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Deoxynivalenol (DON), a type B trichothecene mycotoxin contaminating grain, 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 with 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), GDNF family receptor α-like (GFRAL). Interestingly, this analysis showed that a cell surface receptor for DON, a 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 with WT littermates. Thus, GLP-1 signaling and GFRAL signaling and neurons are not required for DON-induced visceral illness or anorexia.

Human variants of the adapter protein SH2B1 are associated with severe childhood obesity, hyperphagia, and insulin resistance—phenotypes mimicked by mice lacking Sh2b1. SH2B1β and γ isoforms are expressed ubiquitously, whereas SH2B1α and δ isoforms are expressed primarily in the brain. Restoring SH2B1β driven by the neuron-specific enolase promoter largely reverses the metabolic phenotype of Sh2b1-null mice, suggesting crucial roles for neuronal SH2B1β in energy balance control. Here we test this hypothesis by using CRISPR/Cas9 gene editing to delete the β and γ isoforms from the neurons of mice (SH2B1βγ neuron-specific knockout [NKO] mice) or throughout the body (SH2B1βγ knockout [KO] mice). While parameters of energy balance were normal in both male and female SH2B1βγ NKO mice, food intake, body weight, and adiposity were increased in male (but not female) SH2B1βγ KO mice. Analysis of long-read single-cell RNA seq data from wild-type mouse brain revealed that neurons express almost exclusively the α and δ isoforms, whereas neuroglial cells express almost exclusively the β and γ isoforms. Our work suggests that neuronal SH2B1β and γ are not primary regulators of energy balance. Rather, non-neuronal SH2B1β and γ in combination with neuronal SH2B1α and δ suffice for body weight maintenance. While SH2B1β/γ and SH2B1α/δ share some functionality, SH2B1β/γ appears to play a larger role in promoting leanness.

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.

Several discrete groups of feeding-regulated neurons in thenucleus tractus solitarius(NTS) suppress food intake, including aversion-promoting neurons that expressCck(NTSCckcells) and distinctLepr- andCalcr-expressing neurons (NTSLeprand NTSCalcrcells, respectively) that suppress food intake without promoting aversion. To test synergies among these cell groups we manipulated multiple NTS cell populations simultaneously. We found that activating multiple sets of NTS neurons (e.g., NTSLeprplus NTSCalcr(NTSLC), or NTSLCplus NTSCck(NTSLCK)) suppressed feeding more robustly than activating single populations. While activating groups of cells that include NTSCckneurons promoted conditioned taste avoidance (CTA), NTSLCactivation produced no CTA despite abrogating feeding. Thus, the ability to promote CTA formation represents a dominant effect, but activating multiple non-aversive populations additively suppresses food intake without provoking aversion. Although silencing multiple NTS neuron groups augmented food intake and body weight more dramatically than silencing single populations, feeding activated many non-NTSLCKneurons and silencing NTSLCKneurons failed to blunt the weight loss response to vertical sleeve gastrectomy (VSG). Hence, while each of these NTS neuron populations plays crucial and additive roles in the control of energy balance, as-yet undefined cell types must make additional contributions to the control of feeding and the response to VSG.

Leptin exerts its biological actions by activating LepRb. LepRb signaling impairment and leptin resistance are believed to cause obesity. 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 pro-obesity 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, accompanied by decreased food intake and increased fat thermogenesis. Leptin stimulated hypothalamic Stat3 phosphorylation and weight loss to a significantly higher level in SlugΔLepRb than in Slugf/f mice even before their body weight divergence. Conversely, hypothalamic LepRb neuron-specific overexpression of Slug, mediated by AAV-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 LepRb promoter histone 3 lysine-27 methylations, repressive epigenetic marks, and increased LepRb mRNA levels in the hypothalamus. Collectively, these results unravel a previously-unrecognized hypothalamic neuronal Slug/epigenetic reprogramming/leptin resistance axis that promotes energy imbalance, obesity, and metabolic disease.

