Placental nutrient transporters adapt during persistent maternal hypoglycaemia in rats.

Maternal malnutrition is associated with decreased nutrient transfer to the foetus, which may lead to foetal growth restriction, predisposing children to a variety of diseases. However, regulation of placental nutrient transfer during decreased nutrient availability is not fully understood. In the present study, the aim was to investigate changes in levels of placental nutrient transporters accompanying maternal hypoglycaemia following different durations and stages of gestation in rats. Maternal hypoglycaemia was induced by insulin-infusion throughout gestation until gestation day (GD)20 or until end of organogenesis (GD17), with sacrifice on GD17 or GD20. Protein levels of placental glucose transporters GLUT1 (45/55 kDa isotypes) and GLUT3, amino acid transporters SNAT1 and SNAT2, and insulin receptor (InsR) were assessed. On GD17, GLUT1-45, GLUT3, and SNAT1 levels were increased and InsR levels decreased versus controls. On GD20, following hypoglycaemia throughout gestation, GLUT3 levels were increased, GLUT1-55 showed the same trend. After cessation of hypoglycaemia at end of organogenesis, GLUT1-55, GLUT3, and InsR levels were increased versus controls, whereas SNAT1 levels were decreased. The increases in levels of placental nutrient transporters seen during maternal hypoglycaemia and hyperinsulinemia likely reflect an adaptive response to optimise foetal nutrient supply and development during limited availability of glucose.

Prolonged insulin-induced hypoglycaemia reduces ß-cell activity rather than number in pancreatic islets in non-diabetic rats.

Pancreatic ß-cells have an extraordinary ability to adapt to acute fluctuations in glucose levels by rapid changing insulin production to meet metabolic needs. Although acute changes have been characterised, effects of prolonged metabolic stress on ß-cell dynamics are still unclear. Here, the aim was to investigate pancreatic ß-cell dynamics and function during and after prolonged hypoglycaemia. Hypoglycaemia was induced in male and female rats by infusion of human insulin for 8 weeks, followed by a 4-week infusion-free recovery period. Animals were euthanized after 4 or 8 weeks of infusion, and either 2 days and 4 weeks after infusion-stop. Total volumes of pancreatic islets and ß-cell nuclei, islet insulin and glucagon content, and plasma c-peptide levels were quantified. Prolonged hypoglycaemia reduced c-peptide levels, islet volume and almost depleted islet insulin. Relative ß-cell nuclei: total pancreas volume decreased, while being unchanged relative to islet volume. Glucagon: total pancreas volume decreased during hypoglycaemia, whereas glucagon: islet volume increased. Within two days after infusion-stop, plasma glucose and c-peptide levels normalised and all remaining parameters were fully reversed after 4 weeks. In conclusion, our findings indicate that prolonged hypoglycaemia inactivates ß-cells, which can rapidly be reactivated when needed, demonstrating the high plasticity of ß-cells even following prolonged suppression.

Preclinical safety evaluations supporting pediatric drug development with biopharmaceuticals: strategy, challenges, current practices.

Evaluation of pharmaceutical agents in children is now conducted earlier in the drug development process. An important consideration for this pediatric use is how to assess and support its safety. This article is a collaborative effort of industry toxicologists to review strategies, challenges, and current practice regarding preclinical safety evaluations supporting pediatric drug development with biopharmaceuticals. Biopharmaceuticals include a diverse group of molecular, cell-based or gene therapeutics derived from biological sources or complex biotechnological processes. The principles of preclinical support of pediatric drug development for biopharmaceuticals are similar to those for small molecule pharmaceuticals and in general follow the same regulatory guidances outlined by the Food and Drug Administration and European Medicines Agency. However, many biopharmaceuticals are also inherently different, with limited species specificity or immunogenic potential which may impact the approach taken. This article discusses several key areas to aid in the support of pediatric clinical use, study design considerations for juvenile toxicity studies when they are needed, and current practices to support pediatric drug development based on surveys specifically targeting biopharmaceutical development.

Inner histopathologic changes and disproportionate zone volumes in foetal growth plates following gestational hypoglycaemia in rats.

