| 1. |
- Gäreskog, Mattias, et al.
(författare)
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Altered Protein Kinase C Activation Associated with Rat Embryonic Dysmorphogenesis
- 2004
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Ingår i: Pediatric Research. - 0031-3998 .- 1530-0447. ; 56:6, s. 849-857
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Tidskriftsartikel (refereegranskat)abstract
- It has been suggested that protein kinase C (PKC) is involved in the etiology of diabetic complications. The aim of the present study was to investigate the putative involvement of different PKC isoforms (alpha, beta1, beta 2, gamma, delta, epsilon, and zeta) in the embryopathy of diabetic rat pregnancy. Embryos were collected from normal and diabetic rats and assayed for PKC activity, PKC mRNA levels, and PKC protein distribution on gestational d 10 and 11. Embryos of diabetic rats showed markers of increased activity of PKC-alpha, PKC-beta1, PKC-gamma, PKC-delta, and PKC-zeta compared with embryos of normal rats on d 10. In addition, the malformed embryos had further increased PKC-gamma, and PKC-delta activity markers compared with nonmalformed embryos of diabetic rats on gestational d 10. In contrast, maternal diabetes caused only two alterations in PKC activity markers on gestational d 11, i.e. both PKC-alpha and PKC-zeta were decreased in embryos of diabetic rats. We found increased mRNA levels of PKC-beta 1 and PKC-zeta on d 10 in embryos of diabetic rats and decreased mRNA levels of PKC-gamma on d 11 in embryos of diabetic rats. Malformed embryos from diabetic rats showed increased distribution of PKC-beta 1 and PKC-beta 2 protein in the tissue compared with nonmalformed embryos from diabetic rats and embryos from normal rats. We conclude that diabetic rat embryopathy may be associated with increased activity and enhanced tissue distribution of several PKC isoforms in early organogenesis.
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| 2. |
- Gäreskog, Mattias, et al.
(författare)
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Combined Supplementation of Folic Acid and Vitamin E Diminishes Diabetes-Induced Embryotoxicity in Rats
- 2006
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Ingår i: Birth defects research. Clinical and molecular teratology. - : Wiley. - 1542-0752 .- 1542-0760. ; 76:6, s. 483-490
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Oxidative stress and enhanced apoptosis may be involved in the induction of embryonic dysmorphogenesis in diabetic pregnancy. Administration of folic acid or vitamin E diminishes embryonic dysmorphogenesis. We aimed to evaluate the effect of combined treatment with folic acid and vitamin E on the disturbed development in embryos of diabetic rats. METHODS: Pregnant nondiabetic and diabetic rats were treated with daily injections of 15 mg/kg folic acid or with 5% vitamin E in the diet. A third group received combined treatment. Day 10 and day 11 embryos were evaluated for development and apoptotic profile. RESULTS: We found increased malformations, resorptions, and profound growth retardation in embryos of diabetic rats compared to control embryos. Vitamin E or folic acid alone, or the 2 compounds combined, normalized embryonic demise. Maternal diabetes caused decreased nuclear factor-kappa B (NF-kappa B) activity and B-cell lymphoma 2 (Bcl-2) protein level, and increased Bcl-2-associated x proteins (Bax) in embryos. Supplementation of vitamin E alone normalized the Bax protein level in a diabetic environment. Administration of folic acid to diabetic rats increased NF-kappa B activity and Bcl-2 protein level. Combined treatment normalized Bcl-2 and Bax protein level in a diabetic environment. CONCLUSIONS: Combined supplementation of folic acid and vitamin E to pregnant diabetic rats diminished diabetes-induced malformations and resorptions, concomitant with normalization of apoptotic protein levels. No treatment completely abolished the embryonic demise; therefore, other mechanisms than oxidative stress and apoptosis are likely to be involved in diabetic embryopathy.
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| 3. |
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| 4. |
- Gäreskog, Mattias, et al.
(författare)
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Maternal diabetes in vivo and high glucose concentration in vitro increases apoptosis in rat embryos
- 2007
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Ingår i: Reproductive Toxicology. - : Elsevier BV. - 0890-6238 .- 1873-1708. ; 23:1, s. 63-74
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Tidskriftsartikel (refereegranskat)abstract
- Apoptosis may be involved in diabetes-induced embryonic dysmorphogenesis. We estimated the occurrence of apoptosis in embryos of a rat model for diabetic pregnancy. We found decreased Bcl-2, increased Bax and cleaved Caspase 3 proteins in embryos from diabetic rats. Moreover, we found increased activation of Caspase 3 in cells from embryos previously exposed to a diabetes-like environment (in vivo, in vitro) compared to cells from control embryos, which was normalized by supplementation of N-acetylcysteine or apoptosis inhibitor. We detected increased propidium iodide uptake in embryonic cells exposed to maternal diabetes, a finding confirmed by vital staining. Additionally, we found increased dysmorphogenesis in embryos exposed to a diabetic environment in vivo and in vitro. Exposure to a diabetic milieu during organogenesis increases apoptosis in embryonic cells and dysmorphogenesis in embryos. Enhanced apoptotic rate may have a role in diabetic embryopathy by inducing disturbed embryonic maturation, increased rates of resorptions and congenital malformations.
