Recent studies suggest that obesity is accompanied by chronic systemic low-grade inflammation (Hottamisligil 2006). Indeed, institution of a high-fat diet (HF) results in tissue inflammation caused by recruitment and activation of macrophages, and subsequent local or systemic release of pro-inflammatory cytokines can cause insulin resistance (Odegaard and Chawla 2005; Shoelson et al. 2006). Furthermore, a HF increases the expression of pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6, in the hypothalamus, which suggests that hypothalamic inflammation contributes to the secondary effects of diet-induced obesity (De Souza et al. 2005). Although the mechanisms underlying obesity-associated inflammation in peripheral tissues, such as liver and white adipose tissue, are well characterized, much less is known about the pathogenesis of obesity-induced hypothalamic inflammation.
The hypothalamus plays a central role in regulating food intake. In addition of the satiety center (ventromedial hypothalamus, VMH) and feeding center (lateral hypothalamus, LH), the hypothalamic arcuate (ARC) and paraventricular nucleus (PVN) also participate in regulating feeding behavior. Various neuropeptides have been identified that either inhibit or stimulate feeding behavior (Morley 1987). For example, α-melanocyte-stimulating hormone (α-MSH) and cocaine- and amphetamine-regulated transcript (CART) are anorexigenic whereas neuropeptide Y (NPY) and orexin-A stimulate food intake (Stanley and Leibowitz 1985; Kristensen et al. 1998; Sakurai et al. 1998).
The spleen is the largest lymphoid organ in the body and plays an important role in host immune function and blood filtration via the removal and destruction of aged or damaged erythrocytes and other blood cells (Dameshek 1955). Splenic gene expression of pro-inflammatory cytokines, such as TNF-α and IL-6, is decreased in the setting of obesity (Lamas et al. 2004). In contrast, IL-10, which is synthesized within multiple organs, including the spleen, is a potent anti-inflammatory cytokine that inhibits the synthesis of pro-inflammatory cytokines. Large amounts of IL-10 are produced from activated B-cells that mature in the marginal zone of the spleen. Recent studies suggest that IL-10-producing B-cells play a regulatory role in suppressing harmful immune responses (Pestka et al. 2004).
Based on these findings, the present study investigated the hypothesis that obesity suppresses the synthesis of IL-10 in the spleen, thereby resulting in chronic hypothalamic inflammation and altering feeding-related neuropeptides in the hypothalamus.
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- Research design and methods
Obesity is associated with insulin resistance, diabetes, dyslipidemia, and hypertension. Collectively, these conditions comprise metabolic syndrome, which is believed to involve a low-grade chronic pro-inflammatory state (Festa et al. 2001). However, the primary cause of obesity-induced inflammation is not well understood. This is the first study to characterize the impact of splenic dysfunction because of HF-induced obesity and SPX in rats with respect to the hypothalamus.
Our research showed that HF feeding increased the percentage of abnormal erythrocytes in peripheral blood and down-regulated the expression of CD20, a surface molecule present on B-cells in the spleen. These results indicate that HF feeding reduces the ability of spleen to filter out abnormal erythrocytes and the expression of B-cells that play a large role in the immune response including IL-10 synthesis. The inability of the spleen to filter out abnormal erythrocytes and to synthesize cytokines is considered to represent splenic hypofunction. In addition, the present findings imply that HF-induced obesity leads to splenic hypofunction by promoting oxidative stress and apoptosis in the spleen, which is consistent with prior studies showing that the antioxidant, N-acetyl cysteine, attenuates lipopolysaccharide (LPS)-induced apoptosis in splenic B-cells (Martin et al. 2000).
Our results indicate that hypophagia and body weight loss after SPX treatment are caused by a SPX-induced hypothalamic inflammatory response associated with an increase in neuronal histamine and IL-1β expression, which is consistent with a previous finding that central administration of IL-1β activates histamine neuron and results in hypophagia (Kang et al. 1995). Microglia, which are the macrophages of the CNS, are activated by various cytokines, including metabolic hormones such as leptin, and produce IL-1β, TNF-α and IL-6 (Pinteaux et al. 2007; Tang et al. 2007; Lafrance et al. 2010). Hence, microglia could be involved in the aggravation of metabolic disorder caused by obesity-induced hypothalamic inflammation. Our results showing that SPX activated microglia in the hypothalamus and worsened HF feeding-induced inflammation in the hypothalamus suggest that microglia trigger inflammation in response to HF feeding, which induces splenic hypofunction.
