Integration of Eukaryotic Energy Metabolism: The Intramitochondrial and Cytosolic Energy States ([ATP]f/[ADP]f[Pi]).

Maintaining a robust, stable source of energy for doing chemical and physical work is essential to all living organisms. In eukaryotes, metabolic energy (ATP) production and consumption occurs in two separate compartments, the mitochondrial matrix and the cytosol. As a result, understanding eukaryotic metabolism requires knowledge of energy metabolism in each compartment and how metabolism in the two compartments is coordinated. Central to energy metabolism is the adenylate energy state ([ATP]/[ADP][Pi]). ATP is synthesized by oxidative phosphorylation (mitochondrial matrix) and glycolysis (cytosol) and each compartment provides the energy to do physical work and to drive energetically unfavorable chemical syntheses. The energy state in the cytoplasmic compartment has been established by analysis of near equilibrium metabolic reactions localized in that compartment. In the present paper, analysis is presented for energy-dependent reactions localized in the mitochondrial matrix using data obtained from both isolated mitochondria and intact tissues. It is concluded that the energy state ([ATP]f/[ADP]f[Pi]) in the mitochondrial matrix, calculated from the free (unbound) concentrations, is not different from the energy state in the cytoplasm. Corollaries are: (1) ADP in both the cytosol and matrix is selectively bound and the free concentrations are much lower than the total measured concentrations; and (2) under physiological conditions, the adenylate energy states in the mitochondrial matrix and cytoplasm are not substantially different.

Impact of sodium glucose linked cotransporter-2 inhibition on renal microvascular oxygen tension in a rodent model of diabetes mellitus.

BACKGROUND: The mechanisms whereby inhibitors of sodium-glucose linked cotransporter-2 (SGLT2) exert their nephroprotective effects in patients with diabetes are incompletely understood but have been hypothesized to include improved tissue oxygen tension within the renal cortex. The impact of SGLT2 inhibition is likely complex and region specific within the kidney. We hypothesize that SGLT2 inhibitors have differential effects on renal tissue oxygen delivery and consumption in specific regions of the diabetic kidney, including the superficial cortex, containing SGLT2-rich components of proximal tubules, versus the deeper cortex and outer medulla, containing predominantly SGLT1 receptors. METHODS: We measured glomerular filtration rate (GFR), microvascular kidney oxygen tension (Pk O2 ), erythropoietin (EPO) mRNA, and reticulocyte count in diabetic rats (streptozotocin) treated with the SGLT2 inhibitor, dapagliflozin. Utilizing phosphorescence quenching by oxygen and an intravascular oxygen sensitive probe (Oxyphor PdG4); we explored the effects of SGLT2 inhibition on Pk O2 in a region-specific manner, in vivo, in diabetic and non-diabetic rats. Superficial renal cortical or deeper cortical and outer medullary Pk O2 were measured utilizing excitations with blue and red light wavelengths, respectively. RESULTS: In diabetic rats treated with dapagliflozin, measurement within the superficial cortex (blue light) demonstrated no change in Pk O2 . By contrast, measurements in the deeper cortex and outer medulla (red light) demonstrated a significant reduction in Pk O2 in dapagliflozin treated diabetic rats (p = 0.014). Consistent with these findings, GFR was decreased, hypoxia-responsive EPO mRNA levels were elevated and reticulocyte counts were increased with SGLT2 inhibition in diabetic rats (p < 0.05 for all). CONCLUSIONS: These findings indicate that microvascular kidney oxygen tension is maintained in the superficial cortex but reduced in deeper cortical and outer medullary tissue, possibly due to the regional impact of SGLT-2 inhibition on tissue metabolism. This reduction in deeper Pk O2 had biological impact as demonstrated by increased renal EPO mRNA levels and circulating reticulocyte count.

Metabolic Homeostasis in Life as We Know It: Its Origin and Thermodynamic Basis.

Living organisms require continuous input of energy for their existence. As a result, life as we know it is based on metabolic processes that extract energy from the environment and make it available to support life (energy metabolism). This metabolism is based on, and regulated by, the underlying thermodynamics. This is important because thermodynamic parameters are stable whereas kinetic parameters are highly variable. Thermodynamic control of metabolism is exerted through near equilibrium reactions that determine. (1) the concentrations of metabolic substrates for enzymes that catalyze irreversible steps and (2) the concentrations of small molecules (AMP, ADP, etc.) that regulate the activity of irreversible reactions in metabolic pathways. The result is a robust homeostatic set point (-ΔGATP) with long term (virtually unlimited) stability. The rest of metabolism and its regulation is constrained to maintain this set point. Thermodynamic control is illustrated using the ATP producing part of glycolysis, glyceraldehyde-3-phosphate oxidation to pyruvate. Flux through the irreversible reaction, pyruvate kinase (PK), is primarily determined by the rate of ATP consumption. Change in the rate of ATP consumption causes mismatch between use and production of ATP. The resulting change in [ATP]/[ADP][Pi], through near equilibrium of the reactions preceding PK, alters the concentrations of ADP and phosphoenolpyruvate (PEP), the substrates for PK. The changes in ADP and PEP alter flux through PK appropriately for restoring equality of ATP production and consumption. These reactions appeared in the very earliest lifeforms and are hypothesized to have established the set point for energy metabolism. As evolution included more metabolic functions, additional layers of control were needed to integrate new functions into existing metabolism without changing the homeostatic set point. Addition of gluconeogenesis, for example, resulted in added regulation to PK activity to prevent futile cycling; PK needs to be turned off during gluconeogenesis because flux through the enzyme would waste energy (ATP), subtracting from net glucose synthesis and decreasing overall efficiency.

