An In Silico and an In Vitro Inhibition Analysis of Glycogen Phosphorylase by Flavonoids, Styrylchromones, and Pyrazoles

Glycogen phosphorylase (GP) is a key enzyme in the glycogenolysis pathway. GP inhibitors are currently under investigation as a new liver-targeted approach to managing type 2 diabetes mellitus (DM). The aim of the present study was to evaluate the inhibitory activity of a panel of 52 structurally related chromone derivatives; namely, flavonoids, 2-styrylchromones, 2-styrylchromone-related derivatives [2-(4-arylbuta-1,3-dien-1-yl)chromones], and 4- and 5-styrylpyrazoles against GP, using in silico and in vitro microanalysis screening systems. Several of the tested compounds showed a potent inhibitory effect. The structure–activity relationship study indicated that for 2-styrylchromones and 2-styrylchromone-related derivatives, the hydroxylations at the A and B rings, and in the flavonoid family, as well as the hydroxylation of the A ring, were determinants for the inhibitory activity. To support the in vitro experimental findings, molecular docking studies were performed, revealing clear hydrogen bonding patterns that favored the inhibitory effects of flavonoids, 2-styrylchromones, and 2-styrylchromone-related derivatives. Interestingly, the potency of the most active compounds increased almost four-fold when the concentration of glucose increased, presenting an IC50 < 10 µM. This effect may reduce the risk of hypoglycemia, a commonly reported side effect of antidiabetic agents. This work contributes with important considerations and provides a better understanding of potential scaffolds for the study of novel GP inhibitors.

Impact of Glyphosate Exposure on Glycogen Metabolic Enzymes in the Liver of Adult Male Rats

Background: Glyphosate is a broad spectrum herbicide and desiccant. Diabetes is a group of metabolic diseases resulting due to deficiency in insulin secretion. Chronic hyperglycemia will lead to long term damage and failure of different organs like eyes, kidneys, nerves etc. Liver is the major site for gluconeogenesis and a lot of glycolytic enzymes will be involved. Expression of Glycogen synthase and glycogen phosphorylase, the glycolytic enzymes are studied in this research. Aim: To determine whether glyphosate exposure is detrimental to the glycogen metabolic enzymes (Glycogen synthase and phosphorylase) in the liver of adult male rats. Materials and Methods: The following study was done on albino rats of wistar strain, and was approved by the institutional animal ethics committee. They were fed with a rat pellet diet. In our study the rats were divided into 4 groups with 6 rats in each and were subjected to glyphosate orally with different dosage in each group and mRNA expression analysis of glycogen related enzymes was done after a span of 16 weeks. The data were analyzed statistically by a one way analysis of variance (ANOVA) followed by Duncan’s multiple range test was used to see the statistical significance among the group. The results with p<0.05 level were considered to be statistically significant. Results: The present result showed that the mRNA expression of glycogen synthase significantly reduced (P<0.05) and mRNA expression of glycogen phosphorylase activity increased significantly with an increased dose of glyphosate (P<0.05) to that of control. Conclusion: Exposure to glyphosate causes detrimental changes in the glycolytic enzymes glycogen synthase and glycogen phosphorylase leading to diabetes.

Therapeutic Targets Underlying the Evolution of Potential Anti-Diabetic Agents: A Comprehensive Review

