M. Todd King

Sir2 Regulates Skeletal Muscle Differentiation as a Potential Sensor of the Redox State

Sir2 is a NAD(+)-dependent histone deacetylase that controls gene silencing, cell cycle, DNA damage repair, and life span. Prompted by the observation that the [NAD(+)]/[NADH] ratio is subjected to dynamic fluctuations in skeletal muscle, we have tested whether Sir2 regulates muscle gene expression and differentiation. Sir2 forms a complex with the acetyltransferase PCAF and MyoD and, when overexpressed, retards muscle differentiation. Conversely, cells with decreased Sir2 differentiate prematurely. To inhibit myogenesis, Sir2 requires its NAD(+)-dependent deacetylase activity. The [NAD(+)]/[NADH] ratio decreases as muscle cells differentiate, while an increased [NAD(+)]/[NADH] ratio inhibits muscle gene expression. Cells with reduced Sir2 levels are less sensitive to the inhibition imposed by an elevated [NAD(+)]/[NADH] ratio. These results indicate that Sir2 regulates muscle gene expression and differentiation by possibly functioning as a redox sensor. In response to exercise, food intake, and starvation, Sir2 may sense modifications of the redox state and promptly modulate gene expression.

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.

Nutritional Ketosis Alters Fuel Preference and Thereby Endurance Performance in Athletes

Ketosis, the metabolic response to energy crisis, is a mechanism to sustain life by altering oxidative fuel selection. Often overlooked for its metabolic potential, ketosis is poorly understood outside of starvation or diabetic crisis. Thus, we studied the biochemical advantages of ketosis in humans using a ketone ester-based form of nutrition without the unwanted milieu of endogenous ketone body production by caloric or carbohydrate restriction. In five separate studies of 39 high-performance athletes, we show how this unique metabolic state improves physical endurance by altering fuel competition for oxidative respiration. Ketosis decreased muscle glycolysis and plasma lactate concentrations, while providing an alternative substrate for oxidative phosphorylation. Ketosis increased intramuscular triacylglycerol oxidation during exercise, even in the presence of normal muscle glycogen, co-ingested carbohydrate and elevated insulin. These findings may hold clues to greater human potential and a better understanding of fuel metabolism in health and disease.

A Ketone Ester Diet Increases Brain Malonyl-CoA and Uncoupling Proteins 4 and 5 while Decreasing Food Intake in the Normal Wistar Rat

Three groups of male Wistar rats were pair fed NIH-31 diets for 14 days to which were added 30% of calories as corn starch, palm oil, or R-3-hydroxybutyrate-R-1,3-butanediol monoester (3HB-BD ester). On the 14th day, animal brains were removed by freeze-blowing, and brain metabolites measured. Animals fed the ketone ester diet had elevated mean blood ketone bodies of 3.5 mm and lowered plasma glucose, insulin, and leptin. Despite the decreased plasma leptin, feeding the ketone ester diet ad lib decreased voluntary food intake 2-fold for 6 days while brain malonyl-CoA was increased by about 25% in ketone-fed group but not in the palm oil fed group. Unlike the acute effects of ketone body metabolism in the perfused working heart, there was no increased reduction in brain free mitochondrial [NAD+]/[NADH] ratio nor in the free energy of ATP hydrolysis, which was compatible with the observed 1.5-fold increase in brain uncoupling proteins 4 and 5. Feeding ketone ester or palm oil supplemented diets decreased brain l-glutamate by 15–20% and GABA by about 34% supporting the view that fatty acids as well as ketone bodies can be metabolized by the brain.

A rapid, enzymatic assay for the measurement of inorganic pyrophosphate in animal tissues

A simple, rapid enzymatic assay for the determination of inorganic pyrophosphate in tissue and plasma has been developed using the enzyme pyrophosphate--fructose-6-phosphate 1-phosphotransferase (EC 2.7.1.90) which was purified from extracts of Propionibacterium shermanii. The enzyme phosphorylates fructose-6-phosphate to produce fructose-1,6-bisphosphate using inorganic pyrophosphate as the phosphate donor. The utilization of inorganic pyrophosphate is measured by coupling the production of fructose-1,6-bisphosphate with the oxidation of NADH using fructose-bisphosphate aldolase (EC 4.1.2.13), triosephosphate isomerase (EC 5.3.1.1), and glycerol-3-phosphate dehydrogenase (NAD+)(EC 1.1.1.8). The assay is completed in less than 5 min and is not affected by any of the components of tissue or plasma extracts. The recovery of pyrophosphate added to frozen tissue powder was 97 +/- 1% (n = 4). In this assay the change in absorbance is linearly related to the concentration of inorganic pyrophosphate over the curvette concentration range of 0.1 microM to 0.1 mM.

Substrate signaling by insulin: a ketone bodies ratio mimics insulin action in heart.

