The maintenance of mitochondrial activity in hypothalamic neurons is determinant to the control of energy homeostasis in mammals. Disturbs in the mitochondrial proteostasis can trigger the mitonuclear imbalance and mitochondrial unfolded protein response (UPRmt) to guarantee the mitochondrial integrity and function. However, the role of mitonuclear imbalance and UPRmt in hypothalamic cells are unclear. Combining the transcriptomic analyses from BXD mice database and in vivo experiments, we demonstrated that physical training alters the mitochondrial proteostasis in the hypothalamus of C57BL/6J mice. This physical training elicited the mitonuclear protein imbalance, increasing the mtCO-1/Atp5a ratio, which was accompanied by high levels of UPRmt markers in the hypothalamus. Also, physical training increased the maximum mitochondrial respiratory capacity in the brain. Interestingly, the transcriptomic analysis across several strains of the isogenic BXD mice revealed that hypothalamic mitochondrial DNA-encoded genes were negatively correlated with body weight and several genes related to the orexigenic response. As expected, physical training reduced body weight and food intake. Interestingly, we found an abundance of mt-CO1, a mitochondrial DNA-encoded protein, in NPY-producing neurons in the lateral hypothalamus nucleus of exercised mice. Collectively, our data demonstrated that physical training altered the mitochondrial proteostasis and induced the mitonuclear protein imbalance and UPRmt in hypothalamic cells.

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.

The study is aimed at investigating if PUFA supplementation could prevent the effects of a short-term HFD on α7nAChR expression and on the severity of sepsis. Swiss mice were used for the in vivo experiments. For the in vitro experiments, we used a microglia cell line (BV-2) and a hepatoma cell line (Hepa-1c1c7) derived from mice. The animals were either fed standard chow, fed a short-term HFD (60%), or given supplementation with omega-3 fatty acid (2 g/kg or 4 g/kg bw) for 17 days, followed by a short-term HFD. Endotoxemia was induced with an intraperitoneal (i.p.) lipopolysaccharide injection (LPS, 5 or 12 mg/kg), and sepsis was induced by subjecting the animals to cecal ligation and puncture (CLP). BV-2 and Hepa-1c1c7 cells were treated with LPS (100 and 500 ng/mL, respectively) for 3 hours. RT-PCR or Western blotting was used to evaluate α7nAChR expression, inflammatory markers, DNMT1, and overall ubiquitination. LPS and HFD reduced the expression of α7nAChR and increased the expression of inflammatory markers. Omega-3 partially prevented the damage caused by the HFD to the expression of α7nAChR in the bone marrow and hypothalamus, decreased the inflammatory markers, and reduced susceptibility to sepsis-induced death. Exposing the BV-2 cells to LPS increased the protein content of DNMT1 and the overall ubiquitination and reduced the expression of α7nAChR. The inflammation induced by LPS in the BV-2 cell decreased α7nAChR expression and concomitantly increased DNMT1 expression and the ubiquitinated protein levels, indicating the participation of pre- and posttranscriptional mechanisms.

Activation of cholinergic receptor (α7nAChR) in hypothalamus can modulates inflammatory response and they are colocalized with POMC and NPY neurons. Activation of hypothalamic nAChR modulated NPY and POMC expression and reduced food intake. Maternal obesity promoted reduction of nAChR in hypothalamus of the offspring and increased food intake. Thus, the reduction of hypothalamic α7nAChR in offspring may be related to obesity development induced by maternal consumption of high fat diet during pregnancy and lactation.

Studies has shown that the consumption of a high-fat diet induces hypothalamic and systemic inflammation. It is also known that nicotinic acetylcholine receptors (nAChRs) are involved in cholinergic anti-inflammatory response, in which the α7-subunit is the most expressive subtype doing this control in immune cells. This study investigated the effects of a short-term high-fat diet on modulating the anti-inflammatory pathway mediated by α7 receptor. 8-weeks-old Swiss mice consumed either short-term (3 days) high-fat diet (HFD-60% of fatty acids) or standard-chow. Both groups were submitted to either a CLP surgery, to induce polymicrobial sepsis, or an abdominal incision for exposure of the cecum, serving as the control group. We concluded that the consumption of a high-fat diet even for a short period is already capable of increasing hypothalamic anti-inflammatory markers and inhibiting the anti-inflammatory pathway .