2. MATERIALS AND METHODS
Animals and diets. Design and genomic validation of the Zip14-KO
(Zip14Ϫ/Ϫ
) mice have been described previously (3, 5, 33). Briefly,
Zip14 heterozygous mice were used to establish a breeding colony to
generate wild-type (WT; Zip14ϩ/ϩ
) and KO mice. For the experi-
ments described herein, female KO and WT mice were used between
8 and 16 wk of age. Mice were provided with ad libitum access to a
commercial chow rodent diet (7912, with 60 mg zinc/kg provided by
ZnO; Harlan-Teklad, Indianapolis, IN) and tap water.
Animal treatments. In experiments modeling acute inflammation,
lipopolysaccharide (LPS; E. coli serotype 055:B5; Sigma, St. Louis,
MO) in phosphate-buffered saline (PBS) was administered at 2.0
mg/kg via intraperitoneal (ip) injection. Mice received LPS injections
up to 18 h prior to euthanasia. Control animals received ip injections
of PBS. In other experiments, fasted mice were gavaged with 65
Zn (2
Ci/mouse in 250 l of saline) and euthanized 3 h postgavage to
determine the tissue distribution. Specific activity of the 65
Zn (Perkin-
Elmer, Waltham, MA) when used was 4.4 mCi/mg. Tissue accumu-
lation of 65
Zn was measured via ␥-scintillation spectrometry. For the
antibiotic experiment, ultrapure (MiliQ, Billerica, MA) drinking water
was supplemented with neomycin (0.5 mg/ml) and ampicillin (1
mg/ml) for 4 wk prior to tissue collection. Mice were anesthetized
using isoflurane (Baxter, Deerfield, IL) prior to injection or gavage.
Euthanasia was conducted via cardiac puncture. Blood from cardiac
puncture was collected into a clot activator microgel barrier collection
tube (Capiject; Terumo Medical, Somerset, NJ). Serum was separated
via centrifugation and stored at Ϫ80°C prior to further analysis. All
harvested tissues (intestine, muscle, liver, and adipose) were snap-
frozen in liquid nitrogen and stored at Ϫ80°C prior to further pro-
cessing. All studies described herein used intra-abdominal white fat
pads (parameterial fat pads) that lay along the uterine horn. Unless
otherwise specified, tissues were homogenized in assay-specific buf-
fers using a Bullet blender with zirconium oxide beads (Next Ad-
vance, Averill Park, NY). All animal protocols were approved by the
University of Florida Institutional Animal Care and Use Committee.
Cell culture. A well-established embryonic mouse fibroblast cell
line (3T3-L1; ATCC, Manassas, VA) was used to model adipocyte
growth and differentiation. Cells were cultured in growth medium
(DMEM;4.5 g/l glucose, 15% FBS, and 1% pencillin-streptomycin;
all from Corning, Manassas, VA). After two passages, subconfluent
primary cultures were trypsinized and plated into 12-well plates for
experimentation. Transient knockdown of ZIP14 was carried out with
HiPerfect (Qiagen, Valencia, CA). 3T3-L1 cells were grown to 90%
confluence, and cells were treated with 25 nM siRNA (Darmacon,
Pittsburgh, PA) for 48 h prior to incubation with differentiation
medium with or without LPS (10 ng/ml). Thereafter, cells were
exposed to differentiation medium (DMEM; 4.5 g/l glucose, 10%
FBS, and 1% pencillin-streptomycin supplemented with 1.7 M
insulin, 1 M dexamethasone, and 0.5 mM isobutylmethylxanthine).
Cells were exposed to differentiation medium for three days prior to
postdifferentiation medium (DMEM; 4.5 g/l glucose, 10% FBS, and
1% pencillin-streptomycin). The entire experimental period from
differentiation to collection was 8 days. For experiments which
involved LPS, cells were plated into their experimental dishes (pas-
sage 3), and 10 ng/ml LPS was supplemented to the medium the next
day. Thereafter, cells were exposed to LPS throughout the entire
experimental course through differentiation; medium was changed
every 2 days.
To confirm our findings in 3T3L1 cells, ear mesenchymal stem
cells (EMSC) were collected from WT and KO mice, as described
previously by Rim et al. (42). Briefly, ears were minced and digested
in medium containing collagenase type I (Worthington, Lakewood,
NJ). Prior to the cell suspension being filtered (100 m; Fisher
Scientific, Suwanee, GA) minced tissue and collagenase medium were
placed in a 37°C shaking water bath for 1 h. Filtered cells were
pelleted through centrifugation (360 g, 5 min, room temperature), and
red blood cells were lysed. Isolated cells were plated in 100-mm petri
dishes in the previously described growth medium. Cells were al-
lowed to expand for 3–5 days, and then these subconfluent primary
cultures were trypsinized and plated into 24-well plates for experi-
mentation. At visually confirmed confluence the EMSC were stimu-
lated into adipocytes with previously described differentiation me-
dium. After 3 days in differentiation medium, cells were maintained in
postdifferentiation media. The entire experimental period from differ-
entiation to collection was 10 days.
