Homeostatic mechanisms in mammals respond to hormones and nutrients to maintain blood glucose levels within a narrow range. Caloric restriction causes many changes in glucose metabolism and extends lifespan; however, how this metabolism is connected to the ageing process is largely unknown. We show here that the Sir2 homologue, SIRT1--which modulates ageing in several species--controls the gluconeogenic/glycolytic pathways in liver in response to fasting signals through the transcriptional coactivator PGC-1alpha. A nutrient signalling response that is mediated by pyruvate induces SIRT1 protein in liver during fasting. We find that once SIRT1 is induced, it interacts with and deacetylates PGC-1alpha at specific lysine residues in an NAD(+)-dependent manner. SIRT1 induces gluconeogenic genes and hepatic glucose output through PGC-1alpha, but does not regulate the effects of PGC-1alpha on mitochondrial genes. In addition, SIRT1 modulates the effects of PGC-1alpha repression of glycolytic genes in response to fasting and pyruvate. Thus, we have identified a molecular mechanism whereby SIRT1 functions in glucose homeostasis as a modulator of PGC-1alpha. These findings have strong implications for the basic pathways of energy homeostasis, diabetes and lifespan.

Nutrient control of glucose homeostasis through a complex of PGC-1alpha and SIRT1.

Reactive oxygen species (ROS) and other radicals are involved in a variety of biological phenomena, such as mutation, carcinogenesis, degenerative and other diseases, inflammation, aging, and development. ROS are well recognized for playing a dual role as deleterious and beneficial species. The objectives of this review are to describe oxidative stress phenomena, terminology, definitions, and basic chemical characteristics of the species involved; examine the biological targets susceptible to oxidation and the defense mechanisms of the organism against these reactive metabolites; and analyze methodologies, including immunohistochemical markers, used in toxicological pathology in the visualization of oxidative stress phenomena. Direct detection of ROS and other free radicals is difficult, because these molecules are short-lived and highly reactive in a nonspecific manner. Ongoing oxidative damage is, thus, generally analyzed by measurement of secondary products including derivatives of amino acids, nuclei acids, and lipid peroxidation. Attention has been focused on electrochemical methods based on voltammetry measurements for evaluating the total reducing power of biological fluids and tissues. This approach can function as a tool to assess the antioxidant-reducing profile of a biological site and follow changes in pathological situations. This review thus includes different topics essential for understanding oxidative stress phenomena and provides tools for those intending to conduct study and research in this field.

https://journals.sagepub.com/doi/pdf/10.1080/01926230290166724

Oxidation of biological systems: oxidative stress phenomena, antioxidants, redox reactions, and methods for their quantification.

Dimethylbiguanide inhibits cell respiration via an indirect effect targeted on the respiratory chain complex I.

Vasodilation and increased blood flow are characteristic early vascular responses to acute hyperglycemia and tissue hypoxia. In hypoxic tissues these vascular changes are linked to metabolic imbalances associated with impaired oxidation of NADH to NAD+ and the resulting increased ratio of NADH/NAD+. In hyperglycemic tissues these vascular changes also are linked to an increased ratio of NADH/NAD+, in this case because of an increased rate of reduction of NAD+ to NADH. Several lines of evidence support the likelihood that the increased cytosolic ratio of free NADH/NAD+ caused by hyperglycemia, referred to as pseudohypoxia because tissue partial pressure oxygen is normal, is a characteristic feature of poorly controlled diabetes that mimics the effects of true hypoxia on vascular and neural function and plays an important role in the pathogenesis of diabetic complications. These effects of hypoxia and hyperglycemia-induced pseudohypoxia on vascular and neural function are mediated by a branching cascade of imbalances in lipid metabolism, increased production of superoxide anion, and possibly increased nitric oxide formation.

Hyperglycemic Pseudohypoxia and Diabetic Complications

Clock:BMAL1 and NPAS2:BMAL1 are heterodimeric transcription factors that control gene expression as a function of the light-dark cycle. Although built to fluctuate at or near a 24-hour cycle, the clock can be entrained by light, activity, or food. Here we show that the DNA-binding activity of the Clock:BMAL1 and NPAS2:BMAL1 heterodimers is regulated by the redox state of nicotinamide adenine dinucleotide (NAD) cofactors in a purified system. The reduced forms of the redox cofactors, NAD(H) and NADP(H), strongly enhance DNA binding of the Clock:BMAL1 and NPAS2:BMAL1 heterodimers, whereas the oxidized forms inhibit. These observations raise the possibility that food, neuronal activity, or both may entrain the circadian clock by direct modulation of cellular redox state.

https://science.sciencemag.org/content/sci/293/5529/510.full.pdf

Regulation of clock and NPAS2 DNA binding by the redox state of NAD cofactors.

