Search

Objective: Conditional gene targeting has been extensively used for in vivo analysis of gene function in β-cell biology. The objective of this study was to examine whether mouse transgenic Cre lines, used to mediate β-cell- or pancreas-specific recombination, also drive Cre expression in the brain.Research Design and Methods: Transgenic Cre lines driven by Ins1, Ins2, and Pdx1 promoters were bred to R26R reporter strains. Cre activity was assessed by β-galactosidase or yellow fluorescent protein expression in the pancreas and the brain. Endogenous Pdx1 gene expression was monitored using Pdx1(tm1Cvw) lacZ knock-in mice. Cre expression in β-cells and co-localization of Cre activity with orexin-expressing and leptin-responsive neurons within the brain was assessed by immunohistochemistry.Results: All transgenic Cre lines examined that used the Ins2 promoter to drive Cre expression showed widespread Cre activity in the brain, whereas Cre lines that used Pdx1 promoter fragments showed more restricted Cre activity primarily within the hypothalamus. Immunohistochemical analysis of the hypothalamus from Tg(Pdx1-cre)(89.1Dam) mice revealed Cre activity in neurons expressing orexin and in neurons activated by leptin. Tg(Ins1-Cre/ERT)(1Lphi) mice were the only line that lacked Cre activity in the brain.Conclusions: Cre-mediated gene manipulation using transgenic lines that express Cre under the control of the Ins2 and Pdx1 promoters are likely to alter gene expression in nutrient-sensing neurons. Therefore, data arising from the use of these transgenic Cre lines must be interpreted carefully to assess whether the resultant phenotype is solely attributable to alterations in the islet β-cells. [ABSTRACT FROM AUTHOR]

Objective: Leptin acts via its receptor (LepRb) to signal the status of body energy stores. Leptin binding to LepRb initiates signaling by activating the associated Janus kinase 2 (Jak2) tyrosine kinase, which promotes the phosphorylation of tyrosine residues on the intracellular tail of LepRb. Two previously examined LepRb phosphorylation sites mediate several, but not all, aspects of leptin action, leading us to hypothesize that Jak2 signaling might contribute to leptin action independently of LepRb phosphorylation sites. We therefore determined the potential role in leptin action for signals that are activated by Jak2 independently of LepRb phosphorylation (Jak2-autonomous signals).Research Design and Methods: We inserted sequences encoding a truncated LepRb mutant (LepRb(Delta65c), which activates Jak2 normally, but is devoid of other LepRb intracellular sequences) into the mouse Lepr locus. We examined the leptin-regulated physiology of the resulting Delta/Delta mice relative to LepRb-deficient db/db animals.Results: The Delta/Delta animals were similar to db/db animals in terms of energy homeostasis, neuroendocrine and immune function, and regulation of the hypothalamic arcuate nucleus, but demonstrated modest improvements in glucose homeostasis.Conclusions: The ability of Jak2-autonomous LepRb signals to modulate glucose homeostasis in Delta/Delta animals suggests a role for these signals in leptin action. Because Jak2-autonomous LepRb signals fail to mediate most leptin action, however, signals from other LepRb intracellular sequences predominate. [ABSTRACT FROM AUTHOR]

The adipocyte-derived hormone, leptin, signals the status of body energy stores to the central nervous system to regulate appetite and energy expenditure. A specific long-form leptin receptor (LepRb), a type I cytokine receptor, mediates leptin action on LepRb-expressing neurons in the brain. Leptin binding to LepRb activates the associated Janus kinase-2 (Jak2) tyrosine kinase to promote the phosphorylation of Jak2 and three residues on LepRb; each of these sites mediates a distinct aspect of downstream LepRb signaling, with differing physiologic functions. Tyr(1138) --> STAT3 signaling suppresses feeding, but is not required for a number of other leptin actions. Tyr(985) binds SH2-containing tyrosine phosphatase-2 and suppressor of cytokine signaling-3 and primarily mediates the attenuation of LepRb signaling in vivo. The role for Tyr(1077), the major regulator of signal transducer and activator of transcription-5 (STAT5) during leptin signaling, in the physiologic response to leptin remains unclear, although the obese phenotype of animals deleted for STAT5 in the brain suggests the potential importance of this signaling pathway. Leptin also modulates a number of other signaling pathways in the brain, including PI 3-kinase, mammalian target of rapamycin and AMP-dependent protein kinase; the pathways by which leptin controls these signals remain unclear, however, and may involve some indirect mechanisms. Important issues regarding leptin action and LepRb signaling in the future include not only the more thorough analysis of intracellular signaling pathways, but the neural substrate by which leptin acts, as most major populations of LepRb neurons remain poorly studied. [ABSTRACT FROM AUTHOR]

The combination of decreasing food intake and increasing energy expenditure represents a powerful strategy for counteracting cardiometabolic diseases such as obesity and type 2 diabetes 1 . Yet current pharmacological approaches require conjugation of multiple receptor agonists to achieve both effects 2-4 , and so far, no safe energy-expending option has reached the clinic. Here we show that activation of neurokinin 2 receptor (NK2R) is sufficient to suppress appetite centrally and increase energy expenditure peripherally. We focused on NK2R after revealing its genetic links to obesity and glucose control. However, therapeutically exploiting NK2R signalling has previously been unattainable because its endogenous ligand, neurokinin A, is short-lived and lacks receptor specificity 5,6 . Therefore, we developed selective, long-acting NK2R agonists with potential for once-weekly administration in humans. In mice, these agonists elicit weight loss by inducing energy expenditure and non-aversive appetite suppression that circumvents canonical leptin signalling. Additionally, a hyperinsulinaemic-euglycaemic clamp reveals that NK2R agonism acutely enhances insulin sensitization. In diabetic, obese macaques, NK2R activation significantly decreases body weight, blood glucose, triglycerides and cholesterol, and ameliorates insulin resistance. These findings identify a single receptor target that leverages both energy-expending and appetite-suppressing programmes to improve energy homeostasis and reverse cardiometabolic dysfunction across species., Competing Interests: Competing interests T.M., J.H.E., J.B.H., D.P.C., M.B.F.G., M.T.-C., T.W.S. and Z.G.-H. work or have worked in some capacity for Embark Laboratories ApS, a company developing therapeutics for the treatment of T2D and obesity. P.K. is a consultant for Embark Laboratories ApS. The use of and chemical composition for NK2R agonists are patented by the University of Copenhagen and Embark Laboratories ApS, respectively. R.J.S. has received research support from Novo Nordisk, Fractyl, AstraZeneca, Congruence Therapeutics, Eli Lilly, Bullfrog AI, Glycsend Therapeutics and Amgen. R.J.S. has served as a paid consultant for Novo Nordisk, Eli Lilly, CinRx, Fractyl, Structure Therapeutics, Crinetics and Congruence Therapeutics. R.J.S. has equity in Calibrate, Rewind and Levator Therapeutics. The other authors declare no competing interests., (© 2024. The Author(s).)

The brain augments glucose production during fasting, but the mechanisms are poorly understood. Here, we show that Cckbr -expressing neurons in the ventromedial hypothalamic nucleus (VMN Cckbr cells) prevent low blood glucose during fasting through sympathetic nervous system (SNS)-mediated augmentation of adipose tissue lipolysis and substrate release. Activating VMN Cckbr neurons mobilized gluconeogenic substrates without altering glycogenolysis or gluconeogenic enzyme expression. Silencing these cells (Cckbr TetTox animals) reduced fasting blood glucose, impaired lipolysis, and decreased circulating glycerol (but not other gluconeogenic substrates) despite normal insulin, counterregulatory hormones, liver glycogen, and liver gluconeogenic gene expression. Furthermore, β3-adrenergic adipose tissue stimulation in Cckbr TetTox animals restored lipolysis and blood glucose. Hence, VMN Cckbr neurons impact blood glucose not by controlling islet or liver physiology, but rather by mobilizing gluconeogenic substrates. These findings establish a central role for hypothalamic and SNS signaling during normal glucose homeostasis and highlight the importance of gluconeogenic substrate mobilization during physiologic fasting., Competing Interests: Conflict of Interests Statement: MGM receives research support from AstraZeneca, Eli Lilly, and Novo Nordisk. OAM has received grant support from Regeneron Pharmaceuticals, Inc., CombiGene AB, and Rejuvenate Bio. The authors declare that they have no other conflicts of interest.

