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Diabetes Volume 64, January 2015

Xavier S. Revelo,1 Sue Tsai,1 Helena Lei,1 Helen Luck,1 Magar Ghazarian,1 Hubert Tsui,2 Sally Y. Shi,1
Stephanie Schroer,1 Cynthia T. Luk,1 Gloria H.Y. Lin,3 Tak W. Mak,3 Minna Woo,1,4 Shawn Winer,1,5
and Daniel A. Winer15

Perforin Is a Novel Immune


Regulator of Obesity-Related
Insulin Resistance

OBESITY STUDIES

Diabetes 2015;64:90103 | DOI: 10.2337/db13-1524

Obesity-related insulin resistance is associated with an


inux of pathogenic T cells into visceral adipose tissue
(VAT), but the mechanisms regulating lymphocyte balance in such tissues are unknown. Here we describe an
important role for the immune cytotoxic effector molecule perforin in regulating this process. Perforindecient mice (Prf1null) show early increased body
weight and adiposity, glucose intolerance, and insulin
resistance when placed on high-fat diet (HFD). Regulatory effects of perforin on glucose tolerance are mechanistically linked to the control of T-cell proliferation and
cytokine production in inamed VAT. HFD-fed Prf1null
mice have increased accumulation of proinammatory
IFN-gproducing CD4+ and CD8+ T cells and M1polarized macrophages in VAT. CD8+ T cells from
the VAT of Prf1 null mice have increased proliferation
and impaired early apoptosis, suggesting a role for
perforin in the regulation of T-cell turnover during
HFD feeding. Transfer of CD8+ T cells from Prf1null
mice into CD8-decient mice (CD8null) resulted in
worsening of metabolic parameters compared with
wild-type donors. Improved metabolic parameters in
HFD natural killer (NK) celldecient mice (NKnull)
ruled out a role for NK cells as a single source of
perforin in regulating glucose homeostasis. The ndings support the importance of T-cell function in
insulin resistance and suggest that modulation of
lymphocyte homeostasis in inamed VAT is one possible avenue for therapeutic intervention.

Obesity-associated inammation of visceral adipose tissue


(VAT) and associated chronic release of proinammatory
cytokines and adipokines are major contributors to the
development of insulin resistance (13). During obesity,
increased activity of proinammatory immune effector
cells overwhelms anti-inammatory regulators, leading to
impaired insulin signaling and insulin resistance. Obesityrelated insulin resistance is associated with a dramatic inux
of macrophages into VAT (4,5) and a shift in their activation
state from an anti-inammatory or M2-polarized state toward a proinammatory phenotype or M1 polarized (6,7).
Recently, other immune cell types have also been implicated
in obesity-associated inammation of VAT, including mast
cells (8), neutrophils (9,10), CD4+ Th1 (11), CD4+ Th17 (12),
CD8+ T cells (13), and B cells (14).
The T-cell receptor repertoire in VAT becomes restricted during high-fat diet (HFD) feeding (11,15),
suggesting a potential role for antigen-specic T-cell
immunity in inamed VAT. Furthermore, interferon
g (IFN-g)secreting CD8 + T cells increase in VAT starting at 2 weeks of HFD prior to the accumulation of
adipose tissue macrophages, and CD8+ T celldecient
mice fed HFD have improved insulin sensitivity and VAT
inammatory microenvironment (13). Adoptive transfer of
CD8+ T cells into CD8-decient mice reverted these effects
and increased the numbers of M1 polarized in VAT (13).
Activated CD8+ T cells in VAT produce increased amounts
of proinammatory cytokines such as IFN-g and G-CSF,

1Division of Cellular and Molecular Biology, Diabetes Research Group, Toronto


General Research Institute, University Health Network, Toronto, Ontario, Canada
2Department of Laboratory Medicine and Pathobiology, University of Toronto,
Toronto, Ontario, Canada
3Department of Immunology, University of Toronto, Toronto, Ontario, Canada
4Division of Endocrinology and Metabolism, Department of Medicine, University
Health Network, Toronto, Ontario, Canada
5Department of Pathology, University Health Network, Toronto, Ontario, Canada

Received 3 October 2013 and accepted 12 July 2014.

Corresponding author: Shawn Winer, shawn.winer@utoronto.ca, or Daniel A. Winer,


dan.winer@uhn.ca.

This article contains Supplementary Data online at http://diabetes


.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-1524/-/DC1.
X.S.R. and S.T. contributed equally to this work.
2015 by the American Diabetes Association. Readers may use this article as
long as the work is properly cited, the use is educational and not for prot, and
the work is not altered.

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associated with HFD feeding (15). Despite evidence of increased numbers and functional activity of pathogenic
CD8+ T cells, the mechanisms by which T-cell homeostasis
is regulated in inamed VAT are largely unknown. Moreover, it is unclear whether adipocyte cell death in VAT,
manifested as a dying cell surrounded by macrophages
(the so-called crown-like structures [CLS]), is dependent
on CD8+ T cellmediated killing mechanisms.
Perforin is a cytotoxic effector molecule primarily
released by CD8+ T cells and natural killer (NK) cells to
eliminate infected or dangerous cells via the perforingranzyme cell death pathway (16). Perforin-mediated cytotoxicity is also involved in the homeostatic regulation of
CD4+ and CD8+ T cells in vivo, in a physiologic function
distinct from pathogen clearance (17). Interestingly, expression of perforin and associated granzymes is increased
in VAT of HFD-fed mice, likely related to increased CD8+
T-cell activation (15). In humans, impaired perforindependent cytotoxic function leads to excessive T-cell activation, severe hyperinammation, and the often fatal
disorder hemophagocytic lymphohistiocytosis (18,19). Indeed, perforin-decient mice are susceptible to increases
in CD8+ T-cell activation and IFN-g production in response to distinct stimuli (20). Notably, this heightened
immune activation seems to be mediated via signaling of
the T-cell receptor and results from increased antigenspecic stimulation (20). Perforin-mediated regulation
of CD8+ T-cell activation and expansion may also include
a regulatory feedback on dendritic cells, which prime or
initiate T-cell responses during an immune response
(21,22).
Since obesity-mediated insulin resistance is associated
with increased perforin expression, increased inammation, and antigen-specic immunity in VAT, we investigated the effects of perforin deciency in diet-induced
obesity. Here, we show that mice lacking perforin
(Prf1null) placed on HFD develop worsened obesity, glucose tolerance, and insulin resistance when compared
with wild-type (WT) controls. These changes are accompanied by dysregulation of T-cell homeostasis in VAT with
increases in T-cell number and cytokine production, local
proliferation, and reduced T-cell apoptosis. Interestingly,
the ability to form CLS in VAT, a hallmark of adipocyte
cell death, was not reduced in Prf1null mice, suggesting
that perforin is not necessary for the formation of CLS
in VAT. These ndings suggest that a dominant role of
perforin in obesity-associated insulin resistance is to restrict
T-cell expansion and activation in inamed VAT, providing
further evidence of a critical role for adaptive immune cells
in governing obesity and glucose homeostasis.
RESEARCH DESIGN AND METHODS
Mice