Calcitonin receptor (Calcr)-expressing neurons of the nucleus tractus solitarius (NTS; CalcrNTS cells) contribute to the long-term control of food intake and body weight. Here, we show that Prlh-expressing NTS (PrlhNTS) neurons represent a subset of CalcrNTS 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 PrlhNTS cells to hypothalamic feeding circuits, we determined the ability of PrlhNTS-mediated signals to overcome enforced activation of AgRP neurons. We found that PrlhNTS neuron activation and Prlh overexpression in PrlhNTS 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., Calcitonin receptor-expressing neurons of the nucleus tractus solitarius contribute to long-term control of food intake and body weight. The authors show that a subset of these cells expresses Prlh and that enhancing Prlh-mediated neurotransmission from the NTS dampens hypothalamically-driven hyperphagia and obesity in mice.

The purpose of the Zero to Five study was to compare automated office blood pressure (AOBP) readings obtained after either 0 or 5 minutes of antecedent rest in relation to the awake ambulatory blood pressure. AOBP is recommended in different jurisdictions following either a 0- or 5-minute rest. This was a prospective, randomized, 2 arm, trial with blinded outcomes, recruiting adult patients referred for ambulatory blood pressure monitoring (ABPM). Participants had an AOBP measurement performed according to clinical practice guidelines with an OMRON HEM-907XL set to measure after 0 or 5 minutes of rest. The primary outcome was the difference between the mean AOBP and mean awake ABPM in the 0-minute wait group versus that in the 5-minute wait group. The study enrolled 618 participants, mean age 57.1 years, 52% women. For the 0-minute rest group, the mean AOBP was 141.2/83.1 (17.1/12.1 mm Hg) and the awake ABPM was 141.3/83.8 (16.1/10.2 mm Hg), with difference −0.02/0.52 (17.4/11.4 mm Hg). For the 5-minute rest group, the mean AOBP was 138.2/81.7 (16.9/12.4 mm Hg) and the awake ABPM was 143.4/83.6 (17.3/10.3 mm Hg), with difference −5.16/−0.8 (18.6/11.6 mm Hg). The difference of differences in systolic blood pressure (AOBP-awake ABPM) for the 0 versus the 5-minute wait group was 5.1 mm Hg (95% CI, 2.3–8.0, P =0.005) with the 0-minute AOBP measurement closest to the awake ABPM. The Zero to Five study demonstrated that a wait time of 0 minute before an AOBP measurement was closer to the daytime ABPM result than a 5-minute wait before the AOBP. Registration: URL: https://www.clinicaltrials.gov ; Unique identifier: NCT03732924.

The present paper provides an update of previous recommendations on Home Blood Pressure Monitoring from the European Society of Hypertension (ESH) Working Group on Blood Pressure Monitoring and Cardiovascular Variability sequentially published in years 2000, 2008 and 2010. This update has taken into account new evidence in this field, including a recent statement by the American Heart association, as well as technological developments, which have occurred over the past 20 years. The present document has been developed by the same ESH Working Group with inputs from an international team of experts, and has been endorsed by the ESH. ispartof: JOURNAL OF HYPERTENSION vol:39 issue:9 pages:1742-1767 ispartof: location:Netherlands status: published

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 innate immune kinase TBK1 (TANK-binding kinase 1) responds to microbial-derived signals to initiate responses against viral and bacterial pathogens. More recent work implicates TBK1 in metabolism and tumorigenesis. The kinase mTOR (mechanistic target of rapamycin) integrates diverse environmental cues to control fundamental cellular processes. Our prior work demonstrated in cells that TBK1 phosphorylates mTOR (on S2159) to increase mTORC1 and mTORC2 catalytic activity and signaling. Here we investigate a role for TBK1-mTOR signaling in control of glucose metabolism in vivo. We find that diet induced obese (DIO) but not lean mice bearing a whole-body “TBK1 resistant” Mtor S2159A knockin allele (MtorA/A) display exacerbated hyperglycemia and systemic insulin resistance with no change in energy balance. Mechanistically, Mtor S2159A knockin in DIO mice reduces mTORC1 and mTORC2 signaling in response to insulin and innate immune agonists, reduces anti-inflammatory gene expression in adipose tissue, and blunts anti-inflammatory macrophage M2 polarization, phenotypes shared by mice with tissue-specific inactivation of TBK1 or mTOR complexes. Tissues from DIO mice display elevated TBK1 activity and mTOR S2159 phosphorylation relative to lean mice. We propose a model whereby obesity-associated signals increase TBK1 activity and mTOR phosphorylation, which boost mTORC1 and mTORC2 signaling in parallel to the insulin pathway, thereby attenuating insulin resistance to improve glycemic control during diet-induced obesity.

