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Insulin Restores Gestational Diabetes Mellitus–Reduced Adenosine Transport Involving Differential Expression of Insulin Receptor Isoforms in Human Umbilical Vein Endothelium

OBJECTIVE To determine whether insulin reverses gestational diabetes mellitus (GDM)–reduced expression and activity of human equilibrative nucleoside transporters 1 (hENT1) in human umbilical vein endothelium cells (HUVECs). RESEARCH DESIGN AND METHODS Primary cultured HUVECs from full-term normal (n = 44) and diet-treated GDM (n = 44) pregnancies were used. Insulin effect was assayed on hENT1 expression (protein, mRNA, SLC29A1 promoter activity) and activity (initial rates of adenosine transport) as well as endothelial nitric oxide (NO) synthase activity (serine1177 phosphorylation, l-citrulline formation). Adenosine concentration in culture medium and umbilical vein blood (high-performance liquid chromatography) as well as insulin receptor A and B expression (quantitative PCR) were determined. Reactivity of umbilical vein rings to adenosine and insulin was assayed by wire myography. Experiments were in the absence or presence of l-NG-nitro-l-arginine methyl ester (l-NAME; NO synthase inhibitor) or ZM-241385 (an A2A-adenosine receptor antagonist). RESULTS Umbilical vein blood adenosine concentration was higher, and the adenosine- and insulin-induced NO/endothelium-dependent umbilical vein relaxation was lower in GDM. Cells from GDM exhibited increased insulin receptor A isoform expression in addition to the reported NO–dependent inhibition of hENT1-adenosine transport and SLC29A1 reporter repression, and increased extracellular concentration of adenosine and NO synthase activity. Insulin reversed all these parameters to values in normal pregnancies, an effect blocked by ZM-241385 and l-NAME. CONCLUSIONS GDM and normal pregnancy HUVEC phenotypes are differentially responsive to insulin, a phenomenon where insulin acts as protecting factor for endothelial dysfunction characteristic of this syndrome. Abnormal adenosine plasma levels, and potentially A2A-adenosine receptors and insulin receptor A, will play crucial roles in this phenomenon in GDM.

RESULTS-Umbilical vein blood adenosine concentration was higher, and the adenosine-and insulin-induced NO/endotheliumdependent umbilical vein relaxation was lower in GDM. Cells from GDM exhibited increased insulin receptor A isoform expression in addition to the reported NO-dependent inhibition of hENT1-adenosine transport and SLC29A1 reporter repression, and increased extracellular concentration of adenosine and NO synthase activity. Insulin reversed all these parameters to values in normal pregnancies, an effect blocked by ZM-241385 and L-NAME.
CONCLUSIONS-GDM and normal pregnancy HUVEC phenotypes are differentially responsive to insulin, a phenomenon where insulin acts as protecting factor for endothelial dysfunction characteristic of this syndrome. Abnormal adenosine plasma levels, and potentially A 2A -adenosine receptors and insulin receptor A, will play crucial roles in this phenomenon in GDM. Diabetes 60:1677-1687, 2011 G estational diabetes mellitus (GDM) is a syndrome associated with maternal hyperglycemia and defective insulin signaling in the placenta (1)(2)(3). GDM leads to fetoplacental vascular endothelial dysfunction (4,5), a condition associated with reduced uptake and subsequent increased extracellular concentration of adenosine, an endogenous purine vasoactive nucleoside, in primary cultured human umbilical vein endothelial cells (HUVECs) (6,7). Even though adenosine is a well-known vasodilator in the human placental circulation (8) and in other vascular beds (9), no reports have addressed the umbilical plasma level of adenosine in GDM. The adenosine concentration in umbilical vein blood is elevated in pregnancies where the mother is affected by diabetes (10). Because plasma adenosine level is mainly regulated by the capacity of the endothelium to take up and metabolize this nucleoside (3,5,11,12), ectonucleosidases are not expressed in HUVECs (12) and the umbilical sera blood content of adenosine deaminase, an enzyme that metabolizes adenosine, is comparable in GDM and in normal pregnancies (13); a crucial role for nucleoside transporters has been proposed in this cell type in GDM (3,6,14). In HUVECs under physiologic conditions, adenosine uptake is mediated by equilibrative human nucleoside transporters 1 (hENT1), which is inhibited by #1 mmol/L nitrobenzylthioinosine (NBTI) and 2 (hENT2), which is inhibited by .1 mmol/L NBTI and cross-inhibited by hypoxanthine (6,7,15). Interestingly, because GDM is associated with nitric oxide (NO)-dependent reduced hENT1-adenosine transport in HUVECs (7), changes in expression and/or activity of hENT1 could result in altered physiologic plasma adenosine concentration, leading to endothelial dysfunction in this syndrome.
