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Departments of Nutrition and Food Science and
*
Internal Medicine, University of Kentucky and the Lexington Veterans Administration Medical Center, Lexington, KY 40506 and
Bristol-Myers Squibb, Lawrenceville Campus, Princeton, NJ
3To whom correspondence should be addressed. E-mail: lgaetke{at}uky.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • leptin • IL-2–deficient mice • inflammatory bowel disease • inflammation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The causes of this decreased intake include anorexia, food avoidance due to symptoms (e.g., pain, nausea or obstruction) or side effects of medications. Of these, anorexia has been identified as the most important etiologic factor (1
).
The anorexia that occurs in IBD may be due to the increased production of proinflammatory cytokines, such as tumor necrosis factor-
(TNF-), interleukin (IL)-1 and IL-6. Interleukin-2–deficient (IL-2-/-) mice have colitis with an associated wasting syndrome and elevated production of multiple proinflammatory cytokines, including interferon-
(IFN-), IL-1, IL-6 and TNF- by components of the gut and its immunoregulatory cells (2
–4
). The colitis and wasting syndrome in this model can be ameliorated by conditions that decrease proinflammatory cytokine production such as growth in a germ-free environment (5
), blockade of interleukin-12 (IL-12) (6
) or blocking of the OX40 TNF receptor analog on CD4+ cells (7
). Although these studies link wasting with inflammatory cytokines, the mechanisms remain unknown.
Recently, leptin has emerged as a potential mediator of inappropriate satiety in inflammatory states, such as IBD (8
,9
). Leptin secreted by the adipocyte plays a central role in food intake and energy balance. Serum leptin concentration rapidly declines with food restriction (10
,11
). This decline in the serum leptin concentration communicates an energy deficit and appears to trigger a wide range of adaptive responses to minimize loss of body weight, including increased food-seeking behavior, decreased basal metabolism, decreased spontaneous activity and reduced fertility. Low leptin concentrations may be the principal mechanism to prevent wasting in the setting of reduced food intake (12
). We and others have shown that lipopolysaccharide (LPS) and the cytokines, IL-1, TNF-, leukemia inhibitory factor and probably IL-6, elevate serum leptin (8
,9
). Additional studies have supported the potential role of TNF- in modulating leptin in inflammation (13
). Similarly, IL-1 has been shown to play a role in the elevation of leptin with inflammation (14
).
The purpose of this study was to investigate the hypothesis that IL-2-/- mice, a model of chronic IBD with elevated proinflammatory cytokines, have dysregulated leptin concentrations contributing to inappropriate satiety and reduced food intake.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Heterozygous IL-2tm1Hor mutant C57BL/6J mice breeders purchased from the Jackson Laboratory (Bar Harbor, ME) and their offspring were housed in standard plastic cages (22 ± 0.5°C) with a 12-h light:dark cycle and had free access to water and unpurified mouse food (Harlan Teklad Laboratory diet #8604, Madison, WI). This diet contains (g/kg): 466.4 carbohydrate, 244.8 protein, 44.0 fat, 36.9 fiber and 78.4 ash. Weanlings (3 wk old) were genotyped using a 3-primer polymerase chain reaction protocol provided by the Jackson Laboratory. This study was approved and performed in accordance with the guidelines for the care and use of laboratory animals with both the Internal Animal Care and Use Committee (IACUC) and the Veterans Administration Medical Center in Lexington, KY.
Male mice (8 wk old) were divided into three individually housed groups: 1) homozygous IL-2tm1Hor mutants (IL-2-/-) with free access to food (n = 7); 2) matched wild-type (IL-2+/+) litter mates when possible (or age-matched IL-2+/+ from other litters) as control mice with free access to food (n = 7); and 3) control IL-2+/+ mice pair-fed (PF IL-2+/+) (n = 6). The pair-fed, control IL-2+/+ mice were given an amount of food equivalent to that consumed by their age-matched, paired IL-2-/- mouse during the previous 24 h. Mice were fed at 0900 h and the pair-fed mice were noted to consume it rapidly.
After 2 wk, the mice in the fed state were anesthetized with methoxyfurane and blood collected by orbital puncture at 0800 h. Mice were then deprived of food for 24 h, anesthetized and exsanguinated by cardiac puncture (starved state). Carcasses were collected for body fat and total protein analyses as previously described (15
,16
).
Isolation and culture of peritoneal macrophages.
