The Zinc Transporter, Slc39a7 (Zip7) Is Implicated in Glycaemic Control in Skeletal Muscle Cells
Slc39a7 (Zip7) Is Implicated in Glycaemic Control in Skeletal Muscle Cells. PLoS
ONE 8(11): e79316. doi:10.1371/journal.pone.0079316
The Zinc Transporter, Slc39a7 (Zip7 ) Is Implicated in Glycaemic Control in Skeletal Muscle Cells
Stephen A. Myers 0
Alex Nield 0
Guat-Siew Chew 0
Mark A. Myers 0
Barbara Bardoni, CNRS UMR7275, France
0 1 Collaborative Research Network and the School of Health Sciences, University of Ballarat , Mount Helen Campus, Victoria , Australia , 2 School of Health Sciences, University of Ballarat , Mount Helen Campus, Victoria , Australia
Dysfunctional zinc signaling is implicated in disease processes including cardiovascular disease, Alzheimer's disease and diabetes. Of the twenty-four mammalian zinc transporters, ZIP7 has been identified as an important mediator of the 'zinc wave' and in cellular signaling. Utilizing siRNA targeting Zip7 mRNA we have identified that Zip7 regulates glucose metabolism in skeletal muscle cells. An siRNA targeting Zip7 mRNA down regulated Zip7 mRNA 4.6-fold (p = 0.0006) when compared to a scramble control. This was concomitant with a reduction in the expression of genes involved in glucose metabolism including Agl, Dlst, Galm, Gbe1, Idh3g, Pck2, Pgam2, Pgm2, Phkb, Pygm, Tpi1, Gusb and Glut4. Glut4 protein expression was also reduced and insulin-stimulated glycogen synthesis was decreased. This was associated with a reduction in the mRNA expression of Insr, Irs1 and Irs2, and the phosphorylation of Akt. These studies provide a novel role for Zip7 in glucose metabolism in skeletal muscle and highlight the importance of this transporter in contributing to glycaemic control in this tissue.
Funding: This study was funded by a School of Health Sciences Seeding Grant, University of Ballarat, Victoria Australia. The funders had no role in study design,
data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
. These authors contributed equally to this work.
Cellular zinc storage, release and distribution are controlled by
a family of zinc transporters and metallothioneins. In mammals
two families of zinc transporters exist: the zinc efflux (Slc30/ZnT)
and the zinc influx (Slc39/ZIP) proteins . ZnT proteins
transport zinc out of the cell or into subcellular compartments in
the presence of high cytoplasmic zinc. In contrast, ZIP proteins
transport zinc into the cell or out of subcellular compartments
when cytosolic zinc is low or depleted .
There is increasing interest in the importance of zinc
transporters in diseases associated with dysfunctional cellular
signaling. In particular, a significant 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 signaling
pathway activity was observed . The ZnT7 null mice were
susceptible to diet-induced glucose intolerance and insulin
resistance and this was associated with a decrease in the expression
of the insulin receptor, insulin receptor substrate 2 and Akt1 .
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 hyperglycemia . Moreover, ZnT8
plays a critical role in the synthesis and secretion of insulin and
therefore represents a pharmacological target for treating disorders
of insulin secretion including diabetes .
Zinc mediates its effects through two mechanisms; early zinc
signaling (EZS) and late zinc signaling (LZS) . LZS occurs
several hours after an extracellular signaling event and depends on
changes in the expression of zinc-related molecules such as zinc
transporters and metallothioneins [6,7]. In contrast, EZS occurs
minutes after an extracellular stimulus and does not involve
transcriptional-dependent changes [6,7]. Zinc signaling
mechanisms are involved in eliciting an increase in intracellular zinc
concentrations 2 the zinc wave phenomenon . Thus, in this
situation zinc acts as a second messenger that activates pathways
associated with cellular signaling. In fact, zinc has been
categorized as an insulin-mimetic with several groups examining
the role of its mimetic activity on glucose  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 .
Endogenous ZIP7 is predominately localized to the Golgi apparatus ,
the ER , or both  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 multiple 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 .
Given the role of ZIP7 in modulating zinc flux, and the role of
zinc as an insulin mimetic in cellular processes, we propose that
ZIP7 may also be implicated in metabolic processes associated
with glycaemic control. Here we report evidence for a novel role
for Zip7 in modulating glycaemic control in skeletal muscle cells.
We find that the attenuation of Zip7mRNA in C2C12 skeletal
muscle cells modulates genes involved in carbohydrate metabolism
and glycogen synthesis. These studies demonstrate a previously
unprecedented role for Zip7 in regulating glycaemic control in
skeletal muscle and provide a platform to further explore the
potential of this transporter in skeletal muscle insulin resistance.
Materials and Methods
Proliferating mouse C2C12 myoblasts in all experiments were
cultured and maintained in DMEM supplemented with 10% Fetal
Bovine Serum and physiological zinc concentrations (20 mM
ZnSO4), (Life Technologies, Mulgrave, Victoria, Australia).
Differentiation of myoblasts into post-mitotic, multi-nucleated
myotubes was induced by mitogen withdrawal (i.e. DMEM
supplemented with 20 mM Zn SO4 and 2% 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 . The time course experiments on
differentiated C2C12 skeletal muscle cells were performed over 60
minutes in the presence of 10 nM insulin, 20 mM ZnSO4 and
10 mM pyrithione (see Figures S1 and S2).
RNA Extraction and cDNA Synthesis
Mouse quadriceps muscle was a kind gift from Dr. Paul
Lewandowski, Deakin University, Australia 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) according to the manufacturers protocol. Total RNA
was then treated with 2 U of DNase1 for 30 min at 37uC 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). A
High Capacity cDNA Synthesis kit was used to synthesize cDNA
from 2 mg of total RNA using random hexamers according to the
manufacturers instructions (Life Technologies). The cDNA was
diluted to 400 ml in nuclease-free water and stored at 220uC.
Mouse glucose metabolism and zinc transporter arrays
The Mouse Glucose Metabolism RT2 Profiler PCR Array was
purchased from SA Biosciences, Qiagen; Catalogue No. 330321
PAMM-006ZA. This array profiles the expression of 84 genes
involved in the regulation and enzymatic pathways of glucose and
glycogen metabolism (Table S1). The Zinc Transporter RT2
Profiler Custom PCR Array (Qiagen) contained the genes for the
two zinc transporter families, Slc30a1-10 and Slc39a1-14.
