Gene deletion of the PACAP/VIP receptor, VPAC2R, alters glycemic responses during metabolic and psychogenic stress in adult female mice

Pituitary adenylate cyclase‐activating polypeptide (PACAP) and the homologous peptide, vasoactive intestinal peptide (VIP), participate in glucose homeostasis using insulinotropic and counterregulatory processes. The role of VIP receptor 2 (VPAC2R) in these opposing actions needs further characterization. In this study, we examined the participation of VPAC2R on basal glycemia, fasted levels of glucoregulatory hormones and on glycemia responses during metabolic and psychogenic stress using gene‐deleted (Vipr2−/−) female mice. The mean basal glycemia was significantly greater in Vipr2−/− in the fed state and after an 8‐h overnight fast as compared to wild‐type (WT) mice. Insulin tolerance testing following a 5‐h fast (morning fast, 0.38 U/kg insulin) indicated no effect of genotype. However, during a more intense metabolic challenge (8 h, ON fast, 0.25 U/kg insulin), Vipr2−/− females displayed significantly impaired insulin hypoglycemia. During immobilization stress, the hyperglycemic response and plasma epinephrine levels were significantly elevated above basal in Vipr2−/−, but not WT mice, in spite of similar stress levels of plasma corticosterone. Together, these results implicate participation of VPAC2R in upregulated counterregulatory processes influenced by enhanced sympathoexcitation. Moreover, the suppression of plasma GLP‐1 levels in Vipr2−/− mice may have removed the inhibition on hepatic glucose production and the promotion of glucose disposal by GLP‐1. qPCR analysis indicated deregulation of central gene markers of PACAP/VIP signaling in Vipr2−/−, upregulated medulla tyrosine hydroxylase (Th) and downregulated hypothalamic Vip transcripts. These results demonstrate a physiological role for VPAC2R in glucose metabolism, especially during insulin challenge and psychogenic stress, likely involving the participation of sympathoadrenal activity and/or metabolic hormones.

demonstrate that VIP and PACAP and their receptors have complex actions on glucose metabolism.Importantly, VIP derivatives that selectively bind to VPAC2R are promising hypoglycemic drugs that act to promote glucose-stimulated insulin secretion and enhance glucose disposal. 21,28However, additional physiological roles of VPAC2R in glucose homeostasis in vivo have not been fully elucidated and warrants further study.
2][33] PACAP regulates epinephrine secretion to promote hepatic glucose output and thereby can counter-regulate glucosestimulated insulin hypoglycemia, suggesting that PACAP can contribute to glucose homeostasis in a biphasic manner appropriate to the physiological conditions. 34In PACAP gene-deleted mice, insulininduced metabolic stress (hypoglycemia) is lethal due to inadequate adrenal epinephrine secretion. 33Adequate epinephrine secretion requires PACAP-induced upregulation of transcripts encoding the adrenomedullary catecholamine-synthesizing enzymes tyrosine hydroxylase (Th) and phenylethanolamine N-methyltransferase (Pnmt); this is prevented in a PACAP gene-deleted mouse model during restraint stress. 35Several lines of evidence suggest that by modulating the release of epinephrine, glucagon, and insulin from the adrenal gland and pancreas, respectively, PACAP, and to a lesser extent, VIP, can promote sympathetic and parasympathetic function to regulate appropriate glucose responses and maintain homeostasis. 24,34Therefore, whether PACAP/VIP and its receptors participate in sympathoadrenal (SA) activation in response to insulin-induced hypoglycemic and psychogenic stress 33,36 is poorly understood and understudied, especially in females. 36ucidating central PACAP/VIP signaling may be critical for understanding peripheral glucose homeostasis under physiological and disease states.For example, intracerebral application of VPAC2R (but not PAC1R) agonists stimulates hepatic glucose production via sympathetic innervation of the liver. 37In the brain, PACAP is a neurotransmitter localized at stress-transducing central nuclei, including the paraventricular nucleus (PVN) of hypothalamus, an area critical for the regulation of energy homeostasis and pre-autonomic control of metabolic function. 369][40][41][42][43][44][45] Specifically, intra-RVLM PACAP causes PAC1/VPAC2 receptor-mediated sympathoexcitation in rats. 41n this study, we hypothesized that VPAC2R participates in glucose responses to metabolic and psychogenic stress and influences the expression of gene markers of PACAP/VIP signaling in central brain areas associated with its insulinotropic and SA functions.2][53] Compared to WT controls, Vipr2 À/À female mice displayed hyperglycemia during fed and one of the fasted conditions, impaired insulin-induced hypoglycemia, and exaggerated hyperglycemia and plasma epinephrine triggered by restraint stress, implicating upregulated glucose counterregulatory processes.Gene markers for SA but not hypothalamo-pituitary-adrenal (HPA) axis were significantly altered in corresponding brain areas involved in VIP/PACAP signaling.Our findings are consistent with a physiological role of VPAC2 receptors in regulating glucose homeostasis during metabolic and psychogenic stress, which likely involves the participation of SA activity and/or metabolic hormones.

