Responses Of Hypothalamic Neurons Research Articles

Investigating the role of an immediate early gene FOS as a potential regulator of autophagic response to hypoglycemia in embryonic hypothalamic neurons.

Hypoglycemia-associated autonomic failure (HAAF) is a well-established complication of diabetes. Although HAAF has serious outcomes such as recurrent morbidity, coma, and death, the mechanisms of HAAF and its pathological components are largely unknown. Our previous studies have revealed that hypoglycemia is associated with the upregulation of an immediate early gene - FOS. In addition, it is documented that glucose deprivation activates neuronal autophagic activities. Therefore, the present study aimed to identify the role of FOS and one of the core components of the autophagy pathway, Beclin-1 (encoded by the BECN1 gene), in the regulation of autophagic mechanisms in embryonic hypothalamic neurons in response to hypoglycemic conditions. Embryonic Mouse Hypothalamic Cell Line N39 (mHypoE-N39 or N39) was cultured in reduced concentrations of glucose (2000, 900, 500, and 200 mg/L). Gene and protein expression, as well as immunofluorescence studies on autophagy were conducted under different reduced glucose concentrations in N39 hypothalamic neurons with and without FOS and BECN1 gene knockdowns (KD). The outcomes of the present study have demonstrated a significant increase in autophagosome formation and subsequent lysosomal degradation in the hypothalamic neurons in response to reduced glucose concentrations. This hypoglycemic response appears to be lowered to a similar extent in the FOS KD and BECN1 KD cells, albeit insignificantly from the negative control, is indicative of the involvement of FOS in the autophagic response of hypothalamic neurons to hypoglycemia. Moreover, the KD cells exhibited a change in morphology and reduced cell viability compared with the control cells. Our findings suggest that reduced FOS expression could potentially be associated with impaired autophagic activities that are dependent on BECN1, which could lead to decreased or blunted hypothalamic activation in response to hypoglycemia, and this, in turn, may contribute to the development of HAAF.

17β-estradiol promotes acute refeeding in hungry mice via membrane-initiated ERα signaling

ObjectiveEstrogen protects animals from obesity through estrogen receptor α (ERα), partially by inhibiting overeating in animals fed ad libitum. However, the effects of estrogen on feeding behavior in hungry animals remain unclear. In this study, we examined the roles of 17β-estradiol (E2) and ERα in the regulation of feeding in hungry female animals and explored the underlying mechanisms. MethodsWild-type female mice with surgical depletion of endogenous estrogens were used to examine the effects of E2 supplementation on acute refeeding behavior after starvation. ERα-C451A mutant mice deficient in membrane-bound ERα activity and ERα-AF20 mutant mice lacking ERα transcriptional activity were used to further examine mechanisms underlying acute feeding triggered by either fasting or central glucopenia (induced by intracerebroventricular injections of 2-deoxy-D-glucose). We also used electrophysiology to explore the impact of these ERα mutations on the neural activities of ERα neurons in the hypothalamus. ResultsIn the wild-type female mice, ovariectomy reduced fasting-induced refeeding, which was restored by E2 supplementation. The ERα-C451A mutation, but not the ERα-AF20 mutation, attenuated acute feeding induced by either fasting or central glucopenia. The ERα-C451A mutation consistently impaired the neural responses of hypothalamic ERα neurons to hypoglycemia. ConclusionIn addition to previous evidence that estrogen reduces deviations in energy balance by inhibiting eating at a satiated state, our findings demonstrate the unexpected role of E2 that promotes eating in hungry mice, also contributing to the stability of energy homeostasis. This latter effect specifically requires membrane-bound ERα activity.

