Emma Carter

Kinetics, safety and tolerability of (R)-3-hydroxybutyl (R)-3-hydroxybutyrate in healthy adult subjects.

Induction of mild states of hyperketonemia may improve physical and cognitive performance. In this study, we determined the kinetic parameters, safety and tolerability of (R)-3-hydroxybutyl (R)-3-hydroxybutyrate, a ketone monoester administered in the form of a meal replacement drink to healthy human volunteers. Plasma levels of β-hydroxybutyrate and acetoacetate were elevated following administration of a single dose of the ketone monoester, whether at 140, 357, or 714 mg/kg body weight, while the intact ester was not detected. Maximum plasma levels of ketones were attained within 1-2h, reaching 3.30 mM and 1.19 mM for β-hydroxybutyrate and acetoacetate, respectively, at the highest dose tested. The elimination half-life ranged from 0.8-3.1h for β-hydroxybutyrate and 8-14 h for acetoacetate. The ketone monoester was also administered at 140, 357, and 714 mg/kg body weight, three times daily, over 5 days (equivalent to 0.42, 1.07, and 2.14 g/kg/d). The ketone ester was generally well-tolerated, although some gastrointestinal effects were reported, when large volumes of milk-based drink were consumed, at the highest ketone monoester dose. Together, these results suggest ingestion of (R)-3-hydroxybutyl (R)-3-hydroxybutyrate is a safe and simple method to elevate blood ketone levels, compared with the inconvenience of preparing and consuming a ketogenic diet.

Fundus Autofluorescence in the Abca4(-/-) Mouse Model of Stargardt Disease-Correlation With Accumulation of A2E, Retinal Function, and Histology

PURPOSE To investigate fundus autofluorescence (AF) characteristics in the Abca4(-/-) mouse, an animal model for AMD and Stargardt disease, and to correlate findings with functional, structural, and biochemical assessments. METHODS Blue (488 nm) and near-infrared (790 nm) fundus AF images were quantitatively and qualitatively analyzed in pigmented Abca4(-/-) mice and wild type (WT) controls in vivo. Functional, structural, and biochemical assessments included electroretinography (ERG), light and electron microscopic analysis, and A2E quantification. All assessments were performed across age groups. RESULTS In Abca4(-/-) mice, lipofuscin-related 488 nm AF increased early in life with a ceiling effect after 6 months. This increase was first paralleled by an accumulation of typical lipofuscin granules in the retinal pigment epithelium (RPE). Later, lipofuscin and melanin granules decreased in number, whereas melanolipofuscin granules increased. This increase in melanolipofuscin granules paralleled an increase in melanin-related 790 nm AF. Old Abca4(-/-) mice revealed a flecked fundus AF pattern at both excitation wavelengths. The amount of A2E, a major lipofuscin component, increased 10- to 12-fold in 6- to 9-month-old Abca4(-/-) mice compared with controls, while 488 nm AF intensity only increased 2-fold. Despite pronounced lipofuscin accumulation in the RPE of Abca4(-/-) mice, ERG and histology showed a slow age-related thinning of the photoreceptor layer similar to WT controls up to 12 months. CONCLUSIONS Fundus AF can be used to monitor lipofuscin accumulation and melanin-related changes in vivo in mouse models of retinal disease. High RPE lipofuscin may not adversely affect retinal structure or function over prolonged time intervals, and melanin-related changes (melanolipofuscin formation) may occur before the decline in retinal function.

Novel ketone diet enhances physical and cognitive performance

Ketone bodies are the most energy‐efficient fuel and yield more ATP permole of substrate than pyruvate and increase the free energy released from ATP hydrolysis. Elevation of circulating ketones via high‐fat, low carbohydrate diets has been used for the treatment of drug‐refractory epilepsy and for neuro degenerative diseases, such as Parkinsons disease. Ketones may also be beneficial for muscle and brain in times of stress, such as endurance exercise. The challenge has been to raise circulating ketone levels by using a palatable diet without altering lipid levels. We found that blood ketone levels can be increased and cholesterol and triglycerides decreased by feeding rats a novel ketone ester diet: chow that is supplemented with (R)‐3‐hydroxybutyl (R)‐3‐hydroxybutyrate as 30% of calories. For 5 d, rats on the ketone diet ran 32% further on a tread mill than did control rats that ate an isocaloric diet that was supplemented with either corn starch or palmoil (P < 0.05). Ketone‐fed rats completed an 8‐ arm radial maze test 38% faster than did those on the other diets, making more correct decisions before making a mistake (P < 0.05). Isolated, perfused hearts fromrats that were fed the ketone diet had greater free energy available from ATP hydrolysis during increased work than did hearts from rats on the other diets as shown by using [31P]‐ NMRspectroscopy. The novelketone diet, therefore, improved physical performance and cognitive function in rats, and its energy‐sparing properties suggest that it may help to treat a range of human conditions with metabolic abnormalities.—Murray, A. J., Knight, N. S., Cole, M. A., Cochlin, L. E., Carter, E., Tchabanenko, K., Pichulik, T., Gulston, M.K., Atherton, H. J., Schroeder, M.A., Deacon, R.M. J., Kashiwaya, Y., King, M.T., Pawlosky, R., Rawlins, J. N. P., Tyler, D. J., Griffin, J. L., Robertson, J., Veech, R. L., Clarke, K. Novel ketone diet enhances physical and cognitive performance. FASEB J. 30, 4021–4032 (2016). www.fasebj.org

