Glycogen Phosphorylase Activity Research Articles

Structural characterization of the catalytic γ and regulatory β subunits of phosphorylase kinase in the context of the hexadecameric enzyme complex.

In the tightly regulated glycogenolysis cascade, the breakdown of glycogen to glucose-1-phosphate, phosphorylase kinase (PhK) plays a key role in regulating the activity of glycogen phosphorylase. PhK is a 1.3 MDa hexadecamer, with four copies each of four different subunits (α, β, γ and δ), making the study of its structure challenging. Using hydrogen-deuterium exchange, we have analyzed the regulatory β subunit and the catalytic γ subunit in the context of the intact non-activated PhK complex to study the structure of these subunits and identify regions of surface exposure. Our data suggest that within the non-activated complex the γ subunit assumes an activated conformation and are consistent with a previous docking model of the β subunit within the cryoelectron microscopy envelope of PhK.

Exposures to arsenite and methylarsonite produce insulin resistance and impair insulin-dependent glycogen metabolism in hepatocytes.

Environmental exposure to inorganic arsenic (iAs) has been shown to disturb glucose homeostasis, leading to diabetes. Previous laboratory studies have suggested several mechanisms that may underlie the diabetogenic effects of iAs exposure, including (i) inhibition of insulin signaling (leading to insulin resistance) in glucose metabolizing peripheral tissues, (ii) inhibition of insulin secretion by pancreatic β cells, and (iii) dysregulation of the methylation or expression of genes involved in maintenance of glucose or insulin metabolism and function. Published studies have also shown that acute or chronic iAs exposures may result in depletion of hepatic glycogen stores. However, effects of iAs on pathways and mechanisms that regulate glycogen metabolism in the liver have never been studied. The present study examined glycogen metabolism in primary murine hepatocytes exposed in vitro to arsenite (iAs3+) or its methylated metabolite, methylarsonite (MAs3+). The results show that 4-h exposures to iAs3+ and MAs3+ at concentrations as low as 0.5 and 0.2µM, respectively, decreased glycogen content in insulin-stimulated hepatocytes by inhibiting insulin-dependent activation of glycogen synthase (GS) and by inducing activity of glycogen phosphorylase (GP). Further investigation revealed that both iAs3+ and MAs3+ inhibit insulin-dependent phosphorylation of protein kinase B/Akt, one of the mechanisms involved in the regulation of GS and GP by insulin. Thus, inhibition of insulin signaling (i.e., insulin resistance) is likely responsible for the dysregulation of glycogen metabolism in hepatocytes exposed to iAs3+ and MAs3+. This study provides novel information about the mechanisms by which iAs exposure impairs glucose homeostasis, pointing to hepatic metabolism of glycogen as one of the targets.

Immunochemical Study of the Effect of F2Glc on Glycogen Synthase Translocation and Glycogen Synthesis in Isolated Rat Hepatocytes.

The compound 2-deoxy-2-fluoro-α-D-glucopyranosyl fluoride (F2Glc), which is a nonmetabolized superior glucose analogue, is a potent inhibitor of glycogen phosphorylase and pharmacological properties are reported. Glycogen phosphorylase (GP) and glycogen synthase (GS) are responsible of the degradation and synthesis, respectively, of glycogen which is a polymer of glucose units that provides a readily available source of energy in mammals. GP and GS are two key enzymes that modulate cellular glucose and glycogen levels; therefore, these proteins are suggested as potential targets for the treatment of diseases related to glycogen metabolism disorders. We studied by Western Blot technique that F2Glc decreased GP activity, and we also showed that F2Glc did not affect GS activity and its translocation from a uniform cytosolic distribution to the hepatocyte periphery, which is crucial for glycogen synthesis, using immunoblotting and immunofluorescence labeling techniques. F2Glc specifically inhibits glycogenolysis pathway and permits a greater deposition of glycogen. These observations open up the possibility of further develop drugs that act specifically on GP. The ability to selectively inhibit GP, which is a key enzyme for the release of glucose from the hepatic glycogen reserve, may represent a new approach for the treatment of hyperglycemia in type 2 diabetes.

Dietary starch types affect liver nutrient metabolism of finishing pigs.