Elevated extracellular D-glucose reduces hENT1 expression and activity in a NO-dependent manner in HUVECs, effects reversed by insulin; however, this hormone reduced hENT1 expression and activity in HUVECs cultured at physiologic concentrations of D-glucose (16). Because the deleterious effect of GDM on placental endothelial function most likely results from maternal and fetal hyperglycemia (1,2,17,18) and supraphysiologic umbilical vein blood hyperinsulinemia is detected in GDM (2,19,20), we hypothesize that this hormone will reverse GDM-associated reduced hENT1 expression and activity in HUVECs. Interestingly, because insulin resistance is associated with increased expression of A 2B -adenosine receptors in monocyte-derived macrophages from subjects with type 2 diabetes (21), we expect adenosine receptors to play a role in the effect of insulin in HUVECs in GDM. Our results show that reduced adenosine transport in HUVECs from GDM pregnancies is reversed by insulin to values in cells from normal pregnancies, where insulin receptor isoforms A (IR-A) and B (IR-B) and A 2Aadenosine receptors may play a role.

RESULTS
Patients and newborns. The study included 44 patients with normal pregnancies and 44 GDM patients treated with diet ( Table 1). All pregnancies were singleton, and pregnant women were normotensive, nonsmoking, did not consume alcohol or drugs, and were without intrauterine infection or any other medical or obstetrical complications. Adenosine transport. To check insulin effect on ENTslike activity, we assayed insulin and NBTI (ENTs inhibitor) effect on transport. GDM reduced overall adenosine transport. The effect was reversed by insulin but unaltered by NBTI (Fig. 1A). Insulin increased, but NBTI reduced, overall transport in cells from normal pregnancies. NBTI blocked the insulin effect in both cell types. Insulin reduced hENT1-adenosine transport in normal pregnancies; however, it was reversed in GDM to values in normal pregnancies in the absence of insulin (Fig. 1B). Insulin stimulation of overall transport was concentration-dependent ( Fig. 1C), and the insulin required was higher in GDM than in normal pregnancies (Table 2). To test whether NOS activity was involved, L-NAME (general NOS inhibitor) was used. L-NAME increased overall adenosine transport to comparable values in both cell types in the absence of insulin; however, insulin increased overall adenosine transport only in cells from normal pregnancies (Fig. 1C). Insulin reduced hENT1adenosine transport in a concentration-dependent manner in normal pregnancies, but it was increased in GDM (Fig. 1D), requiring more insulin in GDM than in normal pregnancies ( Table 2). In the absence of insulin, hENT1adenosine transport was higher, reaching comparable values in the presence of L-NAME, which was unaltered by insulin in both cell types. NOS activity and eNOS expression. To assess the role of eNOS in insulin effect, NOS activity and eNOS expression in response to insulin was assayed. Insulin increased L-citrulline formation in normal pregnancies but reversed GDM-associated increase on L-citrulline formation to values in normal pregnancies ( Fig. 2A). Equally, insulin increased L-NAME-inhibitable L-citrulline formation in normal pregnancies to values in GDM in the absence of this hormone. However, the total and L-NAME-inhibitable L-citrulline formation in GMD was partially (63 6 5%) reduced by insulin. More insulin was required in GDM than in normal pregnancies ( Table 2). Relative eNOS phosphorylation versus total eNOS protein abundance was increased in normal pregnancies, but GDM increase in eNOS phosphorylation was reversed by insulin to values in normal pregnancies in the absence of this hormone (Fig. 2B). The estimated insulin effectiveness (i.e., IC 50 GDM/SC 50 Normal) in this phenomenon was similar in GDM compared with normal pregnancies (Table 2). Insulin caused a higher increase (2.7 6 0.2-fold) in phosphorylated compared with total eNOS protein abundance in cells from normal