Hypothalamic α7 nicotinic acetylcholine receptor (α7nAChR) is downregulated by TNFα-induced Let-7 overexpression driven by fatty acids.

The α7nAChR is crucial to the anti-inflammatory reflex, and to the expression of neuropeptides that control food intake, but its expression can be decreased by environmental factors. We aimed to investigate whether microRNA modulation could be an underlying mechanism in the α7nAchR downregulation in mouse hypothalamus following a short-term exposure to an obesogenic diet. Bioinformatic analysis revealed Let-7 microRNAs as candidates to regulate Chrna7, which was confirmed by the luciferase assay. Mice exposed to an obesogenic diet for 3 days had increased Let-7a and decreased α7nAChR levels, accompanied by hypothalamic fatty acids and TNFα content. Hypothalamic neuronal cells exposed to fatty acids presented higher Let-7a and TNFα levels and lower Chrna7 expression, but when the cells were pre-treated with TLR4 inhibitor, Let-7a, TNFα, and Chrna7 were rescued to normal levels. Thus, the fatty acids overload trigger TNFα-induced Let-7 overexpression in hypothalamic neuronal cells, which negatively regulates α7nAChR, an event that can be related to hyperphagia and obesity predisposition in mice.

In vivo maternal and in vitro BPA exposure effects on hypothalamic neurogenesis and appetite regulators.

In utero exposure to the ubiquitous plasticizer, bisphenol A (BPA) is associated with offspring obesity. As food intake/appetite is one of the critical elements contributing to obesity, we determined the effects of in vivo maternal BPA and in vitro BPA exposure on newborn hypothalamic stem cells which form the arcuate nucleus appetite center. For in vivo studies, female rats received BPA prior to and during pregnancy via drinking water, and newborn offspring primary hypothalamic neuroprogenitor (NPCs) were obtained and cultured. For in vitro BPA exposure, primary hypothalamic NPCs from healthy newborns were utilized. In both cases, we studied the effects of BPA on NPC proliferation and differentiation, including putative signal and appetite factors. Maternal BPA increased hypothalamic NPC proliferation and differentiation in newborns, in conjunction with increased neuroproliferative (Hes1) and proneurogenic (Ngn3) protein expression. With NPC differentiation, BPA exposure increased appetite peptide and reduced satiety peptide expression. In vitro BPA-treated control NPCs showed results that were consistent with in vivo data (increase appetite vs satiety peptide expression) and further showed a shift towards neuronal versus glial fate as well as an increase in the epigenetic regulator lysine-specific histone demethylase1 (LSD1). These findings emphasize the vulnerability of stem-cell populations that are involved in life-long regulation of metabolic homeostasis to epigenetically-mediated endocrine disruption by BPA during early life.

Maternal bisphenol A exposure alters rat offspring hepatic and skeletal muscle insulin signaling protein abundance.

BACKGROUND: The obesogenic and diabetogenic effects of the environmental toxin bisphenol A during critical windows of development are well recognized. Liver and skeletal muscle play a central role in the control of glucose production, utilization, and storage. OBJECTIVES: We hypothesized that maternal bisphenol A exposure disrupts insulin signaling in rat offspring liver and skeletal muscle. We determined the protein expression of hepatic and skeletal muscle insulin signaling molecules including insulin receptor beta, its downstream target insulin receptor substrate 1 and glucose transporters (glucose transporter 2, glucose transporter 4), and hepatic glucose-regulating enzymes phosphoenolpyruvate carboxykinase and glucokinase. STUDY DESIGN: Rat dams had ad libitum access to filtered drinking water (control) or drinking water with bisphenol A from 2 weeks prior to mating and through pregnancy and lactation. Offspring litters were standardized to 4 males and 4 females and nursed by the same dam. At weaning, bisphenol A exposure was removed from all offspring. Glucose tolerance was tested at 6 weeks and 6 months. Liver and skeletal muscle was collected from 3 week old and 10 month old offspring for protein expression (Western blot) of insulin receptor beta, insulin receptor substrate 1, glucose transporter 2, glucose transporter 4, phosphoenolpyruvate carboxykinase, and glucokinase. RESULTS: Male, but not female, bisphenol A offspring had impaired glucose tolerance at 6 weeks and 6 months. Both male and female adult offspring had higher glucose-stimulated insulin secretion as well as the ratio of stimulated insulin to glucose. Male bisphenol A offspring had higher liver protein abundance of the 200 kDa insulin receptor beta precursor (2-fold), and insulin receptor substrate 1 (1.5-fold), whereas glucose transporter 2 was 0.5-fold of the control at 3 weeks of age. In adult male bisphenol A offspring, the abundance of insulin receptor beta was higher (2-fold) and glucose transporter 4 was 0.8-fold of the control in skeletal muscle. In adult female bisphenol A offspring, the skeletal muscle protein abundance of glucose transporter 4 was 0.4-fold of the control. CONCLUSION: Maternal bisphenol A had sex- and tissue-specific effects on insulin signaling components, which may contribute to increased risk of glucose intolerance in offspring. Glucose transporters were consistently altered at both ages as well as in both sexes and may contribute to glucose intolerance. These data suggest that maternal bisphenol A exposure should be limited during pregnancy and lactation.

