- Email: [email protected]
A reappraisal on the ability of leptin to induce fever
Physiology & Behavior 97 (2009) 430–436
Contents lists available at ScienceDirect
Physiology & Behavior j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / p h b
A reappraisal on the ability of leptin to induce fever Alexandre A. Steiner a,b,⁎, Catherine M. Krall a,b, Elaine Liu a a b
Department of Pharmaceutical Sciences, Albany College of Pharmacy and Health Sciences, Albany, NY, USA Research Service, Stratton Veterans Affairs Medical Center, Albany, NY, USA
a r t i c l e
i n f o
Article history: Received 23 January 2009 Received in revised form 13 March 2009 Accepted 16 March 2009 Keywords: Fever Body temperature Inflammation Cytokines Leptin Adipose tissue
a b s t r a c t Leptin is often regarded as a mediator of fever, even though an in-depth analysis of the dose-dependent effects of leptin on body temperature (Tb), pro-inflammatory cytokines, and circulating leptin has never been performed. In the present study, such an analysis was performed in rats that were food deprived (lower baseline levels of leptin) or free feeding (higher baseline levels of leptin). In a relatively cool environment (22 °C), rats deprived of food for 24 h exhibited mild (~0.5 °C) hypothermia. Leptin infusion (250 μg/kg iv) elevated the Tb of the food-deprived rats to a normothermic level, an effect that peaked (120 min postinfusion) when plasma leptin was at a level (~ 8 ng/mL) often found in leptin-responsive subjects. Increasing the leptin dose to 1000 μg/kg did not produce any further (febrile) elevation in the Tb of food-deprived rats. The anti-hypothermic effect of leptin in food-deprived rats was not associated with any rise in the plasma levels of the pro-inflammatory cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-6. In freefeeding rats kept in a cooler (22 °C) or warmer (28 °C) environment, leptin infusion failed to alter Tb or to produce any surge in plasma TNF-α or IL-6, even when the dose infused (3500 μg/kg iv) resulted in excessive, non-physiological rises in plasma leptin (~ 542 ng/mL at 30 min; ~ 75 ng/mL at 120 min postinfusion). In contrast, free-feeding rats in the same experimental set-up were able to respond to a low dose (2 μg/kg iv) of IL-1β with a typical biphasic fever, which was associated with surges in plasma TNF-α and IL-6. Collectively, our data show that an acute rise in plasma leptin to a level within or fairly above the physiological range does not induce fever. These results challenge the idea that leptin may be a mediator of fever. © 2009 Elsevier Inc. All rights reserved.
1. Introduction Leptin is an adipocyte-derived hormone known to play critical roles in energy homeostasis [1–4]. In the fed state, tonic stimulation of hypothalamic structures by leptin promotes satiety and metabolic energy expenditure [5–9]. In the fasted or food-deprived state, decreased leptin production contributes to suppression of satiety and energy expenditure [10–14]. In addition to its classical roles in energy homeostasis, leptin may modulate immune function [15–17], but the exact roles of leptin in immunomodulation and immune-brain communication are far from being defined. Leptin is often [18–21] referred to as a pro-inflammatory cytokine that can induce a febrile rise in deep body temperature (Tb). This view is largely based on a study by Luheshi et al. [22], in which injection of recombinant leptin induced a cytokine-mediated fever in rats. Although this effect has been reproduced in one study [23], it has not been universally found [24]. Even if leptin can, in fact, induce fever, the question as to whether leptin-induced fever is associated with physiologically relevant rises
in the circulating level of leptin has never been addressed. (Physiologically relevant concentrations of leptin in the plasma range from 0.5 to 20 ng/mL in lean subjects [25,26] known to be responsive to leptin [10–13].) In order to determine whether acute, physiologically relevant rises in circulating leptin are capable of inducing pro-inflammatory cytokines and fever, we simultaneously evaluated the dose-dependent effects of leptin on Tb, pro-inflammatory cytokines, and circulating leptin in rats that were food deprived (lower baseline levels of leptin) or free feeding (higher baseline levels of leptin). The pro-inflammatory cytokines measured were tumor necrosis factor (TNF)-α and interleukin (IL)-6. TNF-α is the earliest cytokine to surge in the circulation after an immune challenge [27], it has the ability to induce fever at low doses [28], and it may mediate the fever induced by low doses of bacterial products [29,30]. IL-6 surges in the circulation subsequently to TNF-α [27], and it has been shown to mediate fever in a variety of experimental models [31]. 2. Materials and methods
⁎ Corresponding author. Department of Pharmaceutical Sciences, Albany College of Pharmacy & Health Sciences, 106 New Scotland Avenue, Albany, NY 12208, USA. Tel.: +518 694 7154; fax: +518 694 7037. E-mail address: [email protected] (A.A. Steiner). 0031-9384/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2009.03.018
2.1. Animals Male Wistar rats were obtained from the Charles River Laboratories plant in Raleigh, NC, USA. Rats were initially caged in pairs; after
A.A. Steiner et al. / Physiology & Behavior 97 (2009) 430–436
surgery, they were caged individually. The rat colony room was maintained at a temperature of 21–24 °C and a 12:12 h light-dark cycle (lights on at 07:00 AM). Each rat was handled 5 min per day for 8 days; during a handling session, a rat was habituated to being dressed with an infusion harness that was used later in the experiments. The rats weighed 290–360 g at the time of the experiments. Each rat was used in an experiment once and euthanized with sodium pentobarbital (100 mg/kg iv) immediately thereafter. All procedures were conducted under a protocol approved by the Animal Care and Use Committee of the Stratton Veterans Affairs Medical Center. 2.2. Surgical preparation Five days before an experiment, rats were subjected to one or more of the following surgical procedures: intravenous catheterization, intra-arterial catheterization, and/or implantation of miniature temperature datalogger. All procedures were performed aseptically under anesthesia (ketamine–xylazine–acepromazine, 80:5:1 mg/kg ip) and antibiotic protection (enrofloxacin, 1.1 mg/kg sc). Ketamine was obtained from Bioniche Pharma (Lake Forest, IL, USA), xylazinefrom IVX Animal Health (St. Joseph, MO, USA), acepromazine from Boehringer Ingelheim Vetmedica (St. Joseph, MO, USA), and enrofloxacin from Bayer Animal Health (Shawnee Mission, KS, USA). Rats were maintained on an operating board warmed to 37 °C by a Deltaphase isothermal pad (Braintree Scientific, Braintree, MA, USA). For intravenous catheterization, a small longitudinal incision was made on the right ventral surface of the neck. The right jugular vein was exposed, freed from its surrounding connective tissue, and ligated. A 3-Fr polyurethane catheter (Instech Laboratories, Plymouth Meeting, PA, USA) filled with heparinized (50 U/mL) saline was passed into the superior vena cava through the jugular vein and secured in place with ligatures. The distal end of the catheter was closed with a stainless steel plug, tunneled under the skin, and exteriorized at the nape. The intravenous catheter was used for drug administration during an experiment. For intra-arterial catheterization, the right carotid artery was isolated and clamped by a microclip. The tip of a PE-50 catheter filled with heparinized saline was placed into the artery, the clip was removed, and the catheter was moved towards the aorta. The catheter was secured in place with ligatures. The distal end of the catheter was heat-sealed and exteriorized at the nape. The intra-arterial catheter was used for blood collection during an experiment. For datalogger implantation, a midline laparotomy was performed and a spacer was used to keep the abdominal muscles separated. The rat was then positioned in lateral decubitus, and a SubCue miniature datalogger (Calgary, AB, Canada) was attached to the internal side of the dorsolateral abdominal wall with 4-0 silk sutures. The dataloggers recorded abdominal temperature (an index of Tb) every 2 min. After the surgical wounds were sutured with 4-0 silk, rats were transferred to an environmental chamber (model NQ1; Environmental Growth Chambers, Chagrin Falls, OH, USA) set to 28.0 °C, where they remained until complete recovery from anesthesia (3–4 h postsurgery). The intravenous catheters were flushed with heparinized saline every other day; the intra-arterial catheters were flushed daily. 2.3. Experimental set-up A rat in its home cage was placed in the environmental chamber (Environmental Growth Chambers), inside which the thermal environment was controlled: the ambient temperature was maintained at 22.0 ± 0.1 °C or 28.0 ± 0.1 °C depending on the experiment (see below), whereas relative air humidity was always kept at 50 ± 5%. The chamber contained fluorescent lamps that operated according to the light-dark cycle. After the rat was equipped with a Covance Infusion Harness (Instech Laboratories), the intravenous catheter was extended with a length of PE-50 tubing filled with saline, and the
431
