Structure Of Glycogen Research Articles

William Joseph Whelan. 14 November 1924 — 5 June 2021

William Whelan was an exceptionally celebrated scientist recognized equally for his work in research, education, publishing and international scientific organizations. He studied organic chemistry at the University of Birmingham. He taught at the University College of North Wales and the University of London and became head of the Department of Biochemistry of the Royal Free Hospital School of Medicine, University of London, in 1964. In 1967 he moved to the United States and served as professor and chairman of the Department of Biochemistry and Molecular Biology of the University of Miami Medical School for the rest of his career. Bill is known for his pioneering research on the structure of starch and glycogen. He discovered that glycogen contained the protein glycogenin, an enzyme with primer function in the synthesis of glycogen. This discovery filled a crucial gap in understanding the biosynthetic process that transforms glucose into glycogen. Bill's contributions, including his innovative methods for isolating and characterizing molecular structures like glycogen, have since become established scientific practices, and he established some of the fundamental concepts and nomenclature used in the carbohydrate field today.

Molecular exploration of the diurnal alteration of glycogen structural fragility and stability in time-restricted-feeding mouse liver

The structure of glycogen α particles in healthy mouse liver has two states: stability and fragility. In contrast, glycogen α particles in diabetic liver present consistent fragility, which may exacerbate hyperglycemia. Currently, the molecular mechanism behind glycogen structural alteration is still unclear. In this study, we characterized the fine molecular structure of liver glycogen α particles in healthy mice under time-restricted feeding (TRF) mode during a 24-h cycle. Then, differentially expressed genes (DEGs) in the liver during daytime and nighttime were revealed via transcriptomics, which identified that the key downregulated DEGs were mainly related to insulin secretion in daytime. Furthermore, GO annotation and KEGG pathway enrichment found that negative regulation of the glycogen catabolic process and insulin secretion process were significantly downregulated in the daytime. Therefore, transcriptomic analyses indicated that the structural stability of glycogen α particles might be correlated with the glycogen degradation process via insulin secretion downregulation. Further molecular experiments confirmed the significant upregulation of glycogen phosphorylase (PYGL), phosphorylated PYGL (p-PYGL), and glycogen debranching enzyme (AGL) at the protein level during the daytime. Overall, we concluded that the downregulation of insulin secretion in the daytime under TRF mode facilitated glycogenolysis, contributing to the structural stability of glycogen α-particles.

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 β 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.

The Synergistic Effect of GH13 and GH57 GBEs of Petrotoga mobilis Results in α-Glucan Molecules with a Higher Branch Density.

Glycogen is a biopolymer consisting of glycosyl units, with a linear backbone connected by α-1,4-linkages and branches attached via α-1,6-linkages. In microorganisms, glycogen synthesis involves multiple enzymes, with glycogen branching enzymes (GBEs) being vital for creating α-1,6-linkages. GBEs exist in two families: glycoside hydrolase (GH) 13 and GH57. Some organisms possess either a single GH13 or GH57 GBE, while others, such as Petrotoga mobilis, have both types of GBEs. In this study, the simultaneous use of a GH13 and GH57 GBE each from Petrotoga mobilis for α-glucan modification was investigated using a linear maltodextrin substrate with a degree of polymerization of 18 (DP18). The products from modifications by one or both GBEs in various combinations were analyzed and demonstrated a synergistic effect when both enzymes were combined, leading to a higher branch density in the glycogen structure. In this cooperative process, PmGBE13 was responsible for creating longer branches, whereas PmGBE57 hydrolyzed these branches, resulting in shorter lengths. The combined action of the two enzymes significantly increased the number of branched chains compared to when they acted individually. The results of this study therefore give insight into the role of PmGBE13 and PmGBE57 in glycogen synthesis, and show the potential use of both enzymes in a two-step modification to create an α-glucan structure with short branches at a high branch density.