Age-matched C57BL/6 (Jax 664), C57BL/6 Prf1null mice


(Jax 2407, derived using a BL/6 embryonic stem cell line),
and C57BL/6 CD8null mice (Jax 2665, backcrossed to
C57BL/6 for at least 13 generations) were purchased

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from The Jackson Laboratory. NKnull mice were a gift of


Dr. Tak Mak (University Health Network), and analysis of
these mice involved littermate controls. Mice were maintained in a pathogen-free, temperature-controlled, and
12-h light and dark cycle environment. Mice received either a normal control diet (NCD) or HFD (60 kcal % fat;
Research Diets) beginning at 6 weeks of age. All mice were
males and age matched between groups. All experiments
were approved by the Institutional Animal Care and Use
Committee of University Health Network.
RNA Isolation and Quantitative Real-Time PCR

Total RNA was isolated from epididymal fat pads using


the RNeasy Plus Mini Kit (Qiagen) and reverse transcribed using random primers by M-MLV (Invitrogen).
RT-PCR was performed using SYBR Green master mix
(Applied Biosystems) to determine the amounts of fatty
acid binding protein 4 (FABP4), CCAAT/enhancer-binding
protein-a (CEBP-a), peroxisome proliferator-activated
receptor-g (PPAR-g), and sterol regulatory elementbinding
protein (SREBP) mRNA. Each sample was run in triplicate,
and the level of gene expression was normalized to the
housekeeping gene 18s. Primer sets are listed in Supplementary Table 1. Changes in gene expression were calculated by the 2(2DDC(T)) method using mean cycle
threshold (CT) values. Fold changes in gene expression
were assessed by calculating the difference between the
CT values of 18s and that of the target gene (DCT). Expression of 18s did not differ between WT and Prf1null mice
(P . 0.20). The mean DCT were subtracted from individual
DCT values to obtain DDCT. Fold changes in gene expression were calculated using the equation 22DDCT.
Metabolic Cage Studies

Mice were placed in automated metabolic cages for 48 h.


Airow was held at 0.5 L/min, and metabolic activity was
assessed using indirect calorimetry recording maximal O2
consumption (VO2), CO2 production (VCO2), and heat
production normalized to body weight. Respiratory exchange ratio was calculated as VCO2/VO2. Ambulatory
activity was assessed by the breaking of infrared laser
beams in the x-y plane. Energy expenditure was adjusted
for body mass using ANCOVA (23). Data are the average
between light and dark measurements.
Metabolic Studies and Histology

Glucose tolerance tests (GTTs), insulin tolerance tests


(ITTs), and measurements of serum insulin and fat cell
diameter were performed as previously described (11).
Mice were fasted overnight for GTTs and fasting insulin.
For GTTs, we administered 1 g $ kg21 of D-glucose intraperitoneally and measured blood glucose levels at indicated time points. For ITTs, mice were fasted for 6 h, insulin
was administered intraperitoneally at 1.5 units $ kg21, and
blood glucose concentrations were determined at indicated time points. VAT was stained with hematoxylineosin, and CLS, dened as adipocytes enveloped by several
macrophages (24), were counted by two blinded observers
(D.A.W. and S.W.). Liver triglyceride concentrations were

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Perforin, Obesity, and Insulin Resistance

determined using a coupled enzyme assay according to the


manufacturers recommendations (Sigma-Aldrich).
Acute Insulin Response and Western Blotting

Mice were fasted overnight and injected intraperitoneally


with 1.5 units $ kg21 of insulin. VAT, muscle, and livers
were harvested 15 min after acute insulin injection and
snap frozen in liquid nitrogen. Tissues were lysed on ice
using the complete radioimmunoprecipitation assay buffer
(Santa Cruz Biotechnology), and their protein concentration was determined using the Pierce BCA Protein Assay
(Thermo Scientic). Protein lysates were separated by SDSPAGE and immunoblotted with antibodies (Cell Signaling
Technology) against total and Ser473-phosphorylated Akt
(pAkt), and GAPDH. The blotted bands were visualized
with a MicroChemi chemiluminescent imaging system
(FroggaBio) and quantitated using the GelQuant analysis
software (FroggaBio).
Processing of Immune Cells from VAT, Spleen, Bone
Marrow, and Lymph Nodes

VAT immune cells were isolated as previously described


(11). For cytokine measurements, 3.0 3 105 VAT stromal
vascular cells (SVCs) were cultured for 1216 h in RPMI
with 10% FCS and penicillin-streptomycin antibiotics.
Bone marrow (BM) cells were obtained by ushing one
femur and one tibia with PBS using a 26-gauge needle,
dissociated into a single-cell suspension, hemolyzed, and
counted. To purify immune cells from spleens and axillary
lymph nodes (LNs), we mechanically dissociated the organs
on 70-mm nylon cell strainers followed by red blood cell
lysis and cell washing.
Cytokine Measurement

