Experimental study on the antifatigue effect of icariin.

Fatigue is a serious disturbance to human health, especially in people who have a severe disease such as cancer, or have been infected with COVID-19. Our research objective is to evaluate the anti-fatigue effect and mechanism of icariin through a mouse experimental model. Mice were treated with icariin for 30 days and anti-fatigue effects were evaluated by the weight-bearing swimming test, serum urea nitrogen test, lactic acid accumulation and clearance test in blood and the amount of liver glycogen. The protein expression levels of adenosine monophosphate-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC1-α) in the skeletal muscle of mice in each group were measured by western blotting. Results showed that icariin prolonged the weight-bearing swimming time of animals, reduced the serum urea nitrogen level after exercise, decreased the blood lactic acid concentration after exercise and increased the liver glycogen content observably. Compared to that in the control group, icariin upregulated AMPK and PGC1-α expression in skeletal muscle. Icariin can improve fatigue resistance in mice and its mechanism may be through improving the AMPK/PGC-1α pathway in skeletal muscle to enhance energy synthesis, decreasing the accumulation of metabolites and slowing glycogen consumption and decomposition.

Study on Anti-fatigue Effects and Mechanisms of Polysaccharide from Paris polyphylla.

The objectives of this study were to investigate the anti-fatigue effects of Paris polyphylla polysaccharide component 1 (PPPm-1) and explore its mechanisms. A mouse model of exercise-induced fatigue was established by weight-bearing swimming to observe the effects of different concentrations of PPPm-1 on weight-bearing swimming time. The anti-fatigue effect of PPPm-1 was determined by the effects of contraction amplitude, contraction rate, and diastolic rate of the frog gastrocnemius muscle in vivo before and after infiltration with 5 mg/mL PPPm-1. The effects of PPPm-1 on the contents of blood lactate, serum urea nitrogen, hepatic glycogen, muscle glycogen in the exercise fatigue model of mice, and acetylcholine (ACh) content and acetylcholinesterase (AChE) activity at the junction of the frog sciatic nerve-gastrocnemius under normal physiological, and Na+-K+-ATPase and Ca2+-Mg2+-ATPase activities of the frog gastrocnemius were determined by enzyme-linked immunosorbent assay (ELISA), to investigate the anti-fatigue mechanisms of PPPm-1. The results showed that PPPm-1 could significantly prolong the weight-bearing swimming time in mice (P < 0.01), decrease the contents of blood lactate and serum urea nitrogen, increase the contents of the hepatic glycogen and muscle glycogen of mice after exercise fatigue compared with those of the control group, and there was extremely significant difference in most indicators (P < 0.01). The 5 mg/mL of PPPm-1 could significantly promote the contraction amplitude, contraction rate, and relaxation rate of the gastrocnemius muscle in the frogs, and the content of ACh at the junction of the frog sciatic nerve-gastrocnemius (P < 0.01), but it had obvious inhibitory effetc on the activity of AChE at the junction of the frog sciatic nerve-gastrocnemius (P < 0.01). PPPm-1 could increase the Na+-K+-ATPase and Ca2+-Mg2+-ATPase activities of gastrocnemius in the frogs (for Ca2+-Mg2+-ATPase, P < 0.01). The above results suggested that the PPPm-1 had a good anti-fatigue effect, and its main mechanisms were related to improving endurance and glycogen reserve, reducing glycogen consumption, lactate and serum urea nitrogen accumulation, and promoting Ca2+ influx.

Microbiome structure and function in parallel full-scale aerobic granular sludge and activated sludge processes.

Aerobic granular sludge (AGS) and conventional activated sludge (CAS) are two different biological wastewater treatment processes. AGS consists of self-immobilised microorganisms that are transformed into spherical biofilms, whereas CAS has floccular sludge of lower density. In this study, we investigated the treatment performance and microbiome dynamics of two full-scale AGS reactors and a parallel CAS system at a municipal WWTP in Sweden. Both systems produced low effluent concentrations, with some fluctuations in phosphate and nitrate mainly due to variations in organic substrate availability. The microbial diversity was slightly higher in the AGS, with different dynamics in the microbiome over time. Seasonal periodicity was observed in both sludge types, with a larger shift in the CAS microbiome compared to the AGS. Groups important for reactor function, such as ammonia-oxidising bacteria (AOB), nitrite-oxidising bacteria (NOB), polyphosphate-accumulating organisms (PAOs) and glycogen-accumulating organisms (GAOs), followed similar trends in both systems, with higher relative abundances of PAOs and GAOs in the AGS. However, microbial composition and dynamics differed between the two systems at the genus level. For instance, among PAOs, Tetrasphaera was more prevalent in the AGS, while Dechloromonas was more common in the CAS. Among NOB, Ca. Nitrotoga had a higher relative abundance in the AGS, while Nitrospira was the main nitrifier in the CAS. Furthermore, network analysis revealed the clustering of the various genera within the guilds to modules with different temporal patterns, suggesting functional redundancy in both AGS and CAS. KEY POINTS: • Microbial community succession in parallel full-scale aerobic granular sludge (AGS) and conventional activated sludge (CAS) processes. • Higher periodicity in microbial community structure in CAS compared to in AGS. • Similar functional groups between AGS and CAS but different composition and dynamics at genus level.