Renal microvascular oxygen tension during hyperoxia and acute hemodilution assessed by phosphorescence quenching and excitation with blue and red light.

PURPOSE: The kidney plays a central physiologic role as an oxygen sensor. Nevertheless, the direct mechanism by which this occurs is incompletely understood. We measured renal microvascular partial pressure of oxygen (PkO2) to determine the impact of clinically relevant conditions that acutely change PkO2 including hyperoxia and hemodilution. METHODS: We utilized two-wavelength excitation (red and blue spectrum) of the intravascular phosphorescent oxygen sensitive probe Oxyphor PdG4 to measure renal tissue PO2 in anesthetized rats (2% isoflurane, n = 6) under two conditions of altered arterial blood oxygen content (CaO2): 1) hyperoxia (fractional inspired oxygen 21%, 30%, and 50%) and 2) acute hemodilutional anemia (baseline, 25% and 50% acute hemodilution). The mean arterial blood pressure (MAP), rectal temperature, arterial blood gases (ABGs), and chemistry (radiometer) were measured under each condition. Blue and red light enabled measurement of PkO2 in the superficial renal cortex and deeper cortical and medullary tissue, respectively. RESULTS: PkO2 was higher in the superficial renal cortex (~ 60 mmHg, blue light) relative to the deeper renal cortex and outer medulla (~ 45 mmHg, red light). Hyperoxia resulted in a proportional increase in PkO2 values while hemodilution decreased microvascular PkO2 in a linear manner in both superficial and deeper regions of the kidney. In both cases (blue and red light), PkO2 correlated with CaO2 but not with MAP. CONCLUSION: The observed linear relationship between CaO2 and PkO2 shows the biological function of the kidney as a quantitative sensor of anemic hypoxia and hyperoxia. A better understanding of the impact of changes in PkO2 may inform clinical practices to improve renal oxygen delivery and prevent acute kidney injury.

RéSUMé: OBJECTIF: Les reins jouent un rôle physiologique central en tant que détecteurs d'oxygène. Cependant, le mécanisme direct de ce rôle n'est pas complètement compris. Nous avons mesuré la pression partielle d'oxygène microvasculaire rénal (PkO2) afin de déterminer l'impact de conditions pertinentes d'un point de vue clinique qui modifient de façon aiguë la PkO2, y compris l'hyperoxie et l'hémodilution. MéTHODE: Nous avons utilisé l'excitation à deux longueurs d'onde (spectres rouge et bleu) de la sonde phosphorescente, sensible à l'oxygène, intravasculaire Oxyphor PdG4 afin de mesurer la PO2 dans le tissu rénal de rats sous anesthésie (isoflurane 2 %, n = 6) dans deux conditions de contenu en oxygène du sang artériel (CaO2) altéré : 1) hyperoxie (fraction d'oxygène inspiré 21 %, 30 % et 50 %) et 2) anémie par hémodilution aiguë (valeurs de base, hémodilution aiguë 25 % et 50 %). La tension artérielle moyenne (TAM), la température rectale, les gaz sanguins artériels et la chimie (radiomètre) ont été mesurés dans chacune des conditions. Les lumières bleue et rouge ont permis de mesurer la PkO2 dans le cortex rénal superficiel et les tissus cortical et médullaire plus profonds, respectivement. RéSULTATS: La PkO2 était plus élevée dans le cortex rénal superficiel (~ 60 mmHg, lumière bleue) comparativement au cortex rénal plus profond et à la zone médullaire extérieure (~ 45 mmHg, lumière rouge). L'hyperoxie a entraîné une augmentation proportionnelle des valeurs de PkO2, alors que l'hémodilution a diminué la PkO2 microvasculaire de façon linéaire tant dans les régions rénales superficielles que plus profondes. Dans les deux cas (lumières bleue et rouge), la PkO2 était corrélée au CaO2 mais pas à la TAM. CONCLUSION: La relation linéaire observée entre le CaO2 et la PkO2 montre la fonction biologique du rein en tant que détecteur quantitatif de l'hypoxie anémique et de l'hyperoxie. Une meilleure compréhension de l'impact des changements de la PkO2 pourrait guider les pratiques cliniques afin d'améliorer la distribution d'oxygène aux reins et prévenir l'insuffisance rénale aiguë.

