Vitamin D actions in neurons require the PI3K pathway for both enhancing insulin signaling and rapid depolarizing effects.

Abstract

Despite correlations between low vitamin D levels and diabetes incidence/severity, supplementation with vitamin D has not been widely effective in improving glucose parameters. This may be due to a lack of knowledge regarding how low vitamin D levels physiologically affect glucose homeostasis. We have previously shown that the brain may be a critical area for vitamin D-mediated action on peripheral glucose levels. However, the mechanisms for how vitamin D acts in the brain are unknown. We utilized a multimodal approach to determine the mechanisms by which vitamin D may act in the brain. We first performed an unbiased search (RNA-sequencing) for pathways affected by vitamin D. Vitamin D (1,25-dihydroxyvitamin D3; 1,25D3) delivered directly into the third ventricle of obese animals differentially regulated multiple pathways, including the insulin signaling pathway. The insulin signaling pathway includes PI3K, which is important in the brain for glucose regulation. Since others have shown that vitamin D acts through the PI3K pathway in non-neuronal cells (muscle and bone), we hypothesized that vitamin D may act in neurons through a PI3K-dependent pathway. In a hypothalamic cell-culture model (GT1–7 cells), we demonstrate that 1,25D3 increased phosphorylation of Akt in the presence of insulin. However, this was blocked with pre-treatment of wortmannin, a PI3K inhibitor. 1,25D3 increased gene transcription of several genes within the PI3K pathway, including Irs2 and p85, without affecting expression of InsR or Akt. Since we had previously shown that 1,25D3 has significant effects on neuronal function, we also tested if the PI3K pathway could mediate rapid actions of vitamin D. We found that 1,25D3 increased the firing frequency of neurons through a PI3K-dependent mechanism. Collectively, these data support that vitamin D enhances insulin signaling and neuronal excitability through PI3K dependent processes which involve both transcriptional and membrane-initiated signaling events.

1. Introduction

Vitamin D deficiency has been linked to both weight gain and insulin resistance [1,2]. However, the causal link between these disorders is not well described. Part of this discrepancy is likely due to the complicated nature of vitamin D signaling. Vitamin D is ingested or synthesized in the skin as a prohormone, mainly cholecalciferol (also known as D3). D3 then undergoes two hydroxylation events, one to make 25-hydroxyvitamin D (25OHD) and a subsequent hydroxylation to make 1α,25-dihydroxyvitamin D3 (1,25D3). What is fairly unique to the vitamin D pathway, is that clinical diagnosis of vitamin D deficiency is made by measuring a precursor, 25OHD, but the end effects of vitamin D occur through 1,25D3 acting through the vitamin D receptor (VDR). VDRs are present in multiple organs of the body and are reported to control thousands of genes [3,4]. Thus, there are a variety of unexplored pathways by which vitamin D may regulate glucose levels.

In vitro data shows that β-cells from VDR-null mice have normal insulin secretion capacity [5], which would imply that it is possible the effects of VDR loss in animals on glucose homeostasis may occur through other tissues. We have previously shown that the active form of vitamin D, 1,25D3 (also known as calcitriol), can improve whole body insulin sensitivity in obese animals through action in the brain [6]. We also demonstrated that knockdown of vitamin D receptors in the paraventricular nucleus of the hypothalamus resulted in glucose intolerance in obese male mice [6]. Of note, both exogenous administration of vitamin D and knockdown of vitamin D receptors affected physiological parameters only in animals on a high-fat diet; neither had any effect on chow-fed animals [6]. However, the molecular mechanisms underpinning vitamin D-mediated alterations in glucose homeostasis through action in the brain in obese animals are still unclear.

In this study, we investigated how vitamin D affects neuronal function and cellular processes. We utilized 1) unbiased pathway analysis on RNA-sequencing results of hypothalamic tissue from animals treated with vitamin D in vivo, 2) hypothalamic cell culture, and 3) ex vivo electrophysiological studies. We show here that vitamin D has genomic and non-genomic actions mediated through a common pathway and discuss how these are biologically relevant.