The Effects of Resistance Exercise Training on Skeletal Muscle Metabolism and Insulin Resistance Development in Female Rodents with Type 1 Diabetes.

The etiology of insulin resistance (IR) development in type 1 diabetes mellitus (T1DM) remains unclear; however, impaired skeletal muscle metabolism may play a role. While IR development has been established in male T1DM rodents, female rodents have yet to be examined in this context. Resistance exercise training (RT) has been shown to improve IR and is associated with a lower risk of hypoglycemia onset in T1DM compared to aerobic exercise. The purpose of this study was to investigate the effects of RT on IR development in female T1DM rodents. Forty Sprague Dawley eight-week-old female rats were divided into four groups: control sedentary (CS; n = 10), control trained (CT; n = 10), T1DM sedentary (DS; n = 10), and T1DM trained (DT; n = 10). Multiple low-dose streptozotocin injections were used to induce T1DM. Blood glucose levels were maintained in the 4-9 mmol/l range with intensive insulin therapy. CT and DT underwent weighted ladder climbing 5 days/week for six weeks. Intravenous glucose tolerance tests (IVGTT) were conducted on all animals following the six-week period. Results demonstrate that DS animals exhibited significantly increased weekly blood glucose measures compared to all groups including DT (p < 0.0001), despite similar insulin dosage levels. This was concomitant with a significant increase in insulin-adjusted area under the curve following IVGTT in DS (p < 0.05), indicative of a reduction in insulin sensitivity. Both DT and DS exhibited greater serum insulin concentrations compared to CT and CS (p < 0.05). DS animals also exhibited significantly greater glycogen content in white gastrocnemius muscle compared to CS and DT (p < 0.05), whereas DT and DS animals exhibited greater p-Akt: Akt ratio in the white vastus lateralis muscle and citrate synthase activity in the red vastus lateralis muscle compared to CS and CT (p < 0.05). These results indicate that female rodents with T1DM develop poor glycemic control and IR which can be attenuated with RT, possibly related to differences in intramyocellular glycogen content.

Spexin acts as a novel glucose-lowering factor in grass carp (Ctenopharyngodon idella).

At present, the physiological roles of various hormones in fish glucose metabolism have been elucidated. Spexin, a 14-amino acids polypeptide, is highly conserved in many species and has functions such as reducing body weight and improving insulin resistance. In this paper, the open reading frame (ORF) of spx21 in grass carp (Ctenopharyngodon idella) was cloned, and the tissue distribution of spx1 and spx2, their direct and indirect regulatory effects on glucose metabolism of grass carp were investigated. The ORF of spx2 gene in grass carp was 279 bp in length. Moreover, spx1 was highly expressed in the adipose tissue, while spx2 was highly expressed in the brain. In vitro, SPX1 and SPX2 showed opposite effects on the glycolytic pathway in the primary hepatocytes. In vivo, intraperitoneal injection of SPX1 and SPX2 significantly reduced serum glucose levels and increased hepatopancreas glycogen contents. Meanwhile, SPX1 and SPX2 promoted the expression of key genes of glycolysis (pk) and glycogen synthesis (gys) in the hepatopancreas at 3 h post injection. As for indirect effects, 1000 nM SPX1 and SPX2 significantly increased insulin-mediated liver type phosphofructokinase (pfkla) mRNA expression and enhanced the inhibitory effects of insulin on glucose-6-phosphatase (g6pase), phosphoenolpyruvate carboxykinase (pepck), glycogen phosphorylase L (pygl) mRNA expression. Our results show that SPX1 and SPX2 have similar indirect effects on the regulation of glucose metabolism that enhance insulin activity, but they exhibit opposite roles in terms of direct effects.

Treatment of infantile-onset Pompe disease in a rat model with muscle-directed AAV gene therapy.