Programmed hyperphagia in offspring of obese dams: Altered expression of hypothalamic nutrient sensors, neurogenic factors and epigenetic modulators.

Maternal overnutrition results in programmed offspring obesity, mediated in part, by hyperphagia. This is remarkably similar to the effects of maternal undernutrition on offspring hyperphagia and obesity. In view of the marked differences in the energy environment of the over and under-nutrition exposures, we studied the expression of select epigenetic modifiers associated with energy imbalance including neurogenic factors and appetite/satiety neuropeptides which are indicative of neurogenic differentiation. HF offspring were exposed to maternal overnutrition (high fat diet; HF) during pregnancy and lactation. We determined the protein expression of energy sensors (mTOR, pAMPK), epigenetic factors (DNA methylase, DNMT1; histone deacetylase, SIRT1/HDAC1), neurogenic factors (Hes1, Mash1, Ngn3) and appetite/satiety neuropeptides (AgRP/POMC) in newborn hypothalamus and adult arcuate nucleus (ARC). Despite maternal obesity, male offspring born to obese dams had similar body weight at birth as Controls. However, when nursed by the same dams, male offspring of obese dams exhibited marked adiposity. At 1 day of age, HF newborn males had significantly decreased energy sensors, DNMT1 including Hes1 and Mash1, which may impact neuroprogenitor cell proliferation and differentiation. This is consistent with increased AgRP in HF newborns. At 6 months of age, HF adult males had significantly increased energy sensors and decreased histone deactylases. In addition, the persistent decreased Hes1, Mash1 as well as Ngn3 are consistent with increased AgRP and decreased POMC. Thus, altered energy sensors and epigenetic responses which modulate gene expression and adult neuronal differentiation may contribute to hyperphagia and obesity in HF male offspring.

Maternal obesity and high-fat diet program offspring metabolic syndrome.

OBJECTIVE: We determined the potential programming effects of maternal obesity and high-fat (HF) diet during pregnancy and/or lactation on offspring metabolic syndrome. STUDY DESIGN: A rat model of maternal obesity was created using an HF diet prior to and throughout pregnancy and lactation. At birth, pups were cross-fostered, thereby generating 4 paradigms of maternal diets during pregnancy/lactation: (1) control (Con) diet during pregnancy and lactation (Con/Con), (2) HF during pregnancy and lactation (HF/HF), (3) HF during pregnancy alone (HF/Con), and (4) HF during lactation alone (Con/HF). RESULTS: Maternal phenotype during pregnancy and the end of lactation evidenced markedly elevated body fat and plasma corticosterone levels in HF dams. In the offspring, the maternal HF diet during pregnancy alone programmed increased offspring adiposity, although with normal body weight, whereas the maternal HF diet during lactation increased both body weight and adiposity. Metabolic disturbances, particularly that of hyperglycemia, were apparent in all groups exposed to the maternal HF diet (during pregnancy and/or lactation), although differences were apparent in the manifestation of insulin resistant vs insulin-deficient phenotypes. Elevated systolic blood pressure was manifest in all groups, implying that exposure to an obese/HF environment is disadvantageous for offspring health, regardless of pregnancy or lactation periods. Nonetheless, the underlying mechanism may differ because offspring that experienced in utero HF exposure had increased corticosterone levels. CONCLUSION: Maternal obesity/HF diet has a marked impact on offspring body composition and the risk of metabolic syndrome was dependent on the period of exposure during pregnancy and/or lactation.

