By
From * The Surgical Metabolism Section, Surgery Branch, National Cancer Institute, National
Institutes of Health, Bethesda, Maryland 20892; and Division of Endocrinology, Beth Israel
Hospital, Boston, Massachusetts 02215
Several inflammatory cytokines, most notably tumor necrosis factor (TNF) and IL-1, induce anorexia and loss of lean body mass, common manifestations of acute and chronic inflammatory conditions. In C57BL/6 female mice, the administration of TNF, IL-1, and, to a lesser extent, leukemia inhibitory factor (LIF), produced a prompt and dose-dependent increase in serum leptin levels and leptin mRNA expression in fat. IL-10, IL-4, ciliary neurotrophic factor, and IL-2, cytokines not known to induce anorexia or decrease food intake, had no effect on leptin gene expression or serum leptin levels. After administration of Escherichia coli lipopolysaccharide (LPS), leptin gene expression and leptin levels were increased. These findings suggest that leptin levels may be one mechanism by which anorexia is induced during acute inflammatory conditions.
Anorexia and loss of lean body mass are hallmark manifestations of acute or chronic disease including infection or cancer. The role of TNF, IL-1, IL-6, and leukemia
inhibitory factor (LIF) as endogenous mediators of the
host response to infection or malignancy has been extensively studied (1). Chronic administration of TNF or
LIF to mice (6, 9, 10), or IL-1 to rats (2, 11) results in
significant anorexia and weight loss. Mice engrafted with
Chinese hamster ovary (CHO) cells genetically engineered to produce TNF, IL-6, or LIF demonstrate profound wasting
comparable to chronic starvation or cancer cachexia (12).
Alternatively, anti-TNF antibodies and a receptor antagonist
against IL-1 ameliorate the manifestations of cancer cachexia
in mice (15, 16). Also, cachectic patients with HIV-related
lymphoma treated with anti-IL-6 antibodies demonstrate
weight gain without change in tumor burden (17).
Though evidence has mounted that inflammatory diseases
mediate energy and weight dysregulation through their
associated cytokines, the mechanisms are unknown. Many
recent studies have suggested that the ob gene product,
leptin, may play a central role in energy regulation (18, 19).
This protein is expressed specifically in white adipose tissue
(18), is strongly correlated with total body fat mass (20, 21),
and when given to lean or ob/ob mice reduces food intake
and in the ob/ob mice increases basal metabolic rate leading
to weight loss (22). Therefore, we investigated the
hypothesis that inflammatory cytokines might elevate leptin, a potential explanation for their anorectic effects.
Animals.
Female C57BL/6 mice (10-12 wk/18-22 g) were
housed (5/cage) and raised on open formula rat/mouse ration (Zeigler, Gardners, PA) and water ad libitum, with a 12 h light-dark cycle beginning at 6:30 a.m. All experiments were conducted in compliance with the Animal Care and Use Committee of the National
Institute of Health. In the diurnal variation experiment animals had
free access to food. In all other experiments, food was withdrawn 2 h
before onset of the dark cycle. In the refeeding experiment only,
food was reintroduced after 7-7.5 h of fasting. Animals were killed at indicated timepoints for retroperitoneal fat and serum harvest.
Reagents.
The following cytokines were obtained: mTNF- Cytokine Response Studies.
After a 7-h fast, mice were given an
i.p. injection of 0.2 ml of a control carrier solution of PBS (Biofluids, Rockville, MD) with 0.5% endotoxin-free fatty acid-poor BSA
(Calbiochem-Novabiochem, La Jolla, CA) or LPS, or one of the
indicated cytokines.
Leptin/Serum Levels.
Leptin levels were measured by RIA as
described (20, 21) using the procedure as directed by Linco (St.
Charles, MO), except that all reagents were used at one-half recommended volume.
Reverse Transcriptase-PCR.
Total RNA was extracted from
frozen fat tissue samples by the guanidinum-thiocyanate/CsCl
method. An ob gene cDNA probe for Northern blotting was
generated as follows. First strand cDNA was synthesized from
3 µg of total RNA derived from an untreated control group, using
Moloney murine leukemia virus reverse transcriptase (GIBCO
BRL, Gaithersburg, MD) and oligo(dT) priming according to the
directions of the manufacturer. The reaction was carried out at
40°C for 1 h in a final volume of 60 µl PCR mix. Amplification primers were synthesized using the mouse ob gene sequence:
sense, 5 Northern Blot Analysis.
Total RNA was extracted from frozen
fat tissue pooled from each experimental group as described above.
Equal amounts of total RNA (25 µg/lane) were subjected to gel
electrophoresis using 1% agarose gels containing 0.6 M formaldehyde. RNA was blotted onto Duralon-UV (Strategene, Inc., La
Jolla, CA) nylon membranes and ultraviolet cross-linked. Hybridization and autoradiography were performed using standard techniques. Equal loading of RNA was confirmed by ethidium bromide staining of the agarose gel or hybridization of nylon
membranes with a chicken Statistical Methods.
Individual comparisons were evaluated with
Student's t test using the StatviewTM program.