The adipose-derived hormone leptin acts via its receptor (LepRb) in the brain to control energy balance. A potentially unidentified population of GABAergic hypothalamic LepRb neurons plays key roles in the restraint of food intake and body weight by leptin. To identify markers for candidate populations of LepRb neurons in an unbiased manner, we performed single-nucleus RNA-Seq of enriched mouse hypothalamic LepRb cells, identifying several previously unrecognized populations of hypothalamic LepRb neurons. Many of these populations displayed strong conservation across species, including GABAergic Glp1r-expressing LepRb (LepRbGlp1r) neurons, which expressed more Lepr than other LepRb cell populations. Ablating Lepr from LepRbGlp1r cells provoked hyperphagic obesity without impairing energy expenditure. Similarly, improvements in energy balance caused by Lepr reactivation in GABA neurons of otherwise Lepr-null mice required Lepr expression in GABAergic Glp1r-expressing neurons. Furthermore, restoration of Glp1r expression in LepRbGlp1r neurons in otherwise Glp1r-null mice enabled food intake suppression by the GLP1R agonist, liraglutide. Thus, the conserved GABAergic LepRbGlp1r neuron population plays crucial roles in the suppression of food intake by leptin and GLP1R agonists.

Deoxynivalenol (DON), a type B trichothecene mycotoxin contaminating grains, promotes nausea, emesis and anorexia. With DON exposure, circulating levels of intestinally derived satiation hormones, including glucagon-like peptide 1 (GLP-1) are elevated. To directly test whether GLP-1 signaling mediates the effects of DON, we examined the response of GLP-1 or GLP-1R-deficient mice to DON injection. We found comparable anorectic and conditioned taste avoidance learning responses in GLP-1/GLP-1R deficient mice compared to control littermates, suggesting that GLP-1 is not necessary for the effects of DON on food intake and visceral illness. We then used our previously published data from translating ribosome affinity purification with RNA sequencing (TRAP-seq) analysis of area postrema neurons that express the receptor for the circulating cytokine growth differentiation factor (GDF15), growth differentiation factor a-like (GFRAL). Interestingly, this analysis showed that a cell surface receptor for DON, calcium sensing receptor (CaSR), is heavily enriched in GFRAL neurons. Given that GDF15 potently reduces food intake and can cause visceral illness by signaling through GFRAL neurons, we hypothesized that DON may also signal by activating CaSR on GFRAL neurons. Indeed, circulating GDF15 levels are elevated after DON administration but both GFRAL knockout and GFRAL neuron-ablated mice exhibited similar anorectic and conditioned taste avoidance responses compared to WT littermates. Thus, GLP-1 signaling and GFRAL signaling and neurons are not required for DON-induced visceral illness or anorexia.

Leptin exerts its biological actions by activating the long-form leptin receptor (LepRb). LepRb signaling impairment and leptin resistance are believed to cause obesity. The transcription factor Slug - also known as Snai2 - recruits epigenetic modifiers and regulates gene expression by an epigenetic mechanism; however, its epigenetic action has not been explored in leptin resistance. Here, we uncover a proobesity function of neuronal Slug. Hypothalamic Slug was upregulated in obese mice. LepRb+ cell-specific Slug-knockout (SlugΔLepRb) mice were resistant to diet-induced obesity, type 2 diabetes, and liver steatosis and experienced decreased food intake and increased fat thermogenesis. Leptin stimulated hypothalamic Stat3 phosphorylation and weight loss to a markedly higher level in SlugΔLepRb than in Slugfl/fl mice, even before their body weight divergence. Conversely, hypothalamic LepRb+ neuron-specific overexpression of Slug, mediated by AAV-hSyn-DIO-Slug transduction, induced leptin resistance, obesity, and metabolic disorders in mice on a chow diet. At the genomic level, Slug bound to and repressed the LepRb promoter, thereby inhibiting LepRb transcription. Consistently, Slug deficiency decreased methylation of LepRb promoter H3K27, a repressive epigenetic mark, and increased LepRb mRNA levels in the hypothalamus. Collectively, these results unravel what we believe to be a previously unrecognized hypothalamic neuronal Slug/epigenetic reprogramming/leptin resistance axis that promotes energy imbalance, obesity, and metabolic disease.

Body weight and adiposity represent biologically controlled parameters that are influenced by a combination of genetic, developmental and environmental variables. Although the hypothalamus plays a crucial role in matching caloric intake with energy expenditure to achieve a stable body weight, it is now recognized that neuronal circuits in the hindbrain not only serve to produce nausea and to terminate feeding in response to food consumption or during pathological states, but also contribute to the long-term control of body weight. Additionally, recent work has identified hindbrain neurons that are capable of suppressing food intake without producing aversive responses like those associated with nausea. Here we review recent advances in our understanding of the hindbrain neurons that control feeding, particularly those located in the area postrema and the nucleus tractus solitarius. We frame this information in the context of new atlases of hindbrain neuronal populations and develop a model of the hindbrain circuits that control food intake and energy balance, suggesting important areas for additional research., (© 2022. Springer Nature Limited.)

Growth differentiation factor 15 (GDF15), a TGFβ superfamily cytokine, acts through its receptor, cell line-derived neurotrophic factorfamily receptor α-like (GFRAL), to suppress food intake and promote nausea. GDF15 is broadly expressed at low levels but increases in states of disease such as cancer, cachexia, and sepsis. Whether GDF15 is necessary for inducing sepsis-associated anorexia and body weight loss is currently unclear. To test this we used a model of moderate systemic infection in GDF15KO and GFRALKO mice with lipopolysaccharide (LPS) treatment to define the role of GDF15 signaling in infection-mediated physiologic responses. Since physiological responses to LPS depend on housing temperature, we tested the effects of subthermoneutral and thermoneutral conditions on eliciting anorexia and inducing GDF15. Our data demonstrate a conserved LPS-mediated increase in circulating GDF15 levels in mouse, rat, and human. However, we did not detect differences in LPS-induced anorexia between WT and GDF15KO or GFRALKO mice. Furthermore, there were no differences in anorexia or circulating GDF15 levels at either thermoneutral or subthermoneutral housing conditions in LPS-treated mice. These data demonstrate that GDF15 is not necessary to drive food intake suppression in response to moderate doses of LPS. NEW & NOTEWORTHY Although many responses to LPS depend on housing temperature, the anorexic response to LPS does not. LPS results in a potent and rapid increase in circulating levels of GDF15 in mice, rats, and humans. Nevertheless, GDF15 and its receptor (GFRAL) are not required for the anorexic response to systemic LPS administration. The anorexic response to LPS likely involves a myriad of complex physiological alterations.