4-oxo-1,4-dihydrocinnoline Derivative with Phosphatase 1B Inhibitor Activity Enhances Leptin Signal Transduction in Hypothalamic Neurons

Most of regulatory effects of leptin on feeding behavior and energy metabolism are implemented via the hypothalamic leptin system. Attenuation of its activity leads to hyperphagia, obesity and other metabolic disorders. To prevent these pathological conditions, it is necessary to develop approaches aimed at restoring the leptin system. Most promising of them is creating efficient inhibitors of protein phosphotyrosine phosphatase PTP1B, a negative regulator of leptin signaling. The aim of this work was to synthesize a new compound, ethyl-3-(hydroxymethyl)-4-oxo-1,4-dihydrocinnoline-6-carboxylate (PI-04), a 4-oxo-1,4-dihydrocinnoline derivative with a PTP1B inhibitor activity, and to study its effect on IRS2- and STAT3-dependent leptin pathways in culture of hypothalamic neurons isolated from 18-day-old rat embryos. It was shown that PI-04 enhances the stimulatory effect of leptin on phosphorylation of the IRS2 protein at the Ser731 residue and of the STAT3 transcriptional factor at the Tyr705 residue, suggesting a potentiation of functional responses of hypothalamic neurons to leptin in the presence of this compound. The potentiating effect of PI-04 on leptin signaling was implemented at micromolar concentrations when this substance had virtually no effect on neuronal survival. The data obtained are promising in terms of creating phosphatase PTP1B inhibitors based on 4-oxo-1,4-dihydrocinnoline, as well as the possibility of their further use to prevent and correct metabolic disorders caused by attenuated activity of the hypothalamic leptin system.

Effects of intranasal insulin application on the hypothalamic BOLD response to glucose ingestion

The hypothalamus is a crucial structure in the brain that responds to metabolic cues and regulates energy homeostasis. Patients with type 2 diabetes demonstrate a lack of hypothalamic neuronal response after glucose ingestion, which is suggested to be an underlying cause of the disease. In this study, we assessed whether intranasal insulin can be used to enhance neuronal hypothalamic responses to glucose ingestion. In a randomized, double-blinded, placebo-controlled 4-double cross-over experiment, hypothalamic activation was measured in young non- diabetic subjects by determining blood-oxygen-level dependent MRI signals over 30 minutes before and after ingestion of 75 g glucose dissolved in 300 ml water, under intranasal insulin or placebo condition. Glucose ingestion under placebo condition lead to an average 1.4% hypothalamic BOLD decrease, under insulin condition the average response to glucose was a 2.2% decrease. Administration of water did not affect the hypothalamic BOLD responses. Intranasal insulin did not change circulating glucose and insulin levels. Still, circulating glucose levels showed a significant dampening effect on the BOLD response and insulin levels a significant strengthening effect. Our data provide proof of concept for future experiments testing the potential of intranasal application of insulin to ameliorate defective homeostatic control in patients with type 2 diabetes.

A Life without Hunger: The Ups (and Downs) to Modulating Melanocortin-3 Receptor Signaling

Melanocortin neurons conserve body mass in hyper- or hypo-caloric conditions by conveying signals from nutrient sensors into areas of the brain governing appetite and metabolism. In mice, melanocortin-3 receptor (MC3R) deletion alters nutrient partitioning independently of hyperphagia, promoting accumulation of fat over muscle mass. Enhanced rhythms in insulin and insulin-responsive metabolic genes during hypocaloric feeding suggest partial insulin resistance and enhanced lipogenesis. However, exactly where and how MC3Rs affect metabolic control to alter nutrient partitioning is not known. The behavioral phenotypes exhibited by MC3R-deficient mice suggest a contextual role in appetite control. The impact of MC3R-deficiency on feeding behavior when food is freely available is minor. However, homeostatic responses to hypocaloric conditioning involving increased expression of appetite-stimulating (orexigenic) neuropeptides, binge-feeding, food anticipatory activity (FAA), entrainment to nutrient availability and enhanced feeding-related motivational responses are compromised with MC3R-deficiency. Rescuing Mc3r transcription in hypothalamic and limbic neurons improves appetitive responses during hypocaloric conditioning while having minor effects on nutrient partitioning, suggesting orexigenic functions. Rescuing hypothalamic MC3Rs also restores responses of fasting-responsive hypothalamic orexigenic neurons in hypocaloric conditions, suggesting actions that sensitize fasting-responsive neurons to signals from nutrient sensors. MC3R signaling in ventromedial hypothalamic SF1(+ve) neurons improves metabolic control, but does not restore appetitive responses or nutrient partitioning. In summary, desensitization of fasting-responsive orexigenic neurons may underlie attenuated appetitive responses of MC3R-deficient mice in hypocaloric situations. Further studies are needed to identify the specific location(s) of MC3Rs controlling appetitive responses and partitioning of nutrients between fat and lean tissues.