2. Methods

2.1. Animals:

Animals were housed at Baylor College of Medicine (BCM) on a 12 hour light/dark cycle with ad libitum access to water and food. Animals were on a high-fat diet (HFD; 40% high-fat butter diet, Research Diets, New Brunswick, NJ) to induce obesity for either 8 weeks (mice) or 26 weeks (rats) prior to euthanasia. Studies used adult, male Long-Evans rats for RNA-sequencing studies (Harlan, Indianapolis, IN) to generate possible mechanistic hypothesis underlying our previous data demonstrating beneficial effects of exogenous vitamin D on glucose tolerance. For electrophysiology studies [7], it was necessary to switch to a mouse model in order to take advantage of genetic models to identify paraventricular hypothalamic neurons (Sim1-Cre/Rosa26-tdTOMATO mice). Animal numbers are stated in the figure legends. All studies were approved by BCM Institutional Animal Care and Use Committee as applicable.

2.2. Surgeries:

In order to deliver vitamin D directly into the brain, intracerebroventricular cannulas were placed into the third ventricle (i3vt) in rats at 9-10 weeks of age. Cannulas were surgically implanted into i3vt as previously described [8]; coordinates: 2.2A/P, 7.8D/V, as determined by the atlas of Paxinos and Watson [9,10].

2.3. Drugs:

For all vitamin D studies, we used 1,25D3 (17936–1mg, Sigma Aldrich, St. Louis, MO). 1,25D3 was dissolved in hydroxypropyl-β-cyclodextrin (THPB-EC, CTD, Inc., Alachua, FL) for in vivo experiments as previously described [6] and in DMSO for cell culture experiments and electrophysiology experiments. A cyclodextrin vehicle [11] was chosen for in vivo work due to known toxic effects of DMSO on neurons at high/prolonged concentrations [12–14]. For in vivo studies, THPB-EC was used as the control and for cell culture and electrophysiology studies, we used DMSO as a control.

2.4. RNA-sequencing:

Male, Long-Evans rats were injected i3vt with 2 μL of 1,25D3 (0.1 μg) or vehicle (THPB-EC) two hours prior to euthanasia (n=3/group). This dose of 1,25D3 was based on our previous research showing that this dose produced an improvement in glucose tolerance and peripheral insulin sensitivity [6]. They were anesthetized with isoflurane, decapitated, and the hypothalamus dissected and immediately frozen in liquid nitrogen. Tissue samples were stored at −80 degrees C until use. Samples were homogenized and RNA extracted utilizing Qiagen Mini Lipid kit (#74804, Qiagen, Germantown, MD) and Qiagen RNeasy Mini Kits (#74104). A double-stranded DNA library was created using 250 ng of total RNA (measured by picogreen), preparing the fragments for hybridization onto a flowcell. First, cDNA was created using the fragmented 3’ poly(A) selected portion of total RNA and random primers. During second strand synthesis, dTTP is replaced with dUTP which quenches the second strand during amplification, thereby achieving strand specificity. Libraries were created from the cDNA by first blunt ending the fragments, attaching an adenosine to the 3’ end and finally ligating unique adapters to the ends. The ligated products were then amplified using 15 cycles of PCR. The resulting libraries were quantitated using the NanoDrop spectrophotometer and fragment size assessed with the Agilent Bioanalyzer. A qPCR quantitation was performed on the libraries to determine the concentration of adapter ligated fragments using Applied Biosystems ViiA7 Real-Time PCR System and a KAPA Library Quant Kit. Using the concentration from the ViiA7 qPCR machine above, 29pM of equimolarly pooled library was loaded onto three lanes of a high output v4 flowcell (Illumina p/n PE-401-4001) and amplified by bridge amplification using the Illumina cBot machine (cBot protocol: PE_HiSeq_Cluster_Kit_v4_cBot_recipe_v9.0). PhiX Control v3 adapter-ligated library (Illumina p/n 15017666) is spiked-in at 2% by weight to ensure balanced diversity and to monitor clustering and sequencing performance. A paired-end 100 cycle run was used to sequence the flowcell on a HiSeq 2500 Sequencing System (Illumina p/n FC-401-4003). RNA sequencing results were first trimmed for low quality reads using TrimGalore (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/). Data was next mapped using HISAT2 [15] onto the mouse genome build UCSC rn6 and gene expression quantified using readCounts [16] against the GENCODE [17] model. Differentially expressed genes were determined using the R packages RUVSeq [18] and DESeq2 [19], with significance achieved for fold change exceeding 1.25x and FDR-adjusted p-value<0.05. Differentially expressed genes were analyzed for enriched pathways and processes using Gene Set Enrichment (GSEA [20])(Broad Institute) data analysis, and GSEA implementation at the Molecular Signature Database (MSigDB [21]) (http://www.broadinstitute.org/gsea/msigdb), with significance achieved for FDR-adjusted p-value<0.2.

