Association of increased renal Cyp24a1 gene expression with low plasma 1,25-dihydroxyvitamin D levels in rats with streptozotocin-induced diabetes

Abstract

Decreases in plasma vitamin D concentrations have been reported in diabetes, although the mechanism involved in this decrease is unclear. Here, we investigated the association between Cyp24a1, a vitamin D catabolic enzyme, and abnormalities in vitamin D metabolism in streptozotocin-induced diabetes rats, an animal model of type 1 diabetes. Plasma 1,25-dihydroxyvitamin D [1,25(OH)₂D] levels were significantly lower in streptozotocin-induced diabetes rats and renal Cyp24a1 mRNA expression levels were increased. Western blotting analysis of streptozotocin-induced diabetes rats kidney tissues with anti-CYP24A1 antibody showed a strong signal around 40 kDa, which differs from the predicted 50-55 kDa molecular weight for full-length Cyp24a1 and could represent the Cyp24a1-splicing variant that lacks exons 1 and 2. We observed high levels of renal Cyp24a1-splicing variant mRNA expression in streptozotocin-induced diabetes rats. We also confirmed transcriptional up-regulation of endogenous Cyp24a1 mRNA expression through glucocorticoid receptors by glucocorticoid in opossum kidney proximal cells. Taken together, our results indicated that high Cyp24a1 expression levels may play a role in the decrease of plasma 1,25(OH)₂D levels in streptozotocin-induced diabetes rats. High plasma corticosterone levels in diabetes may affect transcriptional regulation to promote increases in Cyp24a1 expression.

Keywords: CYP24A1; CYP24A1-SV; diabetes; streptozotocin; vitamin D.

Fig. 1

Plasma levels of calcium, phosphate and vitamin D-associated factors in STZ rats. Levels of plasma (A) calcium, (B) phosphate, (C) 1,25(OH)₂D, (D) 25(OH)D, (E) PTH and (F) FGF23 were measured in plasma collected from the abdominal aorta of control and STZ rats. Values are expressed as means ± SEM. *p<0.05, **p<0.01 vs control. NS, not significant.

Fig. 2

Osteocalcin and Trap-5b levels in plasma from STZ rats. Levels of plasma (A) osteocalcin and (B) Trap-5b were measured in plasma collected from the abdominal aorta of control and STZ rats. Values are expressed as means ± SEM. *p<0.05 vs control. NS, not significant.

Fig. 3

Expression levels of renal Cyp27b1, Cyp24a1, Trpv5 and duodenal Trpv6 mRNA in STZ rats. Relative mRNA levels for (A) Cyp27b1, (B) Cyp24a1, (C) Trpv5 in the kidney and (D) Trpv6 in the duodenum of control and STZ rats as measured by RT-PCR and/or quantitative real-time PCR. (B) Analysis performed using primer Cyp24a1 Ex2-Ex4 (Table 1). Values are expressed as means ± SEM. *p<0.05, **p<0.01 vs control.

Fig. 4

Renal Cyp24a1 protein expression in STZ rats, and effect of insulin treatment on Cyp24a1 expression. Cyp24a1 protein expression in the kidney from control and STZ rats was determined by western blot analysis using anti-CYP24A1 antibody. (A) Whole cell protein (30 µg) was used for the analysis and for (B) peptide neutralization tests, which were used to verify an anti-CYP24A1 antibody specificity. (C) Levels of Cyp24a1 protein expression in kidney cells fractionated into cytosolic (10 µg), mitochondrial (10 µg) and nuclear fractions (5 µg). (D) Whole cell protein (15 µg) was used for the analysis. STZ + Insulin rats were treated subcutaneously with 2 U insulin twice daily from Day 4 to Day 9.

Fig. 5

Plasma 1,25(OH)₂D levels and renal Cyp24a1 and Cyp24a1-SV mRNA expression in STZ rats. (A) Plasma 1,25(OH)₂D levels, (B) renal Cyp24a1 and (C) renal Cyp24a1-SV mRNA levels were measured in control and STZ rats. STZ + Insulin rats were treated subcutaneously with 2 U insulin twice daily from Day 4 to Day 9. Relative Cyp24a1 and Cyp24a1-SV mRNA levels in kidney were measured by quantitative real-time PCR. The analysis were performed using (B) primer Cyp24a1 Ex2-Ex4 and (C) primer Cyp24a1-SV Int2-Ex4 (Table 1). Values are expressed as means ± SEM. *p<0.05, **p<0.01. NS, not significant.

Fig. 6

Effect of DEX on Cyp24a1 expression in OK-P cells. (A) OK-P cells were incubated with ethanol (EtOH) or 10⁻⁸, 10⁻⁷, 10⁻⁶ M of DEX for 24 h. (B) OK-P cells were incubated with EtOH, 10⁻⁷ DEX or 10⁻⁸ M 1,25(OH)₂D₃ for 22 h. Cyp24a1 protein expression in whole cell protein (30 µg) was determined by western blot analysis using anti-CYP24A1 antibody. (C) OK-P cells were incubated with or without 1 µg/ml Act. D for 30 min, and then the cells were incubated with EtOH or 10⁻⁷ M DEX. (D) OK-P cells were incubated with (closed squares) or without (open circles) 10⁻⁶ M RU 486 and EtOH or 10⁻¹¹, 10⁻⁹, 10⁻⁷, 10⁻⁵ M DEX for 24 h. (A, C, D) Relative Cyp24a1 mRNA levels were measured by quantitative real-time PCR.