No abstract

Sepsis is one of the leading causes of death in hospitalized patients and the chronic and low-grade inflammation observed in obesity seems to worsen susceptibility and morbidity of infections. However, little is known with respect to a short-term high-fat diet (HFD) and its role in the development of sepsis. Here, we show for the first time, that short-term HFD consumption impairs early nicotinic acetylcholine receptor α7 subunit (α7nAChR)- mediated signaling, one of the major components of the cholinergic anti-inflammatory pathway, with a focus on hypothalamic inflammation and innate immune response. Mice were randomized to a HFD or standard chow (SC) for 3 days, and sepsis was subsequently induced by a lethal intraperitoneal (i.p.) injection of lipopolysaccharide (LPS) or by cecal ligation and puncture (CLP) surgery. In a separate experiment, both groups received LPS (i.p.) or LPS (i.p.) in conjunction with the selective α7nAChR agonist, PNU-282987 (i.p. or intracerebroventricular; i.c.v.), and were sacrificed 2 h after the challenge. Short-term HFD consumption significantly reduced the α7nAChR mRNA and protein levels in the hypothalamus and liver ( p < 0.05). Immunofluorescence microscopy demonstrated lower cholinergic receptor nicotinic α7 subunit (α7nAChR)+ cells in the arcuate nucleus (ARC) (α7nAChR+ cells in SC = 216 and HFD = 84) and increased F4/80+ cells in the ARC (2.6-fold) and median eminence (ME) (1.6-fold), which can contribute to neuronal damage. Glial fibrillary acidic protein (GFAP)+ cells and neuronal nuclear antigen (NeuN)+ cells were also increased following consumption of HFD. The HFD-fed mice died quickly after a lethal dose of LPS or following CLP surgery (2-fold compared with SC). The LPS challenge raised most cytokine levels in both groups; however, higher levels of TNF-α (Spleen and liver), IL-1β and IL-6 (in all tissues evaluated) were observed in HFD-fed mice. Moreover, PNU-282987 administration (i.p. or i.c.v.) reduced the levels of inflammatory markers in the hypothalamus following LPS injection. Nevertheless, when the i.c.v. injection of PNU-282987 was performed the anti-inflammatory effect was much smaller in HFD-fed mice than SC-fed mice. Here, we provide evidence that a short-term HFD impairs early α7nAChR expression in central and peripheral tissues, contributing to a higher probability of death in sepsis.

Scope: Coconut oil (CO) diets remain controversial due to the possible association with metabolic disorder and obesity. This study investigates the metabolic effects of a low amount of CO supplementation. Methods and Results: Swiss male mice are assigned to be supplemented orally during 8 weeks with 300 µL of water for the control group (CV), 100 or 300 µL of CO (CO100 and CO300) and 100 or 300 µL of soybean oil (SO; SO100 and SO300). CO led to anxious behavior, increase in body weight gain, and adiposity. In the hypothalamus, CO and SO increase cytokines expression and pJNK, pNFKB, and TLR4 levels. Nevertheless, the adipose tissue presented increases macrophage infiltration, TNF-and IL-6 after CO and SO consumption. IL-1B and CCL2 expression, pJNK and pNFKB levels increase only in CO300. In the hepatic tissue, CO increases TNF-and chemokines expression. Neuronal cell line (mHypoA-2/29) exposed to serum from CO and SO mice shows increased NFKB migration to the nucleus, TNF-, and NFKBia expression, but are prevented by inhibitor of TLR4 (TAK-242). Conclusions: These results show that a low-dose CO changes the behavioral pattern, induces inflammatory pathway activation, TLR4 expression in healthy mice, and stimulates the pro-inflammatory response through a TLR4-mediated mechanism.

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LpS treatment. To induce inflammatory response, mice were treated with LPS diluted in sterile saline and administered intraperitoneally (IP) once a day for three days (1 mg kg −1 bw-IP). Mice were euthanized 2 hours after LPS treatment, and fragments of liver were collected, froze in liquid nitrogen and stored at −80 °C until processing. In vitro experiments. Hepatoma cell line, Hepa-1c1c7 (ATCC ® CRL-2026 ™), derived from mice was used to evaluate the ability of the cholinergic pathway to modulate AKT phosphorylation induced by insulin. Cells were cultivated in alpha modified Eagle's medium (αMEM; Invitrogen, USA) supplemented with 10% foetal bovine serum (Invitrogen, USA) and 1% penicillin (100 U/mL)/streptomycin (100 µg/mL) (Invitrogen, USA) at 37 °C and 5% CO 2. Cells were treated with 500 µM palmitate (palmitic acid from Sigma-Aldrich at 500 μM was first diluted in NaOH conjugated to BSA (3:1) for 45 minutes at 37 °C) for 3 hours in 6-well culture plate. The protein content was extracted and analysed by Western blotting. When necessary, 1 µM PNU-282987 (P6499-10MG; Sigma-Aldrich, Brazil) or 1 µM nicotine (N0267-100MG; Sigma-Aldrich, Brazil) was added to the medium for 15 minutes after palmitate treatment. To evaluate the insulin signalling, cells were treated with 100 nM insulin (Humulin, Eli Lilly and Company, USA) for 10 minutes after the 3 hours of palmitate treatment.