extension was passed through a stainless steel spring that linked the harness to a swivel (Instech Laboratories) mounted on the top of the cage. The catheter extension was connected to the bottom portion of the swivel. Another saline-filled PE-50 extension was connected to the top portion of the swivel, passed through a port in the chamber wall, and connected to the infusion syringe located outside of the chamber. The dead volume of the infusion line was 0.4 mL. If present, the intraarterial catheter was extended with a length of PE-50 tubing filled with saline. The arterial extension was passed through the harness and spring but not connected to the swivel; the distal end of the extension was plugged and attached with adhesive tape to the outer surface of the spring, just below the swivel. At the time of blood collection, the arterial line was accessed by briefly (b2 min) opening the chamber door. During this period, the ambient temperature fluctuated by no more than 0.5 °C, and the rats did not display any behavioral sign of stress such as startle responses, ‘freezing’ behavior, or abrupt changes in exploratory behavior. 2.4. Experimental drugs Recombinant mouse leptin (endotoxin residue b0.01%) was a gift from Amylin Pharmaceuticals (San Diego, CA, USA). The mouse leptin has 96% sequence identity with the rat leptin [32], and it is fully able to activate leptin receptors in rats [33–35]. A stock solution of leptin (5000 μg/mL) in saline was aliquoted and stored at − 80 °C. Immediately before an infusion, the stock was warmed to room temperature and diluted with saline to a final concentration of 50, 125, 500, or 1750 μg/mL. As in previous studies [34,36], the leptin solution was administered intravenously as a bolus injection of 1 mL/kg followed by an infusion of 1 mL/kg over 1 h, so that the total dose of leptin administered was 100, 250, 1000, or 3500 μg/kg. Recombinant rat IL-1β (endotoxin residue b0.01%) purchased from Sigma-Aldrich (St. Louis, MO, USA) was also employed in the study. Aliquots of the IL-1β stock solution (200 μg/mL) were stored at −80 °C. The aliquots were warmed to room temperature, diluted with saline to a concentration of 2 μg/mL, and the resulting solution was bolus injected intravenously (1 mL/kg), to deliver an IL-1β dose of 2 μg/kg. 2.5. Experimental design In Experiment 1, the biological activity of the leptin preparation was assessed based on the established effects of leptin on feeding behavior [1–4]. Rats implanted with intravenous catheters were habituated to consuming powdered chow from spill-free feeding chambers (Nalgene Labware, Rochester, NY, USA) for 4 days. The rats were then placed in the environmental chamber at an ambient temperature (22 °C) to which they had been exposed during housing; the rats stayed in the chamber until the end of the experiment. After overnight habituation to the chamber, the rats were food deprived for 24 h (starting at 10 AM); an elevated grid floor was placed inside the cage to prevent coprophagy; drinking water was available ad libitum. During the last hour of the food-deprivation period, the rats were infused with leptin or saline, as described above. The feeding chambers containing powdered chow were then made available for 1 h, during which the number of feeding episodes (“visits” to feeding chamber) was recorded using infrared monitors (Respironics-MiniMitter, Bend, OR, USA). The mass of chow consumed was also measured. Experiment 2 was conducted to determine if leptin can induce fever in food-deprived rats. Because the influence of food deprivation on Tb is best viewed when ambient temperature is subneutral [37], this experiment was conducted at an ambient temperature of 22 °C, which is known to be subneutral for rats in a virtually identical experimental setting [38]. Rats previously implanted with intravenous catheters and temperature dataloggers were placed in the environmental chamber (22 °C). After overnight habituation, the rats were
432
A.A. Steiner et al. / Physiology & Behavior 97 (2009) 430–436
food deprived (starting at 10 AM); an elevated grid floor was placed inside the cage to prevent coprophagy; drinking water was available ad libitum. During the 24th hour of food deprivation, leptin or saline were infused. The rats then continued without access to food for 4 additional hours, after which they were euthanized and the Tb data stored in the dataloggers were transferred to a computer. A separate set of rats was used for determination of the plasma levels of TNF-α, IL-6, and leptin. These rats had been implanted with intravenous and intra-arterial catheters but not dataloggers. They were subjected to the same experimental procedures, and arterial blood samples were collected 30 and 120 min after the end of the leptin/saline infusion; these time points were selected based on the dynamics of the Tb responses (see Results for details). Experiment 3 was conducted to determine if leptin can induce fever in free-feeding rats. The design was identical to that of Experiment 2, except that rats in Experiment 3 were not food deprived. Additionally, Experiment 3 was conducted not only at 22 °C but also at 28 °C, since the febrile response of free-feeding rats to bacterial products and cytokines may be more manifest when the environment is warmer [39]. In Experiment 4, we sought to show that a typical febrigenic cytokine, IL-1β, induces fever in our experimental setting. Rats previously implanted with intravenous catheters and temperature dataloggers were placed in the environmental chamber at an ambient temperature of 22 °C, where they had ad libitum access to chow and water for the entire experiment. After overnight habituation, the rats were intravenously bolus injected with IL-1β or saline. Four hours later, the rats were euthanized, and the Tb data stored in the dataloggers were transferred to a computer. A separate set of rats was used for determination of the plasma levels of TNF-α and IL-6. These rats had been implanted with intravenous and intra-arterial catheters but not dataloggers. Arterial blood samples were collected 30 and 90 min after the IL-1β/saline injection; these time points were selected based on the dynamics of the Tb responses (see Results for details). 2.6. Blood processing and assays Each blood sample (0.5 mL) was collected into a 1-mL syringe and immediately transferred to an EDTA-coated Eppendorf tube. The tube was centrifuged (6000 ×g, 10 min, 4 °C), and the resulting plasma was stored at − 80 °C. The levels of TNF-α, IL-6, and leptin in the plasma samples were determined using Quantikine enzyme immunoassays (R&D Systems, Minneapolis, MN, USA). In each assay, all samples were run simultaneously, in duplicate. The detection limits of the assays were 5 pg/mL for TNF-α, 21 pg/mL for IL-6, and 22 pg/mL for leptin. 2.7. Data analyses Tb and plasma leptin values were compared across treatments and time points by two-way ANOVA. The results of the TNF-α or IL-6 assays were analyzed based on Chi-square comparisons of the number of samples containing detectable/non-detectable levels of these cytokines. The number of feeding episodes and the mass of food consumed were compared across treatments by one-way ANOVA. All analyses were performed using Statistica Advanced 8.0 (StatSoft, Tulsa, OK, USA). The level of significance was set at P b 0.05. Data are reported as means ± SE. 3. Results 3.1. Experiment 1: Effects of leptin on feeding behavior (biological activity test) During the first hour following a 24-h food deprivation and a 1-h intravenous infusion of saline, feeding behavior was characterized by an average of 65 (±6) feeding episodes and 5.4 (±0.6) grams of chow consumed (Fig. 1). Compared to saline, leptin infusion reduced
Fig. 1. Effects of intravenous leptin (doses indicated) or its vehicle (saline) on feeding parameters of rats that were offered food for 1 h after a 24-h food-deprivation period. The rats were maintained at an ambient temperature of 22 °C. The number of animals (n) in each group is indicated. ⁎Significant (P b 0.05) difference in relation to the salineinfused controls.
the number of feeding episodes and the amount of chow consumed in a dose-dependent manner. Whereas the dose of 100 μg/kg was ineffective, doses of 250 and 1000 μg/kg significantly reduced the number of feeding episodes by 29% and 71% and the mass of food consumed by 24% and 40%, respectively. These results confirmed the biological activity of the leptin preparation used and identified the lowest active dose (250 μg/kg) of leptin in our experimental setting. 3.2. Experiment 2: Effects of leptin on the Tb and plasma cytokines of food-deprived rats At an ambient temperature of 22 °C, food deprivation (24 h) resulted in a small (~ 0.5 °C), yet significant decrease in Tb (Fig. 2). The food deprivation-associated hypothermia was unaffected by intravenous infusion of saline or leptin at 100 μg/kg. However, leptin at 250 μg/kg gradually raised the Tb of food-deprived rats to the normothermic level observed before food deprivation. This rise in Tb started at ~30 min and peaked at ~ 120 min after the end of leptin infusion. The plasma levels of TNF-α and IL-6 were not elevated at these points in time (Table 1). The plasma levels of leptin were low (0.4 ng/mL) in food-deprived rats infused with saline, but they averaged 61.7 ng/mL (30 min) and 7.8 ng/mL (120 min) during the Tb response to leptin (Table 1). A higher dose (1000 μg/kg) of leptin also reversed the hypothermia associated with food deprivation, but it did not produce any further (febrile) increase in Tb (Fig. 2). 3.3. Experiment 3: Effects of leptin on the Tb and plasma cytokines of free-feeding rats No change in Tb was observed when free-feeding rats maintained at an ambient temperature of either 22 °C or 28 °C were infused with saline or leptin at a dose as high as 3500 μg/kg (Fig. 3). The highest dose of leptin also failed to produce any detectable increase in the plasma levels of TNF-α and IL-6 (Table 2). The plasma levels of leptin averaged 542 ng/mL at 30 min and 75 ng/mL at 120 min after infusion
A.A. Steiner et al. / Physiology & Behavior 97 (2009) 430–436
433
Fig. 2. Deep body temperature of rats intravenously infused with leptin (doses indicated) or its vehicle (saline) during the 24th hour of food deprivation. The rats were kept at an ambient temperature of 22 °C. The number of animals (n) in each group is indicated. ⁎Significant (P b 0.05) difference in relation to the saline-infused controls.
of the highest leptin dose, as compared to 1.7–1.8 ng/mL after infusion of saline (Table 2).
controls. Plasma IL-6 was predominantly undetectable at 30 min. At 90 min, however, detectable levels of IL-6 were found in 5/6 of the IL1β-treated rats, as compared to 1/6 of the saline-treated controls.
3.4. Experiment 4: Thermoregulatory and cytokine responses to IL-1β 4. Discussion In view of the fact that leptin did not induce fever under any of the experimental conditions tested, it was important to demonstrate that rats in our experimental setting could develop fever in response to a typical pro-inflammatory cytokine. In free-feeding rats maintained at an ambient temperature of 22 °C, saline (iv) caused no change in Tb, whereas a low dose (2 μg/kg iv) of IL-1β triggered a biphasic fever characterized by Tb peaks at 30 and 90 min postinjection (Fig. 4). TNFα was detected in the plasma of all IL-1β-treated rats at both 30 and 90 min, but it was not detected in the plasma of saline-treated Table 1 Plasma concentrations of leptin, TNF-α, and IL-6 in food-deprived rats infused (iv) with saline or leptin. Infusion
Time post-infusion
Plasma concentration Leptin (ng/mL)
TNF-α (pg/mL)
IL-6 (pg/mL)
Saline
30 min 120 min 30 min 120 min
0.4 ± 0.1 (6) 0.4 ± 0.1 (5) 61.7 ± 4.2⁎ (5) 7.8 ± 0.6⁎ (5)
b5.0 b5.0 b5.0 b5.0
b21.0 b21.0 b21.0 b21.0
Leptin 250 μg/kg
(6) (5) (5) (5)
(6) (5) (5) (5)
The number of animals is shown in parentheses. ⁎P b 0.05 compared to saline infusion.