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 from 500 ng of total RNA using
buffer GE at 42uC for 5 min. The RNA was then reverse
transcribed in a total of 20 ml reaction volume for 15 mins at 42uC
then the reaction was stopped by heating the sample at 95uC for
Quantitative Real-time PCR
Quantitative 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 analyzed in 10 ml 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 (Table S2) have
been previously described . Other primers (Table S2) 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
. Note: all primers were rigorously analyzed by BLAST
for target gene specificity and designed to be genomic resistant (i.e.
at least one primer crossed an exon-exon boundary). qPCR was
performed using 4 ml of cDNA template (20 ng) and 40 cycles of
95uC for 15 seconds, 57uC for 15 seconds and 72uC for 20
seconds. The relative level of target gene expression was
normalized to Gapdh, or eukaryotic elongation factor 2 (Eef2) as
described in the results section and associated errors were
calculated using the guidelines described by Bookout and
Primer design to detect endogenous and exogenous
Primers were designed to specifically target endogenous and
exogenous Zip7 mRNA. For the pCMV-Zip7 overexpression
plasmid, we placed the forward primer on the Zip7 mRNA
sequence and the reverse primer on the plasmid C-myc tag (see
Table S2). For the specific amplification of endogenous Zip7 we
designed primers on the 59UTR. This region is omitted on the
pCMV-Zip7 expression plasmid.
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, cDNA (102 ml)
from the RT2 First Strand Kit was added to 1350 ml of 2 x RT2
SYBR Green and 1248 ml of H2O. To each well of the glucose
metabolism array 96-well plate, 25 ml of sample was added. qPCR
was performed on a RealPlex PCR detection system (Eppendorf)
using 45 cycles of amplification consisting 95uC for 15 seconds and
60uC for 1 minute.
Transient transfections of siRNA molecules
The transient transfection of siRNA molecules Zip7 (Catalogue
No: AM16708), Zip1 (Catalogue No: AM16708), Gapdh (Catalogue
No. 4390771) and the scramble control (Catalogue No: AM4635)
(Life Technologies) were performed using RNAiMAX reagent as
instructed by the manufacturer (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
TRIReagent per three wells for RNA extraction or 1 ml of RIPA
Buffer (Thermo Scientific) (containing Halt Protease and
Phosphatase Inhibitor Cocktail; Thermo Scientific) for protein analysis.
Zip7 overexpression plasmid and transient transfection
in C2C12 skeletal muscle cells
A full-length mouse cDNA Zip7 expression plasmid was
obtained from Origene Technologies, Inc (Clone ID
MR216531; Rockville, MD). The pCMV control plasmid was
created by excision of the full-length Zip7 gene by restriction
digest at enzyme sites Sgf1 and Mlu1 followed by end-filling and
blunt-end ligation. Briefly, 1 mg of pCMV-Zip7 plasmid was
digested in the presence of 10X Fast Digest Green Buffer and 1
unit of Sgf1 and Mlu1 restriction enzymes (Fermentas, Thermo
Scientific) for 5 min at 37uC. For the end-filling, approximately
1 mg of pCMV plasmid DNA was incubated with 0.5 mM dNTPs
and 1 unit of Klenow fragment and incubated at 30uC for 15
minutes. The pCMV control plasmid was circularized in the
presence of 2 ml of 10X T4 DNA ligase, 100 ng of plasmid vector
and 1 ml of T4 DNA ligase and incubated at 4uC for
approximately 16 h.
The pCMV-Zip7 and pCMV control plasmid were transiently
transfected into C2C12 skeletal muscle cells using Lipofectamine
2000 (Life Technologies) as instructed by the supplier. Briefly,
C2C12 skeletal muscle cells were grown to 80% confluence and
4 mg of pCMV-Zip7 and the pCMV control vector were mixed
with 5 ml of Lipofectamine 2000 and 500 ml of optimen. Following
a 20 min incubation at RT, the Lipofectamine-DNA reagent was
pipetted onto the C2C12 cells contained in a 6-well plate and
supplemented with 1.5 ml of differentiation media. The cells were
differentiated for 72 hr and subsequent RNA and protein was
extracted as described below.
Protein Extraction and Western Blot
Total cellular protein from the scramble control and the
siRNAZip7 transfected C2C12 cells was isolated by scraping cells with
RIPA buffer (contains protease and phosphatase inhibitor cocktail)
then the samples were place on ice for 1 hr with constant
vortexing every 10 mins. The cells were then sonicated for 5
second pulses for 30 seconds at 50% duty followed by boiling for
10 min. The protein samples were centrifuged at 13,000 rpm for
5 min and the supernatant collected. Total protein concentration
was measured using a BCA kit (BIORAD, Gladesville, New South
Wales) as outlined by the manufacturers instructions.
Total soluble protein (100 mg) from the scramble control and
siRNA-Zip7 transfected C2C12 cell lines was resolved on a 415%
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 either Glut4 (Cell Signaling, 1:2000; Catalog No:
2213), Gapdh (1:5,000; Catalog No: sc-48167) (Santa Cruz
Biotechnology, Santa Cruz, CA), Akt (Cell Signaling 1:5000
Catalog No:9272), pAkt (Cell Signaling, 1:5000, Catalog No:
4058) antibodies. Following 4615 minute washes the membrane
was incubated with either anti-mouse HRP (Cell Signaling,
Catalog No: 7076) for Glut4 (1:5000); anti-Rabbit HRP (Cell
Signaling, Catalog No:7074) for Akt and pAkt (1:5000), and
antigoat 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 visualized by autoradiography or a UVITEC Alliance
digital imaging system (Thermo Fisher Scientific, Victoria
Australia). Note: to assess protein loading consistency, the
membranes were stripped with Restore Plus Western Blot
Stripping Buffer (Thermo Fisher) by incubating the membrane
in the buffer for 15 min at RT. Membranes were subsequently
washes in TBS-Tween and blocked with 5% skim milk before
adding the primary antibody.
Glycogen synthesis assay
The glycogen synthesis assay was performed as described by the
manufacturers (BioVision, Life Research, Scoresby Victoria,
Australia). Briefly, C2C12 cells were transfected with
siRNAZip7 and the scramble control as described above. Following
differentiation, C2C12 cells were treated with 10 nM insulin for
60 minutes. Cell lysates were collected in 200 ml of dH2O on ice
and homogenates were boiled for 5 min to inactivate enzymes.