Epigenomic and metabolic responses of hypothalamic POMC neurons to gestational nicotine exposure in adult offspring.

BackgroundEpidemiological and animal studies have reported that prenatal nicotine exposure (PNE) leads to obesity and type-2 diabetes in offspring. Central leptin-melanocortin signaling via hypothalamic arcuate proopiomelanocortin (POMC) neurons is crucial for the regulation of energy and glucose balance. Furthermore, hypothalamic POMC neurons were recently found to mediate the anorectic effects of nicotine through activation of acetylcholine receptors. Here, we hypothesized that PNE impairs leptin-melanocortinergic regulation of energy balance in first-generation offspring by altering expression of long non-coding RNAs (lncRNAs) putatively regulating development and/or function of hypothalamic POMC neurons.MethodsC57BL/6J females were exposed ad libitum to nicotine through drinking water and crossed with C57BL/6J males. Nicotine exposure was sustained during pregnancy and discontinued at parturition. Offspring development was monitored from birth into adulthood. From the age of 8 weeks, central leptin-melanocortin signaling, diabetes, and obesity susceptibility were assessed in male offspring fed a low-fat or high-fat diet for 16 weeks. Nicotine-exposed and non-exposed C57BL/6J females were also crossed with C57BL/6J males expressing the enhanced green fluorescent protein specifically in POMC neurons. Transgenic male offspring were subjected to laser microdissections and RNA sequencing (RNA-seq) analysis of POMC neurons for determination of nicotine-induced gene expression changes and regulatory lncRNA/protein-coding gene interactions.ResultsContrary to expectation based on previous studies, PNE did not impair but rather enhanced leptin-melanocortinergic regulation of energy and glucose balance via POMC neurons in offspring. RNA-seq of laser microdissected POMC neurons revealed only one consistent change, upregulation of Gm15851, a lncRNA of yet unidentified function, in nicotine-exposed offspring. RNA-seq further suggested 82 cis-regulatory lncRNA/protein-coding gene interactions, 19 of which involved coding genes regulating neural development and/or function, and revealed expression of several previously unidentified metabolic, neuroendocrine, and neurodevelopment pathways in POMC neurons.ConclusionsPNE does not result in obesity and type 2 diabetes but instead enhances leptin-melanocortinergic feeding and body weight regulation via POMC neurons in adult offspring. PNE leads to selective upregulation of Gm15851, a lncRNA, in adult offspring POMC neurons. POMC neurons express several lncRNAs and pathways possibly regulating POMC neuronal development and/or function.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-016-0348-2) contains supplementary material, which is available to authorized users.

Attenuated hypothalamic responses to α-melanocyte stimulating hormone during pregnancy in the rat.

