Interleukin-7 regulates adipose tissue mass and insulin sensitivity in high-fat diet-fed mice through lymphocyte-dependent and independent mechanisms.

Abstract

Although interleukin (IL)-7 is mostly known as a key regulator of lymphocyte homeostasis, we recently demonstrated that it also contributes to body weight regulation through a hypothalamic control. Previous studies have shown that IL-7 is produced by the human obese white adipose tissue (WAT) yet its potential role on WAT development and function in obesity remains unknown. Here, we first show that transgenic mice overexpressing IL-7 have reduced adipose tissue mass associated with glucose and insulin resistance. Moreover, in the high-fat diet (HFD)-induced obesity model, a single administration of IL-7 to C57BL/6 mice is sufficient to prevent HFD-induced WAT mass increase and glucose intolerance. This metabolic protective effect is accompanied by a significant decreased inflammation in WAT. In lymphocyte-deficient HFD-fed SCID mice, IL-7 injection still protects from WAT mass gain. However, IL-7-triggered resistance against WAT inflammation and glucose intolerance is lost in SCID mice. These results suggest that IL-7 regulates adipose tissue mass through a lymphocyte-independent mechanism while its protective role on glucose homeostasis would be relayed by immune cells that participate to WAT inflammation. Our observations establish a key role for IL-7 in the complex mechanisms by which immune mediators modulate metabolic functions.

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Interleukin-7 Regulates Adipose Tissue Mass and Insulin
Sensitivity in High-Fat Diet-Fed Mice through
Lymphocyte-Dependent and Independent Mechanisms
Ste ´phanie Lucas1, Solenne Taront2, Christophe Magnan3, Laurence Fauconnier4, Myriam Delacre1,
Laurence Macia1, Anne Delanoye1, Claudie Verwaerde1, Corentin Spriet5, Pasquine Saule4,
Gautier Goormachtigh4, Laurent He ´liot5, Alain Ktorza3, Jamileh Movassat6, Renata Polakowska7,
Claude Auriault4, Odile Poulain-Godefroy2, James Di Santo8,9, Philippe Froguel2, Isabelle Wolowczuk2*
1Laboratory of Neuroimmunoendocrinology, Institut Fe ´de ´ratif de Recherche (IFR) 142, Univ Lille Nord de France, Institut Pasteur, Lille, France, 2Laboratory of Genomics
and Metabolic Diseases, Centre National de la Recherche Scientifique (CNRS) Unite ´ Mixte de Recherche (UMR) 8199, Univ Lille Nord de France, Institut Pasteur, Lille, France,
3Laboratory of Physiopathology of Nutrition, CNRS UMR 7059, Paris 7 Univ, Paris, France, 4Laboratory of Cellular Immunopathology of Infectious Diseases, CNRS UMR
8527, Institut de Biologie/Institut Pasteur, Lille, France, 5Interdisciplinary Research Institute, CNRS Unite ´ de Service et de Recherche (USR) 3078, Univ Lille Nord de France,
Villeneuve d’Ascq, France, 6Laboratory of Biology and Pathology of the Endocrine Pancreas, CNRS Equipe d’Accueil Conventionne ´e (EAC) 4413, Paris 7 Univ, Paris, France,
7Inserm U837 and Jean-Pierre Aubert Research Center, Centre Hospitalier Universitaire (CHU), Univ Lille Nord de France, Lille, France, 8Innate Immunity Unit, Institut
Pasteur, Paris, France, 9Inserm U668, Paris, France
Abstract
Although interleukin (IL)-7 is mostly known as a key regulator of lymphocyte homeostasis, we recently demonstrated that it
also contributes to body weight regulation through a hypothalamic control. Previous studies have shown that IL-7 is
produced by the human obese white adipose tissue (WAT) yet its potential role on WAT development and function in
obesity remains unknown. Here, we first show that transgenic mice overexpressing IL-7 have reduced adipose tissue mass
associated with glucose and insulin resistance. Moreover, in the high-fat diet (HFD)-induced obesity model, a single
administration of IL-7 to C57BL/6 mice is sufficient to prevent HFD-induced WAT mass increase and glucose intolerance. This
metabolic protective effect is accompanied by a significant decreased inflammation in WAT. In lymphocyte-deficient HFD-
fed SCID mice, IL-7 injection still protects from WAT mass gain. However, IL-7-triggered resistance against WAT inflammation
and glucose intolerance is lost in SCID mice. These results suggest that IL-7 regulates adipose tissue mass through a
lymphocyte-independent mechanism while its protective role on glucose homeostasis would be relayed by immune cells
that participate to WAT inflammation. Our observations establish a key role for IL-7 in the complex mechanisms by which
immune mediators modulate metabolic functions.
Citation: Lucas S, Taront S, Magnan C, Fauconnier L, Delacre M, et al. (2012) Interleukin-7 Regulates Adipose Tissue Mass and Insulin Sensitivity in High-Fat Diet-
Fed Mice through Lymphocyte-Dependent and Independent Mechanisms. PLoS ONE 7(6): e40351. doi:10.1371/journal.pone.0040351
Editor: Orian S. Shirihai, Boston University, United States of America
Received June 22, 2011; Accepted June 7, 2012; Published June 29, 2012
Copyright: ? 2012 Lucas et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The work was supported by the Pasteur Institute in Lille, the Institut Fe ´de ´ratif de Recherche 142 and the Centre National de la Recherche Scientifique.
