Low carbohydrate high fat ketogenic diets on the exercise crossover point and glucose homeostasis.

In exercise science, the crossover effect denotes that fat oxidation is the primary fuel at rest and during low-intensity exercise with a shift towards an increased reliance on carbohydrate oxidation at moderate to high exercise intensities. This model makes four predictions: First, >50% of energy comes from carbohydrate oxidation at ≥60% of maximum oxygen consumption (VO2max), termed the crossover point. Second, each individual has a maximum fat oxidation capacity (FATMAX) at an exercise intensity lower than the crossover point. FATMAX values are typically 0.3-0.6 g/min. Third, fat oxidation is minimized during exercise ≥85%VO2max, making carbohydrates the predominant energetic substrate during high-intensity exercise, especially at >85%VO2max. Fourth, high-carbohydrate low-fat (HCLF) diets will produce superior exercise performances via maximizing pre-exercise storage of this predominant exercise substrate. In a series of recent publications evaluating the metabolic and performance effects of low-carbohydrate high-fat (LCHF/ketogenic) diet adaptations during exercise of different intensities, we provide findings that challenge this model and these four predictions. First, we show that adaptation to the LCHF diet shifts the crossover point to a higher %VO2max (>80%VO2max) than previously reported. Second, substantially higher FATMAX values (>1.5 g/min) can be measured in athletes adapted to the LCHF diet. Third, endurance athletes exercising at >85%VO2max, whilst performing 6 × 800 m running intervals, measured the highest rates of fat oxidation yet reported in humans. Peak fat oxidation rates measured at 86.4 ± 6.2%VO2max were 1.58 ± 0.33 g/min with 30% of subjects achieving >1.85 g/min. These studies challenge the prevailing doctrine that carbohydrates are the predominant oxidized fuel during high-intensity exercise. We recently found that 30% of middle-aged competitive athletes presented with pre-diabetic glycemic values while on an HCLF diet, which was reversed on LCHF. We speculate that these rapid changes between diet, insulin, glucose homeostasis, and fat oxidation might be linked by diet-induced changes in mitochondrial function and insulin action. Together, we demonstrate evidence that challenges the current crossover concept and demonstrate evidence that a LCHF diet may also reverse features of pre-diabetes and future metabolic disease risk, demonstrating the impact of dietary choice has extended beyond physical performance even in athletic populations.

Non-Toxic Metabolic Management of Metastatic Cancer in VM Mice: Novel Combination of Ketogenic Diet, Ketone Supplementation, and Hyperbaric Oxygen Therapy.

The Warburg effect and tumor hypoxia underlie a unique cancer metabolic phenotype characterized by glucose dependency and aerobic fermentation. We previously showed that two non-toxic metabolic therapies - the ketogenic diet with concurrent hyperbaric oxygen (KD+HBOT) and dietary ketone supplementation - could increase survival time in the VM-M3 mouse model of metastatic cancer. We hypothesized that combining these therapies could provide an even greater therapeutic benefit in this model. Mice receiving the combination therapy demonstrated a marked reduction in tumor growth rate and metastatic spread, and lived twice as long as control animals. To further understand the effects of these metabolic therapies, we characterized the effects of high glucose (control), low glucose (LG), ketone supplementation (ßHB), hyperbaric oxygen (HBOT), or combination therapy (LG+ßHB+HBOT) on VM-M3 cells. Individually and combined, these metabolic therapies significantly decreased VM-M3 cell proliferation and viability. HBOT, alone or in combination with LG and ßHB, increased ROS production in VM-M3 cells. This study strongly supports further investigation into this metabolic therapy as a potential non-toxic treatment for late-stage metastatic cancers.

Ketone supplementation decreases tumor cell viability and prolongs survival of mice with metastatic cancer.

