key: cord-0261259-3jmcu83p
authors: Qiu, Jian; Stincic, Todd L.; Bosch, Martha A.; Connors, Ashley M.; Petrie, Stefanie Kaech; Rønnekleiv, Oline K.; Kelly, Martin J.
title: Insulin potentiates the synchronous firing of arcuate nucleus Kiss1 neurons that protects against diet-induced obesity
date: 2020-09-09
journal: bioRxiv
DOI: 10.1101/2020.09.09.289017
sha: ca31727149e52d2d6ffb5329731b72e73372f43b
doc_id: 261259
cord_uid: 3jmcu83p

Kisspeptin neurons in the hypothalamic arcuate nucleus (Kiss1ARH) co-express kisspeptin, neurokinin B, dynorphin and provide an episodic, excitatory drive to gonadotropin-releasing hormone (GnRH) neurons, which is critical for pubertal development and fertility. Previously, we showed that high frequency firing of Kiss1ARH neurons co-releases NKB and dynorphin onto neighboring Kiss1ARH neurons to generate a slow excitatory postsynaptic potential (EPSP) that entrains intermittent, synchronous firing of Kiss1ARH neurons (Qiu et al., 2016). Presently, we discovered that insulin significantly increased the amplitude of the slow EPSP, which we documented is mediated by TRPC5 channels, and augmented synchronous GCaMP6s ([Ca]i) oscillations in Kiss1ARH neurons. Deletion of the endoplasmic reticulum calcium-sensing protein stromal interaction molecule 1 in Kiss1ARH neurons amplified insulin’s actions and protected ovariectomized female mice from developing obesity and glucose intolerance with high-fat dieting. Therefore, insulin appears to be critical for facilitating synchronous firing of Kiss1ARH neurons and coordinating energy homeostasis with fertility.

Multi-unit recordings first done in female rhesus macaques identified synchronous volleys of action potentials 16 in the hypothalamic arcuate nucleus that correlated with pulsatile luteinizing hormone (LH) release (Knobil, 1981;  17 Wilson et al., 1984) . It was later proposed that hypothalamic arcuate kisspeptin (Kiss1 ARH ) neurons were 18 responsible for the "pulse-generator" activity that stimulates pulsatile secretion of gonadotropin releasing 3 releases NKB and dynorphin (Qiu J. et al., 2016) . NKB binds to tachykinin 3 receptor (TacR3) in neighboring 27 Kiss1 ARH neurons to activate canonical transient receptor potential 5 (TRPC5) channels to cause a robust 28 depolarization (slow EPSP), whereas co-released dynorphin feeds back to bind to presynaptic κ-opioid receptors 29 to limit the release of NKB to discrete bursts of activity (Qiu J. et al., 2016) . The co-release of the two peptide 

A key molecule modulating the activity of TRPC channels is stromal-interaction molecule 1 (STIM1), which 67 is localized to the endoplasmic reticulum (ER) membrane of cells, and its N-terminal domain contains an EF-68 hand that protrudes into the lumen of the ER to sense changes in ER Ca 2+ 

Previous results show that insulin receptor (Insr) and Trpc5 mRNA are expressed and involved in sculpting 04 the excitability in Kiss1 ARH neurons (Qiu J. et al., 2011; Qiu X. et al., 2013) , and insulin excites Kiss1 ARH neurons 05 through activation of TPRC5 channels (Qiu J. et al., 2014) . To see if Insr and Trpc5 mRNAs are co-localized in 06 Kiss1 ARH neurons, we harvested 64 Kiss1 ARH neurons from 3 females and did scRT-PCR for Insr and Trpc5 07 6 ( Figure 1C) . The single-cell analysis revealed that Insr transcript was detected in 53% of Kiss1 ARH neurons and 08 GCaMP6s and imaged these neurons using spinning disk confocal microscopy (Figure 2-video supplement 1) .

ER Ca 2+ stores were released by treatment with 2 M thapsigargin (Tg), a blocker of the SERCA 21 (sarcoplasmic/endoplasmic reticulum Ca 2+ ATPase) pump. As expected, Tg treatment of neurons bathed in Ca 2+ -22 free aCSF generated an initial wave of cytoplasmic Ca 2+ release ([Ca 2+ ]i ) as measured by an increase in 23 GCaMP6s activity both in control and Stim1-deleted neurons ( Figure 1D , E and F). As long as neurons were 24 kept in Ca 2+ -free aCSF, the ER stores remained empty, a situation that was presumably sensed by the Ca 2+ 25 sensor STIMs. Upon switching to a normal aCSF containing 2 mM Ca 2+ , an immediate SOCE response was 26 observed as a second wave of cytoplasmic Ca 2+ rise. Consistent with a role for STIM1 regulation, we observed 27 an attenuation of SOCE in Stim1 kko neurons ( Figure 1D , E F and G: ΔF/F0*100 =1274.5 ± 49.4, n = 4, Kiss1 ARH 28 group versus 389.0 ± 86.1, n = 4, Stim1 kko group, which was measured from the 15 minute time point to the peak, 29 unpaired t-test, t(6) = 8.921, p = 0.0001, ***p < 0.005), indicating that STIM1 plays a major role in SOCE after Tg- To test the hypothesis that insulin potentiates the slow EPSP in Stim1 kko neurons to a greater extent than in 88 9 Kiss1 ARH neurons, we pretreated ChR2-mCherry expressing arcuate slices from Stim1 kko mice with insulin.

