Time to drink: Activating lateral hypothalamic area neurotensin neurons promotes intake of fluid over food in a time-dependent manner.

The lateral hypothalamic area (LHA) is essential for ingestive behavior but has primarily been studied in modulating feeding, with comparatively scant attention on drinking. This is partly because most LHA neurons simultaneously promote feeding and drinking, suggesting that ingestive behaviors track together. A notable exception are LHA neurons expressing neurotensin (LHANts neurons): activating these neurons promotes water intake but modestly restrains feeding. Here we investigated the connectivity of LHANts neurons, their necessity and sufficiency for drinking and feeding, and how timing and resource availability influence their modulation of these behaviors. LHANts neurons project broadly throughout the brain, including to the lateral preoptic area (LPO), a brain region implicated in modulating drinking behavior. LHANts neurons also receive inputs from brain regions implicated in sensing hydration and energy status. While activation of LHANts neurons is not required to maintain homeostatic water or food intake, it selectively promotes drinking during the light cycle, when ingestive drive is low. Activating LHANts neurons during this period also increases willingness to work for water or palatable fluids, regardless of their caloric content. By contrast, LHANts neuronal activation during the dark cycle does not promote drinking, but suppresses feeding during this time. Finally, we demonstrate that the activation of the LHANts â†’ LPO projection is sufficient to mediate drinking behavior, but does not suppress feeding as observed after generally activating all LHANts neurons. Overall, our work suggests how and when LHANts neurons oppositely modulate ingestive behaviors.

PDK1-FoxO1 pathway in AgRP neurons of arcuate nucleus promotes bone formation via GHRH-GH-IGF1 axis.

OBJECTIVE: In the hypothalamic arcuate nucleus (ARC), orexigenic agouti-related peptide (AgRP) neurons regulate feeding behavior and energy homeostasis, functions connected to bone metabolism. The 3-phosphoinositide-dependent protein kinase-1 (PDK1) serves as a major signaling molecule particularly for leptin and insulin in AgRP neurons. We asked whether PDK1 in AGRP neurons also contributes to bone metabolism. METHODS: We generated AgRP neuron-specific PDK1 knockout (Agrp Pdk1-/- ) mice and those with additional AgRP neuron-specific expression of transactivation-defective FoxO1 (Agrp Pdk1-/-Δ256Foxo1). Bone metabolism in KO and WT mice was analyzed by quantitative computed tomography (QCT), bone histomorphometry, measurement of plasma biomarkers, and qPCR analysis of peptides. RESULTS: In Agrp Pdk1-/- female mice aged 6 weeks, compared with Agrp Cre mice, both stature and femur length were shorter while body weight was unchanged. Cortical bone mineral density (BMD) and cancellous BMD in the femur decreased, and bone formation was delayed. Furthermore, plasma GH and IGF-1 levels were reduced in parallel with decreased mRNA expressions for GH in pituitary and GHRH in ARC. Osteoblast activity was suppressed and osteoclast activity was enhanced. These changes in stature, BMD and GH level were rescued in Agrp Pdk1-/-Δ256Foxo1 mice, suggesting that the bone abnormalities and impaired GH release were mediated by enhanced Foxo1 due to deletion of PDK1. CONCLUSIONS: This study reveals a novel role of PDK1-Foxo1 pathway of AgRP neurons in controlling bone metabolism primarily via GHRH-GH-IGF-1 axis.

Astrocyte IKKß/NF-κB signaling is required for diet-induced obesity and hypothalamic inflammation.

