Ryan A.V. Bell

Regulation of liver glutamate dehydrogenase by reversible phosphorylation in a hibernating mammal

Glutamate dehydrogenase (GDH) is a key enzyme that links amino acid and carbohydrate metabolism in cells. Regulation is likely most important when organisms are confronted with extreme stresses such as the low environmental temperatures and lack of food associated with winter. Many small mammals, such as Richardsons ground squirrels, Spermophilus richardsonii, cope with these conditions by hibernating. Animals enter long periods of profound torpor where metabolic rate is greatly suppressed, body temperature drops to near-ambient and all metabolic needs must be met from fixed internal body stores of fuels. To investigate how GDH is regulated under these conditions, kinetic properties of GDH were analyzed in liver from euthermic and torpid squirrels, revealing significant differences in V(max), K(m) glutamate, K(a) ADP and inhibition by urea between the two forms of GDH. These data suggested an activation of the glutamate-oxidizing activity of GDH in the hypometabolic state. Subsequent experiments suggested that the molecular basis of the kinetic differences was a change in the protein phosphorylation state of GDH between euthermia and torpor. Specifically, liver GDH appears to be dephosphorylated and activated when animals transition into torpor and this may serve to promote amino acid oxidation to contribute to energy production and gluconeogenesis. This is the first study to show that mammalian liver GDH can be regulated by reversible phosphorylation, providing an important new regulatory mechanism for GDH control.

Purification and Properties of White Muscle Lactate Dehydrogenase from the Anoxia-Tolerant Turtle, the Red-Eared Slider, Trachemys scripta elegans

Lactate dehydrogenase (LDH; E.C. 1.1.1.27) is a crucial enzyme involved in energy metabolism in muscle, facilitating the production of ATP via glycolysis during oxygen deprivation by recycling NAD+. The present study investigated purified LDH from the muscle of 20 h anoxic and normoxic T. s. elegans, and LDH from anoxic muscle showed a significantly lower (47%) K m for L-lactate and a higher V max value than the normoxic form. Several lines of evidence indicated that LDH was converted to a low phosphate form under anoxia: (a) stimulation of endogenously present protein phosphatases decreased the K m of L-lactate of control LDH to anoxic levels, whereas (b) stimulation of kinases increased the K m of L-lactate of anoxic LDH to normoxic levels, and (c) dot blot analysis shows significantly less serine (78%) and threonine (58%) phosphorylation in anoxic muscle LDH as compared to normoxic LDH. The physiological consequence of anoxia-induced LDH dephosphorylation appears to be an increase in LDH activity to promote the reduction of pyruvate in muscle tissue, converting the glycolytic end product to lactate to maintain a prolonged glycolytic flux under energy-stressed anoxic conditions.

Post-translational regulation of PTEN catalytic function and protein stability in the hibernating 13-lined ground squirrel

BACKGROUND The insulin signaling pathway functions as a major regulator of many metabolic and cellular functions, and has been shown to be reversibly suppressed in many species during hibernation. This study characterized the regulation of PTEN phosphatase, a negative regulator of the insulin receptor network, over the torpor-arousal cycle of hibernation in the skeletal muscle of Ictidomys tridecemlineatus. METHODS Western blotting and RT-PCR were used to analyze post-translational and transcriptional regulations of PTEN respectively. Enzymatic activities were determined by the malachite green assay, while protein stability was assessed the using pulse-proteolysis method. RESULTS During torpor, the ratio of non-phosphorylated PTEN (S380/T382/T383) was significantly elevated by 1.4-fold during late torpor compared with euthermic controls; this was coupled with an increase in substrate affinity for PIP3 (by 56%) in late torpor. Two proteolytic cleavage PEST motifs were identified in the C-terminus that overlapped with the phosphorylation sites of PTEN; pulse-proteolysis analysis of PTEN protein showed a decrease in protein stability during late torpor (Cm of urea decreased by 21%). Furthermore, the increase in PTEN activity observed was correlated with a decrease in PDK-1 phosphorylation by 32%, suggesting a downstream effect of PTEN activation during torpor. Transcriptional analysis showed that mRNA expression of pten and pdk-1 remain unchanged during hibernation, suggesting post-translation modification as the primary regulatory mechanism of PTEN function. CONCLUSION Phosphorylation plays an important role in the regulation of PTEN enzymatic activity and protein stability. GENERAL SIGNIFICANCE Activation of PTEN during torpor can regulate insulin signaling during periods of low energy state.

