Am J Physiol Endocrinol Metab 292: E1135-E1139, 2007.
First published December 12, 2006; doi:10.1152/ajpendo.00425.2006
0193-1849/07 $8.00
Brown fat UCP1 is not involved in the febrile and thermogenic responses to IL-1
in mice
Yuko Okamatsu-Ogura,1
Naoya Kitao,1
Kazuhiro Kimura,1 and
Masayuki Saito1,2
1Department of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido University; and 2Department of Nutrition, Graduate School of Nursing and Nutrition, Tenshi College, Sapporo, Japan
Submitted 18 August 2006
; accepted in final form 8 December 2006
 |
ABSTRACT
|
|---|
The activity of brown adipose tissue (BAT), a site of nonshivering metabolic thermogenesis, has been reported to increase after interleukin (IL)-1
/lipopolysaccharide injection. To clarify the possible contribution of BAT thermogenesis to whole body febrile response, we investigated febrile and thermogenic response to IL-1 using mice deficient in uncoupling protein-1 (UCP1), a key molecule for BAT thermogenesis. In wild-type (WT) mice, IL-1 injection (5 µg/kg ip) increased body temperature (+1.82°C at 20 min), decreased physical activity (–37% at 1 h), and produced a slight and insignificant rise (+15% at 1 h) in oxygen consumption (
O2). O2 dependent on metabolic thermogenesis (
O2 thermogenesis) calculated by correcting the effect of physical activity was increased after IL-1 injection (726 ± 200 ml·h–1·kg–1 at 1 h). Almost the same responses were observed in UCP1-deficient mice, showing 638 ± 87 ml·h–1·kg–1 of O2 thermogenesis at 1 h. In contrast, CL316,243, a selective activator of BAT thermogenesis, increased body temperature, decreased physical activity, and produced a significant rise in O2 in WT mice, showing 1,229 ± 35 ml·h–1·kg–1 of O2 thermogenesis at 1 h. These changes were not observed in UCP1-deficient mice. These results, conflicting with a previously proposed idea of a role of BAT in fever, suggest a minor contribution of BAT thermogenesis to IL-1-induced fever. In support of this, we found no effect of IL-1 on triglyceride content and UCP1 mRNA level in BAT, in contrast with apparent effects of CL316,243.
uncoupling protein-1; oxygen consumption; body temperature; knockout mouse; interleukin-1
PROINFLAMMATORY CYTOKINES, such as interleukin (IL)-1 released by activated immune cells during the host response to infection or inflammation, have potent effects on the brain. When administrated systemically or directly into the brain, these cytokines induce symptoms of illness, including fever, activation of hypothalamic-pituitary-adrenal axis, anorexia, and depressed behavior (3, 7, 12, 17, 26). Similar effects are observed when endogenous cytokines are released in response to administration of lipopolysaccharide (LPS) from gram-negative bacteria.
Fever, an increase in body temperature that occurs in response to infection or inflammation, is generated by a number of physiological processes that cause increases in both heat conservation and production (thermogenesis). The increased thermogenesis in fever was proved by the fact that IL-1 or LPS injection increases whole body oxygen consumption (O2) (2, 13, 21).
As the peripheral mechanisms responsible for the thermogenesis, the contribution of brown adipose tissue (BAT) has been suggested (5). BAT is a tissue specified for metabolic heat production and has a significant role in cold- and diet-induced thermogenesis (4, 18). BAT thermogenesis is principally dependent on the activation of uncoupling protein-1 (UCP1), which uncouples oxidative phosphorylation in mitochondria to dissipate the electrochemical gradient as heat. The activity of UCP1 is controlled by the sympathetic nerves to BAT, mainly through the -adrenergic mechanism. The activator of sympathetic nerves, -adrenergic receptor (AR), also induces lipolysis in BAT (20), and the produced fatty acids are used as substrate for UCP1 thermogenesis (14).
