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Targeting the T-type calcium channel Cav3.2 in GABAergic arcuate nucleus neurons to treat obesity

Objective Cav3.2, a T-type low voltage-activated calcium channel widely expressed throughout the central nervous system, plays a vital role in neuronal excitability and various physiological functions. However, the effects of Cav3.2 on energy homeostasis remain unclear. Here, we examined the role of Cav3.2 expressed by hypothalamic GABAergic neurons in the regulation of food intake and body weight in mice and explored the underlying mechanisms. Methods Male congenital Cana1h (the gene coding for Cav3.2) global knockout (Cav3.2KO) mice and their wild type (WT) littermates were first used for metabolic phenotyping studies. By using the CRISPR-Cas9 technique, Cav3.2 was selectively deleted from GABAergic neurons in the arcuate nucleus of the hypothalamus (ARH) by specifically overexpressing Cas9 protein and Cav3.2-targeting sgRNAs in ARH Vgat (VgatARH) neurons. These male mutants (Cav3.2KO-VgatARH) were used to determine whether Cav3.2 expressed by VgatARH neurons is required for the proper regulation of energy balance. Subsequently, we used an electrophysiological patch-clamp recording in ex vivo brain slices to explore the impact of Cav3.2KO on the cellular excitability of VgatARH neurons. Results Male Cav3.2KO mice had significantly lower food intake than their WT littermate controls when fed with either a normal chow diet (NCD) or a high-fat diet (HFD). This hypophagia phenotype was associated with increased energy expenditure and decreased fat mass, lean mass, and total body weight. Selective deletion of Cav3.2 in VgatARH neurons resulted in similar feeding inhibition and lean phenotype without changing energy expenditure. These data provides an intrinsic mechanism to support the previous finding on ARH non-AgRP GABA neurons in regulating diet-induced obesity. Lastly, we found that naringenin extract, a predominant flavanone found in various fruits and herbs and known to act on Cav3.2, decreased the firing activity of VgatARH neurons and reduced food intake and body weight. These naringenin-induced inhibitions were fully blocked in Cav3.2KO-VgatARH mice. Conclusion Our results identified Cav3.2 expressed by VgatARH neurons as an essential intrinsic modulator for food intake and energy homeostasis, which is a potential therapeutic target in the treatment of obesity.

It is well established that the hypothalamus is an essential brain structure for the regulation of feeding behavior and energy homeostasis [12e15]. One of the vital hypothalamic nuclei engaging in this regulation is the arcuate nucleus of the hypothalamus (ARH). Several lines of studies have suggested that ARH neurons that express an amino acid neural transmitter gamma-aminobutyric acid (GABA) play a vital role in promoting food intake and inhibiting energy expenditure [16e18]. Recent studies revealed that GABA transmission mediates the orexigenic effects of classic agouti-related peptide (AgRP) and other GABAergic neurons in the ARH [16,18,19]. Activation of GABAergic, agouti-related protein (AgRP), or non-AgRP GABAergic neurons in the ARH leads to hyperphagia and obesity. In contrast, inhibition of GABAþ, but not AgRP neurons in the ARH reduces agingrelated weight gain and alleviates leptin deficiency-induced obesity [16]. Together, modulation of ARH GABAergic neuron activity is a fundamental mechanism for body weight regulation. There is a potential to prevent obesity by inhibiting ARH GABAergic neurons. Most of the previous studies focused on the effects of Cav3.2 on neuronal firing activity control in the regulation of anxiety [20], memory [20], chronic pain [11], and epilepsy [21e23]. However, the role of Cav3.2 in feeding behavior and energy homeostasis is still not well understood. Here, we first used a germline loss-of-function mouse model to examine the effects of Cav3.2 on feeding behavior and energy balance in a normal chow diet (NCD) or high-fat diet (HFD) fed condition. Mice with Cav3.2 selectively deleted from vesicular GABA transporter (Vgat)-expressing ARH neurons (Vgat ARH ) were used to examine the roles of Cav3.2 expressed by ARH GABA þ neurons in energy homeostasis. We explored the potential Cav3.2-dependent mechanisms in the beneficial metabolic effects mediated by the supplementation of naringenin, a predominant flavanone in a variety of fruits and herbs. Our studies provide compelling evidence to support targeting Cav3.2 expressed by ARH GABA þ neurons in the treatment of obesity and related metabolic diseases.