Analytical procedures. Serum was diluted 1:3 using ultrapure
water, and zinc levels were determined using flame atomic absorption
spectrophotometry (AAS). Adipose tissue was weighed prior to HNO3
digestion (90°C for 3 h) and diluted 1:1 with ultrapure water prior to
AAS analysis. In experiments where total zinc was determined in
cells, 3T3-L1 cells were first cultured in 150-mm plates (Corning).
Cells were collected in ice-cold PBS, and an aliquot was obtained for
total protein determination (Pierce BCA assay; Thermo Fisher Scien-
tific, Waltham, MA). The remaining cell pellet was digested in HNO3
(80°C for 3 h) and diluted 1:1 with ultrapure water. Total zinc was
measured by AAS, and values were normalized to total protein, as
described above. Serum endotoxin was measured as reported previ-
ously (25) using the LAL Chromogenic Endotoxin Quantitation Kit
(Thermo Fisher Scientific), with absorbance read at 407 nm. Serum
and tissue leptin were measured using a mouse/rat-specific ELISA kit
(Alpco, Salem, NH). Tissue was first homogenized in cell extraction
buffer (20 mM Tris·HCl, 1 mM EDTA, and 254 mM sucrose, pH 7.4),
and absorbance was measured at 450 nm (1). Leptin values were
normalized to total protein, as described above. Adipose glucose
content was determined enzymatically with a total glucose assay kit
(Sigma-Aldridge, St. Louis, MO). Prior to analysis, tissues were
homogenized in ultrapure water and diluted 1:2. Absorbance was
measured at 540 nm.
RNA quantification. For RNA isolation, a portion of frozen fat pad
was placed into TRIzol reagent (Life Technologies, Thermo Fisher
Scientific). Early experiments (Fig. 1, A–C) utilized a Polytron
blender to lyse tissue. Subsequent tissues were homogenized as
described previously using the Bullet Blender. cDNA was generated
using the iScript reagents (Bio-Rad, Hercules, CA). Quantitative
real-time PCR was performed using the Real-time PCR Fast SYBR
Green Master Mix and a StepOnePlus Fast Thermocycler (Applied
Biosystems, Thermo Fisher Scientific). Primers to detect specific
mRNAs were designed to span intron/exon boundaries: peroxisome
proliferator-activated receptor-␥ (Ppar␥), 5=-GGAAGACCACTC-
GCATTCCTT-3= and 5=-GTAATCAGCAACCATTGGGTCA-3=;
plasminogen activator inhibitor-1 (Pai-1), 5=-AGGGCTTCATGC-
CCCACTTCTTCA-3= and 5=-GTAGAGGGCATTCACCAGCA-
CCA-3=. All other primer sequences were selected using the qPrim-
erDepot database, http://mouseprimerdepot.nci.nih.gov/, as described
in Cui et al. (17). TBP mRNA was the normalizer for relative
expression, as described previously (3, 5).
Western blotting. Western analysis was performed using polyclonal
rabbit antibodies against ZIP14 developed and affinity purified (Life
Technologies, Thermo Fisher Scientific) as described previously
(3, 5). Akt, phosphorylated Akt (p-Ser473
), hormone-sensitive lipase
(HSL), phosphorylated HSL (Ser660
), IB, phosphorylated IB (p-
Ser32/36
), IR1, phosphorylated IR1 (p-Y1150/1151
), mammalian tar-
get of rapamycin (mTOR), phosphorylated mTOR (Ser2448
), MyD88,
NF-B, phosphorylated NF-B (p65), PPAR␥, preadipocyte factor-1
(PREF-1/DLK-1), SOCS3, STAT3, and phosphorylated STAT3 (p-
Y705
) antibodies were purchased from Cell Signaling Technology
(Boston, MA). F4/80 and zinc ␣2-glycoprotein (ZAG) antibody were
purchased from Santa Cruz Biotechnology (Dallas, TX). Frozen
tissues were processed over liquid nitrogen to prevent freeze thaw and
protein degradation. Tissues were homogenized in RIPA lysis buffer
(Santa Cruz Biotechnology) with 100ϫ protease and phosphatase
inhibitors (Thermo Fisher Scientific), along with phenylmethanesul-
fonyl fluoride (Sigma-Aldrich). Proteins were separated using a 10%
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3. acrylamide gel for SDS-PAGE and transferred to nitrocellulose mem-
branes. Transfer was verified through Ponceau Red staining, and
proteins were visualized through chemiluminescence (SuperSignal
West Pico, Thermo Fisher Scientific) and digital imaging (Protein
Simple, San Jose, CA). Tubulin (Abcam, Cambridge, MA) abundance
was used as the loading control.
