A dedicated hypothalamic oxytocin circuit controls aversive social learning.

To survive in a complex social group, one needs to know who to approach and, more importantly, who to avoid. In mice, a single defeat causes the losing mouse to stay away from the winner for weeks1. Here through a series of functional manipulation and recording experiments, we identify oxytocin neurons in the retrochiasmatic supraoptic nucleus (SOROXT) and oxytocin-receptor-expressing cells in the anterior subdivision of the ventromedial hypothalamus, ventrolateral part (aVMHvlOXTR) as a key circuit motif for defeat-induced social avoidance. Before defeat, aVMHvlOXTR cells minimally respond to aggressor cues. During defeat, aVMHvlOXTR cells are highly activated and, with the help of an exclusive oxytocin supply from the SOR, potentiate their responses to aggressor cues. After defeat, strong aggressor-induced aVMHvlOXTR cell activation drives the animal to avoid the aggressor and minimizes future defeat. Our study uncovers a neural process that supports rapid social learning caused by defeat and highlights the importance of the brain oxytocin system in social plasticity.

mGluR5 in Astrocytes in the Ventromedial Hypothalamus Regulates Pituitary Adenylate Cyclase-Activating Polypeptide Neurons and Glucose Homeostasis.

The ventromedial hypothalamus (VMH) is a functionally heterogeneous nucleus critical for systemic energy, glucose, and lipid balance. We showed previously that the metabotropic glutamate receptor 5 (mGluR5) plays essential roles regulating excitatory and inhibitory transmission in SF1+ neurons of the VMH and facilitating glucose and lipid homeostasis in female mice. Although mGluR5 is also highly expressed in VMH astrocytes in the mature brain, its role there influencing central metabolic circuits is unknown. In contrast to the glucose intolerance observed only in female mice lacking mGluR5 in VMH SF1 neurons, selective depletion of mGluR5 in VMH astrocytes enhanced glucose tolerance without affecting food intake or body weight in both adult female and male mice. The improved glucose tolerance was associated with elevated glucose-stimulated insulin release. Astrocytic mGluR5 male and female mutants also exhibited reduced adipocyte size and increased sympathetic tone in gonadal white adipose tissue. Diminished excitatory drive and synaptic inputs onto VMH Pituitary adenylate cyclase-activating polypeptide (PACAP+) neurons and reduced activity of these cells during acute hyperglycemia underlie the observed changes in glycemic control. These studies reveal an essential role of astrocytic mGluR5 in the VMH regulating the excitatory drive onto PACAP+ neurons and activity of these cells facilitating glucose homeostasis in male and female mice.SIGNIFICANCE STATEMENT Neuronal circuits within the VMH play chief roles in the regulation of whole-body metabolic homeostasis. It remains unclear how astrocytes influence neurotransmission in this region to facilitate energy and glucose balance control. Here, we explored the role of the metabotropic glutamate receptor, mGluR5, using a mouse model with selective depletion of mGluR5 from VMH astrocytes. We show that astrocytic mGluR5 critically regulates the excitatory drive and activity of PACAP-expressing neurons in the VMH to control glucose homeostasis in both female and male mice. Furthermore, mGluR5 in VMH astrocytes influences adipocyte size and sympathetic tone in white adipose tissue. These studies provide novel insight toward the importance of hypothalamic astrocytes participating in central circuits regulating peripheral metabolism.

Anoctamin 4 channel currents activate glucose-inhibited neurons in the mouse ventromedial hypothalamus during hypoglycemia.