The present study is the first to simultaneously evaluate the dosedependent effects of leptin on Tb, pro-inflammatory cytokines, and circulating leptin. The biological activity of the leptin preparation used in our study was demonstrated by the established ability of leptin to suppress food seeking and consumption. Leptin reversed the hypothermia induced by food deprivation at an ambient temperature of 22 °C, an effect that peaked (120 min post-infusion) when plasma leptin was at a level (~8 ng/mL) often found in leptin-responsive subjects [25,26]. This observation agrees with previous studies [11,40] showing that a fall in leptin levels during food deprivation is essential for the associated suppression of basal metabolic rate and, consequently, basal Tb. Leptin administration, however, did not raise Tb to a febrile level in either food-deprived, hypothermic rats kept at 22 °C or in free-feeding, normothermic rats kept at 22 °C or 28 °C. Leptin administration also failed to produce any surge in the plasma levels of cytokines (TNF-α and IL-6) known to be involved in the pathogenesis of fever [31,41]. This was the case even when leptin was infused at a dose (3500 μg/kg iv) that resulted in excessive, non-physiological rises in the plasma levels of leptin. The inability of leptin to induce
434
A.A. Steiner et al. / Physiology & Behavior 97 (2009) 430–436
Fig. 3. Deep body temperature of free-feeding rats intravenously infused with leptin (doses indicated) or its vehicle (saline) at an ambient temperature of 22 °C or 28 °C. The number of animals (n) in each group is indicated.
fever was not due to an inability of the rats to mount a febrile response, because rats in the same experimental set-up were able to respond to a low dose of IL-1β with a typical biphasic fever associated with surges in plasma TNF-α and IL-6. Collectively, these data strongly indicate that an acute rise in plasma leptin to a level within or fairly above the physiological range does not induce fever. Whereas the present study involved a non-stressful procedure of intravenous leptin administration (as evident by the lack of stress hyperthermia in the saline-infused rats), previous studies [22,23] suggesting that leptin can cause fever in free-feeding rats employed stressful procedures of drug administration, which involved handling, temporary restraint, and needle pricking. The doses of leptin shown to cause fever in those studies were also excessively high. When administered systemically, leptin was reported to cause fever at a dose of 3.5 mg/kg [22], which we found to raise plasma leptin to supraphysiological levels for at least 120 min. When administered intracerebroventricularly, leptin was reported to cause fever at doses of 4–5 μg/rat [22,23], which are ~ 10 times higher than the intracerebroventricular doses of leptin needed to affect food intake and energy expenditure [42,43]. Therefore, it is possible that, in association with stress, excessively high doses of leptin might cause fever. Indeed, stress has been reported to enhance inflammatory signaling to the brain [44]. But, anyhow, the effects of excessively high doses of leptin are unlikely to reflect a physiological action of leptin. At persistently (6–24 h) high levels (N50 ng/mL), leptin has been shown to stimulate leukocytes to produce pro-inflammatory cytokines in vitro [45–47]. This action may be relevant for the chronic, lowgrade inflammation seen in obese subjects [48]. However, the same action is unlikely to be relevant for the acute inflammatory response to microbial agents such as bacterial lipopolysaccharide (LPS). Even though circulating leptin can be increased as early as 1 h after administration of LPS [49,50], we presently report that rapid rises in plasma leptin following leptin infusion do not lead to surges in plasma TNF-α and IL-6. In addition, Bik et al. [51] have found no effect of leptin injection on the cytokine response to LPS. Furthermore, it has
been demonstrated that mutant rats and mice that lack leptin or leptin receptors are fully capable to produce pro-inflammatory cytokines in response to LPS [38,52–55]. Some of these mutants (Zucker fa/fa rats and Koletsky f/f rats) have also been shown to respond to LPS with normal fevers in experiments conducted in a warm (28–29 °C) environment [38,56]. Cholecystokinin-1-receptor-deficient rats, which are at least partially leptin-resistant [57], also respond to LPS with fever [58,59]. Corroborating these findings, a recent study has shown that an anti-leptin antibody does not alter the LPS-induced expression of key enzymes (clyclooxygenase-2 and microsomal prostaglandin E synthase-1) involved in the mediation of fever [60]. Since leptin does not seem to activate febrigenic inflammatory signaling, it is important to seek alternative interpretations for reports that leptin neutralization with an antibody attenuates LPS fever in rats
Table 2 Plasma concentrations of leptin, TNF-α, and IL-6 in free-feeding rats infused (iv) with saline or leptin. Infusion
Time post-infusion
Plasma concentration Leptin (ng/mL)
TNF-α (pg/mL)
IL-6 (pg/mL)
Saline
30 min 120 min 30 min 120 min
1.7 ± 0.4 (7) 1.8 ± 0.6 (7) 542.1 ± 62.9 (5)⁎ 75.2 ± 12.6 (5)⁎
b5.0 b5.0 b5.0 b5.0
b21.0 b21.0 b21.0 b21.0
Leptin 3500 μg/kg
(6) (5) (5) (5)
(6) (5) (5) (5)
The number of animals is shown in parentheses. ⁎P b 0.05 compared to saline infusion.
Fig. 4. Effects of intravenous IL-1β (dose indicated) or its vehicle (saline) on the deep body temperature and plasma cytokine levels of free-feeding rats. The rats were maintained at an ambient temperature of 22 °C. The number of animals (n) in each group is indicated. ⁎Significant (P b 0.05) difference in relation to the saline-infused controls.
A.A. Steiner et al. / Physiology & Behavior 97 (2009) 430–436
kept at ambient temperatures of 21–22 °C [61,62]. At these mildly cool ambient temperatures for rats [63,64], the thermoregulatory response to LPS depends on a balance between the actions of febrigenic (increase Tb) and cryogenic (decrease Tb) inflammatory mediators [41,65]. It is, thus, possible that attenuation of fever in the absence of leptin signaling results from overactivation of cryogenic signaling. Because cryogenic signaling lowers Tb by suppressing thermogenesis [66], it does not influence Tb in a warm environment, in which thermogenesis is at its minimum and cannot be further inhibited [66]. Hence, the fact that mutant rats lacking functional leptin receptors respond to LPS with an attenuated fever in a cool environment but with normal fever in a warm environment [38,56] is consistent with an involvement of leptin in cryogenic signaling. Also supporting an involvement of leptin in cryogenic signaling is the finding that the hypothermia induced by a high dose of LPS in a cool environment is largely prolonged in leptin receptor-deficient rats [38]. Such a prolongation of hypothermia was associated with an exaggerated rise in plasma TNF-α [38], a pro-inflammatory cytokine that is febrigenic at lower doses but becomes cryogenic at higher doses [28]. It was also associated with an inability of the rats to activate the antiinflammatory hypothalamo-pituitary-adrenal axis [38]. We have recently proposed that modulation of cryogenic signaling by leptin may link energy balance to host defense [17], but future studies are warranted to address this possibility. Future studies should also investigate whether the crosstalk between energy balance and host defense may involve other adipocyte-derived hormones such as adiponectin [67,68]. In conclusion, the present study shows that non-stressful, acute administration of leptin does not induce pro-inflammatory cytokines and fever in rats, regardless of whether the rats are food deprived (lower baseline levels of leptin) or free feeding (higher baseline levels of leptin), and regardless of the ambient temperature. These results challenge the view that leptin may be a mediator of fever. Acknowledgement We are thankful to Drs. Andrej A. Romanovsky (St. Joseph's Hospital, Phoenix, AZ) and Luciana B. Lopes (Albany College of Pharmacy & Health Sciences, Albany, NY) for their critical comments on the manuscript. The study was supported in part by an Albany College of Pharmacy Scholarship of Discovery Grant to AAS. Recombinant leptin was generously provided by Amylin Pharmaceuticals (San Diego, CA). EL was the recipient of an Albany College of Pharmacy Summer Scholarship Award. References [1] Friedman JM, Halaas JL. Leptin and the regulation of body weight in mammals. Nature 1998;395:763–70. [2] Elmquist JK, Maratos-Flier E, Saper CB, Flier JS. Unraveling the central nervous system pathways underlying responses to leptin. Nat Neurosci 1998;1:445–50. [3] Ahima RS, Flier JS. Leptin. Annu Rev Physiol 2000;62:413–37. [4] Havel PJ. Update on adipocyte hormones: regulation of energy balance and carbohydrate/lipid metabolism. Diabetes 2004;53:S143–51. [5] de Luca C, Kowalski TJ, Zhang Y, Elmquist JK, Lee C, Kilimann MW, et al. Complete rescue of obesity, diabetes, and infertility in db/db mice by neuron-specific LEPR-B transgenes. J Clin Invest 2005;115:3484–93. [6] Coppari R, Ichinose M, Lee CE, Pullen AE, Kenny CD, McGovern RA, et al. The hypothalamic arcuate nucleus: a key site for mediating leptin's effects on glucose homeostasis and locomotor activity. Cell Metab 2005;1:63–72. [7] Scarpace PJ, Matheny M, Zhang Y, Cheng KY, Tumer N. Leptin antagonist reveals an uncoupling between leptin receptor signal transducer and activator of transcription 3 signaling and metabolic responses with central leptin resistance. J Pharmacol Exp Ther 2007;320:706–12. [8] Peters JH, Simasko SM, Ritter RC. Leptin analog antagonizes leptin effects on food intake and body weight but mimics leptin-induced vagal afferent activation. Endocrinology 2007;148:2878–85. [9] Bingham NC, Anderson KK, Reuter AL, Stallings NR, Parker KL. Selective loss of leptin receptors in the ventromedial hypothalamic nucleus results in increased adiposity and a metabolic syndrome. Endocrinology 2008;149:2138–48.
