Induction of Embryonic Dysmorphogenesis by High Glucose Concentration, Disturbed Inositol Metabolism, and Inhibited Protein Kinase C Activity PARRI WENTZEL, * CHRISTIAN R. WENTZEL , MATTIAS B. GA ¨ RESKOG AND ULF J. ERIKSSON Department of Medical Cell Biology, Uppsala University, Biomedical Center, SE-751 23 Uppsala, Sweden ABSTRACT Background: Exposure to a diabetic environment causes excess reactive oxygen species (ROS), de- creased prostaglandin E 2 (PGE 2 ) concentration, and increased embryonic maldevelopment. The aim of the present work was to study whether embryonic dysmor- phogenesis is also dependent on alterations of inositol and associated intracellular metabolites. Methods: Day 9 rat embryos were cultured for 24 or 48 hr and evaluated for gene expression. Day 10 and day 11 embryos from normal and diabetic rats were also examined. RT-PCR was used to study embryonic gene expression of protein kinase C (PKC) and cytoso- lic phospholipase A 2 (cPLA 2 ). Results: Embryos exposed to 30 mmol/L glucose (30G), 500 or 750 mol/L of scyllo-inositol (500SI or 750SI) had higher malformation score than control embryos cultured in 10 mmol/L glucose (10G). Adding 1.6 mmol/L inositol to the 30G or 750SI culture medium partly corrected these embryos, and completely normalized 500SI embry- onic development. Adding 0.5 mmol/L N-acetylcysteine (NAC) or 280 nmol/L PGE 2 protected, and failed to pro- tect, the SI-exposed embryos, respectively. 10G em- bryos exposed to the PKC inhibitor GF-109203X dis- played dose-dependent dysmorphogenesis. Addition of 1.6 mmol/L inositol or 0.5 mmol/L NAC to the PKC- inhibitor-exposed 10G embryos largely normalized the outcome, whereas PGE 2 again failed to protect embry- onic development. 30G culture tended to decrease the expression of cPLA 2 after 24 hr in vitro. We also found decreased mRNA levels of cPLA 2 in offspring of diabetic rats on gestational day 10 and of PKC on day 11, as compared with normal offspring. Conclusions: High glucose concentration causes dys- morphogenesis in embryos by an interaction of oxida- tive stress and inositol depletion. Teratology 63:193–201, 2001. © 2001 Wiley-Liss, Inc. INTRODUCTION Diabetes during pregnancy is associated with in- creased risk of growth retardation and congenital mal- formation in the offspring (Pedersen, ’77). Recent esti- mations of the rate of malformation in diabetic pregnancy have yielded a 3– 8-fold increase compared with nondiabetic gestation (Casson et al., ’97; Dunne et al., ’99; Schaefer-Graf et al., ’00). The mechanisms causing the dysmorphogenesis as- sociated with diabetes are not completely clear. Earlier experimental studies have shown that diabetes in vivo and high glucose concentration in vitro cause alter- ations in the arachidonic acid cascade (Goldman et al., ’85), leading to decreased cyclooxygenase-2 (COX-2) gene expression (Wentzel et al., ’99), and lowered con- centration of prostaglandin E 2 (PGE 2 ) in the offspring (Schoenfeld et al., ’95; Piddington et al., ’96; Wentzel et al., ’99). In addition, inositol supplementation to high glucose culture medium has proved beneficial by di- minishing the rate of embryonic dysmorphogenesis (Baker et al., ’90; Hashimoto et al., ’90; Hod et al., ’90), implicating a teratological role for lowered uptake of inositol (Weigensberg et al., ’90) in embryos subjected to raised glucose levels. Furthermore, it has been sug- gested by several investigators that diabetic pregnancy is associated with either increased ROS activity (Eriks- son and Borg, ’91; Eriksson and Borg, ’93), or decreased antioxidative capacity (Trocino et al., ’95). The putative interrelationships between disturbed inositol metabo- lism and other teratological conditions were the subject for the present investigation (Eriksson et al., ’00). One possible combined metabolic pathway could commence by increased extracellular glucose levels causing decreased inositol uptake (Weigensberg et al., ’90; Yorek et al., ’93), decreased inositol concentration (Hod et al., ’86; Sussman and Matschinsky, ’88; Hashi- Grant sponsor: Ernfors Family Fund; Grant sponsor: So ¨derberg Foun- dation; Grant sponsor: Swedish Diabetes Association; Grant sponsor: Novo Nordisk Foundation; Grant sponsor: Swedish Medical Research Council; Grant number: 12X-7475; Grant number: 12X-109. *Correspondence to: Parri Wentzel, Department of Medical Cell Biol- ogy, Uppsala University, Biomedical Center, P.O. Box 571, SE-751 23 Uppsala, Sweden. E-mail: parri.wentzel@neuro.uu.se Received 5 December 2000; Accepted 24 February 2001 TERATOLOGY 63:193–201 (2001) © 2001 WILEY-LISS, INC.