OBJECTIVES:Hypothalamic Fatty Acid Synthase (FASN) plays a critical role in regulating energy balance by influencing food intake and body weight. This study aimed to investigate the neuronal mechanisms by which FASN impacts metabolism, focusing on its role in Pro-Opiomelanocortin (POMC) neurons. METHODS:We used transgenic mouse models with pre- or postnatal deletion of FASN specifically in POMC neurons in male mice. We evaluated changes in adiposity, glucose metabolism and metabolic parameters including food intake, energy expenditure and substrate utilization using metabolic chambers. Changes in neuronal activity were assessed using electrophysiology and further validated by optogenetic stimulation of POMC neurons. Additionally, the role of adrenergic signaling was examined using pharmacological approaches and gene expression analyses. RESULTS:FASN deletion in POMC neurons reduced food intake, decreased adiposity, and altered glucose metabolism. FASN-deficient POMC neurons exhibited increased baseline activity. The developmental stage of FASN deletion influenced its effects on energy expenditure and body weight regulation. Additionally, FASN in POMC neurons was found to be essential for maintaining glucose homeostasis and insulin release via adrenergic signaling. CONCLUSIONS:FASN in POMC neurons plays an age- and neuron-specific role in regulating feeding, energy expenditure, and glucose homeostasis through mechanisms including the sympathetic nervous system. These findings highlight FASN as a potential therapeutic target for metabolic diseases by improving energy expenditure and insulinemia. Given the developmental programming of metabolic outcomes, interventions aimed at modulating FASN activity may have long-lasting benefits in managing metabolic diseases.

Genome-wide association studies (GWAS) in humans and livestock have identified genes associated with metabolic traits. However, the causality of many of these genes on metabolic homeostasis is largely unclear due to a lack of detailed functional analyses. Here we report ligand-dependent corepressor-like (LCoRL) as a metabolic regulator for body weight and glucose homeostasis. Although GWAS data show that LCoRL is strongly associated with body size, glucose homeostasis, and other metabolic traits in humans and livestock, functional investigations had not been performed. We generated Lcorl knockout mice (Lcorl(-/-)) and characterized the metabolic traits. We found that Lcorl(-/-) pups are born smaller than the wild-type (WT) littermates before reaching normal weight by 7 to 9 weeks of age. While aging, Lcorl(-/-) mice remain lean compared to WT mice, which is associated with a decrease in daily food intake. Glucose tolerance and insulin sensitivity are improved in Lcorl(-/-) mice. Mechanistically, this stunted growth is linked to a reduction of circulating levels of IGF-1. The expression of the genes downstream of GH signaling and the genes involved in glucose and lipid metabolism are altered in the liver of Lcorl(-/-) mice. Furthermore, Lcorl(-/-) mice are protected against a high-fat diet challenge and show reduced exercise capacity in an exercise stress test. Collectively, our results are congruent with many of the metabolic parameters linked to the Lcorl locus as reported in GWAS in humans and livestock.

OBJECTIVE:Metamorphosis is a transition from growth to reproduction, through which an animal adopts adult behavior and metabolism. Yet the neural mechanisms underlying the switch are unclear. Here we report that neuronal E93, a transcription factor essential for metamorphosis, regulates the adult metabolism, physiology, and behavior in Drosophila melanogaster.METHODS:To find new neuronal regulators of metabolism, we performed a targeted RNAi-based screen of 70 Drosophila orthologs of the mammalian genes enriched in ventromedial hypothalamus (VMH). Once E93 was identified from the screen, we characterized changes in physiology and behavior when neuronal expression of E93 is knocked down. To identify the neurons where E93 acts, we performed an additional screen targeting subsets of neurons or endocrine cells.RESULTS:E93 is required to control appetite, metabolism, exercise endurance, and circadian rhythms. The diverse phenotypes caused by pan-neuronal knockdown of E93, including obesity, exercise intolerance and circadian disruption, can all be phenocopied by knockdown of E93 specifically in either GABA or MIP neurons, suggesting these neurons are key sites of E93 action. Knockdown of the Ecdysone Receptor specifically in MIP neurons partially phenocopies the MIP neuron-specific knockdown of E93, suggesting the steroid signal coordinates adult metabolism via E93 and a neuropeptidergic signal. Finally, E93 expression in GABA and MIP neurons also serves as a key switch for the adaptation to adult behavior, as animals with reduced expression of E93 in the two subsets of neurons exhibit reduced reproductive activity.CONCLUSIONS:Our study reveals that E93 is a new monogenic factor essential for metabolic, physiological, and behavioral adaptation from larval behavior to adult behavior.