Developmental programming of appetite/satiety.

Obesity is often attributed to a Western lifestyle, a high-fat diet and decreased activity. While these factors certainly contribute to adult obesity, compelling data from our laboratory and others indicate that this explanation is oversimplified. Recent studies strongly argue that maternal/fetal under- or overnutrition predisposes the offspring to become hyperphagic and increases the risk of later obesity. Both infants small for gestational age (SGA) or infants born to obese mothers who consume a high-fat diet are at a markedly increased risk of adult obesity. Specific alterations in the fetal metabolic/energy environment directly influence the development of appetite regulatory pathways. Specifically, SGA infants demonstrate (1) impaired satiety and anorexigenic cell signaling, (2) enhanced cellular orexigenic responses, (3) programmed dysfunction of neuroprogenitor cell proliferation/differentiation, and (4) increased expression of appetite (NPY) versus satiety (POMC) neurons. In both hypothalamic tissue and ex vivo culture, SGA newborns exhibit increased levels of the nutrient sensor SIRT1, signifying reduced energy, whereas maternal high-fat-exposed newborns exhibit reduced levels of pAMPK, signifying energy excess. Via downstream regulation of bHLH neuroproliferation (Hes1) and neurodifferentiation factors (Mash1, Ngn3), neurogenesis is biased toward orexigenic and away from anorexigenic neurons, resulting in excess appetite, reduced satiety and development of obesity. Despite the developmental programming of appetite neurogenesis, the potential for neuronal remodeling raises the opportunity for novel interventions.

Breastfeeding moderates the association of maternal pre-pregnancy nutritional status with offspring body composition at 30 years.

Maternal pre-pregnancy body mass index is positively associated with offspring obesity, even at adulthood, whereas breastfeeding decreases the risk of obesity. The present study was aimed at assessing whether breastfeeding moderates the association of maternal pre-pregnancy body mass index with offspring body composition at adulthood, using data from 3439 subjects enrolled in a southern Brazilian birth cohort. At 30 years of age, maternal pre-pregnancy body mass index was positively associated with offspring prevalence of obesity, abdominal obesity, as well as body mass index and fat and lean mass index. Breastfeeding moderated the association of maternal pre-pregnancy obesity with offspring adiposity at 30 years of age. For those breastfed<6 months, body mass index was 4.13 kg/m2 (95% confidence interval: 2.98; 5.28) higher among offspring of obese mothers, in relation to offspring of normal weight mothers, whereas among those breastfed≥6 months the magnitude of the difference was small [2.95 kg/m2 (95% confidence interval: 1.17; 4.73)], p-value for interaction = 0.03. Concerning obesity, among those who had been breastfed < 6 months, the prevalence of obesity was 2.56 (95% confidence interval: 1.98; 3.31) times higher among offspring of obese mothers. On the other hand, among those who were breastfed ≥ 6 months, the prevalence of obesity was 1.82 (95% confidence interval: 1.09; 3.04) times higher among offspring of obese mothers. Therefore, among overweight mothers breastfeeding for more than 6 months should be supported, as it may mitigate the consequences of maternal overweight on offspring body composition.

High-Fat, High-Calorie Breast Milk in Women with Overweight or Obesity and Its Association with Maternal Serum Insulin Concentration and Triglycerides Levels.

