycin and L-Glutamax (Life Technologies,

Mulgrave, Australia). Cells were grown in 6 well culture dishes for protein collection or

dishes with a thin glass bottom (Mattek Corporation, Ashland, MA) for confocal microscopy.

Culture media was supplemented with 20 µM ZnSO4 throughout all experiments to maintain

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consistent zinc levels. Undifferentiated myoblasts were used as the model in these studies as the mature myotubes would frequently contract when exposed to the laser light of the confocal microscope. The differentiation of myoblasts into mature multinucleated myotubes, which were required for validating the insulin responsiveness of the myoblasts, was induced via four days of mitogen withdrawal using DMEM media containing 2% horse serum. Media was changed every 48 hours.

Transient siRNA transfection

The transient transfection of siRNA molecules for Zip7 (Catalogue No: AM16708) and the scramble control (Catalogue No: AM4635) (Life Technologies) were performed using

RNAiMAX reagent according to the manufacturer’s protocol (Life Technologies). Briefly,

C2C12 cells were transfected in 6-well dishes with 10 nM of siRNA or the scramble control in RNAiMAX reagent and incubated for 48 hours. Cells used for subsequent PCR analysis were collected in Tri Reagent (Sigma Aldrich, Castle Hill, Australia) after differentiation.

Both the scramble and siRNA-Zip7 transfected cell lines to be used for confocal microscopy were also co-transfected with 10 nM of a fluorescently tagged oligonucleotide as a positive transfection control. Cells were kept at a low confluence to avoid unwanted differentiation.

RNA extraction and cDNA synthesis

RNA was extracted from samples in TRI reagent (Sigma) using the phenol/chloroform extraction method. RNA was treated with 2U DNAase for 30 min and 37°C and was purified using a RNA purification kit (Qiagen, Chadstone, Australia). 2 µg of the purified RNA was converted into cDNA using a one-step High Capacity cDNA synthesis kit (Life

Technologies). cDNA was diluted to a concentration of 5ng/µl for PCR.

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Real-time PCR

RealPlex PCR detection system was used to perform qPCR experiments (Eppendorf, North

Ryde, Australia) in triplicate on at least three independent RNA preparations. After cDNA

synthesis, target gene expression levels were analysed in 10 µl reactions with SensiMix

SYBR No-ROX (Bioline, Alexandria, Australia). The level of target gene expression was

normalised to Eef2.

Determining myoblast insulin responsiveness and the effect of impermeable and

permeable zinc on Akt phosphorylation

C2C12 myoblasts and differentiated mature-multinucleated myotubes were treated with 10

nM recombinant human insulin (Life Technologies) for 30 minutes to compare the insulin

responsiveness between undifferentiated and fully mature skeletal muscle cells. This was

performed to determine if the undifferentiated C2C12 cells were an appropriate model for

studying insulin signalling mechanisms.

As a further control measure myoblasts were treated with 20 µM ZnSO4 for half an hour prior

to protein extraction and western blot to ensure that there was no activation of insulin

signalling due to the ZnSO4 in the media.

Cells were treated with 10 nM insulin and 20 µM ZnSO4 alongside 20 µM sodium pyrithione

(NaP) (Sigma Aldrich, St Louis, IL), an ionophore used to permeabilise the cells to zinc [26] to observe the effect of insulin and increased cytosolic zinc on insulin signalling. Cells were collected at 0, 5, 15, 30 and 60 minute time points to identify the time of maximal insulin response. Cells were serum starved for three hours prior to treatment to ensure a low level of basal Akt phosphorylation at the beginning of the time course.

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Protein extraction and Western blot

Total cellular protein from samples was isolated using RIPA buffer (Thermo Scientific,

Melbourne, Australia) with added protease and phosphatase inhibitor. After collection,

samples were left on ice then vigorously vortexed for 20 seconds and centrifuged at 12,000

RCF for 5 minutes at 4°C, after which the supernatant containing total soluble protein was

collected. Total cellular protein concentrations were measured using a BCA assay kit

(BIORAD, Gladesville, Australia) against a known standard of BSA (Thermo Scientific) according to the manufacturer’s instructions.

Total soluble protein (30 µg per sample) was resolved on a 4-15% SDS-PAGE gradient gel

(BIORAD) at 80 V for 1.5 hours and transferred to a nitrocellulose membrane at 100 V for one hour. A SNAP i.d.® 2.0 blotting system (Merk Millipore, Bayswater, Australia) was used to block and probe protein bound membranes according to the manufacturer’s guidelines. Briefly, membranes were blocked in 0.5% skim milk in TBS Tween for 10 minutes. Membranes were probed with 1:2500 dilutions of either pAkt or Akt antibodies

(Cell Signalling) for 20 minutes. Following 4 x 20 second washes in TBS Tween the

membranes were incubated with 1:2500 dilution of horseradish peroxidase (HRP)-linked

anti-Rabbit secondary antibody (Cell Signaling, Danvers, MA) for 10 minutes. An Enhanced

SuperSignal West Pico Substrate kit (Thermo Scientific) was used to generate a

chemiluminescent signal from the HRP tag which was visualised using a UVITEC Alliance

digital imaging system (Thermo Scientific).

Inhibition of insulin signalling using wortmannin

PI3-kinase activity was inhibited via treatment of myoblasts with 10 µM wortmannin (Life

Technologies) for 24 hours [27, 28]. Inhibition of insulin signalling was confirmed via

western blot analysis of Akt phosphorylation after treatment with 10 nM insulin and

subsequent protein extraction as described above. Cells were then plated in imaging dishes

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and treated with wortmannin for confocal microscopy. As a control, cells treated with

DMSO, the solvent used to dissolve the inhibitor, were also analysed.

