Named Series: Diet, Inflammation and the BrainCalorie restriction dose-dependently abates lipopolysaccharide-induced fever, sickness behavior, and circulating interleukin-6 while increasing corticosterone
Introduction
It has been well established that calorie restriction (CR) can prolong lifespan (Weindruch et al., 1986), reduce the occurrence of age-related diseases such as cancer (Matsuzaki et al., 2000), lessen the severity of the neurochemical deficits and motor dysfunction seen in primate models of Parkinson’s disease (Maswood et al., 2004), and attenuate the ‘normal’ immunosenscence seen with age (Mascarucci et al., 2002). To date there has been limited investigation on the impact CR plays on the development of fever and sickness behavior. We have previously demonstrated that a 50% CR for 28 days attenuates sickness behavior in mice (MacDonald et al., 2011). The CR mice demonstrated no increase in body temperature (Tb) normally seen after lipopolysaccharide (LPS) administration, no anorexia or cachexia, and only a limited reduction in locomotor activity.
Examination of hypothalamic mRNA post-LPS indicated an anti-inflammatory bias in the CR mice. This was demonstrated by an attenuated increase of the expression of hypothalamic cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase-1 (mPGES-1) in CR mice at 2 h post-LPS. Hypothalamic suppressor of cytokine signaling 3 (SOCS3) mRNA expression was increased in the CR50% mice (two-fold that of the controls) at 4 h post-LPS, which led to the conclusion that SOCS3 may play an important role in the attenuation in sickness behavior seen in the CR mice (MacDonald et al., 2011). Hypothalamic mRNA levels of the anti-inflammatory cytokine interleukin-10 (IL-10) were also significantly increased in the CR mice 4 h post-LPS. SOCS3 is a known feedback regulator of cytokine production; it is induced by IL-10 (Cassatella et al., 1999), blocks IL-6 signaling (Lang et al., 2003), and aids in the anti-inflammatory actions of IL-10 (Bogdan et al., 1991).
It is important to investigate the peripheral signals that may also play a role in the CR-induced attenuation of fever and sickness behavior, as well as perhaps driving the central changes we previously observed. For example, peripheral administration of IL-10 attenuates fever induced by peripheral administration of LPS, whereas central administration of IL-10 was ineffective (Ledeboar et al., 2002). In addition to IL-10, investigations of peripheral levels of IL-6 can be seen as imperative. IL-6 is critically important for LPS-induced fever; plasma levels of LPS-induced IL-6 are highly correlated with fever (LeMay et al., 1990), and IL-6 deficient mice are unable to develop fevers post-LPS injection (Chai et al., 1996).
Increases in anti-inflammatory compounds such as corticosterone (CORT) may also play a role in attenuating sickness behavior following LPS. CORT is well established to exert anti-inflammatory effects which are achieved by firstly, increasing the transcription of anti-inflammatory cytokines and decreasing the transcription of pro-inflammatory cytokines (Smoak and Cidlowski, 2004); and secondly, by inhibiting the release of cytokines from macrophages (Barnes, 1998). CORT levels are well known to increase moderately after a period of CR (Han et al., 1995, Heiderstadt et al., 2000, Levay et al., 2010), and serum CORT concentration increases dose-dependently with increasing severity of CR, with a significant increase in serum CORT seen in rats after only 21 days at a 12.5% CR (Levay et al., 2010). A 20% CR for 20 days in hamsters increased basal levels of cortisol; however, this was not enough to alter Tb, or circulating cortisol and IL-6 compared to controls post-LPS (Conn et al., 1995). Given that the CR regimen used in this study was relatively modest (20%) and that we have previously demonstrated no increase in Tb post-LPS in mice CR to 50% (MacDonald et al., 2011) it would be interesting to see what effect a 50% CR would have on circulating CORT and IL-6 levels post-LPS.
In addition, to our knowledge a parametric analysis of how different durations of CR may impact on sickness behavior outcomes has not been conducted. It is important to establish the effect differing durations of CR may have on investigatory outcomes (in this case sickness behavior) so that the underlying mechanisms that play a role in the attenuation of sickness behavior seen in our previous studies can be further clarified.
