*2.2. Pregestational Diabetes Resulted in Fetal Growth Restriction and Decreased Placental E*ffi*ciency in Rats*

Fetal weight was significantly decreased in STZ-D rats compared with normal rats, suggesting newborns from severe diabetic mothers presented intrauterine growth restriction (Figure 1C). Nonetheless, there was no significant difference in placental weight (Figure 1D). However, the fetal weight/placental weight ratio was significantly decreased in STZ-D rats compared with normal rats (Figure 1E). The data are summarized in Table 1.

Because the fetal weight/placental weight ratio was considered as an indicator of placental efficiency, our results suggested that severe pregestational diabetes led to decreased placental efficiency, which might contribute to fetal growth delay in diabetic pregnancies.

concentration (mg/dL)

**Figure 1.** Pregestational diabetes led to fetal growth restriction and decreased placental efficiency in streptozotocin-induced diabetes (STZ-D) rats. (**A**) Maternal plasma glucose levels of normal and STZ-D rats (*n* = 13). (**B**) Representative pictures of abnormal growth of the fetus and placentas in STZ-D rats: a, normal placenta; b, STZ-D placenta; c, normal fetus; d, STZ-D fetus. (**C**) Birthweight from normal and STZ-D rats, showing that STZ-D pregnant rats had decreased birthweight compared with normal rats (*n* = 30). (**D**) Placental weight derived from normal and STZ-D rats (*n* = 30). (**E**) Fetal weight/placental weight ratio (*n* = 30) showing that STZ-D rats had decreased fetal to placental weight ratio. Data are expressed as mean ± SD. \*\*\* *p* < 0.001, normal versus STZ-D. **Figure 1.** Pregestational diabetes led to fetal growth restriction and decreased placental efficiency in streptozotocin-induced diabetes (STZ-D) rats. (**A**) Maternal plasma glucose levels of normal and STZ-D rats (*n* = 13). (**B**) Representative pictures of abnormal growth of the fetus and placentas in STZ-D rats: a, normal placenta; b, STZ-D placenta; c, normal fetus; d, STZ-D fetus. (**C**) Birthweight from normal and STZ-D rats, showing that STZ-D pregnant rats had decreased birthweight compared with normal rats (*n* = 30). (**D**) Placental weight derived from normal and STZ-D rats (*n* = 30). (**E**) Fetal weight/placental weight ratio (*n* = 30) showing that STZ-D rats had decreased fetal to placental weight ratio. Data are expressed as mean ± SD. \*\*\* *p* < 0.001, normal versus STZ-D.



The data are shown as mean ± standard deviation (SD).

#### Fetal weight (g) 4.57 ± 0.95 2.19 ± 1.21 30 <0.001 *2.3. Pregestational Diabetes Resulted in Decreased Placental Amino Acid Transporter Expression in Rats*

Placental weight (g) 0.62 ± 0.11 0.65 ± 0.17 30 0.42 Fetal/placental weight ratio 7.60 ± 2.01 5.11 ± 1.59 30 <0.001 The data are shown as mean ± standard deviation (SD). *2.3. Pregestational Diabetes Resulted in Decreased Placental Amino Acid Transporter Expression in Rats*  Expression of placental system L amino acid transporter isoform LAT1 and LAT2 was assayed by quantitative-PCR and Western blot in normal and diabetic pregnant rats. Our results showed that relative mRNA expression of placental LAT1 and LAT2 was significantly reduced in STZ-D rats compared with normal pregnancies (Figure 2A). Protein level of placental LAT1 (0.95 ± 0.03 vs. 0.72 ± 0.07, normal vs. STZ-D, *p* < 0.05) and LAT2 (0.90 ± 0.03 vs. 0.79 ± 0.06, normal vs. STZ-D, *p* < 0.05) were also decreased in STZ-D pregnant rats (Figure 2B). LAT1 and LAT2 expression was also examined by immunohistochemistry staining in placental tissue sections. Consistent with Western blot data, pregestational diabetes caused reduction of LAT1 and LAT2 expression in the placentas (Figure 2C). These findings indicated that down-regulation of placental amino acid transporters was

Expression of placental system L amino acid transporter isoform LAT1 and LAT2 was assayed by quantitative-PCR and Western blot in normal and diabetic pregnant rats. Our results showed that relative mRNA expression of placental LAT1 and LAT2 was significantly reduced in STZ-D rats

closely associated with fetal growth restriction and decreased placental efficiency in the rat model of severe gestational diabetes. closely associated with fetal growth restriction and decreased placental efficiency in the rat model of severe gestational diabetes.