Samples were then centrifuged at 13000 rpm for 5 min and the
supernatant was collected. Samples were then prepared by
performing hydrolysis of glycogen to glucose and then mixed with
OxiRed probe to generate colour (lmax = 570 nm). Note: a glucose
control was also performed in the absence of glucoamylase to
determine background glucose levels. These were subsequently
subtracted from the glycogen readings. The glycogen
concentration in the samples was calculated by C = Ay/Sv where Ay is the
amount of glycogen (mg) in the sample as determined from a
standard curve and Sv is the sample volume (ml).
Data obtained from individual qPCR was assessed by a
Students unpaired t-test on at least three independent biological
replicates. Statistical significance was denoted as the average 6
standard deviation of the mean. Data was considered statistically
significance 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
The Slc39a (Zip) zinc transporters are differentially
expressed in C2C12 skeletal muscle cells and mouse
To determine the expression levels of the Slc39a zinc transporter
family in mouse C2C12 skeletal muscle cells and mouse
quadriceps we utilized 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). Quantitative
realtime PCR (qPCR) was performed and the expression of each zinc
transporter transcript was measured relative to the housekeeping
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 we observed high levels of
expression for all of the Slc39a transporters with the exception of
Slc39a5 (Figure 1B).
Slc39a7 (Zip7) is expressed during C2C12 skeletal muscle
We were most interested in Slc39a7 (Zip7) as this transporter is
predominately localized to the ER and the Golgi apparatus and is
suggested to be involved in the zinc wave and associated cellular
signaling . Accordingly, to elucidate the role of Zip7 in skeletal
muscle we initially investigated the expression profile of this zinc
transporter relative to Eef2 in the mouse C2C12 myoblast cell line.
Proliferating myoblasts can be induced to biochemically and
morphologically differentiate into post-mitotic multinucleated
myotubes by mitogen withdrawal. This transition from a
nonmuscle 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, we
observed that Zip7 mRNA is highly expressed in proliferating
myoblasts and was constitutively expressed during skeletal muscle
cell differentiation when normalized 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 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 , 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 cell line to the
myotube phenotype (Figure 2BD). 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
[22,2931] and confirms that the muscle cells had acquired the
appropriate contractile and metabolic phenotype.
siRNA-Zip7 Expression Represses Endogenous Zip7
mRNA in Skeletal Muscle Cells
To elucidate the biological role of Zip7 in the context of glucose
metabolism we selectively ablated the expression of this
transporter in C2C12 skeletal muscle cells utilizing a siRNA-Zip7 molecule.
An siRNA targeting mouse Gapdh and a scramble sequence that
contains no known homology to the mouse, rat or human genome
were utilized as controls. The siRNA-Gapdh was used to determine
the robustness of the transfection and the ability to successfully
attenuate a specific target gene that is constitutively expressed.
Accordingly, C2C12 cells were transfected with the scramble
control, siRNA-Gapdh or the siRNA-Zip7 and subsequently
differentiated for three days.
Initially, we aimed to validate the specificity and robustness of
the siRNA transfection in C2C12 cells by transfecting an
siRNAGapdh to determine transfection efficacy and siRNA specificity. We
identified a significant reduction in Gapdh mRNA (4-fold,
p = 0.0023) in the siRNA-Gapdh transfected cells compared to
the scramble control (Figure 3A). We then transfected C2C12
skeletal muscle cells with an 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. We observed a
significant reduction in the mRNA levels of Zip7 (4.6-fold,
p = 0.0006) when compared to the scramble control (Figure 3B).
To determine that the attenuation of Zip7 was not due to
differential Eef2 mRNA expression, qPCR was also performed on
Eef2 normalized to Gapdh. No change in the level of Eef2 in the
Zip7-siRNA cell lines were observed when normalized to Gapdh
mRNA (Figure 3C). We also tested the relative expression of Zip7
in siRNA-Gapdh C2C12 cells. There was no change in Zip7 mRNA
expression in the Gapdh reduced C2C12 cell lines (Figure 4D).
Since Zip1 was also highly expressed in C2C12 skeletal muscle
cells (Figure 1) we decided to selectively reduce the expression of
this transporter with an 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. We successfully
attenuated endogenous levels of Zip1 mRNA (approximately
3fold, p = 0.0025) in the C2C12 cell lines (Figure 3E). No change in
endogenous expression of Zip7 mRNA (p = 0.1040) was observed
in the siRNA-Zip1 cell lines (Figure 3F).
The attenuation of Zip7 resulted in no change in other
To determine the expression status of the other zinc transporter
family members in the presence of the Zip7 reduced C2C12 cell
lines we utilized a custom gene array that contains the primer
sequences for the Slc30a/ZnT (110) and Slc39a/Zip (114) family
members. cDNA from the scramble control and the siRNA-Zip7
C2C12 cells were assayed to assess for compensatory changes in
the other family members due to reduced Zip7 mRNA. We
identified that the reduction of Zip7 had 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 attenuation of Zip7 mRNA as expected. We also
Figure 2. Relative expression of Slc39a7 (Zip7) and markers of skeletal muscle differentiation in C2C12 cell lines. A). Slc39a7 (Zip7)
expression relative to Eef2, BD). Markers of skeletal muscle differentiation: myogenin (MyoG) and the troponins 1 and 2 (Tnni1 and Tnni2),
respectively. EG). 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; D13 = day 1 to day 3 of differentiation of myotubes,
respectively. Error bars indicated the 6 SD from three independent biological samples.
Figure 3. Zip7 mRNA is attenuated by si-RNA-Zip7. Relative expression of Gapdh, Zip7, Zip1 and Eef2 in the scramble control and
corresponding siRNA cells, respectively. A). Gapdh relative to Eef2 in siRNA-Gapdh cells B). Zip7 relative to Eef2 in siRNA-Zip7 cells C). Eef2 relative to
Gapdh in siRNA-Zip7 cells D). Zip7 relative to Eef2 in siRNA-Gapdh cells E). Zip1 relative to Eef2 in siRNA-Zip1 cells, and F). Zip7 relative to Eef2 in
siRNAZip1 cells. Error bars indicated the 6 SD from three independent biological samples. **P,0.01, ***P,0.001.
observed a small, but significant reduction in the expression of
Zip13 and Zip14 mRNA (Figure 4B).
To further assess the reduced expression of Zip13 and Zip14 in
the Zip7 reduced C2C12 cells we designed primer pairs specific for
Zip13 and Zip14 to independently test the validity of this
observation. We performed qPCR on Zip13 and Zip14 expression
in the scramble control and siRNA-Zip7 C2C12 cells. No
significant changes in the level of expression for these zinc
transporters were observed (Figure 4C).