435
[10] Doring H, Schwarzer K, Nuesslein-Hildesheim B, Schmidt I. Leptin selectively increases energy expenditure of food-restricted lean mice. Int J Obes Relat Metab Disord 1998;22:83–8. [11] Gavrilova O, Leon LR, Marcus-Samuels B, Mason MM, Castle AL, Refetoff S, et al. Torpor in mice is induced by both leptin-dependent and -independent mechanisms. Proc Natl Acad Sci U S A 1999;96:14623–8. [12] Rosenbaum M, Murphy EM, Heymsfield SB, Matthews DE, Leibel RL. Low dose leptin administration reverses effects of sustained weight-reduction on energy expenditure and circulating concentrations of thyroid hormones. J Clin Endocrinol Metab 2002;87:2391–4. [13] Freeman DA, Lewis DA, Kauffman AS, Blum RM, Dark J. Reduced leptin concentrations are permissive for display of torpor in Siberian hamsters. Am J Physiol Regul Integr Comp Physiol 2004;287:R97–R103. [14] Montez JM, Soukas A, Asilmaz E, Fayzikhodjaeva G, Fantuzzi G, Friedman JM. Acute leptin deficiency, leptin resistance, and the physiologic response to leptin withdrawal. Proc Natl Acad Sci U S A 2005;102:2537–42. [15] Faggioni R, Feingold KR, Grunfeld C. Leptin regulation of the immune response and the immunodeficiency of malnutrition. FASEB J 2001;15:2565–71. [16] Fantuzzi G. Adipose tissue, adipokines, and inflammation. J Allergy Clin Immunol 2005;115:911–9. [17] Steiner AA, Romanovsky AA. Leptin: at the crossroads of energy balance and systemic inflammation. Prog Lipid Res 2007;46:89–107. [18] Luheshi GN, Rummel C. Is programming of weight regulation immune to neonatal inflammation? Am J Physiol Regul Integr Comp Physiol 2007;293:R578–80. [19] Bartfai T, Sanchez-Alavez M, Andell-Jonsson S, Schultzberg M, Vezzani A, Danielsson E, et al. Interleukin-1 system in CNS stress: seizures, fever, and neurotrauma. Ann N Y Acad Sci 2007;1113:173–7. [20] Fabricio AS, Tringali G, Pozzoli G, Melo MC, Vercesi JA, Souza GE, et al. Interleukin-1 mediates endothelin-1-induced fever and prostaglandin production in the preoptic area of rats. Am J Physiol Regul Integr Comp Physiol 2006;290:R1515–23. [21] Pittman QJ. Endothelin—an emerging role in proinflammatory pathways in brain. Am J Physiol Regul Integr Comp Physiol 2006;290:R162–3. [22] Luheshi GN, Gardner JD, Rushforth DA, Loudon AS, Rothwell NJ. Leptin actions on food intake and body temperature are mediated by IL-1. Proc Natl Acad Sci U S A 1999;96:7047–52. [23] Turek VF. The effects of melanocortin agonists and antagonists on leptin-induced fever in rats. J Therm Biol 2004;29:423–30. [24] Kelly JF, Elias CF, Lee CE, Ahima RS, Seeley RJ, Bjorbaek C, et al. Ciliary neurotrophic factor and leptin induce distinct patterns of immediate early gene expression in the brain. Diabetes 2004;53:911–20. [25] Maffei M, Halaas J, Ravussin E, Pratley RE, Lee GH, Zhang Y, et al. Leptin levels in human and rodent: measurement of plasma leptin and ob RNA in obese and weight-reduced subjects. Nat Med 1995;1:1155–61. [26] Considine RV, Sinha MK, Heiman ML, Kriauciunas A, Stephens TW, Nyce MR, et al. Serum immunoreactive-leptin concentrations in normal-weight and obese humans. N Engl J Med 1996;334:292–5. [27] Li Z, Perlik V, Feleder C, Tang Y, Blatteis CM. Kupffer cell-generated PGE2 triggers the febrile response of guinea pigs to intravenously injected LPS. Am J Physiol Regul Integr Comp Physiol 2006;290:R1262–70. [28] Bibby DC, Grimble RF. Temperature and metabolic changes in rats after various doses of tumour necrosis factor α. J Physiol 1989;410:367–80. [29] Nagai M, Saigusa T, Shimada Y, Inagawa H, Oshima H, Iriki M. Antibody to tumor necrosis factor (TNF) reduces endotoxin fever. Experientia 1988;44:606–7. [30] Roth J, Martin D, Storr B, Zeisberger E. Neutralization of pyrogen-induced tumour necrosis factor by its type 1 soluble receptor in guinea-pigs: effects on fever and interleukin-6 release. J Physiol 1998;509:267–75. [31] Leon LR. Cytokine regulation of fever: studies using gene knockout mice. J Appl Physiol 2002;92:2648–55. [32] Ogawa Y, Masuzaki H, Isse N, Okazaki T, Mori K, Shigemoto M, et al. Molecular cloning of rat obese cDNA and augmented gene expression in genetically obese Zucker fatty (fa/fa) rats. J Clin Invest 1995;96:1647–52. [33] Muller G, Ertl J, Gerl M, Preibisch G. Leptin impairs metabolic actions of insulin in isolated rat adipocytes. J Biol Chem 1997;272:10585–93. [34] Haynes WG, Morgan DA, Walsh SA, Mark AL, Sivitz WI. Receptor-mediated regional sympathetic nerve activation by leptin. J Clin Invest 1997;100:270–8. [35] McCowen KC, Chow JC, Smith RJ. Leptin signaling in the hypothalamus of normal rats in vivo. Endocrinology 1998;139:4442–7. [36] Morrison SF. Activation of 5-HT1A receptors in raphe pallidus inhibits leptinevoked increases in brown adipose tissue thermogenesis. Am J Physiol Regul Integr Comp Physiol 2004;286:R832–7. [37] Wang LC. Modulation of maximum thermogenesis by feeding in the white rat. J Appl Physiol 1980;49:975–8. [38] Steiner AA, Dogan MD, Ivanov AI, Patel S, Rudaya AY, Jennings DH, et al. A new function of the leptin receptor: mediation of the recovery from lipopolysaccharide-induced hypothermia. FASEB J 2004;18:1949–51. [39] Romanovsky AA, Almeida MC, Aronoff DM, Ivanov AI, Konsman JP, Steiner AA, et al. Fever and hypothermia in systemic inflammation: recent discoveries and revisions. Front Biosci 2005;10:2193–216. [40] Blumberg MS, Deaver K, Kirby RF. Leptin disinhibits nonshivering thermogenesis in infants after maternal separation. Am J Physiol 1999;276:R606–10. [41] Kluger MJ. Fever: role of pyrogens and cryogens. Physiol Rev 1991;71:93–127. [42] Sindelar DK, Havel PJ, Seeley RJ, Wilkinson CW, Woods SC, Schwartz MW. Low plasma leptin levels contribute to diabetic hyperphagia in rats. Diabetes 1999;48:1275–80. [43] Wang T, Hartzell DL, Rose BS, Flatt WP, Hulsey MG, Menon NK, et al. Metabolic responses to intracerebroventricular leptin and restricted feeding. Physiol Behav 1999;65:839–48.
436
A.A. Steiner et al. / Physiology & Behavior 97 (2009) 430–436
[44] Johnson JD, O'Connor KA, Hansen MK, Watkins LR, Maier SF. Effects of prior stress on LPS-induced cytokine and sickness responses. Am J Physiol Regul Integr Comp Physiol 2003;284:R422–32. [45] Loffreda S, Yang SQ, Lin HZ, Karp CL, Brengman ML, Wang DJ, et al. Leptin regulates proinflammatory immune responses. FASEB J 1998;12:57–65. [46] Zarkesh-Esfahani H, Pockley G, Metcalfe RA, Bidlingmaier M, Wu Z, Ajami A, et al. High-dose leptin activates human leukocytes via receptor expression on monocytes. J Immunol 2001;167:4593–9. [47] Wong CK, Cheung PF, Lam CW. Leptin-mediated cytokine release and migration of eosinophils: implications for immunopathophysiology of allergic inflammation. Eur J Immunol 2007;37:2337–48. [48] Hotamisligil GS, Shargill NS, Spiegelman BM. Adipose expression of tumor necrosis factor-α: direct role in obesity-linked insulin resistance. Science 1993;259:87–91. [49] Mastronardi CA, Yu WH, Rettori V, McCann S. Lipopolysaccharide-induced leptin release is not mediated by nitric oxide, but is blocked by dexamethasone. Neuroimmunomodulation 2000;8:91–7. [50] Mastronardi CA, Srivastava V, Yu WH, Les Dees W, McCann SM. Lipopolysaccharide-induced leptin synthesis and release are differentially controlled by alphamelanocyte-stimulating hormone. Neuroimmunomodulation 2005;12:182–8. [51] Bik W, Wolinska-Witort E, Chmielowska M, Rusiecka-Kuczalek E, Baranowska B. Does leptin modulate immune and endocrine response in the time of LPS-induced acute inflammation? Neuro Endocrinol Lett 2001;22:208–14. [52] Faggioni R, Fuller J, Moser A, Feingold KR, Grunfeld C. LPS-induced anorexia in leptin-deficient (ob/ob) and leptin receptor-deficient (db/db) mice. Am J Physiol 1997;273:R181–6. [53] Faggioni R, Fantuzzi G, Gabay C, Moser A, Dinarello CA, Feingold KR, et al. Leptin deficiency enhances sensitivity to endotoxin-induced lethality. Am J Physiol 1999;276:R136–42. [54] Mancuso P, Gottschalk A, Phare SM, Peters-Golden M, Lukacs NW, Huffnagle GB. Leptin-deficient mice exhibit impaired host defense in Gram-negative pneumonia. J Immunol 2002;168:4018–24. [55] O'Connor JC, Satpathy A, Hartman ME, Horvath EM, Kelley KW, Dantzer R, et al. IL1β-mediated innate immunity is amplified in the db/db mouse model of type 2 diabetes. J Immunol 2005;174:4991–7.
[56] Ivanov AI, Romanovsky AA. Fever responses of Zucker rats with and without fatty mutation of the leptin receptor. Am J Physiol Regul Integr Comp Physiol 2002;282: R311–6. [57] Niimi M, Sato M, Yokote R, Tada S, Takahara J. Effects of central and peripheral injection of leptin on food intake and on brain Fos expression in the Otsuka Long– Evans Tokushima Fatty rat with hyperleptinaemia. J Neuroendocrinol 1999;11: 605–11. [58] Ivanov AI, Kulchitsky VA, Romanovsky AA. Does obesity affect febrile responsiveness? Int J Obes Relat Metab Disord 2001;25:586–9. [59] Ivanov AI, Kulchitsky VA, Romanovsky AA. Role for the cholecystokinin-A receptor in fever: a study of a mutant rat strain and a pharmacological analysis. J Physiol 2003;547:941–9. [60] Rummel C, Inoue W, Sachot C, Poole S, Hubschle T, Luheshi GN. Selective contribution of interleukin-6 and leptin to brain inflammatory signals induced by systemic LPS injection in mice. J Comp Neurol 2008;511:373–95. [61] Sachot C, Poole S, Luheshi GN. Circulating leptin mediates lipopolysaccharideinduced anorexia and fever in rats. J Physiol 2004;561:263–72. [62] Harden LM, du Plessis I, Poole S, Laburn HP. Interleukin-6 and leptin mediate lipopolysaccharide-induced fever and sickness behavior. Physiol Behav 2006;89: 146–55. [63] Gordon CJ. Thermal biology of the laboratory rat. Physiol Behav 1990;47:963–91. [64] Romanovsky AA, Ivanov AI, Shimansky YP. Ambient temperature for experiments in rats: a new method for determining the zone of thermal neutrality. J Appl Physiol 2002;92:2667–79. [65] Szekely M, Romanovsky AA. Pyretic and antipyretic signals within and without fever: a possible interplay. Med Hypotheses 1998;50:213–8. [66] Romanovsky AA. Do fever and anapyrexia exist? Analysis of set point-based definitions. Am J Physiol Regul Integr Comp Physiol 2004;287:R992–5. [67] Pini M, Sennello JA, Chan L, Fantuzzi G. Adiponectin deficiency does not affect the inflammatory response to endotoxin or concanavalin a in mice. Endocrinology 2006;147:5019–22. [68] Teoh H, Quan A, Bang KW, Wang G, Lovren F, Vu V, et al. Adiponectin deficiency promotes endothelial activation and profoundly exacerbates sepsis-related mortality. Am J Physiol Endocrinol Metab 2008;295:E658–64.