2.2. Characterization of feeding behavior and energy homeostasis Cav3.2KO mice and their control littermates were weaned at three weeks of age on a standard chow diet (5.0% fat, #5001, LabDiet) and singly housed afterward. Some mice were switched to an HFD (60% fat, #D12492, Research Diets) at six weeks of age. Body weight and food intake were measured every four days. Body composition was determined using quantitative magnetic resonance (QMR). At the end of monitoring, the mice were deeply anesthetized with inhaled isoflurane and sacrificed. The gonadal white adipose tissue (gWAT), the inguinal white adipose tissue (iWAT), and the interscapular brown adipose tissue (BAT) were isolated and weighed. To characterize the food intake and energy expenditure, we generated another male cohort (Cav3.2KO mice and their control littermates at 12 weeks of age). They were acclimated into the Promethion system (Sable, NV, USA). Mice were housed individually at room temperature (22 C) under an alternating 12:12-h lightedark cycle. After adaptation for three days, O 2 consumption, CO 2 production, and energy expenditure were monitored when mice were fed ad libitum with NCD. The body composition was measured before and after the mice entered the Promethion system. The energy expenditure was corrected per lean body mass.
2.3. CRISRP-Cas9 deletion of Cav3.2 in the ARH GABA þ neurons AAV vectors carrying sgRNAs targeting mouse Cacna1h were designed and constructed by Vector Builder (Chicago, IL). Exon 6 and exon 11 were chosen to be targeted by CRISPR-Cas9. A total of 21 sgRNAs were designed, with seven targeting exon 6 and fourteen targeting exon 11. These sgRNAs were selected using the CRISPR tool (https:// www.sanger.ac.uk/htgt/wge/) with minimal potential off-target effects. To generate sgRNA expression plasmids, oligonucleotides containing gRNA sequence were created and cloned into the final rAAV backbone with mCherry reporter by Gateway recombination. All 21 sgRNAs were screened for onetarget activity using electrophysiology. We used the pCS(puro)-positive plasmid, which expressed a proven positive-sgRNA, as the positive control. The sgRNA#1 (CTGGGCATGTTCCGGCCCTGT) and sgRNA#2 (CTGTTCGGCCAGAAATGCTAC) were selected to target exon 6 and exon 11 of the Cacna1h gene, respectively, due to their relatively high onetarget activity and low off-target potentials. As a control, scramble RNAs (GTGTAGTTCGACCATTCGTG) and (GTTCAG-GATCACGTTACCGC) was used to replace the sgRNA#1 and sgRNA#2, respectively. U6 promoter-sgRNAs, CAG promoter-DIO-mCherry-bGH polyA cassettes were cloned into the plasmid # VB201223-1293enk vector (http://www.addgene.org/61591/) and further verified by full sequencing. The viruses were packaged and produced by the Vector Builder (Chicago, IL). At 12 weeks of age, male Vgat-ires-Cre mice were anesthetized with isoflurane and received stereotaxic injections of AAV-FLEX-saCas9 mixed with AAV-DIO-Cav3.2 sgRN/mCherry into both sides of the ARH (Cav3.2KO-Vgat ARH ; 250 nL/site, À1.7 mm posterior, AE0.25 mm lateral and À5.8 mm ventral to the Bregma, based on Franklin & Paxinos Mouse Brain Atlas). Another group of male Vgatires-Cre mice at the same age received AAV-FLEX-saCas9 mixed with AAV-DIO-scramble sgRN/mCherry injections into the ARH. One week after the virus injections, mice were singly housed and fed a chow diet for 5 weeks. Body weight and food intake were monitored weekly. Body composition was measured at the end of the chow dietfeeding phase. All mice were then switched to HFD feeding for another 6 weeks. To validate accurate and sufficient infection of AAV vectors, all mice were perfused with 10% formalin. Brain sections were cut at 25 mm (5 series) and subjected to histological validation.
Only those mice with mCherry on both sides of the ARH were included in data analyses. In another experiment, a different cohort of male Cav3.2KO-Vgat ARH and control mice were used to test naringenin's effects on feeding behavior. Two weeks after the virus injection, all mice received naringenin intraperitoneal injection for two days (40 mg/kg body weight/ day). Food intake and body weight were measured daily for 7 days.
