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Abstract-D-Glucose infusion and gestational diabetes induce vasodilatation in humans and increase L-arginine Key Words: humans Ⅲ endothelium Ⅲ glucose Ⅲ arginine Ⅲ nitric oxide T he cationic amino acid L-arginine is the substrate for nitric oxide (NO) synthesis via endothelial NO synthase (eNOS) 1 and is taken up primarily by the Na ϩ -independent high-affinity (K m Ϸ100 to 400 mol/L) systems y ϩ /CAT-1 and y ϩ /CAT-2B (where CAT indicates cationic amino acid transporter) in human umbilical vein endothelial cells (HUVECs). 2,3 L-Arginine transport and NO synthesis (Larginine/NO pathway) are increased in HUVECs from patients with gestational diabetes. 2 Interestingly, long-term incubation (24 hours) of HUVECs from normal pregnancies with elevated D-glucose mimics the effect of gestational diabetes on the L-arginine/NO pathway. 4 In addition, elevated D-glucose for 24 hours 4,5 or 5 days 6 increases eNOS gene expression. A recent report shows that D-glucose infusion induces vasodilatation in humans, 7 and in animal models, an elevation of plasma D-glucose results in rapid (seconds to minutes) vasodilatation. 8 -10 Therefore, rapid fluctuations in the D-glucose level are crucial in maintaining human fetal endothelial function. [2][3][4][5]11 D-Glucose activates protein kinase C (PKC), an enzyme involved with long-term stimulation of the L-arginine/NO pathway, 5,12-14 and (within 1 hour) p42 and p44 mitogen-activated protein (MAP) kinases (p42/44 mapk ). 5,14,15 p42/44 mapk activation may itself be dependent on PKC activation and NO synthesis. 5,14 However, the effect of short-term incubation with elevated D-glucose on the endothelial L-arginine/NO pathway has not been investigated. 4,11,16,17 The present study shows that a 2-minute incubation with 25 mmol/L D-glucose increases L-arginine transport and NO synthesis in HUVECs. The underlying cellular mechanisms involve phosphorylation of eNOS at Ser 1177 via phosphatidylinositol 3-kinase (PI3-k) and activation of eNOS and p42/ p44 mapk by D-glucose. 18 Materials and Methods Cell CultureHuman umbilical vein endothelium was isolated (collagenase digestion 0.25 mg/mL) and cultured (37°C, 5% CO 2 , confluent passage 2) in medium 199 containing 5 mmol/L D-glucose, 10% newborn calf serum, 10% fetal calf serum, 3.2 mmol/L L-glutamine, 100 mol/L L-arginine, and 100 U/mL penicillin-streptomycin (primary culture medium). [2][3][4] Before an experiment (24 hours), the incubation medium was changed to serum-free medium 199.

Vascular tone is controlled by the L-arginine/nitric oxide (NO) pathway, and NO bioavailability is strongly affected by hyperglycaemia-induced oxidative stress. Insulin leads to high expression and activity of human cationic amino acid transporter 1 (hCAT-1), NO synthesis and vasodilation; thus, a protective role of insulin on high D-glucose–alterations in endothelial function is likely. Vascular reactivity to U46619 (thromboxane A2 mimetic) and calcitonin gene related peptide (CGRP) was measured in KCl preconstricted human umbilical vein rings (wire myography) incubated in normal (5 mmol/L) or high (25 mmol/L) D-glucose. hCAT-1, endothelial NO synthase (eNOS), 42 and 44 kDa mitogen-activated protein kinases (p42/44mapk), protein kinase B/Akt (Akt) expression and activity were determined by western blotting and qRT-PCR, tetrahydrobiopterin (BH4) level was determined by HPLC, and L-arginine transport (0–1000 μmol/L) was measured in response to 5–25 mmol/L D-glucose (0–36 hours) in passage 2 human umbilical vein endothelial cells (HUVECs). Assays were in the absence or presence of insulin and/or apocynin (nicotinamide adenine dinucleotide phosphate-oxidase [NADPH oxidase] inhibitor), tempol or Mn(III)TMPyP (SOD mimetics). High D-glucose increased hCAT-1 expression and activity, which was biphasic (peaks: 6 and 24 hours of incubation). High D-glucose–increased maximal transport velocity was blocked by insulin and correlated with lower hCAT-1 expression and SLC7A1 gene promoter activity. High D-glucose–increased transport parallels higher reactive oxygen species (ROS) and superoxide anion (O2 •–) generation, and increased U46619-contraction and reduced CGRP-dilation of vein rings. Insulin and apocynin attenuate ROS and O2 •– generation, and restored vascular reactivity to U46619 and CGRP. Insulin, but not apocynin or tempol reversed high D-glucose–increased NO synthesis; however, tempol and Mn(III)TMPyP reversed the high D-glucose–reduced BH4 level. Insulin and tempol blocked the high D-glucose–increased p42/44mapk phosphorylation. Vascular dysfunction caused by high D-glucose is likely attenuated by insulin through the L-arginine/NO and O2 •–/NADPH oxidase pathways. These findings are of interest for better understanding vascular dysfunction in states of foetal insulin resistance and hyperglycaemia.

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The chicken ovary is organized into a hierarchy of yellow yolky follicles that ovulate on successive days. Active or passive immunization of laying hens against testosterone blocks ovulation without affecting follicle development. Testosterone may play a role in pre-ovulatory follicle maturation by stimulating granulosa progesterone production. We assessed whether this stimulus is dose-related and depends on the maturity of the donor follicle, and if it does so by stimulating granulosa cell STAR, P450 cholesterol side-chain cleavage (P450scc), and LH receptor (LHCGR) mRNAs expression. Progesterone production by granulosa cells from F1, F3, and F4 follicles, cultured for 3 h without testosterone was greater in cells collected 11-14 h than 1-4 h after ovulation. These differences in progesterone production were less pronounced after granulosa cells had been cultured for 24 h. Culture of granulosa cells for 3 or 24 h with testosterone (1-100 ng/ml) stimulated progesterone production in cells collected from F4, F3, or F1 follicles 1-4, or 11-14 h after ovulation. Testosterone (0-4000 ng/ml) alone or in combination with LH (0-100 ng/ml) increased progesterone production by F1 granulosa cells, collected 1-4 and 11-14 h after ovulation and cultured for 3 h. Finally, testosterone (10 or 100 ng/ml) increased STAR, P450scc, and LHCGR mRNAs, when added to 3 h cultures of F1 granulosa cells. In conclusion, testosterone stimulates granulosa cell progesterone production in hen pre-ovulatory hierarchical follicles irrespective of maturational state, acting alone or additively with LH. We propose that testosterone promotes granulosa cell maturation to facilitate the pre-ovulatory release of LH.