Cytokines in the serum and supernatants of SVC VAT


cultures were analyzed using the Bio-Plex Pro Mouse
Cytokine Luminex assay (Bio-Rad). Serum adipokines and
cytokines were also measured by ELISA (TNF-a; Biolegend)
as well as through a multiplex ELISA screening kit (Signosis), according to the manufacturers instructions. Serum
cytokines are indicated in pg/mL, or shown as a ratio of
Prf1null to WT levels for the SVC VAT cultures.
Flow Cytometry

Cells were stained for 30 min with uorophore-conjugated


antibodies to CD3 (145-2C11), CD4 (GK1.5), CD8 (53-6.7),
CD45.2 (104), CD44 (1M7), CD62L (MEL-14), CD69
(H1.2F3), CD107a (1D4B), CD11b (M1/70), CD11c (N418),
F4/80 (BM8), Gr-1 (RB68C5), NK1.1 (PK136), IFN-g
(XMG1.2), and interleukin 17 (IL-17) (TC11-18H10.1) using
recommended dilutions (Biolegend). For intracellular staining,
T and NK cells were incubated with a cell stimulation cocktail and Golgistop (eBioscience) for 5 h. Phycoerithrinconjugated antiperforin and isotype control antibodies
were purchased from eBioscience. Annexin V staining kit
and propidium iodide (eBioscience) were used for the detection of early apoptotic CD8+ T cells harvested from the
spleen and VAT. Splenocytes and VAT cells were left untreated whereas thymocytes were incubated with 1 mmol/L

Diabetes Volume 64, January 2015

dexamethasone at 37C for 4 h as a positive control.


Data were acquired on a Fortessa ow cytometer (BD
Biosciences) and analyzed with FlowJo software (Tree
Star).
CD8+ T-Cell Purication and Adoptive Transfer

Splenic CD8+ T cells from WT and Prf1null mice were


puried using negative selection (.95% purity, EasySep;
StemCell Technologies) and injected intraperitoneally
(5 3 106) in 16-week-old, HFD-fed CD8null mice.
In Vivo Incorporation of EdU

Mice were injected intraperitoneally with 4 mg/g body


weight of EdU (Invitrogen) and their VAT, spleens, BM,
and LNs collected 20 h later and processed into single-cell
suspensions. Intracellular EdU was labeled with Alexa
Fluor 488 in a Click-iT reaction (Invitrogen) according
to the manufacturers protocol and measured by ow
cytometry.
Statistical Analyses

Statistical difference between two means was determined


by two-sided unpaired Student t tests. In gure legends
involving multiple experiments from pooled animal tissue, the number of experiments is listed, followed by
the number of mice. Data are presented as means 6
SEM. Statistical signicance was set at ,0.05.
RESULTS

To determine a potential role for perforin in the regulation of inammation and insulin resistance during
diet-induced obesity, we rst assessed perforin expression
by intracellular staining in immune cells in the VAT,
spleen, and BM from mice fed either NCD or HFD. One
of the dominant cells expressing perforin was CD8+ T cells
in the VAT (Fig. 1A) and spleen (Supplementary Fig. 1A).
Compared with NCD controls, HFD-fed mice showed an
increased frequency of perforin+ CD8+ T cells in the VAT
upon anti-CD3/CD28 stimulation (Fig. 1A) and in the
spleen when left unstimulated (Supplementary Fig. 1A).
Perforin expression was also detected in NK cells (NK1.1+
CD32) and in CD4+ Foxp3+ T cells at low levels in all tissues (Supplementary Fig. 1B). In contrast, perforin was
nondetectable in CD4+ Foxp32 T, gd T, CD19+, CD11b+,
and CD11c+ cells (Supplementary Fig. 1B). No differences were detected in the frequency of NK or CD4+
Foxp3+ T cells expressing perforin between NCD- and
HFD-fed mice in the VAT, spleen, and BM (Supplementary Fig. 1B). Next, to assess the function of perforin
in diet-induced obesity, we compared Prf1null with agematched C57BL/6 WT mice fed HFD starting at 6 weeks
of age. Relative to HFD-fed WT mice, Prf1null mice receiving
the same diet had slight but signicant increases in body
weight between 6 and 18 weeks of age; although by 20
weeks of age, this difference was not statistically signicant
(Fig. 1B). To determine the location of this increase in body
weight, organs and adipose tissues from WT and Prf1null
mice were harvested and weighed after 10 and 20 weeks of
HFD feeding. Prf1null mice had increased subcutaneous fat

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Figure 1Perforin deciency results in increased body weight and adiposity. A: Frequency of perforin+ (Prf-1+) cells within the VAT CD8+ Tcell subsets in WT mice fed either an NCD or HFD for 10 weeks (n = 3 experiments, 10 mice each). Perforin expression was determined in
nonstimulated (NS) or CD3/CD28-stimulated (Stim) VAT cells. B: Body weights of WT and Prf1null mice fed HFD between 6 and 26 weeks of
age (n = 20). Organ weights of WT and Prf1null mice fed HFD (n = 6) for 10 (C) or 20 (D) weeks. E: Abdominal adipose volume of WT and
Prf1null mice fed HFD for 10 weeks determined using MRI (n = 5). F: Relative diameter of adipocytes harvested from WT and Prf1null mice fed
HFD for 10 weeks (n = 75 cells from two mice). Representative adipose tissue histology with the scale bar is set at 100 mm (G), and counting
of CLS per 1003 low power eld (LPF) (H) from WT and Prf1null mice fed HFD for 10 weeks (n = 3 mice; 1012 LPF per mouse). Expression
of lipogenic genes FABP4, CEBP-a, PPAR-g, and SREBP, relative to the housekeeping gene 18s in VAT (n = 3) (I) and liver (n = 5) (J) of WT
and Prf1null mice fed HFD for 10 weeks. K: Concentrations of triglycerides in the liver of WT and Prf1null mice fed HFD for 10 weeks (n = 4).
Data (AK) are means 6 SEM. eVAT, epididymal adipose tissue; SAT, subcutaneous adipose tissue. *P < 0.05, signicance of difference
between WT and Prf1null mice.