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| 5. |
- Gäreskog, Mattias, et al.
(författare)
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N-Acetylcysteine and alpha-cyano-4-hydroxycinnamic acid alter protein kinase C (PKC)-delta and PKC-zeta and diminish dysmorphogenesis in rat embryos cultured with high glucose in vitro
- 2007
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Ingår i: Journal of Endocrinology. - : Bioscientifica. - 0022-0795 .- 1479-6805. ; 192:1, s. 207-214
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Tidskriftsartikel (refereegranskat)abstract
- Malformations and growth disturbances are two- to threefold more common in infants of diabetic mothers than in offspring of non-diabetic pregnancy. Several suggestions have emerged to explain the reasons for diabetic embryopathy, including enhanced mitochondrial production of reactive oxygen species leading to altered activation of protein kinase C. This study aimed to evaluate the effect of alpha-cyano-4-hydroxycinnamic acid (CHC) and N-acetylcysteine (NAC) addition on morphology and activity of protein kinase C-delta and protein kinase C-zeta in rat embryos exposed to a high glucose concentration in vitro. Day 9 embryos from normal rats were cultured in 10 or 30 mM glucose concentrations with or without supplementation of CHC, NAC, or protein kinase C inhibitors specific for protein kinase C-delta and protein kinase C-zeta. Embryos were evaluated for malformations, crown rump length, and somite number. Protein kinase C-delta and protein kinase C-zeta activities were estimated by western blot by separating membranous and cytosolic fractions of the embryo. We found increased malformations and growth retardation in embryos cultured in high versus low glucose concentrations. These abnormalities were diminished when CHC and NAC or specific protein kinase C-inhibitors were added to the culture medium. The activities of embryonic protein kinase C-delta and protein kinase C-zeta were increased in the high glucose environment after 24-h culture, but were normalized by the addition of CHC and NAC as well as respective inhibitor to the culture medium. These findings suggest that mitochondrial overproduction of reactive oxygen species is involved in diabetic embryopathy. Furthermore, such overproduction may affect embryonic development, at least partly, by enhancing the activities of protein kinase C-delta and protein kinase C-zeta.
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| 6. |
- Gäreskog, Mattias, 1976-
(författare)
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Teratogenicity Involved in Experimental Diabetic Pregnancy
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Maternal diabetes is associated with increased risk of growth disturbances and congenital malformations. The malformations rate in the offspring of diabetic mothers is 2-3 fold higher compared to infants of nondiabetic mothers. In this thesis we have investigated the role of the protein kinase C (PKC) pathway and the apoptotic machinery in embryopathy. We investigated the involvement of PKC isoforms in the embryopathy of diabetic rat pregnancy. Embryos of diabetic rats showed altered activity and protein distribution of several PKC isoforms compared with embryos of normal rats. Using whole embryo culture we found increased activity of PKC-delta and PKC-zeta after 24h of culture and increased rate of malformations and growth retardation in embryos cultured in high glucose concentration compared to embryos cultured in low glucose concentration. Addition of α-cyano-4-cinnamic acid and N-acetylcysteine to the culture medium normalized malformations and growth retardations whereas specific PKC-inhibitors abolished malformations and partly restored the growth retardations. All treatment normalized glucose-induced increase of PKC activity. Estimated occurrence of apoptosis in embryos of diabetic rats and in embryonic cells exposed to high glucose concentration showed increased rate of pro-apoptotic markers. The increased apoptosis in the high glucose exposed embryonic cells was normalized by supplementation of N-acetylcysteine or apoptosis inhibitor. Treatment with vitamin E and folic acid to diabetic pregnant rats decreased diabetes-induced malformations and resorptions, concomitant with normalization of apoptotic protein levels. These results suggest that oxidative stress is augmented in embryos of diabetic rats and that it also plays a role in the activation of PKC and apoptosis. We used antioxidative treatment with beneficial effect although we could not completely abolish the embryonic demise; this may indicate that other mechanisms are involved in diabetic embryopathy. Further studies are needed to develop multi-nutrient dietary supplement to eliminate embryonic abnormalities induced by maternal diabetes.