blot data, pregestational diabetes caused reduction of LAT1 and LAT2 expression in the placentas (Figure 2C). These findings indicated that down-regulation of placental amino acid transporters was

*Int. J. Mol. Sci.* **2020**, *21*, 1849 4 of 12

compared with normal pregnancies (Figure 2A). Protein level of placental LAT1 (0.95 ± 0.03 vs. 0.72 ± 0.07, normal vs. STZ-D, *p* < 0.05) and LAT2 (0.90 ± 0.03 vs. 0.79 ± 0.06, normal vs. STZ-D, *p* < 0.05) were also decreased in STZ-D pregnant rats (Figure 2B). LAT1 and LAT2 expression was also

**Figure 2.** Pregestational diabetes resulted in decreased placental amino acid transporter expression in STZ-D rats. (**A**) Relative mRNA expression of system L amino acid transporter LAT1 and LAT2 detected by quantitative-PCR in placentas derived from normal and STZ-D pregnant rats (*n* = 3). (**B**) Expression of LAT1 and LAT2 detected by Western blots in placentas from normal and STZ-D rats. The bar graph shows the relative density of protein expression for LAT1 and LAT2 after normalization with β-actin expression in each sample. Data are mean ± SD from six normal and six STZ-D placentas. \**p* < 0.05, \*\**p* < 0.01, normal versus STZ-D. (**C**) Representative immunostaining images of LAT1 and LAT2 expressions in tissue sections from normal and STZ-D placentas. Scale bar, **Figure 2.** Pregestational diabetes resulted in decreased placental amino acid transporter expression in STZ-D rats. (**A**) Relative mRNA expression of system L amino acid transporter LAT1 and LAT2 detected by quantitative-PCR in placentas derived from normal and STZ-D pregnant rats (*n* = 3). (**B**) Expression of LAT1 and LAT2 detected by Western blots in placentas from normal and STZ-D rats. The bar graph shows the relative density of protein expression for LAT1 and LAT2 after normalization with β-actin expression in each sample. Data are mean ± SD from six normal and six STZ-D placentas. \* *p* < 0.05, \*\* *p* < 0.01, normal versus STZ-D. (**C**) Representative immunostaining images of LAT1 and LAT2 expressions in tissue sections from normal and STZ-D placentas. Scale bar, 250 µm.

#### 250 μm. *2.4. Pregestational Diabetes Reduced Placental mTORC1 Activity in Rats*

*2.4. Pregestational Diabetes Reduced Placental mTORC1 Activity in Rats*  To investigate the alterations of placental mTORC1 activity in diabetic pregnancies, expressions of phosphorylated S6 kinase1 (p-S6K1) and eukaryotic translation initiation factor 4E-bingding protein 1 (p-4EBP1), two down-stream regulators of mTORC1, were examined in normal and STZ-D placentas. As shown in Figure 3, placental p-4EBP1(Thr-37/46) (0.87 ± 0.06 vs. 0.69 ± 0.12, normal vs. STZ-D, *p* < 0.05) and p-S6k1(Thr-389) (0.72 ± 0.06 vs. 0.51 ± 0.09, normal vs. STZ-D, *p* < 0.05) expressions were significantly reduced in STZ-D rats compared with normal rats, which suggested To investigate the alterations of placental mTORC1 activity in diabetic pregnancies, expressions of phosphorylated S6 kinase1 (p-S6K1) and eukaryotic translation initiation factor 4E-bingding protein 1 (p-4EBP1), two down-stream regulators of mTORC1, were examined in normal and STZ-D placentas. As shown in Figure 3, placental p-4EBP1(Thr-37/46) (0.87 ± 0.06 vs. 0.69 ± 0.12, normal vs. STZ-D, *p* < 0.05) and p-S6k1(Thr-389) (0.72 ± 0.06 vs. 0.51 ± 0.09, normal vs. STZ-D, *p* < 0.05) expressions were significantly reduced in STZ-D rats compared with normal rats, which suggested that severe pregestational diabetes could decrease placental mTORC1 signaling activity.

that severe pregestational diabetes could decrease placental mTORC1 signaling activity.