Contents lists available at ScienceDirect
Physiology & Behavior j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / p h b
A reappraisal on the ability of leptin to induce fever Alexandre A. Steiner a,b,⁎, Catherine M. Krall a,b, Elaine Liu a a b
Department of Pharmaceutical Sciences, Albany College of Pharmacy and Health Sciences, Albany, NY, USA Research Service, Stratton Veterans Affairs Medical Center, Albany, NY, USA
a r t i c l e
i n f o
Article history: Received 23 January 2009 Received in revised form 13 March 2009 Accepted 16 March 2009 Keywords: Fever Body temperature Inflammation Cytokines Leptin Adipose tissue
a b s t r a c t Leptin is often regarded as a mediator of fever, even though an in-depth analysis of the dose-dependent effects of leptin on body temperature (Tb), pro-inflammatory cytokines, and circulating leptin has never been performed. In the present study, such an analysis was performed in rats that were food deprived (lower baseline levels of leptin) or free feeding (higher baseline levels of leptin). In a relatively cool environment (22 °C), rats deprived of food for 24 h exhibited mild (~0.5 °C) hypothermia. Leptin infusion (250 μg/kg iv) elevated the Tb of the food-deprived rats to a normothermic level, an effect that peaked (120 min postinfusion) when plasma leptin was at a level (~ 8 ng/mL) often found in leptin-responsive subjects. Increasing the leptin dose to 1000 μg/kg did not produce any further (febrile) elevation in the Tb of food-deprived rats. The anti-hypothermic effect of leptin in food-deprived rats was not associated with any rise in the plasma levels of the pro-inflammatory cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-6. In freefeeding rats kept in a cooler (22 °C) or warmer (28 °C) environment, leptin infusion failed to alter Tb or to produce any surge in plasma TNF-α or IL-6, even when the dose infused (3500 μg/kg iv) resulted in excessive, non-physiological rises in plasma leptin (~ 542 ng/mL at 30 min; ~ 75 ng/mL at 120 min postinfusion). In contrast, free-feeding rats in the same experimental set-up were able to respond to a low dose (2 μg/kg iv) of IL-1β with a typical biphasic fever, which was associated with surges in plasma TNF-α and IL-6. Collectively, our data show that an acute rise in plasma leptin to a level within or fairly above the physiological range does not induce fever. These results challenge the idea that leptin may be a mediator of fever. © 2009 Elsevier Inc. All rights reserved.
1. Introduction Leptin is an adipocyte-derived hormone known to play critical roles in energy homeostasis [1–4]. In the fed state, tonic stimulation of hypothalamic structures by leptin promotes satiety and metabolic energy expenditure [5–9]. In the fasted or food-deprived state, decreased leptin production contributes to suppression of satiety and energy expenditure [10–14]. In addition to its classical roles in energy homeostasis, leptin may modulate immune function [15–17], but the exact roles of leptin in immunomodulation and immune-brain communication are far from being defined. Leptin is often [18–21] referred to as a pro-inflammatory cytokine that can induce a febrile rise in deep body temperature (Tb). This view is largely based on a study by Luheshi et al. [22], in which injection of recombinant leptin induced a cytokine-mediated fever in rats. Although this effect has been reproduced in one study [23], it has not been universally found [24]. Even if leptin can, in fact, induce fever, the question as to whether leptin-induced fever is associated with physiologically relevant rises
in the circulating level of leptin has never been addressed. (Physiologically relevant concentrations of leptin in the plasma range from 0.5 to 20 ng/mL in lean subjects [25,26] known to be responsive to leptin [10–13].) In order to determine whether acute, physiologically relevant rises in circulating leptin are capable of inducing pro-inflammatory cytokines and fever, we simultaneously evaluated the dose-dependent effects of leptin on Tb, pro-inflammatory cytokines, and circulating leptin in rats that were food deprived (lower baseline levels of leptin) or free feeding (higher baseline levels of leptin). The pro-inflammatory cytokines measured were tumor necrosis factor (TNF)-α and interleukin (IL)-6. TNF-α is the earliest cytokine to surge in the circulation after an immune challenge [27], it has the ability to induce fever at low doses [28], and it may mediate the fever induced by low doses of bacterial products [29,30]. IL-6 surges in the circulation subsequently to TNF-α [27], and it has been shown to mediate fever in a variety of experimental models [31]. 2. Materials and methods
⁎ Corresponding author. Department of Pharmaceutical Sciences, Albany College of Pharmacy & Health Sciences, 106 New Scotland Avenue, Albany, NY 12208, USA. Tel.: +518 694 7154; fax: +518 694 7037. E-mail address: [email protected] (A.A. Steiner). 0031-9384/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2009.03.018
2.1. Animals Male Wistar rats were obtained from the Charles River Laboratories plant in Raleigh, NC, USA. Rats were initially caged in pairs; after
A.A. Steiner et al. / Physiology & Behavior 97 (2009) 430–436
surgery, they were caged individually. The rat colony room was maintained at a temperature of 21–24 °C and a 12:12 h light-dark cycle (lights on at 07:00 AM). Each rat was handled 5 min per day for 8 days; during a handling session, a rat was habituated to being dressed with an infusion harness that was used later in the experiments. The rats weighed 290–360 g at the time of the experiments. Each rat was used in an experiment once and euthanized with sodium pentobarbital (100 mg/kg iv) immediately thereafter. All procedures were conducted under a protocol approved by the Animal Care and Use Committee of the Stratton Veterans Affairs Medical Center. 2.2. Surgical preparation Five days before an experiment, rats were subjected to one or more of the following surgical procedures: intravenous catheterization, intra-arterial catheterization, and/or implantation of miniature temperature datalogger. All procedures were performed aseptically under anesthesia (ketamine–xylazine–acepromazine, 80:5:1 mg/kg ip) and antibiotic protection (enrofloxacin, 1.1 mg/kg sc). Ketamine was obtained from Bioniche Pharma (Lake Forest, IL, USA), xylazinefrom IVX Animal Health (St. Joseph, MO, USA), acepromazine from Boehringer Ingelheim Vetmedica (St. Joseph, MO, USA), and enrofloxacin from Bayer Animal Health (Shawnee Mission, KS, USA). Rats were maintained on an operating board warmed to 37 °C by a Deltaphase isothermal pad (Braintree Scientific, Braintree, MA, USA). For intravenous catheterization, a small longitudinal incision was made on the right ventral surface of the neck. The right jugular vein was exposed, freed from its surrounding connective tissue, and ligated. A 3-Fr polyurethane catheter (Instech Laboratories, Plymouth Meeting, PA, USA) filled with heparinized (50 U/mL) saline was passed into the superior vena cava through the jugular vein and secured in place with ligatures. The distal end of the catheter was closed with a stainless steel plug, tunneled under the skin, and exteriorized at the nape. The intravenous catheter was used for drug administration during an experiment. For intra-arterial catheterization, the right carotid artery was isolated and clamped by a microclip. The tip of a PE-50 catheter filled with heparinized saline was placed into the artery, the clip was removed, and the catheter was moved towards the aorta. The catheter was secured in place with ligatures. The distal end of the catheter was heat-sealed and exteriorized at the nape. The intra-arterial catheter was used for blood collection during an experiment. For datalogger implantation, a midline laparotomy was performed and a spacer was used to keep the abdominal muscles separated. The rat was then positioned in lateral decubitus, and a SubCue miniature datalogger (Calgary, AB, Canada) was attached to the internal side of the dorsolateral abdominal wall with 4-0 silk sutures. The dataloggers recorded abdominal temperature (an index of Tb) every 2 min. After the surgical wounds were sutured with 4-0 silk, rats were transferred to an environmental chamber (model NQ1; Environmental Growth Chambers, Chagrin Falls, OH, USA) set to 28.0 °C, where they remained until complete recovery from anesthesia (3–4 h postsurgery). The intravenous catheters were flushed with heparinized saline every other day; the intra-arterial catheters were flushed daily. 2.3. Experimental set-up A rat in its home cage was placed in the environmental chamber (Environmental Growth Chambers), inside which the thermal environment was controlled: the ambient temperature was maintained at 22.0 ± 0.1 °C or 28.0 ± 0.1 °C depending on the experiment (see below), whereas relative air humidity was always kept at 50 ± 5%. The chamber contained fluorescent lamps that operated according to the light-dark cycle. After the rat was equipped with a Covance Infusion Harness (Instech Laboratories), the intravenous catheter was extended with a length of PE-50 tubing filled with saline, and the
431
