Glycogen Synthase Expression Research Articles

Euonymus alatus Extract Reduces Insulin Resistance in db/db Mice by Regulating the PI3K–AKT Pathway

In accordance with the usage of Euonymus alatus (EA) as folk medicine in diabetes, the present study employed water and 70% ethanol twig extract to assess its antidiabetic effects in C57BL/KsJ-db/db mice. These effects were then compared with those observed in normal C57BL/6J Jms Slc mice. After 4 weeks of supplementation with 70% ethanolic EA extract or water EA extract by oral gavage at a dose of 500 mg/kg with distilled water (DW) per day, body weight was measured and compared with the diabetic group (Db). HPLC demonstrated that the maximum flavonoids were extracted in the Et.EA extract rather than in the water EA extract. The supplementation of the Et.EA extract significantly increased liver and muscle glycogen content with respect to the Db group. Additionally, the Et.EA extract modulated the expression of glycogen synthase (GS) in the liver and muscles of Db mice, indicating that it plays a promotive role in glycogen synthesis. Mechanistically, Et.EA extract activates insulin receptor substrate (IRS1/IRS2)/phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) in the liver and muscles of Db mice. In conclusion, Et.EA extract attenuates insulin resistance by regulating the expression of metabolic enzymes and signaling pathways.

Synergistic Effects of Chlorogenic Acid and Ganoderma Lucidum Polysaccharide on Hepatic Fat Accumulation

This study aimed to investigate the impact of different doses of chlorogenic acid and Ganoderma lucidum polysaccharide on lipid accumulation using an obese rat model. We utilized blood glucose assays, oil red O staining, and triglyceride content analysis to examine the effects of chlorogenic acid combined with G. lucidum polysaccharide on fat metabolism in rats. We found substantial potential in using chlorogenic acid combined with G. lucidum polysaccharide for weight control and metabolic regulation in obese rats. After a long-term dietary intervention, chlorogenic acid and G. lucidum polysaccharide markedly inhibited the expression of proteins related to fatty acid synthesis, including AMP-activated protein kinase, fatty acid synthase, and SREBP-1c, leading to reduced body weight and decreased hepatic fat deposition. Additionally, the combined treatment substantially affected enzymes and proteins involved in glucose metabolism, reducing glycogen synthase expression and indicating a potential mechanism for weight reduction through glycogen synthesis inhibition. Furthermore, elevated glucose kinase levels indicated that chlorogenic acid and G. lucidum polysaccharide may enhance hepatic glycogen degradation, thereby reducing hepatic fat accumulation. These findings reveal that chlorogenic acid combined with G. lucidum polysaccharide has a synergistic impact, providing a theoretical basis for their potential as a combined therapeutic approach for obesity.

588-P: Probing the Role of Muscle Glycogen Resynthesis on Postexercise AMPK Activation and Metabolic Protein Abundance Using a Novel Genetic Model

Acute exercise can alter the abundance of key metabolic proteins in skeletal muscle. These effects are influenced by postexercise (PEX) refeeding. Muscle glycogen resynthesis is a putative mediator of these refeeding effects. However, it is challenging to test this idea, because glycogen resynthesis is only one of many outcomes with refeeding. Therefore, we devised a novel genetic model to modify muscle expression of glycogen synthase 1 (GS1) using injection of rat skeletal muscle with adeno-associated viral (AAV) vectors for short hairpin RNA targeting GS1 (shRNA-GS1). Contralateral muscles were injected with scrambled shRNA (shRNA-Scr). Rats were sedentary (SED) or exercised (120min swim-exercise). Muscles were collected immediately postexercise (IPEX) and 9-hours PEX (9hPEX), along with time-matched SED controls. The 9hPEX and their SED-control rats were either refed (RF) or not-refed (NRF). Muscles were analyzed for glycogen and phosphorylation and abundance of key metabolic proteins. Muscles injected with shRNA-GS1 vs shRNA-Scr had lower GS1 abundance in all groups. Regardless of genotype, in IPEX vs SED rats: glycogen fell to similarly low values and phosphorylation of AMPK Thr172 (pAMPK) and its substrate ACC Ser79 (pACC) were comparably increased. Muscle glycogen was increased in all groups of 9h-RF rats, but it was lower for shRNA-GS1 vs shRNA-Scr from RF-9hPEX rats. In the NRF-9hPEX rats, pAMPK and pACC were greater for shRNA-GS1 vs shRNA-Scr muscles. Muscle pyruvate dehydrogenase kinase-4 abundance from 9hPEX rats (both NRF and RF) was greater for shRNA-GS1 vs shRNA-Scr. Hexokinase II (HKII) abundance was greater for 9hPEX vs SED in NRF rats regardless of genotype. In RF-9hPEX rats, HKII abundance was greater for shRNA-GS1 vs shRNA-Scr. These results support the idea that some PEX effects on metabolic protein abundance are influenced by muscle GS1 abundance and the extent of glycogen resynthesis Disclosure S.Kwak: None. A.Zheng: None. E.B.Arias: None. H.Wang: None. X.Pan: None. D.Duan: None. Y.Yue: None. G.D.Cartee: None. Funding National Institutes of Health (R01DK071771); University of Michigan