Calcitonin receptor (Calcr)-expressing neurons of the nucleus tractus solitarius (NTS; Calcr NTS cells) contribute to the long-term control of food intake and body weight. Here, we show that Prlh-expressing NTS (Prlh NTS ) neurons represent a subset of Calcr NTS cells and that Prlh expression in these cells restrains body weight gain in the face of high fat diet challenge in mice. To understand the relationship of Prlh NTS cells to hypothalamic feeding circuits, we determined the ability of Prlh NTS -mediated signals to overcome enforced activation of AgRP neurons. We found that Prlh NTS neuron activation and Prlh overexpression in Prlh NTS cells abrogates AgRP neuron-driven hyperphagia and ameliorates the obesity of mice deficient in melanocortin signaling or leptin. Thus, enhancing Prlh-mediated neurotransmission from the NTS dampens hypothalamically-driven hyperphagia and obesity, demonstrating that NTS-mediated signals can override the effects of orexigenic hypothalamic signals on long-term energy balance., (© 2021. The Author(s).)

Growing evidence implicates the brain in the regulation of both immediate fuel availability (for example, circulating glucose) and long-term energy stores (that is, adipose tissue mass). Rather than viewing the adipose tissue and glucose control systems separately, we suggest that the brain systems that control them are components of a larger, highly integrated, 'fuel homeostasis' control system. This conceptual framework, along with new insights into the organization and function of distinct neuronal systems, provides a context within which to understand how metabolic homeostasis is achieved in both basal and postprandial states. We also review evidence that dysfunction of the central fuel homeostasis system contributes to the close association between obesity and type 2 diabetes, with the goal of identifying more effective treatment options for these common metabolic disorders.

The ventromedial hypothalamus (VMH) is a critical neural node that senses blood glucose and promotes glucose utilization or mobilization during hypoglycemia. The VMH neurons that control these distinct physiologic processes are largely unknown. Here, we show that melanocortin 3 receptor ( Mc3R )-expressing VMH neurons (VMH MC3R ) sense glucose changes both directly and indirectly via altered excitatory input. We identify presynaptic nodes that potentially regulate VMH MC3R neuronal activity, including inputs from proopiomelanocortin (POMC)-producing neurons in the arcuate nucleus. We find that VMH MC3R neuron activation blunts, and their silencing enhances glucose excursion following a glucose load. Overall, these findings demonstrate that VMH MC3R neurons are a glucose-responsive hypothalamic subpopulation that promotes glucose disposal upon activation; this highlights a potential site for targeting dysregulated glycemia., Competing Interests: Competing interest statement: NovoNordisk provides research grant support to D.P.O. and M.G.M. Ionis Pharmaceuticals provides grant support to M.G.M., (Copyright © 2021 the Author(s). Published by PNAS.)

The brainstem dorsal vagal complex (DVC) is known to regulate energy balance and is the target of appetite-suppressing hormones, such as glucagon-like peptide 1 (GLP-1). Here we provide a comprehensive genetic map of the DVC and identify neuronal populations that control feeding. Combining bulk and single-nucleus gene expression and chromatin profiling of DVC cells, we reveal 25 neuronal populations with unique transcriptional and chromatin accessibility landscapes and peptide receptor expression profiles. GLP-1 receptor (GLP-1R) agonist administration induces gene expression alterations specific to two distinct sets of Glp1r neurons-one population in the area postrema and one in the nucleus of the solitary tract that also expresses calcitonin receptor (Calcr). Transcripts and regions of accessible chromatin near obesity-associated genetic variants are enriched in the area postrema and the nucleus of the solitary tract neurons that express Glp1r and/or Calcr, and activating several of these neuronal populations decreases feeding in rodents. Thus, DVC neuronal populations associated with obesity predisposition suppress feeding and may represent therapeutic targets for obesity.

The TGFβ cytokine family member, GDF-15, reduces food intake and body weight and represents a potential treatment for obesity. Because the brainstem-restricted expression pattern of its receptor, GDNF Family Receptor α-like (GFRAL), presents an exciting opportunity to understand mechanisms of action for area postrema neurons in food intake; we generated Gfral Cre and conditional Gfral CreERT mice to visualize and manipulate GFRAL neurons. We found infection or pathophysiologic states (rather than meal ingestion) stimulate GFRAL neurons. TRAP-Seq analysis of GFRAL neurons revealed their expression of a wide range of neurotransmitters and neuropeptides. Artificially activating Gfral Cre -expressing neurons inhibited feeding, decreased gastric emptying, and promoted a conditioned taste aversion (CTA). GFRAL neurons most strongly innervate the parabrachial nucleus (PBN), where they target CGRP-expressing (CGRP PBN ) neurons. Silencing CGRP PBN neurons abrogated the aversive and anorexic effects of GDF-15. These findings suggest that GFRAL neurons link non-meal-associated pathophysiologic signals to suppress nutrient uptake and absorption., Competing Interests: Competing interest statement: S.B.J. is an employee of Novo Nordisk. D.P.O., M.G.M., and R.J.S. receive research support from Novo Nordisk. R.J.S. and M.G.M. receive research support from AstraZeneca. R.J.S. receives research support from Pfizer, Kintai, and Ionis. R.J.S. also serves as a paid consultant for Novo Nordisk, Kintai, Ionis, and Scohia. R.J.S. has equity positions in Zafgen and ReDesign Health. All other authors report no conflicts of interest.

Mice lacking SH2B1 and humans with variants of SH2B1 display severe obesity and insulin resistance. SH2B1 is an adapter protein that is recruited to the receptors of multiple hormones and neurotrophic factors. Of the four known alternatively spliced SH2B1 isoforms, SH2B1β and SH2B1γ exhibit ubiquitous expression, whereas SH2B1α and SH2B1δ are essentially restricted to the brain. To understand the roles for SH2B1α and SH2B1δ in energy balance and glucose metabolism, we generated mice lacking these brain-specific isoforms (αδ knockout [αδKO] mice). αδKO mice exhibit decreased food intake, protection from weight gain on standard and high-fat diets, and an adiposity-dependent improvement in glucose homeostasis. SH2B1 has been suggested to impact energy balance via the modulation of leptin action. However, αδKO mice exhibit leptin sensitivity that is similar to that of wild-type mice by multiple measures. Thus, decreasing the abundance of SH2B1α and/or SH2B1δ relative to the other SH2B1 isoforms likely shifts energy balance toward a lean phenotype via a primarily leptin-independent mechanism. Our findings suggest that the different alternatively spliced isoforms of SH2B1 perform different functions in vivo., (© 2020 by the American Diabetes Association.)

Gonadal steroids modulate growth hormone (GH) secretion and the pubertal growth spurt via undefined central pathways. GH-releasing hormone (GHRH) neurons express estrogen receptor α (ERα) and androgen receptor (AR), suggesting changing levels of gonadal steroids during puberty directly modulate the somatotropic axis. We generated mice with deletion of ERα in GHRH cells (GHRH ΔERα ), which displayed reduced body length in both sexes. Timing of puberty onset was similar in both groups, but puberty completion was delayed in GHRH ΔERα females. Lack of AR in GHRH cells (GHRH ΔAR mice) induced no changes in body length, but puberty completion was also delayed in females. Using a mouse model with two reporter genes, we observed that, while GHRH tdTom neurons minimally colocalize with Kiss1 hrGFP in prepubertal mice, ∼30% of GHRH neurons coexpressed both reporter genes in adult females, but not in males. Developmental analysis of Ghrh and Kiss1 expression suggested that a subpopulation of ERα neurons in the arcuate nucleus of female mice undergoes a shift in phenotype, from GHRH to Kiss1, during pubertal transition. Our findings demonstrate that direct actions of gonadal steroids in GHRH neurons modulate growth and puberty and indicate that GHRH/Kiss1 dual-phenotype neurons play a sex-specific role in the crosstalk between the somatotropic and gonadotropic axes during pubertal transition. SIGNIFICANCE STATEMENT Late maturing adolescents usually show delayed growth and bone age. At puberty, gonadal steroids have stimulatory effects on the activation of growth and reproductive axes, but the existence of gonadal steroid-sensitive neuronal crosstalk remains undefined. Moreover, the neural basis for the sex differences observed in the clinical arena is unknown. Lack of ERα in GHRH neurons disrupts growth in both sexes and causes pubertal delay in females. Deletion of androgen receptor in GHRH neurons only delayed female puberty. In adult females, not males, a subset of GHRH neurons shift phenotype to start producing Kiss1. Thus, direct estrogen action in GHRH/Kiss1 dual-phenotype neurons modulates growth and puberty and may orchestrate the sex differences in endocrine function observed during pubertal transition., (Copyright © 2020 the authors.)