pregnancies (Fig. 2C). However, insulin caused a similar reduction in phosphorylated and total eNOS protein abundance in GDM (Fig. 2D). Expression of hENT1. Because of the possibility that the insulin effect was due to altered hENT1 expression, protein abundance and mRNA expression of hENT1 was assayed. Insulin reduced hENT1 protein abundance in HUVECs from normal pregnancies to values in GDM, but reversed GDM-associated reduced hENT1 protein abundance to values in normal pregnancies in the absence of this hormone (Fig. 3A). The insulin requirement was higher in GDM than in normal pregnancies ( Table 2). L-NAME reversed the insulin effect in normal and GDM pregnancies, reaching hENT1 protein abundance levels higher than in the absence of this hormone (Fig. 3B). In addition, L-NAME increased the effect of insulin in GDM pregnancies. Similar responses were obtained for hENT1 mRNA expression ( Fig. 3C and D), and the insulin requirement for this effect was similar in normal and GDM pregnancies ( Table 2). SLC29A1 promoter activity. Having found that insulin and GDM effect are associated with altered hENT1 expression, we then checked whether changes correlated with SLC29A1 expression. Reporter luciferase activity in cells from GDM transfected with pGL3-hENT1 23198 , but not for the pGL3-hENT1 21670 construct, was lower compared with normal pregnancies in the absence of insulin (Fig. 4A). Insulin reduced reporter activity in cells from normal pregnancies transfected with GL3-hENT1 23198 but increased GDM-reduced pGL3-hENT1 23198 reporter activity to values in normal pregnancies in the absence of this hormone.
The potential involvement of NOS activity in SLC29A1 expression was then assayed. The insulin effect in cells from normal pregnancies was blocked by L-NAME, reaching values higher than basal reporter activity in cells from normal pregnancies in the absence of this hormone (Fig. 4B). GDM-reduced promoter activity was reversed by L-NAME to values in normal pregnancies, an effect that was higher in cells co-incubated with insulin and L-NAME. In addition, L-NAME increased promoter activity to similar values in both cell types when co-incubated with insulin and L-NAME. However, no significant changes were seen for pGL3-hENT1 21670 reporter activity under these conditions (Fig. 4C). Extracellular adenosine. Reduced expression of hENT1 results in extracellular adenosine accumulation. Thus, we measured adenosine levels in HUVEC cultures. Extracellular adenosine concentration in cells from GDM pregnancies was higher than in cells from normal pregnancies, an effect blocked by insulin and L-NAME (Fig. 5A). However, insulin increased extracellular adenosine concentration in normal pregnancies, an effect blocked by L-NAME. Parallel assays showed increased adenosine plasma concentration in umbilical vein blood in GDM compared with normal pregnancies. Umbilical vein response to adenosine. Vessel OID was estimated to determine whether the basal tone of human umbilical veins was altered in GDM. GDM was associated with larger umbilical vein rings OID (6.05 6 0.4 mm) compared with normal pregnancies (4.82 6 0.3 mm) under  basal conditions (P , 0.05, n = 18). In addition, because the extracellular adenosine level is higher in HUVECs from GDM, it is feasible that adenosine could modulate umbilical vein reactivity. Thus, we assayed adenosine effect and involvement of adenosine receptors on reactivity of human umbilical vein rings. Adenosine caused relaxation of umbilical vein rings, an effect less effective in GDM compared with normal pregnancies (Fig. 5B), which was endotheliumdependent and was partially reduced by ZM-241385 in vessels from normal pregnancies but was abolished in GDM pregnancies (Fig. 5C). The ZM-241385-inhibitable fraction of adenosine effect was similar (P . 0.05) in normal and GDM pregnancies (61 6 12 vs. 71 6 14%, respectively). Adenosine effect was blocked by L-NAME and caused vasoconstriction higher than the KCl-maximal response in both vessel types (Fig. 5D).