Attenuation of Zip7 mRNA in C2C12 cells is associated
with changes in several genes implicated in glucose
We utilized 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 to assess
potential pathways that are modulated by Zip7 (Table S1). We
observed that the attenuation of Zip7 mRNA 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 = 0.002997), Dlst (Dihydrolipoamide
S-acetyltransferase, p = 0.035894), Galm (Galactose mutarotase,
p = 0.001714), Gbe1 (Glucan-1,4-alpha branching enzyme 1,
p = 0.003227), Idh3g (Isocitrate dehydrogenase 3 NAD+ gamma,
p = 0.015324), Pck2 (Phosphoenolpyruvate carboxykinase 2,
p = 0.002191), Pgam2 (Phosphoglycerate mutase 2, p = 0.031514),
Pgm2 (Phosphoglucomutase 2, p = 0.027981), Phkb (Phosphorylase
kinase beta, p = 0.032247), Pygm (Muscle glycogen phosphorylase,
p = 0.004097), Tpi1 (Triosephosphate isomerase 1, p = 0.021080)
and Gusb (Glucuronidase beta, p = 0.013637) (Table 1 and Table
We further validated several of these genes 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. We observed significant
downregulation in these genes in concordance with the PCR array data
We speculated that given genes implicated in glycogen
metabolism were affected by reduced Zip7 mRNA levels, that
perhaps 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 (glycogenesis) pathways
. Accordingly, qPCR was performed for Glut4 mRNA
expression in the scramble control and the siRNA-Zip7 C2C12
cells. We observed a significant downregulation of Glut4 in the
siRNA-Zip7 cells (p = 0.0096) (Figure 6A). We also tested for Glut4
immunoreactive protein in the scramble control and siRNA-Zip7
C2C12 cells. Accordingly we observed a significant reduction in
immunoreactive Glut4 in the siRNA-Zip7 C2C12 cells compared
to the scramble control (Figure 6B). Gapdh was used as a protein
loading control and showed that similar amounts of total soluble
protein were resolved (Figure 6B).
Reduced Zip7 compromises insulin-induced glycogen
synthesis and phosphorylation of AKT in C2C12 skeletal
Cellular glucose utilization by Glut4 is responsible for
glycogenesis in muscle  and with increasing plasma insulin
concentration, glycogen synthase is activated by insulin and
glycogen synthesis predominates . 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 . To test the efficacy of insulin to induce
phosphorylation of Akt and thus confirm the robustness of the C2C12
skeletal muscle cell line to respond to insulin, skeletal muscle cells
PATHWAY: GLUCOSE METABOLISM
PATHWAY: GLYCOGEN METABOLISM
Phosphoglycerate mutase 2
Triosephosphate isomerase 1
Phosphoenolpyruvate carboxykinase 2 (mitochondrial)
Isocitrate dehydrogenase 3 (NAD+), gamma
Glucan (1,4-alpha-), branching enzyme 1
Muscle glycogen phosphorylase
Fold Up- or Down-Regulation
Fold Up- or Down-Regulation
Fold Up- or Down-Regulation
Fold Up- or Down-Regulation
Fold Up- or Down-Regulation
Fold Up- or Down-Regulation
*P values ,0.05
A Mouse Glucose Metabolism RT2 Profiler PCR Array was utilized to profile the expression of 84 genes involved in the regulation and enzymatic pathways of glucose
and glycogen metabolism. Three independent biological samples were utilized and the data was considered statistically significance when the P value was #0.05.
were treated with 10 nM insulin over 60 mins mins and
subsequent protein was extracted as described in Material and
Methods. We observed that 10 nM of insulin activated pAkt after
5 min followed by a robust phosphorylation of Akt over the
60 min time course (Figure S1), and thus confirmed the validity of
skeletal muscle cell line to respond to insulin treatment (Figure S1).
Similarly, to test whether maintaining the C2C12 line in the
presence of 20 mM ZnSO4 (see Materials and Methods, Cell
culture) affected the phosphorylation status of AKT we treated
cells with ZnSO4 alone and ZnSO4 in the presence of an
ionphore, pyrithione (Figure S2). Accordingly, 10 mM of
pyrithione in the presence of 20 mM ZnSO4 induced a rapid
phosphorylation of AKT within 15 minutes and this increased
further over 30 and 60 minutes of treatment (Figure S2). We did
not observe an increase in AKT phosphorylation in the presence
of ZnSO4 alone and thus confirmed that maintaining our cell
culture system in the presence of 20 mM ZnSO4 had no effect on
Given that Zip7 modulates core genes implicated in glucose
metabolism, we tested whether glycogen synthesis was
compromised in the attenuated Zip7 skeletal muscle cells. We treated the
scramble and siRNA-Zip7 C2C12 cells with 10nM insulin over 60
minutes and performed glycogen synthesis. We observed a
significant reduction in glycogen synthesis in the siRNA-Zip7
when compared to the scramble control (Figure 6C). As expected,
we observed a significant induction of glycogen synthesis on
exposure to insulin in the scramble control cells, however this
effect was blunted in the Zip7-siRNA C2C12 (Figure 6C).
To determine a potential mechanism of action for the reduced
glycogen synthesis in the presence of reduced Zip7 mRNA we
performed qPCR on the insulin receptor (Insr) and the most
predominant isoforms of the insulin receptor substrate molecules
that are expressed in skeletal muscle, insulin receptor substrate 1
(Irs1), and insulin receptor substrate 2 (Irs2) . These substrates
serve as docking molecules for several SH2-containing proteins
and the subsequent activation of downstream signaling molecules
that result in the activation of AKT, which mediates many of
insulins metabolic effects by modulating gluconeogenesis, protein
synthesis and glycogen synthesis . 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 7AC). In order to confirm that the reduction of these key
genes was associated with a reduction in signaling we performed
immunoblot analysis on phosphorylated Akt (pAkt). We observed
a significant reduction in pAkt in the Zip7-siRNA compared to the
scramble control (Figure 7D).
Figure 5. Reduced Zip7 expression alters gene expression of key glucose metabolic genes. AE). Relative expression of Pgm2, Phkb, Pygm
and Geb1 mRNA to Eef2 in the scramble control and the siRNA-Zip7, respectively. Error bars indicated the 6 SD from three independent biological
samples. *P,0.05, **P,0.01.
