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 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.

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.

Maternal High Fat Diet Programs Male Mice Offspring Hyperphagia and Obesity: Mechanism of Increased Appetite Neurons via Altered Neurogenic Factors and Nutrient Sensor AMPK.

Maternal high-fat (HF) is associated with offspring hyperphagia and obesity. We hypothesized that maternal HF alters fetal neuroprogenitor cell (NPC) and hypothalamic arcuate nucleus (ARC) development with preferential differentiation of neurons towards orexigenic (NPY/AgRP) versus anorexigenic (POMC) neurons, leading to offspring hyperphagia and obesity. Furthermore, these changes may involve hypothalamic bHLH neuroregulatory factors (Hes1, Mash1, Ngn3) and energy sensor AMPK. Female mice were fed either a control or a high fat (HF) diet prior to mating, and during pregnancy and lactation. HF male newborns were heavier at birth and exhibited decreased protein expression of hypothalamic bHLH factors, pAMPK/AMPK and POMC with increased AgRP. As adults, these changes persisted though with increased ARC pAMPK/AMPK. Importantly, the total NPY neurons were increased, which was consistent with the increased food intake and adult fat mass. Further, NPCs from HF newborn hypothalamic tissue showed similar changes with preferential NPC neuronal differentiation towards NPY. Lastly, the role of AMPK was further confirmed with in vitro treatment of Control NPCs with pharmacologic AMPK modulators. Thus, the altered ARC development of HF offspring results in excess appetite and reduced satiety leading to obesity. The underlying mechanism may involve AMPK/bHLH pathways.

Programmed Epigenetic DNA Methylation-Mediated Reduced Neuroprogenitor Cell Proliferation and Differentiation in Small-for-Gestational-Age Offspring.

Small-for-gestational age (SGA) human newborns have an increased risk of hyperphagia and obesity, as well as a spectrum of neurologic and neurobehavioral abnormalities. We have shown that the SGA hypothalamic (appetite regulatory site) neuroprogenitor cells (NPCs) exhibit reduced proliferation and neuronal differentiation. DNA methylation (DNA methyltransferase; DNMT1) regulates neurogenesis by maintaining NPC proliferation and suppressing premature differentiation. Once differentiation ensues, DNMT1 preferentially promotes neuronal and inhibits astroglial fate. We hypothesized that the programmed dysfunction of NPC proliferation and differentiation in SGA offspring is epigenetically mediated via DNMT1. Pregnant rats received either ad libitum food (Control) or were 50% food-restricted to create SGA offspring. Primary hypothalamic NPCs from 1 day old SGA and Controls newborns were cultured and transfected with nonspecific or DNMT1-specific siRNA. NPC proliferation and protein expression of specific markers of NPC (nestin), neuroproliferative transcription factor (Hes1), neurons (Tuj1) and astrocytes (GFAP) were determined. Under basal conditions, SGA NPCs exhibited decreased DNMT1 and reduced proliferation and differentiation, as compared to Controls. In both SGA and Controls, DNMT1 siRNA in complete media inhibited NPC proliferation, consistent with reduced expression of nestin and Hes1. In differentiation media, DNMT1 siRNA decreased expression of Tuj1 but increased GFAP. In vivo data replicated these findings. In SGA offspring, impaired neurogenesis is epigenetically mediated, in part, via reduction in DNMT1 expression and suppression of Hes1 resulting in NPC differentiation. It is likely that the maturation of regions beyond the hypothalamus (e.g., cerebral cortex, hippocampus) may be impacted, contributing to poor cognitive and neurobehavioral competency in SGA offspring.

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.

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.

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.

Basal, endogenous leptin is metabolically active in newborn rat pups.