Abstract Disclosure: S. Gahlot: None. A. Caron: None. S. Lee: None. J.K. Elmquist: None. Pro-opiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus (ARH) are critical for the regulation of energy balance and glucose homeostasis. Pomc gene transcription is regulated by the Erythrocyte Transformation Specific (ETS)-domain transcription factors, including Etv1, Etv2, Etv4, and Etv5. Large-scale genome-wide association studies have identified ETV5 genetic variants linked with human obesity and body mass index. Histological studies in adult mouse pituitaries also show the co-localization of ETV5 with POMC and POMC-derived peptides: adrenocorticotropic hormone (ACTH) and alpha-melanocyte stimulating hormone (α-MSH). Furthermore, whole-body Etv5 knockout mice are lean, severely glucose intolerant and hypoinsulinaemic. While these findings suggest a potential role for Etv5 in the control of energy and glucose balance, the role of Etv5 remains unknown. This study aims to determine whether ETV5 binds to the Pomc promoter to alter its expression and regulate energy balance and glucose homeostasis. To assess whether ETV5 binds to the Pomc promoter, we performed chromatin immunoprecipitation with quantitative real-time PCR (ChIP-qPCR) in C57BL/6J mice. ChIP-qPCR determined that Etv5 directly binds to the Pomc promoter. We further performed luciferase reporter assay in mouse neuroblastoma N2a cell lysates to measure the Pomc promoter activity after Etv5 knockdown or overexpression. Luciferase assay revealed that Etv5 knockdown increased, while Etv5 overexpression reduced the Pomc promoter activity in N2a cells. Also, Etv5 overexpression blocked the leptin-induced POMC activation. Further, to determine the role of Etv5 in POMC neurons to regulate energy balance and glucose homeostasis, we used the Cre/loxP system to selectively delete Etv5 from POMC neurons both prenatally and postnatally. Mice were fed either a standard chow or a 60% high-fat diet (HFD), followed by serial body weight, food intake, body composition, metabolic assessment, glucose tolerance and insulin tolerance test measurements. We found that mice with a prenatal deletion of Etv5 in POMC neurons had reduced body weight, reduced lean mass, and were protected from HFD-induced insulin resistance. Similarly, postnatal deletion of Etv5 in POMC neurons resulted in reduced body weight, lean mass and fat mass, and increased insulin sensitivity and energy expenditure in mice of 8 weeks age. We also performed electrophysiological recordings in hypothalamic slices from these mice to assess POMC neuron excitability. We found that loss of Etv5 increased the activity and electrical excitability of POMC neurons. These findings highlight a previously unrecognized role of Etv5 in POMC neurons in the regulation of energy balance and glucose homeostasis. These results also point to the potential therapeutic applications for Etv5 against obesity. Presentation: Thursday, June 15, 2023

Sympathetic regulation of the cardiovascular system is required to maintain cardiac output and vascular tone in response to varied physiological stressors. This includes the release norepinepherine and epinephrine. Nor/epinephrine acts on nine, evolutionarily conserved, adrenergic, G-protein coupled receptors. Three alpha 1 ( Adra1 ), 3 alpha 2 ( Adra2 ), and 3 beta ( Adrb ) receptors. Understanding the distribution and cell-specific function of each of these receptors may lead to new targeted therapies for cardiovascular disease. To delineate the relative roles of each of the 9 adrenergic receptors, we are taking a genetic approach to study these receptors in mice; specifically, by using the Cre-loxP system to target the varied receptors in a tissue-specific manner. Unfortunately, there is a dearth of such tools to study these receptors. We therefore are in the process of generating three sets of tools for each of the 9 adrenergic receptors using the CRISPR-Cas9 method. 1. Adrenergic receptor floxed mice. 2. Adrenergic receptor reactivation (transcriptional blocking) mice and 3. Adrenergic receptor Cre mice. Thus far, we have generated Adra1a , Adra1b , Adra1d , Adra2a , Adrb2 , and Adrb3 floxed mice by inserting loxP sites flanking at least one exon near the 5’ end of the gene. The floxed mice were crossed to a germline-Cre (CMV-Cre), generating mice with whole-body loss of function alleles. Complementing the conditional loss of function alleles, we are also generating reactivation alleles by inserting a loxP-flanked transcriptional blocking cassette (TB) in each of the 9 endogenous adrenergic receptor genes. These TB alleles will allow us to reactivate endogenous expression of each adrenergic receptor in a Cre-dependent manner in an otherwise global knockout background. This model will allow us to identify tissues which are sufficient for the function of each adrenergic receptor. Finally, the ires-Cre models will allow for targeting of each adrenergic receptor-expressing cells with genetic reporters, and physiological studies using chemo and/or optogenetics. These new tools will assist in identifying the site of action and physiological function of various receptor subtypes and provide mechanistic insights into adrenergic receptor signaling.