The involvement of BAT thermogenesis in fever was suggested first by Blatteis (2), who demonstrated endotoxin-induced and -blocker-sensitive rise in BAT temperature in guinea pigs. Subsequently, it was reported that IL-1/LPS injection increased GDP binding to BAT mitochondria, a marker of UCP1 activity (6, 16, 21). It was also shown that blood flow in BAT was increased after IL-1/LPS injection in rats (6). Although all of these previous reports suggest an activation of BAT thermogenesis during fever, its contribution to whole body thermogenic and febrile response has not been determined. Moreover, there are some studies that report conflicting roles of BAT thermogenesis in fever. For example, Jennings and Elia (15) showed that O2 is not changed despite the rise in body temperature after LPS injection in mice. We also failed to detect any stimulatory effect of IL-1 and LPS on norepinephrine turnover in BAT, a biochemical index of sympathetic nerve activity in this tissue (1, 22, 25). Thus, the contribution of BAT thermogenesis in fever is still controversial. To address this, in the present study, we investigated the febrile and thermogenic responses to IL-1, a major endogenous pyrogen, in UCP1-deficient knockout (UCP1-KO) mice and compared them with those in wild-type (WT) mice.
|
MATERIALS AND METHODS
|
|---|
Animals.
UCP1-KO (ucp1–/–) mice on a congenic background of C57BL/6J were generated by backcross matings of heterozygous (+/–) mice on a mixed 129/SvPas and C57BL/6J background with C57BL/6J mice 15 times, kindly given by Dr. L. Kozak (Pennington Biomedical Research Center, Baton Rouge, LA). All WT (ucp1+/+) mice were C57BL/6J. Mice were housed in plastic cages placed in an air-conditioned room at 26°C with a 12:12-h light-dark cycle (lights on at 0700–1900) and given free access to laboratory chow (MF; Oriental Yeast, Tokyo, Japan) and tap water. Male UCP1-KO and WT mice (10–70 wk old) weighing 20–40 g were used. All experiments were performed at 26°C. The experimental procedures and care of animals were approved by the Animal Care and Use Committee of Hokkaido University.
UCP1 mRNA induction and triglyceride content in BAT.
WT mice were injected with recombinant mouse IL-1 (5 µg/kg; PeproTech, London, UK) or PBS intraperitoneally twice at 1000 and 1300. A 3-AR agonist, CL316,243 [CL (0.1 mg/kg); American Cyanamid, Pearl River, NY], was injected once at 1000. Food was deprived at 1000. At 1600, all mice were killed by cervical dislocation, and interscapular BAT was quickly removed and weighed.
Tissue specimens were homogenized in Tris-EDTA buffer (10 mM Tris and 1 mM EDTA), pH 7.4. Triglyceride (TG) was extracted in chloroform-methanol (1:2, vol:vol), and concentration was measured enzymatically with a kit (TG test WAKO; Wako Pure Chemical, Tokyo, Japan) and expressed as milligrams TG per whole BAT.
Another tissue specimen was transferred into RNALater (Invitrogen, Carlsbad, CA). Total RNA was extracted according to the manufacturer's protocol using TRIzol (Invitrogen). UCP1 mRNA level was measured quantitatively by real-time RT-PCR using respective cDNA fragment as a standard and expressed as relative to -actin mRNA level. Briefly, 2 µg of total RNA were reverse transcribed with an oligo(dT) 15-adaptor primer and Moloney murine leukemia virus reverse transcriptase (Invitrogen). Real-time PCR was performed on a fluorescence thermal cycler (LightCycler system; Roche Diagnostics, Mannheim, Germany) using SYBR Green I (Roche Diagnostics) as a double-strand DNA-specific dye according to the manufacturer's protocol. Primers used were 5'-GTG AAG GTC AGA ATG CAA GC-3' and 5'-AGG GCC CCC TTC ATG AGG TC-3' for mouse UCP1, 5'-TCG TTAC CAC AGG CAT TGT GAT-3' and 5'-TGC TCG AAG TCT AGA GCA AC-3' for mouse -actin.
Body temperature.
Fever was induced by intraperitoneal injection of IL-1 (5 µg/kg) to WT and UCP1-KO mice. PBS and CL (0.1 mg/kg) were used in control experiments. Mice were injected with the agents between 1200 and 1400 to eliminate the influence of diurnal variations in body temperature and plasma hormonal levels.