: Type 2 Diabetes is termed as a chronic metabolic syndrome that occurs due to hyperglycaemia. Type 2 diabetes is growing worldwide really fast. Looking at all the circumstances, it was recognized that insulin check and Beta-cell brokenness are essential issues that are responsible for the unforeseen development of diabetes. Drugs for diabetes were found to have glucose level lessening impacts in the body and they were named as Glucagon-like peptide 1, amylin. Amylin is one of the hormones that show its action after having a meal; they have correlated activity to the pancreatic hormones. GLP-1 speedily corrupts via dipeptidyl-peptidase-4 in-vivo that leads to a decline in the said feasibility. Some other efficacious biological medicines that are being used for Type-2 Diabetes are 11beta-HSD-1 inhibitors or rivals in relation to glucocorticoids receptor, some other for reducing hepatic glucose level these are rival of glucagon receptor or maybe inhibitors to glycogen phosphorylase or fructose-1,6-biphosphatase, last but not least mounting urinary discarding of superfluity glucose those are SGLT inhibitors. Moreover, treatments related to Incretin can be glucagon-like peptide-1 [GLP-1] inhibitors and dipeptidyl peptidase 4 [DPP-4] inhibitors. Certain Non-incretin beta-cell stimulants are also in the phase of developments and they are glucokinase activators, G-protein-coupled receptors. These are soothing, antagonistic towards the oxidizing agent. Targets that cause glucose stimulant effect were glucose-6-phosphatase, glycogen phosphorylase and inhibition of same causes vice-versa. Some more novel targets are yet to be found; these superiors will additionally target metabolic ailment, as talking about the present situation it is one novel and primary clinical ailment that individual in the world estimated to suffer from the threat of 2nd type of diabetes.

Human hair follicles operate an internal Cori cycle and modulate their growth via glycogen phosphorylase

Hair follicles (HFs) are unique, multi-compartment, mini-organs that cycle through phases of active hair growth and pigmentation (anagen), apoptosis-driven regression (catagen) and relative quiescence (telogen). Anagen HFs have high demands for energy and biosynthesis precursors mainly fulfilled by aerobic glycolysis. Histochemistry reports the outer root sheath (ORS) contains high levels of glycogen. To investigate a functional role for glycogen in the HF we quantified glycogen by Periodic-Acid Schiff (PAS) histomorphometry and colorimetric quantitative assay showing ORS of anagen VI HFs contained high levels of glycogen that decreased in catagen. qPCR and immunofluorescence microscopy showed the ORS expressed all enzymes for glycogen synthesis and metabolism. Using human ORS keratinocytes (ORS-KC) and ex vivo human HF organ culture we showed active glycogen metabolism by nutrient starvation and use of a specific glycogen phosphorylase (PYGL) inhibitor. Glycogen in ORS-KC was significantly increased by incubation with lactate demonstrating a functional Cori cycle. Inhibition of PYGL significantly stimulated the ex vivo growth of HFs and delayed onset of catagen. This study defines translationally relevant and therapeutically targetable new features of HF metabolism showing that human scalp HFs operate an internal Cori cycle, synthesize glycogen in the presence of lactate and modulate their growth via PYGL activity.

Glycogen phosphorylase revisited: extending the resolution of the R- and T-state structures of the free enzyme and in complex with allosteric activators

The crystal structures of free T-state and R-state glycogen phosphorylase (GP) and of R-state GP in complex with the allosteric activators IMP and AMP are reported at improved resolution. GP is a validated pharmaceutical target for the development of antihyperglycaemic agents, and the reported structures may have a significant impact on structure-based drug-design efforts. Comparisons with previously reported structures at lower resolution reveal the detailed conformation of important structural features in the allosteric transition of GP from the T-state to the R-state. The conformation of the N-terminal segment (residues 7–17), the position of which was not located in previous T-state structures, was revealed to form an α-helix (now termed α0). The conformation of this segment (which contains Ser14, phosphorylation of which leads to the activation of GP) is significantly different between the T-state and the R-state, pointing in opposite directions. In the T-state it is packed between helices α4 and α16 (residues 104–115 and 497–508, respectively), while in the R-state it is packed against helix α1 (residues 22′–38′) and towards the loop connecting helices α4′ and α5′ of the neighbouring subunit. The allosteric binding site where AMP and IMP bind is formed by the ordering of a loop (residues 313–326) which is disordered in the free structure, and adopts a conformation dictated mainly by the type of nucleotide that binds at this site.

