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Articles

Inflammatory Responses to Lipopolysaccharide Are Suppressed in 40% Energy-Restricted Mice

Junko Matsuzaki^*, Mitsuru Kuwamura, Ryoichi Yamaji^*, Hiroshi Inui and Yoshihisa Nakano^*

Departments of ^* Applied Biological Chemistry and Veterinary Science, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan

¹To whom correspondence should be addressed. E-mail: inui{at}biochem.osakafu-u.ac.jp

	ABSTRACT

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To elucidate the suppressive effects of energy restriction on the inflammatory responses to lipopolysaccharide (LPS), mice were divided into a control group (fed 5.0 g diet/d; 71 kJ/d) and a 40% energy-restricted group (fed 3.0 g diet/d; 43 kJ/d) at 8-wk of age. Four weeks later, 25 µg of LPS was intraperitoneally injected. After the LPS injection, interleukin-1ß, interleukin-6 and tumor necrosis factor- were elevated in serums in the 40% energy-restricted mice and in the controls, but the extent of the elevation was significantly lower in the restricted group. The LPS-induced expression of inducible nitric oxide synthase in the liver was significantly suppressed by the energy restriction. In addition, the LPS-induced elevations of serum aspartate and alanine aminotransferase activities, which are indexes of hepatic injury, were also significantly attenuated in the restricted group. Moreover, the extent of LPS-induced alterations in hepatic structure was less in the restricted mice than in controls. Serum corticosterone level in the restricted mice was higher than that in the controls before LPS treatment (P < 0.05). Furthermore, after LPS injection, the significantly higher level of corticosterone was maintained in the restricted mice, although the LPS treatment significantly enhanced the level even in the control group. These results suggest that the extreme inflammatory responses to endotoxin are prevented in the 40% energy-restricted mice, and corticosterone participates in the preventive effects.

KEY WORDS: • energy-restricted mice • lipopolysaccharide • proinflammatory cytokines • inducible nitric oxide synthase • glucocorticoids

	INTRODUCTION

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Energy restriction (ER)² results in a reduced incidence and delayed onset of tumors and other age-related diseases and prolongs the mean and maximal life spans of rats, mice and other species (1 2 3) . It has been proposed that host immune surveillance mechanisms effectively suppress the incidence of tumors in ER animals (4) . In addition, we have found that ER mice have strong antitumor immunity, and in these mice, even if tumor cells are inoculated, tumor growth is suppressed and survival time is prolonged (5) . ER can prevent changes in immunity during aging; the elevation of interleukin (IL)-6 and tumor necrosis factor (TNF)- with age are inhibited by ER in mice (6 ,7) , whereas ER prevents the age-associated decline of the concanavalin A-induced expression of IL-2 in T cells of rats (8) . Furthermore, the edema response of footpads to carrageenan has been reported to be suppressed by ER (9) . Thus, it seems likely that the activation or improvement of immune functions participates in the longevity of life spans in ER animals.

Lipopolysaccharide (LPS) is a key structural component of the outer membrane of Gram-negative bacteria, and the effects of LPS induced in vivo are initiated by its interaction with host cells, in particular, macrophages, and their subsequent release of proinflammatory cytokines, including IL-1, IL-6 and TNF- (10) . LPS and/or cytokines, such as IL-1 and TNF-, activate the transcription of genes associated with inflammatory responses, such as cyclooxygenase and inducible nitric oxide synthase (iNOS) in many kinds of cells including macrophages (11 ,12) . These responses may be important defenses against invading Gram-negative bacteria, but when excessive, such responses to LPS may result in sepsis (10 ,13) . Nitric oxide (NO) produced by iNOS reacts with the superoxide anion radical, generating peroxynitrite anions, hydroxy radicals and hydrogen peroxide, and consequently, oxidative damage occurs in many organs (14 ,15) . These multiple organ dysfunctions are associated with a substantial elevation in mortality (13) . In liver, the activation of Kupffer cells, the resident macrophages, by LPS is a pivotal response during pathogenesis of endotoxin-associated hepatic tissue dysfunction (16 ,17) . The inflammatory and immunomodulating mediators (IL-1, TNF-, NO and oxygen radicals) synthesized and released by Kupffer cells during endotoxemic episodes mediate LPS-induced alterations, such as fluctuations in metabolic pathways and the necrosis observed in the pathological liver (17 18 19) .

Concerning the in vivo responses to infection, ER has been reported to improve the mortality by intraperitoneal challenge with Salmonella in mice (20) . However, effects of ER on acute inflammatory responses to LPS have not yet been elucidated. In this study, we compared 40% ER mice (43 kJ/d) with controls (71 kJ/d) on responses to LPS.

	MATERIALS AND METHODS

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Animal and treatments.