It was shown that body-mass adjusted oxygen consumption in rats was not significantly affected by a 40% CR for two weeks or two months; however, six months CR reduced oxygen consumption by 40% (Bevilacqua et al., 2004). The same study revealed that mitochondrial production of reactive oxygen species was reduced by 53% compared to controls after two weeks, and further increased to a 57% reduction at two months, and a 74% reduction at six months, indicating a dose-dependent response to the duration of CR (Bevilacqua et al., 2004). In contrast, it was found that when comparing a short and long term (specific length not specified) 44% CR, the effect of both of these CR durations were homologous in both direction and level of change in the expression of genes that change with age (Cao et al., 2001). Furthermore, the short term CR reproduced 100% of the same effects as long term CR on urinary protein and stress response gene expression and the short term CR also reproduced 67% of the effects of long term CR on inflammatory response gene expression (Cao et al., 2001). These findings provide mixed messages as to the precise difference between short versus long term CR. It may be that depending on the variables measured that short or long term CR would be beneficial; however, not solely one or the other. The timing of the CR period needs to be optimized for specific investigations.
Previously our laboratory has demonstrated that LPS-induced sickness behavior can be attenuated by a 50% CR for 28 days in mice (MacDonald et al., 2011). The current study aims to extend that finding to another species and also to investigate possible dose-dependent responses to LPS after different durations of CR and peripheral immune related targets. It is hypothesized that a similar attenuation in sickness behavior will be seen in the rats CR to 50% for 28 days, followed by the rats CR for 21 days, and then the rats CR for 14 days will demonstrate the smallest attenuation of sickness behavior. Further, we expect that the peripheral targets investigated (CORT, IL-6 and IL-10) will further elucidate the mediating factors behind attenuated sickness behavior after CR. Given our previous findings we expect that the LPS-induced increase in CORT will be enhanced in the CR animals, the increase in IL-6 will be attenuated, and that IL-10 will be increased in the CR50% rats.
Section snippets
Animals
Ninety-five male Sprague–Dawley rats were procured from Monash SPF animal services (Clayton, Victoria, Australia) and allowed to acclimate to the facility for at least one week. During this period, standard rodent chow (Barastoc, Melbourne, Australia) and water were available ad libitum (AL). At the beginning of experimentation the rats were aged between 9 and 12 weeks old. Rats were individually housed in polypropylene basin cages (30 × 50 × 15 cm) with sawdust and tissues provided as bedding. Rats
Effect CR on body weight
All CR50% groups lost weight, whereas the AL group gained weight (Fig. 1). In the CR50% (21 days and 28 days) groups it became evident that weight loss plateaus at roughly day 21 of the CR period. There was no difference between the groups at baseline [F(3, 37) = 2.00, p = .13].
Effect of 28 days of CR on Tb
On day 28 of the CR period the CR50% rats demonstrated a lower Tb during the dark phase and were similar to the AL rats during the majority of the light phase (Fig. 2). The ANOVA found a significant effect for hour [F(2.54,
Discussion
The present study demonstrated that CR attenuated sickness behavior in a dose-dependent manner in relation to the duration of the CR period, with rats exposed to a 50% CR for 28 days demonstrating a full attenuation of all sickness behavior measures. CR rats also demonstrated enhanced increases in circulating CORT, attenuated increases in circulating IL-6, but no difference in circulating IL-10 post-LPS compared to AL animals. This study replicates some findings we have previously demonstrated
Author contributions
L.M. and S.K. contributed to the conception and design of the experiments. L.M., A.H., A.P, and S.K. collected, analyzed and interpreted the data. L.M. and S.K. drafted the article or revised it critically for important intellectual content. All authors discussed the results and approved the final version of the manuscript.
Acknowledgments
We would like to thank Jim’s Group Pty. Ltd. and the Australian Research Council Grant (LP 0775284) for generously supporting this research financially. We would also like to thank Morgan Radler for her generous time spent editing the final version of the manuscript.
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