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and liver weights 10 weeks after initiation of HFD (Fig.


1C), but no signicant differences in organ weights were
detected at 20 weeks in HFD (Fig. 1D). Due to the increased body weight at 10 weeks of HFD, we focused on
further characterizing adipose tissue at this time. Interestingly, after 10 weeks of HFD, VAT weight trended higher in
Prf1null mice, and this correlated with increased VAT volume by MRI (Fig. 1E and Supplementary Fig. 1C) and
adipocyte size (Fig. 1F and G) in Prf1null mice, compared
with WT control mice. There was no difference in the
number of CLS (Fig. 1G and H) and expression of multiple
genes associated with lipogenesis (Fig. 1I). However, the
livers of Prf1null mice showed upregulated expression of
lipogenic genes (Fig. 1J) and higher levels of triglycerides
(Fig. 1K) compared with WT mice. Despite increased body
weight and adiposity, HFD-fed Prf1null mice had similar
food intake (Supplementary Fig. 1D, left), core body temperature (Supplementary Fig. 1D, right), VO2, VCO2, respiratory exchange ratio, locomotor activity (Supplementary
Fig. 1E), and energy expenditure adjusted for body mass
(Supplementary Fig. 1F), compared with WT controls.
After 10 and 20 weeks of HFD feeding, we performed
GTTs in Prf1null and age-matched WT control mice on the
same diet. Compared with WT mice, Prf1null mice had
higher fasting glucose (Fig. 2A) and generally worsened
glucose tolerance after 10 (Fig. 2B) and 20 weeks of HFD
feeding (Fig. 2C), even in the absence of body weight
differences at 20 weeks. In agreement with these defects
in glucose homeostasis, Prf1null mice fed HFD for 10 and
20 weeks had markedly increased fasting insulin (Fig. 2D)
and worsened insulin sensitivity upon insulin challenge
after 13 weeks of HFD, compared with WT controls
(Fig. 2E). To determine which tissues show lower insulin
sensitivity in Prf1null mice, we measured the protein levels
of total and pAkt in VAT, muscle, and liver harvested
from HFD-fed WT and Prf1null mice after an acute insulin
challenge. Compared with WT controls, all three metabolic tissues in Prf1null mice showed lower pAkt/Akt ratios
(Fig. 2F and Supplementary Fig. 1G), in agreement with
our insulin tolerance ndings. Interestingly, Prf1null mice
maintained on a standard NCD showed increased body
weight between 6 and 26 weeks of age (Fig. 3A) and
a trend for worsened fasting glucose (Fig. 3B) and worsened glucose tolerance (Fig. 3C) but similar fasting insulin
(Fig. 3D), compared with WT controls. These results suggest that perforin may also have potential effects on regulating glucose metabolism under standard caloric intake.
Proinammatory T-cell and macrophage inltration of
VAT directly contributes to obesity-related insulin resistance (3). Thus, to investigate mechanisms responsible
for the worsening in insulin resistance observed in Prf1null
mice during HFD feeding, we determined the immune cell
composition of epididymal VAT, spleens, BM, and LNs
harvested from Prf1null and WT control mice. Prf1null
mice had increased total T cells and CD8+ T-cell accumulation in VAT after 1020 weeks of HFD (Fig. 4A),
whereas no differences in T-cell subsets were detected

Diabetes Volume 64, January 2015

in spleens and BM (Supplementary Fig. 2A). Compared


with WT controls, HFD-fed Prf1null mice had increased
numbers of total T cells and CD4+ cells in the LNs, although no differences were observed in CD8+ cells (Supplementary Fig. 2A). There was no difference in the total
number of macrophages in VAT; however, Prf1null mice
showed a higher percentage of VAT M1-polarized macrophages (CD11b+ F4/80+ CD11c+) after 10 weeks of HFD
feeding, consistent with worsened metabolic parameters
in these mice (Fig. 4B). Interestingly, whereas the T-cell
subset distribution was similar in the spleen, BM, and LNs
between HFD-fed Prf1null and WT mice (Supplementary
Fig. 2B), there was a trend toward a lower CD4-to-CD8
ratio in the VAT of Prf1null mice 10 and 20 weeks after
initiation of HFD (Fig. 4C).
Since no differences in body weights were observed
after 20 weeks of HFD despite worsening metabolic
control in the Prf1null mice, we decided to further investigate immunological parameters at this time point. To
determine the relative distribution of inltrating immune
cell subsets in VAT inammation during HFD feeding,
we assessed the proportion of regulatory forkhead box
P3+ (Foxp3+) T cells in VAT, as well as the proportions
of IFN-gproducing CD4+, CD8+, and NK cells, and IL-17
producing CD4+ and gd+ T cells. No difference was
detected in the percentage of VAT-resident Foxp3+ T cells
between HFD-fed Prf1null and WT control mice (Fig. 4D).
As well, the frequency of Foxp3+ regulatory T cells in LN
and BM CD4+ T cells was similar between HFD-fed Prf1null
and WT control mice (Supplementary Fig. 2C). After HFD
feeding, VAT of Prf1null mice contained increased percentages of CD8+ and CD4+ T (Th1) cells expressing IFN-g
but no differences in the percentage of NK cells producing
IFN-g (Fig. 4E). Increased frequency of IFN-gproducing
CD8+ and CD4+ T cells was also observed in the BM (Supplementary Fig. 2D), but not in LNs (Supplementary Fig.
2E), of HFD-fed Prf1null compared with WT control mice.
Furthermore, the Th17 response was unchanged in
Prf1null mice, as the percentages of CD4+ and gd+ T cells
positive for IL-17 were similar between the two strains in
VAT (Fig. 4F), BM (Supplementary Fig. 2F), and LNs (Supplementary Fig. 2G). Overall, these ndings depict a state
of heightened Th1-mediated inammation in Prf1null mice
in the diet-induced obesity model that is localized predominantly to metabolic tissues such as VAT.
We measured the amounts of proinammatory cytokines secreted by VAT SVCs from Prf1null and WT mice
cultured for 48 h. SVCs from Prf1null produced increased
levels of IL-6, IL-12 (p70), IFN-g, GM-CSF, MCP-1, and
TNF-a compared with SVCs from HFD-fed WT controls,
consistent with increased inammation in VAT from
Prf1null mice (Fig. 5A). We also determined changes in
several serum adipokines using a multiplex screening
kit. Only leptin levels were increased after 10 weeks of
HFD feeding, consistent with increased fat mass (Supplementary Fig. 3A). However, by 20 weeks of HFD, increased amounts of some cytokines were identied.