Overexpression of Zip7 in C2C12 cells induces genes
associated with glucose metabolism
We observed that reduced Zip7 mRNA in C2C12 skeletal
muscle cells was associated with changes in genes implicated in
glucose metabolism. For example, a significant reduction in the
expression of Pgm2, Phkb, Pygm, Gbe1, Glut4, Insr, Irs1 and Irs2 was
observed in the Zip7-siRNA C2C12 cells compared to the
scramble control (see Figures 5, 6, 7). Accordingly, to determine
if by overexpressing Zip7 we could observe the converse effect on
gene expression, we transiently transfected an overexpression Zip7
plasmid (pCMV-Zip7) into C2C12 skeletal muscle cells and after
72 hours collected RNA for subsequent qPCR analysis. We
observed a significant induction in the expression of exogenous
Zip7 in the pCMV-Zip7 expressing C2C12 cells compared to the
pCMV control (Figure 8A and B). To confirm that the major Zip7
mRNA transcript observed was from the overexpression of the
pCMV-Zip7 plasmid we performed PCR using primers that were
specific for the endogenous form of Zip7 mRNA. We observed that
Zip7 mRNA was expressed at relatively much lower levels in both
the pCMV and pCMV-Zip7 transfected cells (Figure 8C)
confirming that the major Zip7 transcript resulted from the
Moreover, we found that the overexpression of Zip7 mRNA
induced the expression of the insulin receptor (Insr); insulin
receptor substrate 1 (Isr1) and insulin receptor substrate 2 (Isr2) (see
Figure 8DF). This was in contrast to Figure 7 where a reduction
in the expression of Zip7 mRNA resulted in reduced expression of
Insr, Isr1 and Isr2. We also observed an increase in Glut4 mRNA in
the pCMV-Zip7 overexpression system, however this result did
not attain significance (p = 0.0590). Similarly, Glut4 protein levels
were not significantly changed in the pCMV-Zip7 overexpression
system when compared to the pCMV control (data not shown).
Intracellular zinc homeostasis is largely regulated by two
families of zinc transporters (ZnTs and ZIPs) that traffic zinc
across biological membranes [35,36]. Dysregulation of zinc
signaling leads to a number of disease states including cancer
[19,37], autoimmune disease [38,39], cardiovascular disease
[40,41] and diabetes . Of this family, ZIP7 is important
in maintaining physiological and cellular zinc homeostasis through
its ability to initiate the zinc wave and provide cytosolic zinc ions
that are involved in cellular signaling processes. Although many
zinc transporters respond to fluctuating zinc levels and alter their
subcellular localization, ZIP7 is an exception and is restricted
constitutively to the membrane of the Golgi apparatus and/or the
endoplasmic reticulum . Furthermore, Zip7 gene
expression and intracellular location are not altered in response to
changes in intracellular zinc status . Studies in breast cancer
cells have elucidated a role for this transporter in cell signaling
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. Here we provide the
first evidence for a metabolic role for Zip7 in modulating glycaemic
control in skeletal muscle and provide support for 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
 suggesting compensatory actions from other family members.
To explore compensatory mechanisms from other zinc
transporters we performed qPCR on all of the family members in the
scramble control and the siRNA-Zip7 C2C12 skeletal muscle cells.
We did not observe major changes in expression of the other
members of the zinc transporters which suggest that the
attenuation of Zip7 has no other effect on these genes. Of the
zinc transporters, it should be emphasized that, in addition to
ZIP13,  ZIP7 is the only other zinc transporter localized to the
Golgi apparatus and not the plasma membrane  and
compensation is therefore unlikely. Given that ZIP7 is localized
exclusively on the Golgi apparatus or the ER , and the fact
that no compensatory changes in the other transporters were
observed in the reduced Zip7 C2C12 cells suggests that this
transporter may be unique in its specialized function in
transporting zinc from the ER or Golgi into the cytosol .
In contrast to the Zip expression profile in C2C12 cells, we also
observed moderate levels of expression for all of the Zip
transporters (except for Zip5) in mouse quadriceps. 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) . Similar studies on
scramble control and siRNA-Zip7 transfected C2C12 skeletal muscle cells. Error bars indicate the 6 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.
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 . Moreover, studies on
protein arginine methyltransferase 3, 4 and 5 (PRMT35) in
mouse skeletal muscle tissue and C2C12 cells found high
expression of PRMT35 in gastrocnemius in comparison to only
high expression of PRMT4 (with no or minimal expression of
PRMT3 and 5, respectively) . 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 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 .
Our 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 S1) were refractory to the
attenuation of Zip7 expression. These data are noteworthy for
several reasons. For example, in skeletal muscle, GLUT4
predominately transports glucose across the plasma membrane
which is further processed by oxidative (glycolysis) or
nonoxidative (glycogenesis) pathways . Thus, the decline in Glut4
protein in the Zip7-reduced 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 implicated in glycolysis (Galm,
Gusb, Pgam2, Pgm2 and Tpi1) and glycogen synthesis (Gbe1, Agl,
Pgm2, Pygm and Phkg2) were reduced in the Zip7 attenuated cells.
In skeletal muscle, these genes play critical roles in the oxidative
and non-oxidative pathways, respectively. This is further
supported by the reduction in the mRNA of the insulin receptor (Insr), and
the insulin receptor substrates 1 and 2 (Isr1 and Isr2) and the
subsequent reduction of basal and insulin-mediated glycogen
storage in C2C12 myotubes when Zip7 expression was attenuated
(see Figure 6).