Cerebrovascular Blood Flow Design and Regulation; Vulnerability in Aging Brain.

Nutrient delivery to the brain presents a unique challenge because the tissue functions as a computer system with in the order of 200,000 neurons/mm3. Penetrating arterioles bud from surface arteries of the brain and penetrate downward through the cortex. Capillary networks spread from penetrating arterioles through the surrounding tissue. Each penetrating arteriole forms a vascular unit, with little sharing of flow among vascular units (collateral flow). Unlike cells in other tissues, neurons have to be operationally isolated, interacting with other neurons through specific electrical connections. Neuronal activation typically involves only a few of the cells within a vascular unit, but the local increase in nutrient consumption is substantial. The metabolic response to activation is transmitted to the feeding arteriole through the endothelium of neighboring capillaries and alters calcium permeability of smooth muscle in the wall resulting in modulation of flow through the entire vascular unit. Many age and trauma related brain pathologies can be traced to vascular malfunction. This includes: 1. Physical damage such as in traumatic injury with imposed shear stress as soft tissue moves relative to the skull. Lack of collateral flow among vascular units results in death of the cells in that vascular unit and loss of brain tissue. 2. Age dependent changes lead to progressive increase in vascular resistance and decrease in tissue levels of oxygen and glucose. Chronic hypoxia/hypoglycemia compromises tissue energy metabolism and related regulatory processes. This alters stem cell proliferation and differentiation, undermines vascular integrity, and suppresses critical repair mechanisms such as oligodendrocyte generation and maturation. Reduced structural integrity results in local regions of acute hypoxia and microbleeds, while failure of oligodendrocytes to fully mature leads to poor axonal myelination and defective neuronal function. Understanding and treating age related pathologies, particularly in brain, requires better knowledge of why and how vasculature changes with age. That knowledge will, hopefully, make possible drugs/methods for protecting vascular function, substantially alleviating the negative health and cognitive deficits associated with growing old.

Ethanol metabolism: The good, the bad, and the ugly.

Throughout the world, ethanol is both an important commercial commodity and a source of major medical and social problems. Ethanol readily passes through biological membranes and distributes throughout the body. It is oxidized, first to acetaldehyde and then to acetate, and finally by the citric acid cycle in virtually all tissues. The oxidation of ethanol is irreversible and unregulated, making the rate dependent only on local concentration and enzyme activity. This unregulated input of reducing equivalents increases reduction of both cytoplasmic and intramitochondrial NAD and, through the latter, cellular energy state {[ATP]/([ADP][Pi])}. In brain, this increase in energy state stimulates dopaminergic neural activity signalling reward and a sense of well being, while suppressing glutamatergic neural activity signalling anxiety and unease. These positive responses to ethanol ingestion are important to social alcohol consumption. Importantly, decreased free [AMP] decreases AMP-dependent protein kinase (AMPK) activity, an important regulator of cellular energy metabolism. Oxidation of substrates used for energy metabolism in the absence of ethanol is down regulated to accommodate the input from ethanol. In liver, chronic ethanol metabolism results in fatty liver and general metabolic dysfunction. In brain, transport of other oxidizable metabolites through the blood-brain barrier and the enzymes for their oxidation are both down regulated. For exposures of short duration, ethanol induced regulatory changes are rapid and reversible, recovering completely when the concentrations of ethanol and acetate fall again. Longer periods of ethanol exposure and associated chronic suppression of AMPK activity activates regulatory mechanisms, including gene expression, that operate over longer time scales, both in onset and reversal. If chronic alcohol consumption is abruptly ended, metabolism is no longer able to respond rapidly enough to compensate. Glutamatergic neural activity adapts to chronic dysregulation of glutamate metabolism and suppression of glutamatergic neural activity by increasing excitatory and decreasing inhibitory amino acid receptors. A point is reached (ethanol dependence) where withdrawal of ethanol results in significant metabolic energy depletion in neurons and other brain cells as well as hyperexcitation of the glutamatergic system. The extent and regional specificity of energy depletion in the brain, combined with hyperactivity of the glutamatergic neuronal system, largely determines the severity of withdrawal symptoms.

Renal tissue Po2 sensing during acute hemodilution is dependent on the diluent.