Proteomic profile and morphological characteristics of skeletal muscle from the fast- and slow-growing yellow perch (Perca flavescens)

The objective of the present study was to compare skeletal muscle proteomic profiles, histochemical characteristics, and expression levels of myogenic regulatory factors (MRFs) between fast- versus slow-growing yellow perch Perca flavescens and identify the proteins/peptides that might play a crucial role in the muscle growth dynamic. Yellow perch were nursed in ponds for 6 weeks from larval stage and cultured in two meter diameter tanks thereafter. The fingerlings were graded to select the top 10% and bottom 10% fish which represented fast- and slow-growing groups (31 yellow perch per each group). Our statistical analyses showed 18 proteins that had different staining intensities between fast- and slow-growing yellow perch. From those proteins 10 showed higher expression in slow-growers, and 8 demonstrated higher expression in fast-growers. Fast-growing yellow perch with a greater body weight was influenced by both the muscle fiber hypertrophy and mosaic hyperplasia compared to slow-growing fish. These hyperplastic and hypertrophic growth in fast-grower were associated with not only metabolic enzymes, including creatine kinase, glycogen phosphorylase, and aldolase, but also myoD and myogenin as MRFs. Overall, the results of the present study contribute to the identification of different expression patterns of gene products in fast- and slow-growing fish associated with their muscle growth.

The Phenotypic and Genetic Spectrum of Glycogen Storage Disease Type VI

Glycogen storage disease type VI (GSD VI) is an autosomal recessive disorder of glycogen metabolism due to mutations in the glycogen phosphorylase gene (PYGL), resulting in a deficiency of hepatic glycogen phosphorylase. We performed a systematic literature review in order to collect information on the clinical phenotypes and genotypes of all published GSD VI patients and to compare the data to those for GSD IX, a biochemically and clinically very similar disorder caused by a deficiency of phosphorylase kinase. A total of 63 genetically confirmed cases of GSD VI with clinical information were identified (median age: 5.3 years). The age at presentation ranged from 5 weeks to 38 years, with a median of 1.8 years. The main presenting symptoms were hepatomegaly and poor growth, while the most common laboratory findings at initial presentation comprised elevated activity of liver transaminases, hypertriglyceridemia, fasting hypoglycemia and postprandial hyperlactatemia. Liver biopsies (n = 37) showed an increased glycogen content in 89.2%, liver fibrosis in 32.4% and early liver cirrhosis in 10.8% of cases, respectively. No patient received a liver transplant, and one successful pregnancy was reported. Our review demonstrates that GSD VI is a disorder with broad clinical heterogeneity and a small number of patients with a severe phenotype and liver cirrhosis. Neither clinical nor laboratory findings allow for a differentiation between GSD VI and GSD IX. Early biochemical markers of disease severity or clear genotype phenotype correlations are missing. Given the overall benign and unspecific phenotype and the need for enzymatic or genetic analyses for confirmation of the diagnosis, GSD VI is likely underdiagnosed. With new treatment approaches in sight, early, pre-symptomatic diagnosis, especially with respect to hepatic cirrhosis, will become even more important.

Optimization and Validation of an In Vitro Standardized Glycogen Phosphorylase Activity Assay

Glycogen phosphorylase (GP) is a key enzyme in the glycogenolysis pathway and a potential therapeutic target in the management of type 2 diabetes. It catalyzes a reversible reaction: the release of the terminal glucosyl residue from glycogen as glucose 1-phosphate; or the transfer of glucose from glucose 1-phosphate to glycogen. A colorimetric method to follow in vitro the activity of GP with usefulness in structure-activity relationship studies and high-throughput screening capability is herein described. The obtained results allowed the choice of the optimal concentration of enzyme of 0.38 U/mL, 0.25 mM glucose 1-phosphate, 0.25 mg/mL glycogen, and temperature of 37 °C. Three known GP inhibitors, CP-91149, a synthetic inhibitor, caffeine, an alkaloid, and ellagic acid, a polyphenol, were used to validate the method, CP-91149 being the most active inhibitor. The effect of glucose on the IC50 value of CP-91149 was also investigated, which decreased when the concentration of glucose increased. The assay parameters for a high-throughput screening method for discovery of new potential GP inhibitors were optimized and standardized, which is desirable for the reproducibility and comparison of results in the literature. The optimized method can be applied to the study of a panel of synthetic and/or natural compounds, such as polyphenols.