Skeletal muscle is particularly important in maintaining glucose
homeostasis because approximately 70-90% of whole body
insulin-mediated induction of glucose uptake occurs in muscle
where it is incorporated into glycogen for storage . 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 metabolism and the fact that there was a reduction in
glycogen synthesis, we also observed a significant decrease in the
phosphorylation status of AKT in the reduced Zip7 expressing
C2C12 cells. This is consistent with recent studies where a siRNA
targeting ZIP7 significantly decreased zinc-induced pAKT after 5
minutes of 20 mM zinc treatment in MCF-7 tamoxifen-resistant
breast cancer cells . Moreover, in a recent study in a Zip9 gene
knockout chicken DT40 cell model, the levels of phosphorylation
of Akt and Erk were significantly reduced . Given the role of
Zip7 in facilitating zinc flux into the cytosol , and the fact that
previous studies have shown that zinc can activate pAKT [8,59], it
will be important to determine whether Zip7 in skeletal muscle
plays a similar role in mediating zinc flux and signaling events that
lead to phosphorylation of AKT and the mobilization of glucose
Zinc is a well-known inhibitor of protein tyrosine phosphatases
(PTPs)  with a reported inhibition constant in the nanomolar
range . Zinc inhibits PTP1B, a cytoplasmic phosphatase that
interacts with the insulin receptor and catalyzes its
dephosphorylation resulting in the attenuation of insulin signaling . Based
on these results, and the fact that the insulin signaling pathway
depends on the status of tyrosine phosphatases and the release of
zinc into the cytosol, we hypothesize that reduced expression of
Zip7 could lead to a reduction in the cytosolic zinc pool that is
available for cellular signaling. For example, in the testes of
diabetic mice treated with the zinc chelator, TPEN, a significant
down-regulation of Akt-mediated glucose metabolism signaling
was observed that was reflected by reduced phosphorylation of Akt
and Gsk-3b . 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 . Furthermore,
in myocytes isolated from the femoral muscle of mice with a ZnT7
knock-out (these mice display low zinc status) there was reduced
insulin signaling pathway activity and these mice were insulin
resistant. This was also congruent with a reduction in the mRNA
expression of Insr, Irs2 and Akt .
Based on the observations that Zip7 plays a crucial role in
facilitating cytosolic zinc flux , and given the role of zinc as a
second messenger that activates pathways associated with cellular
signaling, these studies now show a new role for Zip7 in regulating
the critical gene programs involved in glucose uptake and glycogen
storage in skeletal muscle. In particular, the mRNA
downregulation of Insr, Irs1 and Irs2, in association with reduced
phosphorylation of Akt and reduced Glut4 expression, suggests
that Zip7 activity may be amenable to manipulation as a novel
approach for the treatment of insulin resistance in skeletal muscle.
Figure S1 A. Western blot analysis for insulin-induced
phosphorylation of AKT in C2C12 skeletal muscle cells. C2C12
skeletal muscle cells were differentiated in 2% horse serum for 3
days and then treated in the absence or presence of 10 nM of
insulin for 60 minutes. Total cellular protein was collected and the
presence for immunoreactive pAkt and Akt was assessed. This
immunoblot is a representation of three independent biologically
insulin-treated C2C12 cell preparations. B. Average densitometry
quantification of pAkt/Akt. pAkt quantified by densitometry on
immunoblots from three independent experiments normalized to
total Akt and displayed as the mean 6 SD with significant
(**P#0.01,*** P#0.001) changes over time 0.
Figure S2 Western blot analysis for zinc induced
phosphorylation of AKT in the absence and presence of 10 mM pyrithione in
C2C12 skeletal muscle cells. C2C12 skeletal muscle cells were
differentiated in 2% horse serum for 3 days and then treated in the
presence (+) or absence (-) of 10 mM of pyrithione over 60 minutes.
Total cellular protein was extracted and the presence for
immunoreactive pAKT and AKT was performed by western blot
analysis. This immunoblot represents at least three independent
Primer sequences for the amplification of target genes.
Thank you to Fahima Ahmady (Biomedical Technician, University of
Ballarat) for her help with reagent orders and general laboratory duties.
Conceived and designed the experiments: SM AN GC MAM. Performed
the experiments: SM AN GC. Analyzed the data: SM AN GC MAM.
Contributed reagents/materials/analysis tools: SM MAM. Wrote the
1. Cousins RJ , Liuzzi JP , Lichten LA ( 2006 ) Mammalian Zinc Transport , Trafficking, and Signals . J Biol Chem 281 : 24085 - 24089 .
2. Gaither LA , Eide DJ ( 2001 ) Eukaryotic zinc transporters and their regulation . BioMetals 14 : 251 - 270 .
3. Huang L , Kirschke CP , Lay YAE , Levy LB , Lamirande DE , et al. ( 2012 ) Znt7- null mice are more susceptible to diet-induced glucose intolerance and insulin resistance . J Biol Chem 282 : 37053 - 37063 .
4. Smidt K , Jessen N , Petersen AB , Larsen A , Magnusson N , et al. ( 2009 ) SLC30A3 Responds to Glucose- and Zinc Variations in beta-Cells and Is Critical for Insulin Production and In Vivo Glucose-Metabolism During betaCell Stress. PLoS ONE 4 : e5684 .
5. Chistiakov DA , Voronova NV ( 2009 ) Zn2+-transporter-8: A dual role in diabetes . Biofactors 35 : 356 - 363 .
6. Fukada T , Yamasaki S , Nishida K , Murakami M , Hirano T ( 2011 ) Zinc homeostasis and signaling in health and diseases . J Biol Inorg Chem 16 : 1123 - 1134 .
7. Yamasaki S , Hasegawa A , Hojyo S , Ohashi W , Fukada T , et al. ( 2012 ) A Novel Role of the L-Type Calcium Channel a1D Subunit as a Gatekeeper for Intracellular Zinc Signaling: Zinc Wave . PLoS ONE 7 : e39654 .
8. Taylor KM , Hiscox S , Nicholson RI , Hogstrand C , Kille P ( 2012 ) Protein Kinase CK2 Triggers Cytosolic Zinc Signaling Pathways by Phosphorylation of Zinc Channel ZIP7 . Sci Signal 5 : ra11 .
9. Ilouz R , Kaidanovich O , Gurwitz D , Eldar-Finkelman H ( 2002 ) Inhibition of glycogen synthase kinase-3b by bivalent zinc ions: insight into the insulinmimetic action of zinc . Biochem Biophys Res Commun 295 : 102 - 106 .
10. Moniz T , Amorim MJ , Ferreira R , Nunes A , Silva A , et al. ( 2011 ) Investigation of the insulin-like properties of zinc(II) complexes of 3-hydroxy-4-pyridinones: Identification of a compound with glucose lowering effect in STZ-induced type I diabetic animals . J Inorg Biochem 105 : 1675 - 1682 .
11. Simon SF , Taylor CG ( 2001 ) Dietary Zinc Supplementation Attenuates Hyperglycemia in db/db Mice . Exp Biol Med 226 : 43 - 51 .
12. Wijesekara N , Chimienti F , Wheeler MB ( 2009 ) Zinc, a regulator of islet function and glucose homeostasis . Diabetes Obes Metab 11 : 202 - 214 .
13. Yoshikawa Y , Ueda E , Kojima Y , Sakurai H ( 2004 ) The action mechanism of zinc(II) complexes with insulinomimetic activity in rat adipocytes . Life Sci 75 : 741 - 751 .