OBJECTIVE: The adipocyte-derived hormone leptin regulates food intake and body weight via the activation of JAK-STAT pathway in mammalian adult hypothalamic neurons. To investigate whether endogenous leptin is metabolically active in newborn rat pups, the JAK-STAT leptin signaling pathway was analyzed following leptin antagonist challenge. METHODS: One day old male control pups were injected with either (i) saline, (ii) leptin (10 µg/g, s.c; n=4), (iii) pegylated leptin antagonist (PEG-MLA, 20 µg/g, s.c, n=4), or (iv) leptin plus PEG-MLA. Hypothalamus was dissected from individual pups at 30, 45, and 60 min. Protein expression of ObR, STAT3, pSTAT3, and SOCS3 was analyzed by Western blot. RESULTS: Leptin, but not PEG-MLA, produced a significant increase in hypothalamic pSTAT3 relative to saline treatment. Systemically administered PEG-MLA effectively blocks leptin signal induction of hypothalamic JAK-STAT signaling. The presence of PEG-MLA in combination with leptin attenuated the leptin-induced increase in pSTAT3. CONCLUSIONS: Thus, basal leptin levels are metabolically active in the newborn rats. These results brings new insights in considering the importance of endogenous leptin at birth, especially in low birth weight offspring who may be predisposed to altered neurogenesis and later obesity, and provide potential therapeutic strategies for programmed or diet-induced obesity.

Hypothalamic neurosphere progenitor cells in low birth-weight rat newborns: neurotrophic effects of leptin and insulin.

A low birth-weight (LBW) offspring exhibits reduced hypothalamic neural satiety pathways and dysregulated signaling leading to programmed hyperphagia and adult obesity. Hypothalamic appetite circuits develop during early life, under the influence of neurotrophic hormones (leptin and insulin). Notably, LBW newborns have reduced plasma leptin and insulin levels. As neurons and glia arise from neuronal progenitor cells (NPC), we postulated that a programmed impairment of NPCs may contribute to reduced hypothalamic neural pathway development in a LBW offspring. Control dams received ad libitum food, whereas study dams were 50% food-restricted from pregnancy day 10 to 21 (LBW). At day 1 of age, hypothalamic NPCs were cultured as neurospheres (NS) and treated with leptin/insulin. We analyzed in vitro NPC proliferation and differentiation into neurons/astrocytes, expression of signal molecules promoting proliferation (activated Notch1 and its downstream target, Hes1) and in vivo NPC proliferation and migration. LBW offspring had impaired in vivo evidence of NPC division and migration, and reduced in vitro evidence of proliferation and differentiation to neurons and astrocytes, under basal and stimulated conditions. The reduced Notch1 and Hes1 expression in LBW neurosphere, under both basal and stimulated conditions, suggests a reduced progenitor cell population or reduced cell density within the neurosphere.

Central insulin sensitivity in male and female juvenile rats.

The incidence of juvenile obesity is increasing at an alarming rate. In adults, central insulin administration decreases hypothalamic orexigenic neuropeptides, food intake and body weight more effectively in males than females. Mechanisms regulating energy balance in juvenile animals are inherently different from those in adults due to differences in growth rates and hormonal milieu. Therefore, we sought to determine if central insulin treatment in juvenile rats (4 wk) would have similar sex-dependent effects on food intake as those reported in adult rats. Twenty-four hour food intake was measured following icv saline or insulin (0.01 or 0.1 U) prior to the onset of dark phase of the light cycle. An additional set of animals was used to assess the effects of central insulin on hypothalamic orexigenic (NPY, AgRP) and anorexigenic (POMC) neuropeptide mRNA expression. In both males and females, insulin reduced meal size initially (first 4 h) and later decreased meal frequency (4-24 h) to reduce cumulative food intake. Consistent with this, central insulin decreased hypothalamic NPY and AgRP and increased POMC mRNA expression. In contrast to adult studies, there were no demonstrated sex differences. These studies indicate that juvenile females and males are equally sensitive to central insulin anorexigenic effects, perhaps due to a lack of circulating gonadal hormones. The anorexigenic responsiveness of both genders suggests a potential pharmacologic approach to childhood obesity.

Programmed alterations in hypothalamic neuronal orexigenic responses to ghrelin following gestational nutrient restriction.

