Role of pregnancy and obesity on vitamin D status, transport, and metabolism in baboons

Abstract

Human studies show that obesity is associated with vitamin D insufficiency, which contributes to obesity-related disorders. Our aim was to elucidate the regulation of vitamin D during pregnancy and obesity in a nonhuman primate species. We studied lean and obese nonpregnant and pregnant baboons. Plasma 25-hydroxy vitamin D (25-OH-D) and 1α,25-(OH)₂-D metabolites were analyzed using ELISA. Vitamin D-related gene expression was studied in maternal kidney, liver, subcutaneous fat, and placental tissue using real-time PCR and immunoblotting. Pregnancy was associated with an increase in plasma bioactive vitamin D levels compared with nonpregnant baboons in both lean and obese groups. Pregnant baboons had lower renal 24-hydroxylase CYP24A1 protein and chromatin-bound vitamin D receptor (VDR) than nonpregnant baboons. In contrast, pregnancy upregulated the expression of CYP24A1 and VDR in subcutaneous adipose tissue. Obesity decreased vitamin D status in pregnant baboons (162 ± 17 vs. 235 ± 28 nM for 25-OH-D, 671 ± 12 vs. 710 ± 10 pM for 1α,25-(OH)₂-D; obese vs. lean pregnant baboons, P < 0.05). Lower vitamin D status correlated with decreased maternal renal expression of the vitamin D transporter cubulin and the 1α-hydroxylase CYP27B1. Maternal obesity also induced placental downregulation of the transporter megalin (LRP2), CYP27B1, the 25-hydroxylase CYP2J2, and VDR. We conclude that baboons represent a novel species to evaluate vitamin D regulation. Both pregnancy and obesity altered vitamin D status. Obesity-induced downregulation of vitamin D transport and bioactivation genes are novel mechanisms of obesity-induced vitamin D regulation.

Keywords: adipose tissue; obesity; placenta; pregnancy; renal; vitamin D.

Fig. 1.

Effects of pregnancy and obesity on vitamin D status in a baboon model. Age-matched pregnant (P) and nonpregnant (NP) baboons were fed a control or high-fat diet to induce obesity. Adult female circulating plasma levels of 25-hydroxy vitamin D (25-OH-D; A), 1α,25-(OH)₂-D (B), total calcium (C), intact parathyroid hormone (iPTH; D), fibroblast growth factor 23 (FGF23; E), and phosphate (F) were determined [10 NP females (5 obese and 5 lean) and 10 pregnant females (5 obese and 5 lean)]. Fetal umbilical cord plasma levels of 25-OH-D (G) and total calcium (H) of 16 female fetuses (8 control and 8 obese) and 16 male fetuses (8 control and 8 obese) were also analyzed. Data are shown as means ± SE. *P < 0.05 control vs. obese; †P < 0.05, NP vs. P.

Fig. 2.

Effects of pregnancy and obesity on the adult renal vitamin D system. A–D: renal cortical mRNA levels of vitamin D transporters megalin (LRP2) and cubulin (CUBN) (A), vitamin D-metabolizing enzymes 1α-hydroxylase (CYP27B1) and 24-hydroxylase (CYP24A1) (B), vitamin D receptor (VDR; C), and 25-hydroxylases CYP2R1 and CYP2J2 (D) are shown as pg mRNA over ng of β-actin (ACTB) mRNA. Renal cortical protein expression of LRP2 and CUBN (E) and CYP27B1 and CYP24A1 (F) are shown as relative expression over ACTB expression levels. G: renal VDR protein levels in both nuclear extracts (NE) and chromatin extracts (CH) are expressed relative to TATA-binding protein (TBP) levels. H: representative immunoblots for proteins studied. Vertical lines indicate noncontiguous lanes, and dashed vertical lines indicate that the last 2 lanes were before the previous 2 lanes. Results are shown as means ± SE (n = 5/group). *P < 0.05 control vs. obese; †P < 0.05, nonpregnant (NP) vs. pregnant (P).

Fig. 3.

Effects of pregnancy and obesity on the adult liver vitamin D-related gene expression. Hepatic mRNA levels of vitamin D transporters megalin (LRP2) and cubulin (CUBN) (A), 25-hydroxylases CYP2R1 and CYP2J2 (B), 1α,25-(OH)₂-D metabolizing enzymes 1α-hydroxylase (CYP27B1) and 24-hydroxylase (CYP24A1) (C), and vitamin D receptor (VDR) and vitamin D-binding protein (GC) (D) are shown as pg mRNA over ng of β-actin (ACTB) mRNA. Results are shown as means ± SE (n = 5/group). *P < 0.05 control vs. obese; †P < 0.05, nonpregnant (NP) vs. pregnant (P).

Fig. 4.

Effects of pregnancy and obesity on vitamin D-related gene expression of adult subcutaneous adipose tissue. A–D: adipose tissue mRNA levels of vitamin D transporters megalin (LRP2) and cubulin (CUBN) (A), vitamin D-metabolizing enzymes 1α-hydroxylase (CYP27B1) and 24-hydroxylase (CYP24A1) (B), vitamin D receptor (VDR; C), and 25-hydroxylases CYP2R1 and CYP2J2 (D) are shown as pg of mRNA over ng of β-actin (ACTB) mRNA. E and F: adipose protein expression of CUBN (E) and CYP27B1 and CYP24A1 (F) are shown as relative expression over ACTB expression levels. G: adipose VDR protein levels in both nuclear extracts (NE) and chromatin extracts (CH) are expressed relative to TATA-binding protein (TBP). H: representative immunoblots for proteins studied. Results are shown as means ± SE (n = 5/group). *P < 0.05 control vs. obese; †P < 0.05 nonpregnant (NP) vs. pregnant (P).

Fig. 5.

Vitamin D-related gene expression in placentas of control and obese pregnancies. A–D: placental mRNA levels of vitamin D transporters megalin (LRP2) and cubulin (CUBN) (A), vitamin D-metabolizing enzymes 1α-hydroxylase (CYP27B1) and 24-hydroxylase (CYP24A1) (B), vitamin D receptor (VDR; C), and 25-hydroxylases CYP2R1 and CYP2J2 (D) are shown as pg of mRNA over ng of β-actin (ACTB) mRNA. E and F: placental protein expression of LRP2 (E) and CYP27B1 and CYP24A1 (F) are shown as relative expression over ACTB expression levels. G: placental VDR levels in both nuclear extracts (NE) and chromatin extracts (CH) are expressed relative to the levels of TATA-binding protein (TBP). H: representative immunoblots for E, F, and G. Vertical lines indicate noncontiguous lanes. Results are shown as means ± SE (n = 5/group). *P < 0.05, control vs. obese.

Fig. 6.

Summary of vitamin D regulation by pregnancy and obesity in humans, baboons, and mice. The effect of pregnancy has been determined in comparison with nonpregnancy in age-matched subjects. The effect of obesity has been determined in adults (both males and females) and in pregnant females. CUBN, cubulin; CYP24A1, 24-hydroxylase; CYP27B1, 1α-hydroxylase; FGF23, fibroblast growth factor 23; LRP2, megalin; ND, not determined; 25-OH-D, plasma 25-hydroxy vitamin D; PTH, parathyroid hormone; VDBP, vitamin D-binding protein; VDR, vitamin D receptor. -, Not changed; ↑increased; ↓decreased.