Body temperature was measured in conscious mice by use of a telemetry system (StarMedical, Tokyo, Japan). A transmitter (10 T-T, 15.5 W x 9.0 D x 4.7 H mm; StarMedical) was implanted into the peritoneal cavity of a mouse under anesthesia with 75 mg/kg ketamine and 1 mg/kg medetomidine. After a 7-day recovery period, each mouse was transferred to a plastic cage, and signals from the transmitter were monitored by a receiver (IMT-10 RT; StarMedical) placed under the cage. After a 2-h adaptation period, IL-1, CL, or PBS were injected intraperitonially and body temperature was measured at 10-min intervals for 1 h.
O2 and physical activity. O2 was measured with an O2 metabolism measuring system (model MM102R; Muromachikikai, Tokyo, Japan) in a transparent chamber (50 W x 150 D x 150 H mm) with an air flow of 0.6–0.7 l/min. O2 of each mouse was measured for 1 min at 3-min intervals for 1 h. Cumulative O2 for 1 h was calculated and expressed as milliliters O2 per kilogram body weight. Each mouse was set into the chamber 
6 h before the start of measurement for adaptation. Mice had free access to food and water during the measurement.
At the same time, physical activity of each mouse was continuously recorded by a video recording system (digital video camera NV-GS50K and DVD video recorder DMR-E90H; Panasonic, Osaka, Japan) and assessed later by an investigator who was blind to the treatments. The video records were checked for behaviors such as locomotion, rearing, grooming, and feeding. If one of these behaviors was observed during a 1-min period, physical activity was scored as one count. Behavioral change was checked at 3-min intervals corresponding to the O2 measurement. Cumulative physical activity for 1 h was calculated, giving the maximum possible score over 1 h of 20 counts.
The O2 thermogenesis was calculated as previously reported (19). In brief, by the use of correlation between total O2 and physical activity of the data from PBS-injected mice, the O2 dependent on physical activity (O2 activity) and basal O2 with no physical activity (O2 basal) was calculated. O2 thermogenesis was obtained as O2 thermogenesis = O2 – O2 basal – O2 activity.
Statistical analysis.
All values are expressed as means ± SE. Statistical analysis was performed using analysis of variance followed by post hoc testing by the Fisher's protected least significant difference multiple range test and analysis of covariance.
|
RESULTS
|
|---|
UCP1 induction and TG content in BAT.
To investigate whether BAT is stimulated in parallel with fever induction we examined the effects of IL-1 injection on TG content and UCP1 mRNA level in BAT. Since 3-AR stimulation is established to activate BAT thermogenesis, we used CL, a highly specific 3-AR agonist, in a control experiment. As predicted, CL decreased BAT weight (56% of PBS-injected control) and TG content (29%) (Fig. 1, A and B), and it induced UCP1 mRNA expression remarkably (Fig. 1C). On the other hand, IL-1 injection failed to produce any change in tissue weight, TG content, and UCP1 expression.
Body temperature.
Body temperature response to IL-1, CL, or PBS injection was monitored for 1 h in conscious mice (Fig. 2). Body temperature prior to injection was 36.2 ± 0.26 and 35.9 ± 0.40°C in WT and UCP1-KO mice, respectively, and did not differ between the genotypes. Intraperitoneal injection of PBS caused a small and insignificant increase within 10 min of injection and decreased thereafter in both genotypes. In WT mice, CL injection increased body temperature significantly compared with PBS control, with a maximal rise of 1.19 ± 0.14°C at 20 min (Fig. 2A). In UCP1-KO mice, however, body temperature response was not different between CL- and PBS-injected groups. On the other hand, IL-1 injection caused a remarkable increase both in WT and UCP1-KO mice with a maximal rise of 1.82 ± 0.25 and 1.76 ± 0.40°C at 20 min, respectively (Fig. 2B). There was no difference in the response of body temperature to IL-1 injection between WT and UCP1-KO mice.
O2 and physical activity.
To investigate whether IL-1 increases thermogenesis, O2 and physical activity were measured for 1 h after the injection. O2 in control mice with PBS injection was not different between the genotypes (2,775 ± 184 and 2,889 ± 129 ml·h–1·kg–1 in WT and UCP1-KO mice, respectively) (Fig. 3A). The physical activity of PBS-injected mice tended to be lower in UCP1-KO mice than WT mice (16 ± 1 and 12 ± 1 count/h in WT and UCP1-KO mice), but the difference was insignificant (P = 0.092; Fig. 3B). CL injection increased O2 significantly in WT mice (+27%) but decreased slightly in UCP1-KO mice (–11%). CL injection suppressed physical activity both in WT (–63%) and UCP1-KO mice (–27%), but the effect was statistically significant only in WT mice. IL-1 injection produced a slight and insignificant increase of O2 in WT mice (+15%) but little or no response in UCP1-KO mice (+5%). In contrast, IL-1 injection substantially suppressed physical activity (–37% in WT and –42% in UCP1-KO mice).
