Tetrahydrobiopterin has a glucose-lowering effect by suppressing hepatic gluconeogenesis in an endothelial nitric oxide synthase-dependent manner in diabetic mice

Abstract

Endothelial nitric oxide synthase (eNOS) dysfunction induces insulin resistance and glucose intolerance. Tetrahydrobiopterin (BH4) is an essential cofactor of eNOS that regulates eNOS activity. In the diabetic state, BH4 is oxidized to 7,8-dihydrobiopterin, which leads to eNOS dysfunction owing to eNOS uncoupling. The current study investigates the effects of BH4 on glucose metabolism and insulin sensitivity in diabetic mice. Single administration of BH4 lowered fasting blood glucose levels in wild-type mice with streptozotocin (STZ)-induced diabetes and alleviated eNOS dysfunction by increasing eNOS dimerization in the liver of these mice. Liver has a critical role in glucose-lowering effects of BH4 through suppression of hepatic gluconeogenesis. BH4 activated AMP kinase (AMPK), and the suppressing effect of BH4 on gluconeogenesis was AMPK-dependent. In addition, the glucose-lowering effect and activation of AMPK by BH4 did not appear in mice with STZ-induced diabetes lacking eNOS. Consecutive administration of BH4 in ob/ob mice ameliorated glucose intolerance and insulin resistance. Taken together, BH4 suppresses hepatic gluconeogenesis in an eNOS-dependent manner, and BH4 has a glucose-lowering effect as well as an insulin-sensitizing effect in diabetic mice. BH4 has potential in the treatment of type 2 diabetes.

FIG. 1.

Biopterin dynamics and effects of BH₄ on blood glucose levels in diabetic mice. A–D: BH2 levels and BH₄-to-BH₂ ratio of liver, blood, kidney, and spleen. Values are means ± SE. n = 7. *P < 0.05, **P < 0.01, ***P < 0.001 vs. without STZ. E and F: Fed blood glucose levels were not changed 2 h after injection of BH₄ (20 mg/kg i.p.) to STZ diabetic wild-type mice; fasting blood glucose levels were significantly decreased. Values are means ± SE. n = 8. *P < 0.05 vs. the value of preinjection of saline with BH₄ intraperitoneally; paired t test. No significant difference of fed and fasting blood glucose levels 2 h after intraperitoneal injection of saline to mice with STZ-induced diabetes.

FIG. 2.

Role of liver tissue in glucose-lowering effects of BH₄. A and B: IPGTT to wild-type mice. Blood glucose levels and plasma insulin levels after administration of glucose (2 g/kg i.p.) with or without BH₄ (20 mg/kg). Values are means ± SE (n = 6). C: PTT to wild-type mice. Elevation of blood glucose levels after intraperitoneal administration of pyruvate with BH₄ (20 mg/kg) to wild-type mice was suppressed compared with those without BH₄. Values are means ± SE (n = 6). *P < 0.05 vs. saline. D: In mice with STZ-induced diabetes, mRNA levels of GTPCH I expression were significantly decreased compared with those in nondiabetic wild-type mice liver. Values are means ± SE (n = 5). **P < 0.01 vs. nondiabetic wild-type mice liver. E: In wild-type mice with STZ-induced diabetes, protein expression levels of GTPCH I were significantly decreased compared with those in nondiabetic wild-type mice liver. Values are means ± SE (n = 5). **P < 0.01 vs. nondiabetic wild-type mice liver. F: No significant difference of mRNA expression levels of DHFR in liver was detected between nondiabetic mice and mice with STZ-induced diabetes. Values are means ± SE (n = 10). G: No significant difference of protein expression levels of DHFR in liver was detected between nondiabetic mice and mice with STZ-induced diabetes. Values are means ± SE (n = 5). H and I: In liver tissues of wild-type mice with STZ-induced diabetes treated with BH₄, mRNA levels of PEPCK were significantly decreased compared with those treated without BH₄. The mRNA levels of G6Pase were not changed. Values are means ± SE (n = 6), *P < 0.05 vs. saline. J: Liver tissues of eNOS dimer and monomer expression 2 h after intraperitoneal injection of saline with or without BH₄ (20 mg/kg) to wild-type mice with STZ-induced diabetes. Densitometric analysis of the ratio of eNOS dimer to monomer. Values are means ± SE (n = 5). *P < 0.05 vs. saline. K: In liver tissues of wild-type mice with STZ-induced diabetes treated with BH₄, NO content was significantly increased compared with those treated without BH₄. Values are means ± SE (n = 5). *P < 0.05 vs. saline.

FIG. 3.