Live cell fluorescent probe staining for analysis of zinc flux

C2C12 cells grown to 60% confluence were washed twice in PBS and loaded with 5 µM cell permeable Fluozin-3 (Excitation/Emission: 494/516 nm) (Life Technologies) in 10% FBS

DMEM media containing no phenol red indicator and incubated at 37°C for 30 minutes. Cells

used for visualisation of cytosolic zinc were again washed twice in PBS and loaded with 1

µM of the nuclear stain Hoechst 33342 (Excitation/Emission: 350⁄461 nm) (Life

Technologies) and 5 µg/ml of the plasma membrane stain Alexa Fluor® 594 wheat germ

agglutinin (Excitation/Emission: 590/617 nm) (Life Technologies). Cells were then incubated

at 37°C for 10 minutes before being washed a final two times and adding fresh media

containing 20 µM ZnSO4. Cells were left in the incubator for a final 5 minutes after removal of stains to ensure complete de-esterification of the FluoZin-3 stain. Cells used for pixel intensity analysis of fluorescent intensity were only stained with Fluozin-3 for 30 minutes before washing, adding fresh media and incubating at 37°C for 15 minutes. Cells were then brought to the 37°C confocal microscope incubation chamber and left to equilibrate for a further 15 minutes prior to imaging. 25 mM Hepes buffer was included in the imaging media to buffer pH fluctuations brought on by the change from 5% CO2 in the cell culture incubator

to the conditions of the microscope incubation chamber.

Confocal microscopy

Live cell imaging was performed using a Nikon Eclipse Ti-E C2 confocal microscope system

with an attached 37°C incubation chamber. Cells were imaged every 5 minutes using a 60X

oil emersion objective. In order to capture total cell fluorescence, images were taken over a

15 µm z-range with an image captured every 1 µm. Cells were imaged for 15 minutes prior to

treatment to establish baseline fluorescence. Cells were then treated with either 200 µl of

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fresh culture media or 200 µl of media containing either insulin or the zinc ionophore NaP, at

a final concentration of 10 nM for insulin or 20 µM for NaP. Cells were then imaged for a

further 30 minutes.

Fluorescence intensity analysis

Pixel density analysis of zinc fluorescence obtained from the confocal microscopy

experiments was performed using ImageJ software (http://imagej.nih.gov/ij/index.html).

Multi-image cell z-stacks were collapsed into a single image using Z-projection. Pixel

intensity of the Fluozin-3 fluorescence of each individual cell was measured with the pixel

threshold adjusted to remove background. Cells were imaged for 15 minutes before treatment

and data were normalised to that time point.

Reactive oxygen species analysis

Insulin-mediated ROS production was measured using an Image-iT LIVE Green Reactive

Oxygen Species Detection Kit (Life Technologies) according to the manufacturer’s protocol.

Briefly, Cellular ROS was labelled with 25 µM 5-(and-6)-carboxy-2′,7′- difluorodihydrofluorescein diacetate (carboxy-H2DCFDA), a compound that fluoresces when

oxidised. Cells were co-treated with 10 nM insulin for 30 mins. 2 µM of Hoechst 33342 was

added in the final 5 minutes of incubation. Cells were then washed twice in warm imaging

media before adding a final 1 ml of media prior to imaging. For a positive control, cells

treated with 100 µM tert-butyl hydrogen peroxide (TBHP) for 60 minutes were stained for

ROS as described above.

Results

Characterising insulin response in C2C12 skeletal muscle myoblasts.

The insulin responsiveness of C2C12 myoblasts was tested by treatment with 10 nM insulin

for 30 minutes and western blot analysis of Akt phosphorylation. The myoblasts were

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compared to mature multi-nucleated myotubes that had been differentiated over 4 days. Both

myoblasts and myotubes showed a large increase in pAkt after the 30 minute insulin

treatment (Figure 1 A), indicating that immature muscle cells are insulin sensitive as required

for this mechanistic study. Untreated myotubes were shown to have a much greater basal

level of pAkt compared to the untreated myoblasts.

To confirm that 20 µM of ZnSO4 in culture media did not cause activation of the insulin

signalling pathway, a western blot for Akt phosphorylation showed that treatment of cells

with ZnSO4 for 30 minutes caused no observable change in Akt phosphorylation. Treatment

with both zinc and insulin also showed no difference compared to cells treated with insulin

alone (Figure 1 B). The effect of cell permeable zinc on insulin signalling was also assessed and showed an increase in phosphorylation of Akt due to the increased levels of cytosolic

bioavailable zinc. The maximal response to cell permeable zinc and insulin occurred at one

hour after treatment. A small increase in pAkt was observed after 60 minutes in the samples

only treated with NaP (Figure 2 A), 20 µM of NaP and 20 µM ZnSO4 induced a large

increase in pAkt (B). Treatment with 10 nM insulin also increased pAkt (C) and this was also

greatly enhanced in the presence of zinc and NaP (D).

Insulin induces an increase in cytosolic zinc fluorescence.

The effect of insulin on cytosolic free zinc concentrations in skeletal muscle myoblasts was

assessed. Cells treated with insulin had a visible increase in green fluorescence for 15

minutes after insulin addition (Figure 3). The median pixel intensity for at least four cells per

field of view was quantified using ImageJ software and normalised to the time point of the

first treatment, at 15 minutes after commencing imaging. Fluorescent intensity declined over

the initial 15 minutes due to photobleaching. After addition of insulin, fluorescence intensity

steadily increased for 15 minutes after treatment and then rapidly declined (Figure 4). Cells

treated only with media showed no increase in fluorescence until the addition of NaP at the

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end of the experiment. All experiments were repeated at least three times and the results shown are from one typical experiment. All control cell preparations displayed a steady decrease in fluorescence over time due to photobleaching as a result of the high number of images taken.

Insulin mediated cytosolic zinc increase in skeletal muscle cells was inhibited by wortmannin.

