Objective
Oxidative stress plays a causative role in diabetic embryopathy. We tested whether mitigating oxidative stress, using superoxide dismutase 1 (SOD1) transgenic (Tg) mice, would block hyperglycemia-induced specific protein kinase C (PKC) isoform activation and its downstream cascade.
Study Design
Day 8.5 embryos from nondiabetic wild-type control (NC), diabetic mellitus wild-type (DM), and diabetic SOD1-Tg mice (DM-SOD1-Tg) were used for detection of phosphorylated (p-) PKCα/βII and p-PKCδ, and levels of 2 prominent PKC substrates, phosphorylated myristoylated alanine-rich protein kinase C substrate (MARCKS) and receptor for activated C kinase 1 (RACK1), and lipid peroxidation markers, 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA).
Results
Levels of p-PKCα/βII, p-PKCδ, p-MARCKS, 4-HNE, and MDA were significantly elevated in the DM group compared with those in the NC group and the DM-SOD1-Tg group. The NC and DM-SOD1-Tg groups had comparable levels of these protein and lipid peroxidation markers. RACK1 levels did not differ among the 3 groups.
Conclusion
Mitigating oxidative stress by SOD1 overexpression blocks maternal hyperglycemia–induced activation of specific PKC isoforms and downstream cascades.
Pregestational diabetes is a significant risk factor for congenital malformations including major structure birth defects and neural tube defects (NTDs). Maternal hyperglycemia increases the production of reactive oxygen species and impairs the intracellular antioxidant capability, leading to oxidative stress. Multiple evidence supports that hyperglycemia-induced oxidative stress causes malformations. Both in vivo and in vitro treatments with variety of antioxidants can effectively reduce hyperglycemia-induced NTDs. Overexpression of the antioxidant enzyme, superoxide dismutase 1 (SOD1), in transgenic mice ameliorates maternal diabetes-induced NTDs. Although oxidative stress appears to be a central mechanism underlying hyperglycemia-induced malformations, it is unclear whether the downstream intracellular signals mediate oxidative stress as well.
Oxidative stress activates multiple kinase signaling pathways. The protein kinase C (PKC) family consists of 12 isoforms that involve diverse physiological and pathophysiological functions including cell proliferation, differentiation, and apoptosis. In diabetic embryopathy, prolonged PKC activation is associated with maternal diabetes–induced NTDs. We have further reported that hyperglycemia specifically activates PKCα/βII and PKCδ. Specific pharmacological inhibitors to PKCα, PKCβII, or PKCδ have been shown to significantly reduce hyperglycemia-induced NTDs, strongly implicating that activation of these specific PKC isoforms plays a causative role in the induction of NTDs by hyperglycemia. PKC activation results in lipid peroxidation, which leads to cell membrane damage. PKC activation has been linked to altered arachidonic acid (AA) metabolism during lipidperoxidation.
In diabetic embryopathy, hyperglycemia-induced lipid peroxidation alters cell membrane lipid metabolism by shifting AA metabolism from prostaglandin E2 to isoprostanes. The loss of membrane AA destabilizes the cell membrane structure and function. Conversely, AA supplementation has been shown to reduce the incidence of diabetic embryopathy. In the present study, we wanted to determine whether oxidative stress–induced-specific PKC isoform activation triggers lipid peroxidation, which, in turns, intensifies the degree of oxidative stress in embryos exposed to hyperglycemia.
Besides the differential activation mechanisms of the 12 PKC isoforms, individual PKC isoforms exerts distinct physiological and pathophysiological functions via substrate specificity. A limited number of PKC substrate has been identified. Among the known PKC substrates, myristoylated alanine-rich protein kinase C substrate (MARCKS) is a prominent PKC substrate that primarily resides in neural tissues. Furthermore, it has been reported that MARCKS is a specific substrate of PKCβII and PKCδ.
Another prominent PKC substrate is the receptor for activated C kinase 1 (RACK1), which was originally discovered through its binding to active PKCβII and other classic PKC isoforms. RACK1 participates in multiprotein signaling complexes and can enhance PKC-dependent c-Jun-N-terminal kinase (JNK) activation, which plays a causative role in the induction of diabetic embryopathy. Therefore, in the present study, we assessed the activation and levels of these 2 PKC substrates with a goal of defining the role of PKC substrates in oxidative stress-mediated teratogenicity in diabetic embryopathy.
