A distinct hypothalamus-to-β cell circuit modulates insulin secretion

Abstract

The central nervous system has long been thought to regulate insulin secretion, an essential process in the maintenance of blood glucose levels. However, the anatomical and functional connections between the brain and insulin-producing pancreatic β cells remain undefined. Here, we describe a functional transneuronal circuit connecting the hypothalamus to β cells in mice. This circuit originates from a subpopulation of oxytocin neurons in the paraventricular hypothalamic nucleus (PVN^OXT), and it reaches the islets of the endocrine pancreas via the sympathetic autonomic branch to innervate β cells. Stimulation of PVN^OXT neurons rapidly suppresses insulin secretion and causes hyperglycemia. Conversely, silencing of these neurons elevates insulin levels by dysregulating neuronal signaling and secretory pathways in β cells and induces hypoglycemia. PVN^OXT neuronal activity is triggered by glucoprivation. Our findings reveal that a subset of PVN^OXT neurons form functional multisynaptic circuits with β cells in mice to regulate insulin secretion, and their function is necessary for the β cell response to hypoglycemia.

Keywords: central nervous system; glucoprivation; hypoglycemia; insulin secretion; oxytocin neurons; pancreatic β cells; paraventricular hypothalamic nucleus; pseudorabies tracing; sympathetic innervation; transneuronal circuit.

Figure 1.. Infection of pancreatic islet β-cells with Ba2017 PRV

(A) Structure of Cre-dependent Ba2017 pseudorabies virus. In the presence of Cre recombinase, the floxed sequence is inversed resulting in expression of EGFP and thymidine kinase (TK), a viral protein that promotes efficient replication. (B) Schematic representation of Ba2017 delivery in the pancreas of Ins1-Cre mice. The recombination leads to activation of the virus (proliferation and retrograde transfer) specifically in insulin-expressing β-cells. The “active” form of Ba2017 retrogradely ascends neuronal afferents all the way to the central nervous system. The localization of the virus can be visualized by EGFP fluorescence. (C-E) Representative images of β-cell-specific (insulin antibody staining, magenta) localization of Ba2017 (EGFP fluorescence, green) in Ins1-Cre mice at 24, 48 and 72h after administration (n = 2 mice per condition, 5-10 images per mouse). (F) Representative images of β-cell-specific (insulin antibody staining, magenta) localization of Ba2017 (EGFP fluorescence, green) in C57B16 mice at 72h after Ba2017 administration (n = 2 mice, 5 images per mouse). All sections were counterstained with nuclear marker DAPI (blue). (Scale bar = 50μm)

Figure 2.. EGFP signal in the central nervous system 72h after Ba2017 administration.

(A) Representative images of EGFP fluorescent labeling (green) showing the localization of Ba2017 in autonomic preganglionic neurons within the intermediolateral nucleus of the spinal cord (IML) (T9-12) (left) and in preautonomic neurons within the paraventricular nucleus of the hypothalamus (PVN) (right). All sections were counterstained with nuclear marker DAPI (blue) and the inset is a higher magnification of the boxed area (n = 4, 4-8 images per region per mouse). (Scale bar = 200 μm, IML large; 50 μm IML higher magnification inset; 1 mm PVN large; 100 μm PVN higher magnification inset) (B-I) Representative images of EGFP (green) labeled cells in various brain regions (n = 4, 4-8 images per region per mouse). Dashed areas represent the regions of interest in green identified in the brain section micrographs above each image. (Scale bar = 200 μm) LH= Lateral hypothalamus, SubC= Subcoereleus nucleus, VMH= Ventromedial hypothalamic nucleus, ARC= Arcuate nucleus, DMH= Dorsomedial hypothalamic nucleus, CeA= Central nucleus of the amygdala, PAG= Periaqueductal grey, PBN= Parabrachial nucleus, DMV= Dorsal motor nucleus of the vagus, 3V= 3^rd Ventricle, Aq= Aqueduct.

Figure 3.. PVN^OXT neurons communicate with β-cells and suppress insulin secretion

