Josie M. McConnell

Coenzyme Q10 prevents accelerated cardiac aging in a rat model of poor maternal nutrition and accelerated postnatal growth

Studies in human and animals have demonstrated that nutritionally induced low birth-weight followed by rapid postnatal growth increases the risk of metabolic syndrome and cardiovascular disease. Although the mechanisms underlying such nutritional programming are not clearly defined, increased oxidative-stress leading to accelerated cellular aging has been proposed to play an important role. Using an established rodent model of low birth-weight and catch-up growth, we show here that post-weaning dietary supplementation with coenzyme Q10, a key component of the electron transport chain and a potent antioxidant rescued many of the detrimental effects of nutritional programming on cardiac aging. This included a reduction in nitrosative and oxidative-stress, telomere shortening, DNA damage, cellular senescence and apoptosis. These findings demonstrate the potential for postnatal antioxidant intervention to reverse deleterious phenotypes of developmental programming and therefore provide insight into a potential translatable therapy to prevent cardiovascular disease in at risk humans.

Coenzyme Q10 prevents hepatic fibrosis, inflammation, and oxidative stress in a male rat model of poor maternal nutrition and accelerated postnatal growth

Background: It is well established that low birth weight and accelerated postnatal growth increase the risk of liver dysfunction in later life. However, molecular mechanisms underlying such developmental programming are not well characterized, and potential intervention strategies are poorly defined. Objectives: We tested the hypotheses that poor maternal nutrition and accelerated postnatal growth would lead to increased hepatic fibrosis (a pathological marker of liver dysfunction) and that postnatal supplementation with the antioxidant coenzyme Q10 (CoQ10) would prevent this programmed phenotype. Design: A rat model of maternal protein restriction was used to generate low-birth-weight offspring that underwent accelerated postnatal growth (termed “recuperated”). These were compared with control rats. Offspring were weaned onto standard feed pellets with or without dietary CoQ10 (1 mg/kg body weight per day) supplementation. At 12 mo, hepatic fibrosis, indexes of inflammation, oxidative stress, and insulin signaling were measured by histology, Western blot, ELISA, and reverse transcriptase–polymerase chain reaction. Results: Hepatic collagen deposition (diameter of deposit) was greater in recuperated offspring (mean ± SEM: 12 ± 2 μm) than in controls (5 ± 0.5 μm) (P < 0.001). This was associated with greater inflammation (interleukin 6: 38% ± 24% increase; P < 0.05; tumor necrosis factor α: 64% ± 24% increase; P < 0.05), lipid peroxidation (4-hydroxynonenal, measured by ELISA: 0.30 ± 0.02 compared with 0.19 ± 0.05 μg/mL per μg protein; P < 0.05), and hyperinsulinemia (P < 0.05). CoQ10 supplementation increased (P < 0.01) hepatic CoQ10 concentrations and ameliorated liver fibrosis (P < 0.001), inflammation (P < 0.001), some measures of oxidative stress (P < 0.001), and hyperinsulinemia (P < 0.01). Conclusions: Suboptimal in utero nutrition combined with accelerated postnatal catch-up growth caused more hepatic fibrosis in adulthood, which was associated with higher indexes of oxidative stress and inflammation and hyperinsulinemia. CoQ10 supplementation prevented liver fibrosis accompanied by downregulation of oxidative stress, inflammation, and hyperinsulinemia.

Experimental manipulations of compaction and their effects on the phosphorylation of uvomorulin

Compaction of the eight‐cell stage mouse embryo is a critical event in the generation of different cell types within the preimplantation embryo. Uvomorulin, a member of the cadherin family of cell adhesion molecules, is important during compaction and its phosphorylation increases early in the eight‐cell stage, suggesting that this posttranslational modification may be important for compaction to proceed. We have assessed the importance of the phosphorylation of uvomorulin during compaction by preventing, reversing, or inducing adhesion prematurely. The only condition that affected the overall level of uvomorulin phosphorylation was the prevention of compaction through prolonged exposure of four‐cell embryos to low Ca2−. This treatment reduced the level of uvomorulin phosphorylation in eight‐cell embryos, and perturbed its localization to regions of cell‐cell contact. Thus, whilst the phosphorylation of uvomorulin does not appear to regulate directly uvomorulins adhesive function, it may be associated with the redistribution of uvomorulin during compaction.

Control of the surface expression of uvomorulin after activation of mouse oocytes.