We first examined the characteristics of leptin gene expression and serum levels under simple physiological manipulations, including circadian variation in freely feeding
mice and the response to acute starvation and refeeding. As
previously demonstrated at the mRNA level (25), leptin
levels under conditions of ad libitum feeding were lowest
in the middle of light cycle and highest in the middle of the
dark cycle (Fig. 1), consistent with the well-established diurnal but primarily nocturnal food intake behavior of rodents (26). When animals were fasted beginning 2 h before the night cycle, the nocturnal rise was abolished. When 7-h
fasted animals were refed, there was an exuberant increase
in leptin gene expression and serum levels higher than freely
fed controls and were manifest as early as 3 h after feeding
(Fig. 1).
After a 7-h evening fast, mice were treated with a single
intraperitoneal injection of LPS, or multiple cytokines at doses that have an anorectic effect (Ma, G. and H.R. Alexander,
manuscript in preparation). Fig. 2 shows that leptin levels are
significantly increased by LPS, TNF, IL-1, and LIF in a doserelated manner. LPS and TNF increased leptin levels nearly
five fold to levels greater than that seen in animals acutely
fasted and refed. IL-1 and LIF increased fasting leptin by
approximately twofold, a level similar to that observed in
fed animals. IL-6 had a trend to increase leptin, which did
not reach significance. LPS administration at sublethal (1 and
10 mg/kg) and lethal doses (20 and 30 mg/kg) produced a
dose-dependent increase in ob gene expression in fasted mice (Fig. 3). In this model, 20-30 mg/kg of LPS resulted
in a 30-40% lethality by 72 h after adminstration (data not
shown). These effects were specific, because IL-10 and IL-4,
which generally exhibit anti-inflammatory characteristics,
as well as IL-2 and ciliary neurotrophic factor (CNTF), had
no effect on leptin mRNA expression in retroperitoneal fat
(data not shown) or serum leptin levels (see Fig. 2).
We next examined the time course of effects of TNF
and IL-1 on leptin levels (Fig. 4). After a 7-h fast, mice
were injected with 100 µg/kg of TNF, or 1,000 U of IL-1.
Expression of the leptin gene in retroperitoneal fat increased within 2 h after injection and was maximal 6-8 h
after TNF administration (data not shown), while leptin serum levels reached a maximal three-fold elevation by 7 h,
and subsequently returned toward baseline at 18 h. IL-1 induced a slower increase in leptin to a maximal twofold increase by 10 h, which persisted for at least another 8 h.
The time course and dose-dependent effects of the inflammatory cytokines TNF and IL-1 on leptin gene expression suggest that their anorectic effects may be mediated in part by regulation of leptin gene expression. The
finding that IL-6 and LIF are not as potent as TNF or LPS
in inducing leptin levels or gene expression is not surprising. Although IL-6 and LIF have pleiotropic inflammatory properties, have been detected in various acute and chronic
diseases (2, 10), and have been shown to induce tissue
wasting in hosts bearing implantable tumors secreting either cytokine (13, 14), the evidence that IL-6 or LIF induce anorexia or mediate cachexia in various disease states
is not consistent (27). CNTF, IL-2, IL-4 and IL-10, the
latter two being primarily counterinflammatory cytokines,
have not been shown to have anorectic effects in mice (30-
33) and had no effect on leptin levels or gene expression.
The full syndrome of leptin insufficiency is seen in ob/ob
mice that lack functional leptin. These mice have increased
food-seeking behavior, insulin resistance, hypothermia, decreased sympathetic drive, elevated corticosterone, and infertility, all of which tend to normalize with exogenous
leptin (21, 34, 35). Acute food restriction in mice, associated with low leptin levels, is characterized by the same
abnormalities. In an acutely infected mouse, these abnormalities (except infertility) might be anticipated to impair
survival in the face of bacterial infection. In particular, leptin support of brown fat, basal metabolism, and prevention of hypothermia may be important in preserving the adaptive febrile response. We conjecture that cytokine stimulation of leptin to levels at or above seen in the fed state may
have developed to prevent the abnormalities of leptin deficiency during infection, regardless of the food intake.
The leptin receptor, closely related to gp-130 (heterodimer of IL-6 and LIF), is a member of the class I cytokine
group (36). Thus, it seems quite likely that the leptin system is ancestrally related to the cytokines. In this context, it
is perhaps not surprising that multiple cytokines influence
leptin levels, as we have demonstrated. The possibility of
the reverse, leptin influence on other cytokines, is plausible
based on the wide distribution of some forms of the leptin
receptor (36), including primitive hematopoetic stem cells
and lymphohematopoetic cell lines (37), and deserves investigation.
In summary, we have demonstrated that multiple cytokines
documented to induce anorexia increase leptin, a protein
demonstrated to produce anorexia and confirm similar
findings with LPS (38). These results strongly advance the
hypothesis that cytokine induction of leptin may play a significant role in the anorexia and cachexia of inflammatory
diseases such as infections, collagen vascular disease, and
cancer. If the cytokine-leptin hypothesis is supported by
further studies, it opens a novel approach to combating this significant comorbidity of many common diseases.