Maternal hypoglycaemia throughout gestation until gestation day (GD)20 delays foetal growth and skeletal development. While partially prevented by return to normoglycaemia after completed organogenesis (GD17), underlying mechanisms are not fully understood. Here, we investigated the pathogenesis of these changes and significance of maternal hypoglycaemia extending beyond organogenesis in non-diabetic rats. Pregnant rats received insulin-infusion until GD20 or GD17, with sacrifice on GD20. Hypoglycaemia throughout gestation increased maternal corticosterone levels, which correlated with foetal levels. Growth plates displayed central histopathologic changes comprising disrupted cellular organisation, hypertrophic chondrocytes, and decreased cellular density; expression of pro-angiogenic factors, HIF-1α and VEGF-A increased in surrounding areas. Disproportionately decreased growth plate zone volumes and lower expression of the structural protein MATN-3 were seen, while bone ossification parameters were normal. Ending maternal/foetal hypoglycaemia on GD17 reduced incidence and severity of histopathologic changes and with normal growth plate volume. Compromised foetal skeletal development following maternal hypoglycaemia throughout gestation is hypothesised to result from corticosterone-induced hypoxia in growth plates, where hypoxia disrupts chondrocyte maturation and growth plate structure and volume, decreasing long bone growth. Maternal/foetal hypoglycaemia lasting only until GD17 attenuated these changes, suggesting a pivotal role of glucose in growth plate development.

High hydrostatic pressure treatment of porcine oocytes before handmade cloning improves developmental competence and cryosurvival.

An innovative technique, called the high hydrostatic pressure (HHP) treatment, has been recently reported to improve the cryosurvival of gametes or embryos in certain mammalian species. The aim of the present study was to investigate the in vitro and in vivo developmental competence and cryotolerance of embryos produced by handmade cloning (HMC) after pressure treatment of recipient oocytes. In vitro-matured porcine oocytes were treated with a sublethal hydrostatic pressure of 20 MPa (200 times greater than atmospheric pressure) and recovered for either 1 or 2 h (HHP1 and HHP2 groups, respectively) before they were used for HMC. After 7 days of in vitro culture, blastocyst rates and mean cell numbers were determined. Randomly selected blastocysts were vitrified with the Cryotop method based on minimum volume cooling procedure. The blastocyst rate was higher in the HHP2 group than in the control group (68.2 +/- 4.1% vs. 46.4 +/- 4.2%; p < 0.01), while there was no difference between HHP1 and control group (52.1 +/- 1.2% vs. 49.0 +/- 2.7%; p > 0.05). Similar mean cell numbers of produced blastocysts were obtained in HHP2 and control groups (56 +/- 4 vs. 49 +/- 5; p > 0.05). Subsequent blastocyst vitrification with the Cryotop method resulted in significantly higher survival rate after thawing in the HHP2 group than in the control group (61.6 +/- 4.0% vs. 30.2 +/- 30.9%; p < 0.01). Fifty-six and 57 day 5 to day 7 fresh blastocysts in HHP1 group were transferred into two recipient sows on day 5 of the estrous cycle. One recipient was diagnosed pregnant and gave birth to two healthy piglets by naturally delivery on day 122 of gestation. This pilot study proved that the sublethal HHP treatment of porcine oocytes before HMC results in improved in vitro developmental competence and cryotolerance, and supports embryonic and fetal development as well as pregnancy establishment and maintenance up to the birth of healthy piglets.

Effect of maternal hypoglycaemia during gestation on materno-foetal nutrient transfer and embryo-foetal development: Evidence from experimental studies focused primarily on the rat.

Glucose is the major energy substrate during embryogenesis and the embryo is dependent on glucose from the maternal circulation to ensure normal metabolism and growth. The placenta plays a key role in this nutrient transfer in mammals, both during embryogenesis and after the development of the chorio-allantoic placental circulation. Maternal hypoglycaemia is accompanied by foetal hypoglycaemia and maternal counter-regulatory measures including a priority to keep nutrients in the maternal circulation by restricting their transfer to the foetus. Concomitantly, the foetus initiates its own counter-regulatory attempt to secure nutrients for its development and survival. Despite these measures, there is a general decrease in nutrient transfer to the foetus, which may have severe consequences for foetal development such as malformations and delayed skeletal development.