This study aimed to evaluate the effect of different starch types on liver nutrient metabolism of finishing pigs. In all ninety barrows were randomly allocated to three diets with five replicates of six pigs, containing purified waxy maize starch (WMS), non-waxy maize starch (NMS) and pea starch (PS) (the amylose to amylopectin ratios were 0·07, 0·19 and 0·28, respectively). After 28 d of treatments, two per pen (close to the average body weight of the pen) were weighed individually, slaughtered and liver samples were collected. Compared with the WMS diet, the PS diet decreased the activities of glycogen phosphorylase, phosphoenolpyruvate carboxykinase and the expression of phosphoenolpyruvate carboxykinase 1 in liver (P0·05). Compared with the WMS diet, the PS diet reduced the expressions of glutamate dehydrogenase and carbamoyl phosphate synthetase 1 in liver (P<0·05). PS diet decreased the expression of the insulin receptor, and increased the expressions of mammalian target of rapamycin complex 1 and ribosomal protein S6 kinase β-1 in liver compared with the WMS diet (P<0·05). These findings indicated that the diet with higher amylose content could down-regulate gluconeogenesis, and cause less fat deposition and more protein deposition by affecting the insulin/PI3K/protein kinase B signalling pathway in liver of finishing pigs.

Navel orange peel hydroethanolic extract, naringin and naringenin have anti-diabetic potentials in type 2 diabetic rats

The therapy of Type 2 Diabetes Mellitus (T2DM) stays a challenging issue. During the last decade, there has been an interest in the expansion of anti-diabetic drugs especially those of natural sources. Thus, the aim of this study was to assess the anti-hyperglycemic and the anti-hyperlipidemic effects as well as the anti-oxidant activities of navel orange hydroethanolic extract and its constituting flavonoids naringin and naringenin on nicotineamide (NA)/streptozotocin (STZ)-induced type 2 diabetic rats. To induce T2DM, 16h-fasted rats were intraperitoneally injected with STZ at dose of 50mg/kg body weight (b. w.), 15min after the intraperitoneal administration of NA (120mg/kg b. w.). The NA/STZ-induced type 2 diabetic rats were orally treated with navel orange peel hydroethanolic extract, naringin and narengenin at dose level of 100mg/kg b. w./day for 4 weeks. The treatments with navel orange peel hydroethanolic extract, naringin and narengenin potentially alleviated the lowered serum insulin and C-peptide levels, the depleted liver glycogen content, the elevated liver glucose-6-phosphatase and glycogen phosphorylase activities, the deteriorated serum lipid profile, and the suppressed liver antioxidant defense system of NA/STZ-induced type 2 diabetic rats. The treatments also enhanced the mRNA expression of insulin receptor β-subunit, GLUT4 and adiponectin in adipose tissue of STZ/NA-induced type 2 diabetic rats. In conclusion, the navel orange peel hydroethanolic extract, naringin and naringenin have potent anti-diabetic effects in NA/STZ-induced type 2 diabetic rats via their insulinotropic effects and insulin improving action which in turn may be mediated through enhancing insulin receptor, GLUT4 and adiponectin expression in adipose tissue.

Abstract 435: Adrenergic Deficiency Disrupts Glucose Metabolism and ATP/NADH Production During Embryonic Heart Development