Original Article 2.4. Electrophysiological recordings Male and female Vgat-ires-Cre/Rosa26-EGFP-L10, Vgat-ires-Cre/ Rosa26-EGFP-L10/Cacna1h À/-, and AAV-injected Vgat-ires-Cre (12 weeks of age) were used for electrophysiological recordings. Mice were deeply anesthetized with isoflurane and transcardially perfused with a modified ice-cold sucrose-based cutting solution (pH 7.3) containing 10 mM NaCl, 25 mM NaHCO 3 , 195 mM sucrose, 5 mM glucose, 2.5 mM KCl, 1.25 mM NaH 2 PO 4 , 2 mM Na-Pyruvate, 0.5 mM CaCl 2 , and 7 mM MgCl 2 , bubbled continuously with 95% O 2 and 5% CO 2 [24,25]. The mice were then decapitated, and the entire brain was removed and immediately submerged in the cutting solution. Slices (200 mm) were cut with a Leica VT1000 S microtome vibratome (Leica, Germany). Three brain slices containing the LH, ZI, TN, and ARH were obtained for each animal (Bregma À2.06 mm to À1.46 mm; Interaural 1.74 mme2.34 mm). The slices were recovered for 1 h at 34 C and then maintained at room temperature in artificial cerebrospinal fluid (aCSF, pH 7.3) containing (126 mM NaCl, 2.5 mM KCl, 2.4 mM CaCl 2 , 1.2 mM NaH 2 PO 4 , 1.2 mM MgCl 2 , 5.0 mM glucose, and 21.4 mM NaHCO 3 ) saturated with 95% O 2 and 5% CO 2 before recording [15,25]. Slices were transferred to a recording chamber and allowed to equilibrate for at least 10 min before recording. The slices were superfused at 34 C in oxygenated aCSF at a flow rate of 1.8e2 ml/ min. EGFP or mCherry labeled neurons in the ARH and vlVMH were visualized using epifluorescence and IR-DIC imaging on an upright microscope (Eclipse FN-1, Nikon) equipped with a movable stage (MP-285, Sutter Instrument). For GABA neurons firing activity recording, the patch pipettes with resistances of 3e5 MU were filled with intracellular solution (pH 7.3) containing 128 mM K-Gluconate, 10 mM KCl, 10 mM HEPES, 0.1 mM EGTA, 2 mM MgCl 2 , 0.05 mM Na-GTP and 0.05 mM Mg-ATP [25]. Recordings were made using a MultiClamp 700B amplifier (Axon Instrument), sampled using Digidata 1440A and analyzed offline with pClamp 10.3 software (Axon Instruments). The firing frequency and membrane potential were recorded under current clamp. For voltage-gated calcium current recording, the pipette solution (pH 7.2) contains 135 mM KMeSO3, 4 mM NaCl, 10 mM HEPES, 1 mM MgCl2, 0.5 mM EGTA, 3 mM Mg-ATP, and 0.3 mM Tris-GTP. The voltage-gated calcium currents were evoked by 100-ms pulse delivered every 10 s from À90 mV to À30 mV followed by return to À90 mV holding for 500 ms [26]. Neurons were held at À70 mV between sweeps. Series resistance was monitored during the recording, and the values were generally <10 MU and were not compensated. The liquid junction potential was þ12.5 mV and was corrected after the experiment. Data were excluded if the series resistance increased dramatically during the experiment or without overshoot for action potential. Currents were amplified, filtered at 1 kHz, and digitized at 20 kHz. To examine the naringenin's function on GABA neurons, neurons were recorded under the current-clamp mode in response to a puff treatment of different doses of naringenin or vehicle (1% DMSO) [27]. The values for firing rate were averaged within 2-min bin in the presence or absence of naringenin. Neurons that were inhibited (>10% decrease of firing rate) or neurons that were activated (>10% increase of firing rate) in response to naringenin were identified as responsive. Neurons that showed responses less than 10% change of firing rate were identified as non-responsive. To further test whether naringenin regulates calcium currents via the PKA signal pathway, Vgat ARH neurons were pretreated by Rp-cAMP before the naringenin puff treatment.

Statistical analyses
The data are presented as mean AE SEM (standard error of the mean). Statistical analyses were performed using GraphPad Prism 8.3 to evaluate normal distribution and variations within and among groups. Methods of statistical analyses were chosen based on the design of each experiment and are indicated in figure legends or main text. p < 0.05 was considered to be statistically significant.

Study approval
Care of all animals and procedures were approved by the PBRC and UIC Institutional Animal Care and Use Committee.

