Exogenous Ketones Lower Blood Glucose Level in Rested and Exercised Rodent Models.

Diseases involving inflammation and oxidative stress can be exacerbated by high blood glucose levels. Due to tight metabolic regulation, safely reducing blood glucose can prove difficult. The ketogenic diet (KD) reduces absolute glucose and insulin, while increasing fatty acid oxidation, ketogenesis, and circulating levels of β-hydroxybutyrate (βHB), acetoacetate (AcAc), and acetone. Compliance to KD can be difficult, so alternative therapies that help reduce glucose levels are needed. Exogenous ketones provide an alternative method to elevate blood ketone levels without strict dietary requirements. In this study, we tested the changes in blood glucose and ketone (βHB) levels in response to acute, sub-chronic, and chronic administration of various ketogenic compounds in either a post-exercise or rested state. WAG/Rij (WR) rats, a rodent model of human absence epilepsy, GLUT1 deficiency syndrome mice (GLUT1D), and wild type Sprague Dawley rats (SPD) were assessed. Non-pathological animals were also assessed across different age ranges. Experimental groups included KD, standard diet (SD) supplemented with water (Control, C) or with exogenous ketones: 1, 3-butanediol (BD), βHB mineral salt (KS), KS with medium chain triglyceride/MCT (KSMCT), BD acetoacetate diester (KE), KE with MCT (KEMCT), and KE with KS (KEKS). In rested WR rats, the KE, KS, KSMCT groups had lower blood glucose level after 1 h of treatment, and in KE and KSMCT groups after 24 h. After exercise, the KE, KSMCT, KEKS, and KEMCT groups had lowered glucose levels after 1 h, and in the KEKS and KEMCT groups after 7 days, compared to control. In GLUT1D mice without exercise, only KE resulted in significantly lower glucose levels at week 2 and week 6 during a 10 weeks long chronic feeding study. In 4-month and 1-year-old SPD rats in the post-exercise trials, blood glucose was significantly lower in KD and KE, and in KEMCT groups, respectively. After seven days, the KSMCT group had the most significantly reduced blood glucose levels, compared to control. These results indicate that exogenous ketones were efficacious in reducing blood glucose levels within and outside the context of exercise in various rodent models of different ages, with and without pathology

Human adaptations to multiday saturation on NASA NEEMO

Human adaptation to extreme environments has been explored for over a century to understand human psychology, integrated physiology, comparative pathologies, and exploratory potential. It has been demonstrated that these environments can provide multiple external stimuli and stressors, which are sufficient to disrupt internal homeostasis and induce adaptation processes. Multiday hyperbaric and/or saturated (HBS) environments represent the most understudied of environmental extremes due to inherent experimental, analytical, technical, temporal, and safety limitations. National Aeronautic Space Agency (NASA) Extreme Environment Mission Operation (NEEMO) is a space-flight analog mission conducted within Florida International University's Aquarius Undersea Research Laboratory (AURL), the only existing operational and habitable undersea saturated environment. To investigate human objective and subjective adaptations to multiday HBS, we evaluated aquanauts living at saturation for 9-10 days via NASA NEEMO 22 and 23, across psychologic, cardiac, respiratory, autonomic, thermic, hemodynamic, sleep, and body composition parameters. We found that aquanauts exposed to saturation over 9-10 days experienced intrapersonal physical and mental burden, sustained good mood and work satisfaction, decreased heart and respiratory rates, increased parasympathetic and reduced sympathetic modulation, lower cerebral blood flow velocity, intact cerebral autoregulation and maintenance of baroreflex functionality, as well as losses in systemic bodyweight and adipose tissue. Together, these findings illustrate novel insights into human adaptation across multiple body systems in response to multiday hyperbaric saturation

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

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

Objectives: Exogenous ketones may provide therapeutic benefit in treatment of obesity. Administration of the ketone ester (KE) R,S-1,3-butanediol acetoacetate diester (BD-AcAc2) 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-AcAc2 on energy intake and expenditure in mice.Methods: Forty-two male C57BL/6J mice were randomly assigned to one of seven isocaloric diets (n = 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.Results: Mean energy intake was similar between CON and each concentration of KE, except KE30 which was 12% lower than CON (P < 0.01). KE25 and KE30 had lower body weight and FM compared to CON, while only KE30 had lower LBM (P < 0.03). Adjusted resting and total EE were lower in KE30 compared to CON (P < 0.03), but similar for all other groups.Conclusions: A diet comprised of 30% energy from BD-AcAc2 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

Targeting Cancer-Anorexia Cachexia Syndrome and Septic Inflammatory-Based Atrophy With R/S 1,3 Butanediol Acetoacetate Diester

Cancer anorexia cachexia syndrome (CACS) is a distinct atrophy disease negatively influencing multiple aspects of clinical care and patient quality of life. Although it directly causes 20% of all cancer-related deaths, there are currently no model systems that encompass the entire multifaceted syndrome, nor are there any effective therapeutic treatments. Here, we show that the VM-M3 mouse model of systemic metastasis demonstrates a novel, immunocompetent, logistically feasible, repeatable phenotype with progressive tumor growth, spontaneous metastatic spread, and the full multifaceted CACS with expected sex dimorphisms across tissue wasting. We also demonstrate that the ubiquitin proteasomal degradation pathway was significantly upregulated in association with reduced IGF-1/Insulin and increased FOXO3a activation, but not TNF-α-induced NF-κB activation, driving skeletal muscle atrophy. Additionally, we show that R/S 1,3-butanediol acetoacetate diester (Ketone Diester; KDE) administration shifted systemic metabolism, attenuated tumor burden, reduced tissue catabolism, and mitigated comorbid symptoms in both CACS and cancer-independent atrophy environments. Our findings suggest the ketone diester attenuates multifactorial CACS skeletal muscle atrophy and inflammation-induced tissue catabolism, demonstrating anti-catabolic effects of ketones bodies in multifactorial atrophy