Oral 28-day and developmental toxicity studies of (R)-3-hydroxybutyl (R)-3-hydroxybutyrate

(R)-3-Hydroxybutyl (R)-3-hydroxybutyrate (ketone monoester) has been developed as an oral source of ketones, which may be utilized for energy. In a 28-day toxicity study, Crl:WI (Wistar) rats received diets containing, as 30% of the calories, ketone monoester (12 and 15 g/kg body weight/day for male and female rats, respectively). Control groups received either carbohydrate- or fat-based diets. Rats in the test group consumed less feed and gained less weight than control animals; similar findings have been documented in studies of ketogenic diets. Between-group differences were noted in selected hematology, coagulation, and serum chemistry parameters; however, values were within normal physiological ranges and/or were not accompanied by other changes indicative of toxicity. Upon gross and microscopic evaluation, there were no findings associated with the ketone monoester. In a developmental toxicity study, pregnant Crl:WI (Han) rats were administered 2g/kg body weight/day ketone monoester or water (control) via gavage on days 6 through 20 of gestation. No Caesarean-sectioning or litter parameters were affected by the test article. The overall incidence of fetal alterations was higher in the test group; however, there were no specific alterations attributable to the test substance. The results of these studies support the safety of ketone monoester.

The cardiac sympathetic co-transmitter galanin reduces acetylcholine release and vagal bradycardia: Implications for neural control of cardiac excitability

The autonomic phenotype of congestive cardiac failure is characterised by high sympathetic drive and impaired vagal tone, which are independent predictors of mortality. We hypothesize that impaired bradycardia to peripheral vagal stimulation following high-level sympathetic drive is due to sympatho-vagal crosstalk by the adrenergic co-transmitters galanin and neuropeptide-Y (NPY). Moreover we hypothesize that galanin acts similarly to NPY by reducing vagal acetylcholine release via a receptor mediated, protein kinase-dependent pathway. Prolonged right stellate ganglion stimulation (10 Hz, 2 min, in the presence of 10 μM metoprolol) in an isolated guinea pig atrial preparation with dual autonomic innervation leads to a significant (p < 0.05) reduction in the magnitude of vagal bradycardia (5 Hz) maintained over the subsequent 20 min (n = 6). Immunohistochemistry demonstrated the presence of galanin in a small number of tyrosine hydroxylase positive neurons from freshly dissected stellate ganglion tissue sections. Following 3 days of tissue culture however, most stellate neurons expressed galanin. Stellate stimulation caused the release of low levels of galanin and significantly higher levels of NPY into the surrounding perfusate (n = 6, using ELISA). The reduction in vagal bradycardia post sympathetic stimulation was partially reversed by the galanin receptor antagonist M40 after 10 min (1 μM, n = 5), and completely reversed with the NPY Y2 receptor antagonist BIIE 0246 at all time points (1 μM, n = 6). Exogenous galanin (n = 6, 50–500 nM) also reduced the heart rate response to vagal stimulation but had no effect on the response to carbamylcholine that produced similar degrees of bradycardia (n = 6). Galanin (500 nM) also significantly attenuated the release of 3H-acetylcholine from isolated atria during field stimulation (5 Hz, n = 5). The effect of galanin on vagal bradycardia could be abolished by the galanin receptor antagonist M40 (n = 5). Importantly the GalR1 receptor was immunofluorescently co-localised with choline acetyl-transferase containing neurons at the sinoatrial node. The protein kinase C inhibitor calphostin (100 nM, n = 6) abolished the effect of galanin on vagal bradycardia whilst the protein kinase A inhibitor H89 (500 nM, n = 6) had no effect. These results demonstrate that prolonged sympathetic activation releases the slowly diffusing adrenergic co-transmitter galanin in addition to NPY, and that this contributes to the attenuation in vagal bradycardia via a reduction in acetylcholine release. This effect is mediated by GalR1 receptors on vagal neurons coupled to protein kinase C dependent signalling pathways. The role of galanin may become more important following an acute injury response where galanin expression is increased.