BH₄ suppressed gluconeogenesis and increased AMPKα phosphorylation in hepatocytes isolated from wild-type mice. A: Time course of gluconeogenesis with exposure to BH₄. Suppressing effect on gluconeogenesis by 50 μmol/L BH₄ compared with control was detected after 60 min in hepatocytes isolated from wild-type mice. Values are means ± SE (n = 6). **P < 0.01 vs. control. B: Time course of phosphorylation of AMPKα and ACC upon exposure to BH₄ (50 μmol/L). Both AMPKα and ACC phosphorylation were stimulated after 30 min exposure to BH₄ in hepatocytes isolated from wild-type mice. Data are expressed as fold stimulation over control. Values are means ± SE (n = 3). *P < 0.05, **P < 0.01 vs. control. C: Suppressing effect on gluconeogenesis after 1 h exposure of BH₄ was detected ranging over 50 μmol/L in hepatocytes isolated from wild-type mice. Values are means ± SE (n = 6). **P < 0.01, ***P < 0.001 vs. control. D: Effect of BH₄ on phosphorylation of AMPK and ACC. After 30 min exposure to BH₄, both AMPKα and ACC phosphorylation were increased by BH₄ dose dependently ranging over 50 μmol/L in hepatocytes isolated from wild-type mice. Data are expressed as fold stimulation over control. Values are means ± SE (n = 3). *P < 0.05, **P < 0.01 vs. control. E: With transfection with AMPKα1 siRNA, protein expression of AMPKα was decreased compared with that of transfection with control siRNA. Values are means ± SE (n = 3). ***P < 0.001 vs. control siRNA. F: Transfected with AMPKα1 siRNA, suppressing effect of BH₄ (50 μmol/L) on hepatic glucose production was inhibited. Values are means ± SE (n = 6). ***P < 0.001 vs. values transfected with control siRNA without BH₄. G: Compound C (20 μmol/L), an AMPK inhibitor, abolished the suppressing effect of BH₄ (50 μmol/L) on gluconeogenesis. Values are means ± SE (n = 6). *P < 0.05 vs. values without BH₄ and without compound C.

FIG. 4.

Lack of the effect of BH₄ on suppression of gluconeogenesis in eNOS^−/− mouse hepatocytes. A: BH₄ (50 μmol/L) significantly increased NO production in hepatocytes from wild-type mice. SNP (20 μmol/L) was used as positive control. Values are means ± SE (n = 5). *P < 0.05 vs. control. B: BH₄ (ranging from 10 to 50 μmol/L) increased eNOS phosphorylation at Ser¹¹⁷⁷ in hepatocytes from wild-type mice. Values are means ± SE (n = 5). *P < 0.05 vs. control. C: BH₄ (ranging from 10 to 100 μmol/L) did not suppress gluconeogenesis after 1 h exposure in hepatocytes from eNOS^−/− mice. Values are means ± SE (n = 6). D: After 30 min exposure to BH₄ ranging from 10 to 100 μmol/L, AMPKα and ACC phosphorylation were not increased by BH₄ in hepatocytes from eNOS^−/− mice. Data are expressed as fold stimulation over control. Values are means ± SE (n = 3).

FIG. 5.

Effect of sepiapterin, a BH₄ precursor, on gluconeogenesis and AMPK activation. A: After 1 h exposure, sepiapterin (50 μmol/L) significantly suppressed gluconeogenesis in hepatocytes isolated from wild-type mice. This effect was not observed in hepatocytes isolated from eNOS^−/− mice. Values are means ± SE (n = 6). *P < 0.05 vs. control. B: After 30 min exposure to sepiapterin (50 μmol/L), AMPKα phosphorylation was increased in hepatocytes isolated from wild-type mice. AMPKα phosphorylation was not increased by sepiapterin in hepatocytes isolated from eNOS^−/− mice. Data are expressed as fold stimulation over control. Values are means ± SE (n = 3). **P < 0.01 vs. control.

FIG. 6.

Effects of BH₄ in eNOS^−/− mice with STZ-induced diabetes. A: No significant difference of fasting blood glucose levels 2 h after intraperitoneal injection of saline with or without BH₄ (20 mg/kg) to eNOS^−/− mice with STZ-induced diabetes. Values are means ± SE (n = 7). B: PTT to eNOS^−/− mice. No effects of BH₄ (20 mg/kg) on suppressing hepatic gluconeogenesis were detected in PTT in eNOS^−/− mice. Values are means ± SE (n = 6). C: AMPKα phosphorylation in liver of eNOS^−/− mice with STZ-induced diabetes was not changed by BH₄ administration. Data are expressed as fold stimulation over saline. Values are means ± SE (n = 3). D: AMPKα phosphorylation in liver of wild-type mice with STZ-induced diabetes was significantly increased by BH₄ (20 mg/kg) administration. Data are expressed as fold stimulation over saline. Values are means ± SE (n = 3). **P < 0.01 vs. saline.

FIG. 7.

Effects of BH₄ in ob/ob mice. A: PTT to ob/ob mice with or without single administration of BH₄ (20 mg/kg). Values are means ± SE (n = 6). *P < 0.05 vs. the value of saline. B: Fasting blood glucose levels of ob/ob mice treated with BH₄ (20 mg/kg/day) for 10 days were significantly decreased compared with those treated without BH₄. Values are means ± SE (n = 6). *P < 0.05 vs. the value of saline. C: Fed blood glucose levels in ob/ob mice treated with or without BH₄ for 10 days. P = 0.07 vs. the value of saline. Values are means ± SE (n = 6). D and E: IPGTT to ob/ob mice. Blood glucose levels and plasma insulin levels after administration of glucose (1 g/kg i.p.) with or without BH₄ for 10 days. Values are means ± SE (n = 6). *P < 0.05, **P < 0.01 vs. without BH₄. F: HOMA-IR calculated from fasting blood glucose and insulin levels from IPGTT data in ob/ob mice treated with or without BH₄ for 10 days. Values are means ± SE (n = 6). **P < 0.01 vs. the value of saline. G: Insulin tolerance test (ITT) to ob/ob mice treated with or without BH₄ for 10 days. Values are means ± SE (n = 6). *P < 0.05 vs. the value of saline. H and I: AMPKα, ACC, and Akt phosphorylation in liver tissues of ob/ob mice was increased by 10 days’ administration of BH₄. Data are expressed as fold stimulation over saline. Values are means ± SE (n = 3). *P < 0.05 vs. saline.