OBJECTIVE: Pompe disease (PD) is caused by deficiency of the lysosomal enzyme acid α-glucosidase (GAA), leading to progressive glycogen accumulation and severe myopathy with progressive muscle weakness. In the Infantile-Onset PD (IOPD), death generally occurs <1 year of age. There is no cure for IOPD. Mouse models of PD do not completely reproduce human IOPD severity. Our main objective was to generate the first IOPD rat model to assess an innovative muscle-directed adeno-associated viral (AAV) vector-mediated gene therapy. METHODS: PD rats were generated by CRISPR/Cas9 technology. The novel highly myotropic bioengineered capsid AAVMYO3 and an optimized muscle-specific promoter in conjunction with a transcriptional cis-regulatory element were used to achieve robust Gaa expression in the entire muscular system. Several metabolic, molecular, histopathological, and functional parameters were measured. RESULTS: PD rats showed early-onset widespread glycogen accumulation, hepato- and cardiomegaly, decreased body and tissue weight, severe impaired muscle function and decreased survival, closely resembling human IOPD. Treatment with AAVMYO3-Gaa vectors resulted in widespread expression of Gaa in muscle throughout the body, normalizing glycogen storage pathology, restoring muscle mass and strength, counteracting cardiomegaly and normalizing survival rate. CONCLUSIONS: This gene therapy holds great potential to treat glycogen metabolism alterations in IOPD. Moreover, the AAV-mediated approach may be exploited for other inherited muscle diseases, which also are limited by the inefficient widespread delivery of therapeutic transgenes throughout the muscular system.

HBV DNA polymerase regulates tumor cell glycogen to enhance the malignancy of HCC cells.

BACKGROUND: The essential function of HBV DNA polymerase (HBV-DNA-Pol) is to initiate viral replication by reverse transcription; however, the role of HBV-DNA-Pol in HBV-associated HCC has not been clarified. Glycogen phosphorylase L (PYGL) is a critical regulator of glycogenolysis and is involved in tumorigenesis, including HCC. However, it is unknown whether HBV-DNA-Pol regulates PYGL to contribute to HCC tumorigenesis. METHODS: Bioinformatic analysis, real-time quantitative PCR, western blotting, and oncology functional assays were performed to determine the contribution of HBV-DNA-Pol and PYGL to HCC development and glycolysis. The mechanisms of co-immunoprecipitation and ubiquitination were employed to ascertain how HBV-DNA-Pol upregulated PYGL. RESULTS: Overexpression of HBV-DNA-Pol enhanced HCC progression in vitro and in vivo. Mechanistically, HBV-DNA-Pol interacted with PYGL and increased PYGL protein levels by inhibiting PYGL ubiquitination, which was mediated by the E3 ligase TRIM21. HBV-DNA-Pol competitively impaired the binding of PYGL to TRIM21 due to its stronger binding affinity to TRIM21, suppressing the ubiquitination of PYGL. Moreover, HBV-DNA-Pol promoted glycogen decomposition by upregulating PYGL, which led to an increased flow of glucose into glycolysis, thereby promoting HCC development. CONCLUSIONS: Our study reveals a novel mechanism by which HBV-DNA-Pol promotes HCC by controlling glycogen metabolism in HCC, establishing a direct link between HBV-DNA-Pol and the Warburg effect, thereby providing novel targets for HCC treatment and drug development.

Glycogen metabolism-mediated intercellular communication in the tumor microenvironment influences liver cancer prognosis.

Glycogen metabolism plays a key role in the development of hepatocellular carcinoma (HCC), but the function of glycogen metabolism genes in the tumor microenvironment (TME) is still to be elucidated. Single-cell RNA-seq data were obtained from ten HCC tumor samples totaling 64,545 cells, and 65 glycogen metabolism genes were analyzed by a nonnegative matrix factorization (NMF). The prognosis and immune response of new glycogen TME cell clusters were predicted by using HCC and immunotherapy cohorts from public databases. HCC single-cell analysis was divided into fibroblasts, NT T cells, macrophages, endothelial cells, and B cells, which were separately divided into new cell clusters by glycogen metabolism gene annotation. Pseudo-temporal trajectory analysis demonstrated the temporal differentiation trajectory of different glycogen subtype cell clusters. Cellular communication analysis revealed extensive interactions between endothelial cells with glycogen metabolizing TME cell-related subtypes and different glycogen subtype cell clusters. SCENIC analysis of transcription factors upstream of TME cell clusters with different glycogen metabolism. In addition, TME cell clusters of glycogen metabolism were found to be enriched in expression in CAF subtypes, CD8 depleted, M1, and M2 types. Bulk-seq analysis showed the prognostic significance of glycogen metabolism-mediated TME cell clusters in HCC, while a significant immune response was found in the immunotherapy cohort in patients treated with immune checkpoint blockade (ICB), especially for CAFs, T cells, and macrophages. In summary, our study reveals for the first time that glycogen metabolism mediates intercellular communication in the hepatocellular carcinoma microenvironment while elucidating the anti-tumor mechanisms and immune prognostic responses of different subtypes of cell clusters.