Male Balb/c mice (5 wk old) were obtained from Kiwa Laboratory Animals (Wakayama, Japan). These mice had free access to water and a diet reported by Matsuzaki et al. (5) , which was composed of 20% casein, 64% potato starch, 5% soybean oil, 5% cellulose powder, 4% salt mixture and 2% vitamin mixture.³ Mice were kept at controlled temperature (23 ± 2°C), humidity (60 ± 10%) and lighting (9:00 to 21:00), and housed individually. At 8 wk of age, these mice were randomly divided into control and 40% ER groups. In control mice, 5.0 g of the diet was given daily at 16:00 (71 kJ/d). This amount was determined by a preliminary experiment in which mice consumed the diet ad libitum. The food intake in the mice was 5.2 g/d during wk 8–12 of age. In the 40% ER group, mice were fed 3.0 g daily (43 kJ/d). The diet given daily was almost completely consumed throughout the experiment in both groups. All experimental procedures involving laboratory animals were approved by the Animal Care and Use Committee of Osaka Prefecture University.

LPS (Escherichia coli 055:B5; Difco Laboratory, Detroit, MI) was dissolved in pyrogen-free saline at 50 mg/L, and the ER and control mice at 12 wk of age were intraperitoneally injected with 25 µg of LPS. The LPS challenge was performed at 10:00 h to exclude any effects of the circadian rhythm of hormone levels on cytokine and glucocorticoid levels. At 0, 1, 2, 3 and 6 h after the LPS injection, mice (seven mice in each group at each time point) were anesthetized with ethyl ether, and blood samples were collected by cardiac puncture. The blood was allowed to clot at 4°C for 2 h and centrifuged to obtain serum. In addition, at 0, 6, 12 and 24 h after injection, livers were obtained from anesthetized (ethyl ether) mice. For preparation of histological samples, the livers were treated with 10% buffered paraformaldehyde and embedded in paraffin. Sections (3 µm) were prepared and stained with hematoxylin-eosin.

Determinations of cytokine, glucocorticoid, aminotransferase and glucose levels in serums.

Levels in serums of IL-1ß, IL-6, IL-10 and TNF- were quantified by enzyme-linked immunosorbent assay methods using commercial kits (Cytoscreen Immunoassay Kit; BioScource International, Camarillo, CA). Serum corticosterone level was determined by radioimmunoassay using Corticosterone [¹²⁵I] Assay System (Amersham Pharmacia Biotech, Buckinghamshire, UK). Cytokine and corticosterone levels in serums obtained from unanesthetized mice were measured, and it was thereby confirmed that these measurements were not affected by anesthesia with ethyl ether. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were assayed using commercial kits (Infinity AST and Infinity ALT; Sigma, St. Louis, MO). Serum glucose level was measured by a glucose oxidase method (Blood Glucose Test; Roche Diagnostics, Mannheim, Germany).

Western blot analysis of iNOS.

Livers were homogenized in 20 mmol/L HEPES-KOH buffer (pH 7.4), containing 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L EDTA, 7.3 µmol/L pepstatin A and 4.3 µmol/L leupeptin, at 4°C. After centrifuging at 4,000 x g for 10 min, the supernatant was used as the sample. Sample (75 µg of protein) was subjected to SDS-polyacrylamide gel electrophoresis (7.5% gel) according to Laemmli (21) . Proteins in the gel were transferred to a polyvinylidene difluoride membrane by electroblotting, and the membrane was treated with rabbit anti-iNOS antibodies (ICN Pharmaceuticals, Costa Mesa, CA) and Peroxidase Stain Kit for Immunoblotting (Nacalai Tesque, Kyoto, Japan), and relative amount was estimated by scanning densitometry. Protein content was determined according to Bradford (22) using bovine serum albumin as a standard.

Nitrite determination.

Livers (0.2 g) were homogenized in ice-cold water (2 mL) and centrifuged at 21,000 x g for 20 min at 4°C to obtain the supernatant. The amount of nitrite in the supernatant was measured following the Griess reaction as described previously (23) .

Statistical analyses.

Statistical analyses were performed with GB-Stat 5.4 (Dynamic Microsystems, Silver Spring, MD). Body weights were compared between the ER and control groups during the experiment by two-way ANOVA for repeated measures, and posthoc analyses were performed by Newman-Keuls method. Liver weights and levels of iNOS protein expression were compared between the two groups by one-way ANOVA followed by Scheffé posthoc test. Effects of ER on LPS-induced changes in serum IL-1ß, IL-6, TNF-, AST, ALT and glucose levels and hepatic nitrite concentration were evaluated by two-way ANOVA, and multiple comparisons were performed by Newman-Keuls test. For corticosterone and IL-10 data, values were logarithmically transformed to improve normality and to compensate for unequal variance and were analyzed by two-way ANOVA followed by Newman-Keuls test. All data are shown as means ± SEM, and statistical significance is defined as P < 0.05.

