**3. Discussion**

In the present study, we obtained interesting findings showing that fetal weight and fetal/placental weight ratio was significantly decreased in a rat model of STZ-induced diabetes compared to normal pregnancies, which confirmed that pregestational diabetes could lead to placental dysfunction and fetal intrauterine growth restriction.

The influence of maternal diabetes on fetal and placental growth might be related to the degree of gestational glucose intolerance, which could lead to opposite fetal outcomes. In some studies, an increased birthweight and placental weight was found in pregnancies complicated with mild diabetes [9], but limited fetal intrauterine growth, miscarriage stoics, and malformations were usually reported in pregnancies with severe diabetes [10], which was verified in our study. Weiss's study found that hyperglycemia in vitro could inhibit the proliferation of first-trimester trophoblast cells [11], which suggested that a reduced growth of the placenta might cause the fetal intrauterine growth delay in diabetes.

Placental efficiency, which is defined as fetal/placental weight ratio, is a useful predictor of those placentas that have adapted their nutrient transfer per gram placenta. A smaller ratio usually indicates that the placenta is less functionally efficient. Several studies have found that fetal/placental weight ratio was reduced in rodent models of IUGR [12–14], suggesting an inefficient placenta that failed to adapt its nutrient supply to meet the demands of the growing fetus. Additionally, it has been observed that fetal/placental weight ratios are associated with a variety of human pregnancy syndromes, including preeclampsia, preterm birth, and IUGR [15,16]. Therefore, our findings provide evidence that decreased placental efficiency plays important role in the fetal growth delay in pregestational diabetes mellitus.

Amino acids are crucial for the development of fetal tissue organs, and active transport of amino acid via placental amino acid transporters is the only way for fetuses to obtain amino acids from maternal circulation. Decreased expression or activity of the placental amino acid transporter system has been found in human IUGR or animal models [2,17], but few studies have been performed to investigate the alterations of placental amino acid transporters in pregnancies complicated with diabetes. Our results showed that the expression of placental amino acid transporters was reduced in the rat model of severe diabetes. In addition, using JEG-3 cell line as a human trophoblast cell model, we verified that the hyperglycemia in vitro could down regulate the expression of amino acid transporters in human trophoblast. These findings suggested that restricted fetal growth resulting from maternal diabetes was associated with the decreased placental amino acid transporter expression. Kuruvilla et al. found that the number of system A amino acid transporters on placental microvillus membrane (MVM) was decreased in diabetic pregnancies associated with macrosomia [18]. In contrast, Jansson et al. reported an increased activity of system A amino acid transporters on the placental MVM, regardless of the type of gestational diabetes [19]. The study methods and subjects may have contributed to the difference in the results. Recently, Ericsson et al. found that glucose injection during early gestation in rats had no significant effects on the expression of placental system A amino acid transporter, but that the transport activity of amino acid transporter was significantly decreased [20].

mTORC1 signaling pathway has been considered as a nutrient sensor in the placenta. It has been well studied that placental mTORC1 activity was decreased in IUGR model [6,21], but few studies were taken to investigate the placental mTROC1 changes in pregnancies complicated with diabetes. Our results found that the activity of placental mTORC1 was significantly reduced in a rat model with severe pregestational diabetes. Furthermore, the in vitro study performed in JEG-3 trophoblast cells confirmed that the high glucose could reduce the activity of mTORC1 signaling pathways. These results provided direct evidence revealing the alterations of placental mTORC1 pathway in diabetic pregnancies. Several studies have reported the mTORC1 changes in people with obesity, which is one of the high risks of diabetes. For example, some studies found that mTORC1 activity was promoted in obese people because of high insulin levels or increased inflammatory cytokines [22]. In contrast, Jansson et al. found that obesity could inhibit the activity of placental mTORC1 in a mouse model of obesity [23].

To determine the effects of decreased mTORC1 activity on placental amino acid transporters, we combined rapamycin treatment and gene silencing targeting raptor to inhibit mTORC1 signaling in JEG-3 cells. Our results showed that expression of system L amino acid transporters was reduced in trophoblast, of which mTORC1 signaling pathway was suppressed. It suggested that trophoblast mTORC1 could modulate amino acid transfer across the placenta by regulating the expression of key amino acid transporters. In contrast to Rosario's report that trophoblast mTORC1 regulation of amino acid transporters occurred mainly by modulation of the translocation of specific transporter isoforms between plasma membrane and cell interior [5], our results showed that mTORC1 could regulate the trophoblast amino acid transporter at the translational level.

In summary, our findings have significant impact on abnormal fetal growth in diabetic pregnancies. In the present study, we found that inefficient placenta was associated with limited fetal growth in a rat model of severe gestational diabetes. Furthermore, we found that placental amino acid transporter expression and mTORC1 activity was reduced in this model. We also demonstrated that decreased mTORC1 activity could lead to decreased amino acid transporter expression in placental trophoblast. These results provide evidence that decreased placental mTORC1 activity could lead to reduced placental amino acid transporter expression and, subsequently, contribute to limited fetal growth in severe diabetic pregnancies.