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Figure 2Perforin regulates whole-body glucose and insulin metabolism in mice fed an HFD. A: Fasting glucose of WT and Prf1null mice
fed HFD for 10 (n = 20) or 20 weeks (n = 7). B: Blood glucose concentrations (left) and calculated areas under the curves (AUC, right) during
a GTT on WT and Prf1null mice fed HFD for 10 weeks (n = 20). C: Blood glucose concentrations (left) and calculated areas under the curves
(AUC, right) during a GTT on WT and Prf1null mice fed HFD for 20 weeks (n = 7). D: Fasting insulin of WT and Prf1null mice fed HFD for 10 (n =
20) or 20 weeks (n = 7). E: Blood glucose concentrations (left) and calculated areas under the curves (AUC, right) during an ITT with 1.5 units $ kg21
i.p. insulin in WT and Prf1null mice fed HFD for 13 weeks (n = 5). F: Western blot analysis of total and pAkt in VAT, muscle, and liver from
HFD-fed WT and Prf1null (n = 3) after injection of 1.5 units $ kg21 i.p. insulin. Data (AF) are means 6 SEM. *P < 0.05, signicance of
difference between WT and Prf1null mice.

Serum leptin levels fell in Prf1null mice at 20 weeks of


HFD feeding (Supplementary Fig. 3B), in agreement
with the trend to lower VAT weight observed at this
time point. Using more sensitive detection methods, we

were able to observe additional inammatory cytokines


increased in the serum of Prf1null mice compared with
WT controls, primarily after 20 weeks of HFD feeding
(Fig. 5B). These changes are consistent with the increased

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Figure 3Perforin regulates whole-body glucose tolerance in mice


fed NCD. A: Body weights of WT and Prf1null mice fed NCD between 6 and 26 weeks of age (n = 10). B: Fasting glucose of 14week-old WT and Prf1null mice fed NCD (n = 5). C: Blood glucose
concentrations (left) and calculated areas under the curves (AUC,
right) during a GTT on 14-week-old WT and Prf1null mice fed NCD
(n = 5). D: Fasting insulin of WT and Prf1null mice fed NCD for 20
weeks (n = 5). Data (AD) are means 6 SEM. *P < 0.05, signicance
of difference between WT and Prf1null mice.

inammation and associated worsening of insulin resistance observed in the HFD-fed Prf1null mice.
To determine whether the increased VAT T-cell
numbers in Prf1null mice, particularly in pathogenic
CD8+ T cells, were associated with altered cell turnover,
we assessed apoptotic changes and the proliferative potential of WT and Prf1null T cells after 10 weeks of HFD
feeding. Compared with WT controls, Prf1null mice had
a marked decrease in the percentage of early apoptotic
VAT CD8+ T cells but not splenic CD8+ T cells (Fig. 6A),
suggesting that Prf1null mice have a decreased ability to
regulate CD8+ T-cell numbers in VAT through a reduced
induction of apoptosis. Next, we assessed the effect of
perforin deciency on the proliferative potential of T cells.
Although CD3/CD28-stimulated WT and Prf1null splenic
CD8+ T cells proliferated similarly in vitro (Fig. 6B),
Prf1null mice showed a dramatic increase in the in vivo
proliferation of CD8+ T cells in VAT as determined by EdU
incorporation (Fig. 6C). Unrestrained CD8+ T-cell proliferation in Prf1null mice was only observed in VAT as no
differences were detected between WT and Prf1null mice in
the proliferation of CD8+ T cells in the spleen, LNs,
and BM (Supplementary Fig. 4A). Enhanced in vitro

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proliferation of VAT-resident CD8+ T cells in response


