The SNAT4 isoform of the system A amino acid transporter is functional in human placental microvillous plasma membrane.

Placental system A activity is important for the supply of neutral amino acids needed for fetal growth. There are three system A isoforms: SNAT1, SNAT2 and SNAT4, but the contribution of each to system A-mediated transport is unknown. Here, we have used immunohistochemistry to demonstrate that all three isoforms are present in the syncytiotrophoblast suggesting each plays a role in amino acid transport across the placenta. We next tested the hypothesis that the SNAT4 isoform is functional in microvillous plasma membrane vesicles (MVM) from normal human placenta using a method which exploits the unique property of SNAT4 to transport both cationic amino acids as well as the system A-specific substrate MeAIB. The data show that SNAT4 contribution to system A-specific amino acid transport across MVM is higher in first trimester placenta compared to term (approx. 70% and 33%, respectively, P < 0.01). Further experiments performed under more physiological conditions using intact placental villous fragments suggest a contribution of SNAT4 to system A activity in first trimester placenta but minimal contribution at term. In agreement, Western blotting revealed that SNAT4 protein expression is higher in first trimester MVM compared to term (P < 0.05). This study provides the first evidence of SNAT4 activity in human placenta and demonstrates the contribution of SNAT4 to system A-mediated transport decreases between first trimester and term: our data lead us to speculate that at later stages of gestation SNAT1 and/or SNAT2 are more important for the supply of amino acids required for normal fetal growth.

SNAT4 isoform of system A amino acid transporter is expressed in human placenta.

The system A amino acid transporter is encoded by three members of the Slc38 gene family, giving rise to three subtypes: Na+-coupled neutral amino acid transporter (SNAT)1, SNAT2, and SNAT4. SNAT2 is expressed ubiquitously in mammalian tissues; SNAT1 is predominantly expressed in heart, brain, and placenta; and SNAT4 is reported to be expressed solely by the liver. In the placenta, system A has an essential role in the supply of neutral amino acids needed for fetal growth. In the present study, we examined expression and localization of SNAT1, SNAT2, and SNAT4 in human placenta during gestation. Real-time quantitative PCR was used to examine steady-state levels of system A subtype mRNA in early (6-10 wk) and late (10-13 wk) first-trimester and full-term (38-40 wk) placentas. We detected mRNA for all three isoforms from early gestation onward. There were no differences in SNAT1 and SNAT2 mRNA expression with gestation. However, SNAT4 mRNA expression was significantly higher early in the first trimester compared with the full-term placenta (P < 0.01). We next investigated SNAT4 protein expression in human placenta. In contrast to the observation for gene expression, Western blot analysis revealed that SNAT4 protein expression was significantly higher at term compared with the first trimester (P < 0.05). Immunohistochemistry and Western blot analysis showed that SNAT4 is localized to the microvillous and basal plasma membranes of the syncytiotrophoblast, suggesting a role for this isoform of system A in amino acid transport across the placenta. This study therefore provides the first evidence of SNAT4 mRNA and protein expression in the human placenta, both at the first trimester and at full term.

Activity and expression of Na+/H+ exchanger isoforms in the syncytiotrophoblast of the human placenta.

The purpose of this study was to compare Na+/H+ exchanger (NHE) activity in the microvillous (MVM) and basal (BM) plasma membrane of the human placental syncytiotrophoblast and to determine the relative contribution of various NHE isoforms to this activity. Uptake of 22Na into isolated MVM vesicles in the presence of a H+ gradient, at initial rate, was four- to fivefold higher than that by BM vesicles (214+/-28 vs. 49+/-9 pmol/mg protein per 30 s, respectively, means+/-SEM, n=8, 6, P<0.001). The 22Na uptake by MVM, but not by BM, was reduced in the absence of a H+ gradient and in the presence of 500 microM amiloride. To determine the contribution of NHE1, NHE2 and NHE3 isoforms to NHE activity in MVM, we investigated the effect of amiloride analogues which show isoform selectivity. HOE 694, an analogue selective for NHE1 at low concentrations, inhibited 22Na uptake with an EC50 of 0.13+/-0.05 microM (n=6), whereas S3226, an analogue selective for NHE3 at low concentrations had an EC50 of 3.01+/-0.85 microM (n=5). To investigate this further, we measured recovery of syncytiotrophoblast intracellular pH (pHi) from an acid load using a H+-selective, fluorescent dye (BCECF) loaded into isolated intact placental fragments. This recovery was blocked in the absence of Na+ and the presence of amiloride (500 microM) and concentrations of HOE 694 and S3226 were comparable to those used in vesicle experiments. Overall these data show that under the conditions used NHE activity in the term placental syncytiotrophoblast is absent from BM. NHE activity in the MVM is attributable predominantly to NHE1.

Increased susceptibility to streptozotocin-induced beta-cell apoptosis and delayed autoimmune diabetes in alkylpurine-DNA-N-glycosylase-deficient mice.