extension was passed through a stainless steel spring that linked the harness to a swivel (Instech Laboratories) mounted on the top of the cage. The catheter extension was connected to the bottom portion of the swivel. Another saline-filled PE-50 extension was connected to the top portion of the swivel, passed through a port in the chamber wall, and connected to the infusion syringe located outside of the chamber. The dead volume of the infusion line was 0.4 mL. If present, the intraarterial catheter was extended with a length of PE-50 tubing filled with saline. The arterial extension was passed through the harness and spring but not connected to the swivel; the distal end of the extension was plugged and attached with adhesive tape to the outer surface of the spring, just below the swivel. At the time of blood collection, the arterial line was accessed by briefly (b2 min) opening the chamber door. During this period, the ambient temperature fluctuated by no more than 0.5 °C, and the rats did not display any behavioral sign of stress such as startle responses, ‘freezing’ behavior, or abrupt changes in exploratory behavior. 2.4. Experimental drugs Recombinant mouse leptin (endotoxin residue b0.01%) was a gift from Amylin Pharmaceuticals (San Diego, CA, USA). The mouse leptin has 96% sequence identity with the rat leptin [32], and it is fully able to activate leptin receptors in rats [33–35]. A stock solution of leptin (5000 μg/mL) in saline was aliquoted and stored at − 80 °C. Immediately before an infusion, the stock was warmed to room temperature and diluted with saline to a final concentration of 50, 125, 500, or 1750 μg/mL. As in previous studies [34,36], the leptin solution was administered intravenously as a bolus injection of 1 mL/kg followed by an infusion of 1 mL/kg over 1 h, so that the total dose of leptin administered was 100, 250, 1000, or 3500 μg/kg. Recombinant rat IL-1β (endotoxin residue b0.01%) purchased from Sigma-Aldrich (St. Louis, MO, USA) was also employed in the study. Aliquots of the IL-1β stock solution (200 μg/mL) were stored at −80 °C. The aliquots were warmed to room temperature, diluted with saline to a concentration of 2 μg/mL, and the resulting solution was bolus injected intravenously (1 mL/kg), to deliver an IL-1β dose of 2 μg/kg. 2.5. Experimental design In Experiment 1, the biological activity of the leptin preparation was assessed based on the established effects of leptin on feeding behavior [1–4]. Rats implanted with intravenous catheters were habituated to consuming powdered chow from spill-free feeding chambers (Nalgene Labware, Rochester, NY, USA) for 4 days. The rats were then placed in the environmental chamber at an ambient temperature (22 °C) to which they had been exposed during housing; the rats stayed in the chamber until the end of the experiment. After overnight habituation to the chamber, the rats were food deprived for 24 h (starting at 10 AM); an elevated grid floor was placed inside the cage to prevent coprophagy; drinking water was available ad libitum. During the last hour of the food-deprivation period, the rats were infused with leptin or saline, as described above. The feeding chambers containing powdered chow were then made available for 1 h, during which the number of feeding episodes (“visits” to feeding chamber) was recorded using infrared monitors (Respironics-MiniMitter, Bend, OR, USA). The mass of chow consumed was also measured. Experiment 2 was conducted to determine if leptin can induce fever in food-deprived rats. Because the influence of food deprivation on Tb is best viewed when ambient temperature is subneutral [37], this experiment was conducted at an ambient temperature of 22 °C, which is known to be subneutral for rats in a virtually identical experimental setting [38]. Rats previously implanted with intravenous catheters and temperature dataloggers were placed in the environmental chamber (22 °C). After overnight habituation, the rats were
432
A.A. Steiner et al. / Physiology & Behavior 97 (2009) 430–436
food deprived (starting at 10 AM); an elevated grid floor was placed inside the cage to prevent coprophagy; drinking water was available ad libitum. During the 24th hour of food deprivation, leptin or saline were infused. The rats then continued without access to food for 4 additional hours, after which they were euthanized and the Tb data stored in the dataloggers were transferred to a computer. A separate set of rats was used for determination of the plasma levels of TNF-α, IL-6, and leptin. These rats had been implanted with intravenous and intra-arterial catheters but not dataloggers. They were subjected to the same experimental procedures, and arterial blood samples were collected 30 and 120 min after the end of the leptin/saline infusion; these time points were selected based on the dynamics of the Tb responses (see Results for details). Experiment 3 was conducted to determine if leptin can induce fever in free-feeding rats. The design was identical to that of Experiment 2, except that rats in Experiment 3 were not food deprived. Additionally, Experiment 3 was conducted not only at 22 °C but also at 28 °C, since the febrile response of free-feeding rats to bacterial products and cytokines may be more manifest when the environment is warmer [39]. In Experiment 4, we sought to show that a typical febrigenic cytokine, IL-1β, induces fever in our experimental setting. Rats previously implanted with intravenous catheters and temperature dataloggers were placed in the environmental chamber at an ambient temperature of 22 °C, where they had ad libitum access to chow and water for the entire experiment. After overnight habituation, the rats were intravenously bolus injected with IL-1β or saline. Four hours later, the rats were euthanized, and the Tb data stored in the dataloggers were transferred to a computer. A separate set of rats was used for determination of the plasma levels of TNF-α and IL-6. These rats had been implanted with intravenous and intra-arterial catheters but not dataloggers. Arterial blood samples were collected 30 and 90 min after the IL-1β/saline injection; these time points were selected based on the dynamics of the Tb responses (see Results for details). 2.6. Blood processing and assays Each blood sample (0.5 mL) was collected into a 1-mL syringe and immediately transferred to an EDTA-coated Eppendorf tube. The tube was centrifuged (6000 ×g, 10 min, 4 °C), and the resulting plasma was stored at − 80 °C. The levels of TNF-α, IL-6, and leptin in the plasma samples were determined using Quantikine enzyme immunoassays (R&D Systems, Minneapolis, MN, USA). In each assay, all samples were run simultaneously, in duplicate. The detection limits of the assays were 5 pg/mL for TNF-α, 21 pg/mL for IL-6, and 22 pg/mL for leptin. 2.7. Data analyses Tb and plasma leptin values were compared across treatments and time points by two-way ANOVA. The results of the TNF-α or IL-6 assays were analyzed based on Chi-square comparisons of the number of samples containing detectable/non-detectable levels of these cytokines. The number of feeding episodes and the mass of food consumed were compared across treatments by one-way ANOVA. All analyses were performed using Statistica Advanced 8.0 (StatSoft, Tulsa, OK, USA). The level of significance was set at P b 0.05. Data are reported as means ± SE. 3. Results 3.1. Experiment 1: Effects of leptin on feeding behavior (biological activity test) During the first hour following a 24-h food deprivation and a 1-h intravenous infusion of saline, feeding behavior was characterized by an average of 65 (±6) feeding episodes and 5.4 (±0.6) grams of chow consumed (Fig. 1). Compared to saline, leptin infusion reduced
Fig. 1. Effects of intravenous leptin (doses indicated) or its vehicle (saline) on feeding parameters of rats that were offered food for 1 h after a 24-h food-deprivation period. The rats were maintained at an ambient temperature of 22 °C. The number of animals (n) in each group is indicated. ⁎Significant (P b 0.05) difference in relation to the salineinfused controls.
the number of feeding episodes and the amount of chow consumed in a dose-dependent manner. Whereas the dose of 100 μg/kg was ineffective, doses of 250 and 1000 μg/kg significantly reduced the number of feeding episodes by 29% and 71% and the mass of food consumed by 24% and 40%, respectively. These results confirmed the biological activity of the leptin preparation used and identified the lowest active dose (250 μg/kg) of leptin in our experimental setting. 3.2. Experiment 2: Effects of leptin on the Tb and plasma cytokines of food-deprived rats At an ambient temperature of 22 °C, food deprivation (24 h) resulted in a small (~ 0.5 °C), yet significant decrease in Tb (Fig. 2). The food deprivation-associated hypothermia was unaffected by intravenous infusion of saline or leptin at 100 μg/kg. However, leptin at 250 μg/kg gradually raised the Tb of food-deprived rats to the normothermic level observed before food deprivation. This rise in Tb started at ~30 min and peaked at ~ 120 min after the end of leptin infusion. The plasma levels of TNF-α and IL-6 were not elevated at these points in time (Table 1). The plasma levels of leptin were low (0.4 ng/mL) in food-deprived rats infused with saline, but they averaged 61.7 ng/mL (30 min) and 7.8 ng/mL (120 min) during the Tb response to leptin (Table 1). A higher dose (1000 μg/kg) of leptin also reversed the hypothermia associated with food deprivation, but it did not produce any further (febrile) increase in Tb (Fig. 2). 3.3. Experiment 3: Effects of leptin on the Tb and plasma cytokines of free-feeding rats No change in Tb was observed when free-feeding rats maintained at an ambient temperature of either 22 °C or 28 °C were infused with saline or leptin at a dose as high as 3500 μg/kg (Fig. 3). The highest dose of leptin also failed to produce any detectable increase in the plasma levels of TNF-α and IL-6 (Table 2). The plasma levels of leptin averaged 542 ng/mL at 30 min and 75 ng/mL at 120 min after infusion
A.A. Steiner et al. / Physiology & Behavior 97 (2009) 430–436
433
Fig. 2. Deep body temperature of rats intravenously infused with leptin (doses indicated) or its vehicle (saline) during the 24th hour of food deprivation. The rats were kept at an ambient temperature of 22 °C. The number of animals (n) in each group is indicated. ⁎Significant (P b 0.05) difference in relation to the saline-infused controls.
of the highest leptin dose, as compared to 1.7–1.8 ng/mL after infusion of saline (Table 2).
controls. Plasma IL-6 was predominantly undetectable at 30 min. At 90 min, however, detectable levels of IL-6 were found in 5/6 of the IL1β-treated rats, as compared to 1/6 of the saline-treated controls.
3.4. Experiment 4: Thermoregulatory and cytokine responses to IL-1β 4. Discussion In view of the fact that leptin did not induce fever under any of the experimental conditions tested, it was important to demonstrate that rats in our experimental setting could develop fever in response to a typical pro-inflammatory cytokine. In free-feeding rats maintained at an ambient temperature of 22 °C, saline (iv) caused no change in Tb, whereas a low dose (2 μg/kg iv) of IL-1β triggered a biphasic fever characterized by Tb peaks at 30 and 90 min postinjection (Fig. 4). TNFα was detected in the plasma of all IL-1β-treated rats at both 30 and 90 min, but it was not detected in the plasma of saline-treated Table 1 Plasma concentrations of leptin, TNF-α, and IL-6 in food-deprived rats infused (iv) with saline or leptin. Infusion
Time post-infusion
Plasma concentration Leptin (ng/mL)
TNF-α (pg/mL)
IL-6 (pg/mL)
Saline
30 min 120 min 30 min 120 min
0.4 ± 0.1 (6) 0.4 ± 0.1 (5) 61.7 ± 4.2⁎ (5) 7.8 ± 0.6⁎ (5)
b5.0 b5.0 b5.0 b5.0
b21.0 b21.0 b21.0 b21.0
Leptin 250 μg/kg
(6) (5) (5) (5)
(6) (5) (5) (5)
The number of animals is shown in parentheses. ⁎P b 0.05 compared to saline infusion.