The administration of saturating doses of insulin to the glucose perfused, working rat heart acutely increased activity of the glucose transporter 4, GLUT 4, in the plasma membrane (equilibrating extracellular glucose and intracellular [glucose]), activated glycogen synthase (stimulating the rate of glycogen synthesis), and increased mitochondrial acetyl CoA production by the pyruvate dehydrogenase multienzyme complex. Unexpectedly, insulin increased cardiac hydraulic work but decreased net glycolytic flux and O2 consumption, improving net cardiac efficiency by 28%. These improvements in physiologic performance and metabolic efficiency resulted from reduction of the mitochondrial free [NAD+]/[NADH] and oxidation of mitochondrial [coenzyme Q]/[coenzyme QH2], increasing the energy of the proton gradient between cytosolic and mitochondrial phases and leading to a doubling of the cytosolic free [sigmaATP]/[sigmaADP][sigmaPi]. The acute metabolic effects of insulin were qualitatively duplicated by addition of a ratio of 4 mM D-beta-hydroxybutyrate and 1 mM acetoacetate, and the increase in the efficiency was the same as with addition of insulin. Addition of both insulin and ketones to the glucose perfusate increased the efficiency of cardiac hydraulic work by 35%. The ability of a physiologic ratio of ketone bodies to correct most of the metabolic defects of acute insulin deficiency suggests therapeutic roles for these natural substrates during periods of impaired cardiac performance and in insulin-resistant states.

Relationship of free cytoplasmic pyrophosphate to liver glucose content and total pyrophosphate to cytoplasmic phosphorylation potential

H. A. Krebs has pointed out the importance to metabolic regulation of certain small inorganic ions which are not normally thought of as metabolites. The compounds discussed were CO*, HCOS, H’, NH: and Pi [ 11. It has been appreciated subsequently that Mg*+, while not subject to large intracellular variations under most physiological conditions, [2] has appreciable effects on the apparent equilibrium constants of a number of important intracellular reactions [3,4] and should be included in this list. In this paper we attempt to show that inorganic pyrophosphate (PPi) is another small inorganic ion which plays an important role in the regulation of a number of metabolic processes in mammals as a result of hormonal or dietary changes. Pyrophosphate has long been known to play an important role in intermediary metabolism ln some microorganisms [5] and in Entamoeba histolytica [6], where it acts in place of ATP as an energy source in the phosphorylation of fructose 6-phosphate and ln other key reactions of the glycolytic and gluconeogenic pathways. Pyrophosphate has been shown to be the product of photophosphorylation in Rhodospirillum rubrum [7] and may play a role ln mammalian oxidative phosphorylation [8]. This view has not been generally accepted because of the widespread, but in our view erroneous, belief that pyrophosphate is rapidly hydrolyzed in cells to inorganic orthophosphate. In data not presented here, we have shown that under appropriate conditions, injection of short chain fatty acids can raise hepatic PPi content to 2-3 pmol/g

The energetics of ion distribution: the origin of the resting electric potential of cells.

The relation between the energies of ion movement and ATP hydrolysis is unknown in tissues with widely varying electric potentials. Consequently, we measured the concentration of the nine major inorganic ions in the extra‐ and intracellular phases in heart, liver, and red cells with resting electrical potentials, E N, of ‐86, ‐28, and ‐6 mV, respectively, under six different physiological conditions. We calculated the Nernst electric potential and the energy of ion movement between the phases. We found that the energy of ATP hydrolysis was essentially constant, between ‐54 and ‐58 kJ/mol, in all tissues and conditions. In contrast, as E N decreased, the energies of the Na + and K + gradients decreased, with slopes approximating their valence. The difference between the energies of Na + and K + gradients remained constant at 17 kJ/mol, which is approximately one third of the energy of ATP hydrolysis, demonstrating near‐equilibrium of the Na +/K + ATPase in all tissues under all conditions. All cations, except K +, were pumped out of cells and all anions, except Cl ‐ in liver and red cell, were pumped into cells. We conclude that the energy of ATP was expressed in Na +/K + ATPase and its linked inorganic ion transporters to create a Gibbs‐Donnan near‐equilibrium system, an inherent part of which was the electric potential.

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

The β/α Peak Height Ratio of ATP A MEASURE OF FREE [Mg2+] USING 31P NMR

From 31P NMR measurements made in vitro at 38°C, I = 0.25, pH 5.75-8.5, and calculated free [Mg2+] from 0 to 5 m, we show that, within the physiological range of cytosolic free [Mg2+] from 0.25 to 1.5 m, the chemical shift difference between the α- and β-ATP resonances, δα- and β, changes by only 0.6 ppm. Consequently, we developed new formalisms from known acid and Mg2+ dissociation constants by which the observed chemical shift of Pi, δPi, and the peak height ratio of the β- and α-ATP resonances, hβ- and α, could be related to free [Mg2+] by simultaneous solution of: We found that hβ/α changed 2.5-fold as free [Mg2+] varied from 0.25 to 1.5 m, providing a more sensitive and accurate measure of free cytosolic [Mg2+]. In working rat heart perfused with glucose, free [Mg2+] was 1.0 ± 0.1 from hβ/α and 1.2 ± 0.03 from measured [citrate]/[isocitrate] but 0.51 ± 0.1 from δαβ. Addition of ketone bodies to the perfusate decreased free [Mg2+] estimated from hβ/α to 0.61 ± 0.02 and 0.74 ± 0.11 by [citrate]/[isocitrate] but the estimate from δαβ was unchanged at 0.46 ± 0.04 m. Such differences in estimated free [Mg2+] alter the apparent Keq of the creatine kinase reaction and hence the estimated cytosolic free [ΣADP].