Type 1 diabetes is thought to occur as a result of the loss of insulin-producing pancreatic beta cells by an environmentally triggered autoimmune reaction. In rodent models of diabetes, streptozotocin (STZ), a genotoxic methylating agent that is targeted to the beta cells, is used to trigger the initial cell death. High single doses of STZ cause extensive beta-cell necrosis, while multiple low doses induce limited apoptosis, which elicits an autoimmune reaction that eliminates the remaining cells. We now show that in mice lacking the DNA repair enzyme alkylpurine-DNA-N-glycosylase (APNG), beta-cell necrosis was markedly attenuated after a single dose of STZ. This is most probably due to the reduction in the frequency of base excision repair-induced strand breaks and the consequent activation of poly(ADP-ribose) polymerase (PARP), which results in catastrophic ATP depletion and cell necrosis. Indeed, PARP activity was not induced in APNG(-/-) islet cells following treatment with STZ in vitro. However, 48 h after STZ treatment, there was a peak of apoptosis in the beta cells of APNG(-/-) mice. Apoptosis was not observed in PARP-inhibited APNG(+/+) mice, suggesting that apoptotic pathways are activated in the absence of significant numbers of DNA strand breaks. Interestingly, STZ-treated APNG(-/-) mice succumbed to diabetes 8 months after treatment, in contrast to previous work with PARP inhibitors, where a high incidence of beta-cell tumors was observed. In the multiple-low-dose model, STZ induced diabetes in both APNG(-/-) and APNG(+/+) mice; however, the initial peak of apoptosis was 2.5-fold greater in the APNG(-/-) mice. We conclude that APNG substrates are diabetogenic but by different mechanisms according to the status of APNG activity.

Alkylpurine-DNA-N-glycosylase knockout mice show increased susceptibility to induction of mutations by methyl methanesulfonate.

Alkylpurine-DNA-N-glycosylase (APNG) null mice have been generated by homologous recombination in embryonic stem cells. The null status of the animals was confirmed at the mRNA level by reverse transcription-PCR and by the inability of cell extracts of tissues from the knockout (ko) animals to release 3-methyladenine (3-meA) or 7-methylguanine (7-meG) from 3H-methylated calf thymus DNA in vitro. Following treatment with DNA-methylating agents, increased persistence of 7-meG was found in liver sections of APNG ko mice in comparison with wild-type (wt) mice, demonstrating an in vivo phenotype for the APNG null animals. Unlike other null mutants of the base excision repair pathway, the APNG ko mice exhibit a very mild phenotype, show no outward abnormalities, are fertile, and have an apparently normal life span. Neither a difference in the number of leukocytes in peripheral blood nor a difference in the number of bone marrow polychromatic erythrocytes was found when ko and wt mice were exposed to methylating or chloroethylating agents. These agents also showed similar growth-inhibitory effects in primary embryonic fibroblasts isolated from ko and wt mice. However, treatment with methyl methanesulfonate resulted in three- to fourfold more hprt mutations in splenic T lymphocytes from APNG ko mice than in those from wt mice. These mutations were predominantly single-base-pair changes; in the ko mice, they consisted primarily of AT-->TA and GC-->TA transversions, which most likely are caused by 3-meA and 3- or 7-meG, respectively. These results clearly show an important role for APNG in attenuating the mutagenic effects of N-alkylpurines in vivo.

Importance of the intestine as a site of metformin-stimulated glucose utilization.

1. The intestine has been implicated as a site of increased glucose utilization by the antihyperglycaemic drug, metformin. This study makes a quantitative assessment of this effect. 2. Glucose utilization by the intestine and hind limb region was determined by arterial-venous glucose difference adjusted for blood flow rate in fasted rats receiving a hyperglycaemic hyperinsulinaemic infusion. 3. Intrajejunal administration of metformin, 250 mg kg-1, increased glucose disposal during the infusion procedure, associated with increased glucose utilization in the intestine by 69% and in the hind limb region by 40%. 4. Metformin, 250 mg kg-1, increased glucose disappearance during an intravenous glucose tolerance test. This was accompanied by increased uptake of tritiated 2-deoxy-D-glucose into the intestinal mucosa to a greater extent than into skeletal muscles (per unit wet weight of tissue). 5. The results demonstrate that the intestinal mucosa is a quantitatively important site of increased glucose utilization during the blood glucose-lowering effect of metformin.

Insulin requirement for the antihyperglycaemic effect of metformin.

1. Insulin-dependent diabetic BB/S rats with little or no endogenous insulin were used to determine whether insulin is required for the acute antihyperglycaemic effect of metformin (dimethylbiguanide). 2. Metformin (250 mg kg-1, intrajejunally) did not lower the hyperglycaemia in BB/S rats in the absence of exogenous insulin, but metformin increased by 69% (P < 0.05) the blood glucose-lowering effect of exogenous insulin. 3. Metformin (250 mg kg-1, intrajejunally) improved glucose disposal in rats with a normal insulin response to an intravenous glucose challenge. Plasma glucose disappearance was increased from 0.7 +/- 0.1 to 2.5 +/- 0.1% min-1 (P < 0.05). 3. When the insulin response to glucose was suppressed with somatostatin and diazoxide, metformin improved glucose disposal to a similar extent to that in rats with a normal insulin response. Plasma glucose disappearance was increased from 0.24 +/- 0.02 to 1.0 +/- 0.1% min-1 (P < 0.01). 5. The results indicate that insulin is required for the acute antihyperglycaemic effect of metformin, but the extent of this effect is not proportional to the prevailing insulin concentration.