Indeed, we found that insulin augmented the slow EPSP by 60% beyond the effects of Stim1 deletion on the (Figure 6A, B) . Moreover, the 05 average fat mass of Stim1 kko mice was significantly less that of Kiss1 Cre controls by week 6 (Stim1 kko versus 06 Kiss1 Cre mice: 7.6 ± 0.9 g, n=10 versus 11.4 ± 1.1 g, n=10 ) ( Figure 6C ). The lean mass of Stim1 kko mice was 07 significantly less versus the Kiss1 Cre mice (Stim1 kko versus the Kiss1 Cre mice: 16.9 ± 0.4 g, n=10 versus 18.9 ± 08 0.4 g, n=10) ( Figure 6D ). After 6 weeks, both Stim1 kko and Kiss1 Cre controls were assessed for glucose tolerance 09 using an i.p. glucose tolerance test (see Methods). Both Stim1 kko and Kiss1 Cre females started at relatively the 10 same blood glucose levels after an overnight fast ( Figure 6E , time 0), suggesting similar whole-body homeostatic 11 conditions after fasting. However, Stim1 kko female mice had significantly lower glucose levels after i.p. glucose 12 compared to Kiss1 Cre females, indicating that Stim1 kko females were more glucose tolerant compared to Kiss1 Cre 13 controls. Moreover, Stim1 kko females had a significantly different glucose clearance rate than controls based on 14 the integrated area under the curve (Stim1 kko versus the Kiss1 Cre controls AUC: 20,232 ± 868 mg/dL × min, n = 15 10 6 vs 22,622 ± 624 mg/dL × min, n = 6). Finally, when both groups were euthanized after eight weeks on HFD 16 and tissues harvested for mRNA measurements, both the intrascapular brown adipose tissue (iBAT) and 

The rapid insulin effects were abrogated by PI3K kinase inhibitor wortmannin. Importantly, the enhanced TacR3 50 signaling in Stim1 kko female mice afforded protection against diet-induced obesity and glucose intolerance. 51 52 We first discovered that TRPC5 channels are coupled to metabolic hormone signaling in both POMC and Software) was used to design primers that cross at least one intron-exon boundary. In order to confirm that 08 STIM1 was knocked out, STIM1 primers were designed to include part of exon 2 (see Table 1 ). Single cell PCR 09 conditions were optimized for primer concentration, magnesium concentration and annealing temperature.

Standard curves were generated using hypothalamic cDNA with dilutions from 1:50 to 1:12,800 for primers used 11 for qPCR to determine the efficiency (E = 10 (-1/m) -1; table 1). Primer pairs with efficiencies of 90-100% permit 12 21 the use of the comparative ΔΔCT method for analysis (Livak and Schmittgen, 2001; Pfaffl, 2001 on high fat diet (unpaired, two-tailed t-test for B, t(18) = 3.222, **p = 0.0047; unpaired two-tailed t test for C, t(18) = 19 2.662, *p = 0.0159; unpaired, two-tailed t test for D, t(18) = 3.489, *p = 0.0026). E, six weeks after high fat diet, 20 there was a significant difference in GTTs between the two groups (two-way ANOVA: main effect of treatment 21 (F(1, 9) = 6.282, p = 0.0335), main effect of time (F(4, 36) = 88.01, p < 0.0001) and interaction (F(4, 36) = 3.527, p = 22 0.0158); Kiss1 Cre , n = 6, Stim1 kko , n = 5; post hoc Bonferroni test, **p < 0.01, *p < 0.05). F and G, both 23 interscapular brown adipose tissue (iBAT) and perigonadal adipose tissue (GAT) mass of Stim1 kko were lighter 24 than that of Kiss1 Cre mice on a fat diet after eight weeks (unpaired, two-tailed t test for iBAT, t(18) = 2.127, *p = 25 0.0475; unpaired two-tailed t-test for GAT, t(18) = 2.711, *p = 0.0143). 58 channels thereby engaging these Ca 2+ channels as store-operated channels, which are activated with