Glycogen synthase 1 targeting reveals a metabolic vulnerability in triple-negative breast cancer

BackgroundHypoxia-induced glycogen turnover is implicated in cancer proliferation and therapy resistance. Triple-negative breast cancers (TNBCs), characterized by a hypoxic tumor microenvironment, respond poorly to therapy. We studied the expression of glycogen synthase 1 (GYS1), the key regulator of glycogenesis, and other glycogen-related enzymes in primary tumors of patients with breast cancer and evaluated the impact of GYS1 downregulation in preclinical models.MethodsmRNA expression of GYS1 and other glycogen-related enzymes in primary breast tumors and the correlation with patient survival were studied in the METABRIC dataset (n = 1904). Immunohistochemical staining of GYS1 and glycogen was performed on a tissue microarray of primary breast cancers (n = 337). In four breast cancer cell lines and a mouse xenograft model of triple-negative breast cancer, GYS1 was downregulated using small-interfering or stably expressed short-hairpin RNAs to study the effect of downregulation on breast cancer cell proliferation, glycogen content and sensitivity to various metabolically targeted drugs.ResultsHigh GYS1 mRNA expression was associated with poor patient overall survival (HR 1.20, P = 0.009), especially in the TNBC subgroup (HR 1.52, P = 0.014). Immunohistochemical GYS1 expression in primary breast tumors was highest in TNBCs (median H-score 80, IQR 53–121) and other Ki67-high tumors (median H-score 85, IQR 57–124) (P < 0.0001). Knockdown of GYS1 impaired proliferation of breast cancer cells, depleted glycogen stores and delayed growth of MDA-MB-231 xenografts. Knockdown of GYS1 made breast cancer cells more vulnerable to inhibition of mitochondrial proteostasis.ConclusionsOur findings highlight GYS1 as potential therapeutic target in breast cancer, especially in TNBC and other highly proliferative subsets.

Euglycemia is affected by stress defense factor hepatocyte NRF1, but not NRF2

Hepatocyte stress signaling has been established to alter glucose metabolism and impair systemic glucose homeostasis. In contrast, the role of stress defenses in the control of glucose homeostasis is less understood. Nuclear factor erythroid 2 related factor-1 (NRF1) and −2 (NRF2) are transcription factors that promote stress defense and can exert hepatocyte stress defense programming via complementary gene regulation. To identify whether there are independent or complementary roles of these factors in hepatocytes on glucose homeostasis, we investigated the effect of adult-onset, hepatocyte-specific deletion of NRF1, NRF2, or both on glycemia in mice fed 1–3 weeks with a mildly stressful diet enriched with fat, fructose, and cholesterol. Compared to respective control, NRF1 deficiency and combined deficiency reduced glycemia, in some cases resulting in hypoglycemia, whereas there was no effect of NRF2 deficiency. However, reduced glycemia in NRF1 deficiency did not occur in the leptin-deficient mouse model of obesity and diabetes, suggesting hepatocyte NRF1 support defenses that counteract hypoglycemia but does not promote hyperglycemia. Consistent with this, NRF1 deficiency was associated with reduced liver glycogen and glycogen synthase expression as well as marked alteration to circulating level of glycemia-influencing hormones, growth hormone and insulin-like growth factor-1 (IGF1). Overall, we identify a role for hepatocyte NRF1 in modulating glucose homeostasis, which may be linked to liver glycogen storage and the growth hormone/IGF1 axis.