Cells were treated with the PI3K inhibitor wortmannin to determine if the increased zinc flux observed was a downstream response of insulin signalling activation. Cells treated with 20

µM wortmannin for 24 hours before insulin treatment showed a decrease in pAkt by western blot analysis compared to cells not treated with the inhibitor (Figure 5). Confocal imaging showed that the insulin-stimulated increase in cytosolic zinc was completely inhibited in cells treated with wortmannin (Figure 6). In comparison cells treated with 5 µl of DMSO carrier solution showed an increase in Fluozin-3 fluorescence in response to insulin treatment.

Reduction in Zip7 expression results in reduced insulin-mediated Akt phosphorylation.

The expression of Zip7 was reduced in the myoblast cell line via siRNA transfection. Gene expression analysis via qPCR showed a significant reduction of 70% in Zip7 mRNA levels

(Figure 7) after a 48 hour transfection when compared to the scramble control. Insulin- mediated pAkt in the Zip7-siRNA cells was decreased compared to the scramble control as expected. This demonstrates that the Zip7-siRNA cells were insulin resistant similar to the effect of Zip7-siRNA in differentiated myotubes reported in Chapter 3

Transient Zip7-siRNA transfection results in no change in the insulin-stimulated increase in cytosolic free zinc.

Zip7-siRNA transfected cells were loaded with Fluozin-3 and insulin mediated zinc movement was observed as described above. The transfected cells showed an insulin-

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mediated zinc increase that was similar to the scramble control (Figure 8) and to what was previously observed in untransfected cells.

MT1 and 2 gene expression is increased when Zip7 expression is reduced.

The MTs were investigated due to their role in zinc binding and their properties regarding

ROS-mediated oxidation leading to zinc release. In Zip7-siRNA cells there was a significant increase in the expression of both MT 1 and 2 (Figure 9).

Insulin treatment of C2C12 myoblasts results in increased cellular ROS production.

To explore other mechanisms that may cause zinc flux in cells, the role of cellular ROS was investigated. Myoblasts were stained with the fluorescent ROS marker and treated with 10 nM insulin for half an hour. Insulin treatment resulted in an increase in ROS production compared to the untreated cells (Figure 10). When cells were treated with ammonium tetrathiomolybdate for 24 hours there was a reduction in the amount of ROS production compared to the untreated cells with no discernible increase when treated with insulin. Cells were treated with 50 µM tert-butyl Hydrogen Peroxide for 60 minutes as a positive control.

Discussion

Zinc has insulin mimetic activity and influences the activity of various phosphatases and kinases, however the mechanisms by which endogenous zinc flux can mediate regulation of insulin signalling remain unclear. Cytosolic zinc levels are primarily controlled by metallothioneins and two families of zinc transporters, the 14 Zips and 10 ZnTs, with many of these 24 mammalian transporters having unique tissue expression and cellular localisation.

Zip7, a transporter localised to the endoplasmic reticulum of cells [23] was shown to be activated via extracellular stimuli through CK2-mediated phosphorylation, although a physiological stimulus for activating Zip7 is yet to be determined [22]. Based on PTP1B being inhibited by zinc [29] and that Zip7 silencing results in insulin resistance in C2C12

140

cells (Thesis chapter 3), it was hypothesised that insulin treatment of skeletal muscle cells

would cause a rapid increase in cytosolic zinc via Zip7 activation.

The insulin sensitivity of the undifferentiated myoblast cell model was compared to fully

differentiated myotubes to ensure they were an appropriate insulin responsive model for this

study. The advantage of using myoblasts was their inability to contract during the course of

imaging experiments. Akt phosphorylation was chosen as the method of evaluating insulin

responsiveness as this mechanism is a well characterised aspect of insulin signalling and

commonly used as a marker of pathway activation [30-34]. Undifferentiated C2C12

myoblasts had a lower basal level of pAkt and a comparably larger increase in pAkt in

response to insulin when compared to myotubes (Figure 1 A), the undifferentiated cells are

responsive to insulin and so a suitable model for this study.

The effect of zinc supplementation and insulin were measured to ensure that the 20 µM zinc

used throughout this study was not causing activation of insulin signalling. Exposure to 20

µM ZnSO4 for 30 minutes had no effect on Akt phosphorylation and did not alter insulin-

mediated pAkt (Figure 1 B). When zinc was made cell permeable through co-treatment with

the zinc ionophore NaP (Figure 2 B), a large increase in pAkt was observed compared to cells

treated only with NaP (Figure 2 A). This increase was also larger than that observed in cells

treated with insulin (Figure 2 C), indicating a high level of insulin signalling activation in the

presence of high levels of bioavailable zinc. This experiment provides further confirmation

that cytosolic zinc activates the insulin signalling pathway, as has been reported previously

[3, 35-37]. One study showing ZnCl2 treatment of fibroblasts and adipocytes resulting in an

increase in Akt phosphorylation was able to achieve this by using very high concentrations of zinc (200 µM) [36].

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Confocal imaging of zinc in live C2C12 myoblasts using Fluozin-3 identified an increase in cytosolic zinc levels after insulin treatment (Figure 3), with a gradual increase over 15 minutes after insulin addition (Figure 4 B). Similar results have been seen in mast cells after high affinity IgE receptor stimulation [8]. The fluorescent zinc probe Fluozin-3 can be used

to measure zinc at physiologically relevant nanomolar concentrations [38], although it is

difficult to precisely quantify the amount of zinc present with this type of assay. The detected

increase in cytosolic zinc in response to insulin is expected to be sufficient to inhibit PTP1B

activity, which begins to occur at picomolar concentrations of free zinc [11].

To ensure that the increase in cytosolic zinc was the result of activation of insulin signalling,

cells were treated with the PI3K inhibitor wortmannin. PI3K is an early and essential protein

within the insulin signalling pathway and treatment of cells with this inhibitor results in

decreased activation of molecules downstream of this kinase [39]. The effectiveness of

wortmannin was demonstrated by reduced insulin-mediated phosphorylation of Akt.

Wortmannin also inhibited the insulin-mediated increase in cytosolic zinc indicating that the

mechanisms controlling the increase in cytosolic zinc are activated by insulin treatment and

dependent on molecules downstream of PI3K.