Mouse peritoneal macrophages were isolated as described previously (17
). Each mouse was injected intraperitoneally with 1 mL of sterile PBS containing 10 g/L Bio-gel beads (Bio-Rad Laboratories, Hercules, CA). After 4 d, 10 mL of sterile PBS was injected into the peritoneum and withdrawn. The collected cells were washed once with PBS, once with serum-free Opti-MEM1, and re-suspended in Opti-MEM1. The cells (2 x 104 cells/well) were plated onto 96-well plates and cultured for 45 min to allow adherence. The plates then were washed 3 times with sterile PBS to remove nonadherent cells. The resulting adherent cell population consisted of >95% macrophages as determined by detecting nonspecific esterase activity. For measuring TNF- protein secretion, cells were cultured overnight in serum-free Opti-MEM1 at 37°C in an atmosphere of 10% CO2 and 95% relative humidity.
Serum concentrations of leptin were measured using a mouse leptin RIA kit purchased from Linco Research (St. Louis, MO). The lowest standard was 0.2 µg/L, the interassay CV was 4% and the interassay CV was 6%. TNF- protein was detected in cell culture supernatants using a mouse TNF- ELISA kit from Endogen (Woburn, MA). Serum concentrations of serum amyloid A (SAA), an acute phase protein, were detected using a mouse ELISA kit purchased from Biosource (Camarillo, CA).
Carcasses were exhaustively digested by alcoholic potassium hydroxide hydrolysis at 60°C and body fat calculated from enzymatic determination of glycerol (15
). Total protein was analyzed from the hydrolysate using the Lowery method with bovine serum allbumin as a standard (16
). Carcass weight and composition varied by weight changes at the end of the study and removal of liver and fat samples.
Mean responses for food consumption, total body protein and total body fat were compared among groups using one way ANOVA. Mean responses for body weight were compared by using a repeated-measures ANOVA with groups as a between-animal factor groups and age (8 vs. 10 wk) as a within-animal factor. Mean responses for serum leptin were compared by using a repeated-measures ANOVA with state (fed vs. starved) as the between-animal factor and groups (IL-2-/- vs. PF IL-2+/+ vs IL-2+/+) as the within-animal factor. Adjustment for body fat was done by regressing serum leptin on body fat using an analysis of covariance. In all ANOVA, post-hoc comparison of means was based on Fisher’s protected least significant difference procedure. The proportions of animals with clinical symptoms (diarrhea, rectal bleeding, rectal prolapse) were compared among groups using Fisher’s exact test. Statistical significance was determined at the 0.05 level. All results are expressed as the mean ± SEM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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At 8 wk of age, IL-2-/- mice lacked overt signs of colitis (4
), but had significantly lower body weights than control IL-2+/+ mice (Table 1
). Two groups of IL-2+/+ mice with initially equivalent weight were followed: one with free access to food (IL-2+/+ controls) and another pair-fed the food intake of a paired IL-2-/- mouse the previous day (PF IL-2+/+).
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. The IL-2-/- mice, and thus the PF IL-2+/+ mice, ate approximately half as much food as the IL-2+/+ mice (both, P < 0.0001) resulting in weight loss (IL-2-/-, P < 0.002, PF IL-2+/+, P < 0.0001), reduced protein (IL-2-/-, P < 0.0001; PF IL-2+/+, P < 0.023) and, in particular, reduced body fat (IL-2-/-, P < 0.0001, PF IL-2+/+, P < 0.0005) (Table 1)
.
Repeated measures ANOVA yielded a significant group x state interaction (F = 31.36 based on 2 and 17 df, P < 0.001). A post-hoc comparison of means showed that in the fed state, serum leptin concentrations were lower in IL-2-/- mice compared with the freely fed IL-2+/+ mice (P < 0.02) as expected with the reduced fat mass of the IL-2-/- mice, but were not different from PF IL-2+/+ mice (Fig. 1
). The remarkable finding was that the starved IL-2-/- mice had inappropriately elevated serum leptin concentrations compared with starved IL-2+/+ control mice (P < 0.0001) and starved PF IL-2+/+ mice (P < 0.0001). Within the IL-2-/- group, serum leptin in the starved state was higher than in the fed state (P < 0.0001). Indeed, starved IL-2-/- mice had serum leptin values similar to those observed in fed IL-2+/+ control mice. As expected after 24 h of starving, serum leptin concentrations declined in the IL-2+/+ control mice (P < 0.0001) and the PF IL-2+/+ mice (P < 0.02) compared with the fed state.