| Generation of Vipr2-deleted mice
Vasoactive-intestinal peptide receptor 2 gene-deleted mice (Vipr2 À/À ) were generated and validated as described. 16Briefly, the Vipr2 À/À mice were generated in E14/4 embryonic stem cells by replacing a 132 bp sequence containing the translation start site of the VPAC2R gene with LacZ-Neo cassette.Correct gene targeting was confirmed using RT-qPCR and gene-specific primer for Vipr2 and by radioreceptor autoradiography using the selective VPAC2R ligand Ro 25-1553.
Vipr2 À/À mice displayed similar gross morphology and fertility as WT littermates on a C57BL/6J background.As further validation of the mutation, we examined the expression of VPAC2R in adrenal tissue.
Expression of Vipr2 was detected in WT mice but not Vipr2 À/À mice (data not shown).Vipr2 À/À mice were obtained as a gift of the last remaining colony from the Waschek Lab (University of California, Los Angeles).Twelve female Vipr2 À/À and seven WT littermates were used in this study.Mice were 5.6-6.9months of age at the beginning of the study and body weight was not significantly different between genotypes throughout the duration of the study, which lasted 10 weeks (Supplementary Information S1).

| Animal care and maintenance
Female Vipr2 À/À and C57BL/6 wild-type (WT) control mice were maintained in accordance with the guidelines in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. 54 The experimental timeline is shown in Figure 1.

| Glucose and insulin tolerance tests
To measure glucose tolerance, mice were fasted overnight (ON) for 11 h and glucose (2.0 g/kg b.w.) was administered by i.p. injection (IPGTT).Tail blood ($1 μL) was collected at the end of the fast at t = 0 (fasting blood glucose, FBG), and 15, 30, 60, and 120 min postinjection and blood glucose was measured using a glucose meter F I G U R E 1 Experimental Timeline.Female Vipr2 À/À mice or wild-type littermates (5.6-6.9 months) were subjected to in vivo and ex vivo tests.At sacrifice, mice were euthanized by isoflurane anesthesia and exsanguination, and blood and other tissues were collected for post mortem analyses: ELISA, adrenal epinephrine assay, and adrenal and brain RT-qPCR.CORT, corticosterone; EPI, epinephrine; GLP-1, glucagon-like peptide 1.
(OneTouch Ultra 2, LifeScan Inc.) and corresponding test strips.One week later, an insulin tolerance test (ITT) was performed using Humulin R (Eli Lilly, USA): weak challenge (morning fast, 5 h, 0.38 U/kg) or strong challenge (ON fast, 8 h, 0.25 U/kg).Tail blood was collected at t = 0, 15, 30, 45, 60, 90, and 120 min post-injection and plasma glucose was measured.The area under (AUC) or above the glycemia curve (inverse AUC), obtained during GTT and ITT, respectively, was calculated over 0-120 min post injection.To determine insulin sensitivity in vivo, the percent blood glucose reduction rate after insulin administration, K ITT , was calculated over the first 30 min using the formula (0.693 Â 100) Â t 1/2 .Half-life (t 1/2 ) was calculated from the slope of the blood glucose concentration response during 0-30 min post insulin injection, when plasma glucose concentration declines linearly. 55

| Stress-induced hyperglycemia
Two weeks after ITT, mice were subjected to immobilization stress for 90 min in a quiet, well-ventilated room at 22.7 C using 50 mL clear polystyrene conical tubes which were perforated with numerous air holes for ventilation.Tail blood was sampled while mice were restrained at t = 0, 15, 30, 60, 90 min after the start of restraint and during the following 30-min rest (at t = 120 min). 56,57The area under the stress glycemia curve was calculated over 0-120 min.