Glucose is the basic fuel essential for maintenance of viability and functionality of all cells. However, some neurons - namely, glucose-inhibited (GI) neurons - paradoxically increase their firing activity in low-glucose conditions and decrease that activity in high-glucose conditions. The ionic mechanisms mediating electric responses of GI neurons to glucose fluctuations remain unclear. Here, we showed that currents mediated by the anoctamin 4 (Ano4) channel are only detected in GI neurons in the ventromedial hypothalamic nucleus (VMH) and are functionally required for their activation in response to low glucose. Genetic disruption of the Ano4 gene in VMH neurons reduced blood glucose and impaired counterregulatory responses during hypoglycemia in mice. Activation of VMHAno4 neurons increased food intake and blood glucose, while chronic inhibition of VMHAno4 neurons ameliorated hyperglycemia in a type 1 diabetic mouse model. Finally, we showed that VMHAno4 neurons represent a unique orexigenic VMH population and transmit a positive valence, while stimulation of neurons that do not express Ano4 in the VMH (VMHnon-Ano4) suppress feeding and transmit a negative valence. Together, our results indicate that the Ano4 channel and VMHAno4 neurons are potential therapeutic targets for human diseases with abnormal feeding behavior or glucose imbalance.

Astrocyte Glycogen Is a Major Source of Hypothalamic Lactate in Rats With Recurrent Hypoglycemia.

Lactate is an important metabolic substrate for sustaining brain energy requirements when glucose supplies are limited. Recurring exposure to hypoglycemia (RH) raises lactate levels in the ventromedial hypothalamus (VMH), which contributes to counterregulatory failure. However, the source of this lactate remains unclear. The current study investigates whether astrocytic glycogen serves as the major source of lactate in the VMH of RH rats. By decreasing the expression of a key lactate transporter in VMH astrocytes of RH rats, we reduced extracellular lactate concentrations, suggesting excess lactate was locally produced from astrocytes. To determine whether astrocytic glycogen serves as the major source of lactate, we chronically delivered either artificial extracellular fluid or 1,4-dideoxy-1,4-imino-d-arabinitol to inhibit glycogen turnover in the VMH of RH animals. Inhibiting glycogen turnover in RH animals prevented the rise in VMH lactate and the development of counterregulatory failure. Lastly, we noted that RH led to an increase in glycogen shunt activity in response to hypoglycemia and elevated glycogen phosphorylase activity in the hours following a bout of hypoglycemia. Our data suggest that dysregulation of astrocytic glycogen metabolism following RH may be responsible, at least in part, for the rise in VMH lactate levels. ARTICLE HIGHLIGHTS: Astrocytic glycogen serves as the major source of elevated lactate levels in the ventromedial hypothalamus (VMH) of animals exposed to recurring episodes of hypoglycemia. Antecedent hypoglycemia alters VMH glycogen turnover. Antecedent exposure to hypoglycemia enhances glycogen shunt activity in the VMH during subsequent bouts of hypoglycemia. In the immediate hours following a bout of hypoglycemia, sustained elevations in glycogen phosphorylase activity in the VMH of recurrently hypoglycemic animals contribute to sustained elevations in local lactate levels.

Sexual differentiation of estrogen receptor alpha subpopulations in the ventromedial nucleus of the hypothalamus.

Estrogen receptor (ER) α-expressing neurons in the ventrolateral area of the ventromedial hypothalamus (VMHvl) are implicated in the control of many behaviors and physiological processes, some of which are sex-specific. Recently, three sex-differentiated ERα subpopulations have been discovered in the VMHvl marked by co-expression with tachikinin1 (Tac1), reprimo (Rprm), or prodynorphin (Pdyn), that may subserve specific functions. These markers show sex differences in adulthood: females have many more Tac1/Esr1 and Rprm/Esr1 co-expressing cells, while males have more Pdyn/Esr1 cells. In this study, we sought to understand the development of these sex differences and pinpoint the sex-differentiating signal. We examined developmental changes in the number of Esr1 cells co-expressing Tac1, Rprm or Pdyn using single-molecule in situ hybridization. We found that both sexes have similarly high numbers of Tac1/Esr1 and Rprm/Esr1 cells at birth, but newborn males have many more Pdyn/Esr1 cells than females. However, the number of cells with Tac1/Esr1 and Rprm/Esr1 co-expression markedly decreases by weaning in males, but not females, leading to sex differences in neurochemical expression. Female mice administered testosterone at birth have expression patterns akin to male mice. Thus, a substantial neurochemical reorganization of the VMHvl occurs in males between birth and weaning that likely underlies the previously reported sex differences in behavioral and physiological responses to estrogens in adulthood.