Cancer cells express an abnormal metabolism characterized by increased glucose consumption owing to genetic mutations and mitochondrial dysfunction. Previous studies indicate that unlike healthy tissues, cancer cells are unable to effectively use ketone bodies for energy. Furthermore, ketones inhibit the proliferation and viability of cultured tumor cells. As the Warburg effect is especially prominent in metastatic cells, we hypothesized that dietary ketone supplementation would inhibit metastatic cancer progression in vivo. Proliferation and viability were measured in the highly metastatic VM-M3 cells cultured in the presence and absence of ß-hydroxybutyrate (ßHB). Adult male inbred VM mice were implanted subcutaneously with firefly luciferase-tagged syngeneic VM-M3 cells. Mice were fed a standard diet supplemented with either 1,3-butanediol (BD) or a ketone ester (KE), which are metabolized to the ketone bodies ßHB and acetoacetate. Tumor growth was monitored by in vivo bioluminescent imaging. Survival time, tumor growth rate, blood glucose, blood ßHB and body weight were measured throughout the survival study. Ketone supplementation decreased proliferation and viability of the VM-M3 cells grown in vitro, even in the presence of high glucose. Dietary ketone supplementation with BD and KE prolonged survival in VM-M3 mice with systemic metastatic cancer by 51 and 69%, respectively (p < 0.05). Ketone administration elicited anticancer effects in vitro and in vivo independent of glucose levels or calorie restriction. The use of supplemental ketone precursors as a cancer treatment should be further investigated in animal models to determine potential for future clinical use.

Development and testing of hyperbaric atomic force microscopy (AFM) and fluorescence microscopy for biological applications.

A commercially available atomic force microscopy and fluorescence microscope were installed and tested inside a custom-designed hyperbaric chamber to provide the capability to study the effects of hyperbaric gases on biological preparations, including cellular mechanism of oxidative stress. In this report, we list details of installing and testing atomic force microscopy and fluorescence microscopy inside a hyperbaric chamber. The pressure vessel was designed to accommodate a variety of imaging equipment and ensures full functionality at ambient and hyperbaric conditions (≤85 psi). Electrical, gas and fluid lines were installed to enable remote operation of instrumentation under hyperbaric conditions, and to maintain viable biological samples with gas-equilibrated superfusate and/or drugs. Systems were installed for vibration isolation and temperature regulation to maintain atomic force microscopy performance during compression and decompression. Results of atomic force microscopy testing demonstrate sub-nanometre resolution at hyperbaric pressure in dry scans and fluid scans, in both contact mode and tapping mode. Noise levels were less when measurements were taken under hyperbaric pressure with air, helium (He) and nitrogen (N(2) ). Atomic force microscopy and fluorescence microscopy measurements were made on a variety of living cell cultures exposed to hyperbaric gases (He, N(2) , O(2) , air). In summary, atomic force microscopy and fluorescence microscopy were installed and tested for use at hyperbaric pressures and enables the study of cellular and molecular effects of hyperbaric gases and pressure per se in biological preparations.

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 (H(2)O(2)) 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) O(2), normobaric hyperoxia (0.95 ATA O(2)) or hyperbaric hyperoxia (HBO(2), 3.25 ATA O(2)) for 60 min. H(2)O(2) (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 H(2)O(2). Average membrane roughness (R(a)) of individual cells from each group (n=35 to 45 cells/group) was quantified to assess ultrastructural changes from oxidative stress. The R(a) of the plasma membrane was 34+/-3, 57+/-3 and 63+/-5 nm in 0.20 ATA O(2), 0.95 ATA O(2) and HBO(2), respectively. R(a) was 56+/-7 and 138+/-14 nm in 0.2 and 2 mM H(2)O(2). Similarly, levels of MDA were significantly elevated in cultures treated with hyperoxia and H(2)O(2) and correlated with O(2)-induced membrane blebbing (r(2)=0.93). Coapplication of antioxidant, Trolox-C (150 microM), significantly reduced membrane R(a) and MDA levels during hyperoxia. Hyperoxia-induced H(2)O(2) production increased 189%+/-5% (0.95 ATA O(2)) and 236%+/-5% (4 ATA O(2)) above control (0.20 ATA O(2)). We conclude that MLP and membrane blebbing increase with increasing O(2) 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.