Adipose histology. Collected fat pads (parametrial fat pads) were
fixed in 10% neutral buffered formalin for 24 h prior to paraffin
embedding and sectioning. Slides were hematoxylin and eosin stained
prior to microscopy using a Zeiss Axiovert 100 microscope at ϫ10
magnification. Adipocyte area was measured using ImageJ software
(National Institute of Mental Health, Bethesda, MD). Cell areas were
determined on cells with contiguous borders using the Adiposoft
open-source ImageJ plugin http://fiji.sc/Adiposoft (22). Between 600
and 1,500 cells/image were used. Ten full images per genotype (5
mice, 2 images/mouse) were used to generate cell area images. In cell
experiments where Oil Red O was used, cells were first fixed in 3.3%
formaldehyde and stained in 3 g/ml Oil Red O. Images were
collected using the previously described Zeiss Axiovert 100 at ϫ10
magnification. To quantify staining, Oil Red O was extracted with
isopropanol containing 4% NP-40, and absorbance was measured at
520 nm (62).
Confocal laser-scanning microscopy. Imaging was done with a
Leica TCS SP5 laser-scanning confocal microscope with LAS-AF
imaging software, using a ϫ40 oil objective. For detection of labile
zinc, cells were incubated with FluoZin-3 AM (Invitrogen, Waltham,
MA), as described previously (4). Briefly, cells were incubated in 5
M FluoZin-3 for 30 min, followed by a 30-min incubation in
Locke’s buffer (154 mM NaCl, 5.6 mM KCl, 3.6 mM NaHCO3, 1.2
mM MgSO4, 5.6 mM glucose, 2.5 mM CaCl2, and 10 mM HEPES).
Cells were then stimulated with 40 M ZnCl2, and fluorescence was
measured at 516 nm with excitation at 494 nm. A similar method was
used to quantify FluoZin-3 AM in 12-well plates, with fluorescence
being determined as above. Immediately after fluorescence was read,
the cells were trypsanized and diluted 1:1 with trypan blue to deter-
mine cell number. FluoZin-3 AM relative fluorescent units were
Fig. 2. LPS-induced Zip14 expression is correlated with cytokine expression. A: mice received LPS (2 mg/kg ip) or the same volume (250 l) of saline [control
(CTRL)] 1–18 h before being euthanized. Relative transcript levels of Zip14 and specific cytokines; n ϭ 3 mice/treatment Ϯ SE. B: serum hypozincemia confirms
the acute-phase response. Zinc concentrations in serum and WAT were measured by atomic absorption spectrophotometry (AAS). Adipose zinc values were
normalized to wet tissue weight; n ϭ 3 mice/treatment Ϯ SE. C: Western analysis of ZIP14, lipolytic marker hormone-sensitive lipase (HSL), and differentiation
marker PPAR␥ (peroxisome proliferator-activated receptor-␥) during the acute-phase response in WAT. Each lane is a pooled lysate of 3 mice/treatment. *P Ͻ
0.05; **P Ͻ 0.01; ***P Ͻ 0.0001. P-IR, phosphorylated insulin receptor.
Fig. 1. Expression of Zip14 in white adipose tissue (WAT) is induced by lipopolysaccharide (LPS). A: tissue expression profile of Zip14 transcripts. Transcript
abundance was determined by quantitative PCR, starting with an equal amount of total RNA from each tissue. B: fold change in Zip14 tissue expression 18 h
post-intraperitoneal (ip) injection of LPS (2 mg/kg). C: of the WAT zinc transporters significantly (P Յ 0.05) impacted by LPS, Zip14 had the highest induction.
For all graphs, n ϭ 5 mice/genotype Ϯ SE. DUO, duodenum.
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4. normalized to cell number. To generate confocal images, 3T3-L1 cells
were passaged into two-well chambered coverglass (Thermo Fisher
Scientific) prior to siRNA treatment and a subsequent 8-day differ-
entiation period. Imaging was performed at the University of Florida
Cell & Tissue Analysis Core with a Leica TCS SP5 laser-scanning
confocal microscope, using a ϫ40 oil objective. LAS-AF imaging
software was used for image analysis.
Statistics. Statistical analyses was performed using SAS verison 9.2
(SAS Institute, Cary, NC). The effects of WT vs. KO genotype were
compared using Student’s t-tests. The independent effects of genotype
and antibiotic were analyzed using the Proc Mixed procedure (SAS)
with mouse within treatment as the random effect. Multiple compar-
ison significance was analyzed using the Tukey adjustment. Reported
values represent means Ϯ SE.
RESULTS
Zip14 is highly expressed in WAT during acute inflammation.
Relative Zip14 mRNA abundance in WAT of WT mice is low
compared with that found in duodenum or liver but greater than
that found in muscle (Fig. 1A). Aydemir et al. (3) demonstrated
that the highest induction of liver Zip14 mRNA occurred 18 h
post-ip injection of LPS. Therefore, the 18-h time point was
used to examine the comparative expression of Zip14 across
tissues during LPS challenge (Fig. 1B). Expression of Zip14
mRNA increased two- and 32-fold in liver and WAT, respec-
tively, with LPS administration. A transcript screening of 24
zinc transporters was performed. The transporter mRNAs that
were significantly (P Յ 0.05) altered by LPS are shown (Fig.