Populational heterogeneity and partial migratory origin of the ventromedial hypothalamic nucleus: genoarchitectonic analysis in the mouse.

The ventromedial hypothalamic nucleus (VMH) is one of the most distinctive hypothalamic tuberal structures, subject of numerous classic and modern functional studies. Commonly, the adult VMH has been divided in several portions, attending to differences in cell aggregation, cell type, connectivity, and function. Consensus VMH partitions in the literature comprise the dorsomedial (VMHdm), and ventrolateral (VMHvl) subnuclei, which are separated by an intermediate or central (VMHc) population (topographic names based on the columnar axis). However, some recent transcriptome analyses have identified a higher number of different cell types in the VMH, suggesting additional subdivisions, as well as the possibility of separate origins. We offer a topologic and genoarchitectonic developmental study of the mouse VMH complex using the prosomeric axis as a reference. We analyzed genes labeling specific VMH subpopulations, with particular focus upon the Nkx2.2 transcription factor, a marker of the alar-basal boundary territory of the prosencephalon, from where some cells seem to migrate dorsoventrally into VMH. We also identified separate neuroepithelial origins of a Nr2f1-positive subpopulation, and a new Six3-positive component, as well as subtle differences in origin of Nr5a1 positive versus Nkx2.2-positive cell populations entering dorsoventrally the VMH. Several of these migrating cell types are born in the dorsal tuberal domain and translocate ventralwards to reach the intermediate tuberal domain, where the adult VMH mass is located in the adult. This work provides a more detailed area map on the intrinsic organization of the postmigratory VMH complex, helpful for deeper functional studies of this basal hypothalamic entity.

Contribution of the co-chaperone FKBP51 in the ventromedial hypothalamus to metabolic homeostasis in male and female mice.

OBJECTIVE: Steroidogenic factor 1 (SF1) expressing neurons in the ventromedial hypothalamus (VMH) have been directly implicated in whole-body metabolism and in the onset of obesity. The co-chaperone FKBP51 is abundantly expressed in the VMH and was recently linked to type 2 diabetes, insulin resistance, adipogenesis, browning of white adipose tissue (WAT) and bodyweight regulation. METHODS: We investigated the role of FKBP51 in the VMH by conditional deletion and virus-mediated overexpression of FKBP51 in SF1-positive neurons. Baseline and high fat diet (HFD)-induced metabolic- and stress-related phenotypes in male and female mice were obtained. RESULTS: In contrast to previously reported robust phenotypes of FKBP51 manipulation in the entire mediobasal hypothalamus (MBH), selective deletion or overexpression of FKBP51 in the VMH resulted in only a moderate alteration of HFD-induced bodyweight gain and body composition, independent of sex. CONCLUSIONS: Overall, this study shows that animals lacking and overexpressing Fkbp5 in Sf1-expressing cells within the VMH display only a mild metabolic phenotype compared to an MBH-wide manipulation of this gene, suggesting that FKBP51 in SF1 neurons within this hypothalamic nucleus plays a subsidiary role in controlling whole-body metabolism.

Prolactin-mediated restraint of maternal aggression in lactation.

Aggressive behavior is rarely observed in virgin female mice but is specifically triggered in lactation where it facilitates protection of offspring. Recent studies demonstrated that the hypothalamic ventromedial nucleus (VMN) plays an important role in facilitating aggressive behavior in both sexes. Here, we demonstrate a role for the pituitary hormone, prolactin, acting through the prolactin receptor in the VMN to control the intensity of aggressive behavior exclusively during lactation. Prolactin receptor deletion from glutamatergic neurons or specifically from the VMN resulted in hyperaggressive lactating females, with a marked shift from intruder-directed investigative behavior to very high levels of aggressive behavior. Prolactin-sensitive neurons in the VMN project to a wide range of other hypothalamic and extrahypothalamic regions, including the medial preoptic area, paraventricular nucleus, and bed nucleus of the stria terminalis, all regions known to be part of a complex neuronal network controlling maternal behavior. Within this network, prolactin acts in the VMN to specifically restrain male-directed aggressive behavior in lactating females. This action in the VMN may complement the role of prolactin in other brain regions, by shifting the balance of maternal behaviors from defense-related activities to more pup-directed behaviors necessary for nurturing offspring.

