Targeting 1,25(OH)2D-mediated calcium absorption machinery in proximal colon with calcitriol glycosides and glucuronides
Introduction
Intestinal calcium (Ca) absorption brings Ca from ingested food into the body. It is part of a three-tissue axis, including bone and kidney that regulates whole body Ca homeostasis. High Ca absorption efficiency has been linked to high peak bone mass in adolescents [1,2] and reduced bone loss in adulthood [3,4]. It is also positively correlated to femur BMD, trabecular bone volume (BV/TV), and trabecular thickness in a genetically diverse population of growing mice [5].
Kinetics analysis across a wide range of luminal Ca concentrations demonstrated that intestinal Ca absorption follows both a saturable and a non-saturable pathway [6]. The relationship between these two transport pathways can be modeled mathematically by a Michaelis-Menton-like equation that is modified to include a linear component that reflects passive diffusion through tight junctions. Saturable Ca transport pathway is prevalent in the proximal small intestine and is regulated by nutritional and physiological conditions. Under habitual low to adequate dietary Ca intake, saturable Ca transport will account for >60 % of total Ca absorption in the small intestine [7]. In contrast, non-saturable Ca transport occurs at a constant rate across the intestine lumen (13 % of luminal load per hour) [6,8].
Intestinal Ca absorption was first reported to be dependent on vitamin D in 1937 [9] and other research shows that intestinal Ca absorption efficiency was reduced by 75 % in vitamin D deficient rats [10]. The bulk of the evidence suggests that the effect of vitamin D on Ca absorption is limited to the saturable Ca component and this is due to the induction of three proteins: the apical membrane Ca channel Trpv6, the intracellular Ca binding protein calbindin D9k (encoded by the S100g gene), and the basolateral membrane Ca ATPase PMCA1b [7]. The genes for each of these proteins are transcriptionally regulated by 1,25-dihydroxyvitamin D (1,25(OH)2D)-mediated activation of the vitamin D receptor (VDR) [[11], [12], [13]]. Previously, we showed that expression of a human VDR transgene throughout the intestinal epithelium could restore intestinal Ca absorption and prevent the growth arrest, hypocalcemia, hyperthyroidism, and osteomalacia observed in VDR KO mice [14]. This illustrates the critical role that intestinal VDR and vitamin D-mediated Ca absorption play in the control of whole body Ca metabolism during growth.
While mechanistic studies on Ca absorption have focused on the proximal small intestine, several studies suggest that Ca absorption in the large intestine could be important for whole body Ca metabolism. For instance, Ca absorption was significantly higher for patients with Crohn’s disease when their colon is not removed [15]. In addition, studies in rodents showed that active Ca absorption exists in the colon and cecum [16,17] and that 1,25(OH)2D injection significantly increased absorptive Ca flux and net Ca transport in colon [18]. All these studies indicate that the machinery for vitamin D-mediated intestinal Ca absorption exists in the lower bowel. Furthermore, research by Dhawan et al. [19] has shown that vitamin D-mediated events in the lower bowel are a critical contributor to whole Ca metabolism and bone health. They found that transgenic VDR expression directed to the distal ileum, cecum, and colon was sufficient to prevent the abnormal Ca metabolism normally seen in VDR KO mice. Collectively, these studies suggest the distal intestine could be targeted with 1,25(OH)2D to enhance Ca absorption and bone health. This could be useful for individuals at risk for bone loss due to conditions such as bariatric surgery, short-bowel syndrome, or aging.
Orally administered 1,25(OH)2D does not reach the large intestine because it is rapidly absorbed by the small intestine before it can reach the colon. This also leads to significantly elevated plasma concentrations of the hormone [20]. Therefore, a novel strategy is needed to deliver the hormone specifically to the colon. Solanum glaucophyllum (Sg) is a plant that grows in South America whose consumption causes high blood Ca level, soft tissue calcification, and wasting in grazing ruminant animals [21]. This is due to the presence of 1,25(OH)2D-glycosides in Sg leaf [22,23]. The glycoside forms of 1,25(OH)2D in Sg are biologically inert until the glycosides are cleaved by bacteria with β-glycosidase activity that reside in the rumen [24]. This capacity also exists in the lower intestine of monogastric animals and account for why rodent studies have identified doses of Sg leaf that induce expression of the vitamin D-dependent gene, Cyp24a1, specifically in the colon without causing systemic effects [24]. Similar, colon-targeted effects have also been observed with synthetic, glucuronidated forms of 1,25(OH)2D [20,25].
Here, our goal is to investigate whether the proximal colon has vitamin D-regulated Ca absorption that is dependent upon the presence of the VDR. In addition, we tested whether doses of Sg leaf, as well as a novel, synthetic 1,3-diglucuronide form of calcitriol, could be optimized to upregulate genes controlling intestinal Ca absorption in the proximal colon of mice without having systemic effects.