The childhood obesity epidemic continues to be a challenge. Maternal obesity and excessive infant weight gain are strong predictors of childhood obesity, which itself is a major risk factor for adult obesity. The primary source of nutrition during early life is breast milk, and its composition is impacted by maternal habitus and diet. We thus studied the relationship between maternal BMI, serum lipids and insulin, and breast milk fat and calorie content from foremilk to hindmilk. Women who were exclusively breastfeeding at 7-8 weeks postpartum were BMI classified as Normal (18.5-24.9, n = 9) and women with Overweight/Obese (OW/OB ≥ 25, n = 13). Maternal blood and continuous breast milk samples obtained from foremilk to hindmilk were analyzed, and infant milk intake was assessed. Women with OW/OB had significantly higher milk fat and calorie content in the first foremilk and last hindmilk sample as compared to Normal BMI women. Amongst all women, maternal serum triglycerides, insulin, and HOMA were significantly correlated with foremilk triglyceride concentration, suggesting that maternal serum triglyceride and insulin action contribute to human milk fat content. As the milk fat content of OW/OB women has caloric implications for infant growth and childhood obesity, these results suggest the potential for modulating milk fat content by a reduction in maternal serum lipids or insulin.

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.

Developmental origins of obesity: programmed adipogenesis.

The metabolic syndrome epidemic, including a marked increase in the prevalence of obesity and gestational diabetes mellitus (GDM) among pregnant women, represents a significant public health problem. There is increasing recognition that the risk of adult obesity is clearly influenced by prenatal and infant environmental exposures, particularly nutrition. This tenet is the fundamental basis of developmental programming. Low birth weight, together with infant catch-up growth, is associated with a significant risk of adult obesity. Exposure to maternal obesity, with or without GDM, or having a high birth weight also represents an increased risk for childhood and adult obesity. Animal models have replicated human epidemiologic findings and elucidated potential programming mechanisms that include altered organ development, cellular signaling responses, and epigenetic modifications. Prenatal care has made great strides in optimizing maternal, fetal, and neonatal health, and now has the opportunity to begin interventions which prevent or reduce childhood/adult obesity. Guidelines that integrate optimal pregnancy nutrition and weight gain, management of GDM, and newborn feeding strategies with long-term consequences on adult obesity, remain to be elucidated.

Maternal high-fat diet programs rat offspring hypertension and activates the adipose renin-angiotensin system.

OBJECTIVE: A maternal high-fat diet creates an increased risk of offspring obesity and systemic hypertension. Although the renal renin-angiotensin system (RAS) is known to regulate blood pressure, it is now recognized that the RAS is also activated in adipose tissue during obesity. We hypothesized that programmed offspring hypertension is associated with the activation of the adipose tissue RAS in the offspring of obese rat dams. STUDY DESIGN: At 3 weeks of age, female rats were weaned to a high-fat diet (60% k/cal; n = 6) or control diet (10% k/cal; n = 6). At 11 weeks of age, these rats were mated and continued on their respective diets during pregnancy. After birth, at 1 day of age, subcutaneous adipose tissue was collected; litter size was standardized, and pups were cross-fostered to either control or high-fat diet dams, which created 4 study groups. At 21 days of age, offspring were weaned to control or high-fat diet. At 6 months of age, body fat and blood pressure were measured. Thereafter, subcutaneous and retroperitoneal adipose tissue was harvested from male offspring. Protein expression of adipose tissue RAS components were determined by Western blotting. RESULTS: The maternal high-fat diet induced early and persistent alterations in offspring adipose RAS components. These changes were dependent on the period of exposure to the maternal high-fat diet, were adipose tissue specific (subcutaneous and retroperitoneal), and were exacerbated by a postnatal high-fat diet. Maternal high-fat diet increased adiposity and blood pressure in offspring, regardless of the period of exposure. CONCLUSION: These findings suggest that programmed adiposity and the activation of the adipose tissue RAS are associated with hypertension in offspring of obese dams.

Developmental programming of offspring obesity, adipogenesis, and appetite.

A newly recognized primary cause of the obesity epidemic is the developmental programming effects of infants born to mothers with obesity or gestational diabetes, intrauterine growth-restricted newborns, and offspring exposed to environmental toxins including bisphenol A. The mechanisms which result in offspring obesity include the programming of the hypothalamic appetite pathway and adipogenic signals regulating lipogenesis. Processes include nutrient sensors, epigenetic modifications, and alterations in stem cell precursors of both appetite/satiety neurons and adipocytes which are modulated to potentiate offspring obesity. Future strategies for the prevention and therapy of obesity must address programming effects of the early life environment.