OBJECTIVE: Obesity and high fat diet (HFD) consumption in rodents is associated with hypothalamic inflammation and reactive gliosis. While neuronal inflammation promotes HFD-induced metabolic dysfunction, the role of astrocyte activation in susceptibility to hypothalamic inflammation and diet-induced obesity (DIO) remains uncertain. METHODS: Metabolic phenotyping, immunohistochemical analyses, and biochemical analyses were performed on HFD-fed mice with a tamoxifen-inducible astrocyte-specific knockout of IKKß (GfapCreERIkbkbfl/fl, IKKß-AKO), an essential cofactor of NF-κB-mediated inflammation. RESULTS: IKKß-AKO mice with tamoxifen-induced IKKß deletion prior to HFD exposure showed equivalent HFD-induced weight gain and glucose intolerance as Ikbkbfl/fl littermate controls. In GfapCreERTdTomato marker mice treated using the same protocol, minimal Cre-mediated recombination was observed in the mediobasal hypothalamus (MBH). By contrast, mice pretreated with 6 weeks of HFD exposure prior to tamoxifen administration showed substantially increased recombination throughout the MBH. Remarkably, this treatment approach protected IKKß-AKO mice from further weight gain through an immediate reduction of food intake and increase of energy expenditure. Astrocyte IKKß deletion after HFD exposure-but not before-also reduced glucose intolerance and insulin resistance, likely as a consequence of lower adiposity. Finally, both hypothalamic inflammation and astrocytosis were reduced in HFD-fed IKKß-AKO mice. CONCLUSIONS: These data support a requirement for astrocytic inflammatory signaling in HFD-induced hyperphagia and DIO susceptibility that may provide a novel target for obesity therapeutics.

Endospanin1 affects oppositely body weight regulation and glucose homeostasis by differentially regulating central leptin signaling.

The hypothalamic arcuate nucleus (ARC) is a major integration center for energy and glucose homeostasis that responds to leptin. Resistance to leptin in the ARC is an important component of the development of obesity and type 2 diabetes. Recently, we showed that Endospanin1 (Endo1) is a negative regulator of the leptin receptor (OBR) that interacts with OBR and retains the receptor inside the cell, leading to a decreased activation of the anorectic STAT3 pathway. Endo1 is up-regulated in the ARC of high fat diet (HFD)-fed mice, and its silencing in the ARC of lean and obese mice prevents and reverses the development of obesity. OBJECTIVE: Herein we investigated whether decreased Endo1 expression in the hypothalamic ARC, associated with reduced obesity, could also ameliorate glucose homeostasis accordingly. METHODS: We studied glucose homeostasis in lean or obese mice silenced for Endo1 in the ARC via stereotactic injection of shRNA-expressing lentiviral vectors. RESULTS: We observed that despite being leaner, Endo1-silenced mice showed impaired glucose homeostasis on HFD. Mechanistically, we show that Endo1 interacts with p85, the regulatory subunit of PI3K, and mediates leptin-induced PI3K activation. CONCLUSIONS: Our results thus define Endo1 as an important hypothalamic integrator of leptin signaling, and its silencing differentially regulates the OBR-dependent functions.

Estrogens modulate ventrolateral ventromedial hypothalamic glucose-inhibited neurons.

OBJECTIVE: Brain regulation of glucose homeostasis is sexually dimorphic; however, the impact sex hormones have on specific neuronal populations within the ventromedial hypothalamic nucleus (VMN), a metabolically sensitive brain region, has yet to be fully characterized. Glucose-excited (GE) and -inhibited (GI) neurons are located throughout the VMN and may play a critical role in glucose and energy homeostasis. Within the ventrolateral portion of the VMN (VL-VMN), glucose sensing neurons and estrogen receptor (ER) distributions overlap. We therefore tested the hypothesis that VL-VMN glucose sensing neurons were sexually dimorphic and regulated by 17ß-estradiol (17ßE). METHODS: Electrophysiological recordings of VL-VMN glucose sensing neurons in brain slices isolated from age- and weight-matched female and male mice were performed in the presence and absence of 17ßE. RESULTS: We found a new class of VL-VMN GI neurons whose response to low glucose was transient despite continued exposure to low glucose. Heretofore, we refer to these newly identified VL-VMN GI neurons as 'adapting' or AdGI neurons. We found a sexual dimorphic response to low glucose, with male nonadapting GI neurons, but not AdGI neurons, responding more robustly to low glucose than those from females. 17ßE blunted the response of both nonadapting GI and AdGI neurons to low glucose in both males and females, which was mediated by activation of estrogen receptor ß and inhibition of AMP-activated kinase. In contrast, 17ßE had no impact on GE or non-glucose sensing neurons in either sex. CONCLUSION: These data suggest sex differences and estrogenic regulation of VMN hypothalamic glucose sensing may contribute to the sexual dimorphism in glucose homeostasis.