O2 thermogenesis.
Since the change of physical activity largely affects the energy expenditure, O2 thermogenesis was calculated. The correlation between physical activity and O2 in PBS-injected mice is shown in Fig. 4. There was a highly significant positive correlation in WT (y = 48x + 1,995, n = 30, r2 = 0.587, P < 0.0001) and UCP1-KO mice (y = 65x + 1,969, n = 30, r2 = 0.640, P < 0.0001). Thus, basal O2 basal was calculated as 1,995 and 1,969 ml·h–1·kg–1 and O2 activity as 48 and 65 ml·h–1·kg–1·count–1 in WT and UCP1-KO mice, respectively. Analysis of covariance revealed no difference between the genotypes.
When O2 from CL-injected mice in each genotype was superimposed to that of PBS-injected mice, O2 of WT mice apparently fell above the regression line, whereas that of UCP1-KO mice was comparable (Fig. 4). In contrast, O2 from IL-1-injected mice apparently fell above the regression line in both genotypes. Thus, O2 thermogenesis was calculated as the difference from the regression line (Fig. 5). CL increased O2 thermogenesis in WT mice (1,229 ± 35 ml·h–1·kg–1) but not in UCP1-KO mice (71 ± 103 ml·h–1·kg–1), whereas IL-1 increased O2 thermogenesis similarly in WT and UCP1-KO mice (726 ± 200 and 638 ± 87 ml·h–1·kg–1in WT and UCP1-KO mice, respectively). Thus, IL-1 caused a thermogenic response in UCP1-KO mice in the same way as in WT mice.
|
DISCUSSION
|
|---|
The major objective of this study was to investigate the contribution of BAT thermogenesis to fever using UCP1-KO mice.
First, we examined whether the mouse is an appropriate model for the fever study, because animals other than mice, such as rats or guinea pigs, were used in most previous reports investigating the involvement of BAT thermogenesis in fever (2, 6, 13, 16, 21). IL-1 injection caused a remarkable rise in body temperature in WT mice. However, total O2 was not changed. These responses to IL-1 were quite in contrast with those to CL, a highly specific 3-AR agonist; CL injection both caused a rise in body temperature and increased total O2. Considering that 3-AR is exclusively expressed in brown and white adipocytes, the effect of CL on body temperature is due to the activation of BAT thermogenesis (8, 11, 14).
Fever is generated by a number of physiological processes that cause increases in both heat conservation and production (thermogenesis). In the present study, we found that IL-1 induced fever without any notable effect on total O2, suggesting that IL-1-induced fever is due to an increase in conservation, rather than production, of heat. Consistent with our results, Jennings and Elia (15) reported that LPS injection caused a remarkable rise in body temperature in both rats and mice, but O2 was increased only in rats. They proposed that there is a species difference in response to LPS, and in the case of mice, fever is generated mainly by the reduction of the heat loss more than activation of thermogenesis. In fact, the heat loss from the skin, especially that from the tail, has been claimed to play an important role in regulation of body temperature in mice (10, 23).
However, it is to be noted that we also found that physical activity was largely suppressed in IL-1 injected mice. Since total O2 is much influenced by physical activity (19), we calculated the O2 thermogenesis by correcting the effect of physical activity. This revealed that IL-1 caused a significant increase of thermogenesis (O2 thermogenesis) in mice similar to that reported in rats despite no apparent increase in total O2. Thus, species differences mentioned above may be caused by the different effects on physical activity.