Thermoneutrality effects on developmental programming of obesity.

Developmental programming studies using mouse models have housed the animals at human thermoneutral temperatures (22°C) which imposes constant cold stress. As this impacts energy homeostasis, we investigated the effects of two housing temperatures (22°C and 30°C) on obesity development in male and female offspring of Control and FR dams. Pregnant mice were housed at 22°C (cold-exposed, CE) or 30°C (thermoneutrality, TN) room temperature. At gestational age e10, mice were fed either an ad libitum diet (Control) or were 30% food-restricted (FR) to produce low birth weight newborns. Following delivery, all dams were fed an ad libitum diet and maternal mice continued to nurse their own pups. At 3 weeks of age, offspring were weaned to an ad libitum diet and housed at similar temperatures as their mothers. Body weights and food intake were monitored. At 6 months of age, body composition and glucose tolerance test were determined, after which, brain and adipose tissue were collected for analysis. FR/CE and FR/TN offspring exhibited hyperphagia and were significantly heavier with increased adiposity as compared to their respective Controls. There was sex-specific effects of temperature in both groups. Male offspring at TN were heavier with increased body fat, though the food intake was decreased as compared to CE males. This was reflected by hypertrophic adipocytes and increased arcuate nucleus satiety/appetite ratio. In contrast, female offspring were not impacted by housing temperature. Thus, unlike female offspring, there was a significant interaction of diet and temperature evident in the male offspring with accentuated adverse effects evident in FR/TN males.

Maternal consumption of a high-fat diet modulates the inflammatory response in their offspring, mediated by the M1 muscarinic receptor.

Introduction: High-fat diet (HFD) consumption is associated with various metabolic disorders and diseases. Both pre-pregnancy and maternal obesity can have long-term consequences on offspring health. Furthermore, consuming an HFD in adulthood significantly increases the risk of obesity and metabolic disorders. However, an intriguing phenomenon known as the obesity paradox suggests that obesity may confer a protective effect on mortality outcomes in sepsis. In sepsis, activation of the cholinergic anti-inflammatory pathway (CAP) can help mitigate systemic inflammation. We employed a metabolic programming model to explore the relationship between maternal HFD consumption and offspring response to sepsis. Methods: We fed female mice either a standard diet (SC) or an HFD during the pre-pregnancy, pregnancy, and lactation periods. Subsequently, we evaluated 28-day-old male offspring. Results: Notably, we discovered that offspring from HFD-fed dams (HFD-O) exhibited a higher survival rate compared with offspring from SC-fed dams (SC-O). Importantly, inhibition of the m1 muscarinic acetylcholine receptor (m1mAChR), involved in the CAP, in the hypothalamus abolished this protection. The expression of m1mAChR in the hypothalamus was higher in HFD-O at different ages, peaking on day 28. Treatment with an m1mAChR agonist could modulate the inflammatory response in peripheral tissues. Specifically, CAP activation was greater in the liver of HFD-O following agonist treatment. Interestingly, lipopolysaccharide (LPS) challenge failed to induce a more inflammatory state in HFD-O, in contrast to SC-O, and agonist treatment had no additional effect. Analysis of spleen immune cells revealed a distinct phenotype in HFD-O, characterized by elevated levels of CD4+ lymphocytes rather than CD8+ lymphocytes. Moreover, basal Il17 messenger RNA (mRNA) levels were lower while Il22 mRNA levels were higher in HFD-O, and we observed the same pattern after LPS challenge. Discussion: Further examination of myeloid cells isolated from bone marrow and allowed to differentiate showed that HFD-O macrophages displayed an anti-inflammatory phenotype. Additionally, treatment with the m1mAChR agonist contributed to reducing inflammatory marker levels in both groups. In summary, our findings demonstrate that HFD-O are protected against LPS-induced sepsis, and this protection is mediated by the central m1mAChR. Moreover, the inflammatory response in the liver, spleen, and bone marrow-differentiated macrophages is diminished. However, more extensive analysis is necessary to elucidate the specific mechanisms by which m1mAChR modulates the immune response during sepsis.