Increased appetite and weight gain occurs during pregnancy, associated with development of leptin resistance, and satiety responses to the anorectic peptide α-melanocyte stimulating hormone (α-MSH) are suppressed. This study investigated hypothalamic responses to α-MSH during pregnancy, using c-fos expression in specific hypothalamic nuclei as a marker of neuronal signalling, and in vivo electrophysiology in supraoptic nucleus (SON) oxytocin neurons, as a representative α-MSH-responsive neuronal population that shows a well-characterised α-MSH-induced inhibition of firing. While icv injection of α-MSH significantly increased the number of c-fos-positive cells in the paraventricular, supraoptic, arcuate and ventromedial hypothalamic nuclei in non-pregnant rats, this response was suppressed in pregnant rats. Similarly, SON oxytocin neurons in pregnant rats did not demonstrate characteristic α-MSH-induced inhibition of firing that was observed in non-pregnant animals. Given the known functions of α-MSH in the hypothalamus, the attenuated responses are likely to facilitate adaptive changes in appetite regulation and oxytocin secretion during pregnancy. During pregnancy, a state of positive energy balance develops to support the growing fetus and to deposit fat in preparation for the subsequent metabolic demands of lactation. As part of this maternal adaptation, the satiety response to the anorectic peptide α-melanocyte stimulating hormone (α-MSH) is suppressed. To investigate whether pregnancy is associated with changes in the response of hypothalamic α-MSH target neurons, non-pregnant and pregnant rats were treated with α-MSH or vehicle and c-fos expression in hypothalamic nuclei was then examined. Furthermore, the firing rate of supraoptic nucleus (SON) oxytocin neurons, a known α-MSH responsive neuronal population, was examined in non-pregnant and pregnant rats following α-MSH treatment. Intracerebroventricular injection of α-MSH significantly increased the number of c-fos-positive cells in the paraventricular, arcuate and ventromedial hypothalamic nuclei in non-pregnant rats, but no significant increase was observed in any of these regions in pregnant rats. In the SON, α-MSH did induce expression of c-fos during pregnancy, but this was significantly reduced compared to that observed in the non-pregnant group. Furthermore, during pregnancy, SON oxytocin neurons did not demonstrate the characteristic α-MSH-induced inhibition of firing rate that was observed in non-pregnant animals. Melanocortin receptor mRNA levels during pregnancy were similar to non-pregnant animals, suggesting that receptor down-regulation is unlikely to be a mechanism underlying the attenuated responses to α-MSH during pregnancy. Given the known functions of α-MSH in the hypothalamus, the attenuated responses will facilitate adaptive changes in appetite regulation and oxytocin secretion during pregnancy.

ΔN-TRPV1: A Molecular Co-detector of Body Temperature and Osmotic Stress.

Thirst and antidiuretic hormone secretion occur during hyperthermia or hypertonicity to preserve body hydration. These vital responses are triggered when hypothalamic osmoregulatory neurons become depolarized by ion channels encoded by an unknown product of the transient receptor potential vanilloid-1 gene (Trpv1). Here, we show that rodent osmoregulatory neurons express a transcript of Trpv1 that mediates the selective translation of a TRPV1 variant that lacks a significant portion of the channel's amino terminus (ΔN-TRPV1). The mRNA transcript encoding this variant (Trpv1dn) is widely expressed in the brains of osmoregulating vertebrates, including the human hypothalamus. Transfection of Trpv1dn into heterologous cells induced the expression of ion channels that could be activated by either hypertonicity or by heating in the physiological range. Moreover, expression of Trpv1dn rescued the osmosensory and thermosensory responses of single hypothalamic neurons obtained from Trpv1 knockout mice. ΔN-TRPV1 is therefore a co-detector of core body temperature and fluid tonicity.

Cell type-specific transcriptomics of hypothalamic energy-sensing neuron responses to weight-loss.

Molecular and cellular processes in neurons are critical for sensing and responding to energy deficit states, such as during weight-loss. Agouti related protein (AGRP)-expressing neurons are a key hypothalamic population that is activated during energy deficit and increases appetite and weight-gain. Cell type-specific transcriptomics can be used to identify pathways that counteract weight-loss, and here we report high-quality gene expression profiles of AGRP neurons from well-fed and food-deprived young adult mice. For comparison, we also analyzed Proopiomelanocortin (POMC)-expressing neurons, an intermingled population that suppresses appetite and body weight. We find that AGRP neurons are considerably more sensitive to energy deficit than POMC neurons. Furthermore, we identify cell type-specific pathways involving endoplasmic reticulum-stress, circadian signaling, ion channels, neuropeptides, and receptors. Combined with methods to validate and manipulate these pathways, this resource greatly expands molecular insight into neuronal regulation of body weight, and may be useful for devising therapeutic strategies for obesity and eating disorders.