RESULTS
3.1. Cav3.2KO mice showed decreased body weight and food intake on a NCD To examine the physiological roles of Cav3.2 in the regulation of feeding behavior and energy expenditure, we generated a cohort of male and female Cav3.2 deficient Cana1h À/-(Cav3.2KO) mice and their control littermates (Supplemental Fig. 1A). We measured their food intake and body weight after they were weaned at 3 weeks of age. When fed with a NCD, the Cav3.2KO male mice started to show significant body weight difference at around 7 weeks of age compared to their control littermates ( Figure 1A). The body weight gain and cumulative food intake were consistently decreased at a similar age ( Figure 1BeD). No gender difference was observed in the metabolic effects induced by Cav3.2KO. Female Cav3.2KO also displayed significantly lower body weight and food intake compared to their control littermates (Supplemental Figs. 1BeC). However, we failed to observe any difference in body length of male and female Cav3.2KO mice and their control littermates ( Figure 1H and Supplemental Fig. S1D). The body weight difference was due to the loss of both lean and fat mass as indicated by lower lean and fat mass in male Cav3.2KO mice observed at 9 weeks of age ( Figure 1EeG).

Abnormal energy metabolism in Cav3.2KO mice
To determine the mechanisms underlying the lean phenotypes seen in Cav3.2KO mice, another cohort of NCD-fed male Cav3.2KO males and their control littermates (12 weeks of age) were adapted into the Sable Promethion Systems metabolic chambers to measure energy expenditure. Compared to control mice, Cav3.2KO males showed significantly higher energy expenditure primarily during the light cycle when fed ad libitum ( Figure 1IeK).

Cav3.2KO Mice have an attenuated response to HFD
We then characterized body weight phenotypes in male Cav3.2KO mice switched to an HFD at 6 weeks of age. Consistent with our observations in the NCD-fed condition, HFD-fed male Cav3.2KO mice gained significantly less body weight, which was associated with lower HFD intake, compared to their control littermates ( Figure  2AeD). In particular, HFD-fed Cav3.2KO males started to have a lower body weight as early as 4 days after HFD feeding, and after 48 days of HFD feeding, Cav3.2KO males gained 6.14 g less body weight than controls (about 15% of control body weight) ( Figure  2AeB). This reduced body weight observed in Cav3.2KO males was associated with decreased lean mass, fat mass, and body fluid (Figure 2EeG). Weights of different fat depots, including brown fat (BAT), inguinal white fat (iWAT), and gonadal white fat (gWAT) were significantly lower in Cav3.2 males compared with control littermates (Figure 2HeJ). We found that HFD-induced obesogenic expansion with abnormal adipocyte enlargement (hyperplasia) in both iWAT and gWAT depots was largely attenuated in Cav3.2KO males ( Figure 2K). In addition, we observed that Cav3.2KO restored BAT morphology with fewer lipid droplets compared to control littermates under HFD feeding ( Figure 2K). Together, our data suggest that the loss of Cav3.2 decreases susceptibility to DIO in male mice.
Original Article mCherry and AAV-FLEX-saCAS9 (1:1; control) in both sides of the ARH ( Figure 4A). The injection accuracy and infection efficiency were validated by mCherry fluorescence (Figure. 4B). We first use the ex vivo brain slice patch-clamp method to validate the successful deletion of Cav3.2 in Vgat ARH neurons ( Figure 4C). We found that Vgat ARH neurons from male Cav3.2KO-Vgat ARH mice showed lower firing frequency, hyperpolarized RMP, and smaller voltage-gated calcium current when compared to Vgat ARH neurons from control mice (Figure 4DeH). This is consistent with our earlier observations in Vgat-Cre/EGFP/Cav3.2KO mice and validates the Cav3.2KO-Vgat ARH model.
3.5. Selective knockout of Cav3.2 in Vgat ARH neurons reduces food intake and body weight gain Consistent with the metabolic effects caused by Cav3.2 global KO, selective deletion of Cav3.2 Vgat ARH led to decreased body weight gain and food intake when fed an NCD or an HFD ( Figure 5A,B). Cav3.2KO-Vgat ARH mice also showed reduced food intake and feeding efficiency compared to their control littermates when fed with NCD or HFD (Figure 5CeF). The body weight difference on HFD feeding was attributed mainly to decreases in fat mass, more specifically iWAT and gWAT, but not the other body components (Figure 5GeL). These male Cav3.2KO-Vgat ARH mice showed a similar level of energy expenditure compared to the control mice under NCD feeding before they displayed body weight and lean mass difference ( Supplementary Fig. 3AeC), suggesting a hypophagia-induced weight loss. These results demonstrate that Cav3.2 expressed by Vgat ARH neurons is physiologically relevant for the regulation of feeding behavior and subsequent body weight homeostasis.