2.5. Cell culture:

GT1-7 cells (Mouse hypothalamic GnRH neuronal cells) were purchase from Millipore. The cells were routinely cultured at 37°C in normoxia conditions (5% CO2, 95% air) in High Glucose Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma Cat. No. D6546) supplemented with 10% fetal bovine serum (Life Technologies) and 1% penicillin-streptomycin. Cells were plated at 6x10⁵ cells/cm². At 85-90% confluence, cells were treated with 1,25D3 at 10 nM for 2 or 48 hours. This vitamin D dose was chosen based on other experiments showing optimal results in neuronal cell culture [22]. For experiments requiring insulin, 100 nM insulin was added during the last 20 min. Wortmannin 100 nM was added 10 minutes prior to insulin treatment for selected experiments. Cell culture experiments were done in 3-4 different experimental replicates, with each experimental replicate compromising the average of at least 3 technical replicates.

2.6. Real Time RT-PCR:

Cell RNA was extracted using a Qiagen RNeasy kit. cDNA was isolated and real-time quantitative PCR (qPCR) was performed using a TaqMan 7900 sequence detection system with TaqMan universal PCR master mix and TaqMan gene expression assays (all from Applied Biosystems). Genes were run multiplexed with 2 housekeeping genes, pgk1 and b2m. All samples were run in triplicate. Relative mRNA expression for the genes within the PI3K pathway were calculated relative to the average of the housekeeping genes using the ΔΔCT method [23]. Primers used are listed in Table 1.

Table 1.

Primers for Real-time RT-PCR.

IRS2	Mm03038438
P85	Mm01282781
P55	Mm00725026
PI3KCA	Mm00435673
IRS1	Mm01278327
INSR	Mm01211875
AKT	Mm01331626

2.7. Protein Extraction and Western Blotting:

Protein from cell culture experiments was extracted using NP40 buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP40) supplemented with 1% of protease inhibitor cocktail (#P8340, Sigma-Aldrich ,St. Louis, MO) and phosphatase inhibitor cocktail (#P5726, Sigma-Aldrich, St. Louis, MO). Aliquots of 20 μg of protein were added to Laemmli sample buffer (Bio-Rad, Hercules, CA) and heated at 95 degrees C for 5 minutes. Samples were separated in a 10% SDS-PAGE gel (Bio-Rad, Hercules, CA) and then transferred to a nitrocellulose membrane at 4 degrees C, 60 Volts, for 2 hours. Membranes were blocked in nonfat dry milk for 60 minutes, rinsed 3 times with TBST, and then primary antibodies for Akt (either S473 phospho-Akt, (#9271, Cell Signaling Technology, Danvers, MA) or total Akt (#9272, Cell Signaling Technology, Danvers, MA)) were added (1:1000 in 3% BSA) overnight. The following day, the membranes were washed 3 times with TBST, followed by a 1 h incubation, at room temperature, with goat anti-mouse IRDye-680 and goat anti-rabbit IRDye-800CW secondary antibodies (LI-COR Biotechnology, Lincoln, NE). After incubation membranes were washed, dried, and then visualized on a LI-COR Odyssey CLx (LI-COR Biotechnology, Lincoln, NE). Total Akt and phospho-Akt data were obtained from different loading and running gels. Ponceau S was used for total protein normalization as this has been shown to be superior to other housekeeping genes, such as β-actin or GAPDH [24]. Protein concentrations were quantified by Image J software.