S. L. was supported by a post-doctoral grant from the French Re ´gion Nord/Pas-de-Calais. 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 no conflicting financial, personal, or professional interests.
* E-mail: Isabelle.Wolowczuk@good.ibl.fr
Introduction
Paradoxically, the disproportionate lack of white adipose tissue
(WAT) in lipodystrophy and the excessive gain of WAT in obesity
are both frequently associated with severe insulin resistance [1],
[2]. This highlights the central role for adipose tissue as an
essential organ for proper metabolic regulation.
Dysfunctional adipose tissue could participate in metabolic
disease development notably through abnormal secretion of
adipokines which have been shown to play important roles in
insulin action, energy balance and inflammation [3]. Adipokines
are considered as key players in the orchestration of the adaptive
metabolic changes which occur at different cell and tissue levels.
Indeed, as exemplified by leptin, adipokines can exert endocrine
action on hypothalamic cells involved in food intake and/or
energy expenditure [4]. Other factors, like IL-6 or TNFa, regulate
adipose tissue mass and function, and modulate insulin sensitivity
[5], [6]. Therefore, altered adipokine secretion associated to either
excess or markedly reduced WAT mass may worsen the
progression of the metabolic disease and promote insulin
resistance, contributing to the development of type 2 diabetes
[1]. Recently, immune-derived cytokines have also been shown to
regulate WAT mass and function. Mice deficient in IL-18 show
increased WAT mass and insulin resistance [7], while IL-15
overexpressing mice present reduced WAT mass [8]. IL-7 has
recently been identified as a new adipokine whose expression and
secretion are increased in the obese human adipose tissue [9].
Whether this endogenous production of IL-7 has any physiological
function in adipose tissue and metabolism is still unknown.
IL-7 is a constitutively secreted cytokine primarily produced in
bone marrow and peripheral lymphoid organs [10]. IL-7 is
critically required for lymphocyte development and homeostasis
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[11], as first appreciated from the severe lymphopenia observed in
IL-7 knockout and IL-7R knockout mice [12], [13], and later on in
comparable immune deficiencies in humans who lack either IL-7
or components of its receptor [14], [15]. The key role of IL-7 in
lymphocyte homeostasis was shown to rely on its control of basal
lymphocyte glucose metabolism [16], maintaining high glucose
uptake and expression of GLUT1 therefore allowing adequate
glycolytic flux [17], [18], [19]. Beside its primary function in the
regulation of the activation, growth and survival of lymphoid cells,
it has been suggested that IL-7 can also act on non-lymphoid cells.
Indeed, IL-7 has been shown to induce the production of pro-
inflammatory IL-1, IL-6, IL-8 and TNFa by monocytes [20], to
Figure 1. Reduced white adipose tissue mass and insulin sensitivity in IL-7 overexpressing mice. (A) Perigonadal WAT mass in transgenic
mice and control animals. Results are expressed as mean 6 SEM of 7 WT mice (white bars) and 8 Tg IL-7 mice (black bars). (B) Adipocyte diameter
distribution of 3 WT (white circles) and 3 Tg IL-7 (black circles). (C) Ratio of total triglycerides to total DNA (in pg/ng). Results are expressed as mean 6
SEM of 3 WT mice (white bars) and 5 Tg IL-7 mice (black bars). (D) Glucose tolerance test: Blood samples for glucose level determination were taken
from each individual animal. Results are expressed as mean 6 SEM of 5 WT (white circles) and 5 Tg IL-7 (black circles). (E) Insulin tolerance test:
Glycaemia were individually measured at the indicated times. Data are presented as mean percentage of basal glycemia (t=0 min) from average
value 6 SEM from 5 WT (white circles) and 5 Tg IL-7 (black circles). (F & G) In vivo hyperinsulinaemic-euglycaemic clamps were performed using
[3-3H] glucose and 2-deoxy-D-[1-14C] glucose (2DG) for the estimation of whole body glucose fluxes (F) and tissue glucose uptake (G), respectively.
The uptake of glucose was determined in one red fiber-type of muscle (Soleus, S), one white fiber-type of muscle (Tibialis anterior, TA), inguinal SCAT,
perigonadal WAT, and brown adipose tissue (BAT). Results are expressed as the mean 6 SEM of 5 WT (white bars) and 5 Tg IL-7 (black bars).
Statistically significant differences between the groups are indicated as*p,0.05 and**p,0.01.
doi:10.1371/journal.pone.0040351.g001
IL-7 and White Adipose Tissue in Mice
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promote eosinophil and hippocampal neuron survival [21], [22]
and to regulate osteoclast formation [23]. We recently extended
IL-7 pleiotropic effects to energy balance regulation; indeed we
reported that IL-7 directly targets the hypothalamic arcuate
nucleus and protects mice from the development of monosodium
glutamate-induced obesity [24].