Role of fatty acids in the nervous control of energy balance

Fatty acid (FA)-sensitive neurons are present in the brain, especially the hypothalamus, and play a key role in the neural control of energy and glucose homeostasis including feeding behavior, insulin secretion and action. Subpopulations of neurons in the ventromedial and arcuate hypothalamic nuclei are selectively either inhibited or activated by FA. Molecular effectors of these FA effects include ion channels such as chloride, potassium or calcium. In addition at least half of the FA responses in ventromedial hypothalamic neurons are mediated by interaction with FAT/CD36, a FA translocator/receptor that does not require intracellular metabolism to activate downstream signaling. Recently, an important role of lipoprotein lipase in FA sensing has also been demonstrated not only in hypothalamus, but also in the hippocampus and striatum. Finally, FA overload might impair neural control of energy homeostasis through enhanced ceramide synthesis and may contribute to obesity and/or type 2 diabetes pathogenesis in predisposed subjects.

Physiological and pathophysiological implications of lipid sensing in the brain.

Fatty acid (FA)-sensitive neurons are present in the brain, especially the hypothalamus, and play a key role in the neural control of energy homeostasis. Through neuronal output, FA may modulate feeding behaviour as well as insulin secretion and action. Subpopulations of neurons in the ventromedial and arcuate hypothalamic nuclei are selectively either inhibited or activated by FA. Molecular effectors of these FA effects probably include chloride or potassium ion channels. While intracellular metabolism and activation of the ATP-sensitive K⁺ channel appear to be necessary for some of the signalling effects of FA, at least half of the FA responses in ventromedial hypothalamic neurons are mediated by interaction with FAT/CD36, an FA transporter/receptor that does not require intracellular metabolism to activate downstream signalling. Thus, FA or their metabolites can modulate neuronal activity as a means of directly monitoring ongoing fuel availability by brain nutrient-sensing neurons involved in the regulation of energy and glucose homeostasis. Recently, the role of lipoprotein lipase in FA sensing has also been shown in animal models not only in hypothalamus, but also in hippocampus and striatum. Finally, FA overload might impair neural control of energy homeostasis through enhanced ceramide synthesis and may contribute to obesity and/or type 2 diabetes pathogenesis in predisposed subjects.

Magel2 Is Required for Leptin-Mediated Depolarization of POMC Neurons in the Hypothalamic Arcuate Nucleus in Mice

Prader-Willi Syndrome is the most common syndromic form of human obesity and is caused by the loss of function of several genes, including MAGEL2. Mice lacking Magel2 display increased weight gain with excess adiposity and other defects suggestive of hypothalamic deficiency. We demonstrate Magel2-null mice are insensitive to the anorexic effect of peripherally administered leptin. Although their excessive adiposity and hyperleptinemia likely contribute to this physiological leptin resistance, we hypothesized that Magel2 may also have an essential role in intracellular leptin responses in hypothalamic neurons. We therefore measured neuronal activation by immunohistochemistry on brain sections from leptin-injected mice and found a reduced number of arcuate nucleus neurons activated after leptin injection in the Magel2-null animals, suggesting that most but not all leptin receptor–expressing neurons retain leptin sensitivity despite hyperleptinemia. Electrophysiological measurements of arcuate nucleus neurons expressing the leptin receptor demonstrated that although neurons exhibiting hyperpolarizing responses to leptin are present in normal numbers, there were no neurons exhibiting depolarizing responses to leptin in the mutant mice. Additional studies demonstrate that arcuate nucleus pro-opiomelanocortin (POMC) expressing neurons are unresponsive to leptin. Interestingly, Magel2-null mice are hypersensitive to the anorexigenic effects of the melanocortin receptor agonist MT-II. In Prader-Willi Syndrome, loss of MAGEL2 may likewise abolish leptin responses in POMC hypothalamic neurons. This neural defect, together with increased fat mass, blunted circadian rhythm, and growth hormone response pathway defects that are also linked to loss of MAGEL2, could contribute to the hyperphagia and obesity that are hallmarks of this disorder.