The central nervous system (CNS) coordinates metabolic target organ function to optimize blood glucose levels, while dysregulation of this system leads to impaired glycemia. Attempts to improve glucose control through CNS targeting are hindered by a incomplete understanding of the specific neuronal populations involved and how these cells coordinate peripheral metabolic target organ function. We found that cholecystokinin b receptor-expressing neurons of the ventromedial nucleus of the hypothalamus (VMN; VMNCCKBR neurons) mediate glucose mobilization during counter-regulatory responses to hypoglycemia and regulate glucose levels in normal and diabetic mice. Silencing VMNCCKBR neurons with tetanus toxin (CCKBRTT) attenuates hyperglycemia in ob/ob mice and decreases baseline blood glucose by suppressing hepatic gluconeogenesis despite decreased insulin and elevated glucagon. CCKBRTT mice have elevated hepatic gluconeogenic enzyme expression and normal glycemic responses to exogenous gluconeogenic substrates, suggesting that silencing VMNCCKBR neurons decreases blood glucose through impaired gluconeogenic substrate mobilization. To determine which gluconeogenic substrates contribute to VMNCCKBR-neuron mediated glucose production, we expressed channel rhodopsin in VMNCCKBR neurons to optogenetically activate VMNCCKBR neurons in freely moving mice. In addition to elevating plasma glucose, optogenetic activation resulted in a β3-adrenergic receptor-dependent increase in plasma ketones and glycerol, suggesting that VMNCCKBR neurons increase lipolysis though SNS innervation of white adipose tissue (WAT) . Consistent with this observation, CCKBRTT mice exhibited decreased fasting plasma glycerol and in vivo lipolysis, while β3-agonist treatment elevated these appropriately. Hence, VMNCCKBR neuron-mediated SNS outflow to WAT promotes lipolysis to provide gluconeogenic substrates and support baseline blood glucose in mice. Disclosure A.Affinati: Other Relationship; Novo Nordisk A/S. J.Flak: Research Support; Eli Lilly and Company. N.Kodur: None. A.Hashsham: None. A.J.Tomlinson: None. N.Bozadjieva kramer: None. C.Li: Employee; Novo Nordisk. M.G.Myers: Advisory Panel; Regeneron Pharmaceuticals Inc., Research Support; AstraZeneca, Novo Nordisk. Funding National Institutes of Health (F32DK122660)

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.

Summary 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., Graphical Abstract, Highlights • Dynamic intestinal crypt remodeling induced by short-term Notch inhibition • Intestinal stem cells are maintained but exhibit a transient loss of function • Paneth cells are rapidly lost by apoptotic cell death • A regenerative response replenishes Paneth cells to repair the crypt, Samuelson and colleagues demonstrate that intestinal stem cell (ISC) niche disruption by short-term Notch inhibition induces dynamic crypt cell remodeling. Surprisingly, Paneth cells were rapidly lost by apoptosis. Although ISCs were maintained, they exhibited a transient loss of function. A subsequent crypt regenerative response was observed with activation of multiple stem/progenitor cells to replenish the Paneth cell pool and return to homeostasis.

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 VMN(CCKBR) neurons increased blood glucose. Furthermore, although silencing the broader VMN decreased energy expenditure and promoted weight gain without altering blood glucose levels, silencing VMN(CCKBR) neurons decreased hIepatic glucose production, insulin-independently decreasing blood glucose without altering energy balance. Silencing VMN(CCKBR) neurons also impaired the counterregulatory response to insulin-induced hypoglycemia and glucoprivation and replicated hypoglycemia-associated autonomic failure. Hence, VMN(CCKBR) 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.