	RESULTS

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When 40% ER was initiated in mice at 8 wk of age, body weight was significantly lowered in 1 wk (from 22.6 ± 0.8 g to 19.8 ± 1.2 g). Four weeks later (at 12 wk of age), the weight in the ER group was 80% of the control (19.1 ± 1.0 g vs. 24.4 ± 0.1 g; P < 0.05). The ER-induced body weight change observed in this experiment was consistent with previous reports (5 ,24) . At 12 wk of age, liver weight in the ER mice was also 80% of that in the control mice (0.96 ± 0.08 g vs. 1.19 ± 0.07 g; P < 0.05).

The mice at 12 wk of age were intraperitoneally injected with 25 µg of LPS (the average amount of LPS injected per g body weight was calculated to be 1.31 µg and 1.02 µg in the ER and control mice, respectively), and IL-1ß, IL-6 and TNF- in serums were followed for 6 h (Fig. 1 ). The serum IL-1ß concentration significantly increased in the ER mice and showed a profile similar to that of the control group in the early phase after the LPS challenge (within 2 h). The elevation of the IL-1ß level was suppressed in the ER mice, compared with the controls, in the latter phases (at 3 and 6 h after the injection; P < 0.05). The serum IL-6 and TNF- concentrations were significantly elevated by the LPS challenge, peaked at 3 h in IL-6 and at 2 h in TNF-, and thereafter declined in both the ER and control groups. However, the IL-6 and TNF- levels at the peak points were lower in the ER mice than in the controls (P < 0.05). These results indicate that the amounts of these cytokines synthesized and released by macrophages in response to LPS were less in the ER mice compared with controls, although the amount of LPS injected per body weight was greater in the ER mice. Plasma volume was not affected by ER in mice because the hematocrit in the ER mice (0.45 ± 0.008) was not different from that in controls (0.44 ± 0.006).

View larger version (21K): [in this window] [in a new window]   Figure 1. Changes in IL-1ß (A), IL-6 (B) and TNF- (C) in serum of control and 40% ER mice after the injection of 25 µg of LPS. Values are means ± SEM, n = 7. Values with different letters differ, P < 0.05.

View larger version (15K): [in this window] [in a new window]   Figure 2. The expression level of iNOS in the liver of control and 40% ER mice 6 h after the injection of LPS. A, Representative immunoblots showing iNOS protein. B, Immunoblots of iNOS protein induced by the LPS injection are quantified by scanning densitometry. Values are means ± SEM, n = 7. *P < 0.05.

As has been reported previously (15) , hypoglycemia was induced by the LPS injection in the control mice (data not shown). In contrast, serum glucose level was lower in the ER mice (70% of that in the control mice; P < 0.05) in the normal state (without the LPS injection); however, there was not LPS effect in the ER mice.

Histological changes were examined in the liver of mice injected with LPS. As shown in Figure 3 , 12 and 24 h after the LPS injection, structural alterations, such as hepatocyte hypertrophy and nuclear enlargement, were observed in the control mice. These structural alterations also occurred in the 40% ER mice, but to a lesser extent.

View larger version (97K): [in this window] [in a new window]   Figure 3. Liver histology in 40% ER mice after the injection of LPS. ER (D--F) and control (A–C) mice were injected with 25 µg of LPS, and 0 h (A and D), 12 h (B and E) and 24 h (C and F) after the injection, liver sections were stained with hematoxylin-eosin. The figure presents one of four independent experiments. (Original magnification x 300.)

View larger version (23K): [in this window] [in a new window]   Figure 4. Change in serum corticosterone level in 40% ER mice after the injection of LPS. Values are means ± SEM, n = 7. Values with different letters differ, P < 0.05.

	DISCUSSION

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It is unlikely that the functions of macrophages themselves are lowered by ER because it has been reported that alveolar macrophages isolated from ER rats, compared with those from controls, have a greater phagocytic activity in vitro (27 ,28) . In addition, ER has been reported to enhance the generation of superoxide anion radicals in peritoneal macrophages during phagocytosis of opsonized zymosan in vitro (29) .