The evidence that a reduction in Zip7 expression in skeletal muscle leads to reduced glycogen

synthesis as an outcome of insulin signalling (Thesis Chapter 3) supports the hypothesis that

the observed insulin-mediated increase in cytosolic zinc occurs through the activation of

Zip7. This is further supported by CK2, the proposed activator of Zip7 [22], being an

important signalling molecule in the insulin signalling pathway [25]. Although these two

pieces of evidence are highly persuasive, to confirm that Zip7 is the gatekeeper for cytosolic

zinc in response to insulin, insulin-mediated cytosolic zinc was investigated in cells with

reduced Zip7 expression.

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Zip7 expression was reduced in the C2C12 myoblasts by 70% via transient siRNA

transfection for 48 hours (Figure 7 A). The level of insulin-mediated Akt phosphorylation was reduced in the Zip7-siRNA myoblasts compared to the scramble control (Figure 7 B).

Interestingly there was no apparent difference in the insulin-mediated cytosolic zinc increases in the Zip7-siRNA cells (Figure 8 B) compared to the scramble control (Figure 8 A and C), both of which showed an increase similar to that observed in the untransfected cells. These findings indicate that although Zip7-siRNA transfected cells become insulin resistant and have reduced Akt phosphorylation in response to insulin, the insulin-mediated zinc increase still occurred. This suggests that another mechanism for zinc release in response to insulin is responsible. As there appeared to be no noteworthy compensatory changes in the expression of other Zip family members as measured via qPCR (Chapter 3), other mechanisms of zinc homeostasis were investigated, namely the metal binding MTs.

Although their exact biological role is yet to be fully understood [40], the MTs are known to contribute to the regulation of zinc homeostasis through the sequestering of bioavailable zinc

[41], with MT gene expression being upregulated by heavy metal concentrations and binding capacity being reduced by oxidative stress [42]. Analysis of gene expression of MT1 and 2 revealed a significantly increased expression of both genes in Zip7-siRNA transfected myoblasts relative to the scramble control (Figure 9). One explanation for this is that cytosolic free zinc levels are increased in the Zip7-siRNA transfected cells.

To further explore the cause of the alteration in MT levels in the transfected cells ROS was measured. The relationship between zinc, MTs and ROS production is well characterised [43] and has been proposed to be an important aspect of insulin signalling regulation [44]. The metal binding site of the MTs when oxidised by hydrogen peroxide [17, 18] leads to a release of the metals bound within the thiolate clusters [16]. Fluorescent staining of ROS in C2C12 myoblasts revealed an increase in ROS production in cells treated with 10 nM insulin and an

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even greater increase in cells treated with 100 nM insulin (Figure 10). This finding confirms

previous studies showing that ROS is produced in response to insulin [45-47] and acts as a secondary messenger in the insulin signalling pathway [48] with hydrogen peroxide having insulin-mimetic properties in adipocytes [49]. This finding also further supports the role of oxidation in MTs releasing zinc to act as a modulator of signalling pathways.

This study has shown that treatment of skeletal muscle myoblasts with insulin causes an acute increase in cytosolic zinc levels, which is attenuated by inhibition of PI3K but not after gene silencing of Zip7. If the insulin-mediated increase in cytosolic zinc observed in this study is not occurring through Zip7, then this raises the question of the role of this novel zinc transporter in insulin responsiveness. The silencing of Zip7 via transient siRNA transfection

resulted in numerous cellular changes including reduced insulin-mediated Akt

phosphorylation and glycogen synthesis. This, along with the lack of expressional

compensation from other Zip members indicated an essential role of Zip7 in this pathway. As

the knockdown of Zip7 did not cause an observable change in insulin-mediated zinc release

then Zip7 may not be the gatekeeper for this function in this cell system. Taylor and

associates identified the importance of calcium for the activation of Zip7 [22] and this is

something that requires further exploration in the context of insulin signalling in muscle. The

finding that expression of Mt1 and 2 is increased when Zip7 expression is reduced indicates a

potential relationship between these zinc-associated proteins, with one possibility being a

general disruption of zinc compartmentalisation upon silencing of Zip7.

The lack of an effect of Zip7 silencing on insulin-stimulated zinc release raised the question

of what is causing the zinc release. Increased ROS production in response to insulin treatment

may suggest that the increased zinc is a result of MT oxidation, leading to metal ion release.

The attenuation of insulin-mediated zinc release after wortmannin treatment also supports

this, as insulin mediated ROS production is proposed to occur downstream of PI3K activation

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[50]. Inhibition of PI3K may reduce insulin-mediated ROS production, leading to reduced oxidation of MTs and subsequent zinc release from their thiol clusters. Although the pathways involved require further investigation, this study demonstrates for the first time an increase in cytosolic free zinc in response to insulin so providing evidence for a positive feedback mechanism by which insulin mediates an increase in cytosolic zinc to regulate insulin signalling and provides further evidence supporting the essential role of zinc within this pathway.

145

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38. Zhao, J., Bertoglio, B.A., Gee, K.R., and Kay, A.R. (2008). The zinc indicator

FluoZin-3 is not perturbed significantly by physiological levels of calcium or

magnesium. Cell Calcium 44, 422-426.

39. Okada, T., Kawano, Y., Sakakibara, T., Hazeki, O., and Ui, M. (1994). Essential role

of phosphatidylinositol 3-kinase in insulin-induced glucose transport and antilipolysis

in rat adipocytes. Studies with a selective inhibitor wortmannin. J Biol Chem 269,

3568-3573.

40. Palmiter, R.D. (1998). The elusive function of metallothioneins. Proc Natl Acad Sci U

S A 95, 8428-8430.

41. Suhy, D.A., Simon, K.D., Linzer, D.I.H., and O’Halloran, T.V. (1999).

Metallothionein Is Part of a Zinc-scavenging Mechanism for Cell Survival under

Conditions of Extreme Zinc Deprivation. J Biol Chem 274, 9183-9192.