Epidemiological and genome-wide association studies (GWAS) have shown high correlation between childhood obesity and advance in puberty. Early age at menarche is associated with a series of morbidities, including breast cancer, cardiovascular diseases, type 2 diabetes, and obesity. The adipocyte hormone leptin signals the amount of fat stores to the neuroendocrine reproductive axis via direct actions in the brain. Using mouse genetics, we and others have identified the hypothalamic ventral premammillary nucleus (PMv) and the agouti-related protein (AgRP) neurons in the arcuate nucleus (Arc) as primary targets of leptin action in pubertal maturation. However, the molecular mechanisms underlying leptin's effects remain unknown. Here we assessed changes in the PMv and Arc transcriptional program during leptin-stimulated and typical pubertal development using overlapping analysis of bulk RNA sequecing, TRAP sequencing, and the published database. Our findings demonstrate that dynamic somatodendritic remodeling and extracellular space organization underlie leptin-induced and typical pubertal maturation in female mice., Competing Interests: The authors declare no competing interest., (© 2020 The Author(s).)

Intestinal crypts have great capacity for repair and regeneration after intestinal stem cell (ISC) injury. Here, we define the cellular remodeling process resulting from ISC niche interruption by transient Notch pathway inhibition in adult mice. Although ISCs were retained, lineage tracing demonstrated a marked reduction in ISC function after Notch disruption. Surprisingly, Notch ligand-expressing Paneth cells were rapidly lost by apoptotic cell death. The ISC-Paneth cell changes were followed by a regenerative response, characterized by expansion of cells expressing Notch ligands Dll1 and Dll4, enhanced Notch signaling, and a proliferative surge. Lineage tracing and organoid studies showed that Dll1-expressing cells were activated to function as multipotential progenitors, generating both absorptive and secretory cells and replenishing the vacant Paneth cell pool. Our analysis uncovered a dynamic, multicellular remodeling response to acute Notch inhibition to repair the niche and restore homeostasis. Notably, this crypt regenerative response did not require ISC loss., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

To identify neurons that specifically increase blood glucose from among the diversely functioning cell types in the ventromedial hypothalamic nucleus (VMN), we studied the cholecystokinin receptor B-expressing (CCKBR-expressing) VMN targets of glucose-elevating parabrachial nucleus neurons. Activation of these VMNCCKBR neurons increased blood glucose. Furthermore, although silencing the broader VMN decreased energy expenditure and promoted weight gain without altering blood glucose levels, silencing VMNCCKBR neurons decreased hIepatic glucose production, insulin-independently decreasing blood glucose without altering energy balance. Silencing VMNCCKBR neurons also impaired the counterregulatory response to insulin-induced hypoglycemia and glucoprivation and replicated hypoglycemia-associated autonomic failure. Hence, VMNCCKBR cells represent a specialized subset of VMN cells that function to elevate glucose. These cells not only mediate the allostatic response to hypoglycemia but also modulate the homeostatic setpoint for blood glucose in an insulin-independent manner, consistent with a role for the brain in the insulin-independent control of glucose homeostasis.

Leptin receptor-expressing (LepRb-expressing) neurons of the nucleus tractus solitarius (NTS; LepRbNTS neurons) receive gut signals that synergize with leptin action to suppress food intake. NTS neurons that express preproglucagon (Ppg) (and that produce the food intake-suppressing PPG cleavage product glucagon-like peptide-1 [GLP1]) represent a subpopulation of mouse LepRbNTS cells. Using Leprcre, Ppgcre, and Ppgfl mouse lines, along with Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), we examined roles for Ppg in GLP1NTS and LepRbNTS cells for the control of food intake and energy balance. We found that the cre-dependent ablation of NTS Ppgfl early in development or in adult mice failed to alter energy balance, suggesting the importance of pathways independent of NTS GLP1 for the long-term control of food intake. Consistently, while activating GLP1NTS cells decreased food intake, LepRbNTS cells elicited larger and more durable effects. Furthermore, while the ablation of NTS Ppgfl blunted the ability of GLP1NTS neurons to suppress food intake during activation, it did not impact the suppression of food intake by LepRbNTS cells. While Ppg/GLP1-mediated neurotransmission plays a central role in the modest appetite-suppressing effects of GLP1NTS cells, additional pathways engaged by LepRbNTS cells dominate for the suppression of food intake.

Leptin stimulates the sympathetic nervous system (SNS), energy expenditure, and weight loss; however, the underlying molecular mechanism remains elusive. Here, we uncover Sh2b1 in leptin receptor (LepR) neurons as a critical component of a SNS/brown adipose tissue (BAT)/thermogenesis axis. LepR neuron-specific deletion of Sh2b1 abrogates leptin-stimulated sympathetic nerve activation and impairs BAT thermogenic programs, leading to reduced core body temperature and cold intolerance. The adipose SNS degenerates progressively in mutant mice after 8 weeks of age. Adult-onset ablation of Sh2b1 in the mediobasal hypothalamus also impairs the SNS/BAT/thermogenesis axis; conversely, hypothalamic overexpression of human SH2B1 has the opposite effects. Mice with either LepR neuron-specific or adult-onset, hypothalamus-specific ablation of Sh2b1 develop obesity, insulin resistance, and liver steatosis. In contrast, hypothalamic overexpression of SH2B1 protects against high fat diet-induced obesity and metabolic syndromes. Our results unravel an unrecognized LepR neuron Sh2b1/SNS/BAT/thermogenesis axis that combats obesity and metabolic disease.

