Richard L. Veech

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.

FREEZE-BLOWING: A NEW TECHNIQUE FOR THE STUDY OF BRAIN IN VIVO

Abstract— A new apparatus is described which removes and freezes brains of conscious rats more rapidly than was heretofore possible. The apparatus consists of two probes which are driven simultaneously into the cranial vault of the rat immobilized in a specially constructed restraining cage. When in position, air under pressure enters through one probe and blows the supratentorial portion of the brain tissue (situated between the olfactory bulbs and the superior colliculi) out the other probe and into a thin chamber previously cooled in liquid N2. This method stops brain tissue metabolism more rapidly than the previously‐described methods of microwave irradiation, decapitation into liquid N2, or whole‐animal immersion into liquid N2, as evidenced by the measurement of labile metabolites and redox states. Thus, samples of freeze‐blown brain had higher levels of a‐oxoglutarate, creatine phosphate, pyruvate, glucose and glucose‐6‐phosphate and lower levels of lactate, malate and AMP than brain tissue obtained by the other methods. The free cytoplasmic [NAD+]/[NADH2], [NADP+]/[NADPH2] and [ATP]/[ADP] [HPO42‐] ratios were higher in freeze‐blown samples. These data indicate that more extensive anoxic metabolism occurred when methods other than freeze‐blowing were used. We conclude that the levels of metabolites measured in brain obtained with the freeze‐blowing technique more closely resemble those which occur in vivo.

Insulin, ketone bodies, and mitochondrial energy transduction.

Addition of insulin or a physiological ratio of ketone bodies to buffer with 10 mM glucose increased efficiency (hydraulic work/energy from O2 consumed) of working rat heart by 25%, and the two in combination increased efficiency by 36%. These additions increased the content of acetyl CoA by 9‐ to 18‐fold, increased the contents of metabolites of the first third of the tricarboxylic acid (TCA) cycle 2‐ to 5‐fold, and decreased succinate, axaloacetate, and aspartate 2‐ to 3‐fold. Suc‐ cinyl CoA, fumarate, and malate were essentially unchanged. The changes in content of TCA metabolites resulted from a reduction of the free mitochondrial NAD couple by 2‐ to 10‐fold and oxidation of the mitochondrial coenzyme Q couple by 2‐ to 4‐fold. Cytosolic pH, measured using 31P‐NMR spectra, was invariant at about 7.0. The total intracellular bicarbonate indicated an increase in mitochondrial pH from 7.1 with glucose to 7.2, 7.5, and 7.4 with insulin, ketones, and the combination, respectively. The decrease in Eh7 of the mitochondrial NAD couple, Eh7NAD∗/NADH, from ‐280 to ‐300 mV and the increase in Eh7 of the coenzyme Q couple, Eh7Q/QH2, from ‐4 to +12 mV was equivalent to an increase from ‐53 kJ to ‐60 kJ/2 mol e in the reaction catalyzed by the mitochondrial NADH dehydrogenase multienzyme com‐plex (EC 1.6.5.3). The increase in the redox energy of the mitochondrial cofactor couples paralleled the increase in the free energy of cytosolic ATP hydrolysis, ΔGatp‐ The potential of the mitochondrial relative to the cytosolic phases, Emito/cyto, calculated from ΔGatp and ΔpH on the assumption of a 4 H+ transfer for each ATP synthesized, was ‐143 mV during perfusion with glucose or glucose plus insulin, and decreased to ‐120 mV on addition of ketones. Viewed in this light, the moderate ketosis characteristic of prolonged fasting or type II diabetes appears to be an elegant compensation for the defects in mitochondrial energy transduction associated with acute insulin deficiency or mitochondrial senescence.—Sato, K., Kashiwaya, Y., Keon, C. A., Tsuchiya, N., King, M. T., Radda, G. K., Chance, B., Clarke, K., Veech, R. L. Insulin, ketone bodies, and mitochondrial energy transduction. FASEB J. 9, 651‐658 (1995)

Interleukin 6 alleviates hepatic steatosis and ischemia/reperfusion injury in mice with fatty liver disease

