scholarly journals Adenovirus-mediated delivery into myocytes of muscle glycogen phosphorylase, the enzyme deficient in patients with glycogen-storage disease type V

1994 ◽  
Vol 304 (3) ◽  
pp. 1009-1014 ◽  
Author(s):  
S Baqué ◽  
C B Newgard ◽  
R D Gerard ◽  
J J Guinovart ◽  
A M Gómez-Foix
Keyword(s):  
Muscle Glycogen ◽  
Glycogen Phosphorylase ◽  
Recombinant Adenovirus ◽  
Genetic Diseases ◽  
C2c12 Myoblast ◽  
Mrna Levels ◽  
Phosphorylase Activity ◽  
Muscle Phosphorylase ◽  
The Impact

The feasibility of using adenovirus as a vector for the introduction of glycogen phosphorylase activity into myocytes has been examined. We used the C2C12 myoblast cell line to assay the impact of phosphorylase gene transfer on myocyte glycogen metabolism and to reproduce in vitro the two strategies proposed for the treatment of muscle genetic diseases, myoblast transplantation and direct DNA delivery. In this study, a recombinant adenovirus containing the muscle glycogen phosphorylase cDNA transcribed from the cytomegalovirus promoter (AdCMV-MGP) was used to transduce both differentiating myoblasts and nondividing mature myotube cells. Muscle glycogen phosphorylase mRNA levels and total phosphorylase activity were increased in both cell types after viral treatment although more efficiently in the differentiated myotubes. The increase in phosphorylase activity was transient (15 days) in myoblasts whereas in myotubes higher levels of phosphorylase gene expression and activity were reached, which remained above control levels for the duration of the study (20 days). The introduction of muscle phosphorylase into myotubes enhanced their glycogenolytic capacity. AdCMV MGP-transduced myotubes had lower glycogen levels under basal conditions. In addition, these engineered cells showed more extensive glycogenolysis in response to both adrenaline, which stimulates glycogen phosphorylase phosphorylation, and carbonyl cyanide m-chlorophenylhydrazone, a metabolic uncoupler. In conclusion, transfer of the muscle glycogen phosphorylase cDNA into myotubes confers an enhanced and regulatable glycogenolytic capacity. Thus this system might be useful for delivery of muscle glycogen phosphorylase and restoration of glycogenolysis in muscle cells from patients with muscle phosphorylase deficiency (McArdle's disease).

Regulation of muscle glycogen phosphorylase activity following short-term endurance training

1996 ◽  
Vol 270 (2) ◽  
pp. E328-E335 ◽  
Author(s):  
A. Chesley ◽  
G. J. Heigenhauser ◽  
L. L. Spriet
Keyword(s):  
Endurance Training ◽  
Muscle Glycogen ◽  
Glycogen Phosphorylase ◽  
Citrate Synthase ◽  
Phosphorylase Activity ◽  
Short Term ◽  
Mitochondrial Potential ◽  
Before And After ◽  
Glycogen Phosphorylase Activity ◽  
Muscle Biopsies

The purpose of this study was to examine the regulation (hormonal, substrate, and allosteric) of muscle glycogen phosphorylase (Phos) activity and glycogenolysis after short-term endurance training. Eight untrained males completed 6 days of cycle exercise (2 h/day) at 65% of maximal O2 uptake (Vo2max). Before and after training subjects cycled for 15 min at 80% of Vo2max, and muscle biopsies and blood samples were obtained at 0 and 30 s, 7.5 and 15 min, and 0, 5, 10, and 15 min of exercise. Vo2max was unchanged with training but citrate synthase (CS) activity increased by 20%. Muscle glycogenolysis was reduced by 42% during the 15-min exercise challenge following training (198.8 +/- 36.9 vs. 115.4 +/- 25.1 mmol/kg dry muscle), and plasma epinephrine was blunted at 15 min of exercise. The Phos a mole fraction was unaffected by training. Muscle phosphocreatine utilization and free Pi and AMP accumulations were reduced with training at 7.5 and 15 min of exercise. It is concluded that posttransformational control of Phos, exerted by reductions in substrate (free Pi) and allosteric modulator (free AMP) contents, is responsible for a blunted muscle glycogenolysis after 6 days of endurance training. The increase in CS activity suggests that the reduction of muscle glycogenolysis was due in part to an enhanced mitochondrial potential.