42. Andrews, G.K. (2000). Regulation of metallothionein gene expression by oxidative

stress and metal ions. Biochem Pharmacol 59, 95-104.

43. Maret, W. (2000). The Function of Zinc Metallothionein: A Link between Cellular

Zinc and Redox State. Journal Nutr 130, 1455S-1458S.

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44. Maret, W. (2008). A role for metallothionein in the pathogenesis of diabetes and its

cardiovascular complications. Mol Gen Metab 94, 1-3.

45. Goldstein, B.J., Mahadev, K., Wu, X., Zhu, L., and Motoshima, H. (2005). Role of

insulin-induced reactive oxygen species in the insulin signaling pathway. Antioxid

Redox Signal 7, 1021-1031.

46. Mahadev, K., Zilbering, A., Zhu, L., and Goldstein, B.J. (2001). Insulin-stimulated

hydrogen peroxide reversibly inhibits protein-tyrosine phosphatase 1b in vivo and

enhances the early insulin action cascade. J Biol Chem 276, 21938-21942.

47. Yang, M., Yang, Y., Zhang, S., and Kahn, A.M. (2003). Insulin-stimulated hydrogen

peroxide increases guanylate cyclase activity in vascular smooth muscle.

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Insulin Signalling: Implications for Antioxidant Treatment of Fatty Liver Disease. Int

J Cell Biol 2014, 9.

49. May, J.M., and de Haen, C. (1979). The insulin-like effect of hydrogen peroxide on

pathways of lipid synthesis in rat adipocytes. J Biol Chem 254, 9017-9021.

50. Seo, J.H., Ahn, Y., Lee, S.-R., Yeo, C.Y., and Hur, K.C. (2005). The Major Target of

the Endogenously Generated Reactive Oxygen Species in Response to Insulin

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Figure Legends

Figure 1: C2C12 skeletal muscle myoblasts respond to insulin. C2C12 myoblasts and

terminally differentiated myotubes were treated with 10 nM insulin for 30 minutes. Total

protein was collected and Akt phosphorylation was measured via western blot. Myoblasts

have a lower basal level of pAkt compared to myotubes and both showed a large increase in

Akt phosphorylation after insulin treatment (A). Cells treated with 20 µM ZnSO4 for 30 minutes showed no increase in pAkt compared to untreated cells. Zinc and insulin treatment together showed no difference compared to insulin treatment alone indicating that cell impermeable extracellular zinc does not affect Akt phosphorylation (B).

Figure 2: Cell permeable zinc and insulin treatment increased phosphorylation of Akt.

C2C12 cells were treated with either 20 µM NaP (A), 20 µM NaP and 20 µM ZnSO4 (B), 20

µM NaP and 10 nM insulin (C) or 20 µM NaP, 20 µM ZnSO4 and 10 nM Insulin (D).

Samples were collected at 0, 5, 15, 30 and 60 minute time points for each group. Protein was

extracted from each sample and a western blot was performed to determine Akt

phosphorylation. Cells treated with NaP alone had a minimal increase in pAkt. When cells

were co-treated with zinc and NaP there was a large increase in pAkt, which was much

greater than the response from cells treated with NaP and insulin. Cells treated with NaP,

insulin and zinc together had a large increase in Akt phosphorylation.

Figure 3: Insulin increases cytosolic free zinc levels in C2C12 cells. C2C12 myoblasts

were loaded with 5 µM Fluozin-3 zinc fluorescent stain. The cells were co-stained with wheat

germ agglutinin Alexafluor 594 plasma membrane stain and the nuclear stain Hoechst 33342.

Cells were imaged over 1 hour with fluorescence captured every 5 minutes. Cells were

imaged for 15 minutes without treatment to capture baseline levels and then either 10 nM

insulin or control media was added and imaging was continued for a further 30 minutes.

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Fluozin-3 green fluorescence was seen to visibly increase after insulin was added, peaking 15

minutes after treatment and then gradually declining.

Figure 4: Increased Fluozin-3 fluorescence in C2C12 cells treated with insulin. C2C12

myoblasts stained with 5 µM Fluozin-3 were imaged over time after treatment with fresh media alone or media with 10 nM insulin or 20 µM NaP. Cells were imaged every 5 minutes for 15 minutes prior to treatment to measure baseline cell fluorescence and then imaged for a further 30 minutes. An increase in cytosolic zinc was observed in the cells treated with insulin but no change in fluorescence was observed in the media treated cells (A). Myoblasts treated with media showed a gradual decrease in fluorescence over time, with no increase after treatment (B). A large and rapid increase was observed in the cells treated with NaP (C).

Pixel density of every cell in frame was captured at every time point and the results were normalised to the 15 minute time point where the media, insulin or NaP was added (D). Pixel intensity increased after the addition of insulin, with maximal fluorescence occurring 15 minutes after treatment. Cells treated with media had no increase in fluorescent intensity, but there was a large increase in cells treated with NaP. Error bars indicate ± SD from the normalised fluorescence between all cells in view. The large bright cell observed in the NaP group was not included due to the high level of variability from the other cells in the field.

Figure 5: Wortmannin attenuates insulin-mediated phosphorylation of Akt. Total

protein was extracted from C2C12 myoblasts treated with 10 µM wortmannin for 24 hours

followed by the addition of 10 nM insulin for 30 minutes. Phosphorylation of Akt was

measured via western blot to determine the level of insulin signalling activation. Insulin

mediated pAkt was shown to be slightly decreased in cells treated with wortmannin

compared to the untreated control. There was also a reduction in basal pAkt in wortmannin

treated cells.

