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-', 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, 250mgkg'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.

. SNAT4 isoform of system A amino acid transporter is expressed in human placenta. Am J Physiol Cell Physiol 290: C305-C312, 2006. First published September 7, 2005 doi:10.1152/ajpcell.00258.2005.-The system A amino acid transporter is encoded by three members of the Slc38 gene family, giving rise to three subtypes: Na ϩ -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 Ͻ 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 Ͻ 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. SNAT1; SNAT2; gestational expression; syncytiotrophoblast SYSTEM A IS A UBIQUITOUS Na ϩ -dependent transporter that actively transports small, zwitterionic, neutral amino acids with short, unbranched side chains such as alanine, serine, and glutamine (24). It has a unique ability to transport N-methylated amino acids such as ␣-(methylamino)isobutyric acid (MeAIB) (7). This nonmetabolized amino acid analog has been used extensively to study system A in the placenta (21,27,37).Recent data show that system A is encoded by three different members of the SLC38 gene family (Slc38a1, Slc38a2, and Slc38a4), giving rise to the three subtypes of this Na ϩ -coupled neutral amino acid transporter (SNAT): SNAT1, SNAT2, and SNAT4 (previously referred to as ATA1, ATA2, and ATA3, respectively) (31). SNAT1 was the first isoform to be cloned from rat brain and initially was designated GlnT because of its preference for glutamine as a substrate (49). Cloning of the human homolog as well as SNAT2 and SNAT4 followed (18,19,44,45,50,51). These previous studies showed the three isoforms to be highly homologous: amino acid sequences for SNAT1 and S...

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 3 H-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 singlebase-pair changes; in the ko mice, they consisted primarily of AT3TA and GC3TA 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.Alkylpurine-DNA-N-glycosylase (APNG) is one of a growing list of enzymes responsible for the recognition and excision of altered bases in the first step of the base excision repair pathway (49, 56). In the simplest form of base excision repair, the resulting abasic site is then repaired by the sequential action of an apurinic-apyrimidinic (AP) endonuclease that generates a single-strand break, the removal of the 5Ј-terminal deoxyribose phosphate residue, insertion of a single nucleotide by DNA polymerase ␤, and finally ligation of the repaired patch by DNA ligase I or XRCC1-DNA ligase III (55, 56).Mammalian APNGs have been shown to be active against a wide range of modified bases in vitro, many structurally unrelated to 3-methyladenine (3-meA), the substrate after which the enzyme was first named (32). In particular, APNG appears to be the only glycosylase in mammalian cells that can release hypoxanthine from DNA, a promutagenic base resulting from the spontaneous deamination of adenine (22,47). Likewise, the highly mutagenic adduct 1,N 6 -ethenoadenine (41), which is produced by metabolic products of the environmental hepatocarcinogens vinyl chloride and ethyl carbamate, is released by APNG (22, 48); indeed, according to one report, the recombinant human enzyme reacted 10-to 20-fold more efficiently with this adduct than with 3-meA in an in vitro assay (14). These results, together ...

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.