Impact of particulate matter 2.5 on the liver function of mice.

The aim of this study was to evaluate the impact of particulate matter 2.5 (PM2.5) on liver function at the animal level and to study its impact targets. 60 male and female BALB/c mice of SPF grade, aged 6-8 weeks, were randomly divided into four groups, with 15 mice in each, including the normal saline control group, the PM2.5 low dose group [2 μg/(100 g/d)], the PM2.5 medium dose group [8 μg/(100 g/d)] and the PM2.5 high dose group [16 μg/(100 g/d)]. Each day, 0.9% saline or PM2.5 particles were administered through the nasal route, and samples were taken after 3 weeks of continuous exposure. Hematoxylin-eosin staining (HE) was used to observe the liver damage caused by PM2.5. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were detected by using an automatic biochemical analyzer to detect the content of liver glycogen and blood glucose. Multiple indicators were observed, including plasma tumor necrosis factor (TNF-α) and interleukin-6 (IL-6) levels, oxidative stress response indicators reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD) detection, RT-PCR and Western blot detection of glycogen synthase (GS), glucokinase (GK), nuclear factor erythroid 2-related factor 2 (Nrf2) expression and phosphorylation level of phospho-c-Jun N-terminal kinases (p-JNK). PM2.5 can cause damage to the liver by increasing PM2.5 concentrations, raising the metabolic rate of liver cells, resulting in a substantial amount of inflammatory infiltration and vacuolar degeneration of cells, and increasing the liver/body weight. TNF-α and IL-6 inflammatory factor expression increased (p<0.05). An increase in the serum ALT and AST levels were also observed. The blood glucose of mice increased, whereas the content of liver glycogen declined (p<0.05). ROS, MDA, and SOD levels all increased considerably. PM2.5 can drastically lower the expression of GS and GK, increase the expression of Nrf2, and raise the phosphorylation level of p-JNK (p<0.05). PM2.5 can induce oxidative stress in mouse liver through the Nrf2/JNK pathway, induce liver inflammation in mice, and inhibit glycogen synthesis.

FUT2 promotes the tumorigenicity and metastasis of colorectal cancer cells via the Wnt/β‑catenin pathway.

The incidence of colorectal cancer (CRC), a leading cause of cancer‑related mortality, has increased globally. Fucosyltransferase 2 (FUT2), catalyzing the α1, 2‑linked fucose in mammals, has been reported to be overexpressed in several malignant cancers, including CRC. However, the effects of FUT2 on CRC remain largely unknown. Herein, it was determined that the FUT2 expression levels in CRC tissues were higher than those in adjacent non‑tumor tissues, whereas no association with tumor stage was revealed. The results of biological functional analysis revealed that FUT2 knockdown inhibited the proliferation, migration and invasion of human CRC cells. Moreover, the knockdown of FUT2 arrested the CRC cells at the G0/G1 phase and promoted the apoptosis of human CRC cells. Western blot analysis demonstrated that the expression levels of β‑catenin, C‑myc and cyclin D1 were decreased by FUT2 knockdown in CRC cells, whereas the expression of glycogen synthase kinase‑3β and the phosphorylation levels of β‑catenin were increased. Additionally, Wnt2 was fucosylated by FUT2 in CRC cells. Furthermore, the knockdown of FUT2 inhibited the growth of human CRC invivo. Overall, the findings of the present study suggest that FUT2 may be used as a potential diagnostic biomarker and therapeutic target for CRC treatment.

Single-Atom Ce-N4-C-(OH)2 Nanozyme-Catalyzed Cascade Reaction to Alleviate Hyperglycemia.