A recent report from the American Heart Association stated that automated office blood pressure (AOBP) is preferred for evaluating office blood pressure (BP) because it is more accurate and devoid of white coat effect, which is mostly caused by higher systolic BP readings. However, AOBP has been criticized for being too variable to be used for identifying patients with possible hypertension. We, therefore, compared AOBP with home BP monitoring (HBPM) with respect to variability as determined by their relationship with the gold standard for determining BP status, awake ambulatory BP (ABP). The main focus was on systolic BP. Data on AOBP, HBPM, and awake ABP were collected on 300 patients referred from the community for 24-hour ambulatory BP monitoring. The SD of the difference between mean systolic awake ABP (136.4±11.5) and AOBP (131.2±15.7) was 13.6 mm Hg compared with 13.1 for the SD of the difference ( P =0.52) between the systolic awake ABP and the HBPM (136.7±16.1). Coefficients of correlation were slightly lower for systolic awake ABP versus AOBP ( r =0.54) compared with HBPM ( r =0.60). Coefficients of variation for AOBP (12.0%) and HBPM (11.8%) and variances between AOBP and HBPM were similar. Of the 139 patients with hypertension as defined by a manual office systolic BP ≥140 mm Hg, variability in BP readings as determined by the SDs of the mean difference versus awake ABP were similar ( P =0.56) for AOBP (14.6) and HBPM (13.9). Overall, both systolic AOBP and HBPM exhibited a similar degree of variability as assessed by the various methods.

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., Leptin regulates the sympathetic nervous system, energy expenditure and body weight through incompletely understood mechanisms. Here the authors report that Sh2b1 in leptin receptor positive neurons mediates the ability of leptin to stimulate sympathetic nerve activity in brown adipose tissue, body temperature and cold tolerance.

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 Tyr985, 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., Highlights • We generated 5 novel mouse lines containing COOH-terminal LepRb truncations. • LepRb residues 922–960 are required to control neuronal activity and metabolism. • LepRb residues 1014–1053 represent a novel inhibitory region. • LepRb mediates multiple phosphotyrosine-independent signals that impact physiology.

The adipose-derived hormone leptin acts via its receptor (LepRb) in the brain to control energy balance. A previously 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-sequencing of enriched mouse hypothalamic LepRb cells, as well as with total hypothalamic cells from multiple mammalian species. In addition to identifying known LepRb neuron types, this analysis identified several previously unrecognized populations of hypothalamic LepRb neurons. Many of these populations display strong conservation across species, including GABAergic Glp1r-expressing LepRb (LepRbGlp1r) neurons that express more Lepr and respond more robustly to exogenous leptin than other LepRb populations. Ablating LepRb from these cells provoked hyperphagic obesity without impairing energy expenditure. Conversely, reactivating LepRb in Glp1r-expressing cells decreased food intake and body weight in otherwise LepRb-null mice. Furthermore, LepRb reactivation in GABA neurons improved energy balance in LepRb-null mice, and this effect required the expression of LepRb in GABAergic Glp1r-expressing neurons. Thus, the conserved GABAergic LepRbGlp1r neuron population plays crucial roles in the control of food intake and body weight by leptin.

Summary Here, we present an in-depth protocol for extracting ribosome-bound mRNAs in low-abundance cells of hypothalamic nuclei. mRNAs are extracted from the micropunched tissue using refined translating ribosome affinity purification. Isolated RNAs can be used for sequencing or transcript quantification. This protocol enables the identification of actively translated mRNAs in varying physiological states and can be modified for use in any neuronal subpopulation labeled with a ribo-tag. We use leptin receptor-expressing neurons as an example to illustrate the protocol. For complete details on the use and execution of this protocol, please refer to Han et al. (2020)., Graphical abstract, Highlights • A protocol to isolate actively transcribed RNAs from low-abundance cells in mouse brain • Isolation of translating RNAs using eGFP-labeled L10a ribosomal protein • A procedure to harvest small brain nuclei avoiding RNA degradation, Here, we present an in-depth protocol for extracting ribosome-bound mRNAs in low-abundance cells of hypothalamic nuclei. mRNAs are extracted from the micropunched tissue using refined translating ribosome affinity purification. Isolated RNAs can be used for sequencing or transcript quantification. This protocol enables the identification of actively translated mRNAs in varying physiological states and can be modified for use in any neuronal subpopulation labeled with a ribo-tag. We use leptin receptor-expressing neurons as an example to illustrate the protocol.

