Selected Amino Acids Promote Mouse Pre-implantation Embryo Development in a Growth Factor-Like Manner.

Groups of amino acids, and some selected amino acids, added to media used for culture of pre-implantation embryos have previously been shown to improve development in various ways including survival to the blastocyst stage, increased blastocyst cell number and improved hatching. In this study, we cultured 1-cell mouse embryos for 5 days to the hatching blastocyst stage in isosmotic medium (270 mOsm/kg) at high density (10 embryos/10 µL), where autocrine/paracrine support of development occurs, and low density (1 embryo/100 µL), where autocrine/paracrine support is minimized and development is compromised. When 400 µM L-Pro or 1 mM L-Gln was added to embryos at low density, the percentage of embryos reaching the blastocyst stage and the percentage hatching increased compared to low-density culture without these amino acids, and were now similar to those for embryos cultured at high density without amino acids. When L-Pro or L-Gln was added to embryos at high density, the percentage of embryos reaching the blastocyst stage didn't change but hatching improved. Neither embryo culture density nor the presence of these amino acids had any effect on blastocyst cell number. D-Pro and the osmolytes Gly and Betaine did not improve embryo development in low- or high-density culture indicating the mechanism was stereospecific and not osmotic, respectively. L-Pro- and L-Gln-mediated improvement in development is observed from the 5-cell stage and persists to the blastocyst stage. Molar excess of Gly, Betaine or L-Leu over L-Pro eliminated improvement in development and hatching consistent with them acting as competitive inhibitors of transporter-mediated uptake across the plasma membrane. The L-Pro effect is dependent on mTORC1 signaling (rapamycin sensitive) while that for L-Gln is not. The addition of L-Pro leads to significant nuclear translocation of p-AktS473 at the 2- and 4-cell stages and of p-ERK1/2T202/Y204 nuclear translocation at the 2-, 4-, and 8-cell stages. L-Pro improvement in embryo development involves mechanisms analogous to those seen with Pro-mediated differentiation of mouse ES cells, which is also stereoselective, dependent on transporter uptake, and activates Akt, ERK, and mTORC1 signaling pathways.

European regulation on orphan medicinal products: 10 years of experience and future perspectives.

In 2000, regulation on orphan medicinal products was adopted in the European Union with the aim of benefiting patients who suffer from serious, rare conditions for which there is currently no satisfactory treatment. Since then, more than 850 orphan drug designations have been granted by the European Commission based on a positive opinion from the Committee for Orphan Medicinal Products (COMP), and more than 60 orphan drugs have received marketing authorization in Europe. Here, stimulated by the tenth anniversary of the COMP, we reflect on the outcomes and experience gained in the past decade, and contemplate issues for the future, such as catalysing drug development for the large number of rare diseases that still lack effective treatments.

Lysophosphatidic acid induces Ca2+ mobilization and c-Myc expression in mouse embryonic stem cells via the phospholipase C pathway.

Embryonic stem cells (ESC) are pluripotent and could be maintained in vitro in a self-renewing state indefinitely, at the same time preserving their potential to differentiate towards more specific lineages. Despite the progress in the field, the complex network of signalling cascades involved in the maintenance of the self-renewing and pluripotent state remains not fully understood. In the present study, we have investigated the role of lysophosphatidic acid (LPA), a potent mitogen present in serum, in Ca(2+) signalling and early gene activation in mouse ESC (mESC). In these cells, we detected the expression of the G-protein coupled LPA receptor subtypes LPA(1), LPA(2) and LPA(3). Using fluorescence Ca(2+) imaging techniques, we showed that LPA induced an increase in intracellular Ca(2+) concentration. This increase was also observed in the absence of extracellular Ca(2+), suggesting the involvement of internal stores. Pre-treatment with BAPTA-AM, thapsigargin or U-73122 efficiently blocked this Ca(2+) release, indicating that LPA was evoking Ca(2+) mobilization from the endoplasmic reticulum via the phospholipase C (PLC) pathway. Interestingly, this signalling cascade initiated by LPA was involved in inducing the expression of the Ca(2+)-dependent early response gene c-myc, a key gene implicated in ESC self-renewal and pluripotency. Additionally, LPA increased the proliferation rate of mESC. Our findings therefore outline the physiological role of LPA in mESC.

Gap junctional intercellular communication is required to maintain embryonic stem cells in a non-differentiated and proliferative state.

