Concentration-Dependent Effects of a Dietary Ketone Ester on Components of Energy Balance in Mice

Exogenous ketones may provide therapeutic benefit in treatment of obesity. Administration of the ketone ester (KE) R,S-1,3-butanediol acetoacetate diester (BD-AcAc) decreases body weight in mice, but effects on energy balance have not been extensively characterized. The purpose of this investigation was to explore concentration-dependent effects of BD-AcAc on energy intake and expenditure in mice. Forty-two male C57BL/6J mice were randomly assigned to one of seven isocaloric diets ( = 6 per group): (1) Control (CON, 0% KE by kcals); (2) KE5 (5% KE); (3) KE10 (10% KE); (4) KE15 (15% KE); (5) KE20 (20% KE); (6) KE25 (25% KE); and (7) KE30 (30% KE) for 3 weeks. Energy intake and body weight were measured daily. Fat mass (FM), lean body mass (LBM), and energy expenditure (EE) were measured at completion of the study. Differences among groups were compared to CON using ANOVA and ANCOVA. Mean energy intake was similar between CON and each concentration of KE, except KE30 which was 12% lower than CON ( \u3c 0.01). KE25 and KE30 had lower body weight and FM compared to CON, while only KE30 had lower LBM ( \u3c 0.03). Adjusted resting and total EE were lower in KE30 compared to CON ( \u3c 0.03), but similar for all other groups. A diet comprised of 30% energy from BD-AcAc results in lower energy intake, coincident with lower body weight and whole animal adiposity; while KE20 and KE25 have significantly lower body weight and adiposity effects independent of changes in energy intake or expenditure

Atomic Force Microscopy (AFM) Analysis of Oxidative Stress in Human U87 Glioblastoma Cells Treated with Hyperoxia

Atomic Force Microscopy (AFM) has been used to image membrane surface topography at nanometer resolution. In this study we used the AFM to characterize the physical changes resulting from hyperoxia on cultured human glioblastoma cells (U87). U87 cells were exposed to air (21% O2), hyperoxia (95% O2) or hyperbaric oxygen (HBO2, 3.5ata O2) for 30 min. H2O2 (200µM) was used as a positive control. Following treatment, the cells were fixed with 2% glutaraldehyde for 20 min prior to AFM imaging. Three dimensional surface plots of U87 cells revealed a significant distortion of the plasma membrane (membrane blebbing) caused by HBO2 and H2O2, which is thought to be indicative of lipid peroxidation. Individual cells from each group were analyzed to assess Mean Roughness (Ra) and Maximum Roughness (Rmax). Ra was 40 ± 4 nm in 21% O2, 52 ± 5 nm in 95%O2, 64 ± 7 nm in HBO2, and 91 ± 9 nm in H2O2 treated cells. Rmaxwas 253 ± 27 nm in 21% O2, 302 ± 30 nm in 95% O2, 366 ± 35 nm in HBO2, and 608 ± 39 nm in H2O2 treated cells. In conclusion, these data show that oxidative damage in the plasma membrane is proportional to oxygen concentration. Moreover, the AFM is capable of characterizing subtle changes in membrane topography from oxidative damage

Atomic Force Microscopy (AFM) Analysis of Lipid Peroxidation Following Hyperoxia and Hydrogen Peroxide Treatment in Human U87 Glioblastoma Cells

The atomic force microscope (AFM) is capable of resolving the plasma membrane with nanometer resolution. In this study we used the AFM to characterize hyperoxia-induced oxidative damage to changes in the plasma membranes of cultured human glioma cells (U87). U87 cells were exposed to 0.20 ATA O2, normobaric hyperoxia (0.95 ATA O2) or hyperbaric hyperoxia (HBO2, 3.25 ATA O2) for 30 min. In separate experiments H2O2 (200 µM and 2 mM) was used as a positive control. Malondialdehyde (MDA) was measured to confirm lipid peroxidation. Following treatment, the cells were fixed with 2% glutaraldehyde and scanned in air or fluid. Individual cells from each group (n = 35 to 45 cells/group) were scanned and analyzed to assess average membrane roughness (Ra). The Ra of the plasma membrane was 34 ± 3 nm, 57 ± 3 nm and 63 ± 5 nm in 0.20 ATA O2, 0.95 ATA O2 and HBO2, respectively. In H2O2 treated cells Ra was 28 ± 4 nm, 56 ± 7 nm and 138 ± 14 nm in air (air in 5% CO2), 200 µM and 2 mM H2O2. Co-treatment with antioxidant Trolox C (150 µM) significantly reduced Raduring exposure to hyperoxia and H2O2, suggesting that the amount of membrane blebbing was proportional to the level of oxidative stress. Furthermore, measurement of MDA confirmed that H2O2 and hyperoxia increased lipid peroxidation, suggesting that membrane blebbing is related to oxidative stress. In conclusion, these data demonstrate oxidative damage from lipid peroxidation increases with oxygen concentration