Modulating adult neurogenesis through dietary interventions.

Three areas in the brain continuously generate new neurons throughout life: the subventricular zone lining the lateral ventricles, the dentate gyrus in the hippocampus and the median eminence in the hypothalamus. These areas harbour neural stem cells, which contribute to neural repair by generating daughter cells that then become functional neurons or glia. Impaired neurogenesis leads to detrimental consequences, such as depression, decline of cognitive abilities and obesity. Adult neurogenesis is a versatile process that can be modulated either positively or negatively by many effectors, external or endogenous. Diet can modify neurogenesis both ways, either directly by ways of food-borne molecules, or possibly by the modifications induced on gut microbiota composition. It is therefore critical to define dietary strategies optimal for the maintenance of the stem cell pools.

Circadian rhythms in liver metabolism and disease.

Mounting research evidence demonstrates a significant negative impact of circadian disruption on human health. Shift work, chronic jet lag and sleep disturbances are associated with increased incidence of metabolic syndrome, and consequently result in obesity, type 2 diabetes and dyslipidemia. Here, these associations are reviewed with respect to liver metabolism and disease.

Differential effects of angiopoietin-like 4 in brain and muscle on regulation of lipoprotein lipase activity.

OBJECTIVE: Lipoprotein lipase (LPL) is a key regulator of circulating triglyceride rich lipoprotein hydrolysis. In brain LPL regulates appetite and energy expenditure. Angiopoietin-like 4 (Angptl4) is a secreted protein that inhibits LPL activity and, thereby, triglyceride metabolism, but the impact of Angptl4 on central lipid metabolism is unknown. METHODS: We induced type 1 diabetes by streptozotocin (STZ) in whole-body Angptl4 knockout mice (Angptl4(-/-) ) and their wildtype littermates to study the role of Angptl4 in central lipid metabolism. RESULTS: In type 1 (streptozotocin, STZ) and type 2 (ob/ob) diabetic mice, there is a ~2-fold increase of Angptl4 in the hypothalamus and skeletal muscle. Intracerebroventricular insulin injection into STZ mice at levels which have no effect on plasma glucose restores Angptl4 expression in hypothalamus. Isolation of cells from the brain reveals that Angptl4 is produced in glia, whereas LPL is present in both glia and neurons. Consistent with the in vivo experiment, in vitro insulin treatment of glial cells causes a 50% reduction of Angptl4 and significantly increases LPL activity with no change in LPL expression. In Angptl4(-/-) mice, LPL activity in skeletal muscle is increased 3-fold, and this is further increased by STZ-induced diabetes. By contrast, Angptl4(-/-) mice show no significant difference in LPL activity in hypothalamus or brain independent of diabetic and nutritional status. CONCLUSION: Thus, Angptl4 in brain is produced in glia and regulated by insulin. However, in contrast to the periphery, central Angptl4 does not regulate LPL activity, but appears to participate in the metabolic crosstalk between glia and neurons.

Control of appetite and energy intake by SCFA: what are the potential underlying mechanisms?