2.8. Electrophysiology:

Sim1-Cre/Rosa26-tdTOMATO mice were used for electrophysiological recordings as previously published [25] with the following modifications. Patch pipettes were filled with intracellular solution (adjusted to pH 7.3) containing 128 mM K gluconate, 10 mM KCl, 10 mM HEPES, 0.1 mM EGTA, 2 mM MgCl2, 0.3 mM Na-GTP and 3 mM Mg-ATP. Current clamp was engaged to test neural firing frequency at the baseline and after puff of 1 μM 1,25D3 for 1s. We first puff treated 1 μM 1,25D3 for 500 ms on the tdTOMATO positive neurons within the paraventricular nucleus. Some of the neurons are activated by 1,25D3 and some are not affected. Only those 1,25D3-responsive neurons were further treated with wortmannin (50 μM) [26–28] for 4 min prior to the second puff of 1 μM 1,25D3. The values for firing frequency were averaged within 2-min bin at the baseline or after 1,25D3 treatment.

2.9. Statistical Analysis:

Analysis for RNA-sequencing is described above. Cell culture results are presented as mean ± SE. Results are analyzed by a one-way or two-way ANOVA as appropriate with Tukey’s post-hoc analysis where appropriate. The level of significance was set as p<0.05. Data were analyzed GraphPad Prism version 7.

3. Results

We used sequencing of messenger RNA (mRNA-seq), together with subsequent unbiased pathway analysis to identify key biological pathways that were altered in the hypothalamus after i3vt calcitriol treatment. Many of the top pathways affected by 1,25D3 involved cellular function, neuronal function, and inflammation (Fig. 1). One of the metabolic pathways regulated by vitamin D was the Insulin Signaling pathway, which contains the critical pathway genes for insulin action within cells.

Fig. 1.

Unbiased pathway analysis in hypothalamus of 1,25D3 versus vehicle treated animals. A. Pathway analysis of genes differentially regulated in the hypothalamus of obese rats treated with 1,25D3 (0.1 μg) relative to vehicle treated animals. Pathways are grouped based on overall functions. B. Genes enriched in each of the 5 pathways involved in different Organismal Systems identified in A.

Insulin is well known to decrease hepatic glucose production in the liver, but a lesser known effect of insulin is direct action in the brain to regulate peripheral glucose [29]. One major pathway by which insulin exerts effects is the insulin receptor substrate (IRS)-phosphatidylinositol 3-OH kinase (PI3K) pathway. Loss of this pathway in the hypothalamus impairs the ability of systemic insulin to lower blood glucose levels [30]. Since we had previously shown that i3vt 1,25D3 increases peripheral insulin sensitivity in a manner that is very similar to how central insulin affects peripheral insulin sensitivity [6,31], and since our RNA-seq data showed that 1,25D3 upregulates the insulin signaling pathway within the hypothalamus, we hypothesized that 1,25D3 may increase insulin action directly in neurons. To test this, we utilized a mouse hypothalamic cell line, GT1-7. In GT1-7 cells, 48 hour pretreatment with 1,25D3 augmented insulin-induced phosphorylation of Akt. The insulin sensitizing effect of vitamin D was dependent upon the PI3K pathway as co-treatment of the cells with both vitamin D and the PI3K inhibitor wortmannin blocked the effects of vitamin D on phosphorylation of Akt (Fig. 2 A, B).

Fig. 2.

Vitamin D increases Akt phosphorylation in a PI3K-dependent manner in neurons. A. Representative western blot of phosphorylated Akt in GT1-7 cells after treatment with insulin, 1,25D3, and wortmannin. B. Quantification of pAkt/Akt ratio response to 1,25D3 and insulin, with and without wortmannin. N = 3 experimental replicates. *p < 0.05 vs. same treatment without insulin; #p < 0.05 vs. + insulin DMSO; $p < 0.05 vs. + insulin 2 h 1,25D3.