Acute Hyperoxia Increases Lipid Peroxidation and Induces Plasma Membrane Blebbing in Human U87 Glioblastoma Cells

Atomic force microscopy (AFM), malondialdehyde (MDA) assays, and amperometric measurements of extracellular hydrogen peroxide (H2O2) were used to test the hypothesis that graded hyperoxia induces measurable nanoscopic changes in membrane ultrastructure and membrane lipid peroxidation (MLP) in cultured U87 human glioma cells. U87 cells were exposed to 0.20 atmospheres absolute (ATA) O2, normobaric hyperoxia (0.95 ATA O2) or hyperbaric hyperoxia (HBO2, 3.25 ATA O2) for 60 min. H2O2 (0.2 or 2 mM; 60 min) was used as a positive control for MLP. Cells were fixed with 2% glutaraldehyde immediately after treatment and scanned with AFM in air or fluid. Surface topography revealed ultrastructural changes such as membrane blebbing in cells treated with hyperoxia and H2O2. Average membrane roughness (Ra) of individual cells from each group (n=35 to 45 cells/group) was quantified to assess ultrastructural changes from oxidative stress. The Ra of the plasma membrane was 34±3, 57±3 and 63±5 nm in 0.20 ATA O2, 0.95 ATA O2 and HBO2, respectively. Ra was 56±7 and 138±14 nm in 0.2 and 2 mM H2O2. Similarly, levels of MDA were significantly elevated in cultures treated with hyperoxia and H2O2 and correlated with O2-induced membrane blebbing (r2=0.93). Coapplication of antioxidant, Trolox-C (150 μM), significantly reduced membrane Ra and MDA levels during hyperoxia. Hyperoxia-induced H2O2 production increased 189%±5% (0.95 ATA O2) and 236%±5% (4 ATA O2) above control (0.20 ATA O2). We conclude that MLP and membrane blebbing increase with increasing O2 concentration. We hypothesize that membrane blebbing is an ultrastructural correlate of MLP resulting from hyperoxia. Furthermore, AFM is a powerful technique for resolving nanoscopic changes in the plasma membrane that result from oxidative damage

Ketone esters for treatment of angelman syndrome

The invention concerns a method of treating Angelman Syndrome (AS) in a subject, comprising inducing ketosis in the subject by administering a therapeutically effective amount of a ketone ester, such as an R,S-1,3-butanediol acetoacetate ester, wherein administration of the ketone ester elevates the blood ketone level in the subject. Other aspects of the invention include a method of increasing cognitive function and/or motor function in a subject with AS; and a method of decreasing seizures and increasing the latency to seize in a subject with AS

Superoxide (·O2−) Production in CA1 Neurons of Rat Hippocampal Slices Exposed to Graded Levels of Oxygen