Sensing changes in blood oxygen content ([Formula: see text]) is an important physiological role of the kidney; however, the mechanism(s) by which the kidneys sense and respond to changes in [Formula: see text] are incompletely understood. Accurate measurements of kidney tissue oxygen tension ([Formula: see text]) may increase our understanding of renal oxygen-sensing mechanisms and could inform decisions regarding the optimal fluid for intravascular volume resuscitation to maintain renal perfusion. In some clinical settings, starch solution may be nephrotoxic, possibly due to inadequacy of tissue oxygen delivery. We hypothesized that hemodilution with starch colloid solutions would reduce [Formula: see text] to a more severe degree than other diluents. Anesthetized Sprague-Dawley rats (n = 77) were randomized to undergo hemodilution with either colloid (6% hydroxyethyl starch or 5% albumin), crystalloid (0.9% saline), or a sham procedure (control) (n = 13-18 rats/group). Data were analyzed by ANOVA with significance assigned at P < 0.05. After hemodilution, mean arterial pressure (MAP) decreased marginally in all groups, while hemoglobin (Hb) and [Formula: see text] decreased in proportion to the degree of hemodilution. Cardiac output was maintained in all groups after hemodilution. [Formula: see text] decreased in proportion to the reduction in Hb in all treatment groups. At comparably reduced Hb, and maintained arterial oxygen values, hemodilution with starch resulted in larger decreases in [Formula: see text] relative to animals hemodiluted with albumin or saline (P < 0.008). Renal medullary erythropoietin (EPO) mRNA levels increased more prominently, relative to other hypoxia-regulated molecules (GLUT-1, GAPDH, and VEGF). Our data demonstrate that the kidney acts as a biosensor of reduced [Formula: see text] following hemodilution and that [Formula: see text] may provide a quantitative signal for renal cellular responsiveness to acute anemia. Evidence of a more severe reduction in [Formula: see text] following hemodilution with starch colloid solution suggests that tissue hypoxia may contribute to starch induced renal toxicity.

Hyperbaric oxygen toxicity in brain: A case of hyperoxia induced hypoglycemic brain syndrome.

Hyperbaric oxygen exposure is a recent hazzard for higher animals that originated as humans began underwater construction, exploration, and sports. Exposure can lead to abnormal brain EEG, convulsions, and death, the time to onset of each stage of pathology decreasing with increase in oxygen pressure. We provide evidence that hyperoxia, through oxidative phosphorylation, increases the energy state ([ATP]/[ADP][Pi]) of cells critical to providing glucose to cells behind the blood brain barrier (BBB). Brain cells without an absolute dependence on glucose metabolism; i.e. those having sufficient ATP synthesis using lactate and glutamate as oxidizable substrates, are not themselves very adversely affected by hyperoxia. The increased energy state and decrease in free [AMP], however, suppress glucose transport through the blood brain barrier (BBB) and into cells behind the BBB. Glucose has to pass in sequence through three steps of transport by facilitated diffusion and transporter activity for each step is regulated in part by AMP dependent protein kinase. The physiological role of this regulation is to increase glucose transport in response to hypoxia and/or systemic hypoglycemia. Hyperoxia, however, through unphysiological decrease in free [AMP] suppresses 1) glucose transport through the BBB (endothelial GLUT1 transporters) into cerebrospinal fluid (CSF); 2) glucose transport from CSF into cells behind the BBB (GLUT3 transporters) and (GLUT4 transporters). Cumulative suppression of glucose transport results in local regions of hypoglycemia and induces hypoglycemic failure. It is suggested that failure is initiated at axons and synapses with insufficient mitochondria to meet their energy requirements.

The Central Role of Glucokinase in Glucose Homeostasis: A Perspective 50 Years After Demonstrating the Presence of the Enzyme in Islets of Langerhans.

It is hypothesized that glucokinase (GCK) is the glucose sensor not only for regulation of insulin release by pancreatic ß-cells, but also for the rest of the cells that contribute to glucose homeostasis in mammals. This includes other cells in endocrine pancreas (α- and δ-cells), adrenal gland, glucose sensitive neurons, entero-endocrine cells, and cells in the anterior pituitary. Glucose transport is by facilitated diffusion and is not rate limiting. Once inside, glucose is phosphorylated to glucose-6-phosphate by GCK in a reaction that is dependent on glucose throughout the physiological range of concentrations, is irreversible, and not product inhibited. High glycerol phosphate shuttle, pyruvate dehydrogenase, and pyruvate carboxylase activities, combined with low pentose-P shunt, lactate dehydrogenase, plasma membrane monocarboxylate transport, and glycogen synthase activities constrain glucose-6-phosphate to being metabolized through glycolysis. Under these conditions, glycolysis produces mostly pyruvate and little lactate. Pyruvate either enters the citric acid cycle through pyruvate dehydrogenase or is carboxylated by pyruvate carboxylase. Reducing equivalents from glycolysis enter oxidative phosphorylation through both the glycerol phosphate shuttle and citric acid cycle. Raising glucose concentration increases intramitochondrial [NADH]/[NAD+] and thereby the energy state ([ATP]/[ADP][Pi]), decreasing [Mg2+ADP] and [AMP]. [Mg2+ADP] acts through control of KATP channel conductance, whereas [AMP] acts through regulation of AMP-dependent protein kinase. Specific roles of different cell types are determined by the diverse molecular mechanisms used to couple energy state to cell specific responses. Having a common glucose sensor couples complementary regulatory mechanisms into a tightly regulated and stable glucose homeostatic network.