Although vitamin D has both genomic and non-genomic actions, we focused initially on genomic actions of vitamin D given that the increase in pAkt/Akt occurred after 48 hours of 1,25D3 treatment. Thus, we analyzed the expression level of key genes within the PI3K pathway. We found that 1,25D3 increased the transcription of Irs2, the predominant insulin receptor substrate in the brain (Fig. 3A). Additionally, 1,25D3 treatment increased transcription of the Pik3r1 gene which encodes the p85α regulatory PI3K subunit (Fig. 3B), while decreasing the transcription of the Pik3r3 gene encoding the p55γ subunit (Fig. 3C). There was no difference in gene expression levels of the catalytic subunit of PI3K p110α (Pi3kca), insulin receptor substrate 1 (Irs1), the insulin receptor (Insr), or Akt (Fig. 3D–G). These results are consistent with other studies demonstrating different time courses of vitamin D regulation of transcription vs. protein changes in the same gene [32]. Additionally, vitamin D mediates transcription of different genes in different timing. In myotubes, vitamin D treatment increases VDR mRNA in a bell-shaped manner, with a peak at 8 hours but in the same cells, no effect of treatment is seen on cyp24a1 mrna levels until 16 hours of treatment [33]. Thus, it is possible that vitamin D may affect the levels of transcription of genes within the PI3K pathway at 24 or 36 hours and we did not capture those changes with our timing. Regardless, these results show that vitamin D has specific effects on gene expression in multiple components of the PI3K pathway.

Fig. 3.

Vitamin D regulates transcription of key genes within the PI3K pathway in hypothalamic (GT1–7) cells. GT1–7 cells were treated with 10⁻⁸ M 1,25D3 for 2 or 48 hours. 1,25D3 treatment increased Irs2 and Pik3r1 expression at 2 hours and decreased expression of Pik3r3 without changing expression of Insr, Irs1, Pik3ca, or Akt. All expression levels are relative to the average of Pgk1 and B2m. *p < 0.05.

We have previously shown that vitamin D has rapid depolarizing effects on hypothalamic neurons [6]. Interestingly, in addition to the known effects of the PI3K pathway on insulin signaling, the PI3K pathway is also important in mediating rapid effects of other nuclear hormones [34]. A role for the PI3K pathway in the rapid actions of vitamin D had not been previously explored. To test if the PI3K was important for rapid effects of vitamin D on neuronal function, we measured electrophysiological properties of vitamin D treatment in paraventricular hypothalamic neurons. We chose to investigate the function of vitamin D on paraventricular neurons due to our previous data demonstrating both neuronal activation of that region specifically after vitamin D administration and specific effects of vitamin D on glucose tolerance through VDR within the paraventricular hypothalamus [6]. We confirmed that similar to our previously published results in pro-opiomelanocortin neurons [6], 1,25D3 increased the firing frequency (Fig. 4A, C) and depolarized the resting membrane potential (Fig. 4A, D) in a portion of Sim1 positive neurons within the paraventricular nucleus. Interestingly, we found that pre-treatment of paraventricular hypothalamic neurons with wortmannin was sufficient to block both of these vitamin D-mediated electrophysiological changes (Fig. 4B, C, D). This indicates that vitamin D requires functional PI3K in order to exert depolarizing effects on paraventricular neurons.

Fig. 4.

Vitamin D requires functional PI3K to change electrophysiological properties in paraventricular neurons. A, B. Representative traces of action potential in Sim1 positive neurons treated with 1, 25D3 (1 μM puff) in the absence (A) or presence (B) of a PI3K inhibitor, wortmannin (50 μM). C. 1,25D3 increases firing frequency in Sim1 positive neurons but there is no effect of 1,25D3 in the presence of wortmannin. D. 1,25D3 depolarizes resting membrane potential in these neurons but has no effect in the presence of wortmannin. N=11 neurons per treatment.

4. Discussion

This study demonstrates that vitamin D alters regulation of key insulin pathway genes within the hypothalamus and in hypothalamic cell culture. Additionally, we show that vitamin D requires the PI3K pathway to both enhance insulin signaling and for rapid, non-genomic effects in hypothalamic neurons. We found 1) vitamin D increased the insulin-mediated phosphorylation of Akt in a PI3K-dependent manner; 2) vitamin D altered transcription of key PI3K pathway genes, namely Irs2, Pik3r1 and Pik3r3; and 3) the vitamin-D mediated depolarization of hypothalamic neurons is PI3K dependent. To our knowledge, this is the first report of vitamin D altering insulin signaling within hypothalamic neurons, upregulating Irs2 expression or requiring PI3K for neuronal excitability.