Intrauterine growth restricted (IUGR) offspring exhibit increased appetite and a propensity to adult obesity. Although the rate of newborn catch-up growth may determine the programming of adult obesity,there is little understanding of mechanisms by which orexigenic pathways are modified. Ghrelin is an orexigenic peptide that acts in the hypothalamic arcuate (ARC) and ventromedial (VMH) nuclei.To examine potential programming effects of IUGR, ghrelin's actions on ARC and VMH neurons were studied in brain slices of adult offspring previously subjected to maternal food restriction (FR) during pregnancy (FR/AdLib [ad libitum]) and both pregnancy and lactation (FR/FR). FR/FR offspring demonstrated increased baseline neuronal firing frequency in both ARC and VMH when compared with both FR/AdLib and control offspring.Among FR/AdLib pups that exhibit hyperphagia and obesity, ghrelin excited more and inhibited fewer ARC neurons when compared with either FR/FR or controls.These results provide evidence of programming of orexigenic/anorexigenic mechanisms depending on the nutrient levels during pregnancy and newborn periods.

Programmed hyperphagia due to reduced anorexigenic mechanisms in intrauterine growth-restricted offspring.

Maternal food restriction during pregnancy results in intrauterine growth-restricted (IUGR) newborns with significantly decreased plasma leptin levels. When nursed by ad libitum-fed controls, IUGR offspring exhibit hyperphagia with adult obesity, marked by increased percentage body fat and plasma leptin, suggesting altered anorexigenic pathways. The authors examined leptin signaling pathways and food intake responses to 2 putative anorexic effectors (leptin and sibutramine, a serotonin reuptake inhibitor) in IUGR offspring. From 10 days to term gestation and through lactation, control pregnant rats received ad libitum food, whereas study rats were 50% food restricted. Following birth, litter size was standardized, and all offspring were nursed by control dams. At 3 weeks of age, offspring were weaned to ad libitum laboratory chow. At ages 1 day and 3 weeks, hypothalamic leptin receptor (Ob-Rb) mRNA and total STAT3 protein expression were determined. In addition, phosphorylated STAT3 was measured in 1-day-old offspring administered peripheral leptin. In prepubescent and adult offspring, anorexic effects of leptin and sibutramine were determined. At 1 day of age, IUGR pups showed increased hypothalamic Ob-Rb mRNA and total STAT3 protein expression though reduced leptin activated phosphorylated STAT3. At 3 weeks of age, IUGR offspring had decreased hypothalamic Ob-Rb mRNA expression, although with continued elevated STAT3 protein levels. The IUGR offspring demonstrated resistance to anorexigenic agents, leptin (6 weeks and 6 months), and sibutramine (8 months), as evidenced by less reduction in food intake and less body weight loss than controls. The IUGR offspring demonstrate suppressed leptin-induced STAT3 phosphorylation and impaired anorexigenic response to 2 factors in the central satiety pathway. This reduced anorexigenic function, together with normal or perhaps enhanced orexigenic function, contributes to the development of programmed obesity in IUGR rat offspring.

Unopposed orexic pathways in the developing fetus.

Fetal swallowing has important roles in fetal gastrointestinal development, and perhaps fetal somatic growth and maturation. Ingestive behavioral responses must develop in utero to provide for acquisition of water and food intake during the neonatal period. At birth, the rat, ovine and human fetus have developed mechanisms to acquire food via intact mechanisms of taste, suckling and swallowing. Our preliminary studies suggest that in sheep and likely in human fetuses, putative orexic-mediated ingestive responses are present near term gestation. We hypothesize that both orexic (appetite) and satiety mechanisms develop during the last third of gestation and the related neurotransmitters involved in this process are functional. The potential in utero imprinting of orexic mechanisms may influence infant, childhood and ultimately adult appetite "set-points". Thus, dysfunctional appetite, and perhaps obesity, may result from maternal environmental influences during critical stages of development.