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    • Fig demonstrates placental amino acid

    2022-06-05

    Fig. 7 demonstrates placental amino Amfenac Sodium Monohydrate mg transporter protein concentrations. While no change in the placental SNAT2 is effected in response to a high-fat diet in wt, a trend toward a reduction was seen in glut3 mice in response to a high-fat vs. chow diet (Fig. 7A and B). A high-fat diet did not change placental SNAT4 protein concentrations in both genotypes (Fig. 7A and C). In the case of the placental LAT2, a trend toward a reduction was again noted only in glut3 mice exposed to a high-fat diet (Fig. 7A and D). Given that our placental investigations revealed such transporter expression differences between the two genotypes in their response to a high-fat diet, our next set of experiments focused on the early preimplantation embryonic stage by employing embryonic stem cells (ES cells) of wt and glut3 mice (Fig. 8). We first characterized the expression of Glut1 and Glut3 in ES cells. We confirmed that Glut3 mRNA (Fig. 8A) and protein (Fig. 8B) were reduced in glut3 when compared to wild-type ES cells. No change in Glut1 mRNA (Fig. 8A) or Glut1, Glut4 and Glut8 proteins was noted (Fig. 8B). We also ensured that while Glut1 protein was noted in the ES cells and the embryonic fibroblast (EF) feeder cells necessary for culturing and maintaining ES cells, Glut3 protein was specific to only ES cells being absent in EF cells (Fig. 8C). We then assessed the effect of the glut3+/− genotype on cell survival (Fig. 9A). Under normal culture conditions, wild-type ES cell numbers increased from day 0 to day 8 in a linear manner. In contrast, glut3+/− ES cell numbers began swerving from the wild-type cell numbers by day 4, significantly dropping by day 6 and 8. This observation lends support to the importance of Glut3 protein for embryonic growth. We next examined the effect of specific conditions encountered by preimplantation embryos upon entering the uterus of hyperglycemic/glucose-intolerant mothers (Fig. 9B and C). Per previous reports, preimplantation embryos experience conditions of low glucose and hypoxia in the presence of maternal hyperglycemia [26]. We therefore examined the time-dependent effect of 5 mM glucose (instead of 25 mM) upon ES cellular glucose uptake (Fig. 9B) and Glut1 and Glut3 proteins (Fig. 9C). A time-dependent reduction in glucose uptake is seen in both wild-type and glut3 ES cells, with the latter being consistently lower than the wild-type counterpart (Fig. 9B). Examination under longer durations of 5 mM glucose exposure increased the Glut3 protein amount in the wt when compared to that of 25 mM (0 time point). However, the glut3 ES cells that expressed much lower amounts of Glut3 protein failed to show a similar increase with time (Fig. 9C, upper panel). This time-dependent increase in Glut3 served as a compensatory mechanism to combat the diminishing glucose uptake exhibited by the wild-type (glut3) ES cells seen as early as 10 to 30 min of such exposure (Fig. 9B), but not so in the glut3 ES cells (Fig. 9C, upper panel), explaining the greater reduction in glucose uptake by these ES cells (Fig. 9B). Glut1 protein on the other hand remained unchanged between the two genotypes, perhaps only increasing in both genotypes after 16 h of exposure to 5 mM glucose (Fig. 9C, middle panel). Next, we examined the effect of hypoxia (3% vs. 20%) (Fig. 10A); the latter can also be mimicked by sodium azide that inhibits cytochrome c oxidase, which is the last enzyme in the mitochondrial respiratory electron chain (complex IV) (Fig. 10B). In response to hypoxic exposure, a time-dependent decline in Glut3 protein was noted in wild-type and glut3 ES cells (Fig. 10A, upper panel), and in the case of the Glut1 protein, a time-dependent trend toward an increase was observed in wild-type and glut3 genotypes at 72 h (Fig. 10A, middle panel). In response to inhibition of complex IV of the mitochondrial respiratory electron chain with sodium azide, while a reduction in Glut3 protein was seen, a less severe reduction in Glut1 protein was evident in wild-type and glut3 ES cells (Fig. 10B, upper and middle panels).