Glucose-6-phosphate dehydrogenase regulation in the hepatopancreas of the anoxia-tolerant marine mollusc, Littorina littorea

Glucose-6-phosphate dehydrogenase (G6PDH) gates flux through the pentose phosphate pathway and is key to cellular antioxidant defense due to its role in producing NADPH. Good antioxidant defenses are crucial for anoxia-tolerant organisms that experience wide variations in oxygen availability. The marine mollusc, Littorina littorea, is an intertidal snail that experiences daily bouts of anoxia/hypoxia with the tide cycle and shows multiple metabolic and enzymatic adaptations that support anaerobiosis. This study investigated the kinetic, physical and regulatory properties of G6PDH from hepatopancreas of L. littorea to determine if the enzyme is differentially regulated in response to anoxia, thereby providing altered pentose phosphate pathway functionality under oxygen stress conditions. Several kinetic properties of G6PDH differed significantly between aerobic and 24 h anoxic conditions; compared with the aerobic state, anoxic G6PDH (assayed at pH 8) showed a 38% decrease in Km G6P and enhanced inhibition by urea, whereas in pH 6 assays Km NADP and maximal activity changed significantly between the two states. The mechanism underlying anoxia-responsive changes in enzyme properties proved to be a change in the phosphorylation state of G6PDH. This was documented with immunoblotting using an anti-phosphoserine antibody, in vitro incubations that stimulated endogenous protein kinases versus protein phosphatases and significantly changed Km G6P, and phosphorylation of the enzyme with 32P-ATP. All these data indicated that the aerobic and anoxic forms of G6PDH were the high and low phosphate forms, respectively, and that phosphorylation state was modulated in response to selected endogenous protein kinases (PKA or PKG) and protein phosphatases (PP1 or PP2C). Anoxia-induced changes in the phosphorylation state of G6PDH may facilitate sustained or increased production of NADPH to enhance antioxidant defense during long term anaerobiosis and/or during the transition back to aerobic conditions when the reintroduction of oxygen causes a rapid increase in oxidative stress.

Stable Suppression of Lactate Dehydrogenase Activity during Anoxia in the Foot Muscle of Littorina littorea and the Potential Role of Acetylation as a Novel Posttranslational Regulatory Mechanism

The intertidal marine snail, Littorina littorea, has evolved to withstand extended bouts of oxygen deprivation brought about by changing tides or other potentially harmful environmental conditions. Survival is dependent on a strong suppression of its metabolic rate and a drastic reorganization of its cellular biochemistry in order to maintain energy balance under fixed fuel reserves. Lactate dehydrogenase (LDH) is a crucial enzyme of anaerobic metabolism as it is typically responsible for the regeneration of NAD+, which allows for the continued functioning of glycolysis in the absence of oxygen. This study compared the kinetic and structural characteristics of the D-lactate specific LDH (E.C. 1.1.1.28) from foot muscle of aerobic control versus 24 h anoxia-exposed L. littorea. Anoxic LDH displayed a near 50% decrease in V max (pyruvate-reducing direction) as compared to control LDH. These kinetic differences suggest that there may be a stable modification and regulation of LDH during anoxia, and indeed, subsequent dot-blot analyses identified anoxic LDH as being significantly less acetylated than the corresponding control enzyme. Therefore, acetylation may be the regulatory mechanism that is responsible for the suppression of LDH activity during anoxia, which could allow for the production of alternative glycolytic end products that in turn would increase the ATP yield under fixed fuel reserves.