There have been some reports suggesting the stimulatory effect of IL-1 on BAT thermogenesis, such as increase in GDP binding to mitochondria and blood flow to this tissue (6, 16, 21). In the present study, we reevaluated the effect of IL-1 on some biochemical parameters of BAT. CL induced a remarkable reduction in TG content and a rise in UCP1 mRNA level, consistent with a well-established view of UCP1-dependent degradation of TG in BAT (4, 14). In contrast, IL-1 failed to induce any of these changes. Moreover, we (1, 22, 25) reported previously that IL-1/LPS increases norepinephrine turnover in spleen and lung, but not in BAT, indicating the absence of activation of sympathetic nerve to BAT in fever. Taken together, these results suggest little or no stimulatory effect of IL-1 on BAT thermogenesis.
To obtain more direct evidence for this idea, we investigated whether the thermogenic effect of IL-1 was affected by the ablation of ucp1 gene. IL-1 injection increased body temperature in UCP1-KO mice, in the same way as WT mice, confirming similar febrile response in the two types of mice. Moreover, IL-1 showed no apparent effect on total O2 but a suppressive effect on physical activity in UCP1-KO mice, as in WT mice. Finally, O2 thermogenesis calculated by correcting the effect of physical activity in UCP1-KO mice was found to be comparable to that in WT mice. Thus, IL-1 increased thermogenesis and induced fever in UCP1-KO mice in the same manner as in WT mice. In contrast to IL-1, CL increased thermogenesis and body temperature only in WT mice and not in UCP1-KO mice. These results suggest a minor role of UCP1-dependent BAT thermogenesis in IL-1-induced thermogenesis and fever.
It is to be noted that the present results do not necessarily rule out the possibility of some compensatory thermogenic mechanism in UCP1-KO mice. In fact, it was reported (9) that UCP1-KO mice can maintain their body temperature by shivering under cold circumstances, whereas WT mice maintain their body temperature mainly by BAT thermogenesis. To clarify a precise role of BAT thermogenesis and shivering in fever, additional studies including the measurement of shivering in UCP1-KO mice are needed.
|
GRANTS
|
|---|
This study was supported by a grant-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (no. 15081201).
|
ACKNOWLEDGMENTS
|
|---|
We are grateful to American Cyanamid for providing CL316,243. We thank Dr. Kennedy Makondo for carefully reading the manuscript.
|
FOOTNOTES
|
|---|
Address for reprint requests and other correspondence: M. Saito, Dept. of Nutrition, Graduate School of Nursing and Nutrition, Tenshi College, Sapporo 065-0013, Japan (e-mail: saito{at}tenshi.ac.jp)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
|
REFERENCES
|
|---|
- Akiyoshi M, Shimizu Y, Saito M. Interleukin-1 increases norepinephrine turnover in the spleen and lung in rats. Biochem Biophys Res Commun 173: 1266–1270, 1990.[CrossRef][ISI][Medline]
- Blatteis CM. Effect of propranolol on endotoxin-induced pyrogenesis in newborn and adult guinea pigs. J Appl Physiol 40: 35–39, 1976.[Abstract/Free Full Text]
- Blatteis CM, Sehic E. Cytokines and fever. Ann NY Acad Sci 840: 608–618, 1998.[Abstract/Free Full Text]
- Cannon B, Nedergaard J. Brown adipose tissue: function and physiological significance. Physiol Rev 84: 277–359, 2004.[Abstract/Free Full Text]
- Cannon B, Houstek J, Nedergaard J. Brown adipose tissue. More than an effector of thermogenesis? Ann NY Acad Sci 856: 171–187, 1998.[Abstract/Free Full Text]
- Dascombe MJ, Rothwell NJ, Sagay BO, Stock MJ. Pyrogenic and thermogenic effects of interleukin 1 in the rat. Am J Physiol Endocrinol Metab 256: E7–E11, 1989.[Abstract/Free Full Text]
- Dinarello CA. Interleukin-1. Rev Infect Dis 6: 51–95, 1984.[ISI][Medline]
- Enerbäck S, Jacobsson A, Simpson EM, Guerra C, Yamashita H, Harper ME, Kozak LP. Mice lacking mitochondrial uncoupling protein are cold-sensitive but not obese. Nature 387: 90–94, 1997.[CrossRef][Medline]
- Golozoubova V, Hohtola E, Matthias A, Jacobsson A, Cannon B, Nedergaard J. Only UCP1 can mediate adaptive nonshivering thermogenesis in the cold. FASEB J 15: 2048–2050, 2001.[Free Full Text]
- Gordon CJ. Temperature Regulation in Laboratory Rodents. Cambridge, UK: Cambridge University, 1993.