Section snippets
Dietary preparation
Sg leaf powder, 25-hydroxyvitamin D3-25-β-glucuronide-25(OH)D (β-gluc-25(OH)D), and the 1,3-diglucuronide-1,25(OH)2D were provided by GlycoMyr, Inc. (Ames, IA). Powdered, modified AIN93 G diet (0.5 % Ca, 0.4 % P and 0.2 IU/g vitamin D3) (Research Diet Inc., New Brunswick, NJ) was mixed with 25-hydroxyvitamin D3-25-β-glucuronide-25(OH)D (β-gluc-25(OH)D) (0.247 nmol/g diet) to make a diet base. Previous research has shown that the addition of β-gluc-25(OH)D to a diet containing
Results
We directly measured and compared Ca absorption efficiency at proximal colon and duodenum from the same mouse using an in situ ligated loop method (2 mM Ca, 10 min). In WT mice, the Ca absorption efficiency at duodenum was 30.0 ± 6.7 %. The proximal colon had similar absorptive capacity (26.24 ± 3.67 %) (Fig. 1). However, age matched VDR KO mice had significantly lower Ca absorption efficiency in the duodenum (11.7 ± 4.12 %, p = 0.04 vs WT) and proximal colon (13.75 ± 2.83 %, p = 0.02 vs WT).
Discussion
Our research is important because it confirms that the proximal colon is a site for vitamin D-mediated intestinal Ca absorption and because it demonstrates that glycosylated and glucuronidated forms of 1,25(OH)2D can be used to upregulate elements of the Ca absorption machinery in the proximal colon.
Most of what we know about Ca absorption is from studies on the small intestine. These studies show that 1,25(OH)2D regulates a saturable, transcellular process [6] that predominates under low and
Funding
Supported by NIH grant R01DK112365 to J.C. Fleet, S. Christakos, and M. Verzi.
CRediT authorship contribution statement
H. Jiang: Investigation, Formal analysis, Writing - original draft, Writing - review & editing, Visualization, Project administration. R.L. Horst: Methodology, Resources. N.J. Koszewski: Methodology, Resources. J.P. Goff: Methodology, Resources, Writing - review & editing. S. Christakos: Writing - review & editing, Supervision, Funding acquisition. J.C. Fleet: Conceptualization, Methodology, Writing - original draft, Writing - review & editing, Supervision, Project administration, Funding
Acknowledgements
We thank Dr. Ryan Calvert for his assistance making the experimental diets and with the tissue harvest at the end of the experiment. We thank Dr. Krittikan Chanpaisaeng for assisting with the tissue harvest at the end of the experiment and Ca absorption test.
References (45)
Calcium absorption efficiency and calcium requirements in children and adolescents
Am. J. Clin. Nutr.
(1991)- et al.
Factors that influence peak bone mass formation: a study of calcium balance and the inheritance of bone mass in adolescent females
Am. J. Clin. Nutr.
(1990) Regulation of intestinal calcium and phosphate absorption
- et al.
1, 25-Dihydroxyvitamin D3-glycoside: identification of a calcinogenic principle of Solanium malocoxylon
Life Sci.
(1976) - et al.
Targeted delivery of 1,25-dihydroxyvitamin D3 to colon tissue and identification of a major 1,25-dihydroxyvitamin D3 glycoside from Solanumglaucophyllum plant leaves
J. Steroid Biochem. Mol. Biol.
(2015) - et al.
Vitamin D receptor (VDR) knockout mice reveal VDR-independent regulation of intestinal calcium absorption and ECaC2 and calbindin D9k mRNA
J. Nutr.
(2003) - et al.
Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method
Methods
(2001) - et al.
Calcium solubility, intestinal sojourn time and paracellular permeability codetermine passive calcium absorption in rats
J. Nutr.
(1995) - et al.
Vitamin D dependence of in vivo calcium transport and mucosal calcium binding protein in rat large intestine
Gastroenterology
(1979) - et al.
Nutritional aspects of calcium absorption
J. Nutr.
(1999)
1,25-Dihydroxyvitamin D3-glycoside of herbal origin exhibits delayed release pharmacokinetics when compared to its synthetic counterpart
J. Steroid Biochem. Mol. Biol.
Calcium absorption may be affected after either sleeve gastrectomy or Roux-en-Y gastric bypass in premenopausal women: a 2-y prospective study
Am. J. Clin. Nutr.
Cortical and trabecular deterioration in mouse models of Roux-en-Y gastric bypass
Bone
Weight in infancy and adult calcium absorption as determinants of bone mineral density in adult men: the hertfordshire cohort study
Calcif. Tissue Int.
Does the response of bone mass to calcium supplements depend on calcium absorption efficiency?
Eur. J. Endocrinol.
Gene-by-diet interactions influence calcium absorption and bone density in mice
J. Bone Miner. Res.
An analysis of intestinal calcium transport across the rat intestine
Am. J. Physiol.
In vivo intestinal absorption of calcium in humans
Miner. Electrolyte Metab.
Studies upon the mode of action of vitamin D. III. The influence of vitamin D on the absorption of calcium and phosphorus in the rat
Biochem.J.
Duodenal and ileal calcium absorption in the rat and effects of vitamin D
Am. J. Physiol.
Calcium transporter 1 and epithelial calcium channel messenger ribonucleic acid are differentially regulated by 1,25 dihydroxyvitamin D3 in the intestine and kidney of mice
Endocrinology
Duodenal calcium absorption in vitamin D receptor-knockout mice: functional and molecular aspects
Proc. Natl. Acad. Sci. U. S. A.
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