Effect of resveratrol on insulin action in primary myotubes from lean individuals and individuals with severe obesity.

Resveratrol, a natural polyphenol compound contained in numerous plants, has been proposed as a treatment for obesity-related disease processes such as insulin resistance. However, in humans there are conflicting results concerning the efficacy of resveratrol in improving insulin action; the purpose of the present study was to determine whether obesity status (lean, severely obese) affects the response to resveratrol in human skeletal muscle. Primary skeletal muscle cells were derived from biopsies obtained from age-matched lean and insulin-resistant women with severe obesity and incubated with resveratrol (1 µM) for 24 h. Insulin-stimulated glucose oxidation and incorporation into glycogen, insulin signal transduction, and energy-sensitive protein targets [AMP-activated protein kinase (AMPK), Sirt1, and PGC1α] were analyzed. Insulin-stimulated glycogen synthesis, glucose oxidation, and AMPK phosphorylation increased with resveratrol incubation compared with the nonresveratrol conditions (main treatment effect for resveratrol). Resveratrol further increased IRS1, Akt, and TBC1D4 insulin-stimulated phosphorylation and SIRT1 content in myotubes from lean women, but not in women with severe obesity. Resveratrol improves insulin action in primary human skeletal myotubes derived from lean women and women with severe obesity. In women with obesity, these improvements may be associated with enhanced AMPK phosphorylation with resveratrol treatment.NEW & NOTEWORTHY A physiologically relevant dose of resveratrol increases insulin-stimulated glucose oxidation and glycogen synthesis in myotubes from individuals with severe obesity. Furthermore, resveratrol improved insulin signal transduction in myotubes from lean individuals but not from individuals with obesity. Activation of AMPK plays a role in resveratrol-induced improvements in glucose metabolism in individuals with severe obesity.

Biomolecular fingerprints of the effect of zoledronic acid on prostate cancer stem cells: Comparison of 2D and 3D cell culture models.

Revealing the potential of candidate drugs against different cancer types without disrupting normal cells depends on the drug mode of action. In the current study, the drug response of prostate cancer stem cells (PCSCs) to zoledronic acid (ZOL) grown in two-dimensional (2D) and three-dimensional (3D) culture systems was compared using Fourier transform-infrared (FT-IR) spectroscopy which is a vibrational spectroscopic technique, supporting by biochemical assays and imaging techniques. Based on our data, in 2D cell culture conditions, the ZOL treatment of PCSCs isolated according to both C133 and CD44 cell surface properties induced early/late apoptosis and suppressed migration ability. The CD133 gene expression and protein levels were altered, depending on culture systems. CD133 expression was significantly reduced in 2D cells upon ZOL treatment. FT-IR data revealed that the integrity, fluidity, and ordering/disordering states of the cell membrane and nucleic acid content were altered in both 2D and 3D cells after ZOL treatment. Regular protein structures decrease in 2D cells while glycogen and protein contents increase in 3D cells, indicating a more pronounced cytotoxic effect of ZOL for 2D cells. Untreated 3D PCSCs exhibited an even different spectral profile associated with IR signals of lipids, proteins, nucleic acids, and glycogen in comparison to untreated 2D cells. Our study revealed significant differences in the drug response and cellular constituents between 2D and 3D cells. Exploring molecular targets and/or drug-action mechanisms is significant in cancer treatment approaches; thus, FT-IR spectroscopy can be successfully applied as a novel drug-screening method in clinical research.

Bed-rest and exercise remobilization: Concurrent adaptations in muscle glucose and protein metabolism.

