Sex-Dependent Variations in Hypothalamic Fatty Acid Profile and Neuropeptides in Offspring Exposed to Maternal Obesity and High-Fat Diet.

Maternal obesity and/or high-fat diet (HF) consumption can disrupt appetite regulation in their offspring, contributing to transgenerational obesity and metabolic diseases. As fatty acids (FAs) play a role in appetite regulation, we investigated the maternal and fetal levels of FAs as potential contributors to programmed hyperphagia observed in the offspring of obese dams. Female mice were fed either a control diet (CT) or HF prior to mating, and fetal and maternal blood and tissues were collected at 19 days of gestation. Elevated levels of linoleic acid were observed in the serum of HF dams as well as in the serum of their fetuses. An increased concentration of eicosadienoic acid was also detected in the hypothalamus of female HF-O fetuses. HF-O male fetuses showed increased hypothalamic neuropeptide Y (Npy) gene expression, while HF-O female fetuses showed decreased hypothalamic pro-opiomelanocortin (POMC) protein content. Both male and female fetuses exhibited reduced hypothalamic neurogenin 3 (NGN-3) gene expression. In vitro experiments confirmed that LA contributed to the decreased gene expression of Pomc and Ngn-3 in neuronal cells. During lactation, HF female offspring consumed more milk and had a higher body weight compared to CT. In summary, this study demonstrated that exposure to HF prior to and during gestation alters the FA composition in maternal serum and fetal serum and hypothalamus, particularly increasing n-6, which may play a role in the switch from POMC to NPY neurons, leading to increased weight gain in the offspring during lactation.

Hypothalamic inflammation and the development of an obese phenotype induced by high-fat diet consumption is exacerbated in alpha7 nicotinic cholinergic receptor knockout mice.

Hypothalamic inflammation and metabolic changes resulting from the consumption of high-fat diets have been linked to low grade inflammation and obesity. Inflammation impairs the hypothalamic expression of α7 nicotinic acetylcholine receptor (α7nAChR). The α7nAChR is described as the main component of the anti-inflammatory cholinergic pathway in different inflammation models. To assess whether the reduction in α7nAChR expression exacerbates hypothalamic inflammation induced by a high-fat diet (HFD), were used male and female global α7nAChR knockout mouse line in normal or high-fat diet for 4 weeks. Body weight gain, adiposity, glucose homeostasis, hypothalamic inflammation, food intake, and energy expenditure were evaluated. Insulin sensitivity was evaluated in neuronal cell culture. Consumption of an HFD for 4 weeks resulted in body weight gain and adiposity in male Chrna7-/- mice and the hypothalamus of male Chrna7-/- mice showed neuroinflammatory markers, with increased gene expression of pro-inflammatory cytokines and dysregulation in the nuclear factor kappa B pathway. Moreover, male Chrna7-/- mice consuming an HFD showed alterations in glucose homeostasis and serum of Chrna7-/- mice that consumed an HFD impaired insulin signalling in neuronal cell culture experiments. In general, female Chrna7-/- mice that consumed an HFD did not show the phenotypic and molecular changes found in male mice, indicating that there is sexual dimorphism in the analysed parameters. Thus, receptor deletion resulted in increased susceptibility to hypothalamic inflammation and metabolic damage associated with HFD consumption in male mice.

Activation of the α7 Nicotinic Acetylcholine Receptor Prevents against Microglial-Induced Inflammation and Insulin Resistance in Hypothalamic Neuronal Cells.