Activation of 49 phospholipase Cγ by PI 3-kinase-induced PH domain-mediated membrane targeting

A rapidly acting glutamatergic ARC→PVH satiety circuit postsynaptically 53 regulated by α-MSH

TrpC5 mediates acute leptin and serotonin 56 effects via Pomc neurons

Exploring the pathophysiology of 59 hypogonadism in men with type 2 diabetes: kisspeptin-10 stimulates serum testosterone and LH 60 secretion in men with type 2 diabetes and mild biochemical hypogonadism

Kisspeptin neurons in the arcuate nucleus of the ewe express both 64 dynorphin A and neurokinin B

Differential Roles for STIM1 and 67 STIM2 in Store-Operated Calcium Entry in Rat Neurons

NEUROD2 Regulates Stim1 Expression and Store-Operated Calcium Entry in Cortical Neurons

STIM1 controls neuronal Ca 2+ signaling, mGluR1-74 27 dependent synaptic transmission, and cerebellar motor behavior

Acute effects of leptin require PI3K signalng in hypothalamic proopiomelanocortin neurons in 78 mice

STIM1 carboxyl-terminus 80 activates native SOC, Icrac and TRPC1 channels

Treatment with HC-85 070, a potent inhibitor of TRPC4 and TRPC5, leads to anxiolytic and antidepressant effects in mice

TRPCing around the hypothalamus

Membrane-initiated actions of estradiol that regulate reproduction, energy 90 balance and body temperature

Neuron-Specific and Self-Sustained Calcium Oscillation in the Hypothalamic Arcuate Nucleus of 93

Regulatory Factors of its Synchronization

Effects of kisspeptin-10 on the 96 electrophysiological manifestation of gonadotropin-releasing hormone pulse generator activity in the 97 female rat

Patterns of hypophysiotropic signals and gonadotropin secretion in the rhesus monkey

Rapid, reversible activation of AgRP neurons drives feeding behavior in mice

Minireview: kisspeptin/neurokinin B/dynorphin (KNDy) cells of 04 the arcuate nucleus: a central node in the control of gonadotropin-releasing hormone secretion

Analysis of relative gene expression data using real-time quantitative PCR 07 and the 2 −ΔΔCT method

A toolbox of Cre-dependent optogenetic transgenic mice for light-induced 11 activation and silencing

Stim and Orai 13 proteins in neuronal Ca 2+ signaling and excitability

Metabolic regulation of kisspeptin -the link between energy balance and reproduction

Interactions between kisspeptin and neurokinin B in the control of GnRH secretion in 19 the female rat

Regulation of gonadotropin-22 releasing hormone secretion by kisspeptin/dynorphin/neurokinin B neurons in the arcuate nucleus of 23 the mouse

STIM1 participates in the contractile rhythmicity of HL-1 cells by moderating T-type Ca 2+ channel 27 activity

Dual functions for the endoplasmic reticulum calcium sensors STIM1 and STIM2 in T cell activation and 31 tolerance

Kisspeptin and GnRH 33 pulse generation

A neural circuit underlying the 36 generation of hot flushes

Kisspeptin neurons in the arcuate nucleus of the hypothalamus orchestrate circadian rhythms 40 and metabolism

The CRAC channel activator STIM1 binds and inhibits L-type 42 voltage-gated calcium channels

STIM1 Is Required for Remodeling of the Endoplasmic Reticulum

Microtubule Cytoskeleton in Steering Growth Cones

A new mathematical model for relative quantification in real-time RT-PCR

Estradiol 51 protects proopiomelanocortin neurons against insulin resistance

Rapid signaling of 54 estrogen in hypothalamic neurons involves a novel G-protein-coupled estrogen receptor that activates 55 30 protein kinase C

Rønnekleiv 58 OK, & Kelly MJ. 2006. A G-protein-coupled estrogen receptor is involved in hypothalamic control of 59 energy homeostasis

Estradiol protects neuropeptide Y/agouti-related 62 peptide neurons against insulin resistance in females

Guinea pig kisspeptin neurons are depolarized by 65 leptin via activation of TRPC channels

Leptin excites proopiomelanocortin neurons via activation of 68 TRPC channels

High-frequency 71 stimulation-induced peptide release synchronizes arcuate kisspeptin neurons and excited GnRH 72 neurons

Estrogenic-dependent glutamatergic neurotransmission from kisspeptin neurons governs feeding 75 circuits in females

Insulin and leptin excite anorexigenic pro-77 opiomelanocortin neurones via activation of TRPC5 channels

Insulin excites anorexigenic proopiomelanocortin neurons via activation of canonical transient 81 receptor potential channels