Here we further assessed the effects of IL-7 on metabolism,
focusing on the white adipose tissue. First, we characterized
glucose homeostasis and WAT development in IL-7 transgenic
mice (referred to as Tg IL-7, [25]). The reduced adipose tissue
mass and the impaired glucose homeostasis of IL-7 overexpressing
mice suggested that IL-7 might be a key factor in adipose tissue
biology, as it was suspected from its increased expression in the
obese human tissue [9]. Then, we aimed to analyze whether IL-7
metabolic effects revealed by its constitutive overexpression in
mice could be extended to a more physiopathological context. We
thus analyzed the consequences of acute IL-7 administration on fat
mass accumulation, glucose intolerance and WAT inflammation
in the high-fat diet (HFD)-induced obesity model in C57BL/6
mice. Finally, to distinguish between the lymphocyte-dependent
and -independent metabolic action mechanisms of IL-7, the same
parameters were analyzed in IL-7-treated HFD-fed severe
combined immunodeficient (SCID) animals.
Results
White Adipose Tissue Mass and Sensitivity to Insulin are
Impaired in IL-7 Overexpressing Mice
As a first step to study whether IL-7 might impact on the
adipose tissue, we used IL-7 transgenic mice, in which overex-
pression is driven by the human keratin-14 (K14) promoter (Tg
IL-7; [25]). IL-7 transgenic mice were fertile and healthy, slightly
leaner than littermate wild-type controls (WT) (22.861.4 g and
2562.9 g, respectively; p value=0.12) yet presented similar BMI
(0.28 g/cm2) and comparable consumption of standard diet.
Compared with WT animals, standard diet-fed Tg IL-7 mice
showed a 50% decrease in perigonadal WAT mass (Figure 1A),
whereas brown adipose tissue, liver and spleen showed no
macroscopical differences (data not shown). Histological exami-
nation of WAT sections showed a higher density of smaller
adipocytes in Tg IL-7 mice (data not shown). Indeed, as shown in
Figure 1B, morphometric analysis confirmed that Tg IL-7
adipocytes were smaller than WT adipocytes (64% adipocytes
with a diameter ,30 mm for Tg IL-7 mice vs 36% for WT mice;
p,0.05*). In addition, adipocyte number per microscopical field
was ,3.5-fold higher in Tg than in WT mice (1,5086306 cells/
mm2for Tg IL-7 vs 426635 cells/mm2for WT, respectively; p
value=0.028*). The decrease in adipocyte size was also reflected
by lower triglyceride content in the transgenic adipose tissue
(Figure 1C). The DNA content per mg of tissue was higher for
transgenic WAT compared to WT tissue (0.3460.042 vs
0.2160.025 mg/mg, respectively; p,0.05*). Since the mass of the
tissue was reduced in transgenic animals compared to WT controls
(Figure 1A), the resulting DNA content per fat pad is comparable
between transgenic and control mice. Therefore, the decreased
adiposity in Tg IL-7 mice is primarily due to decreased adipocyte
size with no change in cellularity.
IL-7 overexpression was associated with increased levels of
circulating free fatty acids (FFAs) and decreased levels of
circulating triglycerides with no change in basal insulinemia and
glycemia (Supporting Information, Table S1). Plasma FFAs
concentrations are primarily governed by lipolysis in adipocytes
and elevated FFAs levels are thought to restrict glucose utilization
and induce insulin resistance. Indeed, in vivo glucose clearance was
severely delayed in Tg IL-7 animals compared with WT mice,
indicating glucose intolerance (Figure 1D). In addition, transgenic
mice are insulin resistant since their hypoglycaemic response to
insulin was lower compared to WT mice (Figure 1E). Hyperin-
sulinaemic-euglycaemic clamp experiments in both IL-7 overex-
pressing and control animals confirmed IL-7-induced insulin
resistance: the glucose infusion rate needed to maintain euglyce-
mia in the presence of a constant infusion of insulin was 2-fold
lower in Tg IL-7 mice compared to control mice (13.760.5 mg/
min/kg and 20.560.8 mg/min/kg for Tg IL-7 and WT,
respectively; p value=0.03*) (Figure 1F). In contrast, both groups
exhibited similar hepatic glucose output (8.460.6 mg/min/kg and
8.761 mg/min/kg for Tg IL-7 and WT, respectively) suggesting
that liver insulin sensitivity is not altered in Tg IL-7 mice. Then,
we assessed the insulin-stimulated uptake of 2-deoxy-D glucose
(2DG; a non-metabolized glucose analogue) in skeletal muscle
(soleus, a predominantly red fiber-type muscle, and tibialis anterior, a
predominantly white fiber-type muscle) and in brown and white
adipose tissues (subcutaneous and perigonadal fat, respectively;
SCAT and WAT). Although 2DG uptake was similar in muscle
and brown fat tissue in both groups, the 2DG uptake in WAT was
markedly lower in Tg IL-7 mice compared to WT mice
(Figure 1G). IL-7 overexpression appeared to affect more
perigonadal WAT than inguinal SCAT (respective decreases in
2DG uptake; 80% vs 60%), yet the expression levels of IL-7 have
not been compared between WAT and SCAT. Altogether, these
data support that the glucose uptake is severely perturbed in the
two main white fat depots in IL-7 overexpressing mice.