Metamorphosis is a transition from growth to reproduction, through which an animal adopts adult behavior and metabolism. Yet the mechanisms underlying the switch is unclear. Here we report that neuronal E93 , a transcription factor essential for metamorphosis, regulates the adult metabolism and circadian rhythm in Drosophila melanogaster . When E93 is specifically knocked down in neurons, the flies become hyperphagic and obese with increased energy stores and disrupted circadian rhythms. A screen of Gal4 lines targeting subsets of neurons and endocrine cells identified neurons producing GABA and myoinhibitory peptide (MIP) as the main sites of E93 action. Knockdown of the ecdysone receptor specifically in MIP neurons partly phenocopies the MIP neuron-specific knockdown of E93 suggesting the steroid signal coordinates adult metabolism via E93 . The circadian disruption caused by neuronal knockdown of E93 is also observed when E93 is knockdown in GABA and MIP neurons. Based on these results we suggest that neuronal E93 is a key switch for metabolic transition representing an intersectional node between metabolism and circadian biology.

Environmental light cycles entrain circadian feeding behaviors in animals that produce rhythms in exposure to foodborne bacteria. Here, we show that the intestinal microbiota generates diurnal rhythms in innate immunity that synchronize with feeding rhythms to anticipate microbial exposure. Rhythmic expression of antimicrobial proteins was driven by daily rhythms in epithelial attachment by segmented filamentous bacteria (SFB), members of the mouse intestinal microbiota. Rhythmic SFB attachment was driven by the circadian clock through control of feeding rhythms. Mechanistically, rhythmic SFB attachment activated an immunological circuit involving group 3 innate lymphoid cells. This circuit triggered oscillations in epithelial STAT3 expression and activation that produced rhythmic antimicrobial protein expression and caused resistance to Salmonella Typhimurium infection to vary across the day-night cycle. Thus, host feeding rhythms synchronize with the microbiota to promote rhythms in intestinal innate immunity that anticipate exogenous microbial exposure.

Cannabinoid 1 receptor (CB1R) inverse agonists reduce body weight and improve several parameters of glucose homeostasis. However, these drugs have also been associated with deleterious side effects. CB1R expression is widespread in the brain and in peripheral tissues, but whether specific sites of expression can mediate the beneficial metabolic effects of CB1R drugs, while avoiding the untoward side effects, remains unclear. Evidence suggests inverse agonists may act on key sites within the central nervous system to improve metabolism. The ventromedial hypothalamus (VMH) is a critical node regulating energy balance and glucose homeostasis. To determine the contributions of CB1Rs expressed in VMH neurons in regulating metabolic homeostasis, we generated mice lacking CB1Rs in the VMH. We found that the deletion of CB1Rs in the VMH did not affect body weight in chow- and high-fat diet-fed male and female mice. We also found that deletion of CB1Rs in the VMH did not alter weight loss responses induced by the CB1R inverse agonist SR141716. However, we did find that CB1Rs of the VMH regulate parameters of glucose homeostasis independent of body weight in diet-induced obese male mice. NEW & NOTEWORTHY Cannabinoid 1 receptors (CB1Rs) regulate metabolic homeostasis, and CB1R inverse agonists reduce body weight and improve parameters of glucose metabolism. However, the cell populations expressing CB1Rs that regulate metabolic homeostasis remain unclear. CB1Rs are highly expressed in the ventromedial hypothalamic nucleus (VMH), which is a crucial node that regulates metabolism. With CRISPR/Cas9, we generated mice lacking CB1Rs specifically in VMH neurons and found that CB1Rs in VMH neurons are essential for the regulation of glucose metabolism independent of body weight regulation.