IL-10 is a potent anti-inflammatory cytokine that inhibits the synthesis of pro-inflammatory cytokines. IL-10 is synthesized within multiple organs, including the spleen. We focused on spleen-derived IL-10 because serum levels of IL-10, but not TNF-α, IL-1β, or MCP-1, were significantly decreased in the HF group compared with the Standard group, despite the finding that the expression of all cytokines in the spleen of the HF group were significantly reduced. This suggested that large amounts of serum IL-10 are synthesized in the spleen. Previous studies demonstrated that treatments that reduce Il-10 levels in diet-induced obese mice results in a progression of systemic inflammation and impairs insulin responsiveness (Cintra et al. 2008). Thus, it was hypothesized that the obesity-induced reduction in splenic IL-10 synthesis might lead to an inflammatory response in the hypothalamus and onset of metabolic disorders. In fact, obesity is associated with low IL-10 production capacity (Esposito et al. 2003; Waters et al. 2007). Moreover, we showed that IL-10 treatment reduced the expression of inflammatory cytokines and microglia in the hypothalamus, and improved the hypothalamic TNF-α/IL-10 ratio, indicating that IL-10 treatment attenuates the SPX-induced pro-inflammatory state in the hypothalamus. These observations are consistent with previous data that the administration of IL-10 attenuates inflammation-induced anorexia (Hollis et al. 2010). These findings indicate that splenic hypofunction can induce inflammation in the hypothalamus and that spleen-derived IL-10 may regulate the hypothalamic inflammatory response.
Inflammatory responses in the brain are mainly associated with microglia, which are the major immune effector cells in the CNS (Kreutzberg 1996; Block et al. 2007). IL-10 is produced by microglia in response to LPS treatment and brain injury, and IL-10 production suppresses the production of pro-inflammatory mediators, such as TNF-α and IL-1β from microglia (Mizuno et al. 1994). Considering our findings that hypothalamic IL-10 was endogenously elevated under SPX-induced hypothalamic inflammatory conditions and that IL-10 treatment restored this inflammatory response, IL-10 derived from spleen or the microglia themselves appear to be important anti-inflammatory modulators of glial activation, functioning to maintain a balance between pro- and anti-inflammatory cytokine levels in the hypothalamus. It is therefore possible that in glial cells, IL-10 acts in an autocrine or paracrine manner to down-regulate the synthesis of pro-inflammatory cytokines.
Furthermore, we evaluated neuropeptides involved in the control of feeding. Both α-MSH and NPY are synthesized in the ARC and project to the PVN, VMH and LH (Sahu et al. 1988; Mountjoy and Wong 1997; Elias et al. 1999; Cone 2005). CART is also mainly located in the ARC and is distributed in the PVN and VMH (Koylu et al. 1998). By contrast, orexin-A is mainly produced in the LH, and orexin-containing fibers are distributed throughout the PVN, VMH and ARC (Wang et al. 2003). This study showed that HF feeding accelerated hyperphagia, although all neuropeptides were decreased in the PVN and VMH, a satiety center. Appetite is controlled by a number of central mediators that may interact to regulate the influence of other systems on feeding behavior. It might be suggested that reduction of α-MSH and CART increases appetite, rather than reduction of NPY and orexin-A produces hypophagia. A previous study demonstrated that the peripheral LPS injection sufficient to elicit hypophagia caused (i) increases in mRNA levels of CART and pro-opiomelanocortin, which is cleaved into α-MSH; (ii) no increase in in NPY in the ARC; (iii) increases in α-MSH but not CART or NPY mRNA in the PVN; and (iv) prevented the activation of orexin neurons in the LH (Sergeyev et al. 2001; Becskei et al. 2008). These findings support our results that SPX led to an increase in α-MSH content in all nuclei except for the LH, an increase in CART content in the ARC, a decrease in CART content in the LH, no change in NPY content in any nuclei, and a decrease in orexin-A content in all nuclei. It is interesting to note that α-MSH and CART are co-localized in the ARC and that we observed a differential alteration between α-MSH and CART in the LH, PVN and VMH. This is consistent with a previous report showing a differential expression of pro-opiomelanocortin and CART in adrenalectomized rats (Vrang et al. 2003). Therefore, these data suggest the existence of distinct intracellular regulation of these neuropeptides.