BACKGROUND: Bed-rest (BR) of only a few days duration reduces muscle protein synthesis and induces skeletal muscle atrophy and insulin resistance, but the scale and juxtaposition of these events have not been investigated concurrently in the same individuals. Moreover, the impact of short-term exercise-supplemented remobilization (ESR) on muscle volume, protein turnover and leg glucose uptake (LGU) in humans is unknown. METHODS: Ten healthy males (24 ± 1 years, body mass index 22.7 ± 0.6 kg/m2) underwent 3 days of BR, followed immediately by 3 days of ESR consisting of 5 × 30 maximal voluntary single-leg isokinetic knee extensions at 90°/s each day. An isoenergetic diet was maintained throughout the study (30% fat, 15% protein and 55% carbohydrate). Resting LGU was calculated from arterialized-venous versus venous difference across the leg and leg blood flow during the steady-state of a 3-h hyperinsulinaemic-euglycaemic clamp (60 mU/m2/min) measured before BR, after BR and after remobilization. Glycogen content was measured in vastus lateralis muscle biopsy samples obtained before and after each clamp. Leg muscle volume (LMV) was measured using magnetic resonance imaging before BR, after BR and after remobilization. Cumulative myofibrillar protein fractional synthetic rate (FSR) and whole-body muscle protein breakdown (MPB) were measured over the course of BR and remobilization using deuterium oxide and 3-methylhistidine stable isotope tracers that were administered orally. RESULTS: Compared with before BR, there was a 45% decline in insulin-stimulated LGU (P < 0.05) after BR, which was paralleled by a reduction in insulin-stimulated leg blood flow (P < 0.01) and removal of insulin-stimulated muscle glycogen storage. These events were accompanied by a 43% reduction in myofibrillar protein FSR (P < 0.05) and a 2.5% decrease in LMV (P < 0.01) during BR, along with a 30% decline in whole-body MPB after 2 days of BR (P < 0.05). Myofibrillar protein FSR and LMV were restored by 3 days of ESR (P < 0.01 and P < 0.01, respectively) but not by ambulation alone. However, insulin-stimulated LGU and muscle glycogen storage were not restored by ESR. CONCLUSIONS: Three days of BR caused concurrent reductions in LMV, myofibrillar protein FSR, myofibrillar protein breakdown and insulin-stimulated LGU, leg blood flow and muscle glycogen storage in healthy, young volunteers. Resistance ESR restored LMV and myofibrillar protein FSR, but LGU and muscle glycogen storage remained depressed, highlighting divergences in muscle fuel and protein metabolism. Furthermore, ambulation alone did not restore LMV and myofibrillar protein FSR in the non-exercised contralateral limb, emphasizing the importance of exercise rehabilitation following even short-term BR.

Brain-Type Glycogen Phosphorylase (PYGB) in the Pathologies of Diseases: A Systematic Review.

Glycogen metabolism is a form of crucial metabolic reprogramming in cells. PYGB, the brain-type glycogen phosphorylase (GP), serves as the rate-limiting enzyme of glycogen catabolism. Evidence is mounting for the association of PYGB with diverse human diseases. This review covers the advancements in PYGB research across a range of diseases, including cancer, cardiovascular diseases, metabolic diseases, nervous system diseases, and other diseases, providing a succinct overview of how PYGB functions as a critical factor in both physiological and pathological processes. We present the latest progress in PYGB in the diagnosis and treatment of various diseases and discuss the current limitations and future prospects of this novel and promising target.

Stratification of tumour cell radiation response and metabolic signatures visualization with Raman spectroscopy and explainable convolutional neural network.

Reprogramming of cellular metabolism is a driving factor of tumour progression and radiation therapy resistance. Identifying biochemical signatures associated with tumour radioresistance may assist with the development of targeted treatment strategies to improve clinical outcomes. Raman spectroscopy (RS) can monitor post-irradiation biomolecular changes and signatures of radiation response in tumour cells in a label-free manner. Convolutional Neural Networks (CNN) perform feature extraction directly from data in an end-to-end learning manner, with high classification performance. Furthermore, recently developed CNN explainability techniques help visualize the critical discriminative features captured by the model. In this work, a CNN is developed to characterize tumour response to radiotherapy based on its degree of radioresistance. The model was trained to classify Raman spectra of three human tumour cell lines as radiosensitive (LNCaP) or radioresistant (MCF7, H460) over a range of treatment doses and data collection time points. Additionally, a method based on Gradient-Weighted Class Activation Mapping (Grad-CAM) was used to determine response-specific salient Raman peaks influencing the CNN predictions. The CNN effectively classified the cell spectra, with accuracy, sensitivity, specificity, and F1 score exceeding 99.8%. Grad-CAM heatmaps of H460 and MCF7 cell spectra (radioresistant) exhibited high contributions from Raman bands tentatively assigned to glycogen, amino acids, and nucleic acids. Conversely, heatmaps of LNCaP cells (radiosensitive) revealed activations at lipid and phospholipid bands. Finally, Grad-CAM variable importance scores were derived for glycogen, asparagine, and phosphatidylcholine, and we show that their trends over cell line, dose, and acquisition time agreed with previously established models. Thus, the CNN can accurately detect biomolecular differences in the Raman spectra of tumour cells of varying radiosensitivity without requiring manual feature extraction. Finally, Grad-CAM may help identify metabolic signatures associated with the observed categories, offering the potential for automated clinical tumour radiation response characterization.