Phosphorylation of STAT3 in hypothalamic nuclei is stimulated by lower doses of leptin than are needed to inhibit food intake.

This experiment investigated which hypothalamic nuclei were activated by a dose of leptin that inhibited food intake. Foodnot intake, energy expenditure, respiratory exchange ratio (RER), and intrascapular brown adipose tissue (IBAT) temperature were measured in male and female Sprague Dawley rats for 36 h following an intraperitoneal injection of 0, 50, 200, 500, or 1,000 µg leptin/kg with each rat tested with each dose of leptin in random order. In both males and females, RER and 12-h food intake were inhibited only by 1,000 µg leptin/kg, but there was no effect on energy expenditure or IBAT temperature. At the end of the experiment, phosphorylated signal transducer and activator of transcription 3 (pSTAT3) immunoreactivity was measured 1 h after injection of 0, 50, 500, or 1,000 µg leptin/kg. In male rats, the lowest dose of leptin produced a maximal activation of STAT3 in the Arc and nucleus of the solitary tract (NTS). There was no response in the dorsomedial hypothalamus, but there was a progressive increase in ventromedial nucleus of the hypothalamus (VMH) pSTAT3 with increasing doses of leptin. In female rats, there was no significant change in Arc and pSTAT3 NTS activation was maximal with 500 mg leptin/kg, but only the highest dose of leptin increased VMH pSTAT3. These results suggest that the VMH plays an important role in the energetic response to elevations of circulating leptin but do not exclude the possibility that multiple nuclei provide the appropriate integrated response to hyperleptinemia.NEW & NOTEWORTHY The results of this experiment show that doses of leptin too small to inhibit food intake produce a maximal response to leptin in the arcuate nucleus. By contrast the VMH shows a robust response that correlates with inhibition of food intake. This suggests that the VMH plays an important role in the energetic response to hyperleptinemia.

Rap1 in the VMH regulates glucose homeostasis.

The hypothalamus is a critical regulator of glucose metabolism and is capable of correcting diabetes conditions independently of an effect on energy balance. The small GTPase Rap1 in the forebrain is implicated in high-fat diet-induced (HFD-induced) obesity and glucose imbalance. Here, we report that increasing Rap1 activity selectively in the medial hypothalamus elevated blood glucose without increasing the body weight of HFD-fed mice. In contrast, decreasing hypothalamic Rap1 activity protected mice from diet-induced hyperglycemia but did not prevent weight gain. The remarkable glycemic effect of Rap1 was reproduced when Rap1 was specifically deleted in steroidogenic factor-1-positive (SF-1-positive) neurons in the ventromedial hypothalamic nucleus (VMH) known to regulate glucose metabolism. While having no effect on body weight regardless of sex, diet, and age, Rap1 deficiency in the VMH SF1 neurons markedly lowered blood glucose and insulin levels, improved glucose and insulin tolerance, and protected mice against HFD-induced neural leptin resistance and peripheral insulin resistance at the cellular and whole-body levels. Last, acute pharmacological inhibition of brain exchange protein directly activated by cAMP 2, a direct activator of Rap1, corrected glucose imbalance in obese mouse models. Our findings uncover the primary role of VMH Rap1 in glycemic control and implicate Rap1 signaling as a potential target for therapeutic intervention in diabetes.

Decoding neuronal composition and ontogeny of individual hypothalamic nuclei.