**Figure 3.** Placental mammalian target of rapamycin (mTOR) complex 1 (mTORC1) activity was decreased in STZ-D rats. Protein expression of p-4E-bingding protein 1 (4EBP1) and p-S6 kinase1 (S6K1) was detected by Western blot in placentas from normal and STZ-D pregnant rats. The bar graphs show relative expression after being normalized with β-actin expression in each sample. Gestational diabetes induced a decrease in p-4EBP1 and p-S6K1 expression. The lower panel shows representative blots for p-4EBP1 and p-S6K1. \**p* < 0.05, normal versus STZ-D. Data are from six **Figure 3.** Placental mammalian target of rapamycin (mTOR) complex 1 (mTORC1) activity was decreased in STZ-D rats. Protein expression of p-4E-bingding protein 1 (4EBP1) and p-S6 kinase1 (S6K1) was detected by Western blot in placentas from normal and STZ-D pregnant rats. The bar graphs show relative expression after being normalized with β-actin expression in each sample. Gestational diabetes induced a decrease in p-4EBP1 and p-S6K1 expression. The lower panel shows representative blots for p-4EBP1 and p-S6K1. \* *p* < 0.05, normal versus STZ-D. Data are from six independent experiments.

#### independent experiments. *2.5. Hyperglycemia in Vitro Down-Regulated Amino Acid Transporter Expression and mTORC1 Activity in 2.5. Hyperglycemia in Vitro Down-Regulated Amino Acid Transporter Expression and mTORC1 Activity in JEG-3 Trophoblast Cells*

*JEG-3 Trophoblast Cells*  To study the regulation of hyperglycemia on placental amino acid transporter and mTORC1 activity in vitro, human trophoblast cell line JEG-3 was cultured with different concentrations of glucose, and expression of LAT1, LAT2, p-4EBP1(Thr-37/46), and p-S6k1(Thr-389) was examined by Western blot. Our results showed that LAT1 (1.02 ± 0.09 vs. 0.86 ± 0.06, 5mM vs. 50mM, *p* < 0.05) and LAT2 (0.89 ± 0.05 vs. 0.75 ± 0.06, 5mM vs. 50mM, *p* < 0.05) expression was significantly decreased in JEG-3 cells treated with 50mM glucose compared to the cells cultured with 5mM glucose (Figure 4A), which indicated the fact that high glucose would attenuate amino acid transporter expression in trophoblast cells. p-4EBP1(Thr-37/46) (0.97 ± 0.09 vs. 0.80 ± 0.05, 5mM vs. 50mM, *p* < 0.05) and p-S6k1 (Thr-389) (0.95 ± 0.14 vs. 0.78 ± 0.08, 5mM vs. 50mM, *p* < 0.05) expression was also reduced in cells treated with 50mM glucose compared with the cells cultured with 5mM glucose (Figure 4B), suggesting high glucose in vitro could inhibit mTORC1 activity in placental trophoblast cells. Together with the findings in the STZ-D rats, our data suggest that maternal diabetes could lead to decreased placental amino acid transporter expression and mTORC1 activity, which may contribute to the fetal intrauterine growth restriction in diabetic pregnancies. To study the regulation of hyperglycemia on placental amino acid transporter and mTORC1 activity in vitro, human trophoblast cell line JEG-3 was cultured with different concentrations of glucose, and expression of LAT1, LAT2, p-4EBP1(Thr-37/46), and p-S6k1(Thr-389) was examined by Western blot. Our results showed that LAT1 (1.02 ± 0.09 vs. 0.86 ± 0.06, 5 mM vs. 50 mM, *p* < 0.05) and LAT2 (0.89 ± 0.05 vs. 0.75 ± 0.06, 5 mM vs. 50 mM, *p* < 0.05) expression was significantly decreased in JEG-3 cells treated with 50 mM glucose compared to the cells cultured with 5 mM glucose (Figure 4A), which indicated the fact that high glucose would attenuate amino acid transporter expression in trophoblast cells. p-4EBP1(Thr-37/46) (0.97 ± 0.09 vs. 0.80 ± 0.05, 5 mM vs. 50 mM, *p* < 0.05) and p-S6k1 (Thr-389) (0.95 ± 0.14 vs. 0.78 ± 0.08, 5 mM vs. 50 mM, *p* < 0.05) expression was also reduced in cells treated with 50mM glucose compared with the cells cultured with 5mM glucose (Figure 4B), suggesting high glucose in vitro could inhibit mTORC1 activity in placental trophoblast cells. Together with the findings in the STZ-D rats, our data suggest that maternal diabetes could lead to decreased placental amino acid transporter expression and mTORC1 activity, which may contribute to the fetal intrauterine growth restriction in diabetic pregnancies.

independent experiments.