The present study is the first to simultaneously evaluate the dosedependent effects of leptin on Tb, pro-inflammatory cytokines, and circulating leptin. The biological activity of the leptin preparation used in our study was demonstrated by the established ability of leptin to suppress food seeking and consumption. Leptin reversed the hypothermia induced by food deprivation at an ambient temperature of 22 °C, an effect that peaked (120 min post-infusion) when plasma leptin was at a level (~8 ng/mL) often found in leptin-responsive subjects [25,26]. This observation agrees with previous studies [11,40] showing that a fall in leptin levels during food deprivation is essential for the associated suppression of basal metabolic rate and, consequently, basal Tb. Leptin administration, however, did not raise Tb to a febrile level in either food-deprived, hypothermic rats kept at 22 °C or in free-feeding, normothermic rats kept at 22 °C or 28 °C. Leptin administration also failed to produce any surge in the plasma levels of cytokines (TNF-α and IL-6) known to be involved in the pathogenesis of fever [31,41]. This was the case even when leptin was infused at a dose (3500 μg/kg iv) that resulted in excessive, non-physiological rises in the plasma levels of leptin. The inability of leptin to induce
434
A.A. Steiner et al. / Physiology & Behavior 97 (2009) 430–436
Fig. 3. Deep body temperature of free-feeding rats intravenously infused with leptin (doses indicated) or its vehicle (saline) at an ambient temperature of 22 °C or 28 °C. The number of animals (n) in each group is indicated.
fever was not due to an inability of the rats to mount a febrile response, because rats in the same experimental set-up were able to respond to a low dose of IL-1β with a typical biphasic fever associated with surges in plasma TNF-α and IL-6. Collectively, these data strongly indicate that an acute rise in plasma leptin to a level within or fairly above the physiological range does not induce fever. Whereas the present study involved a non-stressful procedure of intravenous leptin administration (as evident by the lack of stress hyperthermia in the saline-infused rats), previous studies [22,23] suggesting that leptin can cause fever in free-feeding rats employed stressful procedures of drug administration, which involved handling, temporary restraint, and needle pricking. The doses of leptin shown to cause fever in those studies were also excessively high. When administered systemically, leptin was reported to cause fever at a dose of 3.5 mg/kg [22], which we found to raise plasma leptin to supraphysiological levels for at least 120 min. When administered intracerebroventricularly, leptin was reported to cause fever at doses of 4–5 μg/rat [22,23], which are ~ 10 times higher than the intracerebroventricular doses of leptin needed to affect food intake and energy expenditure [42,43]. Therefore, it is possible that, in association with stress, excessively high doses of leptin might cause fever. Indeed, stress has been reported to enhance inflammatory signaling to the brain [44]. But, anyhow, the effects of excessively high doses of leptin are unlikely to reflect a physiological action of leptin. At persistently (6–24 h) high levels (N50 ng/mL), leptin has been shown to stimulate leukocytes to produce pro-inflammatory cytokines in vitro [45–47]. This action may be relevant for the chronic, lowgrade inflammation seen in obese subjects [48]. However, the same action is unlikely to be relevant for the acute inflammatory response to microbial agents such as bacterial lipopolysaccharide (LPS). Even though circulating leptin can be increased as early as 1 h after administration of LPS [49,50], we presently report that rapid rises in plasma leptin following leptin infusion do not lead to surges in plasma TNF-α and IL-6. In addition, Bik et al. [51] have found no effect of leptin injection on the cytokine response to LPS. Furthermore, it has
been demonstrated that mutant rats and mice that lack leptin or leptin receptors are fully capable to produce pro-inflammatory cytokines in response to LPS [38,52–55]. Some of these mutants (Zucker fa/fa rats and Koletsky f/f rats) have also been shown to respond to LPS with normal fevers in experiments conducted in a warm (28–29 °C) environment [38,56]. Cholecystokinin-1-receptor-deficient rats, which are at least partially leptin-resistant [57], also respond to LPS with fever [58,59]. Corroborating these findings, a recent study has shown that an anti-leptin antibody does not alter the LPS-induced expression of key enzymes (clyclooxygenase-2 and microsomal prostaglandin E synthase-1) involved in the mediation of fever [60]. Since leptin does not seem to activate febrigenic inflammatory signaling, it is important to seek alternative interpretations for reports that leptin neutralization with an antibody attenuates LPS fever in rats
Table 2 Plasma concentrations of leptin, TNF-α, and IL-6 in free-feeding rats infused (iv) with saline or leptin. Infusion
Time post-infusion
Plasma concentration Leptin (ng/mL)
TNF-α (pg/mL)
IL-6 (pg/mL)
Saline
30 min 120 min 30 min 120 min
1.7 ± 0.4 (7) 1.8 ± 0.6 (7) 542.1 ± 62.9 (5)⁎ 75.2 ± 12.6 (5)⁎
b5.0 b5.0 b5.0 b5.0
b21.0 b21.0 b21.0 b21.0
Leptin 3500 μg/kg
(6) (5) (5) (5)
(6) (5) (5) (5)
The number of animals is shown in parentheses. ⁎P b 0.05 compared to saline infusion.
Fig. 4. Effects of intravenous IL-1β (dose indicated) or its vehicle (saline) on the deep body temperature and plasma cytokine levels of free-feeding rats. The rats were maintained at an ambient temperature of 22 °C. The number of animals (n) in each group is indicated. ⁎Significant (P b 0.05) difference in relation to the saline-infused controls.
A.A. Steiner et al. / Physiology & Behavior 97 (2009) 430–436
kept at ambient temperatures of 21–22 °C [61,62]. At these mildly cool ambient temperatures for rats [63,64], the thermoregulatory response to LPS depends on a balance between the actions of febrigenic (increase Tb) and cryogenic (decrease Tb) inflammatory mediators [41,65]. It is, thus, possible that attenuation of fever in the absence of leptin signaling results from overactivation of cryogenic signaling. Because cryogenic signaling lowers Tb by suppressing thermogenesis [66], it does not influence Tb in a warm environment, in which thermogenesis is at its minimum and cannot be further inhibited [66]. Hence, the fact that mutant rats lacking functional leptin receptors respond to LPS with an attenuated fever in a cool environment but with normal fever in a warm environment [38,56] is consistent with an involvement of leptin in cryogenic signaling. Also supporting an involvement of leptin in cryogenic signaling is the finding that the hypothermia induced by a high dose of LPS in a cool environment is largely prolonged in leptin receptor-deficient rats [38]. Such a prolongation of hypothermia was associated with an exaggerated rise in plasma TNF-α [38], a pro-inflammatory cytokine that is febrigenic at lower doses but becomes cryogenic at higher doses [28]. It was also associated with an inability of the rats to activate the antiinflammatory hypothalamo-pituitary-adrenal axis [38]. We have recently proposed that modulation of cryogenic signaling by leptin may link energy balance to host defense [17], but future studies are warranted to address this possibility. Future studies should also investigate whether the crosstalk between energy balance and host defense may involve other adipocyte-derived hormones such as adiponectin [67,68]. In conclusion, the present study shows that non-stressful, acute administration of leptin does not induce pro-inflammatory cytokines and fever in rats, regardless of whether the rats are food deprived (lower baseline levels of leptin) or free feeding (higher baseline levels of leptin), and regardless of the ambient temperature. These results challenge the view that leptin may be a mediator of fever. Acknowledgement We are thankful to Drs. Andrej A. Romanovsky (St. Joseph's Hospital, Phoenix, AZ) and Luciana B. Lopes (Albany College of Pharmacy & Health Sciences, Albany, NY) for their critical comments on the manuscript. The study was supported in part by an Albany College of Pharmacy Scholarship of Discovery Grant to AAS. Recombinant leptin was generously provided by Amylin Pharmaceuticals (San Diego, CA). EL was the recipient of an Albany College of Pharmacy Summer Scholarship Award. References [1] Friedman JM, Halaas JL. Leptin and the regulation of body weight in mammals. Nature 1998;395:763–70. [2] Elmquist JK, Maratos-Flier E, Saper CB, Flier JS. Unraveling the central nervous system pathways underlying responses to leptin. Nat Neurosci 1998;1:445–50. [3] Ahima RS, Flier JS. Leptin. Annu Rev Physiol 2000;62:413–37. [4] Havel PJ. Update on adipocyte hormones: regulation of energy balance and carbohydrate/lipid metabolism. Diabetes 2004;53:S143–51. [5] de Luca C, Kowalski TJ, Zhang Y, Elmquist JK, Lee C, Kilimann MW, et al. Complete rescue of obesity, diabetes, and infertility in db/db mice by neuron-specific LEPR-B transgenes. J Clin Invest 2005;115:3484–93. [6] Coppari R, Ichinose M, Lee CE, Pullen AE, Kenny CD, McGovern RA, et al. The hypothalamic arcuate nucleus: a key site for mediating leptin's effects on glucose homeostasis and locomotor activity. Cell Metab 2005;1:63–72. [7] Scarpace PJ, Matheny M, Zhang Y, Cheng KY, Tumer N. Leptin antagonist reveals an uncoupling between leptin receptor signal transducer and activator of transcription 3 signaling and metabolic responses with central leptin resistance. J Pharmacol Exp Ther 2007;320:706–12. [8] Peters JH, Simasko SM, Ritter RC. Leptin analog antagonizes leptin effects on food intake and body weight but mimics leptin-induced vagal afferent activation. Endocrinology 2007;148:2878–85. [9] Bingham NC, Anderson KK, Reuter AL, Stallings NR, Parker KL. Selective loss of leptin receptors in the ventromedial hypothalamic nucleus results in increased adiposity and a metabolic syndrome. Endocrinology 2008;149:2138–48.