| Measurement of plasma corticosterone and epinephrine
Three weeks after stress-induced hyperglycemia, tail blood (30-80 μL) was obtained immediately following a 1-h restraint session (fed state) and collected into heparinized hematocrit tubes and stored on ice.Plasma epinephrine was measured using a commercial competitive high sensitive ELISA kit (Eagle Biosciences, ADU39-K01).After blood collection, plasma was separated via refrigerated centrifugation, stabilized with the Sample Stabilizer reagent provided in the kit and stored at À80 C until further use.Within 10-14 days of plasma collection, 8-30 μL of plasma was removed from each sample and used for the epinephrine assay.This assay is specifically designed for mouse/rat plasma and had a sensitivity of 1.6 pg/mL.The same plasma samples were diluted 10-fold and corticosterone (CORT) was measured using a commercial competitive ELISA kit (Abcam, AB108821).The assay had a sensitivity of 0.30 ng/mL in a standard range of 0.39-100 ng/mL.The unknown plasma epinephrine and CORT concentrations of test samples were determined by plotting their absorbance values and interpolating the concentration using a 4-parameter-logarithmic standard curve.

| Trihydroxyindole catecholamine assay
Epinephrine content in adrenal glands was measured using a modification of the trihydroxyindole method as described. 58Using this method of catecholamine oxidation at 0 C, Kelner and colleagues obtained values for epinephrine content in bovine chromaffin cell lysates that were nearly identical to those measured using HPLC/electrochemical determination. 59Briefly, adrenal tissue homogenates were centrifuged at 15,000 Â g at 0 C for 15 min.Sample supernatant was incubated with 10% acetic acid.Then 0.25% K 2 Fe(CN) 6 was added to each sample and the mixture was incubated at 0 C for 20 min.The oxidation reaction was stopped by the addition of NaOH solution containing alkaline ascorbate.Fluorescence emission was determined at 520 nm using a fluorescence plate reader (GloMax, Promega, USA).
Each sample yielded mean fluorescence intensity units that were converted into epinephrine concentration expressed as μg/g adrenal wet weight using calibration standards and polynomial curve fitting.

| Quantitative polymerase chain reaction analysis
RT-qPCR was used to quantitate mRNA transcripts for the following genes: Th, Crh, Adcyap1, Adcyap1r1, Vip, and Slc17a6 (encodes VGLUT2).DNA oligonucleotide PCR primers were custom-or predesigned (Integrated DNA Technologies, Coralville, IA, USA).Custom primers were designed to meet several criteria using NCBI Primer Blast.Primers were used to reverse transcribe RNA template to complementary DNA (cDNA) using RT-PCR (iScript kit, # 1708890, Bio-Rad Laboratories, Hercules, CA, USA) and ran on an electrophoresis gel.Only primers that gave single-band amplicons in the presence of RT and that matched the base length of the predicted target were selected.In addition, the selected primers yielded 87.7%-104.2%efficiency on RT-qPCR (Table 1).For some custom-designed primers, a increments melt curve analysis.To rule out extraneous nucleic acid contamination and primer dimer formation, no-template controls were included in each experiment.Additionally, to rule out presence of genomic DNA, negative RT controls, which contained the complete RNA synthesis reaction components without the addition of the enzyme reverse transcriptase (RT) were included.RT-qPCR experiments were performed in adherence to MIQE guidelines 60 (Supporting Information S2).

Expression of GOI was measured relative to the reference gene,
ActB, and differential gene expression was determined compared to WT using the Pfaffl method. 61Vipr2 gene deletion did not affect the stability of the ActB reference gene (data not shown).Mean (±s.e.m.) cycle quantification (Cq) values for medulla were 18.81 ± 0.34 and 18.79 ± 0.22 and for hypothalamus were 18.33 ± 0.20 and 18.38 ± 0.15 and for adrenal were 17.90 ± 0.27 and 17.93 ± 0.39 for WT and Vipr2 À/À mice, respectively.Since Vipr2 gene deletion produced an apparent reduction in adrenal weight, adrenal data are presented as both relative gene expression and copy number/ng total RNA per mg of fresh adrenal tissue obtained at sacrifice.To calculate copy number, for each sample, the corresponding ng dsDNA was determined for the mean sample Cq using the GOI standard curve.The value was inserted into the following equation that uses Avogadro's number, length of amplicon (in bp), and average mass of 1 bp dsDNA (660 ng) to calculate the mean absolute transcripts value (copy number): T A B L E 1 RT-PCR primer sequences, efficiencies, and PCR products.