Regulation of glycogenolysis in human muscle at rest and during exercise

1982 ◽  
Vol 53 (3) ◽  
pp. 708-715 ◽  
Author(s):  
D. Chasiotis ◽  
K. Sahlin ◽  
E. Hultman
Keyword(s):  
Glycogen Phosphorylase ◽  
Total Activity ◽  
Human Muscle ◽  
Phosphorylase Activity ◽  
Michaelis Constant ◽  
Bicycle Exercise ◽  
Muscle Phosphorylase ◽  
Glycogen Utilization ◽  
Main Factors

The regulation of glycogenolysis in human muscle during isometric and dynamic exercise has been investigated. Total glycogen phosphorylase and synthase activities were unchanged during exercise. The fraction of phosphorylase in the alpha form at rest was estimated to be 20%, but the data indicate that the in vivo activity was low and critically dependent on the concentration of inorganic phosphate (Pi) in the muscle. Phosphorylase alpha increased initially 2.4-fold during isometric contraction and 1.6-fold during maximal bicycle exercise but reverted to or below the resting value at fatigue/exhaustion. At rest synthase I was 1713;48% of the total activity but decreased during exercise to about half of this value. The reciprocal changes in phosphorylase and synthase correlate with the enhanced rate of glycogenolysis during exercise. Michaelis constant (Km) for Pi was 27 mmol . l-1 for phosphorylase alpha and 7 mmol . l-1 for alpha + b. From consideration of the changes in Pi during exercise (to 20–30 mmol . l–1) it was concluded that Pi is one of the main factors determining phosphorylase activity and provides a link between phosphocreatine breakdown and glycogen utilization in muscle.

scholarly journals A Theacrine-Based Supplement Increases Cellular NAD+ Levels and Affects Biomarkers Related to Sirtuin Activity in C2C12 Muscle Cells In Vitro

Nutrients ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 3727
Author(s):  
Petey W. Mumford ◽  
Shelby C. Osburn ◽  
Carlton D. Fox ◽  
Joshua S. Godwin ◽  
Michael D. Roberts
Keyword(s):  
Cell Line ◽  
Mitochondrial Biogenesis ◽  
C2c12 Myoblast ◽  
Mrna Levels ◽  
Nicotinamide Phosphoribosyltransferase ◽  
Protein Levels ◽  
Myoblast Cell Line ◽  
Rodent Cell ◽  
Myoblast Cell

There is evidence in rodents to suggest that theacrine-based supplements modulate tissue sirtuin activity as well as other biological processes associated with aging. Herein, we examined if a theacrine-based supplement (termed NAD3) altered sirtuin activity in vitro while also affecting markers of mitochondrial biogenesis. The murine C2C12 myoblast cell line was used for experimentation. Following 7 days of differentiation, myotubes were treated with 0.45 mg/mL of NAD3 (containing ~2 mM theacrine) for 3 and 24 h (n = 6 treatment wells per time point). Relative to control (CTL)-treated cells, NAD3 treatments increased (p < 0.05) Sirt1 mRNA levels at 3 h, as well as global sirtuin activity at 3 and 24 h. Follow-up experiments comparing 24 h NAD3 or CTL treatments indicated that NAD3 increased nicotinamide phosphoribosyltransferase (NAMPT) and SIRT1 protein levels (p < 0.05). Cellular nicotinamide adenine dinucleotide (NAD+) levels were also elevated nearly two-fold after 24 h of NAD3 versus CTL treatments (p < 0.001). Markers of mitochondrial biogenesis were minimally affected. Although these data are limited to select biomarkers in vitro, these preliminary findings suggest that a theacrine-based supplement can modulate select biomarkers related to NAD+ biogenesis and sirtuin activity. However, these changes did not drive increases in mitochondrial biogenesis. While promising, these data are limited to a rodent cell line and human muscle biopsy studies are needed to validate and elucidate the significance of these findings.