Cardiac energy demands increase during embryonic development, requiring activation of oxidative phosphorylation to convert ADP to ATP in mitochondria. We have recently shown that adrenergic hormones are required to maintain sufficient cardiac energy metabolism during embryonic development, but the specific mechanism(s) underlying this regulation are not known. Mouse embryos lacking the adrenergic hormones, norepinephrine (NE) and epinephrine (EPI), due to targeted loss of the dopamine β-hydroxylase ( Dbh ) gene, have markedly (&gt;50-fold) decreased steady-state ATP/ADP ratios. Rates of ATP synthesis and hydrolysis did not differ between adrenergic-deficient and competent embryos suggesting the enzymatic machinery required for ATP production/consumption is functional. We hypothesized that adrenergic-deficient embryonic hearts are metabolically starved of nutrients leading to energy depletion. To identify changes in metabolism in adrenergic-deficient hearts, we performed LC-MS metabolomics, which showed decreases in all nucleotide triphosphates (NTPs) and NAD(H) confirming energy depletion. Additionally, products of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glucose-6-phosphate dehydrogenase (G-6-PDH), and pyruvate dehydrogenase (PDH) were significantly diminished compared to controls, suggesting impaired activity. Enzymatic activities of GAPDH, G-6-PDH, PDH, and glycogen phosphorylase (GP); a well-known enzyme under adrenergic regulation, were measured from the rate of NAD(P)H production. GAPDH, G-6-PDH, and GP activities were significantly decreased (~80%, 40%, and 70% reduction, respectively) compared to controls. Interestingly, GAPDH, G-6-PDH, and GP protein levels, examined by western blot, did not differ from adrenergic-competent controls, thereby suggesting that adrenergic hormones regulate posttranslational activity of these enzymes. These results indicate that mitochondria are metabolically starved due to impairments in glycogenolysis, glycolysis, and pentose phosphate pathways. These findings reveal new mechanistic insights into global adrenergic regulation of major metabolic pathways during embryonic heart development.

Effect of Marsilea quadrifolia (L.) on carbohydrate metabolic enzymes in alloxan induced diabetic rats

Marsilea quadrifolia is an indigenous medicinal plant and it has a folk reputation in central and southern India as hypoglycemic agent. The present study was aimed to evaluate the effect of methanolic leaf and stem extracts of Marsilea quadrifolia on carbohydrate metabolic enzymes in alloxan induced diabetic rats. In this study, the blood glucose level was increased in alloxan induced diabetic rats when compared to normal control rats. The decreased level of blood glucose was observed in diabetic rats treated with leaf and stem of M. quadrifolia. The activities of hexokinase, pyruvate kinase, lactate dehydrogenase, glucose-6-phosphatase, fructose 1,6-bisphosphatase and glucose-6-phosphate dehydrogenase in liver and kidney and glycogen content, glycogen synthase and glycogen phosphorylase in liver were analysed. Glibenclamide was used as a reference drug in the present investigation. The activities of hexokinase, pyruvate kinase and glucose-6-phosphate dehydrogenase were significantly decreased and the activities of lactate dehydrogenase, glucose-6-phosphatase and fructose 1,6-bisphosphatase were significantly increased in liver and kidney of alloxan induced diabetic rats when compared to normal control rats. Oral administration of methanolic leaf and stem extracts of M. quadrifolia (300 mg/kg bw) to diabetic rats for 45 days showed significantly reverted levels of above mentioned enzymes. The levels of glycogen and glycogen synthase in liver were significantly decreased and the glycogen phosphorylase activity was significantly increased in diabetic rats when compared to normal control rats. The diabetic rats treated with leaf and stem extracts of M. quadrifolia reverted the levels of glycogen, glycogen synthase and glycogen phosphorylase to near normal. The present study clearly indicates that the methanolic leaf and stem extracts of M. quadrifolia possesses potent antidiabetic activity.

Molecular mechanisms of action of styrene toxicity in blood plasma and liver.