Female tilapia, Oreochromis sp. mobilised energy differently for growth and reproduction according to living environment.

This study was conducted to investigate the energy mobilisation preference and ionoregulation pattern of female tilapia, Oreochromis sp. living in different environments. Three different treatments of tilapia as physiology compromising model were compared; tilapia cultured in recirculating aquaculture system (RAS as Treatment I-RAS), tilapia cultured in open water cage (Treatment II-Cage) and tilapia transferred from cage and cultured in RAS (Treatment III-Compensation). Results revealed that tilapia from Treatment I and III mobilised lipid to support gonadogenesis, whilst Treatment II tilapia mobilised glycogen as primary energy for daily exercise activity and reserved protein for growth. The gills and kidney Na+/K+ ATPase (NKA) activities remained relatively stable to maintain homeostasis with a stable Na+ and K+ levels. As a remark, this study revealed that tilapia strategized their energy mobilisation preference in accessing glycogen as an easy energy to support exercise metabolism and protein somatogenesis in cage culture condition, while tilapia cultured in RAS mobilised lipid for gonadagenesis purposes.

A novel genetic model provides a unique perspective on the relationship between postexercise glycogen concentration and increases in the abundance of key metabolic proteins after acute exercise.

Some acute exercise effects are influenced by postexercise (PEX) diet, and these diet-effects are attributed to differential glycogen resynthesis. However, this idea is challenging to test rigorously. Therefore, we devised a novel genetic model to modify muscle glycogen synthase 1 (GS1) expression in rat skeletal muscle with an adeno-associated virus (AAV) short hairpin RNA knockdown vector targeting GS1 (shRNA-GS1). Contralateral muscles were injected with scrambled shRNA (shRNA-Scr). Muscles from exercised (2-hour-swim) and time-matched sedentary (Sed) rats were collected immediately postexercise (IPEX), 5-hours-PEX (5hPEX), or 9-hours-PEX (9hPEX). Rats in 5hPEX and 9hPEX experiments were refed (RF) or not-refed (NRF) chow. Muscles were analyzed for glycogen, abundance of metabolic proteins (pyruvate dehydrogenase kinase 4, PDK4; peroxisome proliferator-activated receptor γ coactivator-1α, PGC1α; hexokinase II, HKII; glucose transporter 4, GLUT4), AMP-activated protein kinase phosphorylation (pAMPK), and glycogen metabolism-related enzymes (glycogen phosphorylase, PYGM; glycogen debranching enzyme, AGL; glycogen branching enzyme, GBE1). shRNA-GS1 versus paired shRNA-Scr muscles had markedly lower GS1 abundance. IPEX versus Sed rats had lower glycogen and greater pAMPK, and neither of these IPEX-values differed for shRNA-GS1 versus paired shRNA-Scr muscles. IPEX versus Sed groups did not differ for abundance of metabolic proteins, regardless of GS1 knockdown. Glycogen in RF-rats was lower for shRNA-GS1 versus paired shRNA-Scr muscles at both 5hPEX and 9hPEX. HKII protein abundance was greater for 5hPEX versus Sed groups, regardless of GS1 knockdown or diet, and despite differing glycogen levels. At 9hPEX, shRNA-GS1 versus paired shRNA-Scr muscles had greater PDK4 and PGC1α abundance within each diet group. However, the magnitude of PDK4 or PGC1α changes was similar in each diet group regardless of GS1 knockdown although glycogen differed between paired muscles only in RF-rats. In summary, we established a novel genetic approach to investigate the relationship between muscle glycogen and other exercise effects. Our results suggest that exercise-effects on abundance of several metabolic proteins did not uniformly correspond to differences in postexercise glycogen.

Citrus limon (L.) Osbeck Fruit Peel Extract Attenuates Carbon Tetrachloride-Induced Hepatocarcinogenesis in Sprague-Dawley Rats.