1. The concentrations of the oxidized and reduced substrates of the lactate-, β-hydroxybutyrate- and glutamate-dehydrogenase systems were measured in rat livers freeze-clamped as soon as possible after death. The substrates of these dehydrogenases are likely to be in equilibrium with free NAD+ and NADH, and the ratio of the free dinucleotides can be calculated from the measured concentrations of the substrates and the equilibrium constants (Holzer, Schultz & Lynen, 1956; Bucher & Klingenberg, 1958). The lactate-dehydrogenase system reflects the [NAD+]/[NADH] ratio in the cytoplasm, the β-hydroxybutyrate dehydrogenase that in the mitochondrial cristae and the glutamate dehydrogenase that in the mitochondrial matrix. 2. The equilibrium constants of lactate dehydrogenase (EC 1.1.1.27), β-hydroxybutyrate dehydrogenase (EC 1.1.1.30) and malate dehydrogenase (EC 1.1.1.37) were redetermined for near-physiological conditions (38°; I0·25). 3. The mean [NAD+]/[NADH] ratio of rat-liver cytoplasm was calculated as 725 (pH7·0) in well-fed rats, 528 in starved rats and 208 in alloxan-diabetic rats. 4. The [NAD+]/[NADH] ratio for the mitochondrial matrix and cristae gave virtually identical values in the same metabolic state. This indicates that β-hydroxybutyrate dehydrogenase and glutamate dehydrogenase share a common pool of dinucleotide. 5. The mean [NAD+]/[NADH] ratio within the liver mitochondria of well-fed rats was about 8. It fell to about 5 in starvation and rose to about 10 in alloxan-diabetes. 6. The [NAD+]/[NADH] ratios of cytoplasm and mitochondria are thus greatly different and do not necessarily move in parallel when the metabolic state of the liver changes. 7. The ratios found for the free dinucleotides differ greatly from those recorded for the total dinucleotides because much more NADH than NAD+ is protein-bound. 8. The bearing of these findings on various problems, including the following, is discussed: the number of NAD+–NADH pools in liver cells; the applicability of the method to tissues other than liver; the transhydrogenase activity of glutamate dehydrogenase; the physiological significance of the difference of the redox states of mitochondria and cytoplasm; aspects of the regulation of the redox state of cell compartments; the steady-state concentration of mitochondrial oxaloacetate; the relations between the redox state of cell compartments and ketosis.

/pdf/the-redox-state-of-free-nicotinamide-adenine-dinucleotide-in-1jbnpqtsm5.pdf

The Redox State of Free Nicotinamide-Adenine Dinucleotide in the Cytoplasm and Mitochondria of Rat Liver

1. Slices of rat kidney cortex, on incubation in a saline medium, formed d-glucose from the following substances: d-fructose, d-galactose, d-mannose, l-sorbose, l-arabinose, d-xylose, glycerol, myo-inositol, l-iditol, sorbitol, xylitol, ribitol, methylglyoxal, dihydroxyacetone, l-glyceraldehyde, d-glyceraldehyde, dl-glyceraldehyde, dl-glycerate. Values for the rates of glucose formation from these precursors are given. 2. No glucose was formed from l-rhamnose, d-arabitol, d-arabinose, d-ribose, l-fucose, d-lyxose, mannitol, dulcitol, d-glucuronate, propane-1,2-diol and propan-2-ol. 3. The pathways of glucose formation from the various precursors are discussed (Scheme 1). 4. l-Glyceraldehyde inhibited the formation of glucose from d-glyceraldehyde.

Formation of glucose from hexoses, pentoses, polyols and related substances in kidney cortex.