| Vipr2 gene deletion produced fasting hyperglycemia
Pre-diabetes is characterized by abnormally high FBG levels.In order to examine the effect of Vipr2 gene deletion on glucose metabolism, blood glucose levels were examined after 5 (AM), 8 (ON), and 11 (ON) hours of fasting as well as in the ad libitum-fed state.Table 2 illustrates that mean glycemia values for the ad libitum-fed state were significantly greater in Vipr2 À/À than WT mice (Student's t-test: t (17) = 1.78, p = 0.047).This was also the case after an 8 h ON fast.A Holm-Sidak post hoc test for multiple comparisons across genotype revealed a significantly increased hyperglycemia in Vipr2 to normal by 120 min (Figure 2A).There were no group differences in magnitude or duration of glycemia represented as the area under the glucose curve, AUC GTTglucose (Welch's t-test: t (14.6) = 0.15, p = 0.88) (Figure 2B).Interestingly, some individual Vipr2 À/À mice showed peak glycemia at 30 min, whereas all of the WT mice had earlier peak responses occurring at 15 min after injection (Figure 2C, Mann-Whitney U = 24.50,n 1 = 15, n 2 = 15, adjusted p = 0.06).
Correlation and regression analysis for stress glycemia (AUC) versus plasma GLP-1 yielded an apparent negative relationship in WT: r = À0.83,p = 0.04, R 2 = 0.70, p = 0.08 for WT and r = À0.41,p = 0.13, R 2 = 0.17, p = 0.27 for Vipr2 À/À (Figure 8D).These results may indicate that high stress glycemia values are associated with low GLP-1.Because this did not occur in Vipr2 gene-deleted mice, gene deletion may be interfering with GLP-1 glucoregulatory actions.Additionally, GLP-1 was not correlated to impaired insulin hypoglycemia nor to plasma insulin levels suggesting unrelated regulation of these parameters in our gene-deleted mice (Supporting Information S1).

| Vipr2 gene-deletion produced exaggerated stress-induced plasma epinephrine but not CORT
Given that Vipr2 À/À mice displayed exaggerated stress-induced hyperglycemia, we tested for group differences in plasma levels of stress hormones after 1 h of restraint stress.At this time point, mean plasma epinephrine was significantly elevated relative to baseline in Vipr2 À/À but not WT mice.A two-way ANOVA revealed a significant effect of stress (F (1,15) = 9.54, p = 0.008) and genotype (F (1,15) = 5.8, p = 0.03) without a significant stress Â genotype interaction (F (1,15) = 1.42, ns).A Holm-Sidak post-hoc test for multiple comparisons showed a mean elevation in plasma epinephrine in 1 h restraint stress versus sham mice that was significant for Vipr2 À/À (p = 0.006) and apparent for WT (p = 0.25) (Figure 6A).Mean epinephrine values during restraint were significantly elevated in Vipr2 À/À compared to WT (p = 0.04).These results suggest that Vipr2 gene deletion exaggerates SA epinephrine secretion after acute psychogenic stress.To determine if Vipr2 gene F I G U R E 4 Vipr2 gene deletion elevated plasma insulin and lowered GLP-1.Blood collected at sacrifice (fasted state) obtained from Vipr2 À/À and WT female mice was assayed for plasma levels of insulin (A) and glucagon-like peptide 1 (GLP-1) (B) using antigen-specific ELISAs.deletion affects levels of adrenal epinephrine, we compared group mean values at sacrifice.Adrenal epinephrine content was significantly lower in Vipr2 À/À compared to WT mice (Mann-Whitney U = 8, n 1 = 624.3,n 2 = 170.9,p =0.0013) (Figure 6C).This occurred in spite of an apparent reduction in adrenal weight compared to WT (Student's t-test: t (17) = 1.92, p = 0.07) (Figure 6D).Correlation and regression analysis showed no significant relationship between stress hyperglycemia and plasma epinephrine in either group (Supporting Information S1).However, an apparent negative relationship (Vipr2 À/ : r = À0.71,p = 0.06, R 2 = 0.51, p = 0.11) was found between plasma and adrenal epinephrine in the mutants only (Figure 8C).