Molecular structure and characteristics of phytoglycogen, glycogen and amylopectin subjected to mild acid hydrolysis

The structure and properties of phytoglycogen and glycogen subjected to acid hydrolysis was investigated using amylopectin as a reference. The degradation took place in two stages and the degree of hydrolysis was in the following order: amylopectin > phytoglycogen > glycogen. Upon acid hydrolysis, the molar mass distribution of phytoglycogen or glycogen gradually shifted to the smaller and broadening distribution region, whereas the distribution of amyopectin changed from bimodal to monomodal shape. The kinetic rate constant for depolymerization of phytoglycogen, amylopectin, and glycogen were 3.45 × 10−5/s, 6.13 × 10−5/s, and 0.96 × 10−5/s, respectively. The acid-treated sample had the smaller particle radius, lower percentage of α-1,6 linkage as well as higher rapidly digestible starch fractions. The depolymerization models were built to interpret the structural differences of glucose polymer during acid treatment, which would provide guideline to improve the structure understanding and precise application of branched glucan with desired properties.

Stochastic modelling of a three-dimensional glycogen granule synthesis and impact of the branching enzyme.

In humans, glycogen storage diseases result from metabolic inborn errors, and can lead to severe phenotypes and lethal conditions. Besides these rare diseases, glycogen is also associated to widely spread societal burdens such as diabetes. Glycogen is a branched glucose polymer synthesised and degraded by a complex set of enzymes. Over the past 50 years, the structure of glycogen has been intensively investigated. Yet, the interplay between the detailed three-dimensional glycogen structure and the related enzyme activity is only partially characterised and still to be fully understood. In this article, we develop a stochastic coarse-grained and spatially resolved model of branched polymer biosynthesis following a Gillespie algorithm. Our study largely focusses on the role of the branching enzyme, and first investigates the properties of the model with generic parameter values, before comparing it to in vivo experimental data in mice. It arises that the ratio of glycogen synthase over branching enzyme reaction rates drastically impacts the structure of the granule. We deeply investigate the mechanism of branching and parametrise it using distinct lengths. Not only do we consider various possible sets of values for these lengths, but also distinct rules to apply them. We show how combining various values for these lengths finely tunes glycogen macromolecular structure. Comparing the model with experimental data confirms that we can accurately reproduce glycogen chain length distributions in wild type mice. Additional granule properties obtained for this fit are also in good agreement with typically reported values in the experimental literature. Nonetheless, we find that the mechanism of branching must be more flexible than usually reported. Overall, our model provides a theoretical basis to quantify the effect that single enzymatic parameters, in particular of the branching enzyme, have on the chain length distribution. Our generic model and methods can be applied to any glycogen data set, and could in particular contribute to characterise the mechanisms responsible for glycogen storage disorders.

Phenylboronic acid‐functionalized glycogens as dynamic nano‐frameworks for conjugation and activation of carbonic anhydrase

AbstractBiocatalysis is playing an increasingly important role for chemical, pharmaceutical, and food industries, owing to its high efficiency, selectivity and environment‐friendliness. However, it remains challenging to improve the catalytic activity of enzymes since the most frequently used strategies are costly and time‐consuming. In the current study, a new type of glycogen‐based dynamic nano‐frameworks is prepared as additives for the direct activation of carbonic anhydrase (bovine erythrocytes [bCA]). The boronic acid functionalization of the branched glycogen structures can provide NB coordination with bCA, leading to the conjugation and stabilization of the target enzyme. As a result, the glycogen with a 10% substitution degree (Gly‐PBA10) showed a higher activation effect for bCA than that of glycogen with a 20% substitution degree (Gly‐PBA20). Furthermore, the mechanism for interactions between the dynamic frameworks and bCA has been studied through combined techniques of dynamic light scattering, circular dichroism spectroscopy, and isothermal titration calorimetry. The results suggested different protein binding modes and stabilization abilities for the protein secondary structures from these two types of functionalized dynamic frameworks. This study provided new insights into the protein‐polymer interactions that can be beneficial for various biochemical applications.