The hypothalamus plays crucial roles in regulating endocrine, autonomic, and behavioral functions via its diverse nuclei and neuronal subtypes. The developmental mechanisms underlying ontogenetic establishment of different hypothalamic nuclei and generation of neuronal diversity remain largely unknown. Here, we show that combinatorial T-box 3 (TBX3), orthopedia homeobox (OTP), and distal-less homeobox (DLX) expression delineates all arcuate nucleus (Arc) neurons and defines four distinct subpopulations, whereas combinatorial NKX2.1/SF1 and OTP/DLX expression identifies ventromedial hypothalamus (VMH) and tuberal nucleus (TuN) neuronal subpopulations, respectively. Developmental analysis indicates that all four Arc subpopulations are mosaically and simultaneously generated from embryonic Arc progenitors, whereas glutamatergic VMH neurons and GABAergic TuN neurons are sequentially generated from common embryonic VMH progenitors. Moreover, clonal lineage-tracing analysis reveals that diverse lineages from multipotent radial glia progenitors orchestrate Arc and VMH-TuN establishment. Together, our study reveals cellular mechanisms underlying generation and organization of diverse neuronal subtypes and ontogenetic establishment of individual nuclei in the mammalian hypothalamus.

The Role of Mediobasal Hypothalamic PACAP in the Control of Body Weight and Metabolism.

Body energy homeostasis results from balancing energy intake and energy expenditure. Central nervous system administration of pituitary adenylate cyclase activating polypeptide (PACAP) dramatically alters metabolic function, but the physiologic mechanism of this neuropeptide remains poorly defined. PACAP is expressed in the mediobasal hypothalamus (MBH), a brain area essential for energy balance. Ventromedial hypothalamic nucleus (VMN) neurons contain, by far, the largest and most dense population of PACAP in the medial hypothalamus. This region is involved in coordinating the sympathetic nervous system in response to metabolic cues in order to re-establish energy homeostasis. Additionally, the metabolic cue of leptin signaling in the VMN regulates PACAP expression. We hypothesized that PACAP may play a role in the various effector systems of energy homeostasis, and tested its role by using VMN-directed, but MBH encompassing, adeno-associated virus (AAVCre) injections to ablate Adcyap1 (gene coding for PACAP) in mice (Adcyap1MBHKO mice). Adcyap1MBHKO mice rapidly gained body weight and adiposity, becoming hyperinsulinemic and hyperglycemic. Adcyap1MBHKO mice exhibited decreased oxygen consumption (VO2), without changes in activity. These effects appear to be due at least in part to brown adipose tissue (BAT) dysfunction, and we show that PACAP-expressing cells in the MBH can stimulate BAT thermogenesis. While we observed disruption of glucose clearance during hyperinsulinemic/euglycemic clamp studies in obese Adcyap1MBHKO mice, these parameters were normal prior to the onset of obesity. Thus, MBH PACAP plays important roles in the regulation of metabolic rate and energy balance through multiple effector systems on multiple time scales, which highlight the diverse set of functions for PACAP in overall energy homeostasis.

Norepinephrine Regulation of Ventromedial Hypothalamic Nucleus Metabolic-Sensory Neuron 5'-AMP-Activated Protein Kinase Activity: Impact of Estradiol.

The mediobasal hypothalamus (MBH) shapes the neural regulation of glucostasis by 5'-AMP-activated protein kinase (AMPK)-dependent mechanisms. Yet, the neurochemical identity and neuroanatomical distribution of MBH neurons that express glucoprivic-sensitive AMPK remain unclear. The neurotransmitters γ-aminobutyric acid (GABA) and nitric oxide (NO) act within the MBH to correspondingly inhibit or stimulate glucose counter-regulation. The current review highlights recent findings that GABA and NO, neurons located in the ventromedial hypothalamic nucleus (VMN), a distinct important element of the MBH, are direct targets of noradrenergic regulatory signaling, and thereby, likely operate under the control of hindbrain metabolic-sensory neurons. The ovarian hormone estradiol acts within the VMN to govern energy homeostasis. Discussed here is current evidence that estradiol regulates GABA and NO nerve cell receptivity to norepinephrine and moreover, controls the noradrenergic regulation of AMPK activity in each cell type. Future gains in insight on mechanisms underpinning estradiol's impact on neurotransmitter communication between the hindbrain and hypothalamic AMPKergic neurons are expected to disclose viable new molecular targets for the therapeutic simulation of hormonal enhancement of neuro-metabolic stability during circumstances of diminished endogenous estrogen secretion or glucose dysregulation.

Defensive behaviors and brain regional activation changes in rats confronting a snake.