Oxygen dependence of glucose sensing: role in glucose homeostasis and related pathology.

In glucose homeostasis, glucose concentration is sensed by its metabolism through glucokinase (GCK) and oxidative phosphorylation. Because oxidative phosphorylation is an integral part of the sensory system, glucose sensing is necessarily dependent on oxygen pressure. Much of the dependence on oxygen is suppressed by location of glucose sensing cells in tissues with well-regulated blood flow. In healthy individuals the oxygen dependence is primarily observed in response to transient global hypoxia events such as during birth or transition to high altitude. The GCK sensing system is, however, used to control release of both insulin and glucagon, the preeminant hormonal regulators of blood glucose, as well as glucose sensitive neuronal activity. Suppression of oxygen delivery to glucose-sensing cells or interference with regulation of tissue blood flow by either local or systemic causes, stresses the glucose regulatory system. This is true whether the stress is imposed locally, such as by altered oxygen delivery to the pancreas, or globally, as in pulmonary insufficiency or exposure to high altitude. It may be expected that chronic application of this stress predisposes individuals to developing diabetes. Type 2 diabetes is a broad class of diseases characterized by disturbance of glucose homeostasis, i.e., having either hyperglycemia and/or decreased sensitivity to insulin. Given the role of oxidative phosphorylation in glucose sensing, tissue oxygen deprivation may predispose individuals to developing diabetes as well as contributing to the disease itself. This is particularly true in age-related diabetes because the incidence of vascular insufficiency increases markedly with increasing age. NEW & NOTEWORTHY Glucose sensing requires glucose metabolism through glycolysis and oxidative phosphorylation. Dependence of the latter on oxygen concentration imposes an oxygen dependence on glucose sensing. We have used a validated computational model to quantify that dependence. Evidence is presented that tissue oxygenation plays an important role in predisposition of individuals to developing type 2 diabetes and in progression of the disease.

Metabolic homeostasis: oxidative phosphorylation and the metabolic requirements of higher plants and animals.

A model of oxidative phosphorylation and its regulation is presented, which is consistent with the experimental data on metabolism in higher plants and animals. The variables that provide real-time control of metabolic status are: intramitochondrial [NAD+]/[NADH], energy state ([ATP]/[ADP][Pi]), and local oxygen concentration ([O2]). ATP consumption and respiratory chain enzyme content are tissue specific (liver vs. heart muscle), and the latter is modulated by chronic alterations in ATP consumption (i.e., endurance training etc.). ATP consumption affects the energy state, which increases or decreases as necessary to match synthesis with consumption. [NAD+]/[NADH], local [O2], and respiratory chain content determine the energy state at which match of synthesis and utilization is achieved. Tissues vary widely in their ranges of ATP consumption. Expressed as the turnover of cytochrome c, the rates may change little (7 to 12/s) (liver) or a lot (1 to >300/s) (flight muscle of birds, bats, and insects). Ancillary metabolic pathways, including creatine or arginine kinase, glycerol phosphate shuttle, fatty acid, and citric acid cycle dehydrogenases, are responsible for meeting tissue-specific differences in maximal rate and range in ATP utilization without displacing metabolic homeostasis. Intramitochondrial [NAD+]/[NADH], [ATP], and [Pi] are adjusted to keep [ADP] and [AMP] similar for all tissues despite large differences in ranges in ATP utilization. This is essential because [ADP] and [AMP], particularly the latter, have major roles in regulating the activity of many enzymes and signaling pathways (AMP deaminase, AMP dependent protein kinases, etc.) common to all higher plants and animals.NEW & NOTEWORTHY Oxidative phosphorylation has an intrinsic program that sets and stabilizes cellular energy state ([ATP]/[ADP][Pi]), and thereby metabolic homeostasis. A computational model consistent with regulation of oxidative phosphorylation in higher plants and animals is presented. Focus is on metabolism ancillary to oxidative phosphorylation by which it was integrated into preexisting metabolic regulation and adapted by evolution to develop cells and tissues with differing rates of ATP utilization: i.e., liver versus brain versus muscle.

Glutamate dehydrogenase: role in regulating metabolism and insulin release in pancreatic ß-cells.