Proximal Neuropathy and Associated Skeletal Muscle Changes Resembling Denervation Atrophy in Hindlimbs of Chronic Hypoglycaemic Rats.

Peripheral neuropathy is one of the most common complications of diabetic hyperglycaemia. Insulin-induced hypoglycaemia (IIH) might potentially exacerbate or contribute to neuropathy as hypoglycaemia also causes peripheral neuropathy. In rats, IIH induces neuropathy associated with skeletal muscle changes. Aims of this study were to investigate the progression and sequence of histopathologic changes caused by chronic IIH in rat peripheral nerves and skeletal muscle, and whether such changes were reversible. Chronic IIH was induced by infusion of human insulin, followed by an infusion-free recovery period in some of the animals. Sciatic, plantar nerves and thigh muscle were examined histopathologically after four or eight weeks of infusion and after the recovery period. IIH resulted in high incidence of axonal degeneration in sciatic nerves and low incidence in plantar nerves indicating proximo-distal progression of the neuropathy. The neuropathy progressed in severity (sciatic nerve) and incidence (sciatic and plantar nerve) with the duration of IIH. The myopathy consisted of groups of angular atrophic myofibres which resembled histopathologic changes classically seen after denervation of skeletal muscle, and severity of the myofibre atrophy correlated with severity of axonal degeneration in sciatic nerve. Both neuropathy and myopathy were still present after four weeks of recovery, although the neuropathy was less severe. In conclusion, the results suggest that peripheral neuropathy induced by IIH progresses proximo-distally, that severity and incidence increase with duration of the hypoglycaemia and that these changes are partially reversible within four weeks. Furthermore, IIH-induced myopathy is most likely secondary to the neuropathy.

Piglets born from vitrified cloned blastocysts produced with a simplified method of delipation and nuclear transfer.

Successful cryopreservation of porcine embryos offers a promising perspective in the fields of agriculture, animal science, and human medical research. The objective of the present work was to establish a system facilitating the cryopreservation of porcine embryos produced by somatic cell nuclear transfer (SCNT). Several key techniques including micromanipulator-based enucleation, noninvasive delipation, zona-free fusion, and activation were combined with high efficiency. After a partial zona digestion and high-speed centrifugation, 89.8+/-2.1% (mean+/-SEM) of enucleated oocytes were successfully delipated. Delipated cytoplasts were incubated for an additional 0.5 or 2 h before fusion with somatic cells. After activation and 6 days of in vitro culture, no significant difference in the rate of blastocysts per reconstructed embryo was observed between the two groups (33.1+/-1.8% and 26.0+/-4.3% for 0.5 and 2 h recovery time, respectively). Cryopreservation of the blastocysts was performed with a Cryotop device and factory-prepared vitrification and warming solutions. One hundred fifty-five vitrified SCNT embryos were transferred surgically into two recipient sows to test their developmental capacity in vivo. One recipient became pregnant and delivered six piglets. In conclusion, our simplified delipation and SCNT procedure resulted in viable piglets after vitrification and embryo transfer at the blastocyst stage.

Chronic Hyperinsulinaemic Hypoglycaemia in Rats Is Accompanied by Increased Body Weight, Hyperleptinaemia, and Decreased Neuronal Glucose Transporter Levels in the Brain.

The brain is vulnerable to hypoglycaemia due to a continuous need of energy substrates to meet its high metabolic demands. Studies have shown that severe acute insulin-induced hypoglycaemia results in oxidative stress in the rat brain, when neuroglycopenia cannot be evaded despite increased levels of cerebral glucose transporters. Compensatory measures in the brain during chronic insulin-induced hypoglycaemia are less well understood. The present study investigated how the brain of nondiabetic rats copes with chronic insulin-induced hypoglycaemia for up to eight weeks. Brain level of different substrate transporters and redox homeostasis was evaluated. Hyperinsulinaemia for 8 weeks consistently lowered blood glucose levels by 30-50% (4-6 mM versus 7-9 mM in controls). The animals had increased food consumption, body weights, and hyperleptinaemia. During infusion, protein levels of the brain neuronal glucose transporter were decreased, whereas levels of lipid peroxidation products were unchanged. Discontinued infusion was followed by transient systemic hyperglycaemia and decreased food consumption and body weight. After 4 weeks, plasma levels of lipid peroxidation products were increased, possibly as a consequence of hyperglycaemia-induced oxidative stress. The present data suggests that chronic moderate hyperinsulinaemic hypoglycaemia causes increased body weight and hyperleptinaemia. This is accompanied by decreased neuronal glucose transporter levels, which may be leptin-induced.