Complex I inhibition augments dichloroacetate cytotoxicity through enhancing oxidative stress in VM-M3 glioblastoma cells

<div><p>The robust glycolytic metabolism of glioblastoma multiforme (GBM) has proven them susceptible to increases in oxidative metabolism induced by the pyruvate mimetic dichloroacetate (DCA). Recent reports demonstrate that the anti-diabetic drug metformin enhances the damaging oxidative stress associated with DCA treatment in cancer cells. We sought to elucidate the role of metformin’s reported activity as a mitochondrial complex I inhibitor in the enhancement of DCA cytotoxicity in VM-M3 GBM cells. Metformin potentiated DCA-induced superoxide production, which was required for enhanced cytotoxicity towards VM-M3 cells observed with the combination. Similarly, rotenone enhanced oxidative stress resultant from DCA treatment and this too was required for the noted augmentation of cytotoxicity. Adenosine monophosphate kinase (AMPK) activation was not observed with the concentration of metformin required to enhance DCA activity. Moreover, addition of an activator of AMPK did not enhance DCA cytotoxicity, whereas an inhibitor of AMPK heightened the cytotoxicity of the combination. Our data indicate that metformin enhancement of DCA cytotoxicity is dependent on complex I inhibition. Particularly, that complex I inhibition cooperates with DCA-induction of glucose oxidation to enhance cytotoxic oxidative stress in VM-M3 GBM cells.</p></div

DCA cytotoxicity is dependent on oxidative stress.

<p>(a) Ratiometric detection of BOPIDY® 581/591 oxidation as an indicator of lipid peroxidation in VM-M3 cells following 4-hour treatment with DCA ± NAC. (b) Quantification of average TMRE fluorescence intensity following 4-hour DCA treatment ± N-acetylcysteine (NAC). (c) Analysis of VM-M3 viability following 24-hour treatment with DCA. Bars represent fraction of cells stained positively for ethidium homodimer-I (Ethd-1). (d) Evaluation of VM-M3 viability following 24-hour DCA treatment in the presence of modulators of glutathione availability. (a-b) Error bars represent SEM of a single experiment replicated in triplicate (c-d) Error bars represent SEM of three experimental replicates; *p<0.05, **p<0.01, and ***p<0.001.</p

DCA promotes superoxide production and dissipation of ΔΨ_m in VM-M3 cells.

<p>(a) Western blot analysis of p-PDH-E1α (Ser293) and PDH-E1α in VM-M3 lysates following 4-hour treatment with DCA. Densitometric ratio of p-PDH to PDH was determined for each treatment relative to PBS control. (b) Quantification of lactate concentration in culture medium following 24-hour incubation with indicated treatment. (c) Quantification of average MitoSox Red fluorescence intensity as an indication of VM-M3 superoxide production following 1-hour incubation with DCA. (d) Quantification of average tetramethylrhodamine (TMRE) fluorescence intensity as an indication of mitochondrial membrane potential following 4-hour DCA treatment. (b) Error bars represent standard error of the mean (SEM) of three experimental replicates. (c-d) Error bars represent SEM of a single experiment replicated in triplicate; * p<0.05, and ***p<0.001.</p

Complex I inhibition, but not AMPK activation enhances DCA cytotoxicity.

<p>(a) Average VM-M3 superoxide production following 1-hour treatment with DCA and rotenone. (b) Ratiometric detection of BOPIDY® 581/591 oxidation as an indicator of lipid peroxidation in VM-M3 cells following 4-hour treatment with DCA and rotenone ± NAC. (c-e) Analysis of rotenone, AICAR, and metformin ± compound C modulation of DCA cytotoxicity towards VM-M3 cells. (f) In-cell ELISA analysis of p-AMPKα (Thr172), and AMPKα in VM-M3 cells following 4-hour treatment with modulators of AMPK activation. (a, b, f) Error bars represent SEM of a single experiment replicated in triplicate (c-e) Error bars represent SEM of three experimental replicates; **p<0.01 and ***p<0.001.</p

Metformin enhances DCA cytotoxicity.

<p>(a) Western blot analysis of p-PDH-E1α (Ser293) and PDH-E1α in VM-M3 cell lysates following 4-hour treatment with 5mM DCA and 100μM metformin. Densitometric ratio of p-PDH to PDH was determined for each treatment relative to PBS control. (b) Quantification of superoxide production with MitoSox Red following 1-hour treatment with DCA and metformin. (c) Quantification of BOPIDY® 581/591 oxidation as an indicator of lipid peroxidation in VM-M3 cells following 4-hour treatment with DCA and metformin ± NAC. Determination of VM-M3 cell viability following 24-hour treatment with (d) DCA and metformin ± NAC or (e) combinatorial treatment with DCA and metformin in increasing concentrations. (b, c) Error bars represent SEM of a single experiment replicated in triplicate (d, e) Error bars represent SEM of three experimental replicates; *p<0.05, **p<0.01, and ***p<0.001.</p