to VAT coculture was also observed in Prf1null mice, compared with WT controls (Supplementary Fig. 4B). These
ndings suggest that perforin deciency leads to CD8+
accumulation in VAT in part due to diminished apoptosis,
as well as increased proliferation. Moreover, the increased
proliferation of CD8+ T cells in Prf1null mice is unlikely
solely due to an intrinsic abnormality of the cells themselves (Fig. 6B) but is also related to the in vivo environment generated in the inamed VAT of these mice (Fig.
6C).
To assess the activation status of T cells, we determined
the expression of the degranulation (CD107a), activation
(CD44), and effector memory (CD44 and CD62L) markers
in CD4+ and CD8+ T cells in the VAT, spleens, and LNs
from HFD-fed Prf1null and WT controls. Compared with
WT controls, CD8+ T cells from Prf1null mice had increased
expression of the degranulation marker CD107a in VAT
(Fig. 6D) but not in the spleens, BM, and LNs (Supplementary Fig. 4C). No differences were detected in T-cell expression of the activation marker CD44 in VAT (Fig. 6E),
spleen, BM, and LNs (Supplementary Fig. 4D). However,
CD8+ T cells from Prf1null mice are enriched for CD44hi
CD62L2 effector memory cells in the VAT (Fig. 6F) but
not in the spleen (Supplementary Fig. 4E), suggesting
that these cells are primed and poised to respond to inammatory stimuli locally in the VAT.
To identify CD8+ T cellderived perforin in VAT as
a critical regulatory factor governing glucose sensitivity,
we investigated the pathogenicity of perforin-decient
CD8+ T cells derived from Prf1null mice compared with
their perforin-sufcient counterparts via adoptive transfer. HFD-fed CD8null mice were reconstituted intraperitoneally with 1 3 107 CD8+ T cells either from HFD-fed WT
or HFD-fed Prf1null mice. After 2 weeks, CD8+ T cells
from Prf1null and WT mice had similar reconstitution
in the VAT, which was greater than reconstitution rates
in the spleen of recipient CD8null mice (Supplementary
Fig. 5A and B). Despite the lack of differences in body
weight (Fig. 7A) after adoptive transfer, recipients of
CD8+ T cells from Prf1null mice developed worsened glucose tolerance (Fig. 7B) and hyperinsulinemia (Fig. 7C)
compared with recipients of CD8+ T cells from WT controls, suggesting that perforin derived from CD8+ T cells
is at least partially responsible for maintaining normal
glucose homeostasis. Furthermore, recipients of CD8+
T cells from Prf1null had a decreased pAkt-to-Akt protein
ratio in the VAT but not in muscle and liver (Fig. 7D and
Supplementary Fig. 5C) after insulin challenge, suggesting that the adoptive transfer promoted insulin resistance locally in the VAT. No difference in VAT lipogenesis
gene expression was detected between recipients of
CD8+ T cells from Prf1null or WT mice (Fig. 7E). We
also detected no differences in liver weights (Fig. 7F)
or triglyceride levels (Fig. 7G) between recipients of
CD8+ T cells from Prf1null and recipients of CD8+ T cells
from WT mice.

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Figure 4Perforin deciency drives the accumulation of proinammatory immune cells and release of cytokines in VAT. A: Total, CD4+, and
CD8+ T-cell and macrophage (mac) inltration in VAT of WT and Prf1null mice fed HFD for 10 and 20 weeks. B: Frequency of VAT macrophages
(CD11b+ F4/80+) with M1 phenotype (CD11c+) in WT and Prf1null mice after 10 or 20 weeks of HFD feeding. C: CD4+-to-CD8+ ratio in VAT of WT
and Prf1null mice after 10 or 20 weeks of HFD feeding. Prf1null mice tended to have a lower CD4+-to-CD8+ ratio at 10 weeks (P = 0.09) of HFD
feeding. D: Proportion of VAT CD3+ T cells from HFD-fed WT and Prf1null mice positive for CD4 and intracellular Foxp3 (regulatory T cells
[Tregs]). Intracellular staining of IFN-g in CD4+ and CD8+ T cells and NK cells (E) and IL-17 in CD4+ and gd+ T cells (F). Data (AF) show
means 6 SEM of two to three experiments (three to ve pooled mice per group). *P < 0.05, signicance of difference between WT and
Prf1null mice.

In addition to CD8+ T cells, perforin is highly expressed


in NK cells (16). Thus, we next investigated the overall
effect of global knockdown of NK cells on metabolic

parameters in mice. If a strong essential role for NK


cellderived perforin existed in control glucose homeostasis, we would expect similar phenotype metabolic

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Figure 5Perforin deciency promotes the production of proinammatory cytokines in VAT and systemically. A: Fold changes in cytokine
production by SVCs harvested from VAT from WT and Prf1null mice (n = 3 experiments, nine mice). B: Serum levels of inammatory
cytokines in WT and Prf1null mice fed HFD for 10 (left) or 20 (right) weeks (n = 4). Data (A and B) show means 6 SEM. *P < 0.05, signicance
of difference between WT and Prf1null mice.

abnormalities as observed in the Prf1null mice. Thus, we


studied the metabolic response of NKnull and age-matched
WT mice fed HFD for 12 weeks. However, contrary to
HFD-fed Prf1null mice, HFD-fed NKnull mice had decreased
body weight (Fig. 7H) and improvements in glucose tolerance (Fig. 7I) and fasting insulin (Fig. 7J) compared
with age-matched WT mice. These results highlight that
NK cellderived perforin likely does not have a nonredundant role in promoting glucose tolerance.

DISCUSSION

We have identied a novel role for perforin in regulating


inammatory changes during obesity-related insulin resistance. Previous work has identied critical roles for
perforin in promoting inammatory-mediated diseases,
including type 1 diabetes (25), cerebral malaria (26), and
viral myocarditis (27). In diet-induced obesity, a dominant
overarching effect of perforin appears to be protective
against metabolic abnormalities such as insulin resistance.

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Figure 6Impaired apoptosis and heightened local CD8+ T-cell proliferation in the VAT of Prf1null mice. A: Percentage of early apoptotic
splenic and VAT CD8+ T cells and dexamethasone-treated thymocyte control in WT and Prf1null mice fed HFD for 10 weeks determined by
Annexin V staining with propidium iodide. B: Representative histogram (left) and frequency (right) of splenic CD8+ T cells proliferating after
CD3/CD28 activation as determined by a carboxyuorescein succinimidyl ester (CFSE) proliferation assay in WT and Prf1null mice fed HFD
for 10 weeks (n = 4). C: Representative dot plots of CD8+ EdU+ VAT T cells (left) and EdU incorporation of CD4+ and CD8+ T cells in VAT
(right) of WT and Prf1null mice fed HFD for 10 weeks (n = 4) 20 h after EdU injection. D: Frequency of CD8+ T cells expressing CD107a in the
VAT of HFD-fed WT and Prf1null (n = 36) mice. E: Mean uorescence intensity (MFI) of CD44 expression by CD4+ and CD8+ T cells in the
VAT of HFD-fed WT and Prf1null (n = 5) mice. F: Frequency of effector memory (CD44hi CD62L2) CD4+ and CD8+ T cells in the VAT of HFDfed WT and Prf1null (n = 3) mice. Data (AF) are means 6 SEM. *P < 0.05, signicance of difference.