The enzyme-mimicking catalytic activity of single-atom nanozymes has been widely used in tumor treatment. However, research on alleviating metabolic diseases, such as hyperglycemia, has not been reported. Herein, we found that the single-atom Ce-N4-C-(OH)2 (SACe-N4-C-(OH)2) nanozyme promoted glucose absorption in lysosomes, resulting in increased reactive oxygen species production in HepG2 cells. Furthermore, the SACe-N4-C-(OH)2 nanozyme initiated a cascade reaction involving superoxide dismutase-, oxidase-, catalase-, and peroxidase-like activity to overcome the limitations associated with the substrate and produce •OH, thus improving glucose intolerance and insulin resistance by increasing the phosphorylation of protein kinase B and glycogen synthase kinase 3β, and the expression of glycogen synthase, promoting glycogen synthesis to improve glucose intolerance and insulin resistance in high-fat diet-induced hyperglycemic mice. Altogether, these results demonstrated that the novel nanozyme SACe-N4-C-(OH)2 alleviated the effects of hyperglycemia without evident toxicity, demonstrating its excellent clinical application potential.

Polysaccharides from apple pomace exhibit anti-fatigue activity through increasing glycogen content.

The polysaccharides were isolated from apple pomace by hot-water extraction, and their anti-fatigue activity was evaluated in C2C12 muscle myoblasts and male Kunming mice. The purified polysaccharides from apple pomace (PAP) have a molecular weight of 1.74 × 105Da and were composed of mannose, rhamnose, glucose, galactose and arabinose. In C2C12 myoblasts, PAP showed no cytotoxicity in the concentrations of 0-300μg/ml. PAP treatment increased the glycogen content, while the ATP content was not affected in C2C12 myoblasts. Further investigation found that the activity and gene expression of glycogen synthase, rather than glycogen phosphorylase, were upregulated by PAP treatment. The studies in vivo showed that PAP treatment did not affect the food intake and weight again in mice. Importantly, PAP prolonged the exhaustive swimming time, increased hepatic and skeletal muscle glycogen levels, and effectively inhibited the accumulation of blood lactic and blood urea nitrogen in mice. Taken together, the results suggested that PAP exhibit anti-fatigue activity in vitro and in vivo through increasing glycogen content.

Endometrial glycogen metabolism during early pregnancy in mice.

Glucose is critical during early pregnancy. The uterus can store glucose as glycogen but uterine glycogen metabolism is poorly understood. This study analyzed glycogen storage and localization of glycogen metabolizing enzymes from proestrus until implantation in the murine uterus. Quantification of diastase-labile periodic acid-Schiff (PAS) staining showed glycogen in the glandular epithelium decreased 71.4% at 1.5 days postcoitum (DPC) and 62.13% at DPC 3.5 compared to proestrus. In the luminal epithelium, glycogen was the highest at proestrus, decreased 46.2% at DPC 1.5and 63.2% at DPC 3.5. Immunostaining showed that before implantation, glycogen metabolizing enzymes were primarily localized to the glandular and luminal epithelium. Stromal glycogen was low from proestrus to DPC 3.5. However, at the DPC 5.5 implantation sites, stromal glycogen levels increased sevenfold. Similarly, artificial decidualization resulted in a fivefold increase in glycogen levels. In both models, decidualization increased expression of glycogen synthase as determine by immunohistochemistry and western blot. In conclusion, glycogen levels decreased in the uterine epithelium before implantation, indicating that it could be used to support preimplantation embryos. Decidualization resulted in a dramatic increase in stromal glycogen levels, suggesting it may have an important, but yet undefined, role in pregnancy.

Abstract 5813: Glycogen synthesis as potential novel target in triple negative breast cancer: Glycogen synthase 1 expression in human breast cancers and the impact of downregulation on proliferation of preclinical models