Umbilical vein response to insulin. We also assayed insulin effect on reactivity of human umbilical vein rings. Insulin caused concentration-dependent relaxation of umbilical vein rings, an effect less effective in GDM compared with normal pregnancies and abolished by L-NAME (Fig. 6A, Table 2). This inhibitor caused vessel contraction  in all experimental conditions. Insulin effect was also blocked by ZM-241385 in normal or GDM pregnancies, causing similar vasoconstriction in the absence or presence of insulin or L-NAME (Fig. 6B). Expression of insulin receptor isoforms. Because insulin could signal through two isoforms of insulin receptors in HUVECs, we tested whether IR-A and IR-B expression was altered in GDM. IR-A and IR-B mRNA was detectable in HUVECs, with a higher number of mRNA copies for IR-A compared with IR-B (Fig. 6C). In addition, IR-A expression, but not IR-B mRNA expression, was higher in cells from GDM compared with normal pregnancies. Insulin reversed the GDM effect on IR-A mRNA expression but did not alter IR-A mRNA expression in normal pregnancies, and neither altered IR-B mRNA expression in both cell types.

DISCUSSION
This study shows that GDM-associated reduced adenosine transport in HUVECs is reversed by insulin to values in normal pregnancies, involving re-establishment of hENT1 expression and activity. Insulin also reversed GDM-increased eNOS expression and activity, and GDM-reduced SLC29A1 promoter activity to values in normal pregnancies. However, hENT1 activity and expression as well as SLC29A1 promoter activity were reduced by insulin via NO in HUVEC from normal pregnancies. Extracellular adenosine concentrations in HUVECs and in the umbilical vein blood from GDM were higher than in normal pregnancies. GDM was associated with a higher basal OID in umbilical vein rings compared with normal pregnancies. Adenosine and insulin caused endothelium-dependent relaxation of umbilical vein rings that was dependent on NOS activity and involved activation of A 2A -adenosine receptors. HUVECs express IR-A and IR-B isoforms, of which IR-A mRNA expression was higher in GDM compared with normal pregnancies, an effect also reversed by insulin. Altogether, these results suggest that insulin causes differential responses in HUVECs and umbilical vein reactivity, reversing the GDM-associated phenotype to a normal pregnancy-associated phenotype. Adenosine transport. GDM causes NO-dependent reduced hENT1 expression and activity in HUVEC (7), and insulin increases overall adenosine transport in HUVEC from normal pregnancies (16). We here show that insulin restores hENT1-adenosine transport in HUVECs from GDM pregnancies to values in normal pregnancies, suggesting a potential beneficial action of insulin on human fetal endothelial cell function in GDM. A similar amount of insulin was required to stimulate overall and hENT1mediated transport (P . 0.05) in cells from normal (SC 50 overall/SC 50 hENT1 = 1.01 6 0.02) and GDM pregnancies (SC 50 overall/SC 50 hENT1 = 1.05 6 0.03), which suggests insulin action could be due to preferential (if not exclusive) modulation of hENT1 rather than other nucleoside membrane transport mechanism(s). However, a potential increased insulin-resistant state of HUVECs from GDM, as reported for the human placenta in GDM (2), is feasible because insulin was more effective in stimulating overall adenosine transport in cells from normal compared with GDM pregnancies (SC 50 GDM/SC 50 normal ;2.12). Because fetal insulin plasma level is increased in GDM (2,19,20), the latter could result as a compensatory mechanism to a defective insulin-induced fetoplacental endothelium signaling.