14. Coulston L , Dandona P ( 1980 ) Insulin-like Effect of Zinc on Adipocytes . Diabetes 29 : 665 - 667
15. Huang L , Kirschke CP , Zhang Y , Yu YY ( 2005 ) The ZIP7 Gene (Slc39a7) Encodes a Zinc Transporter Involved in Zinc Homeostasis of the Golgi Apparatus . J Biol Chem 280 : 15456 - 15463 .
16. Taylor KM , Morgan HE , Johnson A , Nicholson RI ( 2004 ) Structure-function analysis of HKE4, a member of the new LIV-1 subfamily of zinc transporters . Biochem J 377 : 131 - 139 .
17. Taylor KM , Morgan HE , Smart K , Zahari NM , Pumford S , et al. ( 2007 ) The emerging role of the LIV-1 subfamily of zinc transporters in breast cancer . Mol Med 13 : 396 - 406 .
18. Lichten LA , Cousins RJ ( 2009 ) Mammalian Zinc Transporters: Nutritional and Physiologic Regulation . Annu Rev Nutr 29 : 153 - 176 .
19. Hogstrand C , Kille P , Nicholson RI , Taylor KM ( 2009 ) Zinc transporters and cancer: a potential role for ZIP7 as a hub for tyrosine kinase activation . Trends Mol Med 15 : 101 - 111 .
20. Taylor KM , Vichova P , Jordan N , Hiscox S , Hendley R , et al. ( 2008 ) ZIP7- Mediated Intracellular Zinc Transport Contributes to Aberrant Growth Factor Signaling in Antihormone-Resistant Breast Cancer Cells . Endocrinology 149 : 4912 - 4920 .
21. Myers SA , Wang SCM , Muscat GEO ( 2006 ) The Chicken Ovalbumin Upstream Promoter-Transcription Factors Modulate Genes and Pathways Involved in Skeletal Muscle Cell Metabolism . J Biol Chem 281 : 24149 - 24160 .
22. Maxwell MA , Cleasby ME , Harding A , Stark A , Cooney GJ , et al. ( 2005 ) Nur77 Regulates Lipolysis in Skeletal Muscle Cells: Evidence for cross-talk between the beta-adrenergic and an orphan nuclear hormone receptor pathway . J Biol Chem 280 : 12573 - 12584 .
23. Lau P , Nixon SJ , Parton RG , Muscat GEO ( 2004 ) RORa Regulates the Expression of Genes Involved in Skeletal Cells: Caveolin-3 and CPT-1 are direct targets of ROR . J Biol Chem 279 : 36828 - 36840 .
24. Spandidos A , Wang X , Wang H , Seed B ( 2010 ) PrimerBank: a resourse of human and mouse PCR primer pairs for gene expression detection and quantification . Nulc Acids Res 38 : D792 - 799 .
25. Spandidos A , Wang X , WangH, Dragnev S , Thurber T , et al. ( 2008 ) A comprehensive collection of experimentally validated primers for Polymerase Chain Reaction quantitation of murine transcript abundance . BMC Genomics 9 : 6333 .
26. Wang X , Seed B ( 2003 ) A PCR primer bank for quantitative gene expression analysis . Nulc Acids Res 31 :e154; 1 - 8 .
27. Bookout A , Mangelsdorf D ( 2003 ) Quantitative real-time PCR protocol for analysis of nuclear receptor signaling pathways . Nucl Recept Signal 1 : e012 .
28. Muscat GE , Rea S , Downes M ( 1995 ) Identification of a regulatory function for an orphan receptor in muscle: COUP-TF II affects the expression of the myoD gene family during myogenesis . Nucleic Acids Res 23 : 1311 - 1318 .
29. Shimokawa T , Kato M , Ezaki O , Hashimoto S ( 1998 ) Transcriptional regulation of muscle-specific genes during myoblast differentiation . Biochem Biophys Res Commun 246 : 287 - 292 .
30. Lau P , Nixon SJ , Parton RG , Muscat GE ( 2004 ) RORalpha regulates the expression of genes involved in lipid homeostasis in skeletal muscle cells: caveolin-3 and CPT-1 are direct targets of ROR . J Biol Chem 279 : 36828 - 36840 .
31. Zhu C , Hu DL , Liu YQ , Zhang QJ , Chen FK , et al. ( 2011 ) Fabp3 Inhibits Proliferation and Promotes Apoptosis of Embryonic Myocardial Cells . Cell Biochem Biophys . 60 : 259 - 266 .
32. Peppa M , Koliaki C , Nikolopoulos P , Raptis SA ( 2010 ) Skeletal Muscle Insulin Resistance in Endocrine Disease . J Biomed Biotechnol 10 .1155/2010/527850.
33. Biddinger SB , Emanueli B ( 2011 ) Insulin Resistance in the Metabolic Syndrome . In: Metabolic Basis of Obesity, Ahima RS (ed.), Springer New York. Pp. 175 - 198 .
34. Bajaj M , DeFronzo RA ( 2003 ) Metabolic and molecular basis of insulin resistance . J Nucl Cadrdiol 10 : 311 - 323 .
35. Liuzzi JP , Cousins RJ ( 2004 ) Mammalian Zinc Transporters . Annu Rev Nutr 24 : 151 - 172 .
36. Myers SA , Nield A , Myers M ( 2012 ) Zinc Transporters, Mecahnisms of Action and Therapeutic Utility: Implications for Type 2 Diabetes Mellitus . J Nutr Metab doi:10.1155/2012/173712
37. Jayaraman AK , Jayaraman S ( 2011 ) Increased level of exogenous zinc induces cytotoxicity and up-regulates the expression of the ZnT-1 zinc transporter gene in pancreatic cancer cells . J Nutr Biochem 22 : 79 - 88 .
38. Delli AJ , Vaziri-Sani F , Lindblad B , Elding-Larsson H , Carlsson A , et al. ( 2012 ) Zinc Transporter 8 Autoantibodies and Their Association With SLC30A8 and HLA-DQ Genes Differ Between Immigrant and Swedish Patients With Newly Diagnosed Type 1 Diabetes in the Better Diabetes Diagnosis Study . Diabetes 10 : 2556 - 2564 .
39. Kawasaki E , Nakamura K , Kuriya G , Satoh T , Kobayashi M , et al. ( 2011 ) Differences in the humoral autoreactivity to zinc transporter 8 between childhood- and adult-onset type 1 diabetes in Japanese patients . Clin Immunol 138 : 146 - 153 .