1C). With the exception of ZnT3 and ZnT8, all zinc transporter
genes were expressed in WAT. Zip14 had the greatest LPS
induction of the zinc transporters.
LPS-induced Zip14 expression is correlated with upregu-
lated cytokine expression. To compare WAT with our previous
liver findings (3), we next sought to evaluate the induction of
WAT ZIP14 during acute LPS challenge. The endotoxin-
induced acute phase response was shown through increased
expression of Il-6, Tnf␣, and Il-1 mRNA (Fig. 2A). The
induction of Zip14 expression peaked at 6 h post-ip injection
and preceded the 9-h peaks of cytokine transcripts (Fig. 2A).
Fig. 3. Zip14-knockout (KO) mice have enhanced levels of circulating endotoxin, predicating an inflammatory state characterized by adipose hypertrophy and
dampened insulin signaling. A: colorimetrically determined that serum endotoxin was higher in Zip14-KO mice; n ϭ 3/genotype Ϯ SE (left). Proinflammatory
signaling pathways in wild-type (WT) and KO mice are shown in WAT (right). Pooled lysates are depicted; lanes are triplicate repeats. B: expression of cytokines
Il-6, Tnf␣, Il-1, and adiponectin (Adpn) in WT and KO mice. Preadipocyte marker [plasminogen activator inhibitor-1 (Pai-1) and preadipocyte factor-1 (Pref-1)]
and differentiation marker (Ppar␥) are also depicted; n ϭ 28 mice/genotype Ϯ SE. C: serum and WAT leptin concentrations in WT and KO mice; n ϭ 3
mice/genotype Ϯ SE. ELISA values from WAT were normalized to total protein. D: representative hematoxylin and eosin (H & E) images of WAT from WT
and KO adipose. Cell areas are from n ϭ 10 images/genotype Ϯ SE. Bars, 150 m. E: transcripts for key adipogenic enzymes (n ϭ 28 mice/genotype Ϯ SE)
and Western analysis of lipolytic markers (n ϭ 3 mice/genotype). F: Western analysis of insulin signaling (n ϭ 3 mice/genotype). MyD88, myeloid
differentiation primary response gene; mTOR, mammalian target of rapamycin; ZAG, zinc ␣2-glycoprotein.
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5. Serum hypozincemia occurred as expected (3, 5); however,
adipose levels of zinc fluctuated over the course of the LPS
challenge (Fig. 2B). This finding suggests that a redistribution
of zinc occurs within adipose during inflammation. The up-
regulation of cytokine mRNA transcripts coincided with in-
creased ZIP14 protein levels, as shown in the Western blots
from adipose lysates (Fig. 2C). In adipose, the acute-phase
response is associated with increased markers of lipolysis and
decreased markers of differentiation (14, 23, 64). Our data
confirmed findings of others in that the phosphorylation of
HSL (HSL660) was upregulated during endotoxemia. In con-
trast, phosphorylated insulin receptor and PPAR␥ were down-
regulated, which suggested a loss of adipocyte differentiation.
Zip14 KO mice have hypertrophy of adipocytes and greater
leptin production. It is apparent that ZIP14 is highly induced
by endotoxin in WAT. Previously, we established that global
Zip14-KO impaired intestinal barrier function, which precipi-
tates systemic endotoxemia (25). Therefore, we hypothesized
that systemic endotoxemia would enhance the inflammatory
status of adipose tissue. In confirmation of our previous find-
ings, plasma endotoxin levels were greater in KO mice (Fig.