Styrene is an aromatic colorless hydrocarbon available in liquid form and highly volatile. In its pure form, it gives a sweet smell. The primary source of exposure in the environment is from plastic materials, rubber industries, packaging materials, insulations, and fiber glass and carpet industry. Natural sources of styrene include: few metabolites in plants which are transferred through food chain. The current study was designed to evaluate styrene toxicity, including: superoxide dismutase (SOD) and protein carbonyl, oxidative stress, glucose-6-phosphatase (G6Pase), glycogen phosphorylase (GP), and phosphoenolpyruvate carboxykinase (PEPCK) activities, adenosine triphosphate (ATP) to adenosine diphosphate (ADP) ratio, and changes in gene expressions such as glutamate dehydrogenase 1 (GLUD1), glucose transporter 2 (GLUT2), and glucokinase (GCK) in the rat liver tissue. For this purpose, styrene was dissolved in corn oil and was administered via gavage, at doses 250, 500, 1000, 1500, 2000, mg/kg/day per mL and control (corn oil) to each rat with one day off in a week, for 42 days. Plasma SOD and protein carbonyl of plasma were significantly up-regulated in 1000, 1500, and 2000 mg/kg/day styrene administrated groups (P < .001). In addition, styrene caused an increase in lipid peroxidation (LPO) and reactive oxygen species (ROS) in the dose-dependent manners in liver tissue (P < .001). Furthermore, the ferrous reducing antioxidant power (FRAP) and total thiol molecules (TTM) in styrene-treated groups were significantly decreased in liver tissue (P < .001) with increasing doses. In treated rats, styrene significantly increased G6Pase activity (P < .001) and down-regulated GP activity (P < .001) as compared to the control group. The PEPCK activity was significantly raised in a dose-dependent manner (P < .001). The ATP/ADP ratio of live cells was significantly raised by increasing the dose (P < .001). There was significantly an up-regulation of GLUD1 and GCK at 2000 mg/kg group (P < .01) and a down-regulation for GLUT2 at the same dose. While in the rest of group, GLUT2 showed up-regulation of relative fold change. By targeting genes such as GLUD1, GLUT2, and GCK, disruption of hepatic gluconeogenesis, glycogenolysis, and insulin secretory functions are obvious. The present study illustrates that induction of oxidative stress followed by changes in G6Pase, GP, and PEPCK activities and the genes responsible for glucose metabolism are the mechanisms of styrene's action in the liver.

Probing the catalytic site of rabbit muscle glycogen phosphorylase using a series of specifically modified maltohexaose derivatives.

Glycogen phosphorylase (GP) is an allosteric enzyme whose catalytic site comprises six subsites (SG1, SG-1, SG-2, SG-3, SG-4, and SP) that are complementary to tandem five glucose residues and one inorganic phosphate molecule, respectively. In the catalysis of GP, the nonreducing-end glucose (Glc) of the maltooligosaccharide substrate binds to SG1 and is then phosphorolyzed to yield glucose 1-phosphate. In this study, we probed the catalytic site of rabbit muscle GP using pyridylaminated-maltohexaose (Glcα1-4Glcα1-4Glcα1-4Glcα1-4Glcα1-4GlcPA, where GlcPA=1-deoxy-1-[(2-pyridyl)amino]-D-glucitol]; abbreviated as PA-0) and a series of specifically modified PA-0 derivatives (Glc m -AltNAc-Glc n -GlcPA, where m+n=4 and AltNAc is 3-acetoamido-3-deoxy-D-altrose). PA-0 served as an efficient substrate for GP, whereas the other PA-0 derivatives were not as good as the PA-0, indicating that substrate recognition by all the SG1 -SG-4 subsites was important for the catalysis of GP. By comparing the initial reaction rate toward the PA-0 derivatives (V derivative) with that toward PA-0 (V PA-0), we found that the value of V derivative/V PA-0 decreased significantly as the level of allosteric activation of GP increased. These results suggest that some conformational changes have taken place in the maltooligosaccharide-binding region of the GP catalytic site during allosteric regulation.

Oleanolic acid protects against diabetic cardiomyopathy via modulation of the nuclear factor erythroid 2 and insulin signaling pathways.

Oleanolic acid (OL) is a pentacyclic triterpene compound used for the treatment of hepatitis, liver fibrosis and liver cirrhosis. In China, there is no published research on the effect or biological utilization of OL on liver diseases. The aim of the present study was to investigate the protective effects of OL against diabetic cardiomyopathy and its possible mechanism. A rat model of diabetes was established using streptozotocin and the effect of OL on diabetic cardiomyopathy (DCM) was evaluated. The results demonstrated that OL significantly reversed the DCM-induced changes to body weight, heart rate, echocardiography and hemodynamics, phosphorylated-glycogen synthase (GS) and glycogen phosphorylase (GP) activity in diabetic rats (all P<0.01). Treatment of diabetic rats with OL significantly inhibited oxidative stress and activated heme oxygenase (HO)-1/nuclear factor erythroid 2 (Nrf2) signaling in a rat model of diabetes (both P<0.01). The results of the present study indicate that OL protects against DCM through the HO-1/Nrf2 and insulin modulating GS/GP signaling pathways.