40. Patrushev N , Seidel-Rogol B , Salazar G ( 2012 ) Angiotensin II Requires Zinc and Downregulation of the Zinc Transporters ZnT3 and ZnT10 to Induce Senescence of Vascular Smooth Muscle Cells . PLoS ONE 7 : e33211 .
41. Foster M , Samman S ( 2010 ) Zinc and redox signaling: perturbations associated with cardiovascular disease and diabetes mellitus . Antioxid Redox Signal 13 : 1549 - 1573 .
42. Stadler N , Heeneman S , Voo S , Stanley N , Giles GI , et al. ( 2012 ) Reduced metal ion concentrations in atherosclerotic plaques from subjects with Type 2 diabetes mellitus . Atherosclerosis 222 : 512 - 518 .
43. Ferdousi S , Mia AR ( 2012 ) Serum levels of copper and zinc in newly diagnosed type-2 diabetic subjects . Mymensingh Med J 21 : 475 - 478 .
44. Basaki M , Saeb M , Nazifi S , Shamsaei HA ( 2012 ) Zinc, Copper, Iron, and Chromium Concentrations in Young Patients with Type 2 Diabetes Mellitus . Biol Trace Elem Res 148 : 161 - 164 .
45. Ruchi S , Ashok K ( 2011 ) A study of Age Related Decrease in Zinc and Chromium and its Correlations with type 2 Diabetes Mellitus . Res J Chem Environment . 15 : 75 - 80 .
46. Kambe T , Geiser J , Lahner B , Salt DE , Andrews GK ( 2008 ) Slc39a1 to 3 (subfamily II) Zip genes in mice have unique cell-specific functions during adaptation to zinc deficiency . Am J Physiol Regul Integr Comp Physiol . 294 : R1474 - R1481 .
47. Bin BH , Fukada T , Hosaka T , Yamasaki S , Ohashi W , et al. ( 2011 ) Biochemical characterization of human ZIP13 protein: a homo-dimerized zinc transporter involved in the spondylocherio dysplastic Ehlers-Danlos syndrome . J Biol Chem 286 : 40255 - 40265 .
48. Armstrong RB , Phelps RO ( 1984 ) Muscle fiber type composition of the rat hindlimb . Am J Anat 171 : 259 - 272 .
49. Crowther LM , Wang SC , Eriksson NA , Myers SA , Murray LA , et al. ( 2011 ) Chicken ovalbumin upstream promoter-transcription factor II regulates nuclear receptor, myogenic, and metabolic gene expression in skeletal muscle cells . Physiol Genomics 43 : 213 - 227 .
50. Wang SCM , Dowhan DH , Eriksson NA , Muscat GEO ( 2012 ) CARM1/ PRMT4 is necessary for the glycogen gene expression programme in skeletal muscle cells . Biochem J 444 : 323 - 331 .
51. Raichur S , Fitzsimmons RL , Myers SA , Pearen MA , Lau P , et al. ( 2010 ) Identification and validation of the pathways and functions regulated by the orphan nuclear receptor , ROR alpha1 , in skeletal muscle. Nucleic Acids Res 38 : 4296 - 4312 .
52. Wang YX , Zhang CL , Yu RT , Cho HK , Nelson MC , et al. ( 2004 ) Regulation of muscle fiber type and running endurance by PPARdelta . PLoS Biol 2 : e294 .
53. Muscat GE , Wagner BL , Hou J , Tangirala RK , Bischoff ED , et al. ( 2002 ) Regulation of cholesterol homeostasis and lipid metabolism in skeletal muscle by liver X receptors . J Biol Chem . 277 : 40722 - 40728 .
54. Dressel U , Allen TL , Pippal JB , Rohde PR , Lau P , et al. ( 2003 ) The peroxisome proliferator-activated receptor beta/delta agonist, GW501516, regulates the expression of genes involved in lipid catabolism and energy uncoupling in skeletal muscle cells . Mol Endocrinol 17 : 2477 - 2493 .
55. Tanaka T , Yamamoto J , Iwasaki S , Asaba H , Hamura H , et al. ( 2003 ) Activation of peroxisome proliferator-activated receptor delta induces fatty acid beta-oxidation in skeletal muscle and attenuates metabolic syndrome . Proc Natl Acad Sci USA 100 : 15924 - 15929 .
56. Jensen J , Jebens E , Brennesvik EO , Ruzzin J , Soos MA , et al. ( 2006 ) Muscle glycogen inharmoniously regulates glycogen synthase activity, glucose uptake, and proximal insulin signaling . Am J Physiol Endocrinol Metab 290 : E154 - E162 .
57. Petersen KF , Dufour S , Savage DB , Bilz S , Solomon G , et al. ( 2007 ) The role of skeletal muscle insulin resistance in the pathogenesis of the metabolic syndrome . Proc Natl Acad Sci USA 104 : 12587 - 12594 .
58. Taniguchi M , Fukunaka A , Hagihara M , Watanabe K , Kambe T , et al. ( 2013 ) Essential role of the zinc transporter ZIP9/SLC39A9 in regulating the activations of Akt and Erk in B-cell receptor signaling pathway in DT40 cells . Plos One 8 : e58022 .
59. Tang X- h, Shay NF ( 2001 ) Zinc Has an Insulin-Like Effect on Glucose Transport Mediated by Phosphoinositol-3-Kinase and Akt in 3T3-L1 Fibroblasts and Adipocytes . J Nutr 131 : 1414 - 1420 .
60. Brautigan DL , Bornstein P , Gallis B ( 1981 ) Phosphotyrosyl-protein phosphatase. Specific inhibition by Zn . J Biol Chem 256 : 6519 - 6522 .
61. Maret W , Jacob C , Vallee BL , Fischer EH ( 1999 ) Inhibitory sites in enzymes: Zinc removal and reactivation by thionein . Proc Natl Acad Sci USA 96 : 1936 - 1940 .
62. Ma Y -m, Tao R-y , Liu Q , Li J , Tian J- y et al. ( 2011 ) PTP1B inhibitor improves both insulin resistance and lipid abnormalities in vivo and in vitro. Mol and Cell Biochem 357 : 65 - 72 .
63. Zhao Y , Tan Y , Dai J , Wang B , Li B , et al. ( 2012 ) Zinc deficency exacerbates diabetic down-regulation of Akt expression and function in the testis: essential roles of PTEN, PTP1B and TRB3 . J Nutr Biochem 23 : 1018 - 1026 .