| Vipr2 gene-deletion altered gene expression of hypothalamic Vip and brainstem Th
We used PCR analysis to assess mRNA expression of gene markers for PACAP, VIP, sympathetic nervous, and HPA systems in the hypothalamus, medulla oblongata, and adrenal gland.The results reveal a significant downregulation of hypothalamic Vip (Student's ttest: t (17) = 3.8, adjusted p value <0.05) and an apparent upregulation of hypothalamic Adcyap1 (Welch's t-test: t (14.4) = 2.09, adjusted p value = 0.08) in Vipr2 À/À compared to WT (Figure 7).There was no transcript alteration in hypothalamic Crh, a marker of HPA axis activity, in Vipr2 À/À versus WT.In medulla, Vipr2 À/À mice showed a significant upregulation in Th (Welch's t-test: t (12.6) = 2.5, adjusted p value = <0.05)and an apparent upregulation of Vglut2 (Student's ttest: t (14) = 2.2, adjusted p value = 0.09).In adrenal, Vipr2 À/À mice showed an apparent downregulation in Vip (Student's t-test: t (16) = 2.6, adjusted p value = 0.08).Adrenal copy number for mRNA levels indicated no significant group differences for any GOI studied (Adcyap1, Adcyap1r1, Vip, Th).Because adrenal weight may have affected our comparisons, Table 3 also illustrates the adrenal gene copy number normalized to adrenal weight.However, no significant group differences were found.Correlation and regression analysis between stress peak glycemia versus hypothalamus Vip yielded opposite relationships in mutants (positive and apparent) and WT (negative and apparent) (r = 0.45, p =0 .07,R 2 = 0.20, p = 0.14 for Vipr2 À/À ; r = À0.70,p = 0.04, R 2 = 0.49, p = 0.08 for WT) (Figure 8E).However, only WT showed a good linear fit.No significant correlation was measured between stress plasma epinephrine versus hypothalamic Vip expression or between stress glycemia versus medulla Th expression or between plasma epinephrine versus medulla Th expression (Supporting Information S1).
F I G U R E 6 Mild psychogenic stress induced exaggerated epinephrine but not corticosterone secretion in Vipr2 À/À mice.(A) Plasma epinephrine measured after 1 h restraint stress was exaggerated in Vipr2 À/À but not WT female mice.(B) Plasma corticosterone measured after 1 h restraint stress was elevated in both Vipr2 À/À versus WT. (C) Epinephrine content in adrenal glands harvested at necropsy was reduced in PACAP and VIP regulate energy metabolism through central and peripheral actions, which may involve the SA as well as the HPA axes. 7,14,34,37,65,66However, the role of PACAP and VIP receptors during physiological challenges to glucose homeostasis is unclear.Our studies performed on VPAC2R gene-deleted female mice have identified a diabetogenic phenotype that includes fed and fasting hyperglycemia and impaired insulin-induced hypoglycemia compared to WT mice.Additionally, Vipr2 À/À female mice displayed exaggerated stress-induced hyperglycemia and plasma epinephrine levels, suggesting hyperactivity of the SA system in these mice.In support of this, Vipr2 À/À mice displayed reduced adrenal epinephrine content and upregulation of Th in medulla oblongata (and downregulation of Vip in hypothalamus) at rest.In addition, VPAC2R gene deletion produced low plasma GLP-1 and elevated plasma insulin fasting levels.In combination, these data are consistent with a physiological role of VPAC2R in glucose regulation, especially during metabolic and psychogenic stress, likely involving the participation of SA activity and/or metabolic hormones.
We examined the effect of genotype on the fasting glucose response to insulin challenge.A previous study demonstrated that VPAC2R gene-deleted females displayed normal responses, while males showed improved insulin sensitivity after an exogenous insulin challenge (4 h AM fast, 0.75 IU Humulin IP). 67Using a similar paradigm, we also observed no effect of Vipr2 gene deletion on insulin sensitivity in female mice.However, when fast conditions were made more intense (8 h ON, 0.25 IU Humulin), female Vipr2 À/À mice displayed impaired insulin hypoglycemia in spite of normal glucose uptake initially after insulin challenge (K ITT ) compared to WT.Given our GTT results, this is not likely due to less glucose uptake by skeletal muscle, the primary route of glucose disposal. 68Instead, impaired insulin hypoglycemia may result from dramatically low plasma levels of the incretin GLP-1 in the mutant mice leading to less GLP-1-mediated suppression of endogenous glucose production.Furthermore, reduction of circulating GLP-1 may have hindered its facilitation of glucose disposal. 69Given the ability of GLP-1 agonists to control fasting glycemia and glucose tolerance, understanding the role of VPAC2R in regulating GLP-1 may aid in efforts to discover new therapeutic targets for type 2 diabetes (T2D). 70,71Fasted plasma GLP-1 levels were not correlated to impaired insulin hypoglycemia nor to elevated plasma insulin levels found in Vipr2 À/À mice, suggesting unrelated regulation of these parameters in these mice.Similarly, no significant relationship was detected between plasma GLP-1 and insulin glycemia response, stress glycemia, or plasma epinephrine in Vipr2 À/À mice.Little is known about the role of VPAC2R in GLP-1 hormone regulation, although VPAC1R and VIP-deficient mouse phenotypes and PACAP receptor antagonism are associated with increased fasting and postprandial GLP-1 levels. 7,72,73PAC1R-and T A B L E 3 PCR transcripts in adrenal gland.
It is unlikely that impaired hypoglycemia during insulin challenge was a direct result of loss of VPAC2R function, since activation of VPAC2 receptors does not inhibit hepatic glycogenolysis and secretion of the glycogenolytic hormone glucagon. 28In fact, because of their inability in this process, in conjunction with their insulinotropic action, VPAC2 receptors are studied for their potential as a novel target for the treatment of T2D. 28,30More likely is the possibility that, in the absence of VPAC2R in our mutant mice, PACAP-or VIP-mediated activation of the preserved and unchallenged VPAC1 and/or PAC1 receptors, respectively, can promote glucose output via direct hepatic glycogenolytic effects and/or indirectly by stimulating pancreatic secretion of glucagon to counter insulin action. 26,27,75Indeed, deletion of the PAC1R gene produces impaired but viable insulin-induced hypoglycemia and impaired secretion of glucagon. 75Taken together, these data support the view that PACAP and/or VIP, by activating either PAC1 or VPAC receptors, in addition to their insulinotropic action, regulate glucose homeostasis in a complex manner that remains to be fully elucidated.
Another counterregulatory response to insulin hypoglycemia is direct sympathetic and/or indirect SA activation of hepatic glucose output.Like glucagon, epinephrine counters glucose uptake by inhibiting insulin secretion, but also by promoting the liberation of glucose from liver via glycogenolysis and gluconeogenesis. 76Indeed, impaired long-term secretion of epinephrine and depletion of its adrenomedullary stores has been implicated in the lethal hypoglycemic responses to insulin observed in PACAP-deficient mice. 33Under a single prolonged (90 min) restraint, our Vipr2 À/À mice displayed exaggerated hyperglycemia as compared to WT mice.Gene-deleted mice also displayed a markedly elevated plasma epinephrine in response to stress.