Nutrient sensor-mediated programmed nonalcoholic fatty liver disease in low birthweight offspring.

OBJECTIVE: We hypothesized that gestationally programmed nonalcoholic fatty liver disease in low-birthweight offspring is mediated through nutrient sensors nicotinamide adenine dinucleotide+-dependent histone deacetylase (SIRT1) and AMP-activated protein kinase (AMPK). STUDY DESIGN: Pregnant dams received ad libitum food or were 50% food restricted from pregnancy days 10-21 to produce control and low-birthweight newborn offspring, respectively. All pups were nursed by control dams and weaned to ad libitum feed. We determined hepatic SIRT1 and AMPK activities and protein expression of lipid targets in low-birthweight and control fetuses, newborns, and adult offspring (3 months). RESULTS: Low-birthweight fetuses demonstrated increased prenatal hepatic SIRT1 activity, although with increased lipogenesis. After birth, low-birthweight newborn offspring undergo postnatal suppression of hepatic SIRT1 and AMPK activities in conjunction with increased lipogenesis, decreased lipolysis, and increased fat stores. CONCLUSION: These findings suggest that undernutrition stress in utero may program hepatic nutrient sensors to perceive normal postnatal nutrition as a state of nutrient excess with the induction of hepatic lipid storage.

Automation-assisted versus manual reading of cervical cytology (MAVARIC): a randomised controlled trial.

BACKGROUND: The standard for reading cervical cytology is for a cytoscreener to manually search across an entire slide for abnormal cells using a conventional microscope. Automated technology can select fields of view to assess abnormal cells, which allows targeted reading by cytoscreeners. In the Manual Assessment Versus Automated Reading In Cytology (MAVARIC) trial, we compared the accuracy of these techniques for the detection of underlying disease. METHODS: For this randomised controlled trial, women aged 25-64 years undergoing primary cervical screening in Manchester, UK, were randomly assigned (1:2) to receive either manual reading only or paired reading (automation-assisted reading and manual reading), between March 1, 2006, and Feb 28, 2009. In the paired arm, two automated systems were used-the ThinPrep Imaging System and the FocalPoint GS Imaging System. General practices and community clinics were randomised to either ThinPrep or to SurePath (for the FocalPoint system) liquid-based cytology with block randomisation stratified by deprivation index. Samples were then individually randomised to manual reading only or paired reading only. Laboratory staff were unaware of the allocation of each slide and concealment was maintained until the end of the reporting process. The primary outcome was sensitivity of automation-assisted reading relative to manual reading for the detection of underlying cervical intraepithelial neoplasia grade 2 or worse (CIN2+) in the paired arm. This trial is registered, number ISRCTN66377374. FINDINGS: 73,266 liquid-based cytology samples were obtained from women undergoing primary cervical screening; 24,688 allocated to the manual-only arm and 48,578 to the paired-reading arm. Automation-assisted reading was 8% less sensitive than manual reading (relative sensitivity 0·92, 95% CI 0·89-0·95), which was equivalent to an absolute reduction in sensitivity of 6·3%, assuming the sensitivity of manual reading to be 79%. Specificity of automation-assisted reading relative to manual reading increased by 0·6% (1·006, 95% CI 1·005-1·007). INTERPRETATION: The inferior sensitivity of automation-assisted reading for the detection of CIN2+, combined with an inconsequential increase in specificity, suggests that automation-assisted reading cannot be recommended for primary cervical screening.

Modulation of Milk and Lipid Synthesis and Secretion in a3-Dimensional Mouse Mammary Epithelial Cell Culture Model: Effects of Palmitate and Orlistat.