In the present study, we examined behavioral and brain regional activation changes of rats). To a nonmammalian predator, a wild rattler snake (Crotalus durissus terrificus). Accordingly, during snake threat, rat subjects showed a striking and highly significant behavioral response of freezing, stretch attend, and, especially, spatial avoidance of this threat. The brain regional activation patterns for these rats were in broad outline similar to those of rats encountering other predator threats, showing Fos activation of sites in the amygdala, hypothalamus, and periaqueductal gray matter. In the amygdala, only the lateral nucleus showed significant activation, although the medial nucleus, highly responsive to olfaction, also showed higher activation. Importantly, the hypothalamus, in particular, was somewhat different, with significant Fos increases in the anterior and central parts of the ventromedial hypothalamic nucleus (VMH), in contrast to patterns of enhanced Fos expression in the dorsomedial VMH to cat predators, and in the ventrolateral VMH to an attacking conspecific. In addition, the juxtodorsalmedial region of the lateral hypothalamus showed enhanced Fos activation, where inputs from the septo-hippocampal system may suggest the potential involvement of hippocampal boundary cells in the very strong spatial avoidance of the snake and the area it occupied. Notably, these two hypothalamic paths appear to merge into the dorsomedial part of the dorsal premammillary nucleus and dorsomedial and lateral parts of the periaqueductal gray, all of which present significant increases in Fos expression and are likely to be critical for the expression of defensive behaviors in responses to the snake threat.

Norepinephrine regulation of ventromedial hypothalamic nucleus metabolic transmitter biomarker and astrocyte enzyme and receptor expression: Impact of 5' AMP-activated protein kinase.

The ventromedial hypothalamic energy sensor AMP-activated protein kinase (AMPK) maintains glucostasis via neurotransmitter signals that diminish [γ-aminobutyric acid] or enhance [nitric oxide] counter-regulation. Ventromedial hypothalamic nucleus (VMN) 'fuel-inhibited' neurons are sensitive to astrocyte-generated metabolic substrate stream. Norepinephrine (NE) regulates astrocyte glycogen metabolism in vitro, and hypoglycemia intensifies VMN NE activity in vivo. Current research investigated the premise that NE elicits AMPK-dependent adjustments in VMN astrocyte glycogen metabolic enzyme [glycogen synthase (GS); glycogen phosphorylase (GP)] and gluco-regulatory neuron biomarker [glutamate decarboxylase65/67 (GAD); neuronal nitric oxide synthase (nNOS); SF-1] protein expression in male rats. We also examined whether VMN astrocytes are directly receptive to NE and if noradrenergic input regulates cellular sensitivity to the neuro-protective steroid estradiol. Intra-VMN NE correspondingly augmented or reduced VMN tissue GAD and nNOS protein despite no change in circulating glucose, data that imply that short-term exposure to NE promotes persistent improvement in VMN nerve cell energy stability. The AMPK inhibitor Compound C (Cc) normalized VMN nNOS, GS, and GP expression in NE-treated animals. NE caused AMPK-independent down-regulation of alpha2-, alongside Cc-reversible augmentation of beta1-adrenergic receptor protein profiles in laser-microdissected astrocytes. NE elicited divergent adjustments in astrocyte estrogen receptor-beta (AMPK-unrelated reduction) and GPR-30 (Cc-revocable increase) proteins. Outcomes implicate AMPK in noradrenergic diminution of VMN nitrergic metabolic-deficit signaling and astrocyte glycogen shunt activity. Differentiating NE effects on VMN astrocyte adrenergic and estrogen receptor variant expression suggest that noradrenergic regulation of glycogen metabolism may be mediated, in part, by one or more receptors characterized here by sensitivity to this catecholamine.

Effects of myeloid sirtuin 1 deficiency on hypothalamic neurogranin in mice fed a high-fat diet.