Uvomorulin (E-cadherin) is the major cell adhesion molecule responsible for intercellular adhesion in early mouse embryos. In contrast to other cell adhesion molecules, it is not detectable on the cell surface until around 6 h after fertilisation or parthenogenetic activation, at the time when pronuclear formation occurs (Clayton, L., Stinchcombe, S.V. and Johnson, M.H., Zygote 1, 333-44, 1993). In order to investigate this developmental control of surface expression of uvomorulin, we examined the effects of inhibitors of various cellular processes on the appearance of uvomorulin at the oocyte surface, as assessed immunocytochemically. Inhibitors of cytoskeletal assembly (cytochalasin D and nocodazole), protein synthesis (puromycin and anisomycin), and DNA synthesis (aphidicolin) had no effect on surface expression. Brefeldin A, which inhibits intracellular transport and secretion, did prevent surface expression, but monensin did not. The effects of brefeldin were reversible; following 8 h of treatment, recovery of surface expression after removal of brefeldin began within 2 h. The time-course of surface expression post-activation suggested a link with pronuclear formation. However, when pronuclear formation was advanced experimentally using 6-dimethylaminopurine (DMAP), concomitant advancement of surface uvomorulin was not observed. Similarly, surface expression of uvomorulin did not accompany puromycin-induced pronuclear formation in maturing meiotic metaphase 1 (MI) oocytes in vitro. Thus, surface uvomorulin expression does not appear to be linked simply to pronuclear formation. Proteolytic processing of both newly synthesised and total uvomorulin to generate mature molecule from precursor increased within 30 min to 1 h after activation, and also occurred in the continued presence of brefeldin, suggesting that uvomorulin processing appears to be controlled independently of its surface expression.

Staurosporine advances interblastomeric flattening of the mouse embryo.

Staurosporine, an inhibitor of protein kinase activity, causes premature intercellular flattening of blastomeres but does not induce their premature polarisation. The flattening induced is calcium dependent, is reversed transiently at mitosis and requires the continuing presence of the drug. Staurosporine also blocks the decompacting effect of phorbol ester on 8-cell embryos.

Nutritional programming of coenzyme Q: potential for prevention and intervention?

Low birth weight and rapid postnatal growth increases risk of cardiovascular‐disease (CVD); however, underlying mechanisms are poorly understood. Previously, we demonstrated that rats exposed to a low‐protein diet in utero that underwent postnatal catch‐up growth (recuperated) have a programmed deficit in cardiac coenzyme Q (CoQ) that was associated with accelerated cardiac aging. It is unknown whether this deficit occurs in all tissues, including those that are clinically accessible. We investigated whether aortic and white blood cell (WBC) CoQ is programmed by suboptimal early nutrition and whether postweaning dietary supplementation with CoQ could prevent programmed accelerated aging. Recuperated male rats had reduced aortic CoQ [22 d (35±8.4%; P<0.05); 12 m (53±8.8%; P<0.05)], accelerated aortic telomere shortening (P<0.01), increased DNA damage (79±13% increase in nei‐endonucleaseVIII‐like‐1), increased oxidative stress (458±67% increase in NAPDH‐oxidase‐4; P < 0.001), and decreased mitochondrial complex II‐III activity (P<0.05). Postweaning dietary supplementation with CoQ prevented these detrimental programming effects. Recuperated WBCs also had reduced CoQ (74±5.8%; P<0.05). Notably, WBC CoQ levels correlated with aortic telomere‐length (P<0.0001) suggesting its potential as a diagnostic marker of vascular aging. We conclude that early intervention with CoQ in at‐risk individuals may be a cost‐effective and safe way of reducing the global burden of CVDs.–Tarry‐Adkins, J. L., Fernandez‐Twinn, D. S., Chen, J.‐H., Hargreaves, I. P., Martin‐Gronert, M. S., McConnell, J. M., Ozanne, S. E. Nutritional programming of coenzyme Q: potential for prevention and intervention? FASEB J. 28, 5398–5405 (2014). www.fasebj.org

Coenzyme Q10 Prevents Insulin Signaling Dysregulation and Inflammation Prior to Development of Insulin Resistance in Male Offspring of a Rat Model of Poor Maternal Nutrition and Accelerated Postnatal Growth