Abstract Background: Triple-negative breast cancer (TNBC) has a poor clinical prognosis and is characterized by a lack of druggable targets and a hypoxic tumor microenvironment. Hypoxia-induced glycogen accumulation and utilization are involved in cancer proliferation and therapy resistance, making modulation of glycogen metabolism of therapeutic interest. Therefore, we studied expression of glycogen synthase 1 (GYS1, the key regulator of glycogen synthesis) and glycogen stores in publicly available expression data and human breast tumors including TNBC. Also, we studied downregulation of GYS1 in preclinical breast cancer models, focusing on TNBC. Methods: GYS1 mRNA expression and correlations with survival per clinical subtype were assessed in the METABRIC dataset. A tissue micro-array was constructed from primary tumors of 396 breast cancer patients with long-term follow-up, including normal breast control tissue. Triplicate tissue cores were stained immunohistochemically for GYS1, glycogen and the hypoxic marker carbonic anhydrase 9, and with periodic acid-Schiff staining for glycogen. In four TNBC cell lines and an MDA-MB-231 xenograft model, GYS1 protein expression, glycogen content and cell proliferation in normoxia and hypoxia were evaluated +/- GYS1 knockdown by siRNA or shRNA. Sensitivity of shGYS1 cell lines to drugs targeting mitochondria was tested. Results: In the METABRIC dataset (n = 1904), overexpression of GYS1 mRNA was associated with poor overall survival (HR 1.20 [95% CI 1.05 - 1.38]), which was mainly driven by the TNBC patients (n = 299, HR 1.52 [95% CI 1.09 - 2.14]). Immunohistochemically, most primary breast tumors had elevated GYS1 levels and glycogen content compared to normal breast tissue, with subtype specific analyses ongoing. In all breast cancer cell lines, hypoxia induced GYS1 protein expression and increased glycogen content. Acute siRNA-mediated GYS1 knockdown decreased proliferation of (TN)BC cell lines and MDA-MB-231 spheroids in both hypoxia and normoxia. shGYS1 MDA-MB-231 cells had decreased glycogen levels and shGYS1 MDA-MB-231 xenograft growth was impaired, especially in the first six weeks after inoculation. In control xenografts, immunohistochemical GYS1 expression was most pronounced adjacent to the necrotic tumor core, whereas shGYS1 xenografts lacked GYS1 expression. Finally, shGYS1 MDA-MB-231 cells were more sensitive to inhibitors of mitochondrial protein homeostasis, suggesting a potential synergistic approach to overcome eventual metabolic adaptation. Conclusions: GYS1 is overexpressed in primary breast tumors and high mRNA levels correlate with poor survival in TNBC. GYS1 downregulation impairs TNBC proliferation in vitro and in vivo, highlighting glycogen synthesis as potential novel therapeutic target in TNBC. Citation Format: Ellen C. De Heer, Christos E. Zois, Esther Bridges, Mieke C. Zwager, Tineke van der Sluis, Bert van der Vegt, Carolien P. Schröder, Steven de Jong, Adrian L. Harris, Mathilde Jalving. Glycogen synthesis as potential novel target in triple negative breast cancer: Glycogen synthase 1 expression in human breast cancers and the impact of downregulation on proliferation of preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5813.

A comparison between high carbohydrate and high lipid diets reception on the growth, feed utilization and glucose homeostasis of genetically improved farmed tilapia Oreochromis niloticus