Toxicological Effects during and following Persistent Insulin-Induced Hypoglycaemia in Healthy Euglycaemic Rats.

New insulin analogues with a longer duration of action and a 'peakless' pharmacokinetic profile have been developed to improve efficacy, safety and convenience for patients with diabetes. During non-clinical development, according to regulatory guidelines, these analogues are tested in healthy euglycaemic rats rendering them persistently hypoglycaemic. Little is known about the effect of persistent (24 hr/day) insulin-induced hypoglycaemia (IIH) in rats, complicating interpretation of results in pre-clinical studies with new longer-acting insulin analogues. In this study, we investigated the effects of persistent IIH and their reversibility in euglycaemic rats. Histopathological changes in insulin-infused animals included partly reversible axonal and reversible myofibre degeneration in peripheral nerve and skeletal muscle tissue, respectively, as well as reversible pancreatic islet atrophy and partly reversible increase in unilocular adipocytes in brown adipose tissue. Additionally, results suggested increased gluconeogenesis. The observed hyperphagia, the pancreatic, peripheral nerve and skeletal muscle changes were considered related to the hypoglycaemia. Cessation of insulin infusion resulted in transient hyperglycaemia, decreased food consumption and body-weight loss before returning to control levels. The implications for the interpretation of non-clinical studies with long-acting insulin analogues are discussed.

A method for chronological intravital imaging of bovine oocytes during in vitro maturation.

Oocyte maturation is known to affect the chances for successful fertilization, embryonic development, establishment of pregnancy and delivery of a live, healthy, and viable offspring. Two-photon laser scanning microscopy (TPLSM) has previously been used to evaluate early embryonic development without a detectable impairment of subsequent development, but has never been applied to assess mammalian oocytes throughout in vitro maturation (IVM). Visualization of structures within live oocytes during IVM, followed by fertilization and embryo culture, may improve the understanding of oocyte maturation. To visualize structures within bovine oocytes using TPLSM, it is necessary to remove the cumulus cells that normally surround the oocyte during maturation. Repeated visualization of structures within the same oocyte is possible, if movement of the oocyte can be avoided. In this article, we describe the development of a method for repeated intravital imaging of denuded bovine oocytes using an upright TPLSM equipped with a specially constructed incubator. Oocytes were stained with Hoechst 33258, and the nuclear structures were evaluated. Oocyte fertilization rate was not affected by TPLSM exposure, but the developmental capacity of the denuded oocytes was significantly reduced. This is, to our knowledge, the first article describing repeated intravital imaging during mammalian oocyte maturation using TPLSM.

Comparative evaluation of nuclear morphology of equine oocytes aspirated in vivo and stained with Hoechst and orcein.

Nuclear maturation of equine oocytes was assessed immediately after in vivo collection. A double-staining technique (Hoechst and orcein) was used on the same oocytes to visualize nuclear morphology, i.e. to evaluate the chromatin configurations of each oocyte after Hoechst in relation to the nuclear morphology after orcein staining. The proportion of oocytes evaluated as germinal vesicle stages was significantly (p < 0.02) lower after Hoechst (14.5%) than after orcein staining (29.0%), while the incidence of the so-called dense chromatin stage was assessed to be higher (p < 0.05) after Hoechst than after orcein staining (14.5 vs. 6.5%). There was no difference between Hoechst and orcein staining in the incidence of diakinesis and germinal vesicle breakdown stages, respectively (44.9 vs. 42.0%), and the same applied for metaphase I (11.6 vs. 8.0%), metaphase II (7.2 vs. 8.0%) and degenerated stages (7.2 vs. 6.5%). It was concluded that the interpretation of the meiotic stages may differ between Hoechst and orcein staining and in a large proportion of equine oocytes the nuclear border may not be visualized on orcein staining.