Neuronal hypothalamic insulin resistance is implicated in energy balance dysregulation and contributes to the pathogenesis of several neurodegenerative diseases. Its development has been intimately associated with a neuroinflammatory process mainly orchestrated by activated microglial cells. In this regard, our study aimed to investigate a target that is highly expressed in the hypothalamus and involved in the regulation of the inflammatory process, but still poorly investigated within the context of neuronal insulin resistance: the α7 nicotinic acetylcholine receptor (α7nAchR). Herein, we show that mHypoA-2/29 neurons exposed to pro-inflammatory microglial conditioned medium (MCM) showed higher expression of the pro-inflammatory cytokines IL-6, IL-1ß, and TNF-α, in addition to developing insulin resistance. Activation of α7nAchR with the selective agonist PNU-282987 prevented microglial-induced inflammation by inhibiting NF-κB nuclear translocation and increasing IL-10 and tristetraprolin (TTP) gene expression. The anti-inflammatory role of α7nAchR was also accompanied by an improvement in insulin sensitivity and lower activation of neurodegeneration-related markers, such as GSK3 and tau. In conclusion, we show that activation of α7nAchR anti-inflammatory signaling in hypothalamic neurons exerts neuroprotective effects and prevents the development of insulin resistance induced by pro-inflammatory mediators secreted by microglial cells.

Interesterified palm oil increases intestinal permeability, promotes bacterial translocation, alters inflammatory parameters and tight-junction protein genic expression in Swiss mice.

High-fat diets seem to have a negative influence on the development of obesity and the processes associated with low-grade chronic systemic inflammation. In recent years, partial hydrogenated oil, rich in trans isomers, has been associated with deleterious health effects. It has been replaced by interesterified fat (IF). However, there is no evidence whether IF ingestion can exert adverse effects on the intestinal mucosa. Thus, this study aimed to evaluate the effect of IF on the intestinal mucosa of male Swiss mice fed a normal or high-fat diet, focusing on its effects on intestinal permeability and bacterial translocation and its possible damage to the intestinal epithelium. The animals were divided into 4 groups: Control (C) and Interesterified Control (IC) groups (10 En% lipids from unmodified fat or interesterified fat, respectively) and High Fat (HF) and Interesterified High Fat (IHF) groups (45 En% lipids from unmodified fat or interesterified fat, respectively). Compare to C, the IC, HF, and IHF groups presented flattened epithelium, a shorter villi length and a lower percentage of goblet cells, less mucin 2, an increased oxidative stress and more inflammatory cells, higher IL-1ß, IL-17, and IL-23 levels. These groups also presented increased intestinal permeability and gene expression of the protein claudin 2, while JAM-A and claudin 1 gene expression was reduced. IC and IHF increased IL-6 levels while reducing occludin expression. In addition, the IC group also presented a mucosa with lesions of low intensity in the ileum, an increased mucin 5ac, TNF-α levels, and reduced occludin expression in the distal jejunum. Moreover, there was a significant increase in bacterial translocation in the IC group to blood, liver, and lungs, while HF and IHF groups presented bacterial translocation which was restricted to the mesenteric lymph nodes. In summary, our results supported the hypothesis that IF added to a normolipidic diet can be considered harmful or even worse when compared to a HF.

Low-Dose Coconut Oil Supplementation Induces Hypothalamic Inflammation, Behavioral Dysfunction, and Metabolic Damage in Healthy Mice.

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-α 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-α 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-α, 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.

Acute effects of fatty acids on autophagy in NPY neurones.