Regulation of insulin release and glucose homeostasis by pancreatic ß-cells is dependent on the metabolism of glucose by glucokinase (GK) and the influence of that activity on oxidative phosphorylation. Genetic alterations that result in hyperactivity of mitochondrial glutamate dehydrogenase (GDH-1) can cause hypoglycemia-hyperammonemia following high protein meals, but the role of GDH-1 remains poorly understood. GDH-1 activity is strongly inhibited by GTP, to near zero in the absence of ADP, and cooperatively activated ( n = 2.3) by ADP. The dissociation constant for ADP is near 200 µM in vivo, but leucine and its nonmetabolized analog 2-amino-2-norbornane-carboxylic acid (BCH) can activate GDH-1 by increasing the affinity for ADP. Under physiological conditions, as [ADP] increases GDH-1 activity remains very low until ~35 µM (threshold) and then increases rapidly. A model for GDH-1 and its regulation has been combined with a previously published model for glucose sensing that coupled GK activity and oxidative phosphorylation. The combined model (GK-GDH-core) shows that GK activity determines the energy state ([ATP]/[ADP][Pi]) in ß-cells for glucose concentrations > 5 mM ([ADP] < 35 µM). As glucose falls < 5 mM the [ADP]-dependent increase in GDH-1 activity prevents [ADP] from rising above ~70 µM. Thus, GDH-1 dynamically buffers ß-cell energy metabolism during hypoglycemia, maintaining the energy state and the basal rate of insulin release. GDH-1 hyperactivity suppresses the normal increase in [ADP] in hypoglycemia. This leads to hypoglycemia following a high protein meal by increasing the basal rate of insulin release (ß-cells) and decreasing glucagon release (α-cells). NEW & NOTEWORTHY A model of ß-cell metabolism and regulation of insulin release is presented. The model integrates regulation of oxidative phosphorylation, glucokinase (GK), and glutamate dehydrogenase (GDH-1). GDH-1 is near equilibrium under physiological conditions, but the activity is inhibited by GTP. In hypoglycemia, however, GK activity is low and [ADP], a potent activator of GDH-1, increases. Reducing equivalents from GDH dynamically buffers the intramitochondrial [NADH]/[NAD+], and thereby the energy state, preventing hypoglycemia-induced substrate deprivation.

Oxidative phosphorylation: regulation and role in cellular and tissue metabolism.

Oxidative phosphorylation provides most of the ATP that higher animals and plants use to support life and is responsible for setting and maintaining metabolic homeostasis. The pathway incorporates three consecutive near equilibrium steps for moving reducing equivalents between the intramitochondrial [NAD+ ]/[NADH] pool to molecular oxygen, with irreversible reduction of oxygen to bound peroxide at cytochrome c oxidase determining the net flux. Net flux (oxygen consumption rate) is determined by demand for ATP, with feedback by the energy state ([ATP]/[ADP][Pi ]) regulating the pathway. This feedback affects the reversible steps equally and independently, resulting in the rate being coupled to ([ATP]/[ADP][Pi ])3 . With increasing energy state, oxygen consumption decreases rapidly until a threshold is reached, above which there is little further decrease. In most cells, [ATP] and [Pi ] are much higher than [ADP] and change in [ADP] is primarily responsible for the change in energy state. As a result, the rate of ATP synthesis, plotted against [ADP], remains low until [ADP] reaches about 30 µm and then increases rapidly with further increase in [ADP]. The dependencies on energy state and [ADP] near the threshold can be fitted by the Hill equation with a Hill coefficients of about -2.6 and 4.2, respectively. The homeostatic set point for metabolism is determined by the threshold, which can be modulated by the PO2 and intramitochondrial [NAD+ ]/[NADH]. The ability of oxidative phosphorylation to precisely set and maintain metabolic homeostasis is consistent with it being permissive of, and essential to, development of higher plants and animals.

Granulocyte colony-stimulating factor significantly decreases density of hippocampal caspase 3-positive nuclei, thus ameliorating apoptosis-mediated damage, in a model of ischaemic neonatal brain injury.

OBJECTIVES: Ischaemic brain injury is a major complication in patients undergoing surgery for congenital heart disease, with the hippocampus being a particularly vulnerable region. We hypothesized that neuronal injury resulting from cardiopulmonary bypass and associated circulatory arrest is ameliorated by pretreatment with granulocyte colony-stimulating factor (G-CSF), a cytokine and an anti-apoptotic neurotrophic factor. METHODS: In a model of ischaemic brain injury, 4 male newborn piglets were anaesthetized and subjected to deep hypothermic circulatory arrest (DHCA) (cooled to 18°C, DHCA maintained for 60 min, rewarmed and recovered for 8-9 h), while 4 animals received G-CSF (34 µg/kg, intravenously) 2 h prior to the DHCA procedure. At the end of each experiment, the animals were perfused with a fixative, the hippocampus was extracted, cryoprotected, cut and the brain sections were immunoprocessed for activated caspase 3, a pro-apoptotic factor. Immunopositive neuronal nuclei were counted in multiple counting boxes (440 × 330 µm) centred on the CA1 or CA3 hippocampal regions and their mean numbers compared between the different treatment groups and regions. RESULTS: G-CSF pretreatment resulted in significantly lower counts of caspase 3-positive nuclei per counting box in both the CA1 [52.2 ± 9.3 (SD) vs 61.6 ± 8.4, P < 0.001] and CA3 (41.2 ± 6.9 vs 60.4 ± 16.4, P < 0.00002) regions of the hippocampus as compared to DHCA groups. The effects of G-CSF were significant for pyramidal cells of both regions and for interneurons in the CA3 region. CONCLUSIONS: In an animal model of ischaemic brain injury, G-CSF reduces neuronal injury in the hippocampus, thus potentially having beneficial effect on neurologic outcomes.