Igf2 pathway dependency of the Trp53 developmental and tumour phenotypes

Insulin‐like growth factor 2 (IGF2) and the transformation related protein 53 (Trp53) are potent regulators of cell growth and metabolism in development and cancer. In vitro evidence suggests several mechanistic pathway interactions. Here, we tested whether loss of function of p53 leads to IGF2 ligand pathway dependency in vivo. Developmental lethality occurred in p53 homozygote null mice that lacked the paternal expressed allele of imprinted Igf2. Further lethality due to post‐natal lung haemorrhage occurred in female progeny with Igf2 paternal null allele only if derived from double heterozygote null fathers, and was associated with a specific gene expression signature. Conditional deletion of Igf2fl/fl attenuated the rapid tumour onset promoted by homozygous deletion of p53fl/fl. Accelerated carcinoma and sarcoma tumour formation in p53+/− females with bi‐allelic Igf2 expression was associated with reductions in p53 loss of heterozygosity and apoptosis. Igf2 genetic dependency of the p53 null phenotype during development and tumour formation suggests that targeting the IGF2 pathway may be useful in the prevention and treatment of human tumours with a disrupted Trp53 pathway.

Maternal Transmission of a Humanised Igf2r Allele Results in an Igf2 Dependent Hypomorphic and Non-Viable Growth Phenotype

The cation independent mannose 6-phosphate/insulin-like growth factor 2 receptor (IGF2R) functions in the transportation and regulation of insulin-like growth factor 2 (IGF2) and mannose 6-phosphate modified proteins. The relative and specific titration of IGF2 by high affinity binding of IGF2R represents a mechanism that supports the parental conflict theory of genomic imprinting. Imprinting of Igf2 (paternal allele expressed) and Igf2r (maternal allele expressed) arose to regulate the relative supply of both proteins. Experiments in the mouse have established that loss of the maternal allele of Igf2r results in disproportionate growth and peri-natal lethality. In order to systematically investigate the consequences of loss of function and of hypomorphic alleles of Igf2r on growth functions, we introduced a conditional human IGF2R exon 3–48 cDNA into the intron 2 region of murine Igf2r. Here we show that the knock-in construct resulted in over-growth when the humanised Igf2r allele was maternally transmitted, a phenotype that was rescued by either paternal transmission of the humanised allele, expression of a wild-type paternal allele or loss of function of Igf2. We also show that expression of IGF2R protein was reduced to less than 50% overall in tissues previously known to be Igf2 growth dependent. This occurred despite the detection of mouse derived peptides, suggesting that trans-splicing of the knock-in human cDNA with the endogenous maternal mouse Igf2r allele. The phenotype following maternal transmission of the humanised allele resulted in overgrowth of the embryo, heart and placenta with partial peri-natal lethality, suggesting that further generation of hypomorphic Igf2r alleles are likely to be at the borderline of maintaining Igf2 dependent viability.

Epithelial-mesenchymal transition and nuclear β-catenin induced by conditional intestinal disruption of Cdh1 with Apc is E-cadherin EC1 domain dependent

Two important protein-protein interactions establish E-cadherin (Cdh1) in the adhesion complex; homophilic binding via the extra-cellular (EC1) domain and cytoplasmic tail binding to β-catenin. Here, we evaluate whether E-cadherin binding can inhibit β-catenin when there is loss of Adenomatous polyposis coli (APC) from the β-catenin destruction complex. Combined conditional loss of Cdh1 and Apc were generated in the intestine, intestinal adenoma and adenoma organoids. Combined intestinal disruption (Cdh1fl/flApcfl/flVil-CreERT2) resulted in lethality, breakdown of the intestinal barrier, increased Wnt target gene expression and increased nuclear β-catenin localization, suggesting that E-cadherin inhibits β-catenin. Combination with an intestinal stem cell Cre (Lgr5CreERT2) resulted in ApcΔ/Δ recombination and adenoma, but intact Cdh1fl/fl alleles. Cultured ApcΔ/ΔCdh1fl/fl adenoma cells infected with adenovirus-Cre induced Cdh1fl/fl recombination (Cdh1Δ/Δ), disruption of organoid morphology, nuclear β-catenin localization, and cells with an epithelial-mesenchymal phenotype. Complementation with adenovirus expressing wild-type Cdh1 (Cdh1-WT) rescued adhesion and β-catenin membrane localization, yet an EC1 specific double mutant defective in homophilic adhesion (Cdh1-MutW2A, S78W) did not. These data suggest that E-cadherin inhibits β-catenin in the context of disruption of the APC-destruction complex, and that this function is also EC1 domain dependent. Both binding functions of E-cadherin may be required for its tumour suppressor activity.