There has been a long-standing debate regarding the role of peripheral afferents in mediating rapid-onset anorexia among other responses elicited by peripheral inflammatory insults. Thus, the current study assessed the sufficiency of peripheral afferents expressing toll-like receptor 4 (TLR4) to the initiation of the anorexia caused by peripheral bacterial lipopolysaccharide (LPS). We generated a Tlr4 null (Tlr4(LoxTB)) mouse in which Tlr4 expression is globally disrupted by a loxP-flanked transcription blocking (TB) cassette. This novel mouse model allowed us to restore the endogenous TLR4 expression in specific cell types. Using Zp3-Cre and Na(v)1.8-Cre mice, we produced mice that express TLR4 in all cells (Tlr4(LoxTB) X Zp3-Cre) and in peripheral afferents (Tlr4(LoxTB) X Na(v)1.8-Cre), respectively. We validated the Tlr4(LoxTB) mice, which were phenotypically identical to previously reported global TLR4 knock-out mice. Contrary to our expectations, the administration of LPS did not cause rapid-onset anorexia in mice with Na(v)1.8-restricted TLR4. The later result prompted us to identify Tlr4-expressing vagal afferents using in situ hybridization (ISH). In vivo, we found that Tlr4 mRNA was primarily enriched in vagal Na(v)1.8 afferents located in the jugular ganglion that co-expressed calcitonin gene-related peptide (CGRP). In vitro, the application of LPS to cultured Na(v)1.8-restricted TLR4 afferents was sufficient to stimulate the release of CGRP. In summary, we demonstrated using a new mouse model that vagally-expressed TLR4 is selectively involved in stimulating the release of CGRP but not in causing anorexia.

Objective: Histaminergic neurons of the tuberomammillary nucleus (TMN) are wake-promoting and contribute to the regulation of energy homeostasis. Evidence indicates that melanocortin 4 receptors (MC4R) are expressed within the TMN. However, whether the melanocortin system influences the activity and function of TMN neurons expressing histidine decarboxylase (HDC), the enzyme required for histamine synthesis, remains undefined. Methods: We utilized Hdc-Cre mice in combination with whole-cell patch-clamp electrophysiology and in vivo chemogenetic techniques to determine whether HDC neurons receive metabolically relevant information via the melanocortin system. Results: We found that subsets of HDC-expressing neurons were excited by melanotan II (MTII), a non-selective melanocortin receptor agonist. Use of melanocortin receptor selective agonists (THIQ, [D-Trp8]- gamma-MSH) and inhibitors of synaptic transmission (TTX, CNQX, AP5) indicated that the effect was mediated specifically by MC4Rs and involved a glutamatergic dependent presynaptic mechanism. MTII enhanced evoked excitatory post-synaptic currents (EPSCs) originating from electrical stimulation of the perifornical lateral hypothalamic area (PeFLH), supportive of melanocortin effects on the glutamatergic PeFLH projection to the TMN. Finally, in vivo chemogenetic inhibition of HDC neurons strikingly enhanced the anorexigenic effects of intracerebroventricular administration of MTII, suggesting that MC4R activation of histaminergic neurons may restrain the anorexigenic effects of melanocortin system activation. Conclusions: These experiments identify a functional interaction between the melanocortin and histaminergic systems and suggest that HDC neurons act naturally to restrain the anorexigenic effect of melanocortin system activation. These findings may have implications for the control of arousal and metabolic homeostasis, especially in the context of obesity, in which both processes are subjected to alterations. (C) 2020 The Author(s). Published by Elsevier GmbH.

Objective: The sympathetic nervous system (SNS) is a key regulator of the metabolic and endocrine functions of adipose tissue. Increased SNS outflow promotes fat mobilization, stimulates non-shivering thermogenesis, promotes browning, and inhibits leptin production. Most of these effects are attributed to norepinephrine activation of the Gs-coupled beta adrenergic receptors located on the surface of the adipocytes. Evidence suggests that other adrenergic receptor subtypes, including the Gi-coupled alpha 2 adrenergic receptors might also mediate the SNS effects on adipose tissue. However, the impact of acute stimulation of adipocyte Gs and Gi has never been reported. Methods: We harness the power of chemogenetics to develop unique mouse models allowing the specific and spatiotemporal stimulation of adipose tissue Gi and Gs signaling. We evaluated the impact of chemogenetic stimulation of these pathways on glucose homeostasis, lipolysis, leptin production, and gene expression. Results: Stimulation of Gs signaling in adipocytes induced rapid and sustained hypoglycemia. These hypoglycemic effects were secondary to increased insulin release, likely consequent to increased lipolysis. Notably, we also observed differences in gene regulation and ex vivo lipolysis in different adipose depots. In contrast, acute stimulation of Gi signaling in adipose tissue did not affect glucose metabolism or lipolysis, but regulated leptin production. Conclusion: Our data highlight the significance of adipose Gs signaling in regulating systemic glucose homeostasis. We also found previously unappreciated heterogeneity across adipose depots following acute stimulation. Together, these results highlight the complex interactions of GPCR signaling in adipose tissue and demonstrate the usefulness of chemogenetic technology to better understand adipocyte function. (C) 2019 The Authors. Published by Elsevier GmbH.