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). When serum leptin is adjusted for amount of body fat, the adjusted means for serum leptin for each of the three groups were not different in the fed state. In the starved state, the adjusted serum leptin mean increased significantly in the IL-2-/- mice (4.73 ± 0.29 µg/L) from the fed state (3.27 ± 0.29 µg/L, P < 0.0001) and compared with both starved PF IL-2+/+ (2.86 ± 0.29 µg/L, P < 0.0001) and starved IL-2+/+ controls (2.25 ± 0.32 µg/L, P < 0.0001). The adjusted serum leptin dropped for IL-2+/+ controls from the fed state (3.81 ± 0.32 µg/L) to 2.25 ± 0.32 µg/L (P < 0.0001) in the starved state and for the PF IL-2+/+ group from the fed state (3.78 ± 0.3 µg/L) to 2.86 ± 0.3 µg/L, P < 0.02, after 24 h of starving. These data imply that factors other than fat mass are affecting the serum concentration of leptin. The elevated serum leptin concentrations, equivalent to those seen in fed controls, would signal the hypothalamic centers exactly what they do in controls, i.e., complete satiety. This would abolish any leptin-related defense against wasting. Colitis and acute phase response measurements.
At the end of the study (10 wk of age), all of the IL-2-/- mice had diarrhea, and most had evidence of rectal bleeding or prolapse (Table 1)
. Histologic analysis for colitis was not done on the mice in this study. We have reported the histologic analysis of colitis in IL-2-/- mice at 5, 10 and 15 wk of age, and the severity of the colitis worsened as the mice aged, correlating with serum levels of SAA (r = 0.93) and clinical findings (4
).
Because of this high correlation between SAA and the severity of colitis, SAA was used as a marker of the presence of an acute phase response and the degree of inflammation active in IL-2-/- mice in this study. After 2 wk, serum concentrations of SAA were elevated in IL-2-/- mice (240 ± 82 mg/L) compared with both IL-2+/+ mice (<10 mg/L, P < 0.02) and PF IL-2+/+ mice (15.40 ± 4.3 mg/L, P < 0.05). In addition, elicited peritoneal macrophages from IL-2-/- mice spontaneously secreted TNF-, whereas none were detected in macrophages from IL-2+/+ (258 ± 40 vs. <5 pg/106 cells). These data provide indirect evidence supporting a role for proinflammatory cytokines in maintaining inappropriately elevated leptin concentrations in IL-2-/- mice.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Leptin has been examined in rodent models of acute intestinal inflammation. Barbier et al. (19
) measured plasma leptin concentrations in rats acutely treated with trinitrobenzene sulfonic acid resulting in transmural colitis and indomethacin-induced ileitis. One day after induction of injury, plasma leptin concentrations rose three- to fourfold and were associated with decreased food intake and weight loss. The severity of the colitis correlated with plasma leptin concentrations. With resolution of the colitis over the next 2 wk, plasma leptin concentrations declined and food intake increased. Similar results were seen in the indomethacin model. These data supported a role for circulating leptin in the anorexia of acute intestinal inflammation, but the question remained whether leptin played a role in chronic inflammation characteristic of IBD patients.
IL-2-/- mice lack the gene that encodes for the protein, IL-2, and fail to develop appropriate immune tolerance, which leads to an autoimmune-mediated inflammatory process that affects several organs (20
). IL-2-/- mice develop a wasting syndrome associated with hemolytic anemia, colitis and other symptoms similar to chronic intestinal inflammation in humans, including decreased food intake, wasting and weight loss. In this model, proinflammatory cytokines known to stimulate leptin production are elevated. Autenrieth et al. (3
) showed increased expression of mRNA for IL-1, TNF, IL-6 and IFN-. Similarly, we found increased expression of TNF- and IL-1ß mRNA in colon tissue, and colonic tissue levels of IL-1ß protein correlated with the severity of the colitis (4
). Here we demonstrated increased spontaneous secretion of TNF- from elicited peritoneal macrophages, which represent circulating monocytes. Consistent with these observations, IL-2-/- mice had clinical features of colitis at 10 wk of age and increased serum SAA concentrations, reflecting the presence of a systemic inflammatory response. SAA concentrations correlate well with the severity of colitis in IL-2-/- mice, but SAA does not correlate with the severity of liver injury, hypersplenism or anemia that also characterize this mouse model (4
).