Doğu hamam böceği Blatta orientalis (L., 1758) (Blattodea: Blattidae)’in trofositlerindeki makromoleküllerin histokimyasal ve ince yapı analizi

The fat body is a major storage area for glycogen, lipid and protein. The trophocyte is the main cell of fat body and stores these macromolecules. The fat body consists of two parts; peripheral and perivisceral. Peripheral fat body is located below the integument while perivisceral fat body is around the digestive tract. The study was conducted in EGEMIKAL Analysis Laboratory and Histology Laboratory of Ege University between 2018 and 2020. The fat body contents of insects at all stages were examined comparatively in three selected sections through histochemical and ultrastructural studies. We identified macromolecules stored in the trophocytes. Both the granular form of proteins and asterisk structure of glycogen localized around the lipid droplets were observed clearly. It was found that accumulation of protein continued in the trophocytes, but glycogen accumulation decreased considerably in adults compared to all nymphal stages. We also found that larger lipid droplets were stored in the PF fat body, while glycogen and protein accumulation was much higher in the PV fat body. These results may contribute to understanding of the mechanisms underlying activities such as amino acid, nitrogen, lipid and carbohydrate metabolism and protein synthesis in insects.

Laforin targets malin to glycogen in Lafora progressive myoclonus epilepsy.

Glycogen is the largest cytosolic macromolecule and is kept in solution through a regular system of short branches allowing hydration. This structure was thought to solely require balanced glycogen synthase and branching enzyme activities. Deposition of overlong branched glycogen in the fatal epilepsy Lafora disease (LD) indicated involvement of the LD gene products laforin and the E3 ubiquitin ligase malin in regulating glycogen structure. Laforin binds glycogen, and LD-causing mutations disrupt this binding, laforin-malin interactions and malin's ligase activity, all indicating a critical role for malin. Neither malin's endogenous function nor location had previously been studied due to lack of suitable antibodies. Here, we generated a mouse in which the native malin gene is tagged with the FLAG sequence. We show that the tagged gene expresses physiologically, malin localizes to glycogen, laforin and malin indeed interact, at glycogen, and malin's presence at glycogen depends on laforin. These results, and mice, open the way to understanding unknown mechanisms of glycogen synthesis critical to LD and potentially other much more common diseases due to incompletely understood defects in glycogen metabolism.

Comparative transcriptome analysis of diurnal alterations of liver glycogen structure: A pilot study

Molecular mechanisms behind structural alterations between fragile and stable glycogen α particles in liver are not clear yet. In this pilot study, we re-examined the diurnal alterations of glycogen structure from the perspective of liver tissue transcriptome. By comparing the structures of liver glycogen from mice at 12 am, 8 am, 12 pm, and 8 pm (light-on: 6 am; light-off: 6 pm), we re-confirmed that the liver glycogen was fragile at 12 am and 8 am and stable at 12 pm and 8 pm as previously reported. The structural differences of glycogen particles at 12 am and 12 pm were thoroughly compared via transcriptomics. Differentially expressed genes (DEGs) with statistical significance were identified, while expression level of the gene ppp1r3g (log2Fold_Change = −6.368, P-value = 2.89E−04) that encoded PPP1R3G with glycogen binding domain was most significantly changed, which provided preliminary clues to the structural alterations of glycogen α particles during the diurnal cycle.

The features of liver glycogen fractions in streptozotocin-induced type-I diabetic rats

The ratio of counter-regulatory hormones to insulin is increased in diabetes. This causes a change in the activities of the enzymes involved in glycogen metabolism. Thus, it is expected that the structure of glycogen will be changed reversibly in diabetes mellitus. The current research was done to study whether the fractions of glycogen have been changed in streptozotocin (STZ)-induced diabetes in rats. To this end, one group of 10 male rats received a single injection with STZ and were compared with the control group. Liver glycogen fractions were analyzed for the content of carbohydrates, protein, and organic phosphate. Hyperglycemia and clinical signs of diabetes appeared after two days of STZ- injection and continued up to two weeks. In diabetic rats relative to controls, total glycogen decreased by about 39% (24.4 ± 3.0 mg/g wet weight of liver vs. 39.7 ± 2.2, p = 0.001), and the change happened entirely in acid-soluble glycogen (ASG) fraction (19.1 ± 1.7 vs. 33.4 ± 2.2, p = 0.001), while acid-insoluble glycogen (AIG) increased slightly but not significantly (5.5 ± 1.1, vs. 4.5 ± 0.8, p = 0.5). Diabetes was also associated with the change in the content of glycogen protein and the ratio of ASG to AIG. The content of organic phosphate was 181.4 ± 26.3 and 39.2 ± 7.9 µg/g wet weight of liver for ASG and AIG fractions in control rats and had no significant change in diabetes. In conclusion, diabetes is accompanied by the reduction in liver glycogen carbohydrate and protein of total and soluble fraction but the content of organic phosphate has not been affected.