Phosphoinositide 3-kinase (PI3K) signaling in hypothalamic neurons integrates peripheral metabolic cues, including leptin and insulin, to coordinate systemic glucose and energy homeostasis. PI3K is composed of different subunits, each of which has several unique isoforms. However, the role of the PI3K subunits and isoforms in the ventromedial hypothalamus (VMH), a prominent site for the regulation of glucose and energy homeostasis, is unclear. Here we investigated the role of subunit p110β in steroidogenic factor-1 (SF-1) neurons of the VMH in the regulation of metabolism. Our data demonstrate that the deletion of p110β in SF-1 neurons disrupts glucose metabolism, rendering the mice insulin resistant. In addition, the deletion of p110β in SF-1 neurons leads to the whitening of brown adipose tissues and increased susceptibility to diet-induced obesity due to blunted energy expenditure. These results highlight a critical role for p110β in the regulation of glucose and energy homeostasis via VMH neurons.

Sustained oliguria during fluid resuscitation represents a perplexing problem in patients undergoing therapy for septic acute kidney injury. Here, we tested whether lipopolysaccharide induces filtrate leakage from the proximal tubular lumen into the interstitium, thus disturbing the recovery of urine output during therapy, such as fluid resuscitation, aiming to restore the glomerular filtration rate. Intravital imaging of the tubular flow rate in the proximal tubules in mice showed that lipopolysaccharide did not change the inflow rate of proximal tubule filtrate, reflecting an unchanged glomerular filtration rate, but significantly reduced the outflow rate, resulting in oliguria. Lipopolysaccharide disrupted tight junctions in proximal tubules and induced both paracellular leakage of filtered molecules and interstitial accumulation of extracellular fluid. These changes were diminished by conditional knockout of Toll-like receptor 4 in the proximal tubules. Importantly, these conditional knockout mice showed increased sensitivity to fluid resuscitation and attenuated acute kidney injury. Thus, lipopolysaccharide induced paracellular leakage of filtrate into the interstitium via a Toll-like receptor 4-dependent mechanism in the proximal tubules of endotoxemic mice. Hence, this leakage might diminish the efficacy of fluid resuscitation aiming to maintain renal hemodynamics and glomerular filtration rate.

Leptin is critical for energy balance, glucose homeostasis, and for metabolic and neuroendocrine adaptations to starvation. A prevalent model predicts that leptin's actions are mediated through pro-opiomelanocortin (POMC) neurons that express leptin receptors (LEPRs). However, previous studies have used prenatal genetic manipulations, which may be subject to developmental compensation. Here, we tested the direct contribution of POMC neurons expressing LEPRs in regulating energy balance, glucose homeostasis and leptin secretion during fasting using a spatiotemporally controlled Lepr expression mouse model. We report a dissociation between leptin's effects on glucose homeostasis versus energy balance in POMC neurons. We show that these neurons are dispensable for regulating food intake, but are required for coordinating hepatic glucose production and for the fasting-induced fall in leptin levels, independent of changes in fat mass. We also identify a role for sympathetic nervous system regulation of the inhibitory adrenergic receptor (ADRA2A) in regulating leptin production. Collectively, our findings highlight a previously unrecognized role of POMC neurons in regulating leptin levels.

Obesity is associated with many complications, including type 2 diabetes and painful neuropathy. There is no cure or prevention for obesity-induced pain, and the neurobiology underlying the onset of the disease is still obscure. In this study, we observe that western diet (WD)-fed mice developed early allodynia with an increase of ER stress markers in the sensory neurons of the dorsal root ganglia (DRG). Using cell-specific approaches, we demonstrate that neuronal liver X receptor (LXR) activation delays ER stress and allodynia in WD-fed mice. Our findings suggest that lipid-binding nuclear receptors expressed in the sensory neurons of the DRG play a role in the onset of obesity-induced hypersensitivity. The LXR and lipid-sensor pathways represent a research avenue to identify targets to prevent debilitating complications affecting the peripheral nerve system in obesity.