Effect of season, supplementation and fasting on glycolytic potential and activity of AMP-activated protein kinase, glycogen phosphorylase and glycogen debranching enzyme in grass-fed steers as determined in Longissimus lumborum muscle

Forty grass fed beef steers close to slaughter weight (500kg) were used to study the effects of season (one experiment was carried out in autumn and one in summer, same farm, same design), supplementation (grass-fed only=control or flaked corn supplemented=suppl during four weeks before slaughter) and fasting during lairage (0h or 24h fasting). The supplementation with flaked corn started with 0.5kganimal−1day1, fed individually and increasing up to 1% of body weight (approximately 5kganimal−1day1) during the first week; this amount was kept constant for three more weeks. The concentrations of muscle glycogen (MGC), glucose-6-phosphate+glucose (G6P+Gluc) and lactate (LA), glycolytic potential (GPot), activity of AMP-activated protein kinase (AMPK), glycogen phosphorylase (GP) and glycogen debranching enzyme (GDE) were determined in M. Longissimus lumborum (LL);pH and postmortem temperature at 0.5h and 24h were measured. Biopsies from the LL were taken from each steer at the beginning of each experiment (B1), at 0.5h (B2) and 24h postmortem (B3). For each metabolic substrate/product measured in the muscle samples a linear mixed effect model was fitted. GPot, MGC and GP were higher and GDE was lower (P < 0.05) in autumn than in summer. Carcass temperature at 0.5h and 24h postmortem was lower in autumn than in summer and non-fasted steers had a lower final carcass temperature than those fasted (P < 0.05). Supplementation and no fasting were significant (P < 0.05) factors that helped maintaining a higher MGC and GPot in the steers between B1 (on farm biopsy) and B2 (at slaughter); no fasting also helped in increasing GDE activity postmortem (between B2 and B3).The effects of treatments on glycogen reserves and on the activities of the glycolytic enzymes included were not reflected in the ultimate pH of the carcasses, because no differences in terms of mean pH due to any of the factors studied were found (P > 0.05). Perhaps other substrates/enzymes that take part in muscle glycolysis/glycogenolysis not included in this study should be analyzed in future studies; considering the high individual variability observed, intrinsic factors of cattle, like genetics, should be taken into consideration.

Synthesis and Evaluation of Novel [1,2,4]Triazolo[5,1-c][1,2,4]-triazines and Pyrazolo[5,1-c][1,2,4]triazines as Potential Antidiabetic Agents.

Inhibition of the dipeptidyl peptidase-4 (DPP4) enzyme activity and prevention of advanced glycation end (AGE) products formation represents a reliable approach to achieve control over hyperglycemia and the associated pathogenesis of diabetic vascular complications. In the frames of this research study, several triazolo- and pyrazolotriazines were synthesized and evaluated as inhibitors of AGE products formation, DPP4, glycogen phosphorylase and α-glucosidase activities, as well as AGE cross-link breakers. From the two considered classes of heterocyclic compounds, the pyrazolotriazines showed the highest potency as antiglycating agents and DPP4 inhibitors. Structure-activity relationships (SAR) for these compounds, which can be considered as potential drugs for the treatment of type 2 diabetes, were evaluated.

Synthesis and pharmacological activity of 3-phenoxybenzoic acid derivatives

New esters of N-benzoyl-3-phenoxyphenylcarboxamide acid and N-benzoyl-N′-4-bromophenyl-3-phenoxybenzamidine were synthesized. Some of the synthesized compounds were shown to inhibit the activity of dipeptidyl peptidase-4 and nonenzymatic glycosylation of proteins and manifested antiplatelet and antioxidant properties. The compounds tested did not display the antagonistic effect toward angiotensin II type 1 receptor, did not influence the activity of glycogen phosphorylase and had very little ability to break cross-links of the glycated proteins. The derivatives with the biological activity of two types were found, which can serve as basic molecules in the search for new drug products.

Glycogen Phosphorylase and Glycogen Synthase: Gene Cloning and Expression Analysis Reveal Their Role in Trehalose Metabolism in the Brown Planthopper, Nilaparvata lugens Stål (Hemiptera: Delphacidae).