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Figure 6: Wortmannin treatment of C2C12 myoblasts ablates insulin mediated zinc

increase. Wortmannin treated skeletal muscle myoblasts were loaded with 5 µM fluozin-3 and treated with insulin to observe changes in free zinc. Images were taken every 5 minutes and the pixel intensity of each cell was measured. The wortmannin treated cells showed no insulin-mediated increase in cytosolic zinc over time (A). Cells treated with DMSO, the solvent used as a vehicle for the wortmannin, showed an increase in insulin-mediated zinc

(B). Pixel intensity for each treatment group shows that wortmannin ablated the insulin- mediated zinc increase (C). Error bars indicate ± SD from the normalised fluorescence between all cells in view.

Figure 7: Zip7 silencing via transient siRNA transfection leads to reduced insulin- mediated Akt phosphorylation. RNA was extracted from transfected cells and gene expression was measured via qPCR. The expression of Zip7 was significantly reduced after

48 hour transient transfection with Zip7-siRNA in C2C12 myoblasts compared to cells transfected with a scramble control (*** = P < 0.001) (A). Error bars indicated the ± SD from three independent biological samples. Total cell protein was extracted from Zip7-siRNA and scramble transfected cells treated with 10 nM insulin for 30 minutes after a 48 hour transfection. Samples were probed via immuno-blot for pAkt compared to total Akt levels.

There was a large increase in insulin-mediated pAkt in the scramble transfected cells and this was attenuated in the Zip7-siRNA cells (B).

Figure 8: Zip7 silencing does not affect insulin-stimulated zinc release. Cells transfected with Zip7 or scramble siRNAs for 48 hours were stained with Fluozin-3 and insulin- stimulated zinc release was measured. Both the scramble and Zip7-siRNA transfects cells had a similar response on insulin, showing an increase in zinc levels over time (A). Graphing the normalised fluorescence from the two groups showed a similar increase in fluorescent intensity to what was observed in untransfected cells, although the transfect cells seems to be

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more prone to a rapid loss of fluorescence (B). This experiment was repeated four times, with

the figure showing a typical result. No significant difference in the rate of fluorescence loss could be determined between the two groups. Error bars indicate ± SD from the normalised fluorescence between all cells in view.

Figure 9: Transient Zip7-siRNA transfection results in increased expression of Mts 1 and 2. RNA was extracted from Zip7-siRNA and scramble transfected cells and qPCR was performed to measure the expression of the metallothioneins. MTs 1 and 2 expression was significantly increased in the Zip7-siRNA compared to the scramble control. Error bars indicate ± SD from the normalised fluorescence between all cells in view. ** = P < 0.01, ***

= P < 0.001

Figure 10: Insulin treatment of C2C12 cells results in increased ROS production. C2C12 myoblasts were stained with a fluorescent ROS indicator (green) and treated with insulin.

There was an increase in ROS fluorescence in insulin treated cells compared to the no insulin control. Cells treated with ammonium thiomolybdate had reduced basal ROS compared to the control and no apparent increase after insulin treatment. As a positive control cells were treated with a known inducer of cellular ROS production before staining and imaging. All groups were also labelled with Hoechst (blue) to identify cell nuclei.

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Chapter 6: Discussion and Conclusions

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Overview

The initial hypothesis examined in this thesis was that Zip7 mediates an increase in cytosolic zinc which modulates the insulin signalling pathway. This was tested with three studies. The first study investigated the evolutionary conservation of Zip7, identifying regions with potential functional importance. The second study investigated the role of Zip7 in insulin signalling by reducing the expression of Zip7 and assessing various markers of insulin activity. The third study assessed whether insulin treatment of skeletal muscle resulted in an increase in cytosolic zinc release and whether this was occurring through Zip7.

Zip7 has a high level of evolutionary conservation

The human Zip7 protein sequence was investigated using bioinformatics to discern unique regions or functional motifs across the family of Zip transporters. A major goal was to find evidence for or against the novel role proposed for this transporter in mediating release of intracellular zinc stores in response to extracellular stimuli.

The evolutionary conservation of Zip7 was analysed using alignments of various mammalian and other vertebrate protein sequences. Zip7 was shown to have a very high level of conservation within predicted TMDs, which comprise the metal ion transport pore of Zips

[1]. All eight of the predicted TMDs identified in the human sequence were highly conserved across a range of vertebrate species, with even the distantly related zebrafish sequence having high conservation throughout these regions. Another interesting characteristic gleaned from this was a large concentration of highly conserved histidine residues within the otherwise poorly conserved loop regions. Loop regions of ion transporters are generally poorly conserved [1], suggesting that any areas of strong evolutionary conservation are likely to be important for protein function.

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The 14 human Zips were aligned to identify regions unique to Zip7 among this family of zinc

transporters. Zip7 was found to be most closely related to Zip13, which is also associated

with the ER/Golgi apparatus. However, the histidine content of each transporter showed that

Zip13 had only a single loop histidine residue compared to the 51 found in Zip7 loop regions.

Zip6 and Zip10 were the only Zip transports with a similarly high level of histidine as Zip7.

Histidine in transporter loop regions is thought to bind to zinc, leading to functional changes

in the transporter as a regulatory mechanism. This has been shown in Zip4, where zinc

binding to loop histidine residues resulted in protein degradation [2]. Histidine rich loops

have also been shown to have the opposite effect in Zip1 where zinc binding increases

transport activity [3]. The high level of histidine within the loop regions of Zip7 may potentially contribute to the proposed novel function of this transporter by providing an increased sensitivity to changes in zinc concentrations, limiting the amount of zinc released by this transporter.

The second stage of this study analysed the human Zip7 protein sequence for SLiMs using the ELM database. SLiMs are regions that may contribute to protein function such as sites for protein activation or interaction [4]. When analysed, the human Zip7 protein sequence was found to have two endoplasmic retention sites, both of which were highly conserved in other vertebrate Zip7 sequences. One of these predicted ER anchors was not found in the zebrafish sequence whereas the other was highly conserved across all species. A CK2 phosphorylation

site was also highly conserved in all species, though its location varied in the zebrafish

sequence it was still located in the same loop. Taylor and associates have confirmed that this

site is phosphorylated in human Zip7 [5].