A Non-Degradative Extraction Method for Molecular Structure Characterization of Bacterial Glycogen Particles.

Currently, there exist a variety of glycogen extraction methods, which either damage glycogen spatial structure or only partially extract glycogen, leading to the biased characterization of glycogen fine molecular structure. To understand the dynamic changes of glycogen structures and the versatile functions of glycogen particles in bacteria, it is essential to isolate glycogen with minimal degradation. In this study, a mild glycogen isolation method is demonstrated by using cold-water (CW) precipitation via sugar density gradient ultra-centrifugation (SDGU-CW). The traditional trichloroacetic acid (TCA) method and potassium hydroxide (KOH) method were also performed for comparison. A commonly used lab strain, Escherichia coli BL21(DE3), was used as a model organism in this study for demonstration purposes. After extracting glycogen particles using different methods, their structures were analyzed and compared through size exclusion chromatography (SEC) for particle size distribution and fluorophore-assisted capillary electrophoresis (FACE) for linear chain length distributions. The analysis confirmed that glycogen extracted via SDGU-CW had minimal degradation.

The effect of high-amylose resistant starch on the glycogen structure of diabetic mice

Glycogen is a complex branched glucose polymer found in many tissues and acts as a blood-glucose buffer. In the liver, smaller β glycogen particles can bind into larger composite α particles. In mouse models of diabetes, these liver glycogen particles are molecularly fragile, breaking up into smaller particles in the presence of solvents such as dimethyl sulfoxide (DMSO). If this occurs in vivo, such a rapid enzymatic degradation of these smaller particles into glucose could exacerbate the poor blood-glucose control that is characteristic of the disease. High-amylose resistant starch (RS) can escape digestion in the small intestine and ferment in the large intestine, which elicits positive effects on glycemic response and type 2 diabetes. Here we postulate that RS would help attenuate diabetes-related liver glycogen fragility. Normal maize starch and two types of high-amylose starch were fed to diabetic and non-diabetic mice. Molecular size distributions and chain-length distributions of liver glycogen from both groups were characterized to test glycogen fragility before and after DMSO treatment. Consistent with the hypothesis that high blood glucose is associated with glycogen fragility, a high-amylose RS diet prevented the fragility of liver-glycogen α particles. The diets had no significant effect on the glycogen chain-length distributions.

The Action of Recombinant Human Lysosomal α-Glucosidase (rhGAA) on Human Liver Glycogen: Pathway to Complete Degradation

Glycogen is present in all tissues, but it is primarily stored in the liver and in muscle. As a branched chain carbohydrate, it is broken down by phosphorylase and debrancher enzymes, which are cytoplasmic. It is also degraded by a lysosomal α-glucosidase (GAA) also known as acid α-glucosidase and lysosomal acid α-glucosidase. The deficiency of GAA in patients is known as Pompe disease, and the phenotypes as infantile, juvenile and later onset forms. Pompe disease is treated by enzyme replacement therapy (ERT) with a recombinant form of rhGAA. Following ERT in Pompe mice and human patients there is residual carbohydrate material present in the cytoplasm of cells. The goal of this work is to improve ERT and attempt to identify and treat the residual cytoplasmic carbohydrate. Initial experiments were to determine if rhGAA can completely degrade glycogen. The enzyme cannot completely degrade glycogen. There is a residual glycosylated protein as well as a soluble glycosylated protein, which is a terminal degradation product of glycogen and as such serves as a biomarker for lysosomal glycogen degradation. The glycosylated protein has a very unusual carbohydrate composition for a glycosylated protein: m-inositol, s-inositol and sorbitol as the major carbohydrates, as well as mannitol, mannose, glucose and galactose. This work describes the residual material which likely contains the same protein as the soluble glycosylated protein. The biomarker is present in serum of control and Pompe patients on ERT, but it is not present in the serum of Pompe mice not on ERT. Pompe mice not on ERT have another glycosylated protein in their serum which may be a biomarker for Pompe disease. This protein has multiple glycosylation sites, each with different carbohydrate components. These glycosylated proteins as well as the complexity of glycogen structure are discussed, as well as future directions to try to improve the outcome of ERT for Pompe patients by being able to monitor the efficacy of ERT in the short term and possibly to adjust the timing and dose of enzyme infusions.

Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging of Glycogen In Situ.

Histopathological detection and quantitation of glycogen in situ are important for the assessment of glycogen storage diseases and different types of cancer. The current standard method for defining the regionality of glycogen rely almost exclusively on Periodic Acid-Schiff (PAS) staining, a workflow that lacks specificity and sensitivity. Herein, we describe a new and much improved workflow to detect microenvironmental glycogen in situ using enzyme-assisted matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI). This method provides superior sensitivity and can elucidate the molecular features of glycogen structure, with 50μm spatial resolution for a next-generation histopathological assessment of glycogen.

Liver fibrosis alters the molecular structures of hepatic glycogen

Liver fibrosis (LF) leads to liver failure and short survival. Liver glycogen is a hyperbranched glucose polymer, comprising individual β particles, which can bind together to form aggregated α particles. Glycogen functionality depends on its molecular structure. This study compared the molecular structure of liver glycogen from both LF and healthy rats, and explored underlying mechanisms for observed differences. Glycogen from both groups contained α and β particles; the LF group contained a higher proportion of β particles, with the glycogen containing fewer long chains than seen in the control group. Both glycogen branching enzyme and glycogen phosphorylase showed a significant decrease of activity in the LF group. Transcriptomics and proteomics revealed a functional deficiency of mitochondria in the LF group, which may lead to changes in glycogen structure. These results provide for the first time an understanding of how liver fibrosis affects liver glycogen metabolism and glycogen structure. HypothesisWe hypothesized that the molecular structure of liver glycogen from a rat model of liver fibrosis would be altered compared to the control group.

Structure and Immunomodulatory Activity of Glycogen Derived from Honeybee Larvae (Apis mellifera).

Honeybee larvae have been recognized as nutrient-rich food in many countries. Although glycogen, a storage form of glucose in animals, is synthesized in honeybee larvae, there is no information on the structure of glycan and its biological activity. In this study, we successfully extracted glycogen from honeybee larvae using hot water extraction and investigated the structure and biological activity of glycan. It was found that the molecular weight of glycogen from honeybee larvae is higher than that of glycogen from bovine liver and oysters. In addition, treatment of RAW264.7 cells with glycogen from honeybee larvae resulted in a much higher production of tumor necrosis factor (TNF)-α and interleukin (IL)-6 than treatment with glycogen from either bovine liver or oysters. These results suggest that the high molecular weight glycogen from honeybee larvae is a functional food ingredient with immunomodulatory activity.

Abstract 2819: Mass spectrometry imaging reveals heterogeneous glycogen metabolism in non small cell lung cancer