There is some debate as to the effect of the VDR on glucose homeostasis. While studies have shown that whole-body loss of the VDR impairs glucose tolerance [35], others have shown no difference in glucose tolerance between mice with total body loss of the VDR or transcriptionally inactive VDR [5]. The latter paper showed equivalent glucose tolerance curves between the groups despite a significantly smaller body weight and increased lean mass % in male mice of both VDR modified strains. However, all mice were kept on chow or rescue diets; they were not fed a high-fat diet. This is an important methodological difference as we have shown effects of 1,25D3 administration or paraventricular hypothalamic VDR loss only in an obese animal model, not in lean animals [6]. Similarly, and also intriguing, is that many studies investigating the effects of vitamin D on the PI3K pathway observe effects in high-glucose conditions. Given these observations, our cell culture experiments were also performed in high-glucose conditions. Thus, the environmental milieu of cells is likely very important for the ultimate response to vitamin D. This may have considerable clinical impact given the association between vitamin D deficiency and obesity.

Our data show that vitamin D has synergistic effects on phosphorylation of Akt in the presence of insulin, without significant effects alone. This is consistent with literature in other cell types. Vitamin D treatment has been previously shown to reverse high-glucose-induced decrement in adipocyte PI3K and p-Akt, although no effect of vitamin D treatment was observed in and of itself [36]. Additionally, vitamin D treatment in myotubes attenuates the decrease of p-IRS1 in high-glucose conditions, again without increasing p-IRS1 with vitamin D treatment alone [37]. What is unique to this study, is that we show vitamin D treatment augments insulin action whereas in these other studies, the effect of vitamin D was thought to be secondary to SIRT1, an insulin-independent mediator.

Interestingly, vitamin D may have cell-specific or environment-specific effects. In mouse models of experimental autoimmune encephalomyelitis, mice supplemented with vitamin D have decreased PI3K/AKT pathway gene transcripts in T-cell populations [38]. This may be secondary to a differential expression of different PI3Ks or different downstream effectors (such as FOXO vs. mTOR) in different cell types [39]. Also, vitamin D supplementation of obese mice resulted in increased muscle Irs1 transcription levels but decreased levels in liver tissue [40]. . However, VDR immunoreactivity has been shown to occur in oxytocin-positive neurons [41], and oxytocin neurons respond to glucose and insulin in a PI3K-dependent manner [42]. Given our previous findings regarding the importance of the paraventricular hypothalamus (which contains many oxytocin neurons) to glucose homeostasis, determining the cell-type specific effects of vitamin D will likely be an important future step for research

The Insulin/IRS/PI3K pathway is well known to be important for glucose tolerance. Infusion of insulin into the brain decreases hepatic glucose production [31] and loss of insulin receptors in the brain lead to either glucose intolerance or insulin resistance [29]. Loss of the catalytic unit of PI3K, Pik3ca (P110α), within POMC neurons of the hypothalamus causes increased fasting insulin and glucose levels while also impairing insulin sensitivity [43]. Loss of p110β in the VMH also results in glucose and insulin intolerance [44]. Interestingly, loss of brain Irs2 also impairs glucose homeostasis [45]. Our data show an increase in Irs2 mRNA concentrations after 1,25D3 treatment. Of note, loss of Irs2 in arcuate POMC neurons seems to have little effect on glucose homeostasis, indicating that Irs2 may have effects on glucose homeostasis in other areas of the brain [46]. The possible role of vitamin D on Irs2 in neurons is intriguing to us given that we have shown vitamin D receptors in the paraventricular nucleus, but not the arcuate nucleus, of the hypothalamus are required for glucose homeostasis in male mice [6]. Supporting this, vitamin D deficiency results in decreased Irs2 protein levels in other tissues in mice [47]. Thus, it is possible that one mechanism by which vitamin D controls glucose control in the paraventricular nucleus of the hypothalamus is through regulation of Irs2. Future experiments will be critical to test this hypothesis.

