Gene Expression, Metabolomic, Microbiome, and Calcium Metabolism in Response to Varied Vitamin D Dosages

Vitamin D requires two metabolic conversions, 25-hydroxylation in the liver and
1α-hydroxylation in the kidney, before its hormonal form, 1,25-dihydroxyvitamin D
[1,25(OH)2D], can bind to the vitamin D receptor (VDR) to modulate gene transcription. The
VDR is present in a wide variety of cells and tissues and 1,25(OH)2D via its receptor
directly or indirectly have been reported to have effects on cell cycling and proliferation,
differentiation, apoptosis and the production of cathelicidin, renin and insulin. It is
estimated that VDR activation may regulate directly and/or indirectly the expression of a
very large number of genes (0.5-5% of the total human genome i.e., 100-1250 genes). Early
microarray studies performed in squamous carcinoma and osteoblasts cells revealed that
1,25(OH)2D3 regulated the expression of numerous genes including those implicated in immune
function . Although there have been numerous observations of vitamin D deficiency and its
links to chronic diseases, a study on how a person's vitamin D status and improvement with
vitamin D supplementation affects genomic expression in vivo in humans had not been reported
until recently when we observed in a small pilot study that supplementation of healthy adults
with vitamin D significantly affected the expression of 291 genes that have been linked to
more than 80 different metabolic pathways. This small randomized controlled double blind
study funded by our CTSI and registered in Clinical Trials.gov (NCT01696409) included 2
groups. Group 1 received 400 IUs of vitamin D3 (400 IUs at the time of the study in 2010 was
before the IOM released its new recommendations) and was considered the control group and
group 2 received 2000 IUs of vitamin D3 daily for 2 months during the winter. Comparing gene
expression in these two groups (deficient vs. insufficient or sufficient) led us to study the
effect of vitamin D status as well as vitamin D supplementation on genome-wide gene
expression. To explore gene-expression relationships between and within the 400 IU and 2000
IU groups, principal component analysis (PCA) was performed. We observed the same trend in
gene expression in the subjects who received 400 and 2000 IUs vitamin D3. There was however a
trend for a larger change in the expression of these genes for the group who received 2000
IUs vitamin D3/d compared to the group who received 400 IUs vitamin D3/d. Regarding all
participants, with false discovery rate (FDR) < 0.1, and a 1.5 fold change, 291 genes were
found to have a statistically significant difference in expression from baseline to follow-up
after vitamin D3 supplementation. There was at least a 1.5 fold inhibition of 82 genes (top
~30% of the heat map) whose expression was dramatically reduced and at least a 1.5 fold
induction of 209 genes (bottom ~70% of the heat map) whose expression was significantly
increased after supplementation with either 400 or 2000 IU of vitamin D3 for 2 months. For
verification that genes that we observed to have their expression affected by vitamin D
supplementation did occur expression changes were evaluated by real-time PCR for four genes
including CD83, TNFAIP3, KLF10 and SBDS. The gene expression changes were concordant with
those observed by the microarray analysis.

This subgroup analysis of the baseline gene expression for the 291 genes in the vitamin D
deficient group compared to the vitamin D insufficient/sufficient group revealed that,
expression of 66 genes were significantly different between the two groups (p<0.01 and fold
change>1.5). There was at least a 1.5 fold increase in gene expression (brown-orange) of 14
genes and at least a 1.5 fold decrease in the expression (yellow-white) of 52 genes in the
vitamin D deficient adults compared to those who were vitamin D insufficient or sufficient at
baseline. After vitamin D3 supplementation gene expression in the vitamin D deficient group
was similar to vitamin D insufficient/sufficient group. To learn which of these genes
affected by vitamin D3 supplementation contained VDR binding domains near the transcriptional
start site (TSS), we performed a VDRE analysis. Of the 66 genes that were influenced by at
least 1.5 fold in their expression by the baseline serum 25(OH)D concentration,17 of these
genes that were significantly changed after vitamin D3 supplementation in both deficient and
insufficient/sufficient groups (p<0.01) were selected for VDRE analysis. We found at least
one candidate VDRE in the upstream region within 30 kb of the TSS in these 17 genes. For
example, the candidate VDRE in coatomer protein complex, subunit beta 2 (COPB2), a gene that
was stimulated at least 1.5 fold by vitamin D3 supplementation, had two hexameric binding
motifs associated with the VDRE. Twelve housekeeping genes served as negative controls. There
were no sequences of candidate VDREs in 100 kb upstream of TSS of these housekeeping genes
and the expression of these housekeeping genes after vitamin D3 supplementation was not
changed.