*Int. J. Mol. Sci.* **2020**, *21*, 1849 6 of 12

**Figure 4.** High glucose in vitro down-regulated amino acid transporter expression and mTORC1 activity in JEG-3 trophoblast cells. (**A**) Protein expression of LAT1 and LAT2 detected by Western blot in JEG-3 trophoblast cells cultured with different concentration of glucose. The bar graphs show relative expression after being normalized with β-actin expression in each sample. We found that 50mM glucose induced a significant decrease in LAT1 and LAT2 expression. The lower panel shows representative blots for LAT1 and LAT2. \**p* < 0.05, 5mM versus 50mM. Data are from five independent experiments. (**B**) Protein expression of p-4EBP1 and p-S6K1 detected by Western blot in JEG-3 trophoblast cells cultured with different concentration of glucose. The bar graphs show relative expression after being normalized with β-actin expression in each sample. We found that 50 mM glucose induced a significant decrease in p-4EBP1 and p-S6K1 expression. The lower panel shows representative blots for p-4EBP1 and p-S6K1. \**p* < 0.05, 5mM versus 50mM. Data are from five **Figure 4.** High glucose in vitro down-regulated amino acid transporter expression and mTORC1 activity in JEG-3 trophoblast cells. (**A**) Protein expression of LAT1 and LAT2 detected by Western blot in JEG-3 trophoblast cells cultured with different concentration of glucose. The bar graphs show relative expression after being normalized with β-actin expression in each sample. We found that 50mM glucose induced a significant decrease in LAT1 and LAT2 expression. The lower panel shows representative blots for LAT1 and LAT2. \* *p* < 0.05, 5mM versus 50mM. Data are from five independent experiments. (**B**) Protein expression of p-4EBP1 and p-S6K1 detected by Western blot in JEG-3 trophoblast cells cultured with different concentration of glucose. The bar graphs show relative expression after being normalized with β-actin expression in each sample. We found that 50 mM glucose induced a significant decrease in p-4EBP1 and p-S6K1 expression. The lower panel shows representative blots for p-4EBP1 and p-S6K1. \* *p* < 0.05, 5 mM versus 50 mM. Data are from five independent experiments.

#### *2.6. Inhibition of mTORC1 Activity Resulted in Decreased Amino Acid Transporter Expression in JEG-3 2.6. Inhibition of mTORC1 Activity Resulted in Decreased Amino Acid Transporter Expression in JEG-3 Trophoblast Cells*

*Trophoblast Cells*  To determine the effects of decreased mTORC1 activity on amino acid transporter in placental trophoblast cells, rapamycin, a specific mTORC1 inhibitor, was employed to inhibit mTORC1 activity in JEG-3 cells. Expression of LAT1 and LAT2 was measured by Western blot. As shown in Figure 5A, LAT1 (1.05 ± 0.14 vs. 0.55 ± 0.08, control vs. rapamycin, *p* < 0.01) and LAT2 (0.85 ± 0.12 vs. 0.60 ± 0.08, control vs. rapamycin, *p* < 0.05) expressions were both significantly reduced in rapamycin-treated To determine the effects of decreased mTORC1 activity on amino acid transporter in placental trophoblast cells, rapamycin, a specific mTORC1 inhibitor, was employed to inhibit mTORC1 activity in JEG-3 cells. Expression of LAT1 and LAT2 was measured by Western blot. As shown in Figure 5A, LAT1 (1.05 ± 0.14 vs. 0.55 ± 0.08, control vs. rapamycin, *p* < 0.01) and LAT2 (0.85 ± 0.12 vs. 0.60 ± 0.08, control vs. rapamycin, *p* < 0.05) expressions were both significantly reduced in rapamycin-treated cells compared with control cells.

cells compared with control cells. mTORC1 inhibition was also conducted by gene silencing targeting raptor, a key component of mTORC1. Consistently, protein expressions of LAT1 and LAT2 were significantly decreased in cells treated with raptor small interfering RNAs (siRNAs) compared to scrambled and negative control mTORC1 inhibition was also conducted by gene silencing targeting raptor, a key component of mTORC1. Consistently, protein expressions of LAT1 and LAT2 were significantly decreased in cells treated with raptor small interfering RNAs (siRNAs) compared to scrambled and negative control cells (Figure 5B).