The connection between oxidative stress and PKC activation has not been explored. Because both SOD1 overexpression in vivo and PKC inhibition in vitro reduce hyperglycemia-induced malformations, in the present study, we used SOD1-transgenic (Tg) mice to test whether SOD1 overexpression in vivo blocks hyperglycemia-induced specific PKC isoforms activation and its downstream cascade.
Materials and Methods
Animals and reagents
C57BL/6J mice (median body weight, 22 g) were purchased from Jackson Laboratory (Bar Harbor, ME). Streptozotocin (STZ) from Sigma (St. Louis, MO) was dissolved in sterile 0.1 M citrate buffer (pH 4.5). Sustained-release insulin pellets were purchased from Linplant (Linshin, Canada). SOD1-Tg mice in a C57BL/6J background were revived from frozen embryos by the Jackson Laboratory (stock no. 002298).
Mouse models of diabetic embryopathy
The procedures for animal use were approved by the Institutional Animal Care and Use Committee of the University of Maryland School of Medicine. Eight-week-old wild-type (WT) and SOD1-Tg mice were intravenously injected daily with 75 mg/kg STZ over 2 days to induce diabetes. Once a level of hyperglycemia indicative of diabetes (≥250 mg/dL) was achieved, insulin pellets were subcutaneously implanted in these diabetic mice to restore euglycemia prior to mating. The mice were then mated with male mice of the same respective genotype.
On day 5 of pregnancy (E5), insulin pellets were removed to permit frank hyperglycemia (>250 mg/dL glucose level), so the developing conceptuses would be exposed to a hyperglycemic environment at E7 onward. The WT nondiabetic female mice with vehicle injections and sham operation of insulin pellet implants served as nondiabetic controls. On E8.5, mice were euthanized, and conceptuses were dissected out of the uteri for analysis.
To avoid any redundancy, data of malformation incidences were not collected because it has been published elsewhere. The typical malformations observed in embryos from diabetic WT mice are NTDs.
Western blotting
Western blotting was performed as described by Yang at el. Briefly, embryonic samples were sonicated in 80 μl ice-cold lysis buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 10 mM NaF, 2 mM Na orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 1% Triton X-100) containing a protease inhibitor cocktail (Sigma). Equal amounts of protein (50 μg) were resolved by sodium dodecyl sulfate-polyacrylamide >gel electrophoresis and transferred onto an Immunobilon-P (Millipore, Bedford, MA).
Membranes were incubated for 18 h at 4°C with the following primary antibodies at 1:1000 to 1:2000 dilutions in 5% nonfat milk: antiphosphorylated (p)-PKCα/βII (#9375), anti-p-PKCδ (#9374), anti-p-MARCKS (#2741), anti-RACK1 (#4716), and anti-SOD1 (human specific, #4266), all from Cell Signaling (Beverly, MA); anti- malondialdehyde (MDA) (#442730; EMD, San Diego, CA); anti-4-hydroxynonenal (4-HNE) (#AB5605; Chemicon, Temecula, CA). Membranes were exposed to goat antirabbit, antimouse (Jackson ImmunoResearch Laboratories, West Grove, PA) secondary antibodies.
To ensure that equivalent amounts of protein were loaded among samples, membranes were stripped and probed with a mouse antibody against β-actin (Abcam, Cambridge, MA). Signals were detected using an Amersham enhanced chemiluminescence advance detection kit (GE Healthcare, Piscataway, NJ), and chemiluminescence emitted from the bands was directly captured using a UVP Bioimage EC3 system (UVP, Upland, CA). Densitometric analysis of chemiluminescence signals were performed by VisionWorks LS software (UVP). Images of representative immunoblots were arranged using Adobe Photoshop (San Jose, CA) and Microsoft PowerPoint software (Richmond, CA). All experiments were repeated 3 times with the use of independently prepared tissue lysates.
Statistics
Data are presented as means ± SE. One-way analysis of variance (ANOVA) was performed using SigmaStat 3.5 software (SyStat Software, San Jose, CA). In ANOVA analysis, a Tukey test was used to estimate the significance of the results. Statistical significance was accepted at P < .05.
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