VDR is present in a wide variety of cells and tissues, in particular they are prominent on
cells involved in innate and adaptive immunity; especially CD8+ T lymphocytes.These immune
cells are found throughout the body including parts of the digestive tract. In particular,
the upper GI tract (including the pyloric antrum and duodenum) has the greatest concentration
of CD8+ T cells relative to the rest of the GI tract. Studies have found that those who
increase their vitamin D intake have a reduction in opportunistic pathogens and an increase
in gut microbiome. Through the action of vitamin D on CD8+ T cells, there is an increase
immune response against gammaproteobacteria which allows the gut microbiome to flourish. This
study found that although the upper GI tract generated the greatest change in microbiome
secondary to vitamin D supplementation, the rest of the GI tract and stool also displayed a
change. Gut microbiome is important for general health maintenance. For example, changes in
gut microbiota have been associated with early onset of Type 2 - Diabetes Mellitus (T2DM).
Thus, it is possible to understand the prevalence of VDR expression on immune cells in an
individual by observing the microbiota in the gut/stool.

The objective of this proposed project will be to expand on these preliminary data in a
well-defined group of healthy adults ages 18-50 years with a BMI <30 kg/m2 who are vitamin D
insufficient with a 25(OH)D of < 29 ng/mL

There continues to be debate as to how much vitamin D an adult requires to be vitamin D
sufficient. A multitude of association studies have suggested that improving serum 25(OH)D
>30 ng/mL may reduce risk of many chronic illnesses and improve immune function.
1,25-dihydroxyvitamin D3 [1,25(OH)2D] may regulate at least 2000 genes. Our recent pilot
study evaluating the effect of vitamin D status and vitamin D supplementation on broad gene
expression revealed as many as 291 genes are influenced by vitamin D3 supplementation.

Aim: Define Dynamic Changes in PTH, Broad Gene Expression in Circulating Immune Cells,
metabolomics, and microbiome profile in Response to Varying Doses of Vitamin D
Supplementation.

The hypothesis that will be tested is that as serum 25(OH)D levels increase as a result of
vitamin D supplementation there will be a gradual decline in PTH levels. Furthermore we
expect that there will continue to be alterations in gene expression, metabolomic protein
levels and in the gut microbiome when the dose is increased to 10,000 IUs daily whereas the
PTH levels will plateau at a dose of 4000 IUs daily and remain the same when the dose is
escalated to 10,000 IUs daily.

This study will last 24 weeks and blood, urine, and stool samples will be obtained at
baseline and at 8 week intervals to evaluate the time course for the changes in 25(OH)D, PTH
levels, gene expression, and determine if these changes remain constant or are continuing to
change after vitamin D supplementation. We will conduct a metabolomics profile in blood and
urine as well as determine if there are any microbiome changes in the stool as previously
described. In addition, one arm of the study will include a group who will receive 600 IUs
for the first 8 weeks followed by 4000 IUs for the next 8 weeks followed by 10,000 IUs of
vitamin D3 for the final 8 weeks to determine whether continued increase in vitamin D intake
in the same adult will continue not only to alter gene expression, metabolomics activity, and
microbiome changes, but also influence PTH levels. Although the IOM's UL for vitamin D for
adults is 4000 IU/D they and the Endocrine Society recognized that up to 10,000 IUs/D was
safe in healthy adults for up to 5 months. In addition a recent study reported that adults
taking up to 20,000 IUs/D for at least one year never raised their blood level above 250
nmol/L i.e. 100 ng/mL which is considered by the IOM, Endocrine Society and many reference
laboratories to be the upper limit of normal. Results from this study should provide a new
insight as to how much vitamin D a healthy adult requires to maximize their calcium and bone
metabolism and expression of genes related to the immune system and other biologic pathways
in the blood, urine, and stool.

Inclusion Criteria:

1. Healthy male or female adults

2. Age 18-50 years

3. BMI <30

4. 25-hydroxyvitamin D < 29 ng/mL

5. No medications or disorders that would affect vitamin D metabolism

6. Women must be on birth control and not pregnant based on a negative pregnancy test at
baseline

7. Ability and willingness to give informed consent and comply with protocol requirements

Exclusion Criteria:

1. Ongoing treatment with pharmacologic doses of vitamin D, vitamin D metabolites or
analogues

2. Pregnancy

3. History of elevated serum calcium (>10.6 mg%); that is corrected for albumin
concentration

4. Chronic hepatic or renal failure

5. Supplementation with over the counter formulations of vitamin D2 or vitamin D3

6. Subjects with a history of an adverse reaction to orally administered vitamin D.

7. Subjects who are taking oral Dilantin or glucocorticoids.

8. History of intestinal malabsorption (i.e. cystic fibrosis, fat malabsorption syndrome,
Crohn's Disease, gastric bypass surgery).

9. Inability to give informed consent

10. Vacation plans to warmer climates (Florida, Southern CA, tropics) during study
participation

11. Subjects with any other condition which in the Investigator's judgment would make the
patient unsuitable for inclusion in the study.