Background: Traditional herbal medicine practitioners in the Ashanti region of Ghana use the fruit peels of Citrus limon (L.) Osbeck (C. limon) in preventive and curative treatment of many cancers including liver cancer. This ethnobotanical claim remains to be verified scientifically. Aim of the Study. This study investigated prophylactic hepatoprotective and anti-HCC effects of C. limon peel extract (LPE) in CCl4/olive oil-induced HCC-like rats. Materials and Methods: After preparation of LPE, it was subjected to phytochemical screening using standard phytochemical methods. A total of 30 healthy adult male Sprague-Dawley rats (weighing 150-200 g) were randomly assigned into six groups of 5 rats each. Rats in the control group received olive oil (5 mL/kg ip) twice weekly for 16 weeks. Rats in the model group received CCl4/olive oil (2 mL/kg, ip) twice weekly for 16 weeks. Rats in capecitabine (10 mg/kg po) and LPE (50, 100, and 200 mg/kg po) groups received CCl4/olive oil (2 mL/kg, i.p) in the morning and their respective treatments in the afternoon twice a week for 16 weeks. Rats in all groups had free access to food and water ad libitum. Body weight and survival rates were monitored. Rats were sacrificed under deep anesthesia, blood was collected, and liver and other organs were isolated. Aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), gamma-glutamyltransferase (GGT), prothrombin time, bilirubin, C-reactive protein (CRP), alpha- (α-) fetoprotein (AFP), and liver histology were assessed. Results: Alkaloids, tannins, flavonoids, terpenoids, and saponins were detected in LPE. Model rats demonstrated increased serum levels of AFP, CRP, ALP, GGT, ALT, and AST, prothrombin time, total bilirubin, direct bilirubin, blood lymphocyte, and monocyte counts, but decreased serum albumin and total protein compared to control rats. Unlike the control, model rats demonstrated fat accumulation in periportal and centrilobular hepatocytes and neoplastic transformation. Semiquantitation of periodic acid Schiff- (PAS-) stained liver sections showed decreased glycogen storage in hepatocytes of model rats compared to control rats. Compared to the model, LPE treatment protected against CCl4-induced hepatocarcinogenesis, which was evidenced by decreased AFP, CRP, liver enzymes, total and direct bilirubin, prothrombin time, and blood lymphocyte and monocyte counts; attenuation of fat accumulation; and increased glycogen storage, albumin, and total protein. Conclusion: LPE abates CCl4-induced hepatocarcinogenesis by attenuating liver inflammation and improving metabolic, biosynthetic, and detoxification functions of the liver. The prophylactic hepatoprotective and anti-hepatocarcinogenic effects of LPE are attributable to its phytochemical composition raising hopes of finding potential anticancer bioactive compounds from C. limon fruit peels.

Epac2 activation mediates glucagon-induced glucogenesis in primary rat hepatocytes.

AIMS/INTRODUCTION: Glucagon plays an essential role in hepatic glucogenesis by enhancing glycogen breakdown, inducing gluconeogenesis, and suppressing glycogenesis. Moreover, glucagon increases cyclic adenosine monophosphate (cAMP) levels, thereby activating protein kinase A (PKA) and cAMP guanine nucleotide exchange factor (also known as Epac). Although the function of PKA in the liver has been studied extensively, the function of hepatic Epac is poorly understood. The aim of this study was to elucidate the role of Epac in mediating the action of glucagon on the hepatocytes. MATERIALS AND METHODS: Epac mRNA and protein expression, localization, and activity in the hepatocytes were analyzed by reverse transcription polymerase chain reaction, western blotting, immunofluorescence staining, and Rap1 activity assay, respectively. Additionally, we investigated the effects of an Epac-specific activator, 8-CPT, and an Epac-specific inhibitor, ESI-05, on glycogen metabolism in isolated rat hepatocytes. Further mechanisms of glycogen metabolism were evaluated by examining glucokinase (GK) translocation and mRNA expression of gluconeogenic enzymes. RESULTS: Epac2, but not Epac1, was predominantly expressed in the liver. Moreover, 8-CPT inhibited glycogen accumulation and GK translocation and enhanced the mRNA expression of gluconeogenic enzymes. ESI-05 failed to reverse glucagon-induced suppression of glycogen storage and partially inhibited glucagon-induced GK translocation and the mRNA expression of gluconeogenic enzymes. CONCLUSIONS: Epac signaling plays a role in mediating the glucogenic action of glucagon in the hepatocytes.

Structural abnormality of hepatic glycogen in rat liver with diethylnitrosamine-induced carcinogenic injury.