High-fat diet (HFD) feeding is deleterious to hypothalamic tissue, leading to inflammation and lipotoxicity, as well as contributing to central insulin resistance. Autophagy is a process that restores cellular homeostasis by degrading malfunctioning organelles and proteins. Chronic HFD-feeding down-regulates hypothalamic autophagy. However, the effects of short-term HFD-feeding and the saturated fatty acid palmitate (PA) on hypothalamic autophagy and in neurones that express neuropeptide Y (NPY) and agouti-related peptide remains unknown. Therefore, we assessed hypothalamic autophagy after 1 and 3 days of HFD-feeding. We also injected PA i.c.v and analysed the modulation of autophagy in hypothalamic tissue. Both interventions resulted in changes in autophagy-related gene profiles without significant differences in protein content of p62 and LC3B-II, markers of the autophagy pathway. When we assessed native NPY neurones in brain slices from PA-treated animals, we observed increased levels of Atg7 and LC3B protein in response to PA treatment, indicating the induction of autophagy. We then tested the direct effects of fatty acids using the immortalised hypothalamic NPY-expressing neuronal cell model mHypoE-46. We found that PA, but not palmitoleate (PO) (a monounsaturated fatty acid), was able to induce autophagy. Co-treatment with PA and PO was able to block the PA-mediated induction of autophagy, as assessed by flow cytometry. When the de novo ceramide synthesis pathway was blocked with myriocin pre-treatment, we observed a decrease in PA-mediated induction of autophagy, although there was no change with the toll-like receptor 4 inhibitor, TAK-242. Taken together, these findings provide evidence that saturated and unsaturated fatty acids can differentially regulate hypothalamic autophagy and that ceramide synthesis may be an important mediator of those effects. Understanding the mechanisms by which dietary fats affect autophagy in neurones involved in the control of energy homeostasis will provide potential new pathways for targeting and containing the obesity epidemic.

Interesterified palm oil impairs glucose homeostasis and induces deleterious effects in liver of Swiss mice.

BACKGROUND: Interesterified fats have largely replaced the partially hydrogenated oils which are the main dietary source of trans fat in industrialized food. This process promotes a random rearrangement of the native fatty acids and the results are different triacylglycerol (TAG) molecules without generating trans isomers. The role of interesterified fats in metabolism remains unclear. We evaluated metabolic parameters, glucose homeostasis and inflammatory markers in mice fed with normocaloric and normolipidic diets or hypercaloric and high-fat diet enriched with interesterified palm oil. METHODS: Male Swiss mice were randomly divided into four experimental groups and submitted to either normolipidic palm oil diet (PO), normolipidic interesterified palm oil diet (IPO), palm oil high-fat diet (POHF) or interesterified palm oil high-fat diet (IPOHF) during an 8 weeks period. RESULTS: When compared to the PO group, IPO group presented higher body mass, hyperglycemia, impaired glucose tolerance, evidence of insulin resistance and greater production of glucose in basal state during pyruvate in situ assay. We also observed higher protein content of hepatic PEPCK and increased cytokine mRNA expression in the IPO group when compared to PO. Interestingly, IPO group showed similar parameters to POHF and IPOHF groups. CONCLUSION: The results indicate that substitution of palm oil for interesterified palm oil even on normocaloric and normolipidic diet could negatively modulate metabolic parameters and glucose homeostasis as well as cytokine gene expression in the liver and white adipose tissue. This data support concerns about the effects of interesterified fats on health and could promote further discussions about the safety of the utilization of this unnatural fat by food industry.

Modulation of hypothalamic S6K1 and S6K2 alters feeding behavior and systemic glucose metabolism.

The mTOR/S6Ks signaling is one of the intracellular pathways important for metabolic control, acting both peripherally and centrally. In the hypothalamus, mTOR/S6Ks axis mediates the action of leptin and insulin and can modulate the expression of neuropeptides. We analyzed the role of different S6Ks isoforms in the hypothalamic regulation of metabolism. We observed decreased food intake and decreased expression of agouti-related peptide (AgRP) following intracerebroventricular (icv) injections of adenoviral-mediated overexpression of three different S6Ks isoforms. Moreover, mice overexpressing p70-S6K1 in undefined periventricular hypothalamic neurons presented changes in glucose metabolism, as an increase in gluconeogenesis. To further evaluate the hypothalamic role of a less-studied S6K isoform, p54-S6K2, we used a Cre-LoxP approach to specifically overexpress it in AgRP neurons. Our findings demonstrate the potential participation of S6K2 in AgRP neurons regulating feeding behavior.

Short-Term High-Fat Diet Consumption Reduces Hypothalamic Expression of the Nicotinic Acetylcholine Receptor α7 Subunit (α7nAChR) and Affects the Anti-inflammatory Response in a Mouse Model of Sepsis.