Short-Term Caloric Restriction Normalizes Hypothalamic Neuronal Responsiveness to Glucose Ingestion in Patients With Type 2 Diabetes

The hypothalamus is critically involved in the regulation of feeding. Previous studies have shown that glucose ingestion inhibits hypothalamic neuronal activity. However, this was not observed in patients with type 2 diabetes. Restoring energy balance by reducing caloric intake and losing weight are important therapeutic strategies in patients with type 2 diabetes. We hypothesized that caloric restriction would have beneficial effects on the hypothalamic neuronal response to glucose ingestion. Functional magnetic resonance imaging was performed in 10 male type 2 diabetic patients before and after a 4-day very-low-calorie diet (VLCD) at a 3.0 Tesla scanner using a blood oxygen level–dependent technique for measuring neuronal activity in the hypothalamus in response to an oral glucose load. Hypothalamic signals were normalized to baseline value, and differences between the pre- and postdiet condition were tested using paired t tests. Pre-VLCD scans showed no response of the hypothalamus to glucose intake (i.e., no signal decrease after glucose intake was observed). Post-VLCD scans showed a prolonged signal decrease after glucose ingestion. The results of the current study demonstrate that short-term caloric restriction readily normalizes hypothalamic responsiveness to glucose ingestion in patients with type 2 diabetes.

Maternal Consumption of High-Fat Diet Disturbs Hypothalamic Neuronal Function in the Offspring: Implications for the Genesis of Obesity

Obesity is a consequence of the loss of the coordinated control of caloric intake and energy expenditure. The homeostasis of this system and, thus, the maintenance of body mass stability depends on the activity of hypothalamic neurons, which are controlled by hormones, nutrients, environmental temperature, and physical activity, providing signals that play pivotal roles in the modulation of hunger, body temperature, motility, and storage of surplus energy (1). Leptin is the most important factor regulating the physiological response to changes in the body energy stores (2). Progressive loss of hypothalamic neuronal response to leptin is a hallmark of obesity, a condition known as hypothalamic resistance to leptin (3). This phenomenon has been characterized at the clinical and molecular levels in experimental models and described, by indirect means, in obese humans (4). As for systemic insulin resistance in type 2 diabetes, hypothalamic resistance to leptin is a consequence of the activation of an inflammatory response in the hypothalamus (5, 6). All of the currently known molecular mechanisms of leptin resistance are activated by inflammatory signals, which target the transduction of the leptin signal by a number of different means. Thus, suppressor of cytokine signaling-3 (SOCS3) can physically block the leptin receptor signal transduction and can also accelerate the proteasome degradation of important downstream targets of this pathway (7); inhibitor of B kinase, C-Jun Nterminal kinase, and protein kinase Ccan catalyze the inhibitory serine phosphorylation of intermediaries of the leptin and insulin signaling systems in the hypothalamus and can also induce the transcription of additional inflammatory genes that boost inflammation in the region (8– 10); and finally, protein tyrosine phosphatase 1B can dephosphorylate and inactivate proteins of the leptin and insulin signaling cascade (11, 12). However, it was only recently that we learned how these inflammatory pathways are triggered. The excessive dietary consumption of saturated fats, one of the most important environmental factors contributing to the global increase of obesity, leads to the activation of signal transduction through Toll-like receptor 4 (TLR4), a receptor of the innate immune system, and the induction of endoplasmic reticulum stress, a cellular mechanism of response to different types of stimuli that can disrupt the physiological protein folding capacity of the endoplasmic reticulum (9, 13). Both these mechanisms can activate inflammation and eventually apoptosis in different cell systems. So far, most studies in this field have focused on adult obesity. However, there are a number of experimental and human studies showing that maternal consumption of high-fat diets during gestation increase the risk for obesity, diabetes, and other components of the metabolic syndrome in the offspring (14, 15). In this issue of Endocrinology, Eva Rother and her colleagues (16) from the University Hospital of Cologne in Germany have evaluated the outcomes of maternal high-fat feeding during gestation and lactation on hypothalamic gene expression in the offspring. According to the experimental approach employed in the study, lean female mice were introduced to a diet containing 35% fat at detection of pregnancy and were maintained on this diet throughout gestation and lactation until

Maternal Diabetes Compromises the Organization of Hypothalamic Feeding Circuits and Impairs Leptin Sensitivity in Offspring