Growing evidence confirms associations between glycogen metabolic re-wiring and the development of liver cancer. Previous studies showed that glycogen structure changes abnormally in liver diseases such as cystic fibrosis, diabetes, etc. However, few studies focus on glycogen molecular structural characteristics during liver cancer development, which is worthy of further exploration. In this study, a rat model with carcinogenic liver injury induced by diethylnitrosamine (DEN) was successfully constructed, and hepatic glycogen structure was characterized. Compared with glycogen structure in the healthy rat liver, glycogen chain length distribution (CLD) shifts towards a short region. In contrast, glycogen particles were mainly present in small-sized ß particles in DEN-damaged carcinogenic rat liver. Comparative transcriptomic analysis revealed significant expression changes of genes and pathways involved in carcinogenic liver injury. A combination of transcriptomic analysis, RT-qPCR, and western blot showed that the two genes, Gsy1 encoding glycogen synthase and Gbe1 encoding glycogen branching enzyme, were significantly altered and might be responsible for the structural abnormality of hepatic glycogen in carcinogenic liver injury. Taken together, this study confirmed that carcinogenic liver injury led to structural abnormality of hepatic glycogen, which provided clues to the future development of novel drug targets for potential therapeutics of carcinogenic liver injury.

A functional mini-GDE transgene corrects impairment in models of glycogen storage disease type III.

Glycogen storage disease type III (GSDIII) is a rare inborn error of metabolism affecting liver, skeletal muscle, and heart due to mutations of the AGL gene encoding for the glycogen debranching enzyme (GDE). No curative treatment exists for GSDIII. The 4.6 kb GDE cDNA represents the major technical challenge toward the development of a single recombinant adeno-associated virus-derived (rAAV-derived) vector gene therapy strategy. Using information on GDE structure and molecular modeling, we generated multiple truncated GDEs. Among them, an N-terminal-truncated mutant, ΔNter2-GDE, had a similar efficacy in vivo compared with the full-size enzyme. A rAAV vector expressing ΔNter2-GDE allowed significant glycogen reduction in heart and muscle of Agl-/- mice 3 months after i.v. injection, as well as normalization of histology features and restoration of muscle strength. Similarly, glycogen accumulation and histological features were corrected in a recently generated Agl-/- rat model. Finally, transduction with rAAV vectors encoding ΔNter2-GDE corrected glycogen accumulation in an in vitro human skeletal muscle cellular model of GSDIII. In conclusion, our results demonstrated the ability of a single rAAV vector expressing a functional mini-GDE transgene to correct the muscle and heart phenotype in multiple models of GSDIII, supporting its clinical translation to patients with GSDIII.

Fluorescent periodic acid Schiff-like staining combined with standard cytologic staining of the same cytologic specimen may facilitate cytopathologic diagnosis.

BACKGROUND: Periodic acid Schiff (PAS) staining which detects glycogen and mucosubstances is frequently used as an ancillary method for an accurate cytopathologic diagnosis. Unfortunately, cytologic slides for PAS stain are not routinely prepared. Aqueous 7-amino-4-methylcoumarin (AMC) is colorless and transparent under bright field illumination but exhibits strong fluorescence under ultraviolet (UV) light and can be used as a Schiff reagent. We recently reported that combining [author: Please define (H&E) in the first occurrence if necessary.]H&E and AMC is useful for histopathologic diagnosis of various disease conditions. In this study, we investigated whether standard cytologic staining (Papanicolaou [Pap] and Giemsa) combined with AMC was useful for cytopathologic analysis. METHODS: Specimens of non-neoplastic human tissues and archived cytologic specimens of various disease conditions were stained with a combination of Pap and AMC (Pap/AMC) or Giemsa and AMC (Giemsa/AMC). RESULTS: The addition of AMC had no significant effect on Pap or Giemsa staining, and the cytomorphology under bright field microscopy was perfectly preserved. The AMC fluorescent signals observed under UV light were intense and the staining pattern was identical to that obtained by PAS staining. Diastase digestion differentiated glycogen from other AMC-positive elements. The efficacy of using Pap/AMC and Giemsa/AMC for archived cytologic specimens was demonstrated in several diseases including cases of endometrial carcinoma, adenoid cystic carcinoma, metastatic signet-ring cell carcinoma, candidiasis, and trichomoniasis. CONCLUSION: Pap/AMC and Giemsa/AMC are useful in aiding cytopathologic diagnosis especially when the information gained from PAS staining is critical and cytologic specimens for PAS are not available.