3.6. Naringenin extract reduces food intake and body weight gain by inhibiting Vgat ARH neurons in a Cav3.2-dependent mechanism Naringenin, a flavonoid present in sweet oranges and grapefruit, was recently found to improve glucose and lipid homeostasis and mitigate adipose tissue inflammation in rodents and humans [27,38e40]. Naringenin has been shown to interact with voltage-gated ion channels and potentially block VGCCs, including Cav3. 2 [41e43]. We speculated that Cav3.2 expressed by Vgat ARH neurons partially mediates the beneficial metabolic effects of naringenin. To test this, we examined the direct electrophysiological effects of naringenin on Vgat ARH neurons and tested whether Cav3.2 is required for these effects. We found that .2KO mice after 8 weeks of HFD feeding (n ¼ 7 in control and n ¼ 5 in Cav3.2KO groups). (K) H&E staining of gWAT, iWAT, and BAT tissue. Results are presented as mean AE SEM. *, P < 0.05, **, P < 0.01, ***, P < 0.001, ****, P < 0.0001 in two-way ANOVA analysis followed by post hoc Sidak tests. ####, P < 0.0001 in unpaired t-tests. naringenin dose-dependently inhibited Vgat ARH neurons ( Supplementary   Fig. 4AeD). 30 mM naringenin was sufficient to block firing activity, reduce RMP, and decrease the voltage-gated calcium current (Figure 6AeE) in control mice. Naringenin stimulated Cl À secretion in colonic epithelium via a signaling pathway involving the cAMPdependent protein kinase (PKA) [44]. Native T-type (Cav3.1, Cav3.2, and Cav3.3) channels are differentially regulated by PKA [45e47]. We speculated that Cav3.2 acts through PKA signaling to regulate Vgat ARH neural activity. To directly test this speculation, we pretreated Vgat ARH neurons with 10 mM Rp-cAMP (a membrane-permeable PKA inhibitor) [45] for 10 min and then puff treated Vgat ARH neurons with 30 mM naringenin. The results showed that naringenin failed to decrease firing activity, RMP, and the voltage-gated calcium current in the presence of Rp-cAMP (Supplementary Figure 4 E-G), suggesting a PKA-dependent mechanism. More importantly, the inhibitory effects of naringenin on Vgat ARH neurons were fully blocked in Cav3.2KO-Vgat ARH mice ( Figure 6AeB, and FeH), suggesting a Cav3.2-dependent inhibition on Vgat ARH neurons. Together, our results indicate that naringenin inhibits Vgat ARH neurons by regulating the Cav3.2 channel via the PKA signal pathway to reduce voltage-gated calcium current. Considering the metabolic effects of Cav3.2 and the bloodebrain barrier permeability of naringenin [48], we hypothesize that circulating naringenin acts on Cav3.2 expressed by Vgat ARH neurons to regulate energy homeostasis. We found that while two days of naringenin injection (40 mg/kg, once/day, intraperitoneal) significantly decreased food intake and body weight in male control mice, these inhibitory effects of naringenin were blocked in male Cav3.2KO-Vgat ARH mice. Intracerebroventricular (ICV) injection of naringenin (1 mM, 1.5 ml) in DIO mice reduced HFD intake during dark time compared to the vehicle injection (Supplemental Fig. 4EeF). Together, these data support that Cav3.2 expressed by Vgat ARH neurons is required for the hypophagia and body weight lowering effects of naringenin.