IL-7 Receptor is Mostly Expressed in the Stromal Vascular
Fraction of C57BL/6 WAT
IL-7 activates target cells through its heterodimeric receptor (IL-
7R; containing CD127 (IL-7Ra chain) coupled with CD132
(common c chain: cc)) [26]. To further characterize the expression
level of IL-7 and IL-7R chains in insulin-sensitive tissues, real-time
quantitative PCR analysis was performed on the adipocyte
fraction and the stromal vascular fraction (SVF) isolated from
the perigonadal WAT of C57BL/6 mice, and compared to the
expression in skeletal muscles, liver and hypothalamus. It showed
that IL-7 and IL-7Ra were expressed at higher levels in both
WAT cell fractions than in the other tested metabolic tissues
(Figure 2). Moreover, whereas IL-7Ra mRNA was mostly found
expressed in the SVF which contains immune, endothelial and
progenitor cells [27], the cc chain was well-detected in both
fractions, yet at a higher level in the SVF (Figure 2A). IL-7 mRNA
was equally expressed in the SVF fraction and the adipocyte
fraction (Figure 2B).
These data suggest that WAT could be both a source and a
target of IL-7 and strengthen that IL-7 could participate in the
regulation of WAT functionality in metabolic diseases.
A Single Administration of IL-7 Protects C57BL/6 Mice
from High-fat Diet-induced WAT Mass Increase and
Glucose Intolerance
At that stage of our work, we hypothesized that IL-7 might
impact on WAT mass increase and metabolic dysfunctions
associated with the development of obesity, such as insulin and
glucose intolerance. A protective role of IL-7 against obesity and
insulin resistance has been suggested in IL-7-treated mice after
pharmacological destruction of hypothalamus by monosodium
glutamate [24] or gold thioglucose (IW, unpublished observation).
Thus we assessed the consequences of a single subcutaneous
injection of recombinant murine IL-7 in the more pathophysio-
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logical model of high-fat diet (HFD)-induced obesity. A unique
administration of IL-7, at 2 weeks of diet, protected mice from
HFD-induced body weight gain (Figure 3A). Food intake was not
altered by IL-7 treatment (data not shown). A glucose tolerance
test on overnight-fasted animals showed that, in contrast to PBS-
treated HFD-fed (PBS/HFD) control mice, IL-7/HFD mice were
not glucose intolerant after 5 weeks (Figure 3B) nor after 12 weeks
(data not shown) of HFD feeding. This protective effect of IL-7 on
HFD-induced glucose intolerance was also effective in condition of
shorter duration of food removal (i.e. 6 hours over morning instead
of overnight starvation period; data not shown). However, IL-7
injection had no effect on glycemia control in standard diet (SD)-
fed animals. Moreover, the acute and early IL-7 injection
protected mice from the HFD-induced increase of perigonadal
WAT mass and, although less significantly, inguinal SCAT mass.
Morphology of the brown adipose tissue (BAT) did not differ
between the different experimental groups and its mass was not
increased by the HFD feeding in the IL-7-treated mice (Figure 3C).
Protective Effects of IL-7 Against HFD-induced Obesity
are Associated with Decreased WAT Inflammation
One of the key features linking obesity and the development of
insulin resistance is the chronic, low-grade inflammatory state of
the WAT [28], [29] that is characterized by macrophage
accumulation [30], [31] and activation [32], [33] which results
in the secretion of various cytokines that eventually alter tissue
insulin responsiveness. This critical process is preceded by the
infiltration of lymphocytes which promote the recruitment and the
activation of macrophages [34], [35]. Therefore, we compared the
expression level of typical markers of immune cells and
inflammatory mediators in the perigonadal WAT of mice from
the different experimental groups of mice.
HFD feeding resulted in increased expression of macrophage
markers Emr1 and CD68 (Figure 4A), TNFa, monocyte
chemotactic protein-1 (MCP-1) and the activated-macrophage
marker Nos2 (Figure 4B). The expression of all these markers was
reduced in the WAT of IL-7-treated mice. The expression of the
T-lymphocyte marker CD3e or of the T-cell subset markers CD4
and CD8 was not altered in the WAT of PBS/HFD and IL-7/
HFD mice after 16 weeks of diet (data not shown). The expression
of the B-cell markers CD19 and CD20 were markedly decreased
in the WAT of IL-7/HFD mice compared to PBS/HFD mice
(Figure 4A). It is noticeable that in SD feeding conditions, IL-7
injection led to a significant decrease in CD68 and CD20 mRNA
expression (markers specific for, respectively, macrophages and B-
lymphocytes).
A Single Administration of IL-7 Protects Immunodeficient
SCID Mice from High-fat Diet-induced WAT Mass Increase
but Not Against the Development of Glucose Intolerance
and WAT Inflammation
To determine if the metabolic effects of IL-7 could be ascribed
to any impact of the interleukin on lymphocytes, we then tested
the consequences of acute IL-7 injection at the beginning of a
HFD study in T- and B-cell deficient SCID mice.
As shown by Figure 5A, HFD feeding did not alter body weight
in SCID mice, yet it led to a 2.5-fold increase in perigonadal fat
mass. The administration of IL-7 in HFD-fed SCID mice
significantly impaired this increase in WAT mass. The different
experimental groups were similar regarding BAT mass and
morphology.