To determine if there were any SLiM variations or combinations unique to Zip7 compared to

the other 13 Zip family members, each transporter proteins sequence was analysed for SLiMs

and the results were tabulated based on whether the SLiM was located within a cytosolic or

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extracellular loop region. SLiMs predicted within TMDs were excluded due to these regions being inaccessible to protein interactions. Many of the transporters appeared to have a similar profile of phosphorylation sites, even if they were located within a different loop region.

Interestingly, two other Zip members containing a high level of loop histidine, Zip6 and 10, both contained a breast cancer type 1 susceptibility protein encoding gene (Brca1) interaction site which was absent from Zip7. Both Zip6 and Zip10 have been associated with breast cancer [6, 7] and have been shown to move from intracellular organelles to the plasma membrane in response to changes in zinc levels, while Zip7 remains associated with the ER under these conditions [8]. Zip6 and 10 also lacked the endoplasmic retention motifs found in the Zip7 sequence which may explain this difference.

Although there were other Zips with endoplasmic retention sites or high histidine content, the combination of these two attributes was unique to Zip7. These findings strengthen the case put forward for Zip7 having a novel role among the Zip transporters in modulating ER zinc release in response to extracellular stimuli.

Zip7 knockdown reduces skeletal muscle insulin sensitivity

The second study investigated the effect of reducing Zip7 expression in C2C12 skeletal muscle myotubes on cellular insulin sensitivity. It was hypothesised that if Zip7 is the primary mediator of rapid zinc release from cellular stores, then reduced expression of this transporter would lead to reduced zinc release in to the cytosol, so allowing greater PTP1B- mediated insulin receptor dephosphorylation and so insulin resistance.

The expression of Zip7 was reduced via siRNA transfection. Initially, the samples were analysed for any compensatory changes in the expression of other Zips or ZnTs. Among the

Zips, there was a change in Zip3 expression, though this transporter had low expression relative to Zip7. There were no changes in the expression of the 10 ZnTs. This suggests that

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Zip7 has a unique function which, when lost, cannot be compensated for by changes in other

zinc transporters.

The Zip7 attenuated cells were investigated for markers of insulin resistance and were found

to have reduced expression of genes associated with glucose metabolism along with a

concomitant reduction in Akt phosphorylation. Insulin-stimulated glycogen synthesis and

Glut4 protein levels were also decreased compared to the scramble control cells when Zip7

expression was reduced. These findings indicate that Zip7 knockdown in skeletal muscle

results in an insulin resistant state.

The results of this investigation support the overall hypothesis that Zip7 is essential for

efficient glucose disposal possibly through a response to extracellular stimuli as a regulatory

mechanism for controlling insulin sensitivity. Given that skeletal muscle is essential for

insulin-mediated glucose uptake in humans [9], insulin resistance as a result of Zip7 knockdown indicates an essential role for this transporter that warrants further investigation.

Although these findings strongly suggest a relationship between Zip7 and insulin signalling

an increase in Zip7-mediated zinc flux in response to insulin would be required to confirm

this.

Insulin treatment stimulates increased cytosolic zinc in skeletal muscle

The aim of the third study was to determine if insulin stimulates an increase in cytosolic zinc

and to understand the mechanisms controlling this function. It was hypothesised that insulin

treatment of C2C12 cells would cause an increase in cytosolic zinc and that this increase

would be mediated by Zip7 activation. Insulin was proposed to stimulate a release of stored

zinc as a positive feedback mechanism through the inhibition of phosphatases that negatively

regulate insulin signalling.

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When C2C12 cells were treated with insulin, an increase in cytosolic zinc was observed,

revealing a potential mechanism whereby insulin self regulates its activity via modulation of

zinc homeostasis. Due to the proposed novel role of Zip7 as a zinc flux gatekeeper initially

reported in breast cancer cell lines [5, 10], these findings further support the hypothesis that

this transporter is essential for insulin signalling in skeletal muscle.

To confirm that the observed zinc increase was occurring as a result of insulin signalling

activation the cells were treated with the PI3-K inhibitor wortmannin. PI3-Ks are required for

the synthesis of phosphatidylinositols, which are essential second messengers in the insulin

signalling pathway [11]. Wortmannin treatment of C2C12 cells attenuated the insulin-

stimulated zinc increase, suggesting that zinc release occurs downstream of PI3-kinase activation within the insulin signalling pathway.

These findings show that insulin signalling activation leads to an increase in cytosolic zinc through PI3-K activity. While zinc is known to stimulate insulin and growth factor signalling via the PI3K pathway, an increase in cytosolic free zinc in response to insulin-mediated PI3K activation has not previously been demonstrated experimentally. This finding indicates a positive feedback mechanism for the regulation of insulin signalling.

Zip7 silencing does not alter insulin-mediated zinc release

This result raised the question of Zip7 involvement in mediating the insulin-stimulated increase in cytosolic zinc. To test for a role for Zip7 cells with reduced Zip7 expression were treated with insulin and zinc flux was measured.

Insulin treatment of cells with reduced Zip7 expression resulted in an increase in cytosolic zinc, with no observable change compared to the control cells. This suggests that the mechanism of insulin-mediated zinc increase does not occur through Zip7 activation. This finding indicated that although Zip7 silencing results in insulin resistance, the cause may not

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be through a loss of insulin-mediated zinc release. This raised many questions regarding the

function of Zip7 in this pathway and also suggested that further investigation into mechanisms of cellular zinc release was required.

ROS and zinc signalling

Although the knockdown of Zip7 in skeletal muscle resulted in decreased insulin-stimulated phosphorylation of Akt, there was no discernible effect on the insulin-mediated zinc flux. As

shown in chapter 4 there was no significant compensatory change in the expression of any

Zips or ZnTs in response to Zip7 knockdown, therefore other mechanisms controlling cellular

zinc homeostasis were investigated, namely the MTs. MTs are well known to buffer heavy

metal concentrations through binding to free cytosolic ions, reducing their bioavailability to

prevent toxicity [12] and hence Mt gene expression is regulated by heavy metal concentrations [13]. Another interesting attribute of MTs relevant to this study is that they have been shown to release bound zinc when oxidised by reactive oxygen and reactive nitrogen species (ROS and RNS respectively) [14, 15]. This occurs via the oxidation of the zinc-bounds thiol clusters leading to the release of free zinc ions [16]. Due to these findings, insulin-stimulated ROS production was investigated using live cell fluorescent microscopy.