DISCUSSION
T-type calcium channels (Cav3.1, Cav3.2, and Cav3.3) play a vital role in the neuronal burst-firing activities in many brain regions [49e51], representing a promising pharmacological target for various diseases or behavioral disorders. However, little is known about the specific functions of these channels in the regulation of energy homeostasis. Here, by using congenital KO mice, we report for the first time that one isoform of T-type calcium channels, Cav3.2, plays an essential role in the regulation of body weight and adiposity by modulating feeding behavior and energy expenditure. Data generated from Cav3.2KO-Vgat ARH mice further demonstrated that the metabolic effects of Cav3.2 are at least partly mediated by modulating spontaneous AP firing and RMP of GABAergic neurons in the ARH. We also provided evidence to support a mediating role of Cav3.2 expressed by Vgat ARH neurons in the beneficial metabolic effects of naringenin, a flavonoid present in citrus fruits, which has been discovered as a potential blocker for Ttype calcium channels, including Cav3.2. Together, our data suggest that Cav3.2 expressed by Vgat ARH neurons is a physiological modulator for food intake and energy homeostasis, serving as a prospective therapeutic target in the treatment of obesity. The best characterized orexigenic neural population in the ARH is the AgRP-expressing neurons. AgRP neurons co-express genes for neuropeptide Y (NPY) [52] and GABA [28] and use AgRP, NPY, and GABA as neurotransmitters to regulate feeding. These neuropeptides play distinct but complementary roles in the regulation of feeding; either GABA or NPY is required for the rapid stimulation of feeding [14]. NPY contributes to maintaining the sustaining hunger selectively on the timescale of a meal [53]. It was recently discovered that AgRP neurons are inhibited within seconds by the sensory detection of food and can drive feeding via a long-lasting hunger signal [54]. This unusual mechanism for controlling behavior is mediated through NPY [53], which extends the duration of AgRP neurons' behavioral effects for tens of minutes beyond the window of their immediate firing. NPY may mediate these sustaining orexigenic effects by acting directly on postsynaptic targets of AgRP neurons to cause durable changes in their activity [55]. Conversely, the neuropeptide AgRP, through action on melanocortin 4 receptor, is sufficient to induce long-lasting potentiation of feeding behavior that persists for days. These prolonged orexigenic effects require several hours to emerge when endogenous AgRP release is stimulated [14,56]. By working together, these neuropeptides contribute to the regulation of temporally distinct phases of eating. Consistent with the rapid orexigenic effects of GABA released from AgRP neurons, the GABAergic neurons in the ARH potently regulate short-term feeding behavior, as previously reported in the mouse models with neuronal activity acutely manipulated [57e59]. However, the intracellular mechanisms intrinsically modulating ARH GABAergic neurons firing activity and subsequent food intake are unclear. Here we combined Vgat-ires-Cre mice and targeted delivery of AAV vectors carrying Cav3.2 gRNA and Cas9 to selectively knockout Cav3.2 from GABAergic neurons in ARH. Cav3.2KO-Vgat ARH significantly inhibited the firing activity of ARH GABAergic neurons. In line with the regulatory effects of ARH GABAergic neurons on food intake, we also found Cav3.2KO-Vgat ARH mice showed phenotypes of hypophagia and associated body weight loss, suggesting a pivotal role of Cav3.2-Vgat ARH in the regulation of feeding behavior. Although our stereotaxic targeting to the Vgat ARH neurons is precise because of the selective Vgat expression in the ARH structure, we observe a small number of dorsal medial hypothalamic (DMH) neurons in some animals with a viral infection. We did not observe any differences in food intake and body weight in these animals compared to ARH selectively targeted mice. Thus, we believe that the feeding behavior and body weight changes in these "leaking" mice were mainly attributed to the selective deletion of Cav3.2 in ARH GABAergic neurons. However, a role of Cav3.2 expressed by DMH neurons in body weight regulation cannot be completely ruled out. Emerging evidence has shown a direct link between the activity of ion channels and systemic energy homeostasis, including central regulation of food intake [15,60e62], energetic balance [63e65], hormone release and response [66e68], as well as the adipocyte cell proliferation [69e72]. Consistently, we found that Cav3.2KO-Vgat ARH resulted in hyperpolarization and hypophagia-induced body weight loss, suggesting an important role of the central Ca 2þ ion channel in energy balance regulation. It has been extensively reported that intracellular Ca 2þ appears to be involved in metabolic derangements, including obesity and insulin resistance [73]. Cav3.2KO-Vgat ARH likely modulates the calcium influx to change the firing activity of Vgat ARH neurons and food intake. In supporting this point of view, the RMP and calcium