As already mentioned, LPS induces microglia-mediated synthesis and release of pro-inflammatory cytokines in the brain. Previous research found that central injection of IL-10 attenuated the body weight loss induced by the central administration of LPS, which is consistent with the present result that IL-10 treatment abolished the SPX-induced body weight loss. This latter phenomenon was probably mediated by SPX-enhanced hypophagia and energy expenditure (Bluthėet al. 1999). Thus, we evaluated the effect of IL-10 on the alteration of hypothalamic neuropeptides induced by SPX. The present study revealed that α-MSH and orexin-A levels in each nucleus of the HF-fed SPX group returned to those of the HF-fed Sham group after treatment with exogenous IL-10, supporting the concept that spleen-derived IL-10 acts as an anti-inflammatory cytokine in the hypothalamus.
To further understand the influence of spleen-derived IL-10 protection, IL-10KO mice were used to determine whether IL-10 deficiency would affect SPX-induced inflammation in the hypothalamus. We observed that food intake and body weight were decreased in IL-10KO mice. This was probably caused by the inflammatory condition in the hypothalamus, as previous findings showed IL-10KO mice have spontaneous weight loss (Kühn et al. 1993). However, SPX-induced reduction in food intake, body weight, and pro-inflammatory responses were not seen in IL-10KO mice, despite the fact that IL-10 treatment increased food intake and body weight in SPX-treated IL-10 KO mice, and suppressed pro-inflammatory responses in both SPX-treated wild-type mice and IL-10KO mice. These results indicate that SPX has little effect on feeding behavior and hypothalamic inflammatory responses in IL-10KO mice and that spleen-derived IL-10 may affect the regulation of hypothalamic inflammation. We showed that the levels of inflammatory cytokines in the hypothalamus were increased in the obesity group when compared with control group, but were considerably lower in the obesity group than in the SPX-treated obesity group. Considering the finding that obesity causes a reduction in splenic IL-10 and that splenic IL-10 plays an important role in the prevention of hypothalamic inflammation, we suggest that spleen-derived IL-10 may be a therapeutic target in the management of the complications of obesity, including hypothalamic inflammation.
Hypothalamic inflammation may exert a paradoxical effect on energy metabolism. For example, hypothalamic inflammation induced by obesity results in hyperphagia and body weight gain, while hypothalamic inflammation induced in response to a systemic or local inflammatory process (e.g. bacterial sepsis) results in anorexia and weight loss. Prior studies have demonstrated that, in the context of sepsis, IL-10 is produced mainly by peritoneal neutrophils, while splenic leukocytes produce comparatively little IL-10 (Kasten et al. 2010). We demonstrated that a reduction of spleen-derived IL-10 resulted in activation of microglia and induction of hypothalamic inflammation, which may explain why anorexia and body weight occur in the setting of sepsis-induced hypothalamic inflammation. Furthermore, we also found that hypothalamic inflammation leads to catabolism after SPX, but results in anabolism in the context of obesity. A recent study showed that TNF-α can exert a dual effect in the hypothalamus, depending on the dose employed; central injection of high dose TNF-α had an anorexigenic effect, whereas central injection of low dose TNF-α had an orexigenic effect (Arruda et al. 2011). The present study may imply that mild reduction of spleen-derived IL-10 by HF feeding causes hyperphagia, whereas s severe reduction of splenic IL-10 by SPX causes hypophagia. Thus, it is assumed that the effects of energy metabolism change sharply with the differences in the IL-10/TNF-α ratio as well as the TNF-α level in the hypothalamus. A recent study demonstrated that intrahypothalamic infusion of recombinant IL-10 blocked IKK/NF-κB signaling and endoplasmic reticulum stress and restored Akt and STAT3 phosphorylation, promoting anti-obesity. This suggests that modulation of hypothalamic IL-10 expression could constitute a promising alternative to reduce hypothalamic inflammation and endoplasmic reticulum stress related to obesity (Ropelle et al. 2010). A plausible hypothesis to explain this paradox of the hypothalamic inflammation proposes that the ability to synthesize IL-10 from the spleen is the first target of HF-induced hypothalamic inflammation and that this paradox is a result of difference in the magnitude of IL-10 induction from the spleen.
Although additional work is needed to understand why obesity elicits inflammatory responses in the hypothalamus, the results of this study indicate that a more comprehensive understanding of the interactions between obesity and the spleen may help to identify new approaches for the prevention and treatment of obesity and metabolic syndrome.