These results suggest that defunctionalization of VPAC2R may result in hyperglycemia via PACAP/VIP-mediated sympathetic activation. 33,34,77Our results prompt us to speculate that exaggerated stress hyperglycemia in Vipr2 À/À mice is either due to removal of potentially inhibitory actions of VPAC2R on sympathetic activation of the liver and/or adrenal and/or due to the unopposed stimulatory actions of VPAC1R and PAC1R on the liver. 26,27,75In any case, in the mutants, we found an apparent negative association between adrenal epinephrine versus plasma epinephrine, suggesting that circulating epinephrine responses to psychogenic stress were not compensated for by enhanced adrenal epinephrine biosynthesis.Furthermore, exaggerated stress glycemia in Vipr2 À/À was not likely due to an overly active HPA axis, since both genotypes displayed similar elevated stress levels of CORT, which contributes to hepatic glucose output and is regulated by PACAP and VIP. 66CAP and VIP are co-transmitters with acetylcholine at the adrenomedullary synapse, where sympathetic regulation of epinephrine occurs in response to stress, secondary to the induction of Th. 78 Using in vivo restraint stress, Stroth and colleagues showed that PACAP gene deletion significantly reduces, but does not abolish, the upregulation of adrenal Th and Pnmt, suggesting that both PACAP and VIP signaling contribute to catecholamine biosynthesis and secretion.35 Given that application of VIP and PACAP to in vitro adrenal slices stimulates catecholamine release via the activation of all three VIP/PACAP receptor subtypes, 79 our findings that gene deletion of Vipr2 promoted stress-induced epinephrine release and exaggerated stress-induced hyperglycemia may represent an inhibitory central action by VPAC2R and/or upregulated stimulatory activity of the preserved PAC1 receptors.80 In support of the former, Inglott and colleagues reported that activation of VPAC receptors, in contrast to that of PAC1 receptors, decreased splanchnic sympathetic nerve activity when activated by intrathecal VIP.81 It should be noted that baseline levels of adrenal mRNA transcripts for Adcyap1r1 and Adcyap1 were not significantly altered in Vipr2 À/À mice, ruling out compensatory changes in PACAP or PAC1 receptor in upregulating adrenal epinephrine and consequent hyperglycemia.Additional mechanistic experiments will need to be undertaken to decipher the role of VPAC2R in hyperglycemia responses to psychogenic stress.
Centrally, VPAC2R may have a differential contribution to hepatic glucose output and glucose disposal.For example, VPAC2Rs in the RVLM of medulla mediate sympathoexcitation induced by PACAP. 41other study by Yi and colleagues, demonstrated the participation of central VPAC2R on pre-autonomic neurons in the PVN of the hypothalamus in promoting sympathetic stimulation of hepatic glucose production. 37PACAP-immunoreactive pre-autonomic neurons in the PVN tightly regulate peripheral glucose homeostasis via projections to the brainstem.These brainstem neurons include the adrenergic C1 group pre-sympathetic neurons in medulla. 52,53In spite of the sympathomimetic actions of VPAC2R in the PVN, we found that transcript modifications in Vipr2 À/À mice are consistent with SA activation: upregulation of medulla Th and downregulation of hypothalamic Vip.
Although further clarification on the actions of central VPAC2Rs is needed, these central gene alterations may help explain the elevated hyperglycemic responses seen during insulin challenge and psychogenic stress in Vipr2 À/À mice.Vipr2 À/À mice also displayed baseline hyperglycemia in fed and fasted states (8 h ON fast), likely due to unchallenged VPAC1R-mediated hepatic glucose production [26][27][28] with additional contribution of an enhanced basal metabolic rate. 67rrelation and regression analyses of peak stress glycemia versus hypothalamic Vip expression indicated a negative relationship.Interestingly, this occurred in WT only, suggesting that other factors may influence this relationship in Vipr2 À/À .
The combined results of the insulin glycemia and stress-induced glycemia assays provide a more complete phenotype of Vipr2 À/À mice.To determine if the metabolic profiles in Vipr2 VPAC2R contributes to physiological responses to metabolic and psychogenic challenges in several ways.[85] 4.1 | Strengths and limitations to the study The strength of the current study is a more complete characterization of the physiological, and homeostatic effects of VPAC2R gene deletion using a null murine model.Our findings indicate participation of VPAC2R in the regulation of glucose homeostasis under metabolic and psychogenic stress.A limited number of mice were used and, therefore, all mice were required for all assays.To minimize potential confounds, we allowed 1-3 weeks between assays.All mice necessarily received restraint stress to measure hyperglycemia responses.
However, only a subset within each genotype group received stress in the following assay measuring plasma epinephrine.Importantly, correlation/regression analysis conducted on the values of consecutive assays did not indicate a predictive relationship on outcomes in both genotype groups arguing against the possibility of procedurally biased outcomes.
Although we did not measure feeding patterns in our mutant mice, their reported circadian deficit should not have impacted the timing of feeding and of glycemia values in fed and fasted states since mice were housed under a steady light: dark cycle.Harmar and colleagues showed that upon exposure to continuous dim red light VPAC2R mutant males failed to express circadian clock genes and exhibited grossly disrupted rhythms of activity. 16However, there was little disruption in the presence of a normal 12-h light:dark cycle, that is, only a short lag in activity onset when responding to the dark periods and similar periods of rhythmicity.
One weakness of the current study is its reliance on a transgenic animal model, although it provides an alternative to pharmacological manipulation of PACAP/VIP receptors which, up to now, has relied on only moderately selective drugs.Moreover, using the transgenic approach, we were unable to evaluate whether VPAC2R-mediated effects were due to the potential binding of either PACAP or VIP, for which the receptor has equal affinity. 86In addition, VPAC2R gene deletion produced unexpected changes in the gene expression of central PACAP/VIP systems, namely upregulation of Adcyap1 in the hypothalamus (apparent).This change, in combination with upregulation of Th in medulla (significant), downregulation of Vip in hypothalamus (significant) and adrenal gland (apparent) may have contributed to the phenotypic effects observed.
Vipr2 À/À female mice displayed impaired insulin hypoglycemia and exaggerated stress hyperglycemia.The potential relationship between these two phenotypes is unclear and requires further study.
Correlation/regression analysis indicated a positive apparent relationship between insulin glycemia responses (inverse AUC) and stress glycemia only in Vipr2 À/À mice, suggesting that abnormally elevated Mice were housed in standard polycarbonate plastic cages (3-4 per cage) with corn cob bedding, unless otherwise noted.Food pellets (Laboratory Rodent Diet 5001, LabDiet, USA) and municipal tap water were provided ad libitum except as required during the experimental period.Temperature was maintained at 21.1-22.8C and relative humidity at 20%-70% under a 12-12 h photoperiod (lights on from 7:00 a.m. to 7:00 p.m.).All experiments were approved by the IACUC at the University of California Riverside (AUP# 20170026 and 20200018) and the University of California Los Angeles (AUP# 93-007).Experiments were conducted between 9:00 a.m.-4:00 p.m., unless otherwise noted.
temperature gradient was used to optimize efficiency at an ideal annealing temperature.The primer concentration ranged from 200 to 500 nM.RT-qPCR was performed on RNA (10 ng) samples, run in triplicate, on a CFX Connect or CFX96 (Bio-Rad Laboratories, Hercules, CA, USA) thermocycler with the Luna Universal one-step qPCR Master Mix (E3005; New England Biolabs, Ispwich, MA, USA).Amplification reactions for genes of interest (GOI) were performed in 50 cycles of the following cycling protocol: reverse transcription 55 C/10 min; RT deactivation 95 C/1 min; per cycle 95 C/10 s denaturation, 60 C or 55 C/30 s anneal/extension; 65-95 C in 0.5 C, 5 s Vipr2 À/À displayed reduced glycemia after a 5 h AM fast when compared to ad libitum-fed state (baseline).In contrast, WT values remained similar to their corresponding baseline.A RM two-way mixed effects ANOVA revealed a statistically significant effect of treatment (F (3,51) = 5.24; p = 0.003) and genotype (F (1,17) = 3.52; p = 0.08) but not interaction (treatment Â genotype F (3,51) = 1.65, ns).