current amplitude were decreased after Cav3.2KO from the Vgat ARH neurons. Acute naringenin treatment hyperpolarized Vgat ARH neurons via inhibition of Cav3.2-mediated calcium currents, however, these data do not entirely exclude the possibility of a calcium influxindependent mechanism mediated by the Cav3.2 ion channel in Vgat ARH neurons. Two different mouse models were used in this study to investigate the metabolic function of Cav3.2. We found Cav3.2KO dramatically reduced body weight, associated with decreased food intake and increased energy expenditure. CRISPR-mediated Cav3.2KO-Vgat ARH also resulted in decreases in food intake and body weight. However, we failed to observe any changes in energy expenditure of Cav3.2KO-Vgat ARH mice, suggesting a Vgat ARH -independent mechanism. Compared to the control mice, the increased energy expenditure induced by Cav3.2KO is mainly in the light phase. This data indicated that Cav3.2 regulates energy expenditure in a circadian-dependent way via some non-Vgat ARH neurons. The gene coding Cav3.2 is strongly expressed in the suprachiasmatic nucleus (SCN) [74,75]. Given that voltage-gated Ca 2þ currents contribute to the diurnal properties of action potential firing in SCN neurons [76] and that SCN neurons interact with ARH neurons to regulate energy balance [77,78], Cav3.2 expressed by SCN neurons may modulate energy expenditure in a circadian-dependent manner. A further study focusing on the metabolic effects of Cav3.2 in SCN neurons is warranted. The regulatory effects of Cav3.2 on energy expenditure may also work through peripheral mechanisms. The Cav3.2 encoding gene expresses in cells from many peripheral tissues, including the adipocytes from WAT and BAT [79], myocytes from skeletal muscle [80] and smooth muscle [81], sensory dorsal root ganglion (DRG) neurons, and spinal dorsal horn (SDH) neurons, but not sympathetic neurons [82]. Cav3.2 has been shown to play essential roles in the physiological functions of excitable tissues, i.e., neurons and muscle. For clarification, Cav3.2 expressed by the neurons in the DRG and SDH contributes to inflammatory hyperalgesia and chronic pain [83,84], Cav3.2 expressed by the skeletal muscle contributes to the maintenance of muscle mass and normal function [80], CaV3.2 in the smooth muscle hyperpolarizes and relaxes arteries [85,86]. Conversely, no direct evidence exists to support a physiological function of Cav3.2 expressed by adipocytes. More accurately, the physiological roles of ion channels inside adipose tissue are poorly characterized, due to the fact that adipocytes are non-excitable cells without the ability to generate action potential as neurons or muscles. However, RMP does exist in adipocytes and is maintained by the complement of ion channels. Whereas most biologists would perceive that the RMP is primarily about excitability, the recent data identified a range of critical roles that the RMP has in various cell types beyond the action potential [87]. There is emerging evidence supporting the connection between adipocyte RMP and adipose tissue metabolic function. In a recent Cell paper [79], Chen et al. have identified a critical K þ -Ca 2þ -adrenergic signaling axis hyperpolarizing brown adipocytes to dampen thermogenesis and maintain tissue homeostasis. This finding reveals an electrophysiological regulatory mechanism of adipocyte function. In line with this crucial observation, we have found that the RMP of adipocytes was metabolically influenced by body weight in both humans and mice (data not shown), suggesting a vital role of adipocyte RMP in adipose tissue function. Considering the regulatory effects of Cav3.2 on RMP, we hypothesize that Cav3.2 expressed by BAT adipocytes plays an essential role in the regulation of energy expenditure. Currently, our laboratory is testing this hypothesis by selectively deleting Cav3.2 from UCP1-positive BAT adipocytes. The energy expenditure inhibitory phenotype has been previously observed in a Vgat ARH chronic inhibition mouse model, which was generated by overexpression of an inward rectifying potassium channel, Kir2.1, selectively in the Vgat ARH neurons (Kir2.1OE-Vgat ARH ) [16]. This discrepancy suggests an ion channel-dependent inhibition on energy expenditure, which may be attributed to different levels of hyperpolarization in these two models. Although both Cav3.2KO-Vgat ARH and Kir2.1OE-Vgat ARH deceased spontaneous AP firing and RMP, Kir2.1OE-Vgat ARH showed much higher inhibitory effects compared to Cav3.2KO-Vgat ARH (Kir2.1OE-Vgat ARH : À50.6 mV/-70.5 mV in RMP; Cav3.2KO-Vgat ARH : À52.7 mV/-58.2 mV in RMP). The same phenotype on feeding was observed in both global Cav3.2KO mice and Cav3.2KO-Vgat ARH mice, despite the wide distribution of Cav3.2s in the brain. According to the distribution data from the published Cav3.2 EGFP knock-in mice, Cav3.2 is highly expressed in the hippocampus, cortex, amygdala, hypothalamus, cerebellum, and hindbrain [82]. Our data showed that the loss of Cav3.2 in Vgat ARH neurons reduced the firing activity in Vgat ARH neurons, which we believe potentially contributes to the feeding behavior changes in both global and Vgat ARH neuron-specific Cav3.2 KO mice. However, our data do not exclude the potential regulatory effects of Cav3.2 expressed by other brain regions on food intake. It has been shown by many human and animal studies that hippocampus [88], cortex [89], amygdala [90e .2KO Vgat ARH mice after 8 weeks of HFD feeding (n ¼ 5 in control and n ¼ 5 in Vgat ARH Cav3.2KO groups). All data are presented as mean AE SEM. *, P < 0.05, **, P < 0.01, ***, P < 0.001, ****, P < 0.0001 in two-way ANOVA analysis followed by post hoc Sidak tests. ####, P < 0.0001 in unpaired ttests. 