In recent years, there has been a renewed interest in the role of dietary fibre in obesity management. Much of this interest stems from animal and human studies which suggest that an increased intake of fermentable fibre can suppress appetite and improve weight management. A growing number of reports have demonstrated that the principal products of colonic fermentation of dietary fibre, SCFA, contribute to energy homeostasis via effects on multiple cellular metabolic pathways and receptor-mediated mechanisms. In particular, over the past decade it has been identified that a widespread receptor system exists for SCFA. These G-protein-coupled receptors, free fatty acid receptor (FFAR) 2 and FFAR3 are expressed in numerous tissue sites, including the gut epithelium and adipose tissue. Investigations using FFAR2- or FFAR3-deficient animal models suggest that SCFA-mediated stimulation of these receptors enhances the release of the anorectic hormones peptide tyrosine tyrosine and glucagon-like peptide-1 from colonic L cells and leptin from adipocytes. In addition, the SCFA acetate has recently been shown to have a direct role in central appetite regulation. Furthermore, the SCFA propionate is a known precursor for hepatic glucose production, which has been reported to suppress feeding behaviour in ruminant studies through the stimulation of hepatic vagal afferents. The present review therefore proposes that an elevated colonic production of SCFA could stimulate numerous hormonal and neural signals at different organ and tissue sites that would cumulatively suppress short-term appetite and energy intake.

Circadian rhythms in liver metabolism and disease

Mounting research evidence demonstrates a significant negative impact of circadian disruption on human health. Shift work, chronic jet lag and sleep disturbances are associated with increased incidence of metabolic syndrome, and consequently result in obesity, type 2 diabetes and dyslipidemia. Here, these associations are reviewed with respect to liver metabolism and disease.

Irs2 and Irs4 synergize in non-LepRb neurons to control energy balance and glucose homeostasis.

Insulin receptor substrates (Irs1, 2, 3 and Irs4) mediate the actions of insulin/IGF1 signaling. They have similar structure, but distinctly regulate development, growth, and metabolic homeostasis. Irs2 contributes to central metabolic sensing, partially by acting in leptin receptor (LepRb)-expressing neurons. Although Irs4 is largely restricted to the hypothalamus, its contribution to metabolic regulation is unclear because Irs4-null mice barely distinguishable from controls. We postulated that Irs2 and Irs4 synergize and complement each other in the brain. To examine this possibility, we investigated the metabolism of whole body Irs4(-/y) mice that lacked Irs2 in the CNS (bIrs2(-/-)·Irs4(-/y)) or only in LepRb-neurons (Lepr (∆Irs2) ·Irs4 (-/y) ). bIrs2(-/-)·Irs4(-/y) mice developed severe obesity and decreased energy expenditure, along with hyperglycemia and insulin resistance. Unexpectedly, the body weight and fed blood glucose levels of Lepr (∆Irs2) ·Irs4 (-/y) mice were not different from Lepr (∆Irs2) mice, suggesting that the functions of Irs2 and Irs4 converge upon neurons that are distinct from those expressing LepRb.

Arcuate AgRP neurons mediate orexigenic and glucoregulatory actions of ghrelin.

The hormone ghrelin stimulates eating and helps maintain blood glucose upon caloric restriction. While previous studies have demonstrated that hypothalamic arcuate AgRP neurons are targets of ghrelin, the overall relevance of ghrelin signaling within intact AgRP neurons is unclear. Here, we tested the functional significance of ghrelin action on AgRP neurons using a new, tamoxifen-inducible AgRP-CreER(T2) transgenic mouse model that allows spatiotemporally-controlled re-expression of physiological levels of ghrelin receptors (GHSRs) specifically in AgRP neurons of adult GHSR-null mice that otherwise lack GHSR expression. AgRP neuron-selective GHSR re-expression partially restored the orexigenic response to administered ghrelin and fully restored the lowered blood glucose levels observed upon caloric restriction. The normalizing glucoregulatory effect of AgRP neuron-selective GHSR expression was linked to glucagon rises and hepatic gluconeogenesis induction. Thus, our data indicate that GHSR-containing AgRP neurons are not solely responsible for ghrelin's orexigenic effects but are sufficient to mediate ghrelin's effects on glycemia.

Ablation of TrkB expression in RGS9-2 cells leads to hyperphagic obesity.