3A). We first sought to characterize the phenotypic profile of
KO adipose. Enhanced expression of MyD88 (an adaptor
protein that facilitates LPS induction of IL-6), NF-B, and
STAT3 is indicative of WAT inflammation in the KO mice
(Fig. 3A, right). In agreement, the steady-state levels of Il-6,
Tnf␣, and Il-1 mRNAs were greater in WAT from the KO
mice (Fig. 3B). Similarly, upregulation of Pref-1 (29, 58) and
Pai-1 (35) mRNAs combined with downregulation of Ppar␥ in
the WAT suggests a proinflammatory state, a characteristic of
preadipocytic cells (Fig. 3B). Adiponectin (Adpn) expression, a
hormone positively correlated with insulin sensitivity, was
reduced with KO. Leptin protein levels were significantly (P Ͻ
0.05) greater in both KO serum and WAT (Fig. 3C). Moreover,
cell areas of KO adipocytes were on average 40% larger than
WT adipocytes (Fig. 3D). KO adipose had increased expres-
sion of key adipogenic enzyme transcripts and altered lipid
homeostasis (Fig. 3E). Compared with WT mice, steady-state
lipolysis markers, p-HSL (HSL660), and ZAG (39) were re-
duced in WAT from KO mice. The observed dyslipidemia was
accompanied by reduced PPAR␥ levels (Fig. 3E). Overall, the
KO mutation appeared to enhance adiposity and depress adi-
pocyte differentiation. An evaluation of steady-state insulin
signaling revealed that KO WAT had decreased phosphoryla-
Fig. 4. Zip14 KO enhances LPS-induced inflammation. The effects of the KO are reversed with oral antibiotics (AB). A: WT and KO mice were administered
LPS (2 mg/kg ip) for 6 h. Zip14, Tnf␣, and Il-1 mRNAs were measured by quantitative PCR. Serum and WAT IL-6 concentrations were measured by ELISA;
n ϭ 3 mice/treatment Ϯ SE. In the absence of LPS, baseline IL-6 levels in either genotype were not detected (ND). B: WT and KO mice were provided AB
in drinking water (0.5 mg/ml neomycin and 1 mg/ml ampicillin) for 4 wk. WAT expression of Il-6, Tnf␣, and Il-1 mRNAs in WT and KO mice with AB
treatment; n ϭ 5 mice/treatment Ϯ SE. C: H & E images of WAT were used to measure cell areas; n ϭ 10 images/treatment Ϯ SE. Bars, 150 m. D: Western
analysis of WAT insulin signaling in WT and KO mice with AB treatment; n ϭ 3 mice/treatment.
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6. tion of the insulin receptor (IR), protein kinase B (Akt), and
mTOR (Fig. 3F) (9). These data show that KO adipose dem-
onstrates a phenotype of dampened insulin signaling. Further-
more, data in Fig. 3F also show that the macrophage marker
(F4/80) is not different between genotypes, suggesting minimal
influence of macrophage infiltration (59) on our observed KO
phenotype.
The Zip14 phenotype is enhanced with LPS challenge and
minimized with oral antibiotics. Given the systemic endotox-
emia in the KO, we hypothesized that the phenotype would be
exacerbated by acute LPS and improved with antibiotics (AB).
The highest induction of Zip14 mRNA during our LPS time
course occurred 6 h post-ip injection (Fig. 1C). Therefore, we
used the 6-h LPS response as the point of comparison between
genotypes. The loss of Zip14 caused higher induction of WAT
cytokine transcripts (Fig. 4A, left) along with serum IL-6 (Fig.
4A, right). These findings suggest that ZIP14 may serve as a
negative regulator of cytokine induction. In contrast to the
exacerbating effects of LPS, the impact of endotoxin-induced
inflammation was reduced with AB. Expression of Il-6 and
Tnf␣ mRNAs in KO WAT was reduced with AB (Fig. 4B).
Furthermore, AB prevented the adipocyte hypertrophy seen in
KO WAT (Fig. 4C). The AB treatment also normalized insulin
signaling between the genotypes (Fig. 4D).
Upregulated cytokine pathways due to the KO mutation lead
to impaired capacity to differentiate. In an effort to eliminate
the effect of circulating endotoxin on KO adipose, mesenchy-
mal stem cells were cultured in the absence of LPS. Stem cells
were derived from WT and KO ears and differentiated into
adipocytes. Zip14-KO was confirmed with quantitative PCR
(Fig. 5A). We hypothesized that KO cells would exhibit en-
hanced steady-state cytokine signaling in culture. Cytokine
mRNA expression (Il-6 and Il-1) was higher in fully differ-
entiated KO cells. Mt1 expression was not significantly differ-
ent between genotype but tended to be lower in KO cells.
Additionally, KO preadipocytic markers Pref-1 (29, 58) and
Pai-1 (35) were upregulated (Fig. 5A). Western analysis of
both PPAR␥ and PREF-1 (Fig. 5B) confirmed our KO tissue
findings (Figs. 3, B and E, and 4D). Although the same
antibody was used for cells and tissue, persistent bands appear
in the Western blots of primary mesenchymal stem cells (Fig.
5B). However, this same persistent banding was again noted in
siRNA-treated 3T3-L1 cells, which may serve as our negative
control for cellular ZIP14 expression (Fig. 6F). Oil Red O
staining also demonstrated that cultured KO cells are more
preadipocytic and have a lower capacity to differentiate and
accumulate lipids (Fig. 5C, top). It was not until cells were
cultured in LPS that KO cells began to overproduce lipids (Fig.
Fig. 5. Stem cells from Zip14-KO mice are inflammatory and preadipocytic in culture. Primary stem cells from WT and KO mice were cultured in 3T3-L1 growth
media. Upon confluence, cells were placed in differentiation medium and cultured for 10 days. A: transcript levels of Zip14, cytokine (Il-6, Tnf␣, and Il-1), Mt1,
and preadipocytic markers (Pref-1 and Pai-1) were measured; n ϭ 3 pooled wells Ϯ SE. B: Western analysis of the preadipocytic markers in WT and KO cells;
n ϭ 3 pooled wells Ϯ SE. C: accumulation of lipids in WT- and KO-derived cells, as measured by Oil Red O (Ϯ 10 ng/ml LPS for 10 days). D: Oil Red O
was eluted from cell cultures and measured colorimetrically and normalized to protein; n ϭ 3 wells/treatment Ϯ SE.