- Grujic D, Susulic VS, Harper ME, Himms-Hagen J, Cunningham BA, Corkey BE, Lowell BB. Beta3-adrenergic receptors on white and brown adipocytes mediate beta3-selective agonist-induced effects on energy expenditure, insulin secretion, and food intake. A study using transgenic and gene knockout mice. J Biol Chem 272: 17686–17693, 1997.[Abstract/Free Full Text]
- Gwosdow AR, Kumar MS, Bode HH. Interleukin 1 stimulation of the hypothalamic-pituitary-adrenal axis. Am J Physiol Endocrinol Metab 258: E65–E70, 1990.[Abstract/Free Full Text]
- Horwitz BA, Hanes GE. Propranolol and pyrogen effects of shivering and nonshivering thermogenesis in rats. Am J Physiol 230: 637–642, 1976.[Abstract/Free Full Text]
- Inokuma K, Okamatsu-Ogura Y, Omachi A, Matsushita Y, Kimura K, Yamashita H, Saito M. Indispensable role of mitochondrial UCP1 for antiobesity effect of 3-adrenergic stimulation. Am J Physiol Endocrinol Metab 290: E1014–E1021, 2006.[Abstract/Free Full Text]
- Jennings G, Elia M. Effect of E. coli endotoxin on temperature, oxygen consumption and brown adipose tissue thermogenesis in rats and mice. Biosci Rep 7: 517–523, 1987.[CrossRef][ISI][Medline]
- Jepson MM, Millward DJ, Rothwell NJ, Stock MJ. Involvement of sympathetic nervous system and brown fat in endotoxin-induced fever in rats. Am J Physiol Endocrinol Metab 255: E617–E620, 1988.[Abstract/Free Full Text]
- Kluger MJ. Fever: role of pyrogens and cryogens. Physiol Rev 71: 93–127, 1991.[Abstract]
- Lowell BB, Spiegelman BM. Towards a molecular understanding of adaptive thermogenesis. Nature 404: 652–660, 2000.[Medline]
- Okamatsu-Ogura Y, Uozumi A, Kitao N, Kimura K, Saito M. Day-night difference in 3-adrenoceptor agonist-induced energy expenditure: contribution of brown fat thermogenesis and physical activity. Obes Res Clin Prac. 1: 61–67, 2007.[CrossRef]
- Robidoux J, Martin TL, Collins S. Beta-adrenergic receptors and regulation of energy expenditure: a family affair. Annu Rev Pharmacol Toxicol 44: 297–323, 2004.[CrossRef][ISI][Medline]
- Rothwell NJ. CRF is involved in the pyrogenic and thermogenic effects of interleukin 1 in the rat. Am J Physiol Endocrinol Metab 256: E111–E115, 1989.[Abstract/Free Full Text]
- Saito M, Akiyoshi M, Shimizu Y. Possible role of the sympathetic nervous system in responses to interleukin-1. Brain Res Bull 27: 305–308, 1991.[CrossRef][ISI][Medline]
- Satinoff E. Neural organization and evolution of thermal regulation in mammals. Science 201: 16–22, 1978.[Abstract/Free Full Text]
- Susulic VS, Frederich RC, Lawitts J, Tozzo E, Kahn BB, Harper ME, Himms-Hagen J, Flier JS, Lowell BB. Targeted disruption of the beta 3-adrenergic receptor gene. J Biol Chem 270: 29483–29492, 1995.[Abstract/Free Full Text]
- Terao A, Oikawa M, Saito M. Tissue-specific increase in norepinephrine turnover by central interleukin-1, but not by interleukin-6, in rats. Am J Physiol Regul Integr Comp Physiol 266: R400–R404, 1994.[Abstract/Free Full Text]
- Uehara A, Gottschall PE, Dahl RR, Arimura A. Interleukin-1 stimulates ACTH release by an indirect action which requires endogenous corticotropin releasing factor. Endocrinology 121: 1580–1582, 1987.[Abstract]
Copyright © 2007 by the American Physiological Society.
TOP
ABSTRACT
MATERIALS AND METHODS
and 
, respective 