In the last three decades protocols for the validation of blood pressure measuring devices have been developed by the US Association for the Advancement of Medical Instrumentation, the British Hypertension Society, the German Hypertension League, the European Society of Hypertension Working Group on blood pressure Monitoring and the International Organization for Standardization. The European Society of Hypertension International Protocol required much smaller sample size than the other protocols, aiming to reduce the time, resources and cost of validation studies and thereby increase the number of validated devices. Given its specifications, the European Society of Hypertension International Protocol was adequate for 'high- and low-accuracy' devices, yet assessment of 'moderate accuracy' devices had high uncertainty with resultant high rate of device failure. Thus, devices validated using the European Society of Hypertension International Protocol should be considered to be as accurate as those validated with the previous Association for the Advancement of Medical Instrumentation or British Hypertension Society protocols. However, the European Society of Hypertension International Protocol did not allow subgroup evaluation (arm sizes, special populations, etc). The mission of the European Society of Hypertension International Protocol to promote the concept of validation has been well achieved, as almost double studies have been published using it than all the other protocols together. However, the maintenance of different validation protocols is confusing and therefore experts from the Association for the Advancement of Medical Instrumentation, European Society of Hypertension International Protocol and International Organization for Standardization have now developed the AAMI/ESH/ISO Universal Standard (ISO 81060-2:2018) as the recommended 21st-century procedure for worldwide application. The European Society of Hypertension Working Group has published a practical guide for using the Universal Standard. It is in the interests of all scientific bodies to propagate the Universal Standard and ensure its wide implementation.

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. Activation of tissue-resident myeloid cells in the brain, known as microglia, is thought to drive obesity-associated hypothalamic dysfunction. The authors of this Perspective present a more nuanced view of microglia, echoing lessons learned from the field of adipose macrophage biology: instead of simply responding to diet-induced damage, microglia are proposed to act as nutrient and environmental sensors that regulate hypothalamic physiology, a role that, if hijacked by chronic overnutrition, can produce disease.

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.

The ventromedial hypothalamic nucleus (VMH) controls diverse behaviors and physiologic functions, suggesting the existence of multiple VMH neural subtypes with distinct functions. Combing translating ribosome affinity purification with RNA-sequencing (TRAP-seq) data with single-nucleus RNA-sequencing (snRNA-seq) data, we identified 24 mouse VMH neuron clusters. Further analysis, including snRNA-seq data from macaque tissue, defined a more tractable VMH parceling scheme consisting of six major genetically and anatomically differentiated VMH neuron classes with good cross-species conservation. In addition to two major ventrolateral classes, we identified three distinct classes of dorsomedial VMH neurons. Consistent with previously suggested unique roles for leptin receptor (Lepr)-expressing VMH neurons, Lepr expression marked a single dorsomedial class. We also identified a class of glutamatergic VMH neurons that resides in the tuberal region, anterolateral to the neuroanatomical core of the VMH. This atlas of conserved VMH neuron populations provides an unbiased starting point for the analysis of VMH circuitry and function.

The ventromedial hypothalamic nucleus (VMH) controls diverse behaviors and physiologic functions, suggesting the existence of multiple VMH neural subtypes with distinct functions. Combing Translating Ribosome Affinity Purification with RNA sequencing (TRAP-seq) data with snRNA-seq data, we identified 24 mouse VMH neuron clusters. Further analysis, including snRNA-seq data from macaque tissue, defined a more tractable VMH parceling scheme consisting of 6 major genetically- and anatomically-differentiated VMH neuron classes with good cross-species conservation. In addition to two major ventrolateral classes, we identified three distinct classes of dorsomedial VMH neurons. Consistent with previously-suggested unique roles for leptin receptor (Lepr)-expressing VMH neurons, Lepr expression marked a single dorsomedial class. We also identified a class of glutamatergic VMH neurons that resides in the tuberal region, anterolateral to the neuroanatomical core of the VMH. This atlas of conserved VMH neuron populations provides an unbiased starting point for the analysis of VMH circuitry and function.