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7. 5C, bottom). Quantification of Oil Red O confirmed our visual
observations (Fig. 5D).
ZIP14-KO alters intracellular zinc, which enhances key
inflammatory pathways. Since the focus of the molecular site
responsible for the KO phenotype is likely related to metal
transport, it was necessary to place those findings within a
function of dyshomeostasis of cellular zinc metabolism. 65
Zn
was administered orally to measure zinc uptake/retention. KO
mice accumulated more zinc (65
Zn) in WAT. Figure 6A shows
that Mt1 mRNA expression, a surrogate measure of cytosolic
zinc, is reduced. Adipogenic 3T3-L1 cells transfected with
siRNA had a pattern of zinc distribution similar to KO tissue.
Specifically Zip14 knockdown increased total zinc in cells,
whereas Mt1 mRNA was decreased (Fig. 6B). Intracellular
labile zinc was visualized with a zinc probe, fluozin-3 AM.
Fluorescence was detected using laser-scanning confocal mi-
croscopy (Fig. 6C). The silencing of Zip14 with siRNA clearly
resulted in increased (P Ͻ 0.0001) vesicular zinc. Transfection
with siRNA increased expression of ZIP8, a transporter that is
a homologue of ZIP14 (Fig. 6D) (31). These in vitro data are
compatible with the hypothesis that less functional cellular zinc
is available in adipocytes of KO mice. This finding is indica-
tive of the “zinc trap” hypothesis associated with Zip14 abla-
tion that we advanced earlier (25) in mouse intestine. Zip14
siRNA clearly resulted in increased expression of Il-6 and Tnf␣
(P Ͻ 0.05) and to a lesser extent Il-1 (Fig. 6E).
Zinc’s ability to inhibit key cytokine pathways is one way in
which zinc may regulate cytokine expression. We hypothe-
sized that zinc trapped within vesicles, through either Zip14
ablation in mice or with siRNA transfection in cells, would
limit the inhibition of key proinflammatory signaling cascades
by zinc. With the exception of A20 (11), Zip14 knockdown
enhanced activation of both the JAK2/STAT3 and NF-B
pathways in 3T3-L1 cells (Fig. 6F). Upregulation of these
pathways depressed the ability of cells to differentiate (upregu-
lation of PREF-1 expression) and increase cytokine expression.
As shown in the model presented in Fig. 7, zinc transported by
ZIP14 appears to impact the TLR4 pathway as early as MyD88
and also influences signaling further downstream, e.g., phos-
phorylated IB␣ (p-IB␣) and NF-B. Zip14 knockdown
Fig. 6. Zip14-KO adipose and 3T3-L1 adipocytes treated with Zip14 siRNA display altered zinc homeostasis and enhanced proinflammatory signaling though
the NF-B and JAK2/STAT3 pathways. A: WT and KO mice received 65
Zn by gavage, and WAT zinc accumulation was calculated from 65
Zn-specific activity.
Mt1 mRNA was measured as an index of available intracellular zinc; n ϭ 10 mice/genotype Ϯ SE. B: 3T3L1 cells were transfected with Zip14 siRNA. Total
zinc (AAS) and Mt1 mRNA; n ϭ 3 plates/group replicated 3 times. C: representative laser-scanning confocal images used to detect labile zinc pools using
FluoZin-3 AM fluorescence. FluoZin-3 AM was quantified and normalized to cell count; n ϭ 24 wells/group replicated twice. D: representative Western blots
show the effect of Zip14 siRNA on ZIP14 and ZIP8 (n ϭ 2 replicated 3 times). E: relative mRNA expression of Zip14, Il-6, Tnf␣, and Il-1 in Zip14
siRNA-transfected 3T3-L1 cells Ϯ 10 ng/ml LPS (n ϭ 3 wells/group Ϯ SE). F: representative Western blots show the effect of Zip14 siRNA on proinflammatory
signaling pathways (n ϭ 4 replicated 3 times). SCR, scrambled; RFU, relative fluorescence units.
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8. limits cytosolic availability of zinc, and inhibition of NF-B is
lifted, allowing enhanced induction of proinflammatory genes
Il-6, Tnf␣, and Il-1 (Fig. 6E). Similarly, Zip14 deficiency
enhanced JAK2 and STAT3 activity (Fig. 3A, right) with a
concomitant stimulation of the Il-6 transcript (Fig. 3B) that
could in turn execute an autocrine response leading to in-
creased leptin production and secretion (Fig. 3C).
