Sepiapterin reverses the changes in gastric nNOS dimerization and function in diabetic gastroparesis

Abstract

Background: We have demonstrated previously that in vivo supplementation of tetrahydrobiopterin (BH₄); a co-factor for neuronal nitric oxide synthase (nNOS) significantly restored delayed gastric emptying and attenuated nitrergic relaxation in diabetic rat. In this study, we have investigated whether supplementation of sepiapterin (SEP), a precursor for BH₄ biosynthesis via salvage pathway restores gastric emptying and nitrergic system in female diabetic rats.

Methods: Diabetic rats (streptozotocin-induced) were supplemented with BH₄ or SEP (20 mg kg⁻¹ body weight). Gastric nitrergic relaxation in the presence or absence of high glucose and SEP were measured by electric field stimulation. Gastric muscular strips from healthy or diabetic female rats were incubated in the presence or absence of high glucose, SEP and/or methotrexate (MTX). Nitric oxide release was measured colorimetrically by NO assay kit. The expression of nNOSα and dimerization was detected by Western blot.

Key results: In vitro studies on gastric muscular tissues showed that MTX, an inhibitor of BH₄ synthesis via salvage pathway, significantly decreased NO release. In vivo treatment with MTX reduced both gastric nitrergic relaxation and nNOSα dimerization. Supplementation of SEP significantly attenuated delayed gastric emptying in diabetic rats. In addition, SEP supplementation restored impaired nitrergic relaxation, gastric nNOSα protein expression, and dimerization in diabetic rats.

Conclusions & inferences: The above data suggests that supplementation of SEP accelerated gastric emptying and attenuated reduced gastric nNOSα expression, and dimerization. Therefore, SEP supplementation is a potential therapeutic option for female patients of diabetic gastroparesis.

Figure 1

Effect of sepiapterin (SEP) treatment on diabetes-induced solid gastric emptying in female rats. Groups (4–6) of diabetic rats received dietary SEP (20 mg kg⁻¹ body wt) daily for 10 days after diabetic induction with single injection of streptozotocin (STZ 55 mg kg⁻¹ body wt; ip). Control group was injected with vehicle (9 mmol citrate buffer) only. The values are mean ± SE for 4–6 animals. Statistical significance was determined by Tukey test after one-way ANOVA. *P < 0.05 compared with control group; ^#P < compared with diabetic |DB) group.

Figure 2

Effect of sepiapterin (SEP) on nitrergic relaxation in diabetic rat gastric muscular tissues in vivo. Nitrergic relaxation was measured following daily exposure to dietary SEP (20 mg kg⁻¹ body wt) for 10 days after diabetic induction with single injection of streptozotocin (STZ 55 mg kg⁻¹ body wt ip). Control group was injected with vehicle (9 mmol citrate buffer) only. Values are mean ± SE (n = 4–6). Statistical significance was determined by Tukey test after one-way ANOVA. *P < 0.05 compared with control group; ^#P < compared with diabetic [DB] group.

Figure 3

(A) Effect of methotrexate (MTX)-induced nitrergic relaxation in female diabetic rats in vivo. Nitrergic relaxation was measured following daily exposure to intraperitoneal injection (i.p) of MTX (3.75 mg kg⁻¹ body wt, two times a day), for 4 days. The values are mean ± SE of 4–6 animals. Statistical significance was determined by Student t-test. *P < 0.05 compared with control group. (B) Effect of MTX-induced neuronal nitric oxide synthaseα (nNOSα) dimcr expression in female diabetic rats in vivo. Western blot was done following daily exposure to MTX (3.75 mg kg⁻¹ body wt, two times a day), for 4 days. Representative immunoblot and densitometric analysis data for nNOSα protein dimerization in female rat gastric antrum. Values are mean ± SE (n = 4). Statistical significance was determined by Tukey test after one-way ANOVA. *P < 0.05 compared with control group ^#P < compared with MTX group. (C) Effect of MTX and sepiapterin (SEP) supplementation on nitric oxide (NO) release in female diabetic rats in vitro. Nitric oxide levels were measured using the NO assay kit following 48 h incubation with MTX (100 μmol L⁻¹), MTX + SEP (100 μmol L⁻¹, 100 μmol L⁻¹). Values are mean ± SE (n = 4–6). Statistical significance was determined by Tukey test after one-way ANOVA. *P < 0.05 compared with control group; ^#P < compared with MTX group.

Figure 4

Effect of sepiapterin (SEP) on neuronal nitric oxide synthaseα (nNOSα) protein expression and nNOSα dimerization of diabetic rat gastric tissues. nNOSα protein expression and nNOSα dimerization was measured following either daily exposure to SEP (20 mg kg⁻¹ body wt) for 10 days or tetrahydrobiopterin (BH₄) supplementation (20 mg kg⁻¹ body wt per day) for 2 weeks after diabetic induction with single injection of STZ (55 mg kg⁻¹ body wt ip). Control group was injected with vehicle (9 mmol citrate buffer) only. (A) Representative immunoblot and densitometric analysis data for nNOSα protein expression in female control rat gastric antrum supplemented with either BH₄ or SEP. (B) Representative immunoblot and densitometric analysis data for nNOSα protein expression in female diabetic rat gastric antrum. (C) Representative immunoblot and densitometric analysis data for nNOSα protein dimerization in female control rat gastric antrum supplemented with either BH₄ or SEP. (D) Representative immunoblot and densitometric analysis data for nNOSα protein dimerization in female diabetic rat gastric antrum. Values are mean ± SE (n = 4). Statistical significance was determined by Tukey test after one-way ANOVA. *P < 0.05 compared with control group; ^#P < compared with diabetic (DB) group.