Effect of the ADAPT strategy on dormant CD133+ colon cancer stem cells (CSC) and molecular complete remission measured by peripheral blood mononuclear (PBMC) CD133 mRNA.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. e14153-e14153
Author(s):  
Edward H. Lin ◽  
Yu Xiazhen ◽  
Xi C He ◽  
Xifeng Wu ◽  
Yang Xie ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Median Survival ◽  
Treatment Protocol ◽  
Mrna Levels ◽  
Peripheral Blood Mononuclear ◽  
Colon Cells ◽  
Ca Ix ◽  
The Impact

e14153 Background: The median survival for patients with unresectable metastatic colorectal cancer (CRC) is ~2 years with modern chemotherapy which yields only 5-10% complete responses (CR) including metastasectomy. Recurrences after CR are very common thanks to presence of dormant CSC that are best targeted by our proposed two-step ADAPT strategy: activate from dormancy and potentiate targeting. We examine this strategy in various CRC models and reviewed the impact on stemess including CD133 mRNA, a circulating CSC marker that predict colon cancer relapse. Methods: Different CRC models (in vitro and in vivo) were interrogated similar to clinical ADAPT treatment protocol using capecitabine (or 5FU) plus celecoxib. We also conducted IRB approved retrospective review of unresectable metastatic CRC patients treated ADAPT therapy and in those who also had PBMC CD133 mRNA measured. Results: Contrary to 5FU, which eliminates proliferating CRC cells via apoptosis but also stimulates stemness, celecoxib preferentially deplete CD133+ colon cells and exert potent stemness inhibition via rapid tumor necrosis by perturbing hypoxia and energy metabolism via CA-IX. Following response to first-line chemotherapy, ADAPT strategy plus radiation improved CR or near CR rate to 49/126 (40%) in unresectable CRC patients whose median survival had reached 92.7 months (95% CI, 53.5 months - not reached). Paradoxically, none surgical CR patients (n= 16) enjoyed 100% 5-year relapse free survival compared to 42% of surgical patients (p = 0.04). The PBMC CD133 mRNA in five long-term CR patients were 0.0024, 0.29, 0.5, 0.56, 2.96 respectively, all below previously reported cutoff value of 4.79 for recurrence and far below CD133 mRNA levels (28, 375, 3997, 15662, 83240) in none CR patients. Conclusions: ADAPT plus radiation preferentially targets colon CSC via hypoxia/CA-IX and improves clinical CR rate and molecular CR as measured by PBMC CD133 mRNA. We are actively interrogating the effects of ADAPT strategies in a phase II study funded by Gateway in CRC patients and in genetic CRC animal models.

Phosphorylase and glycogen levels in skeletal muscle and liver of hibernating and nonhibernating bats

1959 ◽  
Vol 197 (5) ◽  
pp. 1059-1062 ◽  
Author(s):  
Samuel L. Leonard ◽  
William A. Wimsatt
Keyword(s):  
Skeletal Muscle ◽  
Muscle Glycogen ◽  
Liver Glycogen ◽  
Myotis Lucifugus ◽  
Phosphorylase Activity ◽  
Activity Ratios ◽  
Muscle Phosphorylase ◽  
Wet Weight ◽  
Muscle Phosphorylase Activity ◽  
Made In

Determinations of skeletal muscle and liver glycogen concentration and active a and total t phosphorylase activities were made in bats ( Myotis lucifugus) hibernating at 3°–5° and 20 hours after arousal at room temperature. After arousal, liver glycogen was decreased by half and muscle glycogen was increased over twofold. Concomitantly, muscle phosphorylase a was increased, phosphorylase t was unchanged and the ratio a/t was increased. In the liver, phosphorylase a, t and the ratios were increased upon arousal (calculated per unit of wet weight and per mg N). Epinephrine treatment was ineffective in the torpid hibernating bats, but in aroused bats, it decreased muscle and liver glycogen but increased muscle phosphorylase activity ratios only slightly. Histamine was ineffective in the aroused bats. Stimulating aroused bats to fly for short periods consistently resulted in lower muscle glycogen levels and in no change in muscle phosphorylase activity ratios. It is concluded that a) at least part of the increased muscle glycogen in the aroused bats comes from the liver, b) the changes in glycogen levels and phosphorylase activity are in some manner related and c) liver phosphorylase changes upon arousal, unlike that in muscle phosphorylase, involves an increase in total enzyme potential.