Glucocorticoids have antiinflammatory functions (25) and are elevated in serums in response to the injection of LPS (30) . The synthesis of glucocorticoids and proinflammatory cytokines are connected by positive- and negative-feedback loops through the hypothalamic-pituitary-adrenal axis; that is, corticosterone down-regulates the synthesis and activities of proinflammatory cytokines, whereas proinflammatory cytokines, especially IL-1ß, cause a release of adrenocorticotropic hormone from the pituitary gland to elevate corticosterone level (25 ,31) . It has been reported that adrenalectomized mice become extremely sensitive to the lethal effect of LPS, and, in contrast, pretreatment with dexamethasone prevents the lethality of endotoxin (32 ,33) . Although serum corticosterone shows daily periodicity and the daily periodicity is influenced by feeding schedule, it has been reported that the level of elevation in corticosterone before feeding is higher in ER rodents than that in controls (9 ,34 35 36) . Indeed, under our experimental conditions in which mice were fed at 16:00, serum corticosterone in the 40% ER mice was significantly higher just before the LPS injection (at 10:00) than in the controls (Fig. 4) . In addition, as has been reported previously (9) , the relative weight of adrenal glands was significantly greater in the 40% ER mice (0.59 ± 0.03 mg/g body) than in the controls (0.49 ± 0.04 mg/g body). Moreover, the significantly higher level of serum corticosterone was maintained in the ER mice, compared with controls, at all time points examined after the injection of LPS, although the LPS treatment significantly elevated the corticosterone level even in control mice (Fig. 4) . These results suggest that the 40% ER mice have a greater ability to produce glucocorticoids in response to the LPS challenge. The greater ability to produce glucocorticoids may account, at least in part, for the suppressive effect of ER on the LPS-induced release of proinflammatory cytokines. The inflammatory reaction to carrageenan injection in footpads is suppressed by ER in mice, and it was proposed that corticosterone contributes to the suppression (9) .

Structural and functional alterations in the gut epithelium have been observed in ER rodents (37 ,38) . If these alterations result in leaking of LPS into the circulation, these animals exposed to endogenous LPS at a constant low level would have been desensitized to the later injection of LPS. Thus, it is possible that the alterations in the gut epithelium relate to the lower sensitivity of the 40% ER mice to LPS.

The liver is one of the major organs damaged during endotoxemia. During endotoxemic episodes, Kupffer cells, the resident macrophages of the liver, are activated by LPS, leading to the production of proinflammatory cytokines and expression of iNOS, and the activation is a pivotal response during the endotoxin-associated hepatic tissue dysfunctions (17 18 19) . In this experiment, in the 40% ER mice, as well as in controls, iNOS protein was induced in the liver 6 h after the LPS injection, but the expression level of iNOS was significantly lower in the ER mice (Fig. 2) . In addition, LPS-induced NO production in the liver was also lower in the ER mice, compared with the controls (P < 0.05; Table 1 ). The lower level of iNOS induction in the 60% ER mice may be related with the elevated level of corticosterone (Fig. 4) , because glucocorticoids have been shown to inhibit the LPS-induced expression of iNOS mRNA and protein abundance in many organs and tissues (39 ,40) . In contrast, ER would not alter the kinetics of iNOS induction in response to the LPS injection. IL-1ß, IL-6 and TNF-, which are produced and released by macrophages in response to LPS, in serums were elevated in the ER mice and showed similar time changes as observed in the control mice, although the extents of the elevations were significantly less in the ER mice (Fig. 1) . Presumably as a result of the lowered NO production in the liver, LPS-induced elevation of the serum AST and ALT activities, nonspecific and specific markers, respectively, for hepatic parenchymal injury (15) , did not occur in the ER mice (Table 2 ). Furthermore, the extent of LPS-induced histological changes in the liver was also less in the 40% ER mice compared with controls (Fig. 3) .

	FOOTNOTES

 
² Abbreviations used: ALT, alanine aminotransferase; AST, aspartate aminotransferase; ER, energy restriction or energy-restricted; IL, interleukin; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; NO, nitric oxide; TNF, tumor necrosis factor.

³ Vitamin mixture contains 46,600 IU retinyl acetate, 23,300 IU cholecalciferol, 1,200 mg dl -tocopheryl acetate, 6 mg menadione, 59 mg thiamin HCl salt, 59 mg riboflavin, 29 mg vitamin B-6 HCl salt, 0.2 mg vitamin B-12, 588 mg vitamin C, 1 mg D-biotin, 2 mg folic acid, 235 mg pantothenic acid Ca salt, 294 mg nicotinic acid and 1,176 mg inositol in 100 g with a balance with lactose. Mineral mixture contains 0.43 g CaHPO₄^.2H₂O, 34.31 g KH₂PO₄, 25.06 g NaCl, 0.623 g Fe-citrate (Fe 17%), 4.8764 g MgSO₄, 0.02 g ZnCl₂, 0.121 g MnSO₄^.4–5H₂O, 0.156 g CuSO₄^.5H₂O, 0.0005 g KI, 29.29 g CaCO₃ and 0.0025 g (NH₄)₆Mo₇O₂₄^.4H₂O in 100 g with the balance as cellulose powder.

Manuscript received November 20, 2000. Initial review completed December 15, 2000. Revision accepted April 29, 2001.

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