DISCUSSION
Here we show for the first time that deletion of Zip14
impacts adipose function. Although Zip14 was first cloned
from adipogenic 3T3-L1 cells (53), little has been reported
regarding its expression/function in adipose metabolism. In
this report, it is shown that KO alters zinc signaling pathways,
intracellular zinc trafficking, and ultimately adipocyte metab-
olism.
Inflammation, expansion of tissue mass, and recruitment of
inflammatory cells are normal functions of healthy adipose (49,
60). However, a prolonged inflammatory response will even-
tually lead to metabolic alterations within WAT. Acute inflam-
mation impacts ZIP14 expression in a tissue-specific manner
(3, 25). From the experiments described to this point, it has
been established that adipose ZIP14 is highly responsive to
LPS. Zip14 transcript abundance was upregulated 30-fold by
18 h after LPS injection, suggesting a critical role for ZIP14 in
adipose inflammatory response. These data suggest that up-
regulation of Zip14 expression in WAT is kinetically more
rapid than liver expression (3). Furthermore, Zip14 ablation
drastically influences cytokine and leptin production along
with signaling pathway activity in WAT. Upregulated Il-6
production with KO may be responsible for the induction of
leptin secretion (55). We interpret these responses as being
indicative of a greater requirement for ZIP14 and/or zinc
during inflammatory challenge (43). The reversal of the KO
phenotype with antibiotics supports this hypothesis.
The findings with antibiotics are noteworthy in that the
treatment was a cocktail of neomycin and ampicillin. Neomy-
cin in particular, is poorly absorbed through the gastrointestinal
tract (15). Therefore, these antibiotics target intestinal micro-
flora populations directly, a finding that suggests that intestinal
microflora (along with any treatment that alters the microfloral
load, dietary or otherwise) impacts adipose development and
function. In fact, the tie between microflora and adipose has
been established previously by Ley et al. (30). The impact of
KO on the intestinal microfloral load is certainly relevant
considering the proposed links between the microbiome and
metabolic diseases, a finding that we plan to evaluate in further
studies. Relevant to this report is that the in vivo phenotype
was successfully modeled in vitro using LPS alone, confirming
that KO predisposes cells to an inflammatory phenotype that is
exacerbated with LPS.
Increased intestinal permeability increased serum endotoxin
in KO mice (25). This finding was of particular interest, as
chronic exposure to endotoxin leads to metabolic dysfunction.
Metabolic endotoxemia is characterized by atypically high
levels of circulating endotoxin, which produces low-grade,
systemic inflammation (10, 34, 46). This inflammation has
far-reaching physiological implications, including increased
weight gain, hepatic insulin resistance (12, 26, 37), and mac-
rophage recruitment (52, 59), all of which precede the devel-
opment of type 2 diabetes upon the consumption of a high-fat
diet. Luche et al. (35) found that a 28-day LPS pretreatment
caused mice consuming high-fat diets to gain more weight than
cohorts who had not received the LPS conditioning. The
reported increase in body weight was coupled with an in-
creased fat/lean ratio, an influx of small inflammatory preadi-
Fig. 7. Proposed model for increased IL-6
and leptin production caused by global
Zip14 deletion. Increased cytosolic zinc due
to ZIP14 inhibits cytokine signaling. Sys-
temic endotoxemia and/or LPS administration
leads to Toll-like receptor 4 (TLR4) activation
and induction of the NF-B pathway. Without
surface expression of ZIP14, zinc is trapped in
intracellular compartments, where it is unable
to inhibit activities of the NF-B and JAK2/
STAT3 pathways, thereby increasing IL-6 and
leptin production.
E265ZINC TRANSPORTER Zip14 AND ADIPOSE TISSUE REGULATION
AJP-Endocrinol Metab • doi:10.1152/ajpendo.00421.2015 • www.ajpendo.org
9. pocytes, and an overall higher glycemic index (35). Unique to
our KO model, dampened insulin signaling and low-grade
inflammation were coupled with hypertrophy. Enhanced mark-
ers of lipogenesis and decreased markers of lipolysis (8, 38)
were also noted with KO. These markers are suggestive of a
chronic (vs. acute; see Ref. 61) inflammatory state in KO
WAT. Furthermore, these physiological differences occur in
the absence of a dietary intervention such as the feeding of a
high-fat diet.
Inflammatory cytokines are well-known mediators of altered
adipose function and development (12, 48). Furthermore, prea-
dipocytes themselves are able to acquire phagocytic activity
and express macrophage-specific antigens under acute inflam-
matory conditions (13). Using cultures at tiered stages of
differentiation (0, 50, and 90%), Chung et al. (14) elegantly
demonstrated that preadipocytes were the primary producers of
the endogenous cytokines responsible for endotoxin-induced
suppression of insulin-stimulated glucose uptake. It has been
shown previously that Zip14 mRNA is induced during the early
stages of adipocyte differentiation (53). Here, we show that KO
increased preadipocytic markers and enhanced cytokine ex-
pression. The differentiation marker PPAR␥ was downregu-
lated with KO. This finding is relevant, as PPAR␥ is zinc
responsive and has the ability to inactivate STAT3 in myeloma
cells (38, 56, 57). Zip14 KO limits the ability of mesenchymal
stem cells to differentiate and accumulate lipids (Fig. 4).