Figure 2. The IL-7 receptor is mostly expressed in the stromal vascular fraction of C57BL/6 adipose tissue. Expression levels of the
mRNA of the IL-7 receptor subunits (IL-7Ra; black bars, cc; grey bars) (A) and IL-7 (B) in different insulin sensitive tissues and in adipose tissue fractions
isolated from three C57BL/6 male mice, using quantitative PCR. The insulin receptor mRNA levels are shown as control (spotted bars). Thymus was
used as a positive control for the expression of IL-7 and IL-7R.
doi:10.1371/journal.pone.0040351.g002
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In contrast, IL-7 injection did not protect anymore against
HFD-induced glucose intolerance in lymphocyte-deficient SCID
mice (Figure 5B). The same was true 9 weeks after injection (data
not shown). Nevertheless, in SD feeding condition, IL-7-treated
mice exhibited a transitory decrease in glucose clearance 4 weeks
after the administration of IL-7, which was markedly improved
later (data not shown). In addition, IL-7 had no effect on the
perigonadal WAT expression of macrophage, as well as inflam-
matory, specific markers in lymphocyte-deficient HFD-fed SCID
mice (Figure 5C).
Discussion
Interleukin-7 (IL-7) is an immune cytokine that is critical for
lymphocyte development and homeostasis [10], [11], [12], [13],
[14], [15], [16], [17], [18], [19], recently found to be oversecreted
by the visceral adipose tissue in obese subjects [9]. Yet, its potential
role in white adipose tissue (WAT) development and metabolic
functions has never been investigated. Here we describe an
unexpected and dual role for IL-7 in WAT mass and insulin
sensitivity control. In mice, constitutive IL-7 overexpression as well
as IL-7 acute injection during high-fat diet (HFD) feeding reduced
white adipose tissue mass. However, we show that the conse-
quences of IL-7-induced WAT mass decrease on glucose
homeostasis differ in the two models. Indeed, constitutive IL-7
over-expression leads to glucose intolerance and insulin resistance,
traits that are commonly associated with lipodystrophy in both
animals and humans [36]. In IL-7-treated HFD-fed wild-type
animals, the reduced WAT mass is expectedly associated to
protection against glucose intolerance that results from a decreased
inflammation in WAT. Importantly, IL-7 protection against
nutrient excess-induced glucose intolerance is lost in lymphocyte-
deficient animals, suggesting that this protective effect of IL-7
evolves from a lymphocyte-dependent step of the inflammatory
process that develops in the obese tissue.
Even though IL-7 constitutive overexpression is not comparable
to IL-7 acute administration during nutritional excess both animal
models present reduced adipose tissue mass. In addition, each
model brings important clues onto the IL-7 impact in the control
of glucose homeostasis, which may differ according to the
developmental stage, nutritional status or sex. Regarding the
latter, it is conceivable that gender difference between IL-7
overexpressing mice (females) and IL-7-injected animals (males)
may participate to the reported effects on glucose homeostasis.
Indeed, a sexual dimorphism has been described in mice that
affected adipose tissue accumulation and gene expression as well as
insulin sensitivity [37], [38], [39] and sex hormones such as
estrogens have been convincingly reported to alter adipocyte
Figure 3. A single injection of IL-7 protects high-fat diet-fed C576BL/6 mice from obesity and glucose intolerance. (A) Body weight at
16 weeks of diet feeding (white bars; PBS/SD, hatched bars; IL-7/SD, grey bars; PBS/HFD, black bars; IL-7/HFD); (B) An i.p. glucose tolerance test was
performed after 5 weeks of diet feeding (grey squares-continuous line; PBS/SD, grey triangles-dotted line; IL-7/SD, black squares-continuous line; PBS/
HFD, black triangles-dotted line; IL-7/HFD); (C) Masses of perigonadal WAT, inguinal SCAT and interscapular BAT at 16 weeks of diet feeding (white
bars; PBS/SD, hatched bars; IL-7/SD, grey bars; PBS/HFD, black bars; IL-7/HFD); Data are expressed as means 6 SEM of 7 to 9 mice per group.#p,0.05,
##p,0.01,###p,0.001, HFD vs SD within the same injection group;*p,0.05,**p,0.01, IL-7 injection vs PBS injection in the same diet group.
doi:10.1371/journal.pone.0040351.g003
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biology and to impact on obesity-related co-morbidities, such as
insulin resistance [40], [41], [42]. Nevertheless, even if estrous
cycle, estrogen levels, or uterine weights have not been recorded in
IL-7 transgenic female mice, no difference in food intake and/or
meal size (all parameters that are controlled by estrogens, [43],
[44], [45]) could be evidenced in these animals, compared to the
corresponding control mice.
Our study mainly focused on the analysis of the perigonadal
WAT, according to the well-recognized importance of this specific
fat depot regarding lipolysis rate [46], preadipocyte/adipocyte cell
dynamics [47] and inflammatory profile [48]. However, further
investigation is definitely warranted to test whether regional (i.e.
subcutaneous versus visceral) differences exist regarding adipose
tissue responsiveness to IL-7.