Human milk synthesis is impacted by maternal diet, serum composition, and substrate uptake and synthesis by mammary epithelial cells (MECs). The milk of obese/high-fat-diet women has an increased fat content, which promote excess infant weight gain and the risk of childhood/adult obesity. Yet, the knowledge of milk synthesis regulation is limited, and there are no established approaches to modulate human milk composition. We established a 3-dimensional mouse MEC primary culture that recreates the milk production pathway and tested the effects of the major saturated fatty acid in human milk (palmitate) and a lipoprotein lipase inhibitor (orlistat) on triglyceride production. Positive immunostaining confirmed the presence of milk protein and intracellular lipid including milk globules in the cytoplasm and extracellular space. The treatment with palmitate activated "milk" production by MECs (ß-casein) and the lipid pathway (as evident by increased protein and mRNA expression). Consistent with these cellular changes, there was increased secretion of milk protein and triglyceride in MEC "milk". The treatment with orlistat suppressed milk triglyceride production. Palmitate increased milk and lipid synthesis, partly via lipoprotein lipase activation. These findings demonstrate the ability to examine MEC pathways of milk production via both protein and mRNA and to modulate select pathways regulating milk composition in MEC culture.

TNFα-Induced Oxidative Stress and Mitochondrial Dysfunction Alter Hypothalamic Neurogenesis and Promote Appetite Versus Satiety Neuropeptide Expression in Mice.

Maternal obesity results in programmed offspring hyperphagia and obesity. The increased offspring food intake is due in part to the preferential differentiation of hypothalamic neuroprogenitor cells (NPCs) to orexigenic (AgRP) vs. anorexigenic (POMC) neurons. The altered neurogenesis may involve hypothalamic bHLH (basic helix-loop-helix) neuroregulatory factors (Hes1, Mash1, and Ngn3). Whilst the underlying mechanism remains unclear, it is known that mitochondrial function is critical for neurogenesis and is impacted by proinflammatory cytokines such as TNFα. Obesity is associated with the activation of inflammation and oxidative stress pathways. In obese pregnancies, increased levels of TNFα are seen in maternal and cord blood, indicating increased fetal exposure. As TNFα influences neurogenesis and mitochondrial function, we tested the effects of TNFα and reactive oxidative species (ROS) hydrogen peroxide (H2O2) on hypothalamic NPC cultures from newborn mice. TNFα treatment impaired NPC mitochondrial function, increased ROS production and NPC proliferation, and decreased the protein expression of proneurogenic Mash1/Ngn3. Consistent with this, AgRP protein expression was increased and POMC was decreased. Notably, treatment with H2O2 produced similar effects as TNFα and also reduced the protein expression of antioxidant SIRT1. The inhibition of STAT3/NFκB prevented the effects of TNFα, suggesting that TNFα mediates its effects on NPCs via mitochondrial-induced oxidative stress that involves both signaling pathways.

Hepatic Epigenetic Reprogramming After Liver Resection in Offspring Alleviates the Effects of Maternal Obesity.

Obesity has become a public health problem in recent decades, and during pregnancy, it can lead to an increased risk of gestational complications and permanent changes in the offspring resulting from a process known as metabolic programming. The offspring of obese dams are at increased risk of developing non-alcoholic fatty liver disease (NAFLD), even in the absence of high-fat diet consumption. NAFLD is a chronic fatty liver disease that can progress to extremely severe conditions that require surgical intervention with the removal of the injured tissue. Liver regeneration is necessary to preserve organ function. A range of pathways is activated in the liver regeneration process, including the Hippo, TGFß, and AMPK signaling pathways that are under epigenetic control. We investigated whether microRNA modulation in the liver of the offspring of obese dams would impact gene expression of Hippo, TGFß, and AMPK pathways and tissue regeneration after partial hepatectomy (PHx). Female Swiss mice fed a standard chow or a high-fat diet (HFD) before and during pregnancy and lactation were mated with male control mice. The offspring from control (CT-O) and obese (HF-O) dams weaned to standard chow diet until day 56 were submitted to PHx surgery. Prior to the surgery, HF-O presented alterations in miR-122, miR-370, and Let-7a expression in the liver compared to CT-O, as previously shown, as well as in its target genes involved in liver regeneration. However, after the PHx (4 h or 48 h post-surgery), differences in gene expression between CT-O and HF-O were suppressed, as well as in microRNA expression in the liver. Furthermore, both CT-O and HF-O presented a similar regenerative capacity of the liver within 48 h after PHx. Our results suggest that survival and regenerative mechanisms induced by the partial hepatectomy may overcome the epigenetic changes in the liver of offspring programmed by maternal obesity.

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.