We conclude here that increases in pAkt after vitamin D are likely from genomic actions. However, there are reports of non-genomic actions of 1,25D3 increasing phosphorylation of Akt within 5 minutes of incubation in osteoblasts exposed to an apoptotic agent, STSP [48]. While Zhang and Zanello [48] observed increased pAkt sustained from 5 to 60, minutes, levels at 120 minutes were not reported. Thus it is unknown if non-genomic actions of vitamin D are sustained for 2 hours. It is possible that vitamin D has a dual effect on pAkt levels whereby non-genomic actions are present prior to 2 hours and genomic actions later. We observed effects of vitamin D on pAkt only after 48 hours of treatment, supporting a more classic action of the steroid hormone receptor. However, it is possible that a rapid mechanism was present but abated prior to the 2 hour collection.

Several studies have looked at target genes of vitamin D, although few have been performed in the brain and none in the hypothalamus. A previous study using mixed neuron-glial cell culture from brain of mouse embryos at 17 d gestation treated with 1,25D3 for 16 days at a similar dose to our study, underwent RNA GeneChip expression array and found 27 upregulated genes [49]. Of these, only Ncam1 (neural cell adhesion molecule) was also upregulated in our data set. Rats exposed to vitamin D deficiency in utero have decreased gene expression in several genes [50]; of these, Map2, Slc1a3, and Psat1 are increased by vitamin D treatment in our data set. In aged wild type mice treated with vitamin D peripherally, 72-transcripts were found to be related to vitamin D with inflammation accounting for 19 of them [51]. Our data set shows similarity with the Il6 and Grin2a genes. The lack of similarity between the data sets is not surprising given the vast difference in ages (embryos or gestational exposure vs. adults) and the difference in diet (high-fat diet in our study, chow in both the comparators). Additionally, there are gene-specific differences in how vitamin D mediates transcription of genes with regard to timing and concentration. Increased concentrations of vitamin D can cause dose-dependent increases in transcription of one gene while showing a bell-shaped curve in another; time can have a similar effect [22,32,33]. Thus, differences between these data sets and ours may also be related to time and dose-dependent differences.

Since all of these studies are utilizing exogenous administration of vitamin D, it is possible that these results include off-target or supraphysiological effects of vitamin D. Our previous study confirmed that the beneficial effect of exogenous vitamin D on glucose tolerance was unlikely to have occurred through off-target effects since it required the presence of the VDR [6]. Additionally a strength of this study is that multiple different types of experiments in two different species all support the role of the PI3K pathway in vitamin D action within neurons.

Perhaps the most striking finding of this study is that the PI3K is required for non-genomic actions of vitamin D in neurons. The PI3K is also required for spontaneous action potentials after IL-1β treatment in hippocampal neurons [52] and for MC4R-mediated changes in transient outward A-type K⁺ current in trigeminal ganglion neurons [53]. Thus, there is precedent for the PI3K pathway to not only be involved in rapid, neuronal actions. Of note, approximately 70% of Sim1 neurons responded to vitamin D. Since we were not able to identify which Sim1 neurons were VDR⁺ neurons, it is possible that the neurons which did not respond to 1,25D3 either did not contain the VDR or did not contain important second messenger machinery to propagate the effects of 1,25D3 [54]. However, future studies will need to verify this hypothesis as well as further define the molecular mechanisms by which the PI3K modulates rapid actions of vitamin D in neurons.

5. Conclusions

These data demonstrate vitamin D has PI3K-dependent mechanisms to both phosphorylate Akt and increase neuronal excitability. Additionally, we have shown in both cell culture and in vivo mRNA-seq studies that vitamin D can regulate genes within hypothalamic cells and some of these are important mediators of the PI3K pathway. These data provide evidence to support that vitamin D may act within the hypothalamus to increase insulin action. Given the known association between low vitamin D levels and type 2 diabetes, this data set provides intriguing evidence for future studies to determine if vitamin D augmentation of insulin action in the brain is a causal pathway to explain the association of these disorders.

Highlights.

• Vitamin D regulates transcription of several pathways in the hypothalamus, including the insulin signaling pathway.

• Vitamin D augments insulin signaling in neurons and regulates transcription of key genes within the PI3K pathway.

• Vitamin D requires the PI3K pathway for rapid actions in paraventricular neurons.