1. With reference to the post-operative dysfunction of the liver observed after halothane anaesthesia, the effects of the anaesthetic on some metabolic functions were studied in the isolated perfused rat liver. Oxygen uptake, glycolysis, gluconeogenesis and urea synthesis were affected by halothane at a concentration (2.5% of the gas phase) within the range used in clinical anaesthesia. 2. At this concentration of halothane uptake of oxygen was inhibited in livers from both fed and starved rats. 3. In livers from fed rats there was a 16-fold increase in lactate production. This was accompanied by a fivefold decrease in the tissue content of 2-oxoglutarate and a more than twofold decrease in citrate. The calculated [free NAD+]/[free NADH] ratio in both cytoplasm and mitochondria was lower in the halothane-exposed livers than in controls. 4. In livers of starved rats the rate of gluconeogenesis from lactate was decreased by halothane to 30% of the control rate. 5. Halothane inhibited gluconeogenesis from alanine and propionate to the same extent as from lactate, whereas glucose formation from dihydroxyacetone, glycerol, fructose and sorbitol was relatively unaffected. 6. During gluconeogenesis from 10mm-lactate the tissue content of ATP was decreased by 50%, glutamate by 50% and 2-oxoglutarate was decreased eightfold in the halothane-exposed livers. 7. Halothane decreased urea synthesis in the presence of 10mm-NH4Cl and 2mm-ornithine to 15% of the control rate. 8. The inhibitions of gluconeogenesis and urea synthesis were completely abolished within 15min of withdrawal of the anaesthetic. 9. The stimulation of uptake of oxygen brought about by the addition of lactate or precursors of urea was abolished by halothane. 10. Effects on gluconeogenesis similar to those of halothane occurred in livers exposed to the anaesthetic methoxyflurane, although normal rates were not restored on withdrawal of the drug. Other anaesthetic agents tested (ketamine–HCl and trichloroethylene) decreased gluconeogenesis to 66% of the control rate. 11. The inhibitory effects of halothane are consistent with an interference at the stage of the NADH dehydrogenase of the electron-transport chain.

/pdf/the-effects-of-halothane-2-bromo-2-chloro-1-1-1-38n3t4q7vd.pdf

The effects of halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) on glycolysis and biosynthetic processes of the isolated perfused rat liver.

Inter-tissue nitrogen fluxes.

The technique of freeze-clamping has been used to study metabolic changes occurring in the livers of rats in vivo after acute or chronic exposure to halothane (2-bromo-2-chloro-1,1,1,-trifluoroethane), an anesthetic agent in common clinical use. For comparison similar experiments were carried out with methoxyflurane, trichlorethylene, or diethyl ether. In fed animals, exposure to halothane (1.5%, v/v) for 60 min caused glycogenolysis in the liver, with a significant decrease in tissue glycogen and increase in tissue and blood glucose concentrations. These changes were accompanied by a 50% decrease in the concentrations of 2-oxoglutarate and glutamate. The oxidation-reduction states of the NAD couple in both cytoplasm and mitochondria were unaffected by halothane. One hour after withdrawal of halothane the decrease in glycogen persisted, whereas glucose returned to normal within 30 min. On repeated exposure of animals to halothane (six successive periods of anesthesia for 60 min at 48-hr intervals) the glycogenolytic response was less marked, because of glycogen stores were not replenished between periods of anesthesia. At the end of the sixth anesthesia period the levels of 2-oxoglutarate and glutamate were 25% and 40% lower, respectively, than in the unanesthetized controls. Depletion of glycogen persisted for more than 3 days, but less than 4 weeks, after repeated exposure to halothane. None of the liver metabolite changes brought about by halothane in fed rats were found in rats fasted for 24 hr or in livers from rats adapted to a high-fat diet. Glycogenolysis was a metabolic response common to all four anesthetic agents tested. Only halothane brought about decreases in 2-oxoglutarate and glutamate concentrations: a single exposure (30 min) to trichlorethylene (1%, v/v, in O2) caused a 60% increase in 2-oxoglutarate concentration over the unanesthetized control value.

Patricia Lund

Papers

The Redox State of Free Nicotinamide-Adenine Dinucleotide in the Cytoplasm and Mitochondria of Rat Liver

Formation of glucose from hexoses, pentoses, polyols and related substances in kidney cortex.

The effects of halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) on glycolysis and biosynthetic processes of the isolated perfused rat liver.

Inter-tissue nitrogen fluxes.

Effects of halothane and other anesthetic agents on the concentrations of rat liver metabolites in vivo.