IL-7 and IL-7Ra are highly expressed in the murine WAT and
we show that, within the tissue, cells of the stromal vascular
fraction (SVF) are likely the main IL-7 responsive cells, and not
mature adipocytes. Indeed, while IL-7 mRNA is equally expressed
in SVF cells and in mature adipocytes, as initially reported for the
human adipose tissue [9], the specific IL-7Ra chain is mainly
expressed in the SVF whilst less detectable in mature adipocytes.
Among the various cell-types that composed the SVF [49], [50],
adipocyte progenitors could be IL-7 responder cells. Indeed, in the
transgenic mouse model, IL-7 is overexpressed in the WAT (data
not shown), and this sustained exposure of the tissue to IL-7
impacts on adipocyte cell-size. This suggests that IL-7 overex-
pression impairs the adipocyte differentiation process, thereby
contributing to the development of insulin resistance in lipodys-
trophy-like transgenic animals. To support this hypothesis our
preliminary data show that the expression of genes related to
adipogenesis (such as PPARc, C/EBPa and SREBP-1c) is
decreased in the WAT of transgenic mice (data not shown). This
is consistent with the report of decreased adipocyte differentiation
in the adipose tissue of patients with HIV-related lipodystrophy, in
association with insulin resistance [51]. It has been reported that
IL-7 is produced by adipocyte progenitors and that its expression
decreases during adipogenesis [9]. One hypothesis could be that
the constitutive overexpression of IL-7 might have favoured the
emergence and/or maintenance of a population of immature
adipose cells with altered lipid storage and release capacities,
explaining both the reduced adipocyte cell-size and the altered
plasma lipid profile of Tg IL-7 mice.
Beside adipocyte progenitors, the SVF also contains immune
cells that increasingly appear to play a key role in the induction
and progression of the adipose tissue inflammatory state which
contributes to the onset of insulin resistance that occurs in obesity
Figure 4. A single injection of IL-7 reduces WAT inflammation during C57BL/6 HFD feeding. Real-time quantitative PCR analysis of the
perigonadal white adipose tissue from C57BL/6 mice after a unique injection with PBS or IL-7, 16 weeks after SD or HFD feeding. (A) Expression levels
of macrophages (Emr1/F4-80, CD68) and B-cells (CD19, CD20) markers; and (B) expression levels of inflammatory markers (TNFa, MCP-1 and Nos2).
Data are expressed as means 6 SEM of 7 to 9 mice per group (white bars; PBS/SD, hatched bars; IL-7/SD, grey bars; PBS/HFD, black bars; IL-7/HFD).
#p,0.05,##p,0.01,###p,0.001, HFD vs SD within the same injection group;*p,0.05,**p,0.01,***p,0.001, IL-7 injection vs PBS injection within
the same diet group.
doi:10.1371/journal.pone.0040351.g004
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[30], [31], [32], [33], [34], [35], [52], [53], [54]. We confirm
that high-fat diet is associated with increased expression of
macrophage specific markers in the adipose tissue and show that
a single and early injection of IL-7 is sufficient to durably impede
this HFD-induced effect, consequently protecting mice against
glucose intolerance. The fact that acute IL-7 injection does not
impact on any of these parameters in SD-fed mice supports that
a pro-inflammatory and/or energy imbalance, such as the one
triggered by obesity, is necessary for IL-7 to fully exert its
regulatory effect.
The results we obtained in the immunodeficient HFD-SCID
model bring some further clues on how IL-7 protects towards
glucose intolerance in a context of obesity. Indeed, if IL-7-treated
HFD-fed SCID mice still present a significant reduction in WAT
mass, the IL-7 injection was inefficient to maintain glucose
homeostasis and to prevent macrophage recruitment and activa-
tion, as reflected by the analysis of macrophage specific markers.
This leads to conclude that, whereas the effects of IL-7 on WAT
mass are lymphocyte-independent, its protective role against
HFD-induced glucose intolerance requires the presence of
functional lymphocytes. Since we show that IL-7 decreases the
expression of B-lymphocyte specific surface markers in the WAT
of HFD-fed immunocompetent mice, this cell-type might be
proposed as an immune relay of the IL-7 effects on WAT
inflammation. In contrast to macrophages and T-cells, the role of
B-lymphocytes in the development of inflammation and insulin
resistance in WAT is largely unknown, although these cells are
recruited to adipose tissue shortly after the initiation of HFD (e.g.
by 4 weeks) [55] and were recently reported to promote insulin
resistance in mice [56]. Therefore, one could propose that IL-7, by
decreasing the recruitment of these pathogenic B-cells in the
adipose tissue, will limit macrophage recruitment/activation and
thus, inflammation, consequently leading to the observed protec-
tive effect against glucose intolerance. In lymphocyte-deficient
SCID mice, this IL-7-mediated effect on B-cells is lacking, leading
to uncontrolled macrophage infiltration and loss of IL-7 protective
effect against glucose intolerance.