Fatty liver, formerly associated predominantly with excessive alcohol intake, is now also recognized as a complication of obesity and an important precursor state to more severe forms of liver pathology including ischemia/reperfusion injury. No standard protocol for treating fatty liver exists at this time. We therefore examined the effects of 10 days of interleukin 6 (IL‐6) injection in 3 murine models of fatty liver: leptin deficient ob/ob mice, ethanol‐fed mice, and mice fed a high‐fat diet. In all 3 models, IL‐6 injection decreased steatosis and normalized serum aminotransferase. The beneficial effects of IL‐6 treatment in vivo resulted in part from an increase in mitochondrial β oxidation of fatty acid and an increase in hepatic export of triglyceride and cholesterol. However, administration of IL‐6 to isolated cultured steatotic hepatocytes failed to decrease lipid contents, suggesting that the beneficial effects of IL‐6 in vivo do not result from its effects on hepatocytes alone. IL‐6 treatment increased hepatic peroxisome proliferator‐activated receptor (PPAR) α and decreased liver and serum tumor necrosis factor (TNF) α. Finally, 10 days of treatment with IL‐6 prevented the susceptibility of fatty livers to warm ischemia/reperfusion injury. In conclusion, long‐term IL‐6 administration ameliorates fatty livers and protects against warm ischemia/reperfusion fatty liver injury, suggesting the therapeutic potential of IL‐6 in treating human fatty liver disease. Supplementary material for this article can be found on the Hepatology website (http://interscience.wiley.com/jpages/0270‐9139/suppmat/index.html). (HEPATOLOGY 2004;40:933–941.)

MEASUREMENT OF THE RATE OF GLUCOSE UTILIZATION BY RAT BRAIN IN VIVO

Abstract— A method is described by which the rate of glucose utilization by whole brain of conscious rats may be measured. The basis is the uptake of 14C derived front [2‐14C] glucose into the acid‐soluble metabolite pool of brain. Catheters are placed in the femoral artery and vein under light ether anesthesia. After full recovery of consciousness a single intravenous injection of [2‐14C] glucose is given and arterial blood samples taken at intervals. Simultaneous with the last sample the brain is removed and frozen within 1 s. The accumulation of 14C into the acid‐soluble metabilite pool is measured and the rate of glucose utilization is calculated according to the equation:

The effects of intoxicating doses of ethanol upon intermediary metabolism in rat brain.

Abstract— The effect of acute (8‐min) and prolonged (13‐h) exposures to high doses of ethanol upon the intermediary metabolites of rat brain has been studied, with the use of a new freezing technique which minimizes post‐mortem changes. Injection of ethanol (80 mmol/kg body wt) produced general anaesthesia within 8 min after administration. At this time there were increases in the brain contents of glucose, glucose‐6‐phosphate and citrate; there was no change in arterial pCO2. Rats under ethanol anaesthesia for 13 h showed increases in brain contents of glycogen, glucose and glucose 6‐phosphate; and decreases in lactate, pyruvate, α‐oxoglutarate and malate. Under similar experimental conditions, arterial pCO2, increased from 37 to 51 Torr. The changes in levels of metabolites after injection of ethanol were similar to those after administration of many volatile anaesthetic agents or elevation of brain CO2 by other means. Although brain levels of malate and α‐oxoglutarate decreased after prolonged exposure to ethanol, the mitochondrial redox state was maintained. Accordingly, the levels of glutamate and aspartate fell in accordance with the law of mass action. The maintenance of the cytoplasmic and mitochondrial redox states in the brain during ethanol intoxication was in marked contrast to the effects on the liver. We suggest that the different effects observed in brain and liver result from the action of ethanol upon the nerve cell membrane in brain, whereas the primary target in liver is alcohol dehydrogenase.

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.