435
[10] Doring H, Schwarzer K, Nuesslein-Hildesheim B, Schmidt I. Leptin selectively increases energy expenditure of food-restricted lean mice. Int J Obes Relat Metab Disord 1998;22:83–8. [11] Gavrilova O, Leon LR, Marcus-Samuels B, Mason MM, Castle AL, Refetoff S, et al. Torpor in mice is induced by both leptin-dependent and -independent mechanisms. Proc Natl Acad Sci U S A 1999;96:14623–8. [12] Rosenbaum M, Murphy EM, Heymsfield SB, Matthews DE, Leibel RL. Low dose leptin administration reverses effects of sustained weight-reduction on energy expenditure and circulating concentrations of thyroid hormones. J Clin Endocrinol Metab 2002;87:2391–4. [13] Freeman DA, Lewis DA, Kauffman AS, Blum RM, Dark J. Reduced leptin concentrations are permissive for display of torpor in Siberian hamsters. Am J Physiol Regul Integr Comp Physiol 2004;287:R97–R103. [14] Montez JM, Soukas A, Asilmaz E, Fayzikhodjaeva G, Fantuzzi G, Friedman JM. Acute leptin deficiency, leptin resistance, and the physiologic response to leptin withdrawal. Proc Natl Acad Sci U S A 2005;102:2537–42. [15] Faggioni R, Feingold KR, Grunfeld C. Leptin regulation of the immune response and the immunodeficiency of malnutrition. FASEB J 2001;15:2565–71. [16] Fantuzzi G. Adipose tissue, adipokines, and inflammation. J Allergy Clin Immunol 2005;115:911–9. [17] Steiner AA, Romanovsky AA. Leptin: at the crossroads of energy balance and systemic inflammation. Prog Lipid Res 2007;46:89–107. [18] Luheshi GN, Rummel C. Is programming of weight regulation immune to neonatal inflammation? Am J Physiol Regul Integr Comp Physiol 2007;293:R578–80. [19] Bartfai T, Sanchez-Alavez M, Andell-Jonsson S, Schultzberg M, Vezzani A, Danielsson E, et al. Interleukin-1 system in CNS stress: seizures, fever, and neurotrauma. Ann N Y Acad Sci 2007;1113:173–7. [20] Fabricio AS, Tringali G, Pozzoli G, Melo MC, Vercesi JA, Souza GE, et al. Interleukin-1 mediates endothelin-1-induced fever and prostaglandin production in the preoptic area of rats. Am J Physiol Regul Integr Comp Physiol 2006;290:R1515–23. [21] Pittman QJ. Endothelin—an emerging role in proinflammatory pathways in brain. Am J Physiol Regul Integr Comp Physiol 2006;290:R162–3. [22] Luheshi GN, Gardner JD, Rushforth DA, Loudon AS, Rothwell NJ. Leptin actions on food intake and body temperature are mediated by IL-1. Proc Natl Acad Sci U S A 1999;96:7047–52. [23] Turek VF. The effects of melanocortin agonists and antagonists on leptin-induced fever in rats. J Therm Biol 2004;29:423–30. [24] Kelly JF, Elias CF, Lee CE, Ahima RS, Seeley RJ, Bjorbaek C, et al. Ciliary neurotrophic factor and leptin induce distinct patterns of immediate early gene expression in the brain. Diabetes 2004;53:911–20. [25] Maffei M, Halaas J, Ravussin E, Pratley RE, Lee GH, Zhang Y, et al. Leptin levels in human and rodent: measurement of plasma leptin and ob RNA in obese and weight-reduced subjects. Nat Med 1995;1:1155–61. [26] Considine RV, Sinha MK, Heiman ML, Kriauciunas A, Stephens TW, Nyce MR, et al. Serum immunoreactive-leptin concentrations in normal-weight and obese humans. N Engl J Med 1996;334:292–5. [27] Li Z, Perlik V, Feleder C, Tang Y, Blatteis CM. Kupffer cell-generated PGE2 triggers the febrile response of guinea pigs to intravenously injected LPS. Am J Physiol Regul Integr Comp Physiol 2006;290:R1262–70. [28] Bibby DC, Grimble RF. Temperature and metabolic changes in rats after various doses of tumour necrosis factor α. J Physiol 1989;410:367–80. [29] Nagai M, Saigusa T, Shimada Y, Inagawa H, Oshima H, Iriki M. Antibody to tumor necrosis factor (TNF) reduces endotoxin fever. Experientia 1988;44:606–7. [30] Roth J, Martin D, Storr B, Zeisberger E. Neutralization of pyrogen-induced tumour necrosis factor by its type 1 soluble receptor in guinea-pigs: effects on fever and interleukin-6 release. J Physiol 1998;509:267–75. [31] Leon LR. Cytokine regulation of fever: studies using gene knockout mice. J Appl Physiol 2002;92:2648–55. [32] Ogawa Y, Masuzaki H, Isse N, Okazaki T, Mori K, Shigemoto M, et al. Molecular cloning of rat obese cDNA and augmented gene expression in genetically obese Zucker fatty (fa/fa) rats. J Clin Invest 1995;96:1647–52. [33] Muller G, Ertl J, Gerl M, Preibisch G. Leptin impairs metabolic actions of insulin in isolated rat adipocytes. J Biol Chem 1997;272:10585–93. [34] Haynes WG, Morgan DA, Walsh SA, Mark AL, Sivitz WI. Receptor-mediated regional sympathetic nerve activation by leptin. J Clin Invest 1997;100:270–8. [35] McCowen KC, Chow JC, Smith RJ. Leptin signaling in the hypothalamus of normal rats in vivo. Endocrinology 1998;139:4442–7. [36] Morrison SF. Activation of 5-HT1A receptors in raphe pallidus inhibits leptinevoked increases in brown adipose tissue thermogenesis. Am J Physiol Regul Integr Comp Physiol 2004;286:R832–7. [37] Wang LC. Modulation of maximum thermogenesis by feeding in the white rat. J Appl Physiol 1980;49:975–8. [38] Steiner AA, Dogan MD, Ivanov AI, Patel S, Rudaya AY, Jennings DH, et al. A new function of the leptin receptor: mediation of the recovery from lipopolysaccharide-induced hypothermia. FASEB J 2004;18:1949–51. [39] Romanovsky AA, Almeida MC, Aronoff DM, Ivanov AI, Konsman JP, Steiner AA, et al. Fever and hypothermia in systemic inflammation: recent discoveries and revisions. Front Biosci 2005;10:2193–216. [40] Blumberg MS, Deaver K, Kirby RF. Leptin disinhibits nonshivering thermogenesis in infants after maternal separation. Am J Physiol 1999;276:R606–10. [41] Kluger MJ. Fever: role of pyrogens and cryogens. Physiol Rev 1991;71:93–127. [42] Sindelar DK, Havel PJ, Seeley RJ, Wilkinson CW, Woods SC, Schwartz MW. Low plasma leptin levels contribute to diabetic hyperphagia in rats. Diabetes 1999;48:1275–80. [43] Wang T, Hartzell DL, Rose BS, Flatt WP, Hulsey MG, Menon NK, et al. Metabolic responses to intracerebroventricular leptin and restricted feeding. Physiol Behav 1999;65:839–48.
436
A.A. Steiner et al. / Physiology & Behavior 97 (2009) 430–436
[44] Johnson JD, O'Connor KA, Hansen MK, Watkins LR, Maier SF. Effects of prior stress on LPS-induced cytokine and sickness responses. Am J Physiol Regul Integr Comp Physiol 2003;284:R422–32. [45] Loffreda S, Yang SQ, Lin HZ, Karp CL, Brengman ML, Wang DJ, et al. Leptin regulates proinflammatory immune responses. FASEB J 1998;12:57–65. [46] Zarkesh-Esfahani H, Pockley G, Metcalfe RA, Bidlingmaier M, Wu Z, Ajami A, et al. High-dose leptin activates human leukocytes via receptor expression on monocytes. J Immunol 2001;167:4593–9. [47] Wong CK, Cheung PF, Lam CW. Leptin-mediated cytokine release and migration of eosinophils: implications for immunopathophysiology of allergic inflammation. Eur J Immunol 2007;37:2337–48. [48] Hotamisligil GS, Shargill NS, Spiegelman BM. Adipose expression of tumor necrosis factor-α: direct role in obesity-linked insulin resistance. Science 1993;259:87–91. [49] Mastronardi CA, Yu WH, Rettori V, McCann S. Lipopolysaccharide-induced leptin release is not mediated by nitric oxide, but is blocked by dexamethasone. Neuroimmunomodulation 2000;8:91–7. [50] Mastronardi CA, Srivastava V, Yu WH, Les Dees W, McCann SM. Lipopolysaccharide-induced leptin synthesis and release are differentially controlled by alphamelanocyte-stimulating hormone. Neuroimmunomodulation 2005;12:182–8. [51] Bik W, Wolinska-Witort E, Chmielowska M, Rusiecka-Kuczalek E, Baranowska B. Does leptin modulate immune and endocrine response in the time of LPS-induced acute inflammation? Neuro Endocrinol Lett 2001;22:208–14. [52] Faggioni R, Fuller J, Moser A, Feingold KR, Grunfeld C. LPS-induced anorexia in leptin-deficient (ob/ob) and leptin receptor-deficient (db/db) mice. Am J Physiol 1997;273:R181–6. [53] Faggioni R, Fantuzzi G, Gabay C, Moser A, Dinarello CA, Feingold KR, et al. Leptin deficiency enhances sensitivity to endotoxin-induced lethality. Am J Physiol 1999;276:R136–42. [54] Mancuso P, Gottschalk A, Phare SM, Peters-Golden M, Lukacs NW, Huffnagle GB. Leptin-deficient mice exhibit impaired host defense in Gram-negative pneumonia. J Immunol 2002;168:4018–24. [55] O'Connor JC, Satpathy A, Hartman ME, Horvath EM, Kelley KW, Dantzer R, et al. IL1β-mediated innate immunity is amplified in the db/db mouse model of type 2 diabetes. J Immunol 2005;174:4991–7.
[56] Ivanov AI, Romanovsky AA. Fever responses of Zucker rats with and without fatty mutation of the leptin receptor. Am J Physiol Regul Integr Comp Physiol 2002;282: R311–6. [57] Niimi M, Sato M, Yokote R, Tada S, Takahara J. Effects of central and peripheral injection of leptin on food intake and on brain Fos expression in the Otsuka Long– Evans Tokushima Fatty rat with hyperleptinaemia. J Neuroendocrinol 1999;11: 605–11. [58] Ivanov AI, Kulchitsky VA, Romanovsky AA. Does obesity affect febrile responsiveness? Int J Obes Relat Metab Disord 2001;25:586–9. [59] Ivanov AI, Kulchitsky VA, Romanovsky AA. Role for the cholecystokinin-A receptor in fever: a study of a mutant rat strain and a pharmacological analysis. J Physiol 2003;547:941–9. [60] Rummel C, Inoue W, Sachot C, Poole S, Hubschle T, Luheshi GN. Selective contribution of interleukin-6 and leptin to brain inflammatory signals induced by systemic LPS injection in mice. J Comp Neurol 2008;511:373–95. [61] Sachot C, Poole S, Luheshi GN. Circulating leptin mediates lipopolysaccharideinduced anorexia and fever in rats. J Physiol 2004;561:263–72. [62] Harden LM, du Plessis I, Poole S, Laburn HP. Interleukin-6 and leptin mediate lipopolysaccharide-induced fever and sickness behavior. Physiol Behav 2006;89: 146–55. [63] Gordon CJ. Thermal biology of the laboratory rat. Physiol Behav 1990;47:963–91. [64] Romanovsky AA, Ivanov AI, Shimansky YP. Ambient temperature for experiments in rats: a new method for determining the zone of thermal neutrality. J Appl Physiol 2002;92:2667–79. [65] Szekely M, Romanovsky AA. Pyretic and antipyretic signals within and without fever: a possible interplay. Med Hypotheses 1998;50:213–8. [66] Romanovsky AA. Do fever and anapyrexia exist? Analysis of set point-based definitions. Am J Physiol Regul Integr Comp Physiol 2004;287:R992–5. [67] Pini M, Sennello JA, Chan L, Fantuzzi G. Adiponectin deficiency does not affect the inflammatory response to endotoxin or concanavalin a in mice. Endocrinology 2006;147:5019–22. [68] Teoh H, Quan A, Bang KW, Wang G, Lovren F, Vu V, et al. Adiponectin deficiency promotes endothelial activation and profoundly exacerbates sepsis-related mortality. Am J Physiol Endocrinol Metab 2008;295:E658–64.