Hypothalamic inflammation has been known as a contributor to high-fat diet (HFD)-induced insulin resistance and obesity. Myeloid-specific sirtuin 1 (SIRT1) deletion aggravates insulin resistance and hypothalamic inflammation in HFD-fed mice. Neurogranin, a calmodulin-binding protein, is expressed in the hypothalamus. However, the effects of myeloid SIRT1 deletion on hypothalamic neurogranin has not been fully clarified. To investigate the effect of myeloid SIRT1 deletion on food intake and hypothalamic neurogranin expression, mice were fed a HFD for 20 weeks. Myeloid SIRT1 knockout (KO) mice exhibited higher food intake, weight gain, and lower expression of anorexigenic proopiomelanocortin in the arcuate nucleus than WT mice. In particular, KO mice had lower ventromedial hypothalamus (VMH)-specific neurogranin expression. However, SIRT1 deletion reduced HFD-induced hypothalamic neurogranin. Furthermore, hypothalamic phosphorylated AMPK and parvalbumin protein levels were also lower in HFD-fed KO mice than in HFD-fed WT mice. Thus, these findings suggest that myeloid SIRT1 deletion affects food intake through VMH-specific neurogranin-mediated AMPK signaling and hypothalamic inflammation in mice fed a HFD.

CPT1C in the ventromedial nucleus of the hypothalamus is necessary for brown fat thermogenesis activation in obesity.

OBJECTIVE: Carnitine palmitoyltransferase 1C (CPT1C) is implicated in central regulation of energy homeostasis. Our aim was to investigate whether CPT1C in the ventromedial nucleus of the hypothalamus (VMH) is involved in the activation of brown adipose tissue (BAT) thermogenesis in the early stages of diet-induced obesity. METHODS: CPT1C KO and wild type (WT) mice were exposed to short-term high-fat (HF) diet feeding or to intracerebroventricular leptin administration and BAT thermogenesis activation was evaluated. Body weight, adiposity, food intake, and leptinemia were also assayed. RESULTS: Under 7 days of HF diet, WT mice showed a maximum activation peak of BAT thermogenesis that counteracted obesity development, whereas this activation was impaired in CPT1C KO mice. KO animals evidenced higher body weight, adiposity, hyperleptinemia, ER stress, and disrupted hypothalamic leptin signaling. Leptin-induced BAT thermogenesis was abolished in KO mice. These results indicate an earlier onset leptin resistance in CPT1C KO mice. Since AMPK in the VMH is crucial in the regulation of BAT thermogenesis, we analyzed if CPT1C was a downstream factor of this pathway. Genetic inactivation of AMPK within the VMH was unable to induce BAT thermogenesis and body weight loss in KO mice, indicating that CPT1C is likely downstream AMPK in the central mechanism modulating thermogenesis within the VMH. Quite opposite, the expression of CPT1C in the VMH restored the phenotype. CONCLUSION: CPT1C is necessary for the activation of BAT thermogenesis driven by leptin, HF diet exposure, and AMPK inhibition within the VMH. This study underscores the importance of CPT1C in the activation of BAT thermogenesis to counteract diet-induced obesity.

Thioredoxin-1 Overexpression in the Ventromedial Nucleus of the Hypothalamus Preserves the Counterregulatory Response to Hypoglycemia During Type 1 Diabetes in Male Rats.

We previously showed that the glutathione precursor, N-acetylcysteine (NAC), prevented hypoglycemia-associated autonomic failure (HAAF) and impaired activation of ventromedial hypothalamus (VMH) glucose-inhibited (GI) neurons by low glucose after recurrent hypoglycemia (RH) in nondiabetic rats. However, NAC does not normalize glucose sensing by VMH GI neurons when RH occurs during diabetes. We hypothesized that recruiting the thioredoxin (Trx) antioxidant defense system would prevent HAAF and normalize glucose sensing after RH in diabetes. To test this hypothesis, we overexpressed Trx-1 (cytosolic form of Trx) in the VMH of rats with streptozotocin (STZ)-induced type 1 diabetes. The counterregulatory response (CRR) to hypoglycemia in vivo and the activation of VMH GI neurons in low glucose using membrane potential sensitive dye in vitro was measured before and after RH. VMH Trx-1 overexpression normalized both the CRR and glucose sensing by VMH GI neurons in STZ rats. VMH Trx-1 overexpression also lowered the insulin requirement to prevent severe hyperglycemia in STZ rats. However, like NAC, VMH Trx-1 overexpression did not prevent HAAF or normalize activation of VMH GI neurons by low glucose in STZ rats after RH. We conclude that preventing HAAF in type 1 diabetes may require the recruitment of both antioxidant systems.