cells (Figure 5B). LAT1 and LAT2 expression was also examined by immunofluorescence staining in JEG-3 cells treated with raptor siRNAs. Consistent with Western blot data, LAT1 (Figure 6A) and LAT2 (Figure 6B) expressions were reduced in cells cultured with raptor siRNAs compared to negative controls. Our results suggested that amino acid transporter expression was regulated by placental mTORC1 signaling pathway, and that inhibition of mTORC1 activity could lead to decreased expression of amino acid transporter in human trophoblast cells. LAT1 and LAT2 expression was also examined by immunofluorescence staining in JEG-3 cells treated with raptor siRNAs. Consistent with Western blot data, LAT1 (Figure 6A) and LAT2 (Figure 6B) expressions were reduced in cells cultured with raptor siRNAs compared to negative controls. Our results suggested that amino acid transporter expression was regulated by placental mTORC1 signaling pathway, and that inhibition of mTORC1 activity could lead to decreased expression of amino acid transporter in human trophoblast cells.

**Figure 5.** Inhibition of mTORC1 activity resulted in decreased amino acid transporter expression in JEG-3 trophoblast cells. (**A**) Protein expression of LAT1 and LAT2 assayed by Western blot in JEG-3 cells treated with or without rapamycin. The bar graphs show relative expression after being normalized with β-actin expression in each sample. Rapamycin induced a significant decrease in LAT1 and LAT2 expression. Data are from five independent experiments. \**p* < 0.05, \*\**p* < 0.01, control versus rapamycin. (**B**) Expression of LAT1 and LAT2 detected by Western blot in JEG-3 cells treated with scrambled or negative or raptor siRNA. The bar graph shows data from five independent experiments. Raptor siRNA caused a significant reduction in LAT1 and LAT2 expression. \*\**p* < 0.01, scrambled control or negative control versus raptor siRNA. **Figure 5.** Inhibition of mTORC1 activity resulted in decreased amino acid transporter expression in JEG-3 trophoblast cells. (**A**) Protein expression of LAT1 and LAT2 assayed by Western blot in JEG-3 cells treated with or without rapamycin. The bar graphs show relative expression after being normalized with β-actin expression in each sample. Rapamycin induced a significant decrease in LAT1 and LAT2 expression. Data are from five independent experiments. \* *p* < 0.05, \*\**p* < 0.01, control versus rapamycin. (**B**) Expression of LAT1 and LAT2 detected by Western blot in JEG-3 cells treated with scrambled or negative or raptor siRNA. The bar graph shows data from five independent experiments. Raptor siRNA caused a significant reduction in LAT1 and LAT2 expression. \*\* *p* < 0.01, scrambled control or negative control versus raptor siRNA. **Figure 5.** Inhibition of mTORC1 activity resulted in decreased amino acid transporter expression in JEG-3 trophoblast cells. (**A**) Protein expression of LAT1 and LAT2 assayed by Western blot in JEG-3 cells treated with or without rapamycin. The bar graphs show relative expression after being normalized with β-actin expression in each sample. Rapamycin induced a significant decrease in LAT1 and LAT2 expression. Data are from five independent experiments. \**p* < 0.05, \*\**p* < 0.01, control versus rapamycin. (**B**) Expression of LAT1 and LAT2 detected by Western blot in JEG-3 cells treated with scrambled or negative or raptor siRNA. The bar graph shows data from five independent experiments. Raptor siRNA caused a significant reduction in LAT1 and LAT2 expression. \*\**p* < 0.01, scrambled control or negative control versus raptor siRNA.

**Figure 6.** Representative imaging of immunofluorescent staining of LAT1 and LAT2 in JEG-3 trophoblast cells treated with negative or raptor siRNA. (**A**) LAT1 expression; (**B**) LAT2 expression. **Figure 6.** Representative imaging of immunofluorescent staining of LAT1 and LAT2 in JEG-3 trophoblast cells treated with negative or raptor siRNA. (**A**) LAT1 expression; (**B**) LAT2 expression. **Figure 6.** Representative imaging of immunofluorescent staining of LAT1 and LAT2 in JEG-3 trophoblast cells treated with negative or raptor siRNA. (**A**) LAT1 expression; (**B**) LAT2 expression. Consistent with Western blot results, raptor siRNA could reduce LAT1 and LAT2 expression in placental trophoblast.