Regulation of liver glutamate dehydrogenase from an anoxia-tolerant freshwater turtle

Abstract Background: Freshwater turtles, Trachemys scripta elegans, are one of the few vertebrate species capable of surviving prolonged periods without oxygen. Anoxic survival requires numerous physiological and

Insights into the In Vivo Regulation of Glutamate Dehydrogenase from the Foot Muscle of an Estivating Land Snail

Land snails, Otala lactea, survive in seasonally hot and dry environments by entering a state of aerobic torpor called estivation. During estivation, snails must prevent excessive dehydration and reorganize metabolic fuel use so as to endure prolonged periods without food. Glutamate dehydrogenase (GDH) was hypothesized to play a key role during estivation as it shuttles amino acid carbon skeletons into the Krebs cycle for energy production and is very important to urea biosynthesis (a key molecule used for water retention). Analysis of purified foot muscle GDH from control and estivating conditions revealed that estivated GDH was approximately 3-fold more active in catalyzing glutamate deamination as compared to control. This kinetic difference appears to be regulated by reversible protein phosphorylation, as indicated by ProQ Diamond phosphoprotein staining and incubations that stimulate endogenous protein kinases and phosphatases. The increased activity of the high-phosphate form of GDH seen in the estivating land snail foot muscle correlates well with the increased use of amino acids for energy and increased synthesis of urea for water retention during prolonged estivation.

Novel detection method for chemiluminescence derived from the Kinase-Glo luminescent kinase assay platform: Advantages over traditional microplate luminometers

Graphical abstract

Hexokinase regulation in the hepatopancreas and foot muscle of the anoxia-tolerant marine mollusc, Littorina littorea

Hexokinase from the hepatopancreas and foot muscle of Littorina littorea undergoes stable modification of its kinetic and structural properties in response to prolonged oxygen deprivation. In the hepatopancreas, a reduction in the Km glucose for hexokinase from the anoxic animal suggests a more active enzyme form during anoxia. Conversely, in the foot muscle, an increase in Km ATP and a decrease in Vmax for anoxic snail hexokinase were consistent with a less active enzyme form during anoxia. In either case, the molecular basis for the stable modification of hexokinase kinetics is reversible phosphorylation. The activation of endogenous PKC and AMPK increased the Km glucose for anoxic hepatopancreas hexokinase to a value that was similar to the control Km glucose. Alternatively, stimulation of endogenous PKA, PKG, and CamK for control foot muscle hexokinase increased the Km ATP to a value similar to that seen for the anoxic enzyme form. In both tissues, activation of endogenous phosphatases reversed the effects of protein kinases. Dephosphorylation and activation of hepatopancreas hexokinase during anoxia may allow for increased shunting of glucose-6-phosphate into the pentose phosphate pathway, thereby producing reducing equivalents of NADPH needed for antioxidant defense upon tissue re-oxygenation. Conversely, phosphorylation and inhibition of foot muscle hexokinase during anoxia may reflect the decreased need for glucose oxidation during hypometabolism.

Purification and properties of glyceraldehyde-3-phosphate dehydrogenase from the skeletal muscle of the hibernating ground squirrel, Ictidomys tridecemlineatus

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the skeletal muscle of euthermic and torpid Ictidomys tridecemlineatus was purified to electrophoretic homogeneity using a novel method involving Blue-agarose and Phenyl-agarose chromatography. Kinetic analysis of the enzymes isolated from the two conditions suggested the existence of two structurally distinct proteins, with GAPDH V max being 40–60% less for the enzyme from the torpid condition (in both glycolytic and gluconeogenic directions) as compared to the euthermic enzyme form. Thermal denaturation, in part determined by differential scanning fluorimetry, revealed that purified GAPDH from the torpid animals was significantly more stable that the enzyme from the euthermic condition. Mass spectrometry combined with Western blot analyses of purified GAPDH indicate that the cellular GAPDH population is extensively modified, with posttranslational phosphorylation, acetylation and methylation being detected. Global reduction in GAPDH tyrosine phosphorylation during torpor as well as site specific alterations in methylation sites suggests that that the stable changes observed in kinetic and structural GAPDH properties may be due to posttranslational modification of this enzyme during torpor. Taken together, these results suggest a stable suppression of GAPDH (possibly by some reversible posttranslational modification) during ground squirrel torpor, which likely contributes to the overall reduction in carbohydrate metabolism when these animals switch to lipid fuels during dormancy.