Neuronal signaling, plasticity, and pathologies in CA1 hippocampal neurons are all intimately related to the redox environment and, thus tissue oxygenation. This study tests the hypothesis that hyperoxic superfusate (95% O2) causes a time-dependent increase in superoxide anion (·O2−) production in CA1 neurons in slices, which will decrease as oxygen concentration is decreased. Hippocampal slices (400 μm) from weaned rats were incubated with the fluorescent probe dihydroethidium (DHE), which detects intracellular ·O2− production. Slices were loaded for 30 min using 10 μM DHE and maintained using one-sided superfusion or continuously loaded using 2.5 μM DHE and maintained using two-sided superfusion (36°C). Continuous loading of DHE and two-sided superfusion gave the highest temporal resolution measurements of ·O2− production, which was estimated by the increase in fluorescence intensity units (FIUs) per minute (FIU/min ± SE) over 4 h. Superoxide production (2.5 μM DHE, 2-sided superfusion) was greatest in 95% O2 (6.6 ± 0.4 FIU/min) and decreased significantly during co-exposure with antioxidants (100 μM melatonin, 25 μM MnTMPyP) and lower levels of O2 (60, 40, and 20% O2 at 5.3 ± 0.3, 3.3 ± 0.1, and 1.6 ± 0.2 FIU/min, respectively). CA1 cell death after 4 h (ethidium homodimer-1 staining) was greatest in 95% O2 and lowest in 40 and 20% O2. CA1 neurons generated evoked action potentials in 20% O2 for \u3e4 h, indicating viability at lower levels of oxygenation. We conclude that ·O2− production and cell death in CA1 neurons increases in response to increasing oxygen concentration product (= PO2 × time). Additionally, lower levels of oxygen (20–40%) and antioxidants should be considered to minimize superoxide-induced oxidative stress in brain slices

Atomic Force Microscopy (AFM) Analysis of Lipid Peroxidation Following Hyperoxia and Hydrogen Peroxide Treatment in Human U87 Glioblastoma Cells

The atomic force microscope (AFM) is capable of resolving the plasma membrane with nanometer resolution. In this study we used the AFM to characterize hyperoxia-induced oxidative damage to changes in the plasma membranes of cultured human glioma cells (U87). U87 cells were exposed to 0.20 ATA O2, normobaric hyperoxia (0.95 ATA O2) or hyperbaric hyperoxia (HBO2, 3.25 ATA O2) for 30 min. In separate experiments H2O2 (200 µM and 2 mM) was used as a positive control. Malondialdehyde (MDA) was measured to confirm lipid peroxidation. Following treatment, the cells were fixed with 2% glutaraldehyde and scanned in air or fluid. Individual cells from each group (n = 35 to 45 cells/group) were scanned and analyzed to assess average membrane roughness (Ra). The Ra of the plasma membrane was 34 ± 3 nm, 57 ± 3 nm and 63 ± 5 nm in 0.20 ATA O2, 0.95 ATA O2 and HBO2, respectively. In H2O2 treated cells Ra was 28 ± 4 nm, 56 ± 7 nm and 138 ± 14 nm in air (air in 5% CO2), 200 µM and 2 mM H2O2. Co-treatment with antioxidant Trolox C (150 µM) significantly reduced Raduring exposure to hyperoxia and H2O2, suggesting that the amount of membrane blebbing was proportional to the level of oxidative stress. Furthermore, measurement of MDA confirmed that H2O2 and hyperoxia increased lipid peroxidation, suggesting that membrane blebbing is related to oxidative stress. In conclusion, these data demonstrate oxidative damage from lipid peroxidation increases with oxygen concentration

Atomic Force Microscopy (AFM) Analysis of Oxidative Stress in Human U87 Glioblastoma Cells Treated with Hyperoxia

Atomic Force Microscopy (AFM) has been used to image membrane surface topography at nanometer resolution. In this study we used the AFM to characterize the physical changes resulting from hyperoxia on cultured human glioblastoma cells (U87). U87 cells were exposed to air (21% O2), hyperoxia (95% O2) or hyperbaric oxygen (HBO2, 3.5ata O2) for 30 min. H2O2 (200µM) was used as a positive control. Following treatment, the cells were fixed with 2% glutaraldehyde for 20 min prior to AFM imaging. Three dimensional surface plots of U87 cells revealed a significant distortion of the plasma membrane (membrane blebbing) caused by HBO2 and H2O2, which is thought to be indicative of lipid peroxidation. Individual cells from each group were analyzed to assess Mean Roughness (Ra) and Maximum Roughness (Rmax). Ra was 40 ± 4 nm in 21% O2, 52 ± 5 nm in 95%O2, 64 ± 7 nm in HBO2, and 91 ± 9 nm in H2O2 treated cells. Rmaxwas 253 ± 27 nm in 21% O2, 302 ± 30 nm in 95% O2, 366 ± 35 nm in HBO2, and 608 ± 39 nm in H2O2 treated cells. In conclusion, these data show that oxidative damage in the plasma membrane is proportional to oxygen concentration. Moreover, the AFM is capable of characterizing subtle changes in membrane topography from oxidative damage