This study was performed to compare the effects of high carbohydrate and high lipid diets reception on the growth, feed utilization and glucose homeostasis of genetically improved farmed tilapia Oreochromis niloticus. Three isonitrogenous (ca 30.2% protein) diets were formulated with different levels of starch and lipid: the control (CON, 24.2% starch and 6.57% lipid), high carbohydrate diet (HCD, 33.8% starch and 6.91% lipid), and high lipid diet (HLD, 24.0% starch and 16.5% lipid). Tilapia juveniles (initial mean body weight: 32.2 ± 0.2 g/fish) were randomly divided into 12 rectangular tanks (250 L) with 20 fish per tank, and fed the diets to apparent satiation for 8 weeks. The results showed that mean body weight was lower in the HLD fish (183 g/fish) than in the CON (212 g/fish) and HCD fish (206 g/fish). Feeding ratio and feed efficiency ratio were not affected by different treatments. Both dietary starch and lipid provision in excess exhibited poor protein-sparing effect, as protein efficiency ratio and protein productive value were not affected while whole-body protein percentages were lower in the HCD and HLD fish than in the CON fish. High lipid diets reception exerted a greater impact on proximate composition of the whole fish and intraperitoneal fat deposition, while high carbohydrate diets reception exerted a greater impact on liver size through promoting glycogen deposition. Plasma glucose level sharply increased in the HLD fish (7.17 mmol/L) as compared with the CON fish (4.66 mmol/L). Evidences from the molecular level revealed that hyperglycemia of the HLD fish was probably resulted from hepatic glucose output (represented by mRNA levels of glucose transporter 2 and glucose-6-phosphatase catalytic subunit a2) through stimulating gluconeogenesis (the expression of phosphoenolpyruvate carboxykinase 2) rather than glycogenolysis. In addition, high lipid diets reception suppressed hepatic de novo lipogenesis (mRNA level of fatty acid synthase (fas)) along with muscular glucose transport (mRNA level of glucose transporter 4) and glycogenesis (the expression of glycogen synthase 1), which aggravated the hyperglycemia of the HLD fish. In contrast, high carbohydrate diets reception did not affect the expression of representative genes involved with glycolysis and gluconeogenesis, but sharply upregulated the mRNA abundances of lipogenic acetyl-CoA carboxylase α and fas in the liver of tilapia. Except lipid, glycogen concentration also increased in the liver of the HCD fish as compared with the CON fish. Thus, plasma glucose slightly increased in the HCD fish (5.58 mmol/L) as compared with the CON fish. The overall results of this study suggested that tilapia possessed a better capability to tolerate high carbohydrate diets (33.8%) than high lipid diets (16.5%) based on growth performance and the maintenance of glucose homeostasis. This conclusion might have limitations, because the energy density of diets HCD and HLD was different. Further studies are still warranted to compare the glucose homeostasis of tilapia in response to high carbohydrate and high lipid diets reception on an isoenergetic basis.

A Dioxidovanadium Complex cis-[VO2 (obz) py] Attenuates Hyperglycemia in Streptozotocin (STZ)-Induced Diabetic Male Sprague-Dawley Rats via Increased GLUT4 and Glycogen Synthase Expression in the Skeletal Muscle.

Vanadium has demonstrated antihyperglycemic effects in diabetes mellitus (DM) but is, however, associated with toxicity. Therefore, new vanadium complexes envisaged to possess heightened therapeutic potency while rendering less toxicity are being explored. Accordingly, the aim of the study was to investigate the effects of a dioxidovanadium (V) complex, cis-[VO2 (obz) py], on selected glucose metabolism markers in streptozotocin (STZ)-induced diabetic rats. STZ-induced diabetic rats were treated orally with cis-[VO2 (obz) py] (10, 20, and 40 mg/kg) twice every 3rd day for 5 weeks. Blood glucose concentrations, body weight, and food and water intake were monitored weekly, for 5 weeks. Rats were then euthanized after which blood, liver, and muscle tissues were collected for biochemical analysis. The administration of dioxidovanadium complex significantly decreased blood glucose concentrations throughout the 5-week period in comparison with the diabetic control (DC). The attenuation of hyperglycemia was accompanied by an increased glycogen concentration in both liver and muscle tissues in the treated groups. Furthermore, a significant increase was observed in the expression of glucose transporter type 4 (GLUT4) in the skeletal muscle tissues and glycogen synthase in the liver tissues. These findings indicate that our vanadium complex cis-[VO2 (obz) py] may exert antihyperglycemic effects through increased glucose uptake, glycogen synthesis, and increased GLUT4 and glycogen synthase expression.

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.&#x0D; Aim: To determine whether glyphosate exposure is detrimental to the glycogen metabolic enzymes (Glycogen synthase and phosphorylase) in the liver of adult male rats.&#x0D; 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&lt;0.05 level were considered to be statistically significant.&#x0D; Results: The present result showed that the mRNA expression of glycogen synthase significantly reduced (P&lt;0.05) and mRNA expression of glycogen phosphorylase activity increased significantly with an increased dose of glyphosate (P&lt;0.05) to that of control.&#x0D; Conclusion: Exposure to glyphosate causes detrimental changes in the glycolytic enzymes glycogen synthase and glycogen phosphorylase leading to diabetes.

