br Experimental section br Acknowledgments MIN cells were ki

Experimental section
Acknowledgments MIN6 resveratrol were kindly provided by Dr. Junichi Miyazaki, Osaka University. This research was supported in part by the Ministry of Education, Culture, Sports, Science and Technology of Japan (JP16H05099 and JP18H04609 to K.H., and JP16H06574 to T.U.), SENTAN, JST to K.H, and Hirosaki University Institutional Grant to K.Y. K.H. was also supported by a grant JSPS Core-to-Core program, A. Advanced Research Networks.
Introduction High-caloric dietary intake along with escalating sedentary lifestyle has caused an epidemic of obesity and diabetes mellitus [1], [2], [3]. To mimic this human situation, many investigator groups have examined the effect of a high-fat or high-carbohydrate diet in various wild-type animal models from mice to baboons [4], [5], [6]. The effect of a high-fat diet during pregnancy can affect not only the mother but also the offspring immediately and on a long-term basis [7], [8], [9], [10]. Given this paradigm, it behooves us to determine the mechanisms and the impact of such a diet not just on the pregnant state but also on the pregestational stage when the female prepares for pregnancy. In order to have a good pregnancy outcome, it is imperative to ensure good pregestational health. In humans, pregestational body weight and body mass index determine the outcome of pregnancy and health of the offspring [11], [12], [13]. To this end, we hypothesized that a high-fat diet during the pregestational and gestational stages affects placental macronutrient transporter proteins which in turn influence the outcome of the offspring. We tested this hypothesis first in wild-type mice. In addition to the dietary intake of a high-fat diet, prenatal and postnatal programming due to perturbed maternal metabolism has also led to adult-onset adiposity and glucose intolerance in the offspring [14], [15]. While multiple investigations have shown this paradigm in humans and various animal studies [15], [16], [17], [18], we have also demonstrated such an occurrence in genetically modified mice [19], [20]. Employing such genetically modified mice (glut1 and glut3), our previous investigations, while not demonstrating a significant role for glucose transporter isoform 1 (Glut1) [21], revealed a key role for Glut3 in mediating murine transplacental glucose transport [21], [22]. Thus, focusing on glut3 heterozygous null mice, we observed metabolic programming [19], [21]. At 9–11 months of age, while these mice were aging, we encountered obesity, glucose intolerance, insulin resistance and a fatty liver in response to reduced transplacental glucose transport in fueling the developing fetus [23]. These findings supported a metabolic programming effect while mothers and the offspring were maintained on an ad-libitum-fed regular chow diet. Given these previous observations, we put forth a second part to our hypothesis that a high-fat diet in pregestational and gestational glut3 heterozygous mice will exacerbate the outcome of pregnancies and the offspring. To test this second part of the hypothesis, we engaged glut3 mice and exposed them to a high-fat diet during pregestational and gestational stages in females and examined placental macronutrient transporters and body weights of the suckling offspring. Based upon our glut3 placental results that revealed enhanced Glut3 protein expression with enhanced fetal/postnatal size upon pregestational and gestational exposure to a high-fat diet, we subsequently investigated the proliferative growth capacity and glucose uptake in wild-type and glut3 embryonic stem cells under control and differing stressing conditions (low glucose, low oxygen and inhibited oxidative metabolism) encountered by preimplantation embryos as they enter the uterus soon after conception.
Materials and methods
Results Fig. 1 depicts the study design demonstrating introduction of a high-fat diet or regular chow diet 8 weeks prior to mating during the pregestational period in female wt and glut3 mice. The respective diets were continued throughout pregnancy and lactation in the female mice, and at weaning, the offspring from both groups were placed on a regular chow diet.