(Genentech, South San Francisco, CA), hIL-1
(Biologic Response Modifiers Program [BRMP], Frederick, MD), hIL-2 (Cetus, Chiron Corp., Emeryville, CA), mIL-10 (BRMP, Frederick,
MD), mIL-4 (BRMP, Frederick, MD), mLIF (Genentech, South
San Francisco, CA), mCNTF (R&D Systems, Minneapolis, MN),
and mIL-6 (BRMP, Frederick, MD). E. coli lipopolysaccharide (serotype 0127:B8) was purchased from Sigma (St. Louis, MO).
-AATGTGCTGCAGACCCCTGTG-3
, and antisense,
5
-CATTCAGGGCTAACATCCAAC-3
. Hot-start PCR was
performed using the protocol for Ampliwax in the following reaction: 2.5 µl PCR buffer (10×), 10.5 µl sterile water, 5 µl of
each primer (4 µM), 2 µl dNTP mix (10 mM each dNTP) were
mixed in a PCR microfuge tube, followed by a single Ampliwax PCR Gem, heated to 80°C, and allowed to cool at room temperature. On the solidified wax layer, 7.5 µl PCR buffer (10×), 56.5 µl sterile water, 10 µl cDNA template, and 1 µl Taq polymerase
(5 U/µl) were mixed and heated at 95°C for 5 min, followed by
37 cycles using a 1-min denaturation step at 94°C, a 1-min annealing step at 60°C, and a 2-min extension step at 72°C. An additional 8-min extension step at 72°C was added after 37 cycles.
PCR reactions were performed in a thermocycler (Perkin-Elmer
Cetus model 480, Norwalk, CT). A single predicted 500-bp PCR
product was obtained as resolved on a 2% agarose gel. The product was cloned with the Invitrogen T/A cloning kit (San Diego,
CA), produced in quantity, purified, random prime labeled with
P32, and used as a cDNA probe for our Northern blots. A quantity of the product was subjected to restriction enzyme analysis for
confirmation of specificity.
-actin cDNA probe (Oncor, Gaithersburg, MD). Each lane represents RNA from tissue pooled
from five or six mice. Each experiment was done between three
to six times with consistent results.
Fig. 1.
Leptin levels in freely feeding mice at intervals throughout a
24-h period and after short-term fasting and refeeding. The initial point
represents midnight, 5.5 h after beginning the dark cycle in the ad libitum
fed diurnal experiment, 7 h after the commencement of the fast in the
fasting and refeeding experiments, and the beginning of refeeding in the
latter experiment. Each point represents the mean ± SEM of 6-8 individually measured mice. Northern blot shows ob gene expression in adipose
tissue from freely fed mice (control), decreased expression after a 7- or 12-h
fast (starved), and increased 5 h after refeeding groups of mice starved for 7 h
(refed 5 h).
[View Larger Version of this Image (18K GIF file)]
Fig. 2.
Leptin levels after i.p. administration of various cytokines and
LPS. Mice were injected with LPS (mg/kg) or recombinant cytokine in
the doses shown (IL-2, IL-1: U/mouse; TNF, IL-6, LIF, CNTF: µg/kg)
or PBS after a 7-h fast and sera and adipose tissue harvested 5 h later. Further details are in Materials and Methods. Each bar represents the mean ± SEM serum leptin level of 6-7 mice. Significance compared with the
PBS-treated animals is indicated as *P <0.05, **P <0.01.
[View Larger Version of this Image (16K GIF file)]
Fig. 3.
Effect of LPS on ob
gene expression. After a 7-h fast
mice were injected i.p. with LPS
at the doses indicated and adipose tissue harvested 5 h later as
described. ob gene expression is low in fasted animals treated
with PBS and increased in a
dose-dependent manner after LPS.
[View Larger Version of this Image (40K GIF file)]
Fig. 4.
Kinetics of leptin in sera from mice injected with 100 µg/kg
of TNF or 1,000 U of IL-1, after a 7-h fast. All animals were food-deprived
during the experimental period. Each point represents mean ± SEM leptin level of 5-6 mice, except for the 13-h points, which represent 10-14
mice. Compared with fasted controls, significant differences are indicated
as *P <0.05.
[View Larger Version of this Image (16K GIF file)]
Address correspondence to H. Richard Alexander, Jr., Head, Surgical Metabolism Section, Surgery Branch, National Cancer Institute, National Institutes of Health, Building 10, Room 2B17, Bethesda, MD 208921502.
Received for publication 19 August 1996
The authors wish to thank Michael Nishimura, Rudy Pozzatti, and James Gnarra for their technical advice and support. The assistance of Aida Ordoubadi and Aaron Rosenfeld is gratefully acknowledged. We also wish to thank Dr. Carl Grunfeld for valuable discussions and sharing of data before publication. Finally, we thank Barbara Owen for her help in preparation of the manuscript.This work was supported in part by National Institutes of Health grants P30 DK46200 and K08 HL02564. D.J. Rivet III is a Howard Hughes Medical Institute-National Institutes of Health Research Scholar.
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