Insulin treatment of C2C12 cells was shown to stimulate an increase in measureable ROS in

C2C12 myoblasts, matching previous literature describing the link between insulin signalling and ROS production [17-18]. H2O2 is one such product that is produced during insulin signalling activity, which acts as secondary messenger in this pathway [16] and is even

known to have insulin-mimetic properties [19], although ROS has negative effects on insulin

responsiveness at higher concentrations [20]. The SOD enzyme family is essential for the

formation of H2O2 from superoxides [21]. To test the role of SOD in the C2C12 cell system

used here, inhibition of SODs via treatment with TTHB was tested and found to reduce

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insulin-stimulated ROS production, consistent with the role for this enzyme in insulin- stimulated H2O2 production.

A compensatory change in the expression of Mt genes was assessed to determine if there was

a relationship between Zip7 and MT activity. It was found that when Zip7 expression was

reduced via siRNA transfection, there was a subsequent increase in the expression of Mt1 and

2, potentially indicating a relationship between Zip7 activity and MTs. This may also indicate

that the attenuation of Zip7 caused a shift in cytosolic heavy metal concentrations, leading to

a compensatory increase in MT gene transcription. These findings suggest that the insulin-

stimulated zinc release is not via activation of Zip7 to release zinc from the ER, but may

instead be due to the release of zinc bound to MTs being released when thiol clusters are

oxidised by the H2O2 produced in response to insulin signalling activation.

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Figure 3: Insulin-stimulated zinc occurring through ROS production. Superoxide is produced via NADPH oxidase activation in response to insulin signalling activity. The

superoxide is converted to H2O2 via SOD1. The H2O2 oxidises thiol clusters of MTs, leading to the release of bound zinc to inhibit PTP1B as a positive feedback mechanism. The results of this thesis suggest that the release of zinc is dependent on PI3-Kinase activity, but not Akt. Free zinc is also known to inhibit GSK-3, leading to increased glycogen synthesis in the presence of bioavailable zinc.

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Conclusion

This thesis provides new evidence supporting the importance of zinc in the regulation of

insulin signalling in skeletal muscle. Insulin was found to stimulate an increase in cytosolic

zinc levels as a potential positive feedback loop. Interestingly, this insulin-stimulated zinc

release did not appear to be occurring through Zip7 activation, even though cells with

reduced expression of Zip7 were shown to be insulin resistant.

The insulin-mediated increase in cytosolic zinc may be explained through an increased

production of ROS leading to the dissociation of zinc from MTs. The well-established

insulin-stimulated generation of cellular ROS could cause the increased oxidation of MTs and

subsequent release of zinc from thiol clusters, as has been previously proposed, may explain

the findings observed in this study. Further evidence supporting this relationship was the

upregulated expression of Mts 1 and 2 when Zip7 expression was reduced, indicating a

relationship between Zip7 function and MTs. The nature of this relationship is still unknown,

but it could indicate that when Zip7 expression is reduced, intracellular zinc homeostasis is

perturbed leading to a compensatory increase in MT expression to buffer the cells against

uncontrolled increases in cytosolic zinc. In support of this is the lack of compensatory

expression changes in other zinc transporters when Zip7 expression is knocked down, which

indicates a unique role for Zip7 in controlling intracellular zinc homeostasis. In this scenario,

ROS-stimulated zinc release from MTs would still be expected to occur, as was seen in the

results presented in chapter 5.

Another theory based on the results presented here is that Zip7 is activated by stimuli other than phosphorylation by CK2 in response to insulin, as glucose homeostasis has multiple controlling factors. Further work is needed to investigate other possible activators of Zip7.

Due to the insulin mimetic effects of zinc, potential stimuli that cause an acute increase in

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cytosolic zinc through biological mechanisms may have potential therapeutic utility for improving insulin sensitivity and may be a safer option than the use of cell permeable zinc complexes.

Interestingly, the results from Chapter 5 showed that Zip7 attenuation resulted in reduced insulin mediated Akt phosphorylation yet did not change insulin-stimulated zinc flux, however PI3-kinase inhibition resulted in a loss of this zinc flux. This seems to indicate that the alterations in insulin signalling activity by reduced Zip7 expression may be occurring at steps between PI3-K activation and Akt phosphorylation while ROS production is occurring upstream of Akt. Further work is required to fully elucidate where these signalling changes are occurring.

Zip7 is a novel zinc transporter linked to signalling pathway modulation and this thesis provides evidence supporting this in the context of insulin signalling. Though this transporter has been shown to be important in the context of this pathway, evidence suggests that insulin- stimulated zinc release occurs through the oxidation of MTs rather than Zip7 activation.

Future Studies

There are many questions raised by this study. Further investigating the role of the SOD enzymes in regards to superoxide conversion to H2O2 and whether inhibition results in attenuated insulin-mediated zinc release would provide another piece of evidence supporting zinc release via the oxidation of MTs. Reducing the expression of MTs via siRNA transfection would help in elucidating the pathway proposed in this thesis. Another aspect of this study requiring further investigation is the mechanisms of Zip7-siRNA transfection- mediated insulin resistance. Availability of a ratiometric fluorescent indicator for zinc would allow comparison of free zinc concentrations between cell preparations, which would enable measurement of changes in zinc availability in Zip7 knockdown versus control cells. Further

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study into other possible activators of Zip7 is warranted. Exploring the overexpression of

Zip7 in skeletal muscle would be beneficial for investigating the role of this transporter in insulin signalling modulation.

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