To understand hindbrain pathways involved in the control of food intake, we examined roles for calcitonin receptor (CALCR)-containing neurons in the NTS. Ablation of NTS Calcr abrogated the long-term suppression of food intake, but not aversive responses, by CALCR agonists. Similarly, activating Calcr NTS neurons decreased food intake and body weight but (unlike neighboring Cck NTS cells) failed to promote aversion, revealing that Calcr NTS neurons mediate a non-aversive suppression of food intake. While both Calcr NTS and Cck NTS neurons decreased feeding via projections to the PBN, Cck NTS cells activated aversive CGRP PBN cells while Calcr NTS cells activated distinct non-CGRP PBN cells. Hence, Calcr NTS cells suppress feeding via non-aversive, non-CGRP PBN targets. Additionally, silencing Calcr NTS cells blunted food intake suppression by gut peptides and nutrients, increasing food intake and promoting obesity. Hence, Calcr NTS neurons define a hindbrain system that participates in physiological energy balance and suppresses food intake without activating aversive systems., Competing Interests: Declaration of Interests J.L.T. and C.J.R. are past or present employees of MedImmune, LLC and hold stock in AstraZeneca. The other authors declare that they have no conflicts of interest relevant to this manuscript., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Leptin acts via its receptor, LepRb, on specialized neurons in the brain to modulate energy balance and glucose homeostasis. LepRb→STAT3 signaling plays a crucial role in leptin action, but LepRb also mediates an additional as-yet-unidentified signal (Signal 2) that is important for leptin action. Signal 2 requires LepRb regions in addition to those required for JAK2 activation but operates independently of STAT3 and LepRb phosphorylation sites., Methods: To identify LepRb sequences that mediate Signal 2, we used CRISPR/Cas9 to generate five novel mouse lines containing COOH-terminal truncation mutants of LepRb. We analyzed the metabolic phenotype and measures of hypothalamic function for these mouse lines., Results: We found that deletion of LepRb sequences between residues 921 and 960 dramatically worsens metabolic control and alters hypothalamic function relative to smaller truncations. We also found that deletion of the regions including residues 1013-1053 and 960-1013 each decreased obesity compared to deletions that included additional COOH-terminal residues., Conclusions: LepRb sequences between residues 921 and 960 mediate the STAT3 and LepRb phosphorylation-independent second signal that contributes to the control of energy balance and metabolism by leptin/LepRb. In addition to confirming the inhibitory role of the region (residues 961-1013) containing Tyr 985 , we also identified the region containing residues 1013-1053 (which contains no Tyr residues) as a second potential mediator of LepRb inhibition. Thus, the intracellular domain of LepRb mediates multiple Tyr-independent signals., (Copyright © 2020 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Disruption of the adaptor protein SH2B1 (SH2-B, PSM) is associated with severe obesity, insulin resistance, and neurobehavioral abnormalities in mice and humans. Here, we identify 15 SH2B1 variants in severely obese children. Four obesity-associated human SH2B1 variants lie in the Pleckstrin homology (PH) domain, suggesting that the PH domain is essential for SH2B1's function. We generated a mouse model of a human variant in this domain (P322S). P322S/P322S mice exhibited substantial prenatal lethality. Examination of the P322S/+ metabolic phenotype revealed late-onset glucose intolerance. To circumvent P322S/P322S lethality, mice containing a two-amino acid deletion within the SH2B1 PH domain (ΔP317, R318 [ΔPR]) were studied. Mice homozygous for ΔPR were born at the expected Mendelian ratio and exhibited obesity plus insulin resistance and glucose intolerance beyond that attributable to their increased adiposity. These studies demonstrate that the PH domain plays a crucial role in how SH2B1 controls energy balance and glucose homeostasis., (© 2019 by the American Diabetes Association.)

Objectives: Leptin acts via its receptor LepRb on specialized neurons in the brain to modulate food intake, energy expenditure, and body weight. LepRb activates signal transducers and activators of transcription (STATs, including STAT1, STAT3, and STAT5) to control gene expression., Methods: Because STAT3 is crucial for physiologic leptin action, we used TRAP-seq to examine gene expression in LepRb neurons of mice ablated for Stat3 in LepRb neurons (Stat3 LepRb KO mice), revealing the STAT3-dependent transcriptional targets of leptin. To understand roles for STAT proteins in leptin action, we also ablated STAT1 or STAT5 from LepRb neurons and expressed a constitutively-active STAT3 (CASTAT3) in LepRb neurons., Results: While we also found increased Stat1 expression and STAT1-mediated transcription of leptin-regulated genes in Stat3 LepRb KO mice, ablating Stat1 in LepRb neurons failed to alter energy balance (even on the Stat3 LepRb KO background); ablating Stat5 in LepRb neurons also failed to alter energy balance. Importantly, expression of a constitutively-active STAT3 (CASTAT3) in LepRb neurons decreased food intake and body weight and improved metabolic parameters in leptin-deficient (ob/ob) mice, as well as in wild-type animals., Conclusions: Thus, STAT3 represents the unique STAT protein required for leptin action and STAT3 suffices to mediate important components of leptin action in the absence of other LepRb signals., (Copyright © 2019 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Objective: Analogues of GDF15 (Growth Differentiation Factor 15) are promising new anti-obesity therapies as pharmacological treatment with GDF15 results in dramatic reductions of food intake and body weight. GDF15 exerts its central anorexic effects by binding to the GFRAL receptor exclusively expressed in the Area Postrema (AP) and the Nucleus of the Solitary Tract (NTS) of the hindbrain. We sought to determine if GDF15 is an indispensable factor for other interventions that cause weight loss and which are also known to act via these hindbrain regions., Methods: To explore the role of GDF15 on food choice we performed macronutrient intake studies in mice treated pharmacologically with GDF15 and in mice having either GDF15 or GFRAL deleted. Next we performed vertical sleeve gastrectomy (VSG) surgeries in a cohort of diet-induced obese Gdf15-null and control mice. To explore the anatomical co-localization of neurons in the hindbrain responding to GLP-1 and/or GDF15 we used GLP-1R reporter mice treated with GDF15, as well as naïve mouse brain and human brain stained by ISH and IHC, respectively, for GLP-1R and GFRAL. Lastly we performed a series of food intake experiments where we treated mice with targeted genetic disruption of either Gdf15 or Gfral with liraglutide; Glp1r-null mice with GDF15; or combined liraglutide and GDF15 treatment in wild-type mice., Results: We found that GDF15 treatment significantly lowered the preference for fat intake in mice, whereas no changes in fat intake were observed after genetic deletion of Gdf15 or Gfral. In addition, deletion of Gdf15 did not alter the food intake or bodyweight after sleeve gastrectomy. Lack of GDF15 or GFRAL signaling did not alter the ability of the GLP-1R agonist liraglutide to reduce food intake. Similarly lack of GLP-1R signaling did not reduce GDF15's anorexic effect. Interestingly, there was a significant synergistic effect on weight loss when treating wild-type mice with both GDF15 and liraglutide., Conclusion: These data suggest that while GDF15 does not play a role in the potent effects of VSG in mice there seems to be a potential therapeutic benefit of activating GFRAL and GLP-1R systems simultaneously., (Copyright © 2019. Published by Elsevier GmbH.)

Tissue-resident myeloid cells initiate local inflammation in response to infectious or injurious stimuli. Sixteen years ago, macrophages in the adipose tissue (ATMs) were shown to undergo a form of activation in response to diet-induced obesity, thus leading to the conclusion that these macrophages sense a type of pro-inflammatory injury. ATMs are now known to be central to adipose tissue development, plasticity, maintenance and function. Indeed, their involvement in obesity may represent hijacking of these functions. More recently, microglia, 'CNS macrophages', have been shown to accumulate and undergo activation in response to dietary excess in the mediobasal hypothalamus (MBH), and early studies have implicated these cells as injury-responsive mediators of hypothalamic dysfunction. However, microglia are amazingly diverse cells now known to have moment-to-moment sensory functions and to communicate with neighbouring neurons to maintain and shape brain circuitry. Here, we build on this view, detailing our rapidly evolving understanding of microglial heterogeneity in the MBH and their roles as nutrient and environmental sensors. We propose that microglia, instead of simply responding to diet-induced damage, act as critical metabolic regulators that may coordinate a complex cellular network in the MBH. Understanding their roles in hypothalamic development and function should reveal unexpected mechanistic information relevant to important diseases such as obesity.

Leptin, a hormone primarily produced by adipocytes, contributes to the regulation of bone health by modulating bone density, growth and adiposity. Upon leptin binding, multiple sites of the long form of the leptin receptor (LepRb) are phosphorylated to trigger activation of downstream signaling pathways. To address the role of LepRb-signaling pathways in bone health, we compared the effects of three LepRb mutations on bone density, adiposity, and growth in male and female mice. The ∆65 mutation, which lacks the known tyrosine phosphorylation sites, caused obesity and the most dramatic bone phenotype marked by excessive bone adiposity, osteoporosis, and decreased growth, consistent with the phenotype of db/db and ob/ob mice that fully lack leptin receptor signaling. Mutation of LepRb Tyr 1138 , which results in an inability to recruit and phosphorylate signal transducer and activator of transcription 3, also caused obesity, but bone loss and adiposity were more dominant in male mice and no growth defect was observed. In contrast, mutation of LepRb Tyr 985 , which blocks SHP2/SOCS3 recruitment to LepRb and contributes to leptin hypersensitivity, promoted increased femur bone density only in male mice, while marrow adiposity and bone growth were not affected. Additional analyses of vertebral trabecular bone volume indicate that only the Tyr 1138 mutant mice exhibit bone loss in vertebrae. Together, our findings suggest that the phosphorylation status of specific sites of the LepRb contribute to the sex- and location-dependent bone responses to leptin. Unraveling the mechanisms by which leptin responses are sex- and location-dependent can contribute to the development of uniquely targeted osteoporosis therapies., (© 2018 Wiley Periodicals, Inc.)