RNA interference has been used to study insects’ gene function and regulation. Glycogen synthase (GS) and glycogen phosphorylase (GP) are two key enzymes in carbohydrates’ conversion in insects. Glycogen content and GP and GS gene expression in several tissues and developmental stages of the Brown planthopper Nilaparvata lugens Stål (Hemiptera: Delphacidae) were analyzed in the present study, using quantitative reverse-transcription polymerase chain reaction to determine their response to double-stranded trehalases (dsTREs), trehalose-6-phosphate synthases (dsTPSs), and validamycin injection. The highest expression of both genes was detected in the wing bud, followed by leg and head tissues, and different expression patterns were shown across the developmental stages analyzed. Glycogen content significantly decreased 48 and 72 h after dsTPSs injection and 48 h after dsTREs injection. GP expression increased 48 h after dsTREs and dsTPSs injection and significantly decreased 72 h after dsTPSs, dsTRE1-1, and dsTRE1-2 injection. GS expression significantly decreased 48 h after dsTPS2 and dsTRE2 injection and 72 h after dsTRE1-1 and dsTRE1-2 injection. GP and GS expression and glycogen content significantly decreased 48 h after validamycin injection. The GP activity significantly decreased 48 h after validamycin injection, while GS activities of dsTPS1 and dsTRE2 injection groups were significantly higher than that of double-stranded GFP (dsGFP) 48 h after injection, respectively. Thus, glycogen is synthesized, released, and degraded across several insect tissues according to the need to maintain stable trehalose levels.

Priming Effect of a Morning Meal on Hepatic Glucose Disposition Later in the Day.

We used hepatic balance and tracer ([3H]glucose) techniques to examine the impact of “breakfast” on hepatic glucose metabolism later in the same day. From 0–240 min, 2 groups of conscious dogs (n = 9 dogs/group) received a duodenal infusion of glucose (GLC) or saline (SAL), then were fasted from 240–360 min. Three dogs from each group were euthanized and tissue collected at 360 min. From 360–600 min, the remaining dogs underwent a hyperinsulinemic (4× basal) hyperglycemic clamp (arterial blood glucose 146 ± 2 mg/dL) with portal GLC infusion. The total GLC infusion rate was 14% greater in dogs infused with GLC than in those receiving SAL (AUC360–600min 2,979 ± 296 vs. 2,597 ± 277 mg/kg, respectively). The rates of hepatic glucose uptake (5.8 ± 0.8 vs. 3.2 ± 0.3 mg ⋅ kg−1 ⋅ min−1) and glycogen storage (4.7 ± 0.6 vs. 2.9 ± 0.3 mg ⋅ kg−1 ⋅ min−1) during the clamp were markedly greater in dogs receiving GLC compared with those receiving SAL. Hepatic glycogen content was ∼50% greater, glycogen synthase activity was ∼50% greater, glycogen phosphorylase activity was ∼50% lower, and the amount of phosphorylated glycogen synthase was 34% lower, indicating activation of the enzyme, in dogs receiving GLC compared with those receiving SAL. Thus, morning GLC primed the liver to extract and store more glucose in the presence of hyperinsulinemic hyperglycemia later in the same day, indicating that breakfast enhances the liver’s role in glucose disposal in subsequent same-day meals.

The effects and regulatory mechanism of RIP3 on RGC-5 necroptosis following elevated hydrostatic pressure.