Hindbrain catecholamine regulation of ventromedial hypothalamic nucleus glycogen metabolism during acute versus recurring insulin-induced hypoglycemia in male versus female rat

Ventromedial hypothalamic nucleus (VMN) glycogen metabolism affects local glucoregulatory signaling. The hindbrain metabolic-sensitive catecholamine (CA) neurotransmitter norepinephrine controls VMN glycogen phosphorylase (GP)-muscle (GPmm) and -brain (GPbb) type expression in male rats. Present studies addressed the premise that CA regulation of hypoglycemic patterns of VMN glycogen metabolic enzyme protein expression is sex-dimorphic, and that this signal is responsible for sex differences in acclimation of these profiles to recurrent insulin-induced hypoglycemia (RIIH). VMN tissue was acquired by micropunch-dissection from male and female rats pretreated by caudal fourth ventricular administration of the CA neurotoxin 6-hydroxydopamine (6OHDA) before single or serial insulin injection. 6-OHDA averted acute hypoglycemic inhibition of VMN glycogen synthase (GS) and augmentation of GPmm and GPbb protein expression in males, and prevented GPmm and -bb down-regulation in females. Males recovered from antecedent hypoglycemia (AH) exhibited neurotoxin-preventable diminution of baseline GS profiles, whereas acclimated GPmm and -bb expression in females occurred irrespective of pretreatment. RIIH did not alter VMN GS, GPmm, and GPbb expression in vehicle- or 6-OHDA-pretreated animals of either sex. VMN glycogen content was correspondingly unchanged or increased in males versus females following AH; 6-OHDA augmented glycogen mass in AH-exposed animals of both sexes. RIIH did not alter VMN glycogen accumulation in vehicle-pretreated rats of either sex, but diminished glycogen in neurotoxin-pretreated animals. AH suppresses baseline GS (CA-dependent) or GPmm/GPbb (CA-independent) expression in male and female rats, respectively, which corresponds with unaltered or augmented VMN glycogen content in those sexes. AH-associated loss of sex-distinctive CA-mediated enzyme protein sensitivity to hypoglycemia (male: GS, GPmm, GPbb; female: GPmm, Gpbb) may reflect, in part, VMN target desensitization to noradrenergic input.

Effect of Caralluma tuberculata on regulation of carbohydrate metabolizing genes in alloxan-induced rats

Ethnopharmacological relevanceCaralluma tuberculata (C. tuberculata) has traditionally been used in Pakistan and other parts of the world as a folk treatment for diabetes mellitus. A few studies indicated its antihyperglycemic effect, however, the mystery remained unfolded as how did it modify the pathophysiological condition. Aim of studyHence, this study aimed to explore underlying mechanism(s) for its hypoglycemic activity at biochemical and molecular levels. Materials and methodsMethanol extract (ME) of C. tuberculata as well as its hexane (HF) and aqueous (AF) fractions were explored for their effect on total glycogen in liver and skeletal muscle of alloxan-induced rats by spectroscopy. Moreover, the expression of genes related to hepatic carbohydrate metabolizing enzymes was quantified. At molecular level, mRNA expression of glucose transporter 2 (GLUT-2), glycogen synthase (GS), glucokinase (GK), hexokinase 1 (HK-1), pyruvate kinase (PK), glucose 6 phosphate dehydrogenase (G-6-PDH), pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6 phosphatase (G-6-Pase) was determined by using quantitative real time polymerase chain reaction (qRT-PCR) after administration of ME (350 mg), HF(3 mg), AF (10 mg) and metformin (500 mg). The doses were administered twice daily according to per kg of body weight. ResultsA significant reduction in hepatic and skeletal muscle glycogen content was exhibited. The data of qRT-PCR revealed that gene's expression of GLUT-2 was significantly decreased after treatment with ME and HF, whilst it was unaltered by AF, however, a significant decrease was observed in genes corresponding to GS, GK and HK-1 after treatment with ME. Similarly, there was a significant decrease in expression of genes corresponding to GS, GK and HK-1 following treatment with HF. Surprisingly, post-treatment with AF didn't modify the gene's expression of GS and GK, whilst it caused a profound decrease in expression of HK-1 gene. Contrarily, the expression of gene related to PK was significantly up-regulated post-administration with ME, HF and AF. The expression levels of G-6-PDH, however, remained unaltered after treatment with the experimental extract and fractions of the plant. In addition, HF and AF did not cause any modification in PEPCK, whereas ME caused a significant down-regulation of the gene. Treatment with all the extract and fractions of the plant caused a substantial decrease in the gene's expression of PC, while there was a significant increase in the expression of gene related to G-6-Pase. ConclusionThe three experimental extract and fractions caused a substantial decrease in glycogen content in liver and skeletal muscle tissues. The analysis by qRT-PCR showed that glucose transport via GLUT-2 was profoundly declined by ME and HF. The expression of genes related to various metabolic pathways involved in metabolism of carbohydrate in hepatocytes revealed explicitly that the ME, HF and AF decreased the phenomena of glycogenesis and gluconeogenesis. Contrarily, all the extract and fractions of the plant activated glycogenolysis and glycolysis but did not modify the pentose phosphate shunt pathway.