Pluripotent embryonic stem (ES) cells are capable of maintaining a self-renewal state and have the potential to differentiate into derivatives of all three embryonic germ layers. Despite their importance in cell therapy and developmental biology, the mechanisms whereby ES cells remain in a proliferative and pluripotent state are still not fully understood. Here we establish a critical role of gap junctional intercellular communication (GJIC) and connexin43 (Cx43) in both processes. Pharmacological blockers of GJIC and Cx43 down-regulation by small interfering RNA (siRNA) caused a profound inhibitory effect on GJIC, as evidenced by experiments of fluorescence recovery after photobleaching. This deficient intercellular communication in ES cells induced a loss of their pluripotent state, which was manifested in morphological changes, a decrease in alkaline phosphatase activity, Oct-3/4 and Nanog expression, as well as an up-regulation of several differentiation markers. A decrease in the proliferation rate was also detected. Under these conditions, the formation of embryoid bodies from mouse ES cells was impaired, although this inhibition was reversible upon restoration of GJIC. Our findings define a major function of GJIC in the regulation of self-renewal and maintenance of pluripotency in ES cells.

Glucose induces opposite intracellular Ca2+ concentration oscillatory patterns in identified alpha- and beta-cells within intact human islets of Langerhans.

Homeostasis of blood glucose is mainly regulated by the coordinated secretion of glucagon and insulin from alpha- and beta-cells within the islets of Langerhans. The release of both hormones is Ca(2+) dependent. In the current study, we used confocal microscopy and immunocytochemistry to unequivocally characterize the glucose-induced Ca(2+) signals in alpha- and beta-cells within intact human islets. Extracellular glucose stimulation induced an opposite response in these two cell types. Although the intracellular Ca(2+) concentration ([Ca(2+)](i)) in beta-cells remained stable at low glucose concentrations, alpha-cells exhibited an oscillatory [Ca(2+)](i) response. Conversely, the elevation of extracellular glucose elicited an oscillatory [Ca(2+)](i) pattern in beta-cells but inhibited low-glucose-induced [Ca(2+)](i) signals in alpha-cells. These Ca(2+) signals were synchronic among beta-cells grouped in clusters within the islet, although they were not coordinated among the whole beta-cell population. The response of alpha-cells was totally asynchronic. Therefore, both the alpha- and beta-cell populations within human islets did not work as a syncitium in response to glucose. A deeper knowledge of alpha- and beta-cell behavior within intact human islets is important to better understand the physiology of the human endocrine pancreas and may be useful to select high-quality islets for transplantation.

Different metabolic responses in alpha-, beta-, and delta-cells of the islet of Langerhans monitored by redox confocal microscopy.

Blood glucose homeostasis is mainly achieved by the coordinated function of pancreatic alpha-, beta-, and delta-cells, which secrete glucagon, insulin, and somatostatin, respectively. Each cell type responds to glucose changes with different secretion patterns. Currently, considerable information can be found about the signal transduction mechanisms that lead to glucose-mediated insulin release in the pancreatic beta-cell, mitochondrial activation being an essential step. Increases in glucose stimulate the mitochondrial metabolism, activating the tricarboxylic acid cycle and raising the source of redox electron carrier molecules needed for respiratory ATP synthesis. However, little is known about the glucose-induced mitochondrial response of non-beta-cells and its role in the stimulus-secretion coupling process. This limited information is probably a result of the scarcity of these cells in the islet, the lack of identification patterns, and the technical limitations of conventional methods. In this study, we used flavin adenine dinucleotide redox confocal microscopy as a noninvasive technique to specifically monitor mitochondrial redox responses in immunoidentified alpha-, beta-, and delta-cells in freshly isolated intact islets and in dispersed cultured cells. We have shown that glucose provokes metabolic changes in beta- and delta-cell populations in a dose-dependent manner. Conversely, no significant responses were observed in alpha-cells, despite the sensitivity of their metabolism to drugs acting on the mitochondrial function, and their intact ability to develop Ca2+ signals. Identical results were obtained in islets and in cultures of dispersed cells. Our findings indicate metabolic differences in glucose utilization among the alpha-, beta-, and delta-cell populations, which might be important in the signal transduction events that lead to hormone release.

Management of multifactorial idiopathic epilepsy in EL mice with caloric restriction and the ketogenic diet: role of glucose and ketone bodies.