F I G U R E 2
Vipr2 gene deletion did not alter glucose tolerance.(A-C) Mice were fasted for 11 h overnight and tail blood was sampled for glucose before (t = 0 min) and after (t = 15, 30, 60, and 120 min) i.p. injection of 2.0 g/kg glucose (IPGTT).(A) Mean absolute blood glucose concentrations taken during IPGTT in Vipr2 À/À and WT.(B) Corresponding mean values for the integrated area under the glucose curve (AUC GTTglucose ).(C) Latency to maximum glycemia during IPGTT.a Significantly different from corresponding baseline ( a p < 0.01-0.0001).n, 7-12/ group.

F I G U R E 3
Vipr2 gene-deletion impairs insulin hypoglycemia.Absolute blood glucose concentrations before and at t = 15, 30, 45, 60, 90, and 120 min post-injection with insulin for female Vipr2 À/À and WT mice were recorded after weak hypoglycemic stress (A-C) (5 h morning fast, 0.38 U/kg Humulin R) and strong hypoglycemic stress (D-H) (8 h ON fast, 0.25 U/kg Humulin R). (A) Mean absolute blood glucose response to hypoglycemic stress (5 h morning fast, 0.38 U/kg Humulin).(B) Glycemia was analyzed by an inverse integrated area under the ITT glucose curve (AUC ITTglucose ).(C) The corresponding rate constant for percent glucose reduction (K ITT ) was calculated over the initial slope of the ITT glucose response curve from 0 to 30 min post injection.(D) Absolute blood glucose concentrations were taken over the post-injection time course after strong stimulation.(E) Corresponding mean values for the inverse integrated area under the ITT glucose curve (AUC ITTglucose ).(F) The early effects of insulin, represented as K ITT insulin, were measured over the first 30 min post injection.(G) Glucose values taken during ITT are plotted versus time as a percentage of the individual basal level.(H) The corresponding inverse integrated area (AUC) under the percent basal glucose curve (AUC ITTglucose ).*Significantly different versus WT; *p < 0.05; **p < 0.01; a Significantly different from corresponding baseline ( a p < 0.05-0.0001).n, 6-12/group.reflected in the area under the restraint glucose curve (AUC GlucoseRes ) (Student's t-test: t (17) = 3.05, p = 0.007) (Figure 5B).