DECREASED RATE OF GLUCOSE UTILIZATION BY RAT BRAIN IN VIVO AFTER EXPOSURE TO ATMOSPHERES CONTAINING HIGH CONCENTRATIONS OF CO2

—(1) The effects of exposure of rats to increased atmospheric concentrations of CO2 on brain metabolism in vivo were studied. (2) After 2·5 min exposure to an atmosphere of 20% CO2, the rate of glucose utilization by brain decreased from 0·61 μmol/min per g to 0·32 μmol/min per g and remained between 0·3 and 0·4 μmol/min per g for 60 min, the longest interval studied. O2 utilization, calculated from the arteriovenous difference of O2 across the brain and blood flow, was 3·5 μmol/min per g in controls and was 4·7 μmol/min per g after 5 min in the 20% CO2 atmosphere. (3) The concentrations of glucose, glucose 6‐phosphate and aspartate were increased during the first 10 min of CO2 exposure whereas the concentrations of other glycolytic intermediates, tricarboxylic acid cycle intermediates and glutamate were decreased. The amount of endogenous substrate which disappeared during the first 10 min was sufficient, if used to supplement glucose as a fuel, to maintain the O2 consumption at, or slightly above, the control level. Glutamate and lactate were quantitatively the most important energy sources. (4) The mechanism whereby‘CO2 decreased the rate of glucose utilization is uncertain. The initial rise in glucose 6‐phosphate and fall in fructose 1,6‐diphosphate concentrations suggested that an inhibition of phosphofructokinase was responsible. However, after 60 min in 20% CO2, the concentrations of both of these metabolites returned to normal while the rate of glucose utilization remained depressed.

Low doses of alcohol substantially decrease glucose metabolism in the human brain.

Moderate doses of alcohol decrease glucose metabolism in the human brain, which has been interpreted to reflect alcohol-induced decreases in brain activity. Here, we measure the effects of two relatively low doses of alcohol (0.25 g/kg and 0.5 g/kg, or 5 to 10 mM in total body H2O) on glucose metabolism in the human brain. Twenty healthy control subjects were tested using positron emission tomography (PET) and FDG after placebo and after acute oral administration of either 0.25 g/kg, or 0.5 g/kg of alcohol, administered over 40 min. Both doses of alcohol significantly decreased whole-brain glucose metabolism (10% and 23% respectively). The responses differed between doses; whereas the 0.25 g/kg dose predominantly reduced metabolism in cortical regions, the 0.5 g/kg dose reduced metabolism in cortical as well as subcortical regions (i.e. cerebellum, mesencephalon, basal ganglia and thalamus). These doses of alcohol did not significantly change the scores in cognitive performance, which contrasts with our previous results showing that a 13% reduction in brain metabolism by lorazepam was associated with significant impairment in performance on the same battery of cognitive tests. This seemingly paradoxical finding raises the possibility that the large brain metabolic decrements during alcohol intoxication could reflect a shift in the substrate for energy utilization, particularly in light of new evidence that blood-borne acetate, which is markedly increased during intoxication, is a substrate for energy production by the brain.

Measurement of tissue purine, pyrimidine, and other nucleotides by radial compression high-performance liquid chromatography.

A high-performance liquid-chromatographic (HPLC) method for the rapid separation of purine and pyrimidine nucleotides, NAD+, NADP+, FAD, FMN, UDP-Glc, UDP-glucuronate, and ADP-ribose found in neutralized perchloric acid extracts of rat liver is described. Separation was achieved within 26 min on a radially compressed column of Partisil 10-SAX. The column was eluted with a gradient of sodium phosphate and sodium chloride. The sodium phosphate was purified by passage through tandem columns of anion- and cation-exchange resins to remove uv-absorbing impurities. The sensitivity of this procedure is such that an amount of ATP contained in 10 micrograms of liver can be measured. The recoveries of all nucleotides were between 87 and 107%. In extracts of rat liver interfering substances were found to elute with GDP, and UDP eluted with NADP. Consequently, the tissue contents of UDP and GDP were determined in a second run by measuring the increase in UTP and GTP, respectively, following sample pretreatment with pyruvate kinase (PK). The tissue level of NADP+ was calculated as the difference between the total UDP and NADP+ peak and the increase in UTP following PK treatment. In those nucleotides amenable to enzymatic analysis, namely NAD+, AMP, UDP-Glc, UTP, and ATP, the tissue contents measured enzymatically were not significantly different from those determined by HPLC. However, ADP as measured with PK was found to be 15% higher compared to the HPLC determination.