The thermodynamic basis of glucose-stimulated insulin release: a model of the core mechanism.

A model for glucose sensing by pancreatic ß-cells is developed and compared with the available experimental data. The model brings together mathematical representations for the activities of the glucose sensor, glucokinase, and oxidative phosphorylation. Glucokinase produces glucose 6-phosphate (G-6-P) in an irreversible reaction that determines glycolytic flux. The primary products of glycolysis are NADH and pyruvate. The NADH is reoxidized and the reducing equivalents transferred to oxidative phosphorylation by the glycerol phosphate shuttle, and some of the pyruvate is oxidized by pyruvate dehydrogenase and enters the citric acid cycle. These reactions are irreversible and result in a glucose concentration-dependent reduction of the intramitochondrial NAD pool. This increases the electrochemical energy coupled to ATP synthesis and thereby the cellular energy state ([ATP]/[ADP][Pi]). ATP and Pi are 10-100 times greater than ADP, so the increase in energy state is primarily through decrease in ADP The decrease in ADP is considered responsible for altering ion channel conductance and releasing insulin. Applied to the reported glucose concentration-dependent release of insulin by perifused islet preparations (Doliba et al. 2012), the model predicts that the dependence of insulin release on ADP is strongly cooperative with a threshold of about 30 µmol/L and a negative Hill coefficient near -5.5. The predicted cellular energy state, ADP, creatine phosphate/creatine ratio, and cytochrome c reduction, including their dependence on glucose concentration, are consistent with experimental data. The ability of the model to predict behavior consistent with experiment is an invaluable resource for understanding glucose sensing and planning experiments.

Experimental assessment of oxygen homeostasis during acute hemodilution: the integrated role of hemoglobin concentration and blood pressure.

BACKGROUND: Low hemoglobin concentration (Hb) and low mean arterial blood pressure (MAP) impact outcomes in critically ill patients. We utilized an experimental model of "normotensive" vs. "hypotensive" acute hemodilutional anemia to test whether optimal tissue perfusion is dependent on both Hb and MAP during acute blood loss and fluid resuscitation, and to assess the value of direct measurements of the partial pressure of oxygen in tissue (PtO2). METHODS: Twenty-nine anesthetized rats underwent 40% isovolemic hemodilution (1:1) (or sham-hemodilution control, n = 4) with either hydroxyethyl starch (HES) (n = 14, normotensive anemia) or saline (n = 11, hypotensive anemia) to reach a target Hb value near 70 g/L. The partial pressure of oxygen in the brain and skeletal muscle tissue (PtO2) were measured by phosphorescence quenching of oxygen using G4 Oxyphor. Mean arterial pressure (MAP), heart rate, temperature, arterial and venous co-oximetry, blood gases, and lactate were assessed at baseline and for 60 min after hemodilution. Cardiac output (CO) was measured at baseline and immediately after hemodilution. Data were analyzed by repeated measures two-way ANOVA. RESULTS: Following "normotensive" hemodilution with HES, Hb was reduced to 66 ± 6 g/L, CO increased (p < 0.05), and MAP was maintained. These conditions resulted in a reduction in brain PtO2 (22.1 ± 5.6 mmHg to 17.5 ± 4.4 mmHg, p < 0.05), unchanged muscle PO2, and an increase in venous oxygen extraction. Following "hypotensive" hemodilution with saline, Hb was reduced to 79 ± 5 g/L and both CO and MAP were decreased (P < 0.05). These conditions resulted in a more severe reduction in brain PtO2 (23.2 ± 8.2 to 10.7 ± 3.6 mmHg (p < 0.05), a reduction in muscle PtO2 (44.5 ± 11.0 to 19.9 ± 12.4 mmHg, p < 0.05), a further increase in venous oxygen extraction, and a threefold increase in systemic lactate levels (p < 0.05). CONCLUSIONS: Acute normotensive anemia (HES hemodilution) was associated with a subtle decrease in brain tissue PtO2 without clear evidence of global tissue hypoperfusion. By contrast, acute hypotensive anemia (saline hemodilution) resulted in a profound decrease in both brain and muscle tissue PtO2 and evidence of inadequate global perfusion (lactic acidosis). These data emphasize the importance of maintaining CO and MAP to ensure adequacy of vital organ oxygen delivery during acute anemia. Improved methods of assessing PtO2 may provide an earlier warning signal of vital organ hypoperfusion.

Oxidative phosphorylation: unique regulatory mechanism and role in metabolic homeostasis.