Collectively, our findings indicate that the immune cytokine IL-
7 could participate to the development of metabolic diseases
through the modulation of the white adipose tissue. More
specifically, we show here that IL-7 can play a dual role in the
regulation of WAT mass and function. First, IL-7 regulates WAT
mass in a lymphocyte-independent manner. How alteration of the
adipogenesis, lipogenesis or lipolysis processes and/or increased
oxidative capacity of the WAT concur to IL-7 effect on WAT mass
remains to be established. Secondly, in condition of nutrient excess
such as high-fat diet feeding, IL-7 protects durably against
inflammation, and thus glucose intolerance, possibly by regulating
the recruitment of immune cells - the canonical IL-7 responsive
cell-types [10], [11], [12], [13], [14], [15], [16] - to the inflamed
adipose tissue.
Several clinical trials using IL-7 are currently underway in
cancer, HIV, HBV and HCV infections to remediate disease-
associated immune deficits and our results in mice suggest that
increasing IL-7 levels may also affect fat mass development and
insulin sensitivity in the IL-7-treated patients. Moreover, our
Figure 5. A single injection of IL-7 protects SCID mice from HFD-induced obesity but not from glucose intolerance and WAT
inflammation. (A) Body weight and perigonadal WAT and BAT masses of SCID male mice fed during 15 weeks with SD or HFD (white bars; PBS/SD,
hatched bars; IL-7/SD, grey bars; PBS/HFD, black bars; IL-7/HFD); (B) Intraperitoneal glucose tolerance test at 6 weeks of diet feeding (white bars; PBS/
SD, hatched bars; IL-7/SD, grey bars; PBS/HFD, black bars; IL-7/HFD); (C) Expression levels of macrophage (Emr1/F4-80, CD68) and inflammation
(TNFa, MCP-1, Nos2) markers in perigonadal WAT, after 15 weeks of diet feeding, using real-time quantitative PCR (white bars; PBS/SD, hatched bars;
IL-7/SD, grey bars; PBS/HFD, black bars; IL-7/HFD). Data are expressed as means 6 SEM of 8 PBS/SD mice, 6 PBS/IL-7 mice, 3 PBS/HFD mice and 9 IL-7/
PBS mice.#p,0.05,##p,0.01,###p,0.001, HFD vs SD within the same injection group;*p,0.05,***p,0.001, IL-7 injection vs PBS injection in the
same diet group.
doi:10.1371/journal.pone.0040351.g005
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results reinforce the contribution of immune cells in insulin
resistance in which IL-7 may play some important role. It shall
help developing novel immune-based diagnostic and therapeutic
modalities for managing white adipose tissue disorders such as
obesity, lipodystrophy and insulin-resistance.
Materials and Methods
Animals
Heterozygous IL-7 transgenic mice on a C57BL/6J background
expressed murine IL-7 under the control of the human keratin
K14 promoter (Tg IL-7; [25]). Female wild-type littermate mice
(WT) were used as controls. Body mass index was calculated for 8
week-old Tg IL-7 and WT mice. For acute IL-7 administration,
we used C57BL/6J male mice (Janvier Laboratory, Le Genest-St-
Isle, France) or C.B-17 SCID (Pasteur Institute, Lille, France) male
mice.
Diet-induced Obesity and Acute IL-7 Injection
Seven-week-old C57BL/6J and SCID male mice were fed
either with a standard diet (AO4, SAFE, Augy, France) or with a
high-fat diet (HFD 35.8% (wt/wt) fat from lard, Purified Diet
230HF, SAFE) containing, respectively, 5% or 60.6% in kcal of
fat.
After 2 weeks of acclimatization to the diets, C57BL/6J and
SCID mice were once subcutaneously injected with PBS or with
recombinant murine IL-7 (0.3 mg/mouse, Peprotech, Neuilly-sur-
Seine, France). Food intake and body weight were measured
weekly, up to 16 weeks of diet.
Hyperinsulinaemic-euglycaemic Clamp Experiment
Food was removed 5–6 hours before the in vivo protocol on
catheterized animals. Tg IL-7 and WT mice underwent a
120 min-hyperinsulinaemic-euglycaemic clamp study with a
prime continuous infusion of human insulin (Novonordisk,
Copenhagen, Denmark) at a rate of 15 pmol/kg/min to raise
plasma insulinaemia to ,800 pM. Glucose (20%) was infused at
variable rates to maintain euglycaemia. Insulin-stimulated whole-
body glucose flux was estimated using a prime continuous infusion
of HPLC-purified [3-3H] glucose (Amersham PerkinElmer,
Waltham, MA, USA), throughout the clamp procedure. To
estimate insulin-stimulated glucose-transport activity and metab-
olism in skeletal muscles, brown and white adipose tissues
(subcutaneous (i.e. inguinal), and intraabdominal (i.e. perigonadal)),
2-deoxy-D[1-14C]-glucose (2DG; Amersham PerkinElmer) was
administered as a bolus (376104Bq) 45 min before the end of
clamp procedure. In separate experiments, the basal glucose
turnover rates were measured by continuous infusing of [3-3H]
glucose (Amersham PerkinElmer) (746103Bq/min) for 120 min,
and plasma [3H] glucose concentration was determined every 10-
min during the last 30 min.