The paraventricular nucleus of the hypothalamus (PVH) is a heterogeneous collection of neurons that play important roles in modulating feeding and energy expenditure. Abnormal development or ablation of the PVH results in hyperphagic obesity and defects in energy expenditure whereas selective activation of defined PVH neuronal populations can suppress feeding and may promote energy expenditure. Here, we characterize the contribution of calcitonin receptor–expressing PVH neurons (CalcRPVH) to energy balance control. We used Cre-dependent viral tools delivered stereotaxically to the PVH of CalcR2Acre mice to activate, silence, and trace CalcRPVH neurons and determine their contribution to body weight regulation. Immunohistochemistry of fluorescently-labeled CalcRPVH neurons demonstrates that CalcRPVH neurons are largely distinct from several PVH neuronal populations involved in energy homeostasis; these neurons project to regions of the hindbrain that are implicated in energy balance control, including the nucleus of the solitary tract and the parabrachial nucleus. Acute activation of CalcRPVH neurons suppresses feeding without appreciably augmenting energy expenditure, whereas their silencing leads to obesity that may be due in part due to loss of PVH melanocortin-4 receptor signaling. These data show that CalcRPVH neurons are an essential component of energy balance neurocircuitry and their function is important for body weight maintenance. A thorough understanding of the mechanisms by which CalcRPVH neurons modulate energy balance might identify novel therapeutic targets for the treatment and prevention of obesity.

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.

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.

While Cre-dependent viral systems permit the manipulation of many neuron types, some cell populations cannot be targeted by a single DNA recombinase. Although the combined use of Flp and Cre recombinases can overcome this limitation, insufficient recombinase activity can reduce the efficacy of existing Cre+Flp-dependent viral systems. We developed a sensitive dual recombinase-activated viral approach: tTA-driven Recombinase-Guided Intersectional Targeting (tTARGIT) adeno-associated viruses (AAVs). tTARGIT AAVs utilize a Flp-dependent tetracycline transactivator (tTA) ‘Driver’ AAV and a tetracycline response element-driven, Cre-dependent ‘Payload’ AAV to express the transgene of interest. We employed this system in Slc17a6FlpO;LeprCre mice to manipulate LepRb neurons of the ventromedial hypothalamus (VMH; LepRbVMH neurons) while omitting neighboring LepRb populations. We defined the circuitry of LepRbVMH neurons and roles for these cells in the control of food intake and energy expenditure. Thus, the tTARGIT system mediates robust recombinase-sensitive transgene expression, permitting the precise manipulation of previously intractable neural populations., eLife digest The brain contains hundreds of types of neurons, which differ in size, shape and behavior. But neuroscientists often wish to study individual neuronal types in isolation. They are able to do this with the aid of a toolkit made up of two parts: viral vectors and genetically modified mice. Viral vectors are viruses that have been modified so that they are no longer harmful and can instead be used to introduce genetic material into cells on demand. To create a viral vector, the virus’ own genetic material is replaced with a ‘cargo’ gene, such as the gene for a fluorescent protein. The virus is then introduced into a new host such as a mouse. Importantly, the virus only produces the protein encoded by its ‘cargo’ gene if it is inside a cell that also contains one of two specific enzymes. These enzymes are called Cre and Flp. This is where the second part of the toolkit comes in. Mice can be genetically engineered to produce either Cre or Flp exclusively in specific cell types. By introducing a viral vector into mice that produce either Cre or Flp only in one particular type of neuron, researchers can limit the activity of the cargo gene to that neuronal type. But sometimes even this approach is not selective enough. Researchers may wish to limit the activity of the cargo gene to a subpopulation of cells that produce Cre or Flp. Or they may wish to target only Cre- or Flp-producing cells in a small area of the brain, while leaving cells in neighboring areas unaffected. Sabatini et al. have now overcome this limitation by developing and testing a new set of viral vectors that are active only in neurons that produce both Cre and Flp. The vectors are called tTARGIT AAVs and allow researchers to target cells more precisely than was possible with the previous version of the toolkit. Sabatini et al. show tTARGIT AAVs in action by using them to identify a group of neurons that control how much energy mice use and how much food they eat. As well as applying the vectors to their own research on obesity, Sabatini et al. have also made them freely available for other researchers to use in their own projects.