Delayed puberty 84 but normal fertility in mice with selective deletion of insulin receptors from Kiss1 cells

Phosphoinositide 3-kinase 87 regulates phospholipase Cγ-mediated calcium signaling

Dual regulation of voltage-sensitive ion 90 channels by PIP2

Contribution of a membrane estrogen receptor to the estrogenic regulation of 94 body temperature and energy homeostasis

Arcuate Kisspeptin Neurons Coordinate Reproductive Activities with 97

Regulation of arcuate neurons coexpressing kisspeptin, neurokinin 00 B, and dynorphin by modulators of neurokinin 3 and κ-opioid receptors in adult male mice

STIM1 regulates somatic Ca 2+ signals and intrinsic firing properties of cerebellar Purkinje neurons. The 04

The TRPC ion channels: association with Orai1 and STIM1 proteins 06 and participation in capacitative and non-capacitative calcium entry

Insulin: its role in the 09 32 central control of reproduction

The membrane estrogen receptor ligand 12 STX rapidly enhances GABAergic signaling in NPY/AgRP neurons: Role in mediating the anorexigenic 13 effects of 17β-estradiol

CRAC Channels Regulate Gene Expression and Proliferation in Neural Progenitor Cells

Glucagon regulates hepatic kisspeptin to 20 impair insulin secretion

Membrane and nuclear initiated estrogenic regulation of 23 homeostasis

Inhibition of L-Type Ca 2+ 25 channels by TRPC1-STIM1 complex is essential for the protection of dopaminergic neurons. The 26

Impaired 28 kisspeptin signaling decreases metabolism and promotes glucose intolerance and obesity

TRPC channels: Structure, function, 31 regulation and recent advances in small molecular probes

The calcium store 34 sensor, STIM1, reciprocally controls Orai and CaV1.2 channels

Uneven balance of power between hypothalamic peptidergic neurons in the control of 38 feeding

Central electrophysiological 41 correlates of pulsatile luteinizing hormone secretion in the Rhesus monkey

PI3K integrates the action of insulin 44 and leptin on hypothalamic neurons

STIM1 heteromultimerizes TRPC channels to 47 determine their function as store-operated channels

Kisspeptin activation of TRPC4 channels in female 50

GnRH neurons requires PIP2 depletion and cSrc kinase activation

Kisspeptin depolarizes gonadotropin-releasing 53 hormone neurons through activation of TRPC-like cationic channels

Molecular mechanisms that drive estradiol-dependent burst firing of Kiss1 neurons in the rostral 57 periventricular preoptic area

a ChR2-expressing Kiss1 ARH neuron from control Kiss1 mice (B) and in a ChR2-expressing Kiss1 ARH neuron 49 from Stim1 kko mice (C). The lower trace shows the slow EPSP after low-pass filtering from B and C (arrow)

D, summary of the effects of Stim1 deletion on the slow EPSP amplitude. Bar graphs represent 51 the mean ± SEM (Unpaired t-test, t(39) = 2.802, **p = 0.0079). E and F, rapid bath application of senktide

the presence of fast sodium channel blockade (TTX, 1µM) in Kiss1 ARH neurons from 53

Kiss1 Cre and Stim1 kko mice. Vhold = -60 mV. G, summary of the effects of senktide in Kiss1 ARH neurons from 54

Kiss1 Cre and Stim1 kko mice (Unpaired t-test, t(11) = 2.929, *p = 0.0137). Data points represent the mean ± SEM. 55 Cell numbers are indicated

Slow EPSP was induced by a 10-s 20 Hz photostimulation (light intensity 0.9 mW and 59 pulse duration, 10 ms) in a ChR2-expressing Kiss1 ARH neuron

Insulin augments the slow EPSP in Kiss1 ARH neurons from Kiss1 Cre ::Ai32 mice in a PI3K 72 dependent manner and senktide-induced depolarization is antagonized by TRPC5 channel blockers

D, representative traces of the slow EPSPs in the presence or absence of PI3 kinase inhibitor wortmannin

ER) depletion of Ca 2+ . B, however, under physiological conditions in reproductively 60 active females, E2 down-regulates the expression of STIM1, thereby converting the TRPC5 channels to receptor-61 operated channels in Kiss1 ARH neurons. Insulin signals via InsR-IRS-PI3K-PLCγ1 to activate TRPC5 channels, 62 generating a robust inward Na + /Ca 2+ current to depolarize Kiss1 ARH neurons, activating T-type calcium (Cav3.1) 63 channels to greatly increase Kiss1 ARH neuronal excitability. PI3K (p85/p110) will also accelerate the rapid 64 insertion of TPRC5 channels into the plasma membrane