Compared with age-matched WT controls, Prf1null mice


already showed increased body weight at the start of HFD
feeding, and this difference was sustained up to 18
weeks of age. The presence of increased body weight
before HFD initiation in Prf1null mice suggests that perforin may also regulate early body size and growth independent of diet. Indeed, perforin has been shown to
be expressed in some nonimmune cells, such as chondrocytes of articular cartilage, and may be involved in tissue
remodeling (28). Consistently, our ndings indicate that
Prf1null mice fed NCD have increased body weight up to
26 weeks of age.

After 10 weeks of HFD, part of the increase in body


weight was located in liver and adipose tissues. At this
time point, Prf1null mice also had increased VAT volume
and fat cell diameter; however, no changes in expression
of genes controlling lipogenesis were detected, suggesting
that adipocyte hypertrophy is an early manifestation of
perforin deciency. In liver, on the other hand, perforin
deletion resulted in upregulated expression of lipogenic
genes and triglyceride accumulation, likely owing to
hyperinsulinemia-induced increase in uptake and de novo
synthesis of fatty acids, typical of the insulin-resistant state
(29). The increase in body weights and adiposity were not

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Figure 7CD8+ T cellderived perforin regulates glucose homeostasis. Body weights (A), GTT (B), and fasting insulin (C) in HFD-fed
CD8null recipients 2 weeks after adoptive transfer of splenic CD8+ T cells from either WT or Prf1null mice (n = 5). D: Western blot analysis of
total and pAkt in VAT, muscle, and liver from HFD-fed CD8null recipients (n = 3) after injection of 1.5 units $ kg21 i.p. insulin. E: Expression of
lipogenic genes FABP4, CEBP-a, PPAR-g, and SREBP relative to the housekeeping gene 18s in VAT of HFD-fed Ragnull recipients 5 days
after adoptive transfer of splenic CD8+ T cells from either WT or Prf1null mice (n = 3). Liver weight (F) and hepatic triglyceride levels (G) in
HFD-fed CD8null recipients 2 weeks after adoptive transfer of splenic CD8+ T cells from either WT or Prf1null mice (n = 5). Body weight (H),
intraperitoneal GTT (I), and fasting insulin (J) in age-matched WT (n = 15 for GTT and n = 10 for insulin) and NKnull (n = 5 for GTT and n = 2 for
insulin) mice fed HFD for 12 weeks. Data (AI) are means 6 SEM. *P < 0.05, signicance of difference.

accompanied by changes in food intake, metabolic rate,


and activity. The mechanisms by which perforin deciency affects early weight gain and adipocyte hypertrophy is unclear. However, there is evidence that the
immune system can directly regulate these parameters.
For example, certain cytokines, such as interleukin 15
(IL-15) can induce perforin expression in nave CD8+ T
cells (30) and NK cells (31) and can promote weight loss

independent of food intake and lymphocyte function (32).


We also cannot exclude a potential role for direct CD8+ Tcell interactions with adipocytes and perforin-mediated apoptosis of expanding adipocytes in this process.
Interestingly, the effects of perforin deciency on
adipose tissue weight gain may be transient because by
around 20 weeks of HFD feeding, Prf1null mice tended to
have decreased VAT weight, conrmed by falling levels of

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leptin on longer durations of HFD. It is possible that


these effects might be related to excessive T-cell expansion and proinammatory cytokine secretion, including
IFN-g, leading to local insulin resistance and lipolysis of
fat. Other organ weights at 20 weeks of HFD feeding were
unaffected, conrming that perforin inuences body
weight early during the course of HFD feeding.
To assess the effect of perforin on the regulation of
HFD-induced insulin resistance, we then investigated
whole-body glucose homeostasis and insulin sensitivity
of HFD-fed Prf1null and age-matched WT controls. After
HFD feeding, Prf1null mice had worsened fasting hyperglycemia and hyperinsulinemia and showed deteriorated
glucose tolerance and peripheral insulin sensitivity. Inltration of immune cells into adipose tissue is a key event
in the development of insulin resistance during HFDinduced obesity (2,3,33). In particular, IFN-gsecreting
CD8+ T cells and Th1 CD4+ T cells inltrate VAT and
enhance macrophage M1 polarization and proinammatory functions (11,13). These classically activated or M1
macrophages in VAT produce IL-1b, IL-6, and TNF-a,
factors, which can alter insulin receptor signaling in target
tissues, directly contributing to insulin resistance (3). In
the current study, all three metabolic tissues, liver, muscle, and VAT, showed reduced insulin sensitivity in
Prf1null mice.
HFD-fed Prf1null mice displayed increased CD8+ T cells
and IFN-gsecreting Th1 CD4+ T cells in VAT, associated
with VAT proinammatory cytokine production and increased M1 macrophage polarization. This augmented
presence of inammatory T cells in inamed VAT is likely
a direct consequence of perforins critical ability to maintain T-cell homeostasis in inamed tissues (17,20,21)
through downregulation of peripheral T-cell expansion
(34). Consistently, we observed a substantial decrease in
the percentages of early apoptotic CD8+ T cells and an
increase in CD8+ T-cell proliferation, degranulation, and
cytokine production in the VAT of HFD-fed Prf1null mice.
Thus, perforin-mediated control of T-cell homeostasis in
VAT may oppose previously described mechanisms of induced CD8+ T-cell proliferation in VAT, including local
activation of CD8+ T cells by the proinammatory cytokines IL-12 and IL-18 (35). Given the worsened insulin
sensitivity observed in muscle and liver of Prf1null mice,
more studies are needed to characterize the role of perforin in regulating immune cell activation in these metabolic tissues during HFD.
In mouse models of hemophagocytic lymphohistiocytosis, Prf1null mice injected with lymphocytic choriomeningitis virus develop a striking increase in CD8+ and CD4+
T-cell activation and increased antigen presentation by
rare dendritic cells (20,21). Diet-induced obesity is also
a state of increased inammation, especially in metabolic
tissues like VAT. Prf1null mice show an increase in IFN-g
production by VAT T cells as well as antigen-presenting
cell proinammatory cytokines such as IL-12(p70), IL-6,
and TNF-a. During viral clearance, such as in lymphocytic