F I G U R E 8
Correlation analysis between selected assay outcomes.Pearson correlation coefficient (r), coefficient of determination (R 2 ) with corresponding p-values indicate the strength of the relationship between two variables and the prediction of the value of one continuous variable on another, respectively.(A) Inverse AUC ITTglucose (% basal) versus stress glycemia AUC.(B) Nadir insulin glycemic response (% baseline) versus peak stress glycemia.(C) Adrenal epinephrine versus stress plasma epinephrine.(D) Stress hyperglycemia AUC versus plasma GLP-1.(E) Peak stress glycemia versus hypothalamic Vip expression.*Indicates a significant linear fit ( p < 0.05).EPI, epinephrine.
stress hyperglycemia was not positively associated with deficient insulin hypoglycemia.It is unclear what mechanisms underlie this relationship in gene-deleted mice, which show abnormal responses in both outcomes relative to WT.Furthermore, these abnormal glycemia responses did not associate significantly with altered SA control of plasma epinephrine or plasma GLP-1 levels.Furthermore, Vipr2 À/À mice did not show the apparent negative association between hypothalamic Vip and stress glycemia nor between stress glycemia and plasma GLP-1 observed in WT mice, suggesting other interactions likely exist.Nevertheless, our findings provide novel information about the physiological role of VPAC2R in glucose homeostasis under metabolic and psychogenic stress.
, we examined the glycemia response to exogenous insulin during