scholarly journals Time-Dependent Effects of POT1 Knockdown on Proliferation, Tumorigenicity, and HDACi Response of SK-OV3 Ovarian Cancer Cells

2018 ◽  
Vol 2018 ◽  
pp. 1-12 ◽  
Author(s):  
Hua Zhou ◽  
Abdul Mondal ◽  
Aleksandra Dakic ◽  
Lama Alhawas ◽  
Xuefeng Liu ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Cell Proliferation ◽  
Telomerase Activity ◽  
Population Doubling ◽  
Ovarian Cancer Cells ◽  
Human Ovarian Cancer ◽  
Activity Levels ◽  
Mrna Levels ◽  
The Impact

The roles of protection of telomeres 1 (POT1) in human ovarian cancer have not been fully elucidated. Here, we investigated the impact of POT1 knockdown (POT1-KD) on in vitro cell proliferation, tumorigenesis, and histone deacetylase inhibitor (HDACi) response in human ovarian cancer-derived SK-OV3 cells. The POT1 gene was knocked down by infection with POT1 lenti-shRNA. POT1, c-Myc, and hTERT mRNA levels and relative telomere length were determined by qRT-PCR; POT1 protein levels were determined by western blot. The relative telomerase activity levels were detected using qTRAP; cell proliferation was assessed using cumulative population doubling (cPD) experiments. Cell tumorigenicity was evaluated by anchorage-independent cell growth assays, and cell response to HDACi was determined by luminescence cell viability assays. Results indicate that lenti-shRNA-mediated POT1-KD significantly reduced POT1 mRNA and protein expression. POT1-KD immediately downregulated c-Myc expression, which led to the inhibition of cell proliferation, tumorigenesis, and HDACi response. However, after brief suppression, c-Myc expression increased in the medium term, which resulted in enhanced cell proliferation, tumorigenesis, and HDACi response in the POT1-KD cells. Furthermore, we discovered that c-Myc regulated cell proliferation and tumorigenesis via hTERT/telomerase/telomere pathway.

scholarly journals Chlorogenic Acid Functions as a Novel Agonist of PPARγ2 during the Differentiation of Mouse 3T3-L1 Preadipocytes

2018 ◽  
Vol 2018 ◽  
pp. 1-14 ◽  
Author(s):  
Shu-guang Peng ◽  
Yi-lin Pang ◽  
Qi Zhu ◽  
Jing-he Kang ◽  
Ming-xin Liu ◽  
...  
Keyword(s):  
Chlorogenic Acid ◽  
Fruits And Vegetables ◽  
Quantitative Polymerase Chain Reaction ◽  
Lipid Synthesis ◽  
Peroxisome Proliferator ◽  
Mrna Levels ◽  
Related Gene ◽  
Peroxisome Proliferator Activated Receptor ◽  
The Impact

Rosiglitazone (RG) is a well-known activator of peroxisome proliferator-activated receptor-gamma (PPARγ) and used to treat hyperglycemia and type 2 diabetes; however, its clinical application has been confounded by adverse side effects. Here, we assessed the roles of chlorogenic acid (CGA), a phenolic secondary metabolite found in many fruits and vegetables, on the differentiation and lipolysis of mouse 3T3-L1 preadipocytes. The results showed that CGA promoted differentiation in vitro according to oil red O staining and quantitative polymerase chain reaction assays. As a potential molecular mechanism, CGA downregulated mRNA levels of the adipocyte differentiation-inhibitor gene Pref1 and upregulated those of major adipogenic transcriptional factors (Cebpb and Srebp1). Additionally, CGA upregulated the expression of the differentiation-related transcriptional factor PPARγ2 at both the mRNA and protein levels. However, following CGA intervention, the accumulation of intracellular triacylglycerides following preadipocyte differentiation was significantly lower than that in the RG group. Consistent with this, our data indicated that CGA treatment significantly upregulated the expression of lipogenic pathway-related genes Plin and Srebp1 during the differentiation stage, although the influence of CGA was weaker than that of RG. Notably, CGA upregulated the expression of the lipolysis-related gene Hsl, whereas it did not increase the expression of the lipid synthesis-related gene Dgat1. These results demonstrated that CGA might function as a potential PPARγ agonist similar to RG; however, the impact of CGA on lipolysis in 3T3-L1 preadipocytes differed from that of RG.