Interestingly, insulin reduced hENT1-adenosine transport in cells from normal pregnancies, thus suggesting a differential effect of insulin on hENT1 transport activity in HUVECs from normal compared with GDM pregnancies. This finding agrees with the reported insulin effect abolishing GDM-increased L-arginine transport in HUVECs (25), experimental diabetes-increased L-arginine transport in rabbit gastric glands (26) and in rat exocrine pancreas (27), and blocking the high extracellular D-glucose-increased L-arginine transport and eNOS activity in HUVECs from normal pregnancies (23). Thus, the beneficial effect of insulin is not a phenomenon restricted to nucleoside transport or HUVECs. However, nothing more than a cycloheximidedependent, unveiled post-transductional regulation explaining differential mechanism(s) behind the biologic effects of insulin has been reported for these phenomena (3,25,28). NO involvement. GDM-increased NO synthesis and eNOS expression was reversed by insulin to values in normal pregnancies. However, higher NOS activity and eNOS expression in response to insulin was found in normal pregnancies, confirming previous observations in this cell type (16). Thus, the possibility of a potential differential effect of insulin included the capacity of HUVECs to synthesize NO. Interestingly, NOS activity modulation by insulin was paralleled by similar changes in eNOS phosphorylation  at Ser 1177 (i.e., indicative of eNOS activation) (29) in cells from GDM and normal pregnancies, suggesting that modulation of eNOS activity rather than expression is crucial for insulin effect in HUVECs from normal and GDM pregnancies. Insulin-caused eNOS post-transductional modifications result in opposite responses in these two types of cells. However, nothing is yet reported regarding a mechanism for this phenomenon in fetal endothelium (3,28). We recently showed that reduced SLC29A1 gene expression leads to lower hENT1-adenosine transport in HUVECs from GDM via a NO-dependent mechanism, involving activation of the hC/element-binding protein (EPB) homologous protein 10 (CHOP)-C/EBP-a transcription factors complex (7). Because we found that insulin 1) reversed the GDM-reduced SLC29A1 promoter activity of the construct containing hCHOP-C/EBP-a consensus sequence, 2) reversed GDM-increased NO synthesis, and 3) 21845G.T and 21844C.A mutations in SLC29A1 blocks the NO/hCHOP-C/EBPa-dependent GDM repressive effect on expression (7), insulin action likely involves this complex of transcription factors in its potential beneficial effects in GDM.
Interestingly, insulin increased overall but not hENT1adenosine transport in the presence of a NOS inhibitor in cells from normal pregnancies. Thus, insulin would also be triggering NO-independent mechanism(s) modulating other than hENT1-adenosine transport. Because insulin causes NO-independent hENT2 activation in HUVECs from normal pregnancies (16), insulin-increased overall adenosine transport due to NO-independent activation of this nucleoside transporter isoform in HUVECs from normal pregnancies is feasible. Insulin did not alter overall, and neither did hENT1-adenosine transport in GDM pregnancies where NOS activity was inhibited; thus, it is suggested that this transporter will be subjected to modulation by insulin in fetal endothelium from pregnancies with this syndrome.
Interestingly, exposure of primary cultures of HUVECs from GDM to supraphysiologic concentrations of D-glucose (25 mmol/L, 24 h) resulted in insulin insensitivity (25). Thus, because a higher mother-to-fetus D-glucose flux occurs in GDM pregnancies, which therefore exposes fetal endothelium to hyperglycemia in the uterus (1,2,17,18), lack of modulation of hENT1 activity by insulin in cells from GDM may result from a long-term exposure of HUVECs to an abnormal environmental condition. However, this feasibility is unlikely because glycemia in patients was controlled by diet; therefore, maternal hyperglycemia will instead most likely increase in postprandial peaks, perhaps lasting for 2 to 4 h and thus leading to states of  (8 h) in the absence (-) or presence (+) of insulin and/or