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-1ß 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.

Interesterified soybean oil promotes weight gain, impaired glucose tolerance and increased liver cellular stress markers.

Interesterified fats have largely replaced hydrogenated vegetable fat, which is rich in trans fatty acids, in the food industry as an economically viable alternative, generating interest to study their health effects. The aim of this study was to evaluate the effect that interesterification of oils and fat has on lipid-induced metabolic dysfunction, hepatic inflammation and ER stress. Five week-old male Wistar rats were randomly divided into three experimental groups, submitted to either normocaloric and normolipidic diet containing 10% of lipids from unmodified soybean oil (SO) or from interesterified soybean oil (ISO), and one more group submitted to a high fat diet (HFD) containing 60% of fat from lard as a positive control, for 8 or 16 weeks. Metabolic parameters and hepatic gene expression were evaluated. The HFD consumption led to increased body mass, adiposity and impaired glucose tolerance compared to SO and ISO at both time points of diet. However, the ISO group showed an increased body mass gain, retroperitoneal WAT mass, fasting glucose, and impaired glucose tolerance during ipGTT at 16 weeks compared to SO. Moreover, at 8 weeks, hepatic gene expression of Atf3 and Tnf were increased in the ISO group compared to the SO group. Thus, replacement of natural fat with interesterified fat on a normocaloric and normolipidic diet negatively modulated metabolic parameters and resulted in impaired glucose tolerance in rats.

Maternal Consumption of High-fat Diet in Mice Alters Hypothalamic Notch Pathway, NPY Cell Population and Food Intake in Offspring.

Studies show that maternal consumption of a high-fat diet (HFD) can impair the formation of hypothalamic neuronal circuits in mouse offspring. This damage can be mediated by Notch1/Hes5 signaling activation, leading to repression of proneural factors such as Mash1 and Ngn2/3, which are essential for neuronal differentiation and neurogenesis. Thus, we aimed to investigate the effects of maternal HFD consumption during gestation and lactation on the Notch1/Mash1 pathway in the hypothalamus and arcuate nucleus (ARC) of mouse offspring (neonates and 28 days old). Our results showed that maternal HFD consumption increases body weight and adiposity of mouse offspring, accompanied by increased levels of Il-1ß mRNA compared to those in control offspring. We noticed high mRNA levels of Hes5 accompanied by diminished mRNA levels of Ascl1 (Mash1). The number of Mash1-labeled cells in the ARC was diminished in HFD-O. Additionally, the population of NPY neurons was increased in these animals. Mash1 is important for the development of POMC and NPY neurons in the ARC. Therefore, the reduction in Mash1-labeled cells could be related to modification of the NPY neuron population in the ARC. This scenario favors hyperphagia and weight gain, and could be responsible for the development of obesity in adulthood.

Diet-Induced Maternal Obesity Alters Insulin Signalling in Male Mice Offspring Rechallenged with a High-Fat Diet in Adulthood.

Modern lifestyle has resulted in an increase in the prevalence of obesity and its comorbidities in pregnant women and the young population. It has been well established that the consumption of a high-fat diet (HFD) has many direct effects on glucose metabolism. However, it is important to assess whether maternal consumption of a HFD during critical periods of development can lead to metabolic changes in the offspring metabolism. This study evaluated the potential effects of metabolic programming on the impairment of insulin signalling in recently weaned offspring from obese dams. Additionally, we investigated if early exposure to an obesogenic environment could exacerbate the impairment of glucose metabolism in adult life in response to a HFD. Swiss female mice were fed with Standard Chow (SC) or a HFD during gestation and lactation and tissues from male offspring were analysed at d28 and d82. Offspring from obese dams had greater weight gain and higher adiposity and food intake than offspring from control dams. Furthermore, they showed impairment in insulin signalling in central and peripheral tissues, which was associated with the activation of inflammatory pathways. Adipose tissue was ultimately the most affected in adult offspring after HFD rechallenge; this may have contributed to the metabolic deregulation observed. Overall, our results suggest that diet-induced maternal obesity leads to increased susceptibility to obesity and impairment of insulin signalling in offspring in early and late life that cannot be reversed by SC consumption, but can be aggravated by HFD re-exposure.