Brain-derived neurotrophic factor (BDNF) and its cognate receptor, TrkB (tropomyosin receptor kinase B), are widely expressed in the brain where they regulate a wide variety of biological processes, including energy homeostasis. However, the specific population(s) of TrkB-expressing neurons through which BDNF governs energy homeostasis remain(s) to be determined. Using the Cre-loxP recombination system, we deleted the mouse TrkB gene in RGS9-2-expressing cells. In this mouse mutant, TrkB expression was abolished in several hypothalamic nuclei, including arcuate nucleus, dorsomedial hypothalamus, and lateral hypothalamus. TrkB expression was also abolished in a small number of cells in other brain regions, including the cerebral cortex and striatum. The mutant animals developed hyperphagic obesity with normal energy expenditure. Despite hyperglycemia under fed conditions, these animals exhibited normal fasting blood glucose levels and normal glucose tolerance. These results suggest that BDNF regulates energy homeostasis in part through TrkB-expressing neurons in the hypothalamus.

Ghrelin regulates hypothalamic prolyl carboxypeptidase expression in mice.

Hypothalamic Prolyl carboxypeptidase (PRCP) plays a role in the regulation of energy metabolism by inactivating hypothalamic α-melanocyte stimulating hormone (α-MSH) levels and thus affecting melanocortin signaling. Alpha-MSH production is highly regulated both at transcriptional and posttranslational levels. Here we show that fasting induces a hypothalamic-specific up-regulation of Prcp mRNA and protein levels. Since fasting is characterized by elevated circulating ghrelin levels, we tested the effect of peripheral and central administration of ghrelin, and found that ghrelin increases hypothalamic Prcp mRNA expression. No changes in Prcp mRNA levels were detected in ghrelin knockout mice compared to their controls. Finally, ghrelin effect on PRCP expression was ghrelin receptor-mediated. Altogether our data show that ghrelin is a key regulator of hypothalamic PRCP expression, and up-regulation of PRCP by ghrelin may be an additional mechanism to decrease melanocortin signaling.

Deletion of miRNA processing enzyme Dicer in POMC-expressing cells leads to pituitary dysfunction, neurodegeneration and development of obesity.

MicroRNAs (miRNAs) have recently emerged as key regulators of metabolism. However, their potential role in the central regulation of whole-body energy homeostasis is still unknown. In this study we show that the expression of Dicer, an essential endoribonuclease for miRNA maturation, is modulated by nutrient availability and excess in the hypothalamus. Conditional deletion of Dicer in POMC-expressing cells resulted in obesity, characterized by hyperphagia, increased adiposity, hyperleptinemia, defective glucose metabolism and alterations in the pituitary-adrenal axis. The development of the obese phenotype was paralleled by a POMC neuron degenerative process that started around 3 weeks of age. Hypothalamic transcriptomic analysis in presymptomatic POMCDicerKO mice revealed the downregulation of genes implicated in biological pathways associated with classical neurodegenerative disorders, such as MAPK signaling, ubiquitin-proteosome system, autophagy and ribosome biosynthesis. Collectively, our results highlight a key role for miRNAs in POMC neuron survival and the consequent development of neurodegenerative obesity.

Leptin action via LepR-b Tyr1077 contributes to the control of energy balance and female reproduction.

Leptin action in the brain signals the repletion of adipose energy stores, suppressing feeding and permitting energy expenditure on a variety of processes, including reproduction. Leptin binding to its receptor (LepR-b) promotes the tyrosine phosphorylation of three sites on LepR-b, each of which mediates distinct downstream signals. While the signals mediated by LepR-b Tyr1138 and Tyr985 control important aspects of energy homeostasis and LepR-b signal attenuation, respectively, the role of the remaining LepR-b phosphorylation site (Tyr1077) in leptin action has not been studied. To examine the function of Tyr1077, we generated a "knock-in" mouse model expressing LepR-b (F1077), which is mutant for LepR-b Tyr1077. Mice expressing LepR-b (F1077) demonstrate modestly increased body weight and adiposity. Furthermore, females display impairments in estrous cycling. Our results suggest that signaling by LepR-b Tyr1077 plays a modest role in the control of metabolism by leptin, and is an important link between body adiposity and the reproductive axis.