Necroptosis is a type of regulated cell death that has been implicated in various diseases. Receptor-interacting protein 3 (RIP3), a member of the RIP family, is an important mediator of the necroptotic pathway. Cleavage of RIP3 at Asp328 by caspase-8 abolishes the kinase activity of RIP3, which is critical for necroptosis. Moreover, RIP3 is significantly upregulated during the early stages of acute high intra-ocular pressure and oxygen glucose deprivation. In this study, the effects of RIP3 during elevated hydrostatic pressure (EHP) were investigated and the possible mechanism through which caspase-8 regulated RIP3 cleavage was explored. Flow cytometry analysis revealed that the number of EHP-induced necrotic retinal ganglion cell 5 (RGC-5) cells was reduced after RIP3-knockdown. Furthermore, malondialdehyde (MDA) levels and glycogen phosphorylase (PYGL) activity in normal RGC-5 cells were much higher than those in RIP3-knockdown cells after EHP. EHP-induced RGC-5 necrosis was significantly reduced after treatment with butylated hydroxyanisole (BHA), a reactive oxygen species (ROS) scavenger. MDA levels and PYGL activity were lower in normal RGC-5 cells than those in cells with caspase-8 inhibition after EHP. Western blot analysis demonstrated that the RIP3 cleavage product was upregulated in cells with caspase-8 inhibition. Additionally, flow cytometry analysis revealed that the number of EHP-induced necrotic RGC-5 cells was increased after caspase-8 inhibition. Our results suggested that RGC-5 necroptosis following EHP was mediated by RIP3 through induction of PYGL activity and subsequent ROS accumulation. Thus, caspase-8 may participate in the regulation of RGC-5 necroptosis via RIP3 cleavage.

Metabolic adaptations in skeletal muscle after 84 days of bed rest with and without concurrent flywheel resistance exercise.

As metabolic changes in human skeletal muscle after long-term (simulated) spaceflight are not well understood, this study examined the effects of long-term microgravity, with and without concurrent resistance exercise, on skeletal muscle oxidative and glycolytic capacity. Twenty-one men were subjected to 84 days head-down tilt bed rest with (BRE; n = 9) or without (BR; n = 12) concurrent flywheel resistance exercise. Activity and gene expression of glycogen synthase, glycogen phosphorylase (GPh), hexokinase, phosphofructokinase-1 (PFK-1), and citrate synthase (CS), as well as gene expression of succinate dehydrogenase (SDH), vascular endothelial growth factor (VEFG), peroxisome proliferator-activated receptor gamma coactivator-1 (PGC-1α), and myostatin, were analyzed in samples from m. vastus lateralis collected before and after bed rest. Activity and gene expression of enzymes controlling oxidative metabolism (CS, SDH) decreased in BR but were partially maintained in BRE. Activity of enzymes regulating anaerobic glycolysis (GPh, PFK-1) was unchanged in BR. Resistance exercise increased the activity of GPh. PGC-1α and VEGF expression decreased in both BR and BRE. Myostatin increased in BR but decreased in BRE after bed rest. The analyses of these unique samples indicate that long-term microgravity induces marked alterations in the oxidative, but not the glycolytic, energy system. The proposed flywheel resistance exercise was effective in counteracting some of the metabolic alterations triggered by 84-day bed rest. Given the disparity between gene expression vs. enzyme activity in several key metabolic markers, posttranscriptional mechanisms should be explored to fully evaluate metabolic adaptations to long-term microgravity with/without exercise countermeasures in human skeletal muscle.

Glycogen Repletion in Brown Adipose Tissue upon Refeeding Is Primarily Driven by Phosphorylation-Independent Mechanisms.

Glycogen storage in brown adipose tissue (BAT) is generally thought to take place through passive, substrate-driven activation of glycogenesis rather than programmatic shifts favoring or opposing the storage and/or retention of glycogen. This perception exists despite a growing body of evidence suggesting that BAT glycogen storage is actively regulated by covalent modification of key glycogen-metabolic enzymes, protein turnover, and endocrine hormone signaling. Members of one such class of covalent-modification regulators, glycogen-binding Phosphoprotein Phosphatase-1 (PP1)-regulatory subunits (PPP1Rs), targeting PP1 to glycogen-metabolic enzymes, were dynamically regulated in response to 24 hr of starvation and/or 24 hr of starvation followed by ad libitum refeeding. Over-expression of the PPP1R Protein Targeting to Glycogen (PTG), under the control of the aP2 promoter in mice, inactivated glycogen phosphorylase (GP) and enhanced basal- and starvation-state glycogen storage. Total interscapular BAT glycogen synthase and the constitutive activity of GS were conditionally affected. During starvation, glucose-6-phosphate (G-6-P) levels and the relative phosphorylation of Akt (p-Ser-473-Akt) were both increased in PTG-overexpressing (Tg) mice, suggesting that elevated glycogen storage during starvation modifies broader cellular metabolic pathways. During refeeding, Tg and WT mice reaccumulated glycogen similarly despite altered GS and GP activities. All observations during refeeding suggest that the phosphorylation states of GS and GP are not physiologically rate-controlling, despite there being a clear balance of endogenous kinase- and phosphatase activities. The studies presented here reveal IBAT glycogen storage to be a tightly-regulated process at all levels, with potential effects on nutrient sensing in vivo.