Hypothalamic endoplasmic reticulum stress and insulin resistance in offspring of mice dams fed high-fat diet during pregnancy and lactation.

OBJECTIVE: The goal of this study was to determine the presence early of markers of endoplasmic reticulum stress (ERS) and insulin resistance in the offspring from dams fed HFD (HFD-O) or standard chow diet (SC-O) during pregnancy and lactation. MATERIALS/METHODS: To address this question, we evaluated the hypothalamic and hepatic tissues in recently weaned mice (d28) and the hypothalamus of newborn mice (d0) from dams fed HFD or SC during pregnancy and lactation. RESULTS: Body weight, adipose tissue mass, and food intake were more accentuated in HFD-O mice than in SC-O mice. In addition, intolerance to glucose and insulin was higher in HFD-O mice than in SC-O mice. Compared with SC-O mice, levels of hypothalamic IL1-ß mRNA, NFκB protein, and p-JNK were increased in HFD-O mice. Furthermore, compared with SC-O mice, hypothalamic AKT phosphorylation after insulin challenge was reduced, while markers of ERS (p-PERK, p-eIF2α, XBP1s, GRP78, and GRP94) and p-AMPK were increased in the hypothalamic tissue of HFD-O at d28 but not at d0. These damages to hypothalamic signaling were accompanied by increased triglyceride deposits, activation of NFκB, p-JNK, p-PERK and p-eIF2α. CONCLUSION: These point out lactation period as maternal trigger for metabolic changes in the offspring. These changes may occur early and quietly contribute to obesity and associated pathologies in adulthood. Although in rodents the establishment of ARC neuronal projections occurs during the lactation period, in humans it occurs during the third trimester. Gestational diabetes and obesity in this period may contribute to impairment of energy homeostasis.

Western diet modulates insulin signaling, c-Jun N-terminal kinase activity, and insulin receptor substrate-1ser307 phosphorylation in a tissue-specific fashion.

The mechanisms by which diet-induced obesity is associated with insulin resistance are not well established, and no study has until now integrated, in a temporal manner, functional insulin action data with insulin signaling in key insulin-sensitive tissues, including the hypothalamus. In this study, we evaluated the regulation of insulin sensitivity by hyperinsulinemic-euglycemic clamp procedures and insulin signaling, c-jun N-terminal kinase (JNK) activation and insulin receptor substrate (IRS)-1(ser307) phosphorylation in liver, muscle, adipose tissue, and hypothalamus, by immunoprecipitation and immunoblotting, in rats fed on a Western diet (WD) or control diet for 10 or 30 d. WD increased visceral adiposity, serum triacylglycerol, and insulin levels and reduced whole-body glucose use. After 10 d of WD (WD10) there was a decrease in IRS-1/phosphatidylinositol 3-kinase/protein kinase B pathway in hypothalamus and muscle, associated with an attenuation of the anorexigenic effect of insulin in the former and reduced glucose transport in the latter. In WD10, there was an increased glucose transport in adipose tissue in parallel to increased insulin signaling in this tissue. After 30 d of WD, insulin was less effective in suppressing hepatic glucose production, and this was associated with a decrease in insulin signaling in the liver. JNK activity and IRS-1(ser307) phosphorylation were higher in insulin-resistant tissues. In summary, the insulin resistance induced by WD is tissue specific and installs first in hypothalamus and muscle and later in liver, accompanied by activation of JNK and IRS-1(ser307) phosphorylation. The impairment of the insulin signaling in these tissues, but not in adipose tissue, may lead to increased adiposity and insulin resistance in the WD rats.