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choriomeningitis virus models, CD8+ T cells function to


kill dendritic cell populations in a perforin-dependent
manner to limit T-cell activation (21). Collectively, our
results indicate that perforin has a regulatory role in
T-cell turnover, activation, and inammatory cytokine
production in inamed VAT during HFD feeding. A question that remains unanswered is whether there is cross
talk between CD8+ T cells and dendritic cells in VAT to
limit pathological T-cell function during obesity-related
insulin resistance.
Perforin plays a critical role in the autoimmune
destruction of insulin-producing b-cells of the pancreas,
leading to type 1 diabetes. Prf1null mice crossed onto the
nonobese diabetic (NOD) background show markedly reduced disease incidence (25,36). In these mice, islet inammation is not diminished in protected mice but
targeted b-cell killing is impaired (25,36). In diet-induced
obesity, VAT contains a limited T-cell receptor repertoire
of CD4+ and CD8+ T cells, a hallmark of antigen-specic
immunity. Moreover, HFD feeding induces VAT adipocyte
cell death, and dying fat cells become surrounded by macrophages and other immune cells, including prominent
numbers of CD8+ T cells (13). To screen for perforindependent CD8+ T-cell killing of adipocytes as a potential
mechanism of antigen-specic immunity in VAT, we
counted CLS in HFD Prf1null mice and WT controls. Interestingly, HFD Prf1null mice showed no differences in
the ability to form CLS in VAT, suggesting alternative
mechanisms of VAT adipocyte cell death, which might
include Fas (CD95)-mediated apoptosis (37) or activation
of the key proapoptotic molecule Bid (38). More recently,
mechanisms of adipocyte cell death have focused on
pyroptosis, involving the NLRP3 inammasome and
caspase-1 activation (39).
We next investigated whether CD8+ T cells or NK cells
are dominant sources of protective perforin (16). NK cells
play a crucial role in limiting viral replication through
perforin-dependent cytotoxic effects and, along with
CD8+ T cells, are dominant producers of perforin in the
immune system (40). Transfer of CD8+ cells from HFDfed Prf1null to CD8null mice results in worsened glucose
tolerance and hyperinsulinemia. These effects indicate
that the abnormal glucose and insulin metabolism in
Prf1null mice were at least partially triggered by the perforin deciency in CD8+ T cells. Consistently, a high frequency of perforin expression was detected in stimulated
VAT CD8+ T cells. Perforin was also detected in low
amounts in other immune cells in VAT, including CD4+
Foxp3+ T cells and NK cells. Thus, although we have shown
that perforin derived from CD8+ T cells plays an important
role in obesity-related VAT inammation, we cannot entirely exclude that contributions from other immune cell
sources, including CD4+ Foxp3+ T cells and NK cells,
exist to regulate inammation during insulin resistance.
To examine a potential nonredundant role for NK cell
derived perforin in limiting obesity-related insulin resistance, we examined glucose and insulin metabolism of

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HFD-fed NKnull mice, caused by deletion of the Nl3


(E4bp4) gene, a critical transcription factor involved in
NK cell development (41). HFD-fed NKnull mice had
improvements in glucose tolerance and fasting insulin. Although NK cell deciency may inuence diet-induced obesityinduced glucose intolerance and insulin sensitivity via
perforin-independent mechanisms, these results suggest
that NK cells overall are not a nonredundant source of
protective perforin in diet-induced obesityinduced insulin
resistance. Glucose homeostasis in NKnull mice is likely either compensated by perforin from other sources or overshadowed by other pathogenic functions of the NK cell in
promoting glucose intolerance. Furthermore, we acknowledge that some of the improvements observed in glucose
homeostasis in NKnull mice are likely attributable to the
reduction in body weight observed in age-matched mice.
In type 2 diabetic patients, the frequency of blood NK cells
expressing the activation markers NKG2D+ and CD107a+ is
increased in correlation with larger body mass (42). Relative
to subcutaneous depots, obese patients also have increased
frequency of IFN-gproducing CD56+ NK cells in VAT, suggesting that NK cells can inuence the inammatory tone
of human VAT (43). In agreement, our results indicate that
NK cells may have a dominant pathogenic effect on HFDinduced weight gain, glucose intolerance, and insulin resistance in mice. However, additional studies are required to
elicit the exact roles and mechanisms of NK cell function in
the low-grade inammation during diet-induced obesity.
Collectively, our work supports a model whereby
perforin limits expansion, activation, and cytokine production of pathogenic T cells in inamed VAT during DIO.
Because T-cell activation in VAT regulates macrophage
polarization and function, perforin seems to have a key
role in obesity-related chronic inammation. Our ndings
provide further evidence for a role for adaptive immune
cells in insulin resistance and highlight the potential
benet of therapies that target pathogenic T-cell homeostasis and activation in VAT.
Acknowledgments. The authors thank the Spatio-Temporal Targeting
and Amplication of Radiation Response Program (STTARR) facility, in particular
Warren Foltz, for performing the MRIs in this study.
Funding. This work was supported in part by Canadian Institutes of Health
Research grant 119414 (D.A.W.) and Canadian Diabetes Association grants OG3-12-3844 (D.A.W.) and CS-5-12-3886 (D.A.W.). X.S.R. is the recipient of a Banting & Best Diabetes Centre Fellowship (funded by Eli Lilly Canada).
Duality of Interest. No potential conicts of interest relevant to this article
were reported.
Author Contributions. X.S.R., S.T., S.W., and D.A.W. designed and
conducted experiments, analyzed data, and wrote the manuscript. H.Le., H.Lu.,
M.G., S.Y.S., S.S., C.T.L., and G.H.Y.L. performed experiments. H.T., T.W.M., and
M.W. reviewed the manuscript. D.A.W. is the guarantor of this work and, as such,
had full access to all the data in the study and takes responsibility for the integrity
of the data and the accuracy of the data analysis.
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