L-NAME. Culture medium, mixed with 0.5 mol/L acetate buffer, 1 mmol/L adenosine (internal standard), and 10 mmol/L aqueous (50%) chloroacetaldehyde, was incubated (80°C, 1 h), centrifuged (14,000g, 4 min), injected (80 mL) into a fluorescence detector-equipped Isco HPLC system (C 18 reverse-phase column, 5-mm particle size, 10 mmol/L citrate-buffer with 4.5% acetonitrile as mobile phase), and run isocratically (1 mL/min) as described (6,16). Insert shows adenosine concentration in human umbilical vein blood for normal (N) or GDM pregnancies. For umbilical vein blood adenosine, blood samples, mixed with 10 mmol/L erythro-9-(2-hydroxy-3-nonyl) adenine, 10 mmol/L NBTI, 1 mmol/L dilazep, 2 mg/mL indomethacin, and 40 mmol/L O,O'-bis(2-aminoethyl) ethyleneglycol-N,N,N'-N'-tetraacetic acid, taken at birth were centrifuged (14,000g, 1 min), deproteinated (50% trichloroacetic acid), and centrifuged (5 min) to obtain a supernatant that was neutralized (100 mL 3.3 N potassium hydroxide). Adenine nucleotides were extracted (1 mol/L zinc sulfate, saturated barium hydroxide, centrifugation at 14,000g, 5 min), and adenosine was converted to ethenoadenosine (440 mmol/L chloroacetaldehyde, 80°C, 1 h) for HPLC analysis (23). repetitive short-term exposure of HUVECs to elevated D-glucose. In addition, we cannot rule out the possibility that response to insulin of HUVECs from GDM resulted from adaptation to a normal (non-GDM) environment in vitro. However, we speculate on the possibility that chronic short-term exposure to hyperglycemia could be enough to cause a lack or an insufficient response to insulin by the fetal vasculature in the uterus. This is supported by the potential insulin insensitivity suggested in HUVECs from GDM exposed to elevated extracellular D-glucose (25). Vascular reactivity and adenosine levels. Umbilical vein blood adenosine in GDM is higher than in normal pregnancies, correlating with increased extracellular adenosine concentration in cultures of HUVECs from GDM (6). Thus, reduced adenosine transport by the endothelium could, at least in part, be responsible for the elevated umbilical vein blood adenosine level detected in GDM pregnancies. The latter is also a finding complemented by the elevated umbilical cord plasma adenosine reported in pregnancies complicated by maternal diabetes (10) and recently suggested to occur in patients with type 2 diabetes (21). Adenosine caused relaxation of umbilical vein rings from GDM, a response less potent compared with vessels from normal pregnancies; therefore, a tonic and close to maximal vasodilatory effect of adenosine due to a supraphysiologic concentration of this nucleoside on this type of vessel in GDM is suggested. This is supported by results showing that the adenosine receptor antagonist ZM-241385 blocked adenosine effect and increased the basal tone in these vessels. Because adenosine caused vasodilatation in human fetoplacental vasculature (8), A 2A and A 2B adenosine receptors are expressed in HUVECs (30) and are involved in adenosine-induced vasodilatation in the human placenta vasculature (31); a role for these adenosine receptors in the response to adenosine of umbilical veins from normal or GDM pregnancies is likely. This phenomenon could result from reduced adenosine transport because inhibition of hENT1 by NBTI caused vasodilatation in human umbilical vein rings from normal pregnancies (32).
Adenosine-induced vasodilatation most likely required NO because its effect was abolished by L-NAME. Furthermore, L-NAME caused vessel contraction reaching similar values in umbilical vein rings from normal or GDM pregnancies in the absence or presence of adenosine. These findings could result from a maximal inhibition caused by adenosine via increased NO synthesis. In fact, incubation of umbilical vessel rings with ZM-241385 and L-NAME resulted in a similar maximal contraction as seen in response to adenosine and L-NAME, thus supporting this possibility. In addition, because the highly selective A 2Aadenosine receptor antagonist ZM-241385 abolished the adenosine effect, these types of receptors may be involved in this response.