Acute Hyperoxia Increases Lipid Peroxidation and Induces Plasma Membrane Blebbing in Human U87 Glioblastoma Cells

Atomic force microscopy (AFM), malondialdehyde (MDA) assays, and amperometric measurements of extracellular hydrogen peroxide (H2O2) were used to test the hypothesis that graded hyperoxia induces measurable nanoscopic changes in membrane ultrastructure and membrane lipid peroxidation (MLP) in cultured U87 human glioma cells. U87 cells were exposed to 0.20 atmospheres absolute (ATA) O2, normobaric hyperoxia (0.95 ATA O2) or hyperbaric hyperoxia (HBO2, 3.25 ATA O2) for 60 min. H2O2 (0.2 or 2 mM; 60 min) was used as a positive control for MLP. Cells were fixed with 2% glutaraldehyde immediately after treatment and scanned with AFM in air or fluid. Surface topography revealed ultrastructural changes such as membrane blebbing in cells treated with hyperoxia and H2O2. Average membrane roughness (Ra) of individual cells from each group (n=35 to 45 cells/group) was quantified to assess ultrastructural changes from oxidative stress. The Ra of the plasma membrane was 34±3, 57±3 and 63±5 nm in 0.20 ATA O2, 0.95 ATA O2 and HBO2, respectively. Ra was 56±7 and 138±14 nm in 0.2 and 2 mM H2O2. Similarly, levels of MDA were significantly elevated in cultures treated with hyperoxia and H2O2 and correlated with O2-induced membrane blebbing (r2=0.93). Coapplication of antioxidant, Trolox-C (150 μM), significantly reduced membrane Ra and MDA levels during hyperoxia. Hyperoxia-induced H2O2 production increased 189%±5% (0.95 ATA O2) and 236%±5% (4 ATA O2) above control (0.20 ATA O2). We conclude that MLP and membrane blebbing increase with increasing O2 concentration. We hypothesize that membrane blebbing is an ultrastructural correlate of MLP resulting from hyperoxia. Furthermore, AFM is a powerful technique for resolving nanoscopic changes in the plasma membrane that result from oxidative damage

Superoxide (·O2−) Production in CA1 Neurons of Rat Hippocampal Slices Exposed to Graded Levels of Oxygen

Neuronal signaling, plasticity, and pathologies in CA1 hippocampal neurons are all intimately related to the redox environment and, thus tissue oxygenation. This study tests the hypothesis that hyperoxic superfusate (95% O2) causes a time-dependent increase in superoxide anion (·O2−) production in CA1 neurons in slices, which will decrease as oxygen concentration is decreased. Hippocampal slices (400 μm) from weaned rats were incubated with the fluorescent probe dihydroethidium (DHE), which detects intracellular ·O2− production. Slices were loaded for 30 min using 10 μM DHE and maintained using one-sided superfusion or continuously loaded using 2.5 μM DHE and maintained using two-sided superfusion (36°C). Continuous loading of DHE and two-sided superfusion gave the highest temporal resolution measurements of ·O2− production, which was estimated by the increase in fluorescence intensity units (FIUs) per minute (FIU/min ± SE) over 4 h. Superoxide production (2.5 μM DHE, 2-sided superfusion) was greatest in 95% O2 (6.6 ± 0.4 FIU/min) and decreased significantly during co-exposure with antioxidants (100 μM melatonin, 25 μM MnTMPyP) and lower levels of O2 (60, 40, and 20% O2 at 5.3 ± 0.3, 3.3 ± 0.1, and 1.6 ± 0.2 FIU/min, respectively). CA1 cell death after 4 h (ethidium homodimer-1 staining) was greatest in 95% O2 and lowest in 40 and 20% O2. CA1 neurons generated evoked action potentials in 20% O2 for \u3e4 h, indicating viability at lower levels of oxygenation. We conclude that ·O2− production and cell death in CA1 neurons increases in response to increasing oxygen concentration product (= PO2 × time). Additionally, lower levels of oxygen (20–40%) and antioxidants should be considered to minimize superoxide-induced oxidative stress in brain slices