(A) (left) Representative image of β-cell-projecting PVN neurons expressing EGFP (green) co-labeled with oxytocin-NP (magenta) 72 hours after Ba2017 administration White arrows indicate double-labeled cells. Dashed box in the upper left panel represents the area of the magnified image shown in the right panel, (right) Quantification of EGFP⁺ and EGFP⁺/OXT⁺ neurons in the PVN of Ins1-Cre mice 72 hours after Ba2017 administration (n = 4, 4 images per mouse). graph shows mean ± sem. (Scale bar = 50 μm) (B) Schematic representation of DREADD virus injection site with representative image of viral mCherry expression in PVN^OXT neurons. (C) Schematic representation of mCherry control virus injection site with representative image of viral mCherry expression in PVN^OXT neurons. (D) Plasma insulin levels 10 minutes after administration of CNO and/or glucose in male (n = 7 mice) and female (n = 6 mice) PVN^OXT:hM3Dq mice. (E) Grouped % of basal changes of basal plasma insulin levels. All graphs show mean ± sem. **P < 0.01; ratio paired Student’s t-test. (F) Plasma insulin levels 10 minutes after administration of CNO and/or glucose in male (n = 3) or female (n = 5) PVN^OXT:mCherry mice. (G) Grouped % of basal changes of basal plasma insulin levels. All graphs show mean ±sem. **P <0.01; ratio paired Student’s t-test. (H) Representative EM images of β-cells in islets within pancreas sections from PVN^OXT:mCherry (left; n = 4, 25-40 images per mouse) and PVN^OXT:hM3Dq mice (right; n = 4, 25-40 images per mouse) 10 min after co-administration of glucose and CNO. Docked granules: located within 0.2 μm of the plasma membrane. Cyan lines indicate the cell membrane and yellow arrows point to the docked granules. (I) Quantification of number of docked insulin granules per μm of plasma membrane. (n = 4 mice per group). Graph shows mean ± sem. **P < 0.01; two-tailed, unpaired Student’s t-test. (J-K) Glycemic level changes (J) during GTT with or without chemogenetic stimulation of PVN^OXT neurons with CNO (shaded areas represent error bars). Baseline glucose at time of injection (0 min): 89.75 ± 8.797 and 81.50 ± 3.571 respectively, and, (K) Glycemic change area under curve (left) and Δ peak glycemia (right) during GTT after PVN^OXT stimulation via CNO. (n = 8 mice) *P < 0.05, **P < 0.01; paired Student’s t-test.

Figure 4.. PVN^OXT neurons that project to the spinal cord (spPVN^OXT) segments 9-13 suppress insulin secretion.

(A) Schematic representation of retrograde DREADD virus injection in the spinal cord of Oxt-ires-Cxe mice. (B) Representative image of viral mCherry expression (magenta) in preautonomic PVN^OXT neurons (green) (n=9, 1-6 images per mouse). (Scale bar = 200 μm). (C) Plasma insulin levels 10 minutes after administration of CNO and/or glucose in mice expressing the hM3Dq receptor in spPVN^OXT neurons (left males, n = 4; right females, n = 5). (D) Grouped % of basal changes of basal plasma insulin levels. All graphs show mean ± sem. *P < 0.05; ratio paired Student’s t-test.

Figure 5.. PVN^OXT neuron silencing dysregulates insulin secretory apparatus

(A) Schematic representation of YFP and TeNT virus injection site with representative images of viral YFP (n=7, 4 images per mouse) or EGFP (n=7, 4 images per mouse) expression in PVN^OXT neurons, respectively. (B) 24h-fasted and fed plasma insulin (left) and blood glucose (right) levels in PVN^OXT:YFP (n = 7) and PVN^OXT:TeNT (n = 7) mice. Graph shows mean ± sem. *P < 0.05, ***P < 0.001, ns: non-significant; two-tailed, unpaired Student’s t-test. (C) KEGG pathways significantly altered upon PVN^OXT silencing in islets from 24h-fasted mice (n = 4 per group). (D) Heat map of expression patterns of significantly down- or upregulated genes implicated in granule docking (n = 4 per group). (E) Fold change in mRNA levels of genes implicated in regulation of insulin secretion (n = 4 per group). (F) Map of gene products involved in insulin secretion significantly altered in PVN^OXT:TeNT mice (affected genes highlighted in blue; unaffected genes in grey) (n = 4 per group). All data from the RNASeq were selected with a cutoff at *P < 0.05.

Figure 6.. PVN^OXT neurons respond to glucoprivation.

(A) Representative images of PVN sections showing c-fos (green) and oxytocin-neurophysin (magenta) immunostaining after i.p. injection of saline (top; n=4, 4 images per mouse) or 2-DG (400 mg/kg; bottom; n=5, 4 images per mouse). Dashed box in the left panels represents the area of the magnified image shown in the right panels. White arrows indicate double-labeled cells. (B) Quantification of the number cells with double c-fos and OXT labeling in the PVN after saline (n = 4) or 2-DG injection (n = 5) (top). Quantification of the number of cells positive for c-fos staining in the entire PVN after saline (n = 4) or 2-DG injection (n = 5) (bottom). Graphs show mean ± sem. ***P < 0.001; two-tailed, unpaired Student’s t-test (C) Schematic representation and representative image of AAV1-CAG-FLEX-GCaMP6s-WPRE-SV40 virus injection site and fiber implantation, shown as a dashed region (n=8, 4 images per mouse). (D and E) Plot of calcium signals from PVN^OXT neurons (D) and quantification of fluorescence changes in 15-minute increments (E) after i.p. injection of saline or 2-DG (200 mg/kg). (n = 8) Graph shows mean ± sem. **P < 0.01 two-tailed, paired Student’s t-test (F) 1h food intake after the end of GCaMP recording in mice injected with saline or 2-DG (200 mg/kg). (n = 8) ***P <0.001 two-tailed, paired Student’s t-test