Abstract Lung cancer is the leading cause of cancer related death worldwide, with a high mortality rate even when diagnosed at an early stage. Identifying the unique molecular features that drive disease progression within distinct tumor subtypes is a critical step in lung cancer research that could advance our understanding of lung cancer biology, leading to predictive biomarkers and novel therapies. Glycogen is the primary source of carbohydrate storage in most issues, and its degradation products are intimately connection to central carbon metabolism. Recently, aberrant glycogen accumulation has been observed in lung tumors and been reported to promote lung cancer progression. Despite these findings, the full clinical implications of dysregulated glycogen metabolism and its role in lung cancer tumorigenesis remain largely unknown. Due to its unique physiochemical properties, glycogen is extremely difficult to measure biochemically and visualize in situ. Herein, we describe a novel method to image microenvironmental glycogen via enzymatic release of glycogen substrates coupled to matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI). Our assay provides robust information on glycogen structure and phosphate composition with spatial distribution. With this technique, we analyzed glycogen chain length and glycogen phosphate content from tissue microarrays containing over 200 lung cancer patient samples banked at the University of Kentucky with over 10 years of clinical metadata. Using MALDI-MSI, we found significant glycogen accumulation in lung tumor tissue compared to match normal lung across all patients, and found adenocarcinoma (LUAD) samples contained higher glycogen content than squamous cell carcinoma (SCC). Regional analysis by MALDI-MSI revealed SCC primarily accumulates glycogen in the stroma and blood vessels, but not in tumors. Notably, LUAD patient tumors exhibit decreased expression of laforin, a glucan phosphatase that plays a key role in glycogen phosphorylation and glycogen degradation. Low laforin expression also corresponds to poorer overall survival in LUAD patients. In vivo, deletion of Epm2a (the gene that encodes laforin) in KRASG12D/p53-/- mice results in increased tumor burden and accumulation of hyperphosphorylated intratumoral glycogen. Further, A549 LUAD xenografts treated with guaiacol, a glycogen synthase inhibitor, show markedly reduced tumor growth, while guaiacol had no effect on H2030 SCC tumor growth. Given the significant effect of glycogen accumulation on in vivo tumor growth, our data suggest the glycogen metabolic pathway represents a novel therapeutic target specifically for LUAD patients. Together, our study describes a novel method to visualize glycogen structure using MALDI-MSI and reveals unique metabolic features that contribute to LUAD disease progression. Citation Format: Lindsey R. Conroy, Lyndsay EA Young, Harrison A. Clarke, Alexandra E. Stanback, Michael D. Buoncristiani, Christine F. Brainson, Derek B. Allison, Ramon C. Sun, Richard R. Drake. Mass spectrometry imaging reveals heterogeneous glycogen metabolism in non small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2819.

William "Bill" Joseph Whelan, D.Sc., FRS November 14, 1924 to June 5, 2021.

William "Bill" Joseph Whelan, D.Sc., FRS November 14, 1924 to June 5, 2021.

Ethanol extract of mulberry leaves partially restores the composition of intestinal microbiota and strengthens liver glycogen fragility in type 2 diabetic rats

BackgroundMulberry leaf as a traditional Chinese medicine is able to treat obesity, diabetes, and dyslipidemia. It is well known that diabetes leads to intestinal microbiota dysbiosis. It is also recently discovered that liver glycogen structure is impaired in diabetic animals. Since mulberry leaves are able to improve the diabetic conditions through reducing blood glucose level, it would be interesting to investigate whether they have any positive effects on intestinal microbiota and liver glycogen structure.MethodsIn this study, we first determined the bioactive components of ethanol extract of mulberry leaves via high-performance liquid chromatography (HPLC) and liquid chromatography/mass spectrometry (LC/MS). Murine animal models were divided into three groups, normal Sprague-Dawley (SD) rats, high-fat diet (HFD) and streptozotocin (STZ) induced type 2 diabetic rats, and HFD/STZ-induced rats administered with ethanol extract of mulberry leaves (200 mg/kg/day). Composition of intestinal microbiota was analyzed via metagenomics by sequencing the V3-V4 region of 16S rDNAs. Liver glycogen structure was characterized through size exclusion chromatography (SEC). Both Student’s t-test and Tukey’s test were used for statistical analysis.ResultsA group of type 2 diabetic rat models were successfully established. Intestinal microbiota analysis showed that ethanol extract of mulberry leaves could partially change intestinal microbiota back to normal conditions. In addition, liver glycogen was restored from fragile state to stable state through administration of ethanol extract of mulberry leaves.ConclusionsThis study confirms that the ethanol extract of mulberry leaves (MLE) ameliorates intestinal microbiota dysbiosis and strengthens liver glycogen fragility in diabetic rats. These finding can be helpful in discovering the novel therapeutic targets with the help of further investigations.