BACKGROUND: The high fat, low carbohydrate ketogenic diet (KD) was developed as an alternative to fasting for seizure management. While the mechanisms by which fasting and the KD inhibit seizures remain speculative, alterations in brain energy metabolism are likely involved. We previously showed that caloric restriction (CR) inhibits seizure susceptibility by reducing blood glucose in the epileptic EL mouse, a natural model for human multifactorial idiopathic epilepsy. In this study, we compared the antiepileptic and anticonvulsant efficacy of the KD with that of CR in adult EL mice with active epilepsy. EL mice that experienced at least 15 recurrent complex partial seizures were fed either a standard diet unrestricted (SD-UR) or restricted (SD-R), and either a KD unrestricted (KD-UR) or restricted (KD-R). All mice were fasted for 14 hrs prior to diet initiation. A new experimental design was used where each mouse in the diet-restricted groups served as its own control to achieve a 20-23% body weight reduction. Seizure susceptibility, body weights, and the levels of plasma glucose and beta-hydroxybutyrate were measured once/week over a nine-week treatment period. RESULTS: Body weights and blood glucose levels remained high over the testing period in the SD-UR and the KD-UR groups, but were significantly (p < 0.001) reduced in the SD-R and KD-R groups. Plasma beta-hydroxybutyrate levels were significantly (p < 0.001) increased in the SD-R and KD-R groups compared to their respective UR groups. Seizure susceptibility remained high in both UR-fed groups throughout the study, but was significantly reduced after three weeks in both R-fed groups. CONCLUSIONS: The results indicate that seizure susceptibility in EL mice is dependent on plasma glucose levels and that seizure control is more associated with the amount than with the origin of dietary calories. Also, CR underlies the antiepileptic and anticonvulsant action of the KD in EL mice. A transition from glucose to ketone bodies for energy is predicted to manage EL epileptic seizures through multiple integrated changes of inhibitory and excitatory neural systems.

Perspectives on the metabolic management of epilepsy through dietary reduction of glucose and elevation of ketone bodies.

Brain cells are metabolically flexible because they can derive energy from both glucose and ketone bodies (acetoacetate and beta-hydroxybutyrate). Metabolic control theory applies principles of bioenergetics and genome flexibility to the management of complex phenotypic traits. Epilepsy is a complex brain disorder involving excessive, synchronous, abnormal electrical firing patterns of neurons. We propose that many epilepsies with varied etiologies may ultimately involve disruptions of brain energy homeostasis and are potentially manageable through principles of metabolic control theory. This control involves moderate shifts in the availability of brain energy metabolites (glucose and ketone bodies) that alter energy metabolism through glycolysis and the tricarboxylic acid cycle, respectively. These shifts produce adjustments in gene-linked metabolic networks that manage or control the seizure disorder despite the continued presence of the inherited or acquired factors responsible for the epilepsy. This hypothesis is supported by information on the management of seizures with diets including fasting, the ketogenic diet and caloric restriction. A better understanding of the compensatory genetic and neurochemical networks of brain energy metabolism may produce novel antiepileptic therapies that are more effective and biologically friendly than those currently available.

Sexual dysfunction and sudden death in epileptic male EL mice: inheritance and prevention with the ketogenic diet.

PURPOSE: The EL mouse is an animal model for multifactorial idiopathic epilepsy. Although EL mice have been studied extensively for >45 years, the etiology of male sudden death and its relation to seizures have not been defined. Here we investigated the cause of EL male sudden death and its relation to epilepsy. METHODS: For histopathologic analysis, the terminally ill EL mice (n = 15) were killed, and the tissues were fixed. Blood chemical composition was compared between the terminally ill EL (n = 9) and the healthy age-matched EL (n = 17) and DDY (n = 11) males. To determine the effect of the ketogenic diet (KD) on sudden male death, young male EL mice (P30) were randomly separated into two groups that were fed ad libitum with either Agway lab chow (control n = 38) or with the KD (treated, n = 39) for 5 months. The genetic predisposition to sudden death was analyzed in the backcross generation (n = 106) of a cross between EL and the nonepileptic ABP strains. RESULTS: Sudden death coincided with the onset of seizures (70-80 days) and affected 94% of male EL mice by age 300 days. Urethral plugs were observed histologically in 13 of 15 longitudinally sectioned penises. Concentrations of blood urea nitrogen, creatinine, phosphorus, and calcium in the terminally ill mice were significantly elevated when compared with those of healthy animals. None of the mice treated with the KD experienced sudden death, whereas 15 (39%) of the untreated control mice died by age 5 months. The sudden death in male EL mice was inherited as an autosomal recessive sex-limited lethal trait. CONCLUSIONS: The cause of sudden death in male EL mice arises from abnormal ejaculation, which produces a urethral plug with consequent urinary retention and acute severe uremia. The coincident onset of seizures and sudden death in EL males suggests that a sexual dysfunction is associated with epilepsy in this model.