A novel rat model of gestational diabetes induced by intrauterine programming is associated with alterations in placental signaling and fetal overgrowth
Introduction
Gestational diabetes mellitus (GDM) is a common pregnancy disease affecting more than 8% of the pregnant women in many populations, and is associated with short and long-term adverse consequences for the mother and the fetus (Ashwal and Hod, 2015, Pu et al., 2015, Wang et al., 2013). GDM affects the fetal growth pattern, and macrosomia has been observed in 15–45% newborns of GDM mothers (Kc et al., 2015). Alterations in nutrient transfer and oxidative/inflammatory pathways characterize GDM placentas, and have been related to the adverse intrauterine programming (Diaz et al., 2014, Lappas et al., 2011). Indeed, GDM increases the risk of metabolic syndrome and type 2 diabetes in the offspring (Bellamy et al., 2009).
To explore the mechanisms linking GDM to adverse outcomes and study novel intervention strategies animal models relevant to the clinical condition are required. Specifically, a clinically relevant model of GDM should replicate common characteristics of the human condition, including hyperglycemia detected for first time in pregnancy as a result of insulin resistance in combination with some degree of beta cell insufficiency and increased fetal weight. Unfortunately, few such models are currently available (Jawerbaum and White, 2010).
It is well-known that a family history of diabetes is a risk factor for developing GDM (Buchanan and Xiang, 2005). In addition, there is evidence that experimental models of pre-gestational diabetes, obesity and intrauterine growth restriction induce metabolic alterations in the offspring leading to type 2 diabetes (Capobianco et al., 2015, Zambrano and Nathanielsz, 2013). There is a strong association between type 2 diabetes and GDM in the genetic background, and, in several populations, more than 30% of women that had GDM will develop type 2 diabetes within 5 years of delivery (Bellamy et al., 2009, Huopio et al., 2013, Kim et al., 2002).
Fetal overgrowth is a frequent complication of GDM related to the alterations in growth factors and sustained by an increase in placental nutrient transport (Hiden et al., 2009, Jansson et al., 2002, Lappas et al., 2011, Magnusson et al., 2004). Mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase which has been proposed to function as a placenta nutrient sensor (Jansson et al., 2012, Roos et al., 2009). mTOR is the catalytic subunit of two complexes named mTOR Complex 1 (mTORC1) and 2 (mTORC2), and its signaling leads to phosphorylation cascades resulting in an increase in amino acid transport, protein synthesis and cellular proliferation (Bracho-Valdes et al., 2011). Downstream target proteins phosphorylated by mTORC1 and related to increased protein synthesis are p70 S6 kinase 1 (S6K1), the ribosomal protein S6 (rpS6) and the eukaryotic initiation factor 4E-binding protein 1 (4EBP1) (Laplante and Sabatini, 2013). Activation of placental mTORC1 has been observed in association to maternal obesity and GDM and is related to fetal overgrowth (Jansson et al., 2013, Perez-Perez et al., 2013).
mTORC2 signaling has been less studied in the placenta. mTORC2 direct targets include protein kinase Cα (PKCα) and serum- and glucocorticoid-inducible kinase 1 (SGK1) (Heikamp et al., 2014, Oh and Jacinto, 2011). mTORC2 activation is involved in the regulation of cell volume, growth, differentiation and motility in different cell types (Betz and Hall, 2013, Oh and Jacinto, 2011). Furthermore, recent works has demonstrated that mTORC2 signaling is a positive regulator of placental amino acids transport (Rosario et al., 2013).
A common feature of obesity, pre-gestational diabetes, GDM and intrauterine growth restriction is the generation of a pro-oxidative/pro-inflammatory environment, which may provide a mechanism underpinning intrauterine programming of metabolic diseases (Higa and Jawerbaum, 2013, Lappas et al., 2011, Pantham et al., 2015). GDM is characterized by elevated levels of maternal and placental pro-inflammatory cytokines, and increased markers of oxidative stress, nitrative stress and infiltration of immune cells in the placenta (Lappas et al., 2011, Mrizak et al., 2014). In experimental models of diabetes and in women with GDM and type 2 diabetes there are reduced placental expression of peroxisome proliferator activated receptor α (PPARα) and PPARγ (Capobianco et al., 2013, Holdsworth-Carson et al., 2010, Jawerbaum et al., 2004). PPARs are ligand activated transcription factors that transactivate and transrepress multiple genes involved in the regulation of diverse metabolic, developmental and anti-inflammatory pathways (Wahli and Michalik, 2012). Previous experimental work has addressed the relevance of impaired PPARs in diabetic pregnancies and the role of their activation in the prevention of the pro-oxidant and pro-inflammatory intrauterine environment (Jawerbaum and Capobianco, 2011, Kurtz et al., 2014, Martinez et al., 2012).
In this work, we hypothesized that 1) adverse intrauterine programming in an experimental model of mild pre-gestational diabetes (F0) leads to GDM in the female offspring (F1) and, 2) experimental GDM pregnancies are characterized by fetal overweight and placental alterations in regulators of nutrients transfer and a pro-oxidative/pro-inflammatory environment. Thus, we studied the female offspring of control rats compared with the female offspring of rats with pre-gestational diabetes before mating and at term pregnancy. We analyzed metabolic and growth parameters, as well as placental mTOR signaling, lipoperoxidation, nitric oxide production, peroxynitrite-induced damage and PPARs.
Section snippets
Animals
Albino Wistar rats bred in our animal facility were fed ad libitum with commercial rat chow (Asociación Cooperativa Argentina, Buenos Aires, Argentina). As shown in Fig. 1, to induce diabetes in the F0 generation, pups were injected with streptozotocin (90 mg/kg, s.c, Sigma–Aldrich, St. Louis, MO, USA) diluted in citrate buffer (0.05 M, pH 4.5, Sigma–Aldrich) at postnatal day 2, as previously described (Jawerbaum and White, 2010, Kurtz et al., 2014). Control animals were injected with citrate
Metabolic characterization of an experimental model of GDM
We have previously shown that from the fifth month of age the offspring of mild pre-gestational diabetic rats are hyperglycemic and have elevated fasting plasma insulin levels (Capobianco et al., 2015). In this work we found that at three months of age plasma concentrations of glucose, insulin, triglycerides and cholesterol in the offspring from mild pre-gestational diabetic rats were not different when compared to the offspring of control rats (Fig. 2A). In contrast, at term pregnancy, the
Discussion
Gestational diabetes is a common pregnancy pathology, however, the mechanistic understanding of how GDM leads to adverse maternal and fetal outcomes is limited due to the paucity of clinically relevant animal models of GDM (Jawerbaum and White, 2010, Lappas et al., 2011). In this work, we characterized a novel GDM model induced by intrauterine programming leading to maternal and fetal metabolic impairments in the female offspring when pregnant. The changes are consistent with GDM and clinically
Funding
This work was supported by the Agencia Nacional de Promoción Científica y Tecnológica de Argentina and GlaxoSmithKline (PICTO-GSK 2012-0054), by the Agencia Nacional de Promoción Científica y Tecnológica de Argentina (PICT 2014-0411), and by the International Cooperation Grant CONICET–NIH–2014 (AJ-TJ).
Acknowledgements
The authors would like to thank Vet. Marcela Márquez and Tech. Enzo Cuba for the valuable help with animal handling. The authors would like to acknowledge Vanessa Ramirez for her expert technical assistance.
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