Low birth weight and rapid postnatal growth increases the risk of developing insulin resistance and type 2 diabetes in later life. However, underlying mechanisms and potential intervention strategies are poorly defined. Here we demonstrate that male Wistar rats exposed to a low-protein diet in utero that had a low birth weight but then underwent postnatal catch-up growth (recuperated offspring) had reductions in the insulin signaling proteins p110-β (13% ± 6% of controls [P < .001]) and insulin receptor substrate-1 (39% ± 10% of controls [P < .05]) in adipose tissue. These changes were not accompanied by any change in expression of the corresponding mRNAs, suggesting posttranscriptional regulation. Recuperated animals displayed evidence of a proinflammatory phenotype of their adipose tissue with increased IL-6 (139% ± 8% [P < .05]) and IL1-β (154% ± 16% [P < .05]) that may contribute to the insulin signaling protein dysregulation. Postweaning dietary supplementation of recuperated animals with coenzyme Q (CoQ10) (1 mg/kg of body weight per day) prevented the programmed reduction in insulin receptor substrate-1 and p110-β and the programmed increased in IL-6. These findings suggest that postweaning CoQ10 supplementation has antiinflammatory properties and can prevent programmed changes in insulin-signaling protein expression. We conclude that CoQ10 supplementation represents an attractive intervention strategy to prevent the development of insulin resistance that results from suboptimal in utero nutrition.

Twin pregnancy consisting of a complete hydatidiform mole and a fetus: genetic origin determined by DNA typing

A 29 year old woman presented with complaints of intermittent vaginal spotting at 15 weeks of gestation. On examination she had a blood pressure of 186/110 mmHg, ankle oedema, and a large-for-dates uterus. An apparently normal fetus with a biparietal diameter compatible with the gestational age (31 mm) and with active fetal heart motion was seen on ultrasonographic examination. Although a placenta of normal appearance was located at the anterior uterine wall, more than half of the uterine cavity was occupied by a complex echogenic and echolucent mass suggestive of a molar pregnancy. The placenta and the vesicular mass were clearly separable. After appropriate consultation with the woman, the pregnancy was terminated by suction curettage under ultrasound guidance. The aborted fetus was 10 cm in length and weighed approximately 20 g. The placenta weighed 22 g and was free of any vesicular tissue. There were 660 g of vesicular tissue. Purified DNA (3-5 pg) (KO et al. 1991) from each individual and conceptal tissue was digested overnight at 37°C with restriction endonucleases AluI and MboI separately. The digested DNA fragments were separated by electrophoresis on a 0.85% agarose gel at 1.5 voltdcm for 24 h and transferred to filter (Hybond-N, Amersham, UK) by vacuum blot (Hybaid Vacu-Aid vacuum blotting system, Hybaid Ltd UK). Locus-specific probes hMS1, XMS31, hMS43 and phg3 (Wong et al. 1987) were radiolabelled by random oligonucleotide priming to specific activities of loR to 10 cpm/pg. The filter was prehybridised at 65°C for 30 min in solution containing 1 mM EDTA, 0.5 M NaHPO, pH 7.2,7% SDS and 0.2% BSA. Hybridisation took place at 65°C overnight in the solution of 1 x SSC and 6% polyethylene glycol 6000 containing 32P-labelled probe. The post-hybridisation wash was first in 40 mmol NaHPO,, 1% SDS and later to a stringency of 0.1 x SSC and 0.01% SDS at 65°C (Wong et al. 1987). Autoradiography was carried out using X-ray film (Fuji Nif RX, Japan) with an intensifier screen at -70°C for two days. The alleles of parental DNA using MboI digestion and hybridised with ALMS31 were heterozygous and different (Fig. 1). Under the same conditions, the allelcs identified in the fetal tissue were also heterozygous and showed an

Molecular Basis of Cell Cycle Control in Early Mouse Embryos

Publisher Summary This chapter describes recent advances in understanding the molecules—individually and in complexes—that are controlling the events of the cell cycle in model systems, such as yeast and early amphibian, and in transformed cell types, such as HeLa cells. The chapter presents the events that occur in early murine embryos and explains the ways in which probes derived from other systems have been used successfully to study the molecular mechanisms operating in early mouse development. The chapter also discusses the way the repertoire of techniques advances the understanding of the molecular mechanisms underlying the control of the cell cycle in early mammalian development. Yeasts provide good model systems to study cell-cycle control as they are simple single cell eukaryotes having less complicated cell-cycle regulation. The cell cycles of yeast are not exactly identical to those in higher organisms, such as mammals. As with higher eukaryotes, a spindle forms and chromosomes condense, but unlike higher eukaryotes, there is no breakdown of the nuclear envelope and the cell divides by medial fission.