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| 7. |
- Wentzel, Parri, et al.
(författare)
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Decreased cardiac glutathione peroxidase levels and enhanced mandibular apoptosis in malformed embryos of diabetic rats
- 2008
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Ingår i: Diabetes. - : American Diabetes Association. - 0012-1797 .- 1939-327X. ; 57:12, s. 3344-3352
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE: To characterize normal and malformed embryos within the same litters from control and diabetic rats for expression of genes related to metabolism of reactive oxygen species (ROS) or glucose as well as developmental genes. RESEARCH DESIGN AND METHODS: Embryos from nondiabetic and streptozotocin-induced diabetic rats were collected on gestational day 11 and evaluated for gene expression (PCR) and distribution of activated caspase-3 and glutathione peroxidase (Gpx)-1 by immunohistochemistry. RESULTS: Maternal diabetes (MD group) caused growth retardation and an increased malformation rate in the embryos of MD group rats compared with those of controls (N group). We found decreased gene expression of Gpx-1 and increased expression of vascular endothelial growth factor-A (Vegf-A) in malformed embryos of diabetic rats (MDm group) compared with nonmalformed littermates (MDn group). Alterations of messenger RNA levels of other genes were similar in MDm and MDn embryos. Thus, expression of copper zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD), and sonic hedgehog homolog (Shh) were decreased, and bone morphogenetic protein-4 (Bmp-4) was increased, in the MD embryos compared with the N embryos. In MDm embryos, we detected increased activated caspase-3 immunostaining in the first visceral arch and cardiac area and decreased Gpx-1 immunostaining in the cardiac tissue; both findings differed from the caspase/Gpx-1 immunostaining of the MDn and N embryos. CONCLUSIONS: Maternal diabetes causes growth retardation, congenital malformations, and decreased general antioxidative gene expression in the embryo. In particular, enhanced apoptosis of the first visceral arch and heart, together with decreased cardiac Gpx-1 levels, may compromise the mandible and heart and thus cause an increased risk of developing congenital malformation.
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| 8. |
- Wentzel, Parri, et al.
(författare)
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Folic acid supplementation diminishes diabetes- and glucose-induced dysmorphogenesis
- 2005
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Ingår i: Diabetes. - : American Diabetes Association. - 0012-1797 .- 1939-327X. ; 54:2, s. 546-553
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Tidskriftsartikel (refereegranskat)abstract
- Maternal administration of folic acid diminishes the risk of neural tube defects (NTDs) in offspring, but whether folic acid exerts a similar effect in diabetic pregnancy is unknown. The aim was to investigate whether maldevelopment in rat embryos caused by exposure to diabetes in vivo or high-glucose concentrations in vitro is affected by subcutaneous administration of folic acid to the pregnant mother or by adding the compound to the culture medium, respectively. Exposure of embryos to maternal diabetes in vivo or 30 mmol/l glucose in vitro yielded an increased malformation rate (71 and 88% NTD, respectively) and lowered somite number and crown-rump length compared with control embryos. When we injected folic acid into the diabetic pregnant rat, or added 2 mmol/l folic acid to the culture medium with high glucose, the embryonic parameters improved (3 and 5% NTD, respectively). The present work shows that administration of folic acid can diminish diabetes-induced maldevelopment. This suggests that folic acid supplementation may have a role in the prevention of malformations in diabetic pregnancy.
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| 9. |
- Wentzel, Parri, et al.
(författare)
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Induction of embryonic dysmorphogenesis by high glucose concentration, disturbed inositol metabolism, and inhibited protein kinase C activity
- 2001
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Ingår i: Teratology. - : Wiley. - 0040-3709 .- 1096-9926. ; 63:5, s. 193-201
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Exposure to a diabetic environment causes excess reactive oxygen species (ROS), decreased prostaglandin E(2) (PGE(2)) concentration, and increased embryonic maldevelopment. The aim of the present work was to study whether embryonic dysmorphogenesis 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 cytosolic phospholipase A(2) (cPLA(2)). RESULTS: Embryos exposed to 30 mmol/L glucose (30G), 500 or 750 micromol/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 embryonic development. Adding 0.5 mmol/L N-acetylcysteine (NAC) or 280 nmol/L PGE(2) protected, and failed to protect, the SI-exposed embryos, respectively. 10G embryos exposed to the PKC inhibitor GF-109203X displayed 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 embryonic 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 dysmorphogenesis in embryos by an interaction of oxidative stress and inositol depletion.
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