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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)

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.

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.

Between embryonic days 10.5 and 14.5, active proliferation drives rapid elongation of the murine midgut epithelial tube. Within this pseudostratified epithelium, nuclei synthesize DNA near the basal surface and move apically to divide. After mitosis, the majority of daughter cells extend a long, basally oriented filopodial protrusion, building a de novo path along which their nuclei can return to the basal side. WNT5A, which is secreted by surrounding mesenchymal cells, acts as a guidance cue to orchestrate this epithelial pathfinding behavior, but how this signal is received by epithelial cells is unknown. Here, we have investigated two known WNT5A receptors: ROR2 and RYK. We found that epithelial ROR2 is dispensable for midgut elongation. However, loss of Ryk phenocopies the Wnt5a(−/−) phenotype, perturbing post-mitotic pathfinding and leading to apoptosis. These studies reveal that the ligand-receptor pair WNT5A-RYK acts as a navigation system to instruct filopodial pathfinding, a process that is crucial for continuous cell cycling to fuel rapid midgut elongation.