The central melanocortin system plays a crucial role in the control of energy balance. Although the decreased energy expenditure and increased adiposity of melanocortin-3 receptor (Mc3R)-null mice suggest the importance of Mc3R-regulated neurons in energy homeostasis, the roles for specific subsets of Mc3R neurons in energy balance have yet to be determined. Because the lateral hypothalamic area (LHA) contributes to the control of energy expenditure and feeding, we generated Mc3rcre mice to determine the roles of LHA Mc3R (Mc3RLHA) neurons in energy homeostasis. We found that Mc3RLHA neurons overlap extensively with LHA neuron markers that contribute to the control of energy balance (neurotensin, galanin, and leptin receptor) and project to brain areas involved in the control of feeding, locomotion, and energy expenditure, consistent with potential roles for Mc3RLHA neurons in these processes. Indeed, selective chemogenetic activation of Mc3RLHA neurons increased locomotor activity and augmented refeeding after a fast. Although the ablation of Mc3RLHA neurons did not alter food intake, mice lacking Mc3RLHA neurons displayed decreased energy expenditure and locomotor activity, along with increased body mass and adiposity. Thus, Mc3R neurons lie within LHA neurocircuitry that modulates locomotor activity and energy expenditure and contribute to energy balance control.

Background & Aims: Loss of leucine-rich repeat-containing G-protein-coupled receptor 5-positive crypt base columnar cells provides permissive conditions for different facultative stem cell populations to dedifferentiate and repopulate the stem cell compartment. In this study, we used a defensin α4-Cre recombinase (Defa4Cre) line to define the potential of Paneth cells to dedifferentiate and contribute to intestinal stem cell (ISC) maintenance during normal homeostasis and after intestinal injury., Methods: Small intestine and enteroids from Defa4 Cre ;Rosa26 tandem dimer Tomato (tdTomato), a red fluoresent protein, (or Rosa26 Enhanced Yellow Fluorescent Protein (EYFP)) reporter, Notch gain-of-function (Defa4 Cre ;Rosa26 Notch Intracellular Domain (NICD)-ires-nuclear Green Fluorescent Protein (nGFP) and Defa4 Cre ;Rosa26 reverse tetracycline transactivator-ires Enhanced Green Fluorescent Protein (EGFP);TetO NICD ), A Disintegrin and Metalloproteinase domain-containing protein 10 (ADAM10) loss-of-function (Defa4 Cre ;ADAM10 flox/flox ), and Adenomatous polyposis coli (APC) inactivation (Defa4 Cre ;APC flox/flox ) mice were analyzed. Doxorubicin treatment was used as an acute intestinal injury model. Lineage tracing, proliferation, and differentiation were assessed in vitro and in vivo., Results: Defa4 Cre -expressing cells are fated to become mature Paneth cells and do not contribute to ISC maintenance during normal homeostasis in vivo. However, spontaneous lineage tracing was observed in enteroids, and fluorescent-activated cell sorter-sorted Defa4 Cre -marked cells showed clonogenic enteroid growth. Notch activation in Defa4 Cre -expressing cells caused dedifferentiation to multipotent ISCs in vivo and was required for adenoma formation. ADAM10 deletion had no significant effect on crypt homeostasis. However, after acute doxorubicin-induced injury, Defa4 Cre -expressing cells contributed to regeneration in an ADAM10-Notch-dependent manner., Conclusions: Our studies have shown that Defa4 Cre -expressing Paneth cells possess cellular plasticity, can dedifferentiate into multipotent stem cells upon Notch activation, and can contribute to intestinal regeneration in an acute injury model., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

To meet the challenge to human health posed by obesity, a better understanding of the regulation of feeding is essential. Medications targeting 5-hydroxytryptamine (5-HT; serotonin) 2C receptors (htr2c; 5-HT 2C R) improve obesity. Here we probed the functional significance of 5-HT 2C Rs specifically within the brainstem nucleus of the solitary tract (5-HT 2C R NTS ) in feeding behavior. Selective activation of 5-HT 2C R NTS decreased feeding and was sufficient to mediate acute food intake reductions elicited by the 5-HT 2C R agonist obesity medication lorcaserin. Similar to pro-opiomelanocortin neurons expressed within the hypothalamic arcuate nucleus (POMC ARC ), a subset of POMC NTS neurons co-expressed 5-HT 2C Rs and were activated by 5-HT 2C R agonists. Knockdown of POMC NTS prevented the acute appetite-suppressive effect of lorcaserin, whereas POMC ARC knockdown prevented the full anorectic effect. These data identify 5-HT 2C R NTS as a sufficient subpopulation of 5-HT 2C Rs in reducing food intake when activated and reveal that 5-HT 2C R agonist obesity medications require POMC within the NTS and ARC to reduce food intake., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: To date, early developmental ablation of leptin receptor (LepRb) expression from circumscribed populations of hypothalamic neurons (e.g., arcuate nucleus (ARC) Pomc- or Agrp-expressing cells) has only minimally affected energy balance. In contrast, removal of LepRb from at least two large populations (expressing vGat or Nos1) spanning multiple hypothalamic regions produced profound obesity and metabolic dysfunction. Thus, we tested the notion that the total number of leptin-responsive hypothalamic neurons (rather than specific subsets of cells with a particular molecular or anatomical signature) subjected to early LepRb deletion might determine energy balance., Methods: We generated new mouse lines deleted for LepRb in ARC Ghrh Cre neurons or in Htr2c Cre neurons (representing roughly half of all hypothalamic LepRb neurons, distributed across many nuclei). We compared the phenotypes of these mice to previously-reported models lacking LepRb in Pomc, Agrp, vGat or Nos1 cells., Results: The early developmental deletion of LepRb from vGat or Nos1 neurons produced dramatic obesity, but deletion of LepRb from Pomc, Agrp, Ghrh, or Htr2c neurons minimally altered energy balance., Conclusions: Although early developmental deletion of LepRb from known populations of ARC neurons fails to substantially alter body weight, the minimal phenotype of mice lacking LepRb in Htr2c cells suggests that the phenotype that results from early developmental LepRb deficiency depends not simply upon the total number of leptin-responsive hypothalamic LepRb cells. Rather, specific populations of LepRb neurons must play particularly important roles in body energy homeostasis; these as yet unidentified LepRb cells likely reside in the DMH., (Copyright © 2018 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Leptin is a pleiotropic hormone produced by white adipose tissue that regulates appetite and many physiological functions, including the immune response to infection. Genetic leptin deficiency in humans and mice impairs host defenses against respiratory tract infections. Since leptin deficiency is associated with obesity and other metabolic abnormalities, we generated mice that lack the leptin receptor (LepRb) in cells of the myeloid linage (LysM-LepRb-KO) to evaluate its impact in lean metabolically normal mice in a murine model of pneumococcal pneumonia. We observed higher lung and spleen bacterial burdens in LysM-LepRb-KO mice following an intratracheal challenge with Streptococcus pneumoniae. Although numbers of leukocytes recovered from bronchoalveolar lavage fluid did not differ between groups, we did observe higher levels of pulmonary IL-13 and TNFα in LysM-LepRb-KO mice 48 h post infection. Phagocytosis and killing of ingested S. pneumoniae were also impaired in alveolar macrophages (AMs) from LysM-LepRb-KO mice in vitro and were associated with reduced LTB 4 and enhanced PGE 2 synthesis in vitro. Pretreatment of AMs with LTB 4 and the cyclooxygenase inhibitor, indomethacin, restored phagocytosis but not bacterial killing in vitro. These results confirm our previous observations in leptin-deficient ( ob/ob) and fasted mice and demonstrate that decreased leptin action, as opposed to metabolic irregularities associated with obesity or starvation, is responsible for the defective host defense against pneumococcal pneumonia. They also provide novel targets for therapeutic intervention in humans with bacterial pneumonia.