Maternal diabetes is a common complication of pregnancy, and the offspring of diabetic mothers have a higher risk of developing obesity and type 2 diabetes later in life. Despite these observations, the precise biological processes mediating this metabolic programming are not well understood. Here, we explored the consequences of maternal diabetes on the organization of hypothalamic neural circuits involved in the regulation of energy balance. To accomplish this aim, we used a mouse model of maternal insulin deficiency induced by streptozotocin injections. Maternal diabetes was found to be associated with changes in offspring growth as revealed by a significantly higher pre- and postweaning body weight in the offspring of insulin-deficient dams relative to those of control mice. Mice born to diabetic dams also showed increased fasting glucose levels, increased insulin levels, and increased food intake during their adult lives. These impairments in metabolic regulation were associated with leptin resistance during adulthood. Importantly, the ability of leptin to activate intracellular signaling in arcuate neurons was also significantly reduced in neonates born to diabetic dams. Furthermore, neural projections from the arcuate nucleus to the paraventricular nucleus were markedly reduced in the offspring of insulin-deficient dams. Together, these data show that insulin deficiency during gestation has long-term consequences for metabolic regulation. They also indicate that animals born to diabetic dams display abnormally organized hypothalamic feeding pathways that could result from the attenuated responsiveness of hypothalamic neurons to the neurotrophic actions of leptin during neonatal development.

Effect of Ovariectomy on External Urethral Sphincter Activity in Anesthetized Female Rats

The postmenopausal hypoestrogen condition is associated with various lower urinary tract dysfunctions, including frequency, urgency, stress urinary incontinence and recurrent urinary infection. We determined whether hypoestrogen induced lower urinary tract dysfunction after ovariectomy is also associated with an alteration in external urethral sphincter activity. Bilateral ovariectomy was performed in female Sprague-Dawley® rats and sham operated rats served as controls. Transvesical cystometry and external urethral sphincter electromyogram activity were monitored 4, 6 and 12 weeks after sham operation or bilateral ovariectomy and at 6 weeks in bilaterally ovariectomized rats treated with estrogen. The micturition reflex was elicited in sham operated and bilaterally ovariectomized, urethane anesthetized animals. Post-void residual urine increased and voiding efficiency decreased in rats with 4 to 12 weeks of bilateral ovariectomy. The silent period of external urethral sphincter electromyogram activity was shortened significantly and progressively at increased times after bilateral ovariectomy. These effects were prevented by estradiol treatment. As evidenced by shortening of the external urethral sphincter electromyogram silent period in ovariectomized rats, the disruption of coordination between the external urethral sphincter and the detrusor muscle could decrease urine outflow and in turn voiding efficiency. Estrogen replacement reverses these changes, suggesting that the central pathways responsible for detrusor-sphincter coordination are modulated by gonadal hormones.

Ventromedial Hypothalamic Nitric Oxide Production Is Necessary for Hypoglycemia Detection and Counterregulation

OBJECTIVEThe response of ventromedial hypothalamic (VMH) glucose-inhibited neurons to decreased glucose is impaired under conditions where the counterregulatory response (CRR) to hypoglycemia is impaired (e.g., recurrent hypoglycemia). This suggests a role for glucose-inhibited neurons in the CRR. We recently showed that decreased glucose increases nitric oxide (NO) production in cultured VMH glucose-inhibited neurons. These in vitro data led us to hypothesize that NO release from VMH glucose-inhibited neurons is critical for the CRR.RESEARCH DESIGN AND METHODSThe CRR was evaluated in rats and mice in response to acute insulin-induced hypoglycemia and hypoglycemic clamps after modulation of brain NO signaling. The glucose sensitivity of ventromedial nucleus glucose-inhibited neurons was also assessed.RESULTSHypoglycemia increased hypothalamic constitutive NO synthase (NOS) activity and neuronal NOS (nNOS) but not endothelial NOS (eNOS) phosphorylation in rats. Intracerebroventricular and VMH injection of the nonselective NOS inhibitor NG-monomethyl-l-arginine (l-NMMA) slowed the recovery to euglycemia after hypoglycemia. VMH l-NMMA injection also increased the glucose infusion rate (GIR) and decreased epinephrine secretion during hyperinsulinemic/hypoglycemic clamp in rats. The GIR required to maintain the hypoglycemic plateau was higher in nNOS knockout than wild-type or eNOS knockout mice. Finally, VMH glucose-inhibited neurons were virtually absent in nNOS knockout mice.CONCLUSIONSWe conclude that VMH NO production is necessary for glucose sensing in glucose-inhibited neurons and full generation of the CRR to hypoglycemia. These data suggest that potentiating NO signaling may improve the defective CRR resulting from recurrent hypoglycemia in patients using intensive insulin therapy.