Metabolic phenotype of bladder cancer.

Metabolism of bladder cancer represents a key issue for cancer research. Several metabolic altered pathways are involved in bladder tumorigenesis, representing therefore interesting targets for therapy. Tumor cells, including urothelial cancer cells, rely on a peculiar shift to aerobic glycolysis-dependent metabolism (the Warburg-effect) as the main energy source to sustain their uncontrolled growth and proliferation. Therefore, the high glycolytic flux depends on the overexpression of glycolysis-related genes (SRC-3, glucose transporter type 1 [GLUT1], GLUT3, lactic dehydrogenase A [LDHA], LDHB, hexokinase 1 [HK1], HK2, pyruvate kinase type M [PKM], and hypoxia-inducible factor 1-alpha [HIF-1α]), resulting in an overproduction of pyruvate, alanine and lactate. Concurrently, bladder cancer metabolism displays an increased expression of genes favoring the pentose phosphate pathway (glucose-6-phosphate dehydrogenase [G6PD]) and the fatty-acid synthesis (fatty acid synthase [FASN]), along with a decrease of AMP-activated protein kinase (AMPK) and Krebs cycle activities. Moreover, the PTEN/PI3K/AKT/mTOR pathway, hyper-activated in bladder cancer, acts as central regulator of aerobic glycolysis, hence contributing to cancer metabolic switch and tumor cell proliferation. Besides glycolysis, glycogen metabolism pathway plays a robust role in bladder cancer development. In particular, the overexpression of GLUT-1, the loss of the tumor suppressor glycogen debranching enzyme amylo-α-1,6-glucosidase, 4-α-glucanotransferase (AGL), and the increased activity of the tumor promoter enzyme glycogen phosphorylase impair glycogen metabolism. An increase in glucose uptake, decrease in normal cellular glycogen storage, and overproduction of lactate are consequences of decreased oxidative phosphorylation and inability to reuse glucose into the pentose phosphate and de novo fatty acid synthesis pathways. Moreover, AGL loss determines augmented levels of the serine-to-glycine enzyme serine hydroxymethyltransferase-2 (SHMT2), resulting in an increased glycine and purine ring of nucleotides synthesis, thus supporting cells proliferation. A deep understanding of the metabolic phenotype of bladder cancer will provide novel opportunities for targeted therapeutic strategies.

In vivo effects of diabetes, insulin and oleanolic acid on enzymes of glycogen metabolism in the skin of streptozotocin-induced diabetic male Sprague-Dawley rats

The skin is the largest organ in the body and diabetes induces pathologic changes on the skin that affect glucose homeostasis. Changes in skin glycogen and glucose levels can mirror serum glucose levels and thus the skin might contribute to whole body glucose metabolism. This study investigated the in vivo effects of diabetes, insulin and oleanolic acid (OA) on enzymes of glycogen metabolism in skin of type 1 diabetic rats. Diabetic and non-diabetic adult male Sprague–Dawley rats were treated with a single daily dose of insulin (4 IU/kg body weight), OA (80 mg/kg body weight) and a combination of OA + insulin for 14 days. Glycogen phosphorylase (GP) expression; and GP, glycogen synthase (GS) and hexokinase activities as well glycogen levels were evaluated. The results suggest that diabetes lowers hexokinase activity, GP activity and GP expression with no change in GS activity whilst the treatments increased GP expression and the activities of hexokinase, GP and GS except for the GS activity in OA treated rats. Glycogen levels were increased slightly by diabetes as well as OA treatment. In conclusion diabetes, OA and insulin can lead to changes in GS and GP activities in skin without significantly altering the glycogen content. We suggest that the skin may contribute to whole body glucose homeostasis particularly in disease states.