Vasodilatation of umbilical vein rings caused by insulin was lower in preparations from GDM compared with normal pregnancies. This finding could result from reduced sensitivity to insulin in GDM (IC 50 GDM/IC 50 normal = 1.36 6 0.06), agreeing with a potential placental insulin-resistant state in GDM (2,19,20). Insulin required NO synthesis and most likely involved A 2A -adenosine receptor activation to cause vasodilatation because L-NAME and ZM-241385 abolished its effect. ZM-241385 also led to comparable maximal vessel contraction in vessels from normal and GDM pregnancies; therefore, the insulin effect could be due to an increased adenosine level in these preparations. However, this seems feasible only for vessels from normal pregnancies because the extracellular adenosine concentration was increased by insulin in these vessels but was reduced in preparations from GDM. Thus, the insulin effect in umbilical vein rings is different in GDM than in normal pregnancies, and a mechanism other than NO and/or activation of adenosine receptors for insulin effect is suggested for umbilical veins from GDM. A recent report suggests that A 2B -adenosine receptor activation could be a factor reducing insulin sensitivity in mice (21); however, nothing is reported regarding this phenomenon in human endothelium. Potential insulin receptor isoform involvement. Human placenta expresses at least two subtypes of insulin receptors, IR-A and IR-B (4). We here detected the mRNA for IR-A and IR-B in HUVECs from normal and GDM pregnancies, with IR-A being the predominant isoform in normal compared with GDM pregnancies (IR-A/IR-B number of copies in normal vs. GDM ;1.6). Thus, GDM could be associated with a reduced response to insulin because the IR-A isoform predominates in diseases associated with insulin resistance, such as type 2 diabetic patients (33), and in states of insulin resistance associated with higher IR-A/IR-B ratio in skeletal muscles of patients with myotonic dystrophy types 1 (34) and 2 (34,35). In addition, along with the IR-A predominant expression in GDM, the reduced overall and hENT1-adenosine transport, hENT1 protein abundance, mRNA expression, and SLC29A1 promoter activity were equally (P . 0.05) reduced (;60%) in GDM. Thus, these phenomena could result from IR-A overexpression in HUVEC from GDM.
Restoring IR-A mRNA expression in GDM to levels in normal pregnancies by insulin leads to recovery of hENT1like transport activity, supporting the possibility that basal IR-A expression may be required to maintain normal adenosine transport activity. In addition, hENT1 protein abundance and mRNA expression in GDM cells were restored to values in normal cells, suggesting that insulin via IR-A expression modulation could also modulate hENT1 expression in HUVECs. Dysregulation of the splicing of the insulin receptor in target tissues of insulin can occur in patients with insulin resistance, but its role in diabetes is not clear (36). Activation of IR-B by insulin leads to preferential activation of a metabolic rather than a mitogenic signaling pathway in response to activation of IR-B in the Rmouse embryonic fibroblast cell line (37). Thus, a potentially differential cell-signaling pathway triggered by activation of insulin receptor subtypes in HUVECs from GDM (preferentially IR-A) and normal pregnancies is feasible.
In summary, GDM is associated with abnormal handling of adenosine by the human umbilical vein endothelium, a phenomenon that could result in an elevated concentration of this nucleoside in the umbilical vein blood. Umbilical vein rings exhibited a larger internal diameter in GDM than in normal pregnancies, suggesting that in this syndrome, adenosine may induce a tonic umbilical vein dilatation. We speculate on the possibility that the elevated adenosine level in the fetal blood in GDM could be due to reduced hENT1 transport activity by the endothelium. Thus, it is feasible that a correction of the altered activity (and perhaps expression) of this type of nucleoside transporter could help to reduce the chance of the actual outcome of macrosomic newborns in GDM by limiting the resulting oversupply of metabolic substrates to the fetus.
Adenosine and insulin caused A 2A -adenosine receptormediated relaxation of human umbilical vein rings that was lower in GDM compared with normal pregnancies. The GDM-associated increase in extracellular adenosine concentration and the reduction of hENT1-mediated adenosine transport and expression (including SLC29A1 transcriptional activity) in HUVECs were reversed by insulin via a mechanism that included the re-establishment of this syndrome-induced eNOS activity and expression to levels in cells from normal pregnancies. Thus, the GDM phenotype in HUVECs could be reversed to a normal phenotype by insulin through modulation of extracellular adenosine levels and its A 2A -adenosine receptor-mediated biologic effects. In addition, because HUVECs from GDM preferentially express the IR-A isoform of insulin receptors (isoform associated with insulin resistance) (33-35) compared with cells from normal pregnancies, a crucial role for this type of insulin receptor in the fetoplacental vascular endothelial cells from GDM in this phenomenon is suggested.