92], cerebellum [93], and hindbrain [94,95] are all involved in regulating food intake. Cav3.2 expressed by these brain regions may contribute to different aspects of appetite control. These aspects may include declarative memory processes evaluating when, what, and how much to eat (hippocampus), interoception of food craving and executive functioning (cortex), integration of hunger, satiation, and thirst (cerebellum), sensing of peripheral hormone or nutrient fluctuation (hindbrain), and stimulation of stress-related eating (amygdala). Cav3.2 in different brain regions could regulate food intake via multiple interconnected pathways, many of which are unstudied and understudied. Naringenin, a flavonoid belonging to the flavanones subclass, is widely distributed in various herbs and fruits, including grapefruit, citrus fruits, bergamot, tomatoes, cocoa, Greek oregano, water mint, as well as in beans [96]. Dietary naringenin supplementation within a dose range of 150e900 mg is safe in healthy adults according to a recent clinical study [40]. Ingestion of 300 mg naringenin three times/day was found to increase insulin sensitivity and decrease body weight in a recent human case study [27], suggesting naringenin as a promising therapeutic agent. However, the underlying mechanism in which naringenin modulates energy homeostasis is largely unknown. Recently, naringenin has been found to enter bloodebrain barrier (BBB) [97] and acts as a potential blocker for VGCCs, including Cav3.2 [41e43], indicating a possible central mechanism through Cav3.2. In our ex vivo brain slice recording study, we found that naringenin decreases the firing activity of Vgat ARH neurons by inhibiting Cav3.2-mediated calcium currents. In line with these ex vivo observations, the inhibitory effects of naringenin on food intake and body weight were largely attenuated in Cav3.2KO mice, suggesting a Cav3.2-dependent mechanism. Our data support a model that naringenin blocks the Cav3.2 channel expressed by ARH GABAergic neurons to reduce food intake and body weight. These results also strongly argue the therapeutic potential to target Cav3.2 expressed by Vgat ARH neurons in the treatment of obesity. A majority of the rodent studies showed that 12 weeks of naringenin supplementation reduces body weight, increases insulin sensitivity, and improves lipid metabolism without affecting food intake in the obese models [98e100]. This is inconsistent with our observations that naringenin decreases food intake via Cav3.2 expressed by Vgat ARH neurons. This discrepancy may be attributed to different ways of treatment or mouse models: oral supplementation vs. intraperitoneal injection; genetically obese mice vs. wild-type mice. It would also be essential to administer naringenin for a more extended period and determine whether it can induce malaise or conditioned taste aversion. Results are presented as mean AE SEM. **, P < 0.01, ***, P < 0.001, ****, P < 0.0001 in two-way ANOVA analysis followed by post hoc Sidak tests.

CONCLUSIONS
In conclusion, we found a new role of the Cav3.2 ion channel expressed by Vgat ARH neurons in feeding control. In addition to the anorexic actions in animals fed ad libitum, Cav3.2 is also expected to maintain normal energy expenditure. We further provided evidence that the BBB permeable naringenin produces hypophagia-induced body weight loss by inhibiting Vgat ARH neurons via Cav3.2 blockage. These results extend our understanding of Cav3.2's effects on the regulation of feeding behavior and body weight, suggesting Cav3.2 as a promising target for treating obesity and diabetes.

AUTHOR CONTRIBUTIONS
B. F. is the main contributor to the conduct of the study and data collection; J.H., N. P., H.Y., P.L., V.I., J.V., A. C., and C.R., contributed to the conduct of the study; S. Y, J.F., F.G., H.B., H. M., and C. M. contributed to the manuscript writing and data interpretation; P. X. and Y. H. contributed to the study design, data interpretation, and manuscript writing.

ACKNOWLEDGMENTS
This work was supported by grants from NIH (R01 DK123098 to P. X.; P20 GM135002 to Y. H.), DOD (Innovative Grant W81XWH-19-PRMRP-DA to P. X.), DRTC (The Pilot and Feasibility Award DK020595 to P. X.), and NIA (5K99AG065419-02 to CJR). The authors wish to thank the Laboratory of Animal Center of Pennington Biomedical Research Center at Louisiana State University, and The University of Illinois at Chicago for invaluable help in mouse colony maintenance.