Oxidative phosphorylation is the primary source of metabolic energy, in the form of ATP, in higher plants and animals, but its regulation in vivo is not well understood. A model has been developed for oxidative phosphorylation in vivo that predicts behavior patterns that are both distinctive and consistent with experimental measurements of metabolism in intact cells and tissues. A major regulatory parameter is the energy state ([ATP]/[ADP][Pi], where brackets denote concentration). Under physiological conditions, the [ATP] and [Pi] are ~100 times that of [ADP], and most of the change in energy state is through change in [ADP]. The rate of oxidative phosphorylation (y-axis) increases slowly with increasing [ADP] until a threshold is reached and then increases very rapidly and linearly with further increase in [ADP]. The dependence on [ADP] can be characterized by a threshold [ADP] (T) and control strength (CS), the normalized slope above threshold (Δy/(Δx/T). For normoxic cells without creatine kinase, T is ~30 µM and CS is ~10 s-1 Myocytes and cells with larger ranges of rates of ATP utilization, however, have the same [ADP]- and [AMP]-dependent mechanisms regulating metabolism and gene expression. To compensate, these cells have creatine kinase, and hydrolysis/synthesis of creatine phosphate increases the change in [Pi] and thereby CS. Cells with creatine kinase have [ADP] and [AMP], which are similar to cells without creatine kinase, despite the large differences in metabolic rate. 31P measurements in human muscles during work-to-rest and rest-to-work transitions are consistent with predictions of the model.NEW & NOTEWORTHY A model developed for oxidative phosphorylation in vivo is shown to predict behavior patterns that are both novel and consistent with experimental measurements of metabolism in working muscle and other cells. The dependence of the rate on ADP concentration shows a pronounced threshold with a steep, nearly linear increase above the threshold. The threshold determines the homeostatic set point, and the slope above threshold determines how much metabolism changes in response to varied energy demand.

EVIDENCE OF EPSTEIN-BARR VIRUS ASSOCIATION WITH HEAD AND NECK CANCERS: A REVIEW.

Epstein-Barr virus (EBV) is ubiquitous: over 90% of the adult population is infected with this virus. EBV is capable of infecting both B lymphocytes and epithelial cells throughout the body including the head and neck region. Transmission occurs mainly by exchange of saliva. The infection is asymptomatic or mild in children but, in adolescents and young adults, it causes infectious mononucleosis, a self-limiting disease characterized by lethargy, sore throat, fever and lymphadenopathy. Once established, the virus often remains latent and people become lifelong carriers without experiencing disease. However, in some people, the latent virus is capable of causing malignant tumours, such as nasopharyngeal carcinoma and various B- and T-cell lymphomas, at sites including the head, neck and oropharyngeal region. As lymphoma is the second-most common malignant disease of the head, neck and oral region after squamous cell carcinoma, oral health care workers including dentists and specialists have a responsibility to carry out a thorough clinical examination of this anatomical region with a view to identifying and diagnosing lesions that may represent lymphomas. Early detection allows early treatment resulting in better prognosis. The focus of this review is on the morphology, transmission and carcinogenic properties of EBV and clinical and diagnostic aspects of a range of EBV-associated malignancies occurring in the head, neck and oral region. As carcinogenic agents, viruses contribute to a significant proportion of the global cancer burden: approximately 15% of all human cancers, worldwide, are attributable to viruses.1,2 Serologic and epidemiologic studies are providing mounting evidence of an etiologic association between viruses and head and neck malignancies.3 To update oral and maxillofacial surgeons and oral medicine specialists and raise awareness of this association, we recently reviewed the evidence of the etiologic role of human papillomavirus in oral disease.4 In this paper, we review the current state of knowledge of the association of Epstein-Barr virus (EBV) with malignant diseases in the head and neck region.

Regulation of metabolism: the work-to-rest transition in skeletal muscle.

The behavior of oxidative phosphorylation predicted by a model for the mechanism and kinetics of cytochrome c oxidase is compared with the experimentally observed behavior during the work-to-rest transition in skeletal muscle. For both experiment and model, when work stops, the increase in creatine phosphate and decrease in creatine and inorganic phosphate concentrations ([CrP], [Cr], and [Pi]) begin immediately. The rate of change for each is maximal and then progressively slows as the increasing energy state ([ATP]/[ADP][Pi]) suppresses the rate of oxidative phosphorylation. The time courses can be reasonably fitted to single exponential curves with similar time constants. The energy state in the working and resting steady states at constant Po2 are dependent on the intramitochondrial [NAD+]/[NADH], mitochondrial content, and size of the creatine pool ([CrP] + [Cr]). The rate of change in [CrP] is linearly correlated with [CrP] and with [Pi] and [Cr]. The time constant for [CrP] increase in the resting and working steady states, and the rate of decrease in oxygen consumption are similarly dependent on the Po2 in the inspired gas (experimental) or tissue Po2 (model). Myoglobin strongly buffers intracellular Po2 below ∼15 torr, truncating the low end of the oxygen distribution in the tissue and suppressing intra- and intermyocyte oxygen gradients. The predictions of the model are consistent with the experimental data throughout the work/rest transition, providing valuable insights into the regulation of cellular and tissue metabolism.