Region-Specific Suppression of Hypothalamic Responses to Insulin To Adapt to Elevated Maternal Insulin Secretion During Pregnancy.

As part of the adaptation of maternal glucose regulation during pregnancy to ensure glucose provision to the fetus, maternal insulin concentrations become elevated. However, increased central actions of insulin, such as suppression of appetite, would be maladaptive during pregnancy. We hypothesized that central nervous system targets of insulin become less responsive during pregnancy to prevent overstimulation by the increased circulating insulin concentrations. To test this hypothesis, we have measured insulin-induced phosphorylation of Akt (pAkt) in specific hypothalamic nuclei as an index of hypothalamic insulin responsiveness. Despite higher endogenous insulin concentrations following feeding, arcuate nucleus pAkt levels were significantly lower in the pregnant group compared with the nonpregnant group. In response to an intracerebroventricular injection of insulin, insulin-induced pAkt was significantly reduced in the arcuate nucleus and ventromedial nucleus of pregnant rats compared with nonpregnant rats. Similar levels of insulin receptor ß and PTEN, a negative regulator of the phosphoinositide 3-kinase/Akt pathway, were detected in hypothalamic areas of nonpregnant and pregnant rats. In the ventromedial nucleus, however, levels of phosphorylated PTEN were significantly lower in pregnancy, suggesting that reduced inactivation of PTEN may contribute to the attenuated insulin signaling in this area during pregnancy. In conclusion, these results demonstrate region-specific changes in responsiveness to insulin in the hypothalamus during pregnancy that may represent an adaptive response to minimize the impact of elevated circulating insulin on the maternal brain.

Temporal and regional onset of leptin resistance in diet-induced obese mice.

In common forms of obesity, leptin fails to convey its regulatory effect. This so called "leptin resistance" is not well understood, and solving this puzzle is a key to understanding how obesity develops. In the present study, we investigated the temporal and regional onset of leptin resistance in response to a diet enriched with long-chain saturated fatty acids (high-fat diet; HFD) in mice. Mice were exposed to either a low-fat diet (LFD) or a HFD for 4 hours, 24 hours, 10 days and 28 days. Mice in each group received an i.p. injection of either phosphate-buffered saline or leptin and the number of phosphorylated signal transducer and activator of transcription-3 (pSTAT3) immunoreactive (-IR) cells in the arcuate nucleus (ARC), ventromedial nucleus of the hypothalamus (VMH) and dorsomedial nucleus of the hypothalamus (DMH) was analysed 30 or 120 minutes after treatment. In the ARC, as soon as 24 hours of HFD, the molecular leptin response was reduced by 40% (P≤.01). Compared to at 24 hours, after 10 days, the number of leptin-induced pSTAT3-IR cells was elevated after 120 minutes, suggesting a sustained response and a partial return of leptin sensitivity. After 28 days, leptin failed to induce the number of pSTAT3-IR over control levels, suggesting a markedly reduced sensitivity to leptin. In the VMH after 24 hours, we observed a 50% reduction in leptin-induced pSTAT-3-IR cells, followed by a further decline after 10 days. However, after 28 days, there was a significant increase in pSTAT-3-IR cells (P≤.05), indicating partial recovery of leptin sensitivity. By contrast to these two regions, in the DMH, no loss of leptin sensitivity was observed at any time-point. These findings demonstrate that a loss of sensitivity to leptin occurs rapidly after exposure to HFD in the ARC and VMH but not the DMH. However, there appears to be a biphasic pattern of leptin responsiveness, with a partial return of leptin sensitivity occurring after 10 days in the arcuate nucleus, and after 28 days in the VMH. By 28 days, the response to leptin in the arcuate nucleus was completely lost. These findings suggest that the molecular responses to leptin are altered after high-fat feeding in a time- and region-specific manner.