Although this may initially seem counterintuitive, limiting a
preadipocyte’s ability to differentiate precipitates hypertrophic
obesity in humans (24, 49). Hypertrophic obesity is the likely
cause of our previous finding where KO mice had enhanced
liver lipids (3).
Aberrant zinc signaling with Zip14 KO impacts the
growth and development of adipocytes. Here, we report a
phenotype characterized by high tissue zinc but low Mt1
expression. Trayhurn et al. (54) found that adipose expres-
sion of Mt1 was not influenced by subcutaneous zinc injec-
tion. However, experiments conducted with metallothion-
ein-KO (7) revealed that MtϪ/Ϫ
mice had increased visceral
WAT weight coupled with adipocyte hypertrophy and
higher levels of circulating leptin (7). Furthermore, this
phenotype was exacerbated by high-fat feeding (44). Previ-
ous literature has reported conflicting relationships between
serum leptin and fat pad zinc content (2, 41, 51). In humans,
obesity has long been associated with hypozincemia. In
terms of zinc transporters, obesity is correlated with down-
regulated ZnT (Ϫ2, Ϫ3, Ϫ6, and Ϫ8) and ZIP (Ϫ1, Ϫ2, Ϫ3,
Ϫ4, Ϫ5, Ϫ6, and Ϫ7) expression in subcutaneous adipose
(47). Our data shows that Zip14 deletion causes adipose to
act as if it were experiencing a zinc deficiency. Evidence for
that includes the lower Mt1 mRNA levels in WAT of the KO
mice (Fig. 6A). This zinc-deficient status apparently cannot
be overcome by the concomitant upregulation of zinc trans-
porters, most notably ZIP8 (Fig. 6D). The ability of adi-
pocytes to regulate zinc trafficking may determine how
adipose is remodeled during tissue expansion. Furthermore,
ZIP14 appears to lie at the plastic interface of adipose
metabolism and inflammation.
As shown in Fig. 7, our data show that ZIP14 influences
adipocyte cytokine expression through two well-established
signaling pathways, NF-B and JAK2/STAT3 (6, 34, 55, 63).
The adipose mass expresses TLR4 (45) and is a major secre-
tory organ of both leptin and IL-6 (28). Hence, the demonstra-
tion here that both are overproduced with Zip14 deletion points
to ZIP14 acting to provide control over the production of both
mediators. Zip14 deletion enhances phosphorylation of these
signaling pathways and is a likely cause for the upregulation of
cytokines and leptin observed with KO. It should be noted that
the ZIP14 homolog ZIP8 has been shown to inhibit the TLR4
pathway though inhibition of IB kinase (IKK) (32). In this
report, ZIP8 levels are greater with Zip14 ablation, demonstrat-
ing that ZIP8 is unable to compensate for the upregulation of
the NF-B pathway. Therefore, ZIP8-mediated inhibition of
IKK may be of minor significance in our Zip14-KO adipose
model.
In summary, adipocytes with a Zip14 deletion appear to have
an impaired ability to mobilize intracellular zinc. In our model,
limited availability of intracellular zinc disinhibits key cyto-
kine pathways. Adipocytes have enhanced cytokine signaling
and are more preadipocytic with KO. ZIP14 knockdown cells
did not produce excess lipids until they were cultured in LPS
media. These results in part model hypertrophic WAT ob-
served with KO in vivo. Furthermore, the KO phenotype of
hypertrophy was diminished with oral antibiotics, indicating
that low-grade inflammation is necessary to induce enhanced
adiposity. Our results demonstrate that ZIP14 is critical to
cytokine production in adipose tissue and that targeted zinc
transport is critical to adipocyte development.
ACKNOWLEDGMENTS
Scanning confocal microscopy was performed at the University of Florida
Core. We thank Dr. Joseph L. Purswell, US Department of Agriculture/
Agricultural Research Service at Mississippi State University, for assistance
with the ImageJ analysis.
GRANTS
This project was supported by National Institute of Diabetes and Digestive
and Kidney Diseases Grant R01-DK-094244 and the Boston Family Endow-
ment Funds of the University of Florida to R. J. Cousins.
DISCLOSURES
The authors report no conflicts of interest, financial or otherwise.
AUTHOR CONTRIBUTIONS
C.T., T.B.A., and R.J.C. conception and design of research; C.T. and T.B.A.
performed experiments; C.T. analyzed data; C.T., T.B.A., and R.J.C. inter-
preted results of experiments; C.T. prepared figures; C.T., T.B.A., and R.J.C.
drafted manuscript; C.T., T.B.A., and R.J.C. edited and revised manuscript;
R.J.C. approved final version of manuscript.
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