Leptin acts via its receptor (LepRb) to modulate gene expression in hypothalamic LepRb-expressing neurons, thereby controlling energy balance and glucose homeostasis. Despite the importance of the control of gene expression in hypothalamic LepRb neurons for leptin action, the transcriptional targets of LepRb signaling have remained undefined because LepRb cells contribute a small fraction to the aggregate transcriptome of the brain regions in which they reside. We thus employed translating ribosome affinity purification followed by RNA sequencing to isolate and analyze mRNA from the hypothalamic LepRb neurons of wild-type or leptin-deficient ( Lep ob/ob ) mice treated with vehicle or exogenous leptin. Although the expression of most of the genes encoding the neuropeptides commonly considered to represent the main targets of leptin action were altered only following chronic leptin deprivation, our analysis revealed other transcripts that were coordinately regulated by leptin under multiple treatment conditions. Among these, acute leptin treatment increased expression of the transcription factor Atf3 in LepRb neurons. Furthermore, ablation of Atf3 from LepRb neurons (Atf3 LepRb KO mice) decreased leptin efficacy and promoted positive energy balance in mice. Thus, this analysis revealed the gene targets of leptin action, including Atf3 , which represents a cellular mediator of leptin action., (© 2018 by the American Diabetes Association.)

The adipocyte-derived hormone leptin acts via its receptor (LepRb) on central nervous system neurons to communicate the repletion of long-term energy stores, to decrease food intake, and to promote energy expenditure. We generated mice that express Cre recombinase from the calcitonin receptor (Calcr) locus (Calcrcre mice) to study Calcr-expressing LepRb (LepRbCalcr) neurons, which reside predominantly in the arcuate nucleus (ARC). Calcrcre-mediated ablation of LepRb in LepRbCalcrknockout (KO) mice caused hyperphagic obesity. Because LepRb-mediated transcriptional control plays a crucial role in leptin action, we used translating ribosome affinity purification followed by RNA sequencing to define the transcriptome of hypothalamic Calcr neurons, along with its alteration in LepRbCalcrKO mice. We found that ARC LepRbCalcr cells include neuropeptide Y (NPY)/agouti-related peptide (AgRP)/γ-aminobutyric acid (GABA) ("NAG") cells as well as non-NAG cells that are distinct from pro-opiomelanocortin cells. Furthermore, although LepRbCalcrKO mice exhibited dysregulated expression of several genes involved in energy balance, neither the expression of Agrp and Npy nor the activity of NAG cells was altered in vivo. Thus, although direct leptin action via LepRbCalcr cells plays an important role in leptin action, our data also suggest that leptin indirectly, as well as directly, regulates these cells.

Growing evidence implicates neurons that project from the lateral parabrachial nucleus (LPBN) to the hypothalamic ventromedial nucleus (VMN) in a neurocircuit that drives counterregulatory responses to hypoglycemia, including increased glucagon secretion. Among LPBN neurons in this circuit is a subset that expresses cholecystokinin (LPBNCCK neurons) and is tonically inhibited by leptin. Because uncontrolled diabetes is associated with both leptin deficiency and hyperglucagonemia, and because intracerebroventricular (ICV) leptin administration reverses both hyperglycemia and hyperglucagonemia in this setting, we hypothesized that deficient leptin inhibition of LPBNCCK neurons drives activation of this LPBN→VMN circuit and thereby results in hyperglucagonemia. Here, we report that although bilateral microinjection of leptin into the LPBN does not ameliorate hyperglycemia in rats with streptozotocin-induced diabetes mellitus (STZ-DM), it does attenuate the associated hyperglucagonemia and ketosis. To determine if LPBN leptin signaling is required for the antidiabetic effect of ICV leptin in STZ-DM, we studied mice in which the leptin receptor was selectively deleted from LPBNCCK neurons. Our findings show that although leptin signaling in these neurons is not required for the potent antidiabetic effect of ICV leptin, it is required for leptin-mediated suppression of diabetic hyperglucagonemia. Taken together, these findings suggest that leptin-mediated effects in animals with uncontrolled diabetes occur through actions involving multiple brain areas, including the LPBN, where leptin acts specifically to inhibit glucagon secretion and associated ketosis.

Pro-opiomelanocortin (POMC) neurons function as key regulators of metabolism and physiology by releasing prohormone-derived neuropeptides with distinct biological activities. However, our understanding of early events in prohormone maturation in the ER remains incomplete. Highlighting the significance of this gap in knowledge, a single POMC cysteine-to-phenylalanine mutation at position 28 (POMC-C28F) is defective for ER processing and causes early onset obesity in a dominant-negative manner in humans through an unclear mechanism. Here, we report a pathologically important role of Sel1L-Hrd1, the protein complex of ER-associated degradation (ERAD), within POMC neurons. Mice with POMC neuron-specific Sel1L deficiency developed age-associated obesity due, at least in part, to the ER retention of POMC that led to hyperphagia. The Sel1L-Hrd1 complex targets a fraction of nascent POMC molecules for ubiquitination and proteasomal degradation, preventing accumulation of misfolded and aggregated POMC, thereby ensuring that another fraction of POMC can undergo normal posttranslational processing and trafficking for secretion. Moreover, we found that the disease-associated POMC-C28F mutant evades ERAD and becomes aggregated due to the presence of a highly reactive unpaired cysteine thiol at position 50. Thus, this study not only identifies ERAD as an important mechanism regulating POMC maturation within the ER, but also provides insights into the pathogenesis of monogenic obesity associated with defective prohormone folding.

Obesity represents the single most important risk factor for early disability and death in developed societies, and the incidence of obesity remains at staggering levels. CNS systems that modulate energy intake and expenditure in response to changes in body energy stores serve to maintain constant body adiposity; the adipocyte-derived hormone leptin and its receptor (LEPR) represent crucial regulators of these systems. As in the case of insulin resistance, a variety of mechanisms (including feedback inhibition, inflammation, gliosis and endoplasmic reticulum stress) have been proposed to interfere with leptin action and impede the systems that control body energy homeostasis to promote or maintain obesity, although the relative importance and contribution of each of these remain unclear. However, LEPR signalling may be increased (rather than impaired) in common obesity, suggesting that any obesity-associated defects in leptin action must result from lesions somewhere other than the initial LEPR signal. It is also possible that increased LEPR signalling could mediate some of the obesity-associated changes in hypothalamic function.