Fish muscle glycogen phosphorylase

1968 ◽  
Vol 46 (5) ◽  
pp. 423-432 ◽  
Author(s):  
M. Yamamoto
Keyword(s):  
Skeletal Muscle ◽  
Glycogen Phosphorylase ◽  
Active Form ◽  
Maximal Activity ◽  
Fish Muscle ◽  
Salmo Gairdneri ◽  
Rabbit Muscle ◽  
Muscle Phosphorylase

Glycogen phosphorylase b was purified 70- to 90-fold from skeletal muscle of rainbow trout (Salmo gairdneri). The purified enzyme exhibited maximal activity near pH 6.8 at 37°. Of several 5′-nucleotides tested, only 5′-AMP caused stimulation of phosphorylase b. The Km value for glucose-1-phosphate was 10–15 mM, and for 5′-AMP, 0.2–0.4 mM. Glucose (25 mM) and ATP (5 mM) were both inhibitory, but glucose-6-phosphate (5 mM) had no effect. Inactive trout muscle phosphorylase was converted to the active form in vivo by subjecting a fish to physical exercise. The conversion of fish muscle phosphorylase b to a was also catalyzed in vitro with purified rabbit muscle phosphorylase b kinase in the presence of ATP and Mg++. Evidence is presented to indicate the presence of phosphorylase b kinase and phosphorylase phosphatase in trout skeletal muscle.

Regulation of muscle glycogen phosphorylase activity during intense aerobic cycling with elevated FFA

1996 ◽  
Vol 270 (1) ◽  
pp. E116-E125 ◽  
Author(s):  
D. J. Dyck ◽  
S. J. Peters ◽  
P. S. Wendling ◽  
A. Chesley ◽  
E. Hultman ◽  
...  
Keyword(s):  
Muscle Glycogen ◽  
Glycogen Phosphorylase ◽  
O2 Consumption ◽  
Elevated Plasma ◽  
Phosphorylase Activity ◽  
Allosteric Regulator ◽  
Plasma Ffa ◽  
Glycogen Phosphorylase Activity ◽  
Muscle Biopsies ◽  
Allosteric Activator

This study examined muscle glycogenolysis and the regulation of glycogen phosphorylase (Phos) activity during 15 min of cycling at 85% of maximal O2 consumption (VO2max) in control and high free fatty acid (FFA; Intralipid-heparin) conditions in 11 subjects. Muscle biopsies were sampled at rest and 1, 5, and 15 min of exercise, and glycogen Phos transformation state (%Phos alpha), substrate (Pi, glycogen), and allosteric regulator (ADP, AMP, IMP) contents were measured. Infusion of intralipid elevated plasma FFA from 0.32 +/- 0.04 mM at rest to 1.00 +/- 0.04 mM just before exercise and 1.12 +/- 0.10 mM at 14 min of exercise. In the control trial, plasma FFA were 0.36 +/- 0.04 mM at rest and unchanged at the end of exercise (0.34 +/- 0.03 mM). Seven subjects used less muscle glycogen (46.7 +/- 7.6%, mean +/- SE) during the Intralipid trial, and four did not respond. In subjects who spared glycogen, glycogen Phos transformation into the active (alpha) form was unaffected by high FFA except for a nonsignificant reduction during the initial 5 min of exercise. Total AMP and IMP contents were not significantly different during exercise between trials, but total ADP was significantly lower with Intralipid only at 15 min. The calculated free ADP, AMP, and Pi contents were lower with Intralipid but not significantly different. However, when the present results were pooled with the data from a previous study using the same protocol [Dyck et al., Am. J. Physiol. 265 (Endocrinol, Metab. 28): E852-E859, 1993], the free ADP, AMP, and Pi contents of all subjects who spared glycogen (n = 13) were significantly lower at 15 min in the Intralipid trial. The findings suggest that the elevation of plasma FFA during intense cycling spares muscle glycogen by posttransformational regulation of Phos. This may be due to blunted increases in the contents of AMP, an allosteric activator of Phos alpha, and Pi, a substrate for Phos.

Sign in / Sign up

Export Citation Format

Share Document