Intraperitoneal Glucose and Insulin Tolerance Tests
Glucose tolerance tests (GTT) were performed on overnight
fasted mice injected i.p. with D-glucose (2 g/kg body weight,
Sigma-Aldrich) [57], [58]. Glucose levels were measured by tail-tip
bleeding with an automatic glucometer (ACCU-CHEK Performa,
Roche) immediately before and 15, 30, 60, 180 and 360 min after
the glucose injection. For determination of insulin sensitivity (ITT),
mice were fasted for 4 h, and injected i.p. with porcine insulin
(Sigma-Aldrich) (0.75 IU/kg body weight). Blood glucose levels
were measured before and 15, 30, 45, 60, and 75 min after insulin
injection.
Gene Expression Analysis
One microgram of RNA extracted from the white adipose tissue
was treated with Deoxyribonuclease I Amplification grade
(Sigma), before being reverse-transcribed using Verso cDNA Kit
and oligo-dT according to the manufacturer’s instructions
(Thermo Scientific, ABgene, Epsom, UK). Real-time quantitative
PCR was performed on 7900HT Fast real-Time PCR system
using SYBR green chemistry (Applied Biosystems). Primers were
designed using the software Primer Express 1.5 (Applied
Biosystems) and primer sequences are available on request.
GAPDH was used as an internal control to normalize gene
expression. Results are expressed as fold-change compared to the
PBS/SD group of the analyzed cohort.
Characterization of IL-7 and IL-7R mRNA Expression in
Insulin-sensitive Tissues
C57BL/6J male mice (Janvier Laboratory) fed with a standard
diet, were sacrificed at the age of 8 weeks. Liver, skeletal muscles
(soleus and gastrocnemius), hypothalamus and thymus were rapidly
dissected and frozen in liquid nitrogen. Adipocytes and cells of the
stromal vascular fraction (SVF) were isolated from perigonadal
and subcutaneous white adipose tissues after digestion with
collagenase (0.3 U/ml, Serva, Heidelberg, Germany) in DMEM
supplemented with 2% BSA (Sigma). Following a 45 min
digestion, cells were filtered through a 100 mm mesh filter and
centrifuged at 600 g for 8 min. Floating adipocytes were then
isolated from the SVF-cell fraction. Both fractions were washed
twice in DMEM-2% BSA before being frozen in liquid nitrogen.
Total RNA were extracted, reverse-transcribed and analyzed
using real-time quantitative PCR as described above.
Determination of Triglyceride Contents
Perigonadal adipose tissue was stored at 220uC in PBS
containing 20 mM EDTA and 0.1% Triton X-100. Frozen samples
were sonicated in 1 ml chloroform/methanol (2:1 vol) and centri-
fuged so that the chloroform layer could be removed and the upper
phase re-extracted. Pooled upper-phases were dried at 40uC and
solubilized in isopropanol (HPLC Grade; Sigma-Aldrich, Lyon,
France) for triglyceride content measurement (Sigma-Aldrich).
DNA content was determined in the aqueous phase using the
diaminobenzoic acid technique, by spectrophotometry.
Adipose Tissue Morphometric Analysis
Perigonadal adipose tissues were fixed, embedded in paraffin,
sectioned and stained with hematoxylin and eosin. Tissue sections
images were acquired with a Zeiss Axiovert 100 M microscope. A
minimum of 100 non-overlapping images was automatically
acquired by serial scanning. Morphometric analyses were
performed using the Image J software (NIH image, National
Center for Biotechnology Information).
Ethics Statement
Transgenic mice, WT littermate controls, C57BL/6J and C.B-
17 SCID mice were bred and housed in specific pathogenic-free
environment in Pasteur Institute’s animal facilities. All animals
were maintained in a temperature-controlled (2062uC) facility
room with a strict 12-h light/dark and were given free access to
regular food and water, unless otherwise stated.
Breeding, housing and experimentations were carried out
according to the ‘‘Principles of laboratory animal care (NIH
publication no. 85-23, revised 1985; http://grants1.nih.gov/
grants/olaw/references/phspol.htm) as well as to the French and
European guidelines of laboratory animal care (European
IL-7 and White Adipose Tissue in Mice
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Page 9

Communities Council Directive of 1986, 86/609/EEC) and
approved by the Departmental Direction of Veterinary Services
(Prefecture of Lille, France; authorization number: 59-350152).
Statistical Analyses
Data are presented as means 6 SEM. The statistical
significance of comparison between the different experimental
groups was determined using two-way ANOVA test and the
Mann-Whitney nonparametric test (GraphPad Prism software). p
values less than 0.05 were considered statistically significant.
Supporting Information
Table S1
and wild-type controls.
(TIF)
Metabolic parameters in IL-7 transgenic mice
Acknowledgments
The authors thank the staff of the animal facility of the Pasteur Institute in
Lille for transgenic animals husbandry, especially E. Fleubaix. We thank
Drs. J. Coll, D. Dombrowicz and H. Duez for fruitful discussions and
comments on the manuscript, and J. Bertout and Drs. J.-C. Che `vre, N.
Delhem and J. Vicogne for technical assistance.
Author Contributions
Conceived and designed the experiments: SL CM JDS IW. Performed the
experiments: SL ST CM LF MD LM AD CV CS PS IW. Analyzed the
data: SL CM LF LM CV CS PS GG LH AK JM RP CA OP-G JDS PF
IW. Contributed reagents/materials/analysis tools: CM CS LH RP. Wrote
the paper: SL CM JDS PF IW.
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