To examine the potential role of leptin as a factor in the anorexia associated with chronic inflammation, we measured serum leptin concentrations in the fed state and after 24 h of food deprivation. This protocol revealed three key observations. First, serum leptin concentration may be altered by the chronic nutritional state of the animal. Normally, serum leptin concentration declines sharply in the setting of food restriction in both animals and humans (10
,11
). If samples are collected without reference to the fed or starved/food-deprived state, samples in the starved state during inflammation may be elevated and appear "normal" as demonstrated here. Serum leptin concentrations in the starved IL-2-/- mice were not different from fed control mice.
Second, our protocol demonstrated that serum leptin concentration during inflammation may not reflect fat mass. It has been shown that serum leptin concentrations are linearly related to fat mass in mice and humans (18
,21
). Here, there was significant weight loss in IL-2-/- and PF IL-2+/+ mice by the end of the study. When serum leptin was adjusted for body fat, all groups had similar leptin concentrations in the fed state, but leptin increased in the IL-2-/- mice with disproportionately low body fat in the starved state. Adjusted serum leptin in IL-2-/- mice in the starved state also did not differ from fed controls. Thus, in spite of weight loss, when serum leptin in (starved) IL-2-/- mice is compared with (fed) IL-2+/+ controls without reference to fat mass, the IL-2-/- mice would appear to have a "normal" leptin concentration. It was anticipated that pair-fed mice might have reduced leptin in both the fed and starved states because of their reduced body weight and fat mass. This was not observed, possibly for the following two reasons: 1) restricted daytime feeding shifts the diurinal leptin peak to the hours normally postprandial (22
). Thus, we are comparing near trough concentrations in the freely fed mice to what is likely diurnal peak levels in the pair-fed mice. 2) A modest elevation of SAA in the pair-fed group suggests inflammation may elevate leptin relative to fat mass in fed PF IL-2+/+ mice.
Third, it was important to obtain samples for leptin measurement at a similar time point in a 24-h period to minimize the effect of diurnal rhythm. There is a diurnal rhythm of leptin with a nocturnal rise after midnight and a nadir around noon (22
–24
). Any time period other than a complete diurnal cycle would have to consider this phenomenon.
Surprisingly, despite their lower body weight, lean body mass and dramatically reduced body fat, the IL-2-/- mice had only slightly reduced serum leptin concentrations after the nocturnal feeding cycle. Thus in the fed state, the hypothalamus of IL-2-/- mice receives an inappropriate signal suggesting that fat stores are much larger than actually present. In food-deprived IL-2-/- mice, we found that serum leptin concentrations paradoxically rise to concentrations not only higher than food-deprived controls but to concentrations as high as in fed controls. In the setting of reduced food intake either due to a direct effect of inflammatory cytokines or other mechanisms, the normal defense against anorexia of falling leptin concentrations is abolished. However, because leptin concentrations in the fed mice were not above those of controls, and assuming that leptin sensitivity is not altered, it is difficult to argue that leptin is the only explanation for reduced feeding. Grunfeld and co-workers (14
,25
) demonstrated that LPS and its associated proinflammatory cytokines produce anorexia in ob/ob and db/db mice, which lack leptin or its receptor.
These studies may have implications for further study of chronic inflammatory disease models with elevated proinflammatory cytokines, suggesting the following: 1) that careful attention to feeding state and diurnal variation may be necessary to uncover a role for the leptin system; 2) further studies are required to establish a direct link between TNF- and serum leptin concentrations; and 3) further studies are required to identify mechanisms other than leptin that may be responsible for a reduction in food intake.
In summary, serum leptin concentrations in the IL-2-/- mouse model of IBD were lower in the fed state than in either pair-fed or freely fed controls, but showed a paradoxical rise after 24 h of starving compared with a decline in both control groups. Thus, in the face of chronic inflammation, the leptin defense against food reduction was abolished, leaving other mechanisms of wasting unopposed.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported in part by the University of Kentucky Summer Faculty Research Fellowship Program, National Institutes of Health: KO8 DK02401–01A and the Veterans Administration Career Development Award 596522803585003.
4 Present address: Helieh Oz, University of Louisville, Louisville, KY 40292.
5 Present address: Gary Varilek, Gastroenterology Specialties, PC, Lincoln, NB 68503.
6 Abbreviations used: IBD, inflammatory bowel disease; IFN-, interferon-; IL, interleukin; IL-2-/-, interleukin-2 deficient; LPS, lipopolysaccharide; PF, pair-fed; SAA, serum amyloid A; TNF-, tumor necrosis factor-.
Manuscript received 8 October 2001. Initial review completed 4 December 2001. Revision accepted 25 January 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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