Human Umbilical Cord Blood Cells Protect Against Hypothalamic Apoptosis and Systemic Inflammation Response During Heatstroke in Rats

Intravenous administration of human umbilical cord blood cells (HUCBC) has been shown to improve heatstroke by reducing arterial hypotension as well as cerebral ischemia and damage in a rat model. To extend these findings, we assessed both hypothalamic neuronal apoptosis and systemic inflammatory responses in the presence of HUCBCs or vehicle medium immediately after initiation of heatstroke. Anesthetized rats, immediately after the initiation of heat stress, were divided into two groups and given either serum-free lymphocyte medium (0.3mL per rat, intravenously) or HUCBCs (5 x 10(6) in 0.3 mL serum-free lymphocyte medium, intravenously). Another group of rats were exposed to room temperature (26 degrees C) and used as normothermic controls. Heatstroke was induced by exposing the anesthetized rats to a high ambient temperature of 43 degrees C for 68 minutes. After the onset of heatstroke, animals treated with serum-free lymphocyte medium displayed hyperthermia, hypotension, bradycardia, hypothalamic neuronal apoptosis and degeneration, and up-regulation of systemic inflammatory response molecules including serum tumor necrosis factor-alpha, soluble intercellular adhesion molecule-1 and E-selectin. Heatstroke-induced hypotension, bradycardia, hypothalamic neuronal apoptosis and degeneration, and increased systemic inflammatory response molecules were significantly inhibited by HUCBC treatment. Although heatstroke-induced hyperthermia was not affected by HUCBC treatment, the serum levels of the anti-inflammatory cytokine interleukin-10 were significantly increased by HUCBC therapy during hyperthermia. These findings suggest that HUCBC transplantation may prevent the occurrence of heatstroke by reducing hypothalamic neuronal damage and the systemic inflammatory responses.

Age-related LH surge dysfunction correlates with reduced responsiveness of hypothalamic anteroventral periventricular nucleus kisspeptin neurons to estradiol positive feedback in middle-aged rats

Female reproductive aging in rats is characterized by reduced gonadotropin releasing hormone (GnRH) neuronal activation under estradiol positive feedback conditions and a delayed and attenuated luteinizing hormone (LH) surge. The newly identified excitatory neuropeptide kisspeptin is proposed to be a critical mediator of the pubertal transition and the ovarian steroid-induced LH surge. We previously showed that estradiol induces less kisspeptin mRNA expression in the anterior hypothalamus [anatomical location of anteroventral periventricular nucleus (AVPV)] in middle-aged than in young rats and intrahypothalamic infusion of kisspeptin restores LH surge amplitude in middle-aged females. Thus, reduced kisspeptin neurotransmission may contribute to age-related LH surge abnormalities. This study tested the hypothesis that middle-aged females will also exhibit reduced numbers of kisspeptin immunopositive neurons in the AVPV under estradiol positive feedback conditions. Using immunohistochemistry, we demonstrate that middle-aged females primed with ovarian steroids have fewer AVPV kisspeptin immunopositive neurons than young females. Age did not affect kisspeptin mRNA expression in the pituitary, numbers of kisspeptin immunopositive neurons in the arcuate nucleus, or estradiol-dependent reductions in kisspeptin mRNA expression in the posterior hypothalamus (containing the arcuate nucleus). These data strongly suggest that age-related LH surge dysfunction results, in part, from a reduced sensitivity of AVPV kisspeptin neurons to estradiol and hence decreased availability of AVPV kisspeptin neurons to activate GnRH neurons under positive feedback conditions.

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.