Estradiol feedback regulates gonadotropin-releasing hormone (GnRH) neurons and subsequent luteinizing hormone (LH) release. Estradiol acts via estrogen receptor α (ERα)-expressing afferents of GnRH neurons, including kisspeptin neurons in the anteroventral periventricular (AVPV) and arcuate nuclei, providing homeostatic feedback on episodic GnRH/LH release as well as positive feedback to control ovulation. Ionotropic glutamate receptors are important for estradiol feedback, but it is not known where they fit in the circuitry. Estradiol-negative feedback decreased glutamatergic transmission to AVPV and increased it to arcuate kisspeptin neurons; positive feedback had the opposite effect. Deletion of ERα in kisspeptin cells decreased glutamate transmission to AVPV neurons and markedly increased it to arcuate kisspeptin neurons, which also exhibited increased spontaneous firing rate. KERKO mice had increased LH pulse frequency, indicating loss of negative feedback. These observations indicate that ERα in kisspeptin cells is required for appropriate differential regulation of these neurons and neuroendocrine output by estradiol. SIGNIFICANCE STATEMENT The brain regulates fertility through gonadotropin-releasing hormone (GnRH) neurons. Ovarian estradiol regulates the pattern of GnRH (negative feedback) and initiates a surge of release that triggers ovulation (positive feedback). GnRH neurons do not express the estrogen receptor needed for feedback (estrogen receptor α [ERα]); kisspeptin neurons in the arcuate and anteroventral periventricular nuclei are postulated to mediate negative and positive feedback, respectively. Here we extend the network through which feedback is mediated by demonstrating that glutamatergic transmission to these kisspeptin populations is differentially regulated during the reproductive cycle and by estradiol. Electrophysiological and in vivo hormone profile experiments on kisspeptin-specific ERα knock-out mice demonstrate that ERα in kisspeptin cells is required for appropriate differential regulation of these neurons and for neuroendocrine output., (Copyright © 2018 the authors 0270-6474/18/381061-12$15.00/0.)

The innate immune kinase TBK1 initiates inflammatory responses to combat infectious pathogens by driving production of type I interferons. TBK1 also controls metabolic processes and promotes oncogene-induced cell proliferation and survival. Here, we demonstrate that TBK1 activates mTOR complex 1 (mTORC1) directly. In cultured cells, TBK1 associates with and activates mTORC1 through site-specific mTOR phosphorylation (on S2159) in response to certain growth factor receptors (i.e., EGF-receptor but not insulin receptor) and pathogen recognition receptors (PRRs) (i.e., TLR3; TLR4), revealing a stimulus-selective role for TBK1 in mTORC1 regulation. By studying cultured macrophages and those isolated from genome edited mTOR S2159A knock-in mice, we show that mTOR S2159 phosphorylation promotes mTORC1 signaling, IRF3 nuclear translocation, and IFN-β production. These data demonstrate a direct mechanistic link between TBK1 and mTORC1 function as well as physiologic significance of the TBK1-mTORC1 axis in control of innate immune function. These data unveil TBK1 as a direct mTORC1 activator and suggest unanticipated roles for mTORC1 downstream of TBK1 in control of innate immunity, tumorigenesis, and disorders linked to chronic inflammation., (© 2017 The Authors.)

Activation of JAK-STAT signaling has been implicated in the pathogenesis of diabetic kidney disease. An increased expression of JAK-STAT genes was found in kidney glomerular cells, including podocytes, in patients with early diabetic kidney disease. However, it is not known whether increased expression of JAK or STAT isoforms in glomerular cells can lead to worsening nephropathy in the setting of diabetes. Therefore, we overexpressed JAK2 mRNA specifically in glomerular podocytes of 129S6 mice to determine whether this change alone could worsen diabetic kidney disease. A 2-3 fold increase in glomerular JAK2 expression, an increase similar to that found in humans with early diabetic kidney disease, led to substantial and statistically significant increases in albuminuria, mesangial expansion, glomerulosclerosis, glomerular fibronectin accumulation, and glomerular basement membrane thickening, and a significant reduction in podocyte density in diabetic mice. Treatment with a specific JAK1/2 inhibitor for 2 weeks partly reversed the major phenotypic changes of diabetic kidney disease and specifically normalized expression of a number of downstream STAT3-dependent genes implicated in diabetic kidney disease progression. Thus, moderate increases in podocyte JAK2 expression at levels similar to those in patients with early diabetic kidney disease can lead directly to phenotypic and other alterations of progressive diabetic glomerulopathy. Hence, inhibition of these changes by treatment with a JAK1/2 inhibitor suggests that such treatment may help retard progression of early diabetic kidney disease in patients., (Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Adipocytes secrete the hormone leptin to signal the sufficiency of energy stores. Reductions in circulating leptin concentrations reflect a negative energy balance, which augments sympathetic nervous system (SNS) activation in response to metabolically demanding emergencies. This process ensures adequate glucose mobilization despite low energy stores. We report that leptin receptor-expressing neurons (LepRb neurons) in the periaqueductal gray (PAG), the largest population of LepRb neurons in the brain stem, mediate this process. Application of noxious stimuli, which often signal the need to mobilize glucose to support an appropriate response, activated PAG LepRb neurons, which project to and activate parabrachial nucleus (PBN) neurons that control SNS activation and glucose mobilization. Furthermore, activating PAG LepRb neurons increased SNS activity and blood glucose concentrations, while ablating LepRb in PAG neurons augmented glucose mobilization in response to noxious stimuli. Thus, decreased leptin action on PAG LepRb neurons augments the autonomic response to noxious stimuli, ensuring sufficient glucose mobilization during periods of acute demand in the face of diminished energy stores.

Obesity is primarily due to food intake in excess of the body's energetic requirements, intake that is not only associated with hunger but also the incentive value of food. The 5-hydroxytryptamine 2C receptor (5-HT 2C R) is a target for the treatment of human obesity. Mechanistically, 5-HT 2C Rs are positioned to influence both homeostatic feeding circuits within the hypothalamus and reward circuits within the ventral tegmental area (VTA). Here we investigated the role of 5-HT 2C Rs in incentive motivation using a mathematical model of progressive ratio (PR) responding in mice. We found that the 5-HT 2C R agonist lorcaserin significantly reduced both ad libitum chow intake and PR responding for chocolate pellets and increased c-fos expression in VTA 5-HT 2C R expressing γ-aminobutyric acid (GABA) neurons, but not 5-HT 2C R expressing dopamine (DA) neurons. We next adopted a chemogenetic approach using a 5-HT 2C R CRE line to clarify the function of subset of 5-HT 2C receptor expressing VTA neurons in the modulation of appetite and food-motivated behavior. Activation of VTA 5-HT 2C receptor expressing neurons significantly reduced ad libitum chow intake, operant responding for chocolate pellets, and the incentive value of food. In contrast, chemogenetic inhibition of VTA 5-HT 2C receptor expressing neurons had no effect on the feeding behavior. These results indicate that activation of the subpopulation of 5-HT 2C R neurons within the VTA is sufficient to significantly reduce homeostatic feeding and effort-based intake of palatable food, and that this subset has an inhibitory role in motivational processes. These findings are relevant to the treatment of obesity.

Background: Neural networks that regulate binge eating remain to be identified, and effective treatments for binge eating are limited., Methods: We combined neuroanatomic, pharmacologic, electrophysiological, Cre-lox, and chemogenetic approaches to investigate the functions of 5-hydroxytryptamine (5-HT) 2C receptor (5-HT 2C R) expressed by dopamine (DA) neurons in the regulation of binge-like eating behavior in mice., Results: We showed that 5-HT stimulates DA neural activity through a 5-HT 2C R-mediated mechanism, and activation of this midbrain 5-HT→DA neural circuit effectively inhibits binge-like eating behavior in mice. Notably, 5-HT medications, including fluoxetine, d-fenfluramine, and lorcaserin (a selective 5-HT 2C R agonist), act on 5-HT 2C Rs expressed by DA neurons to inhibit binge-like eating in mice., Conclusions: We identified the 5-HT 2C R population in DA neurons as one potential target for antibinge therapies, and provided preclinical evidence that 5-HT 2C R agonists could be used to treat binge eating., (Copyright © 2016 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)