Sex differences in glucoprivic regulation of glycogen metabolism in hypothalamic primary astrocyte cultures: Role of estrogen receptor signaling

Hypoglycemia causes sex-reliant changes in hypothalamic astrocyte glycogen metabolism in vivo. The role of nuclear versus membrane astrocyte estrogen receptors (ER) in glucoprivic regulation of glycogen is unclear. Here, primary hypothalamic astrocyte cultures were treated with selective ER antagonists during glucoprivation to investigate the hypothesis that ER mediate sex-specific glycogen responses to glucoprivation. Results show that glucoprivic down-regulation of glycogen synthase expression is mediated by transmembrane G protein-coupled ER-1 (GPER) signaling in each sex and estrogen receptor (ER)-beta (ERβ) activity in females. Glucoprivic inhibition of glycogen phosphorylase involves GPER and ERβ in females, but ER-independent mechanisms in males. GPER, ERβ, and ER-alpha (ERα) inhibit or stimulate AMPK protein expression in male versus female astrocytes, respectively. Glucoprivic augmentation of phospho-AMPK profiles in male glia was opposed by GPER activation, whereas GPER and ERβ suppress this protein in females. Astrocyte ERα and GPER content was down-regulated in each sex during glucose deficiency, whereas ERβ levels was unaltered (males) or increased (females). Glucoprivation correspondingly elevated or diminished male versus female astrocyte glycogen content; ER antagonism reversed this response in males, but not females. Results identify distinctive ER variants involved in sex-similar versus sex-specific astrocyte protein responses to withdrawal of this substrate fuel. Notably, glucoprivation elicits a directional switch or gain-of-effect of GPER and ERβ on specific glial protein profiles. Outcomes infer that ERs are crucial for glucoprivic regulation of astrocyte glycogen accumulation in males. Alternatively, estradiol may act independently of ER signaling to disassemble this reserve in females.

Effects of Retinoic Acid and Insulin on Glycogen Content and Expression Levels of Glycogenic Proteins in Differentiating L6 Myocytes

Abstract Objectives The skeletal muscle is critical for the control of glucose homeostasis partially through glycogenesis. The effects of retinoic acid (RA) on glycogen content and expressions of key glycogenic proteins without or with insulin in L6 rat myocytes deserve to be studied. Methods L6 myocytes at confluence were induced to differentiation using DMEM with 2% horse serum. Cells were incubated in medium with vehicle control or 1 μM RA without or with 10 nM insulin for 2, 4, and 6 days. Glycogen was extracted and analyzed using α-amyloglucosidase and released glucose assay. Proteins in cell lysates from parallel groups were extracted and subjected to analysis using western blotting. The expression levels of glycogen synthase (GS), phospho-glycogen synthase (Ser 641) (p-GS), glycogen synthase kinase 3-β(GSK3β), and phospho-glycogen synthase kinase 3-β (p-GSK3β) were determined using their specific antibodies, and then quantified with ImageJ software. Results The RA + insulin group had higher glycogen content than the control group on days 2 to 6. A synergistic effect of RA and insulin were observed on day 2. GS expression was synergistically induced in the RA + insulin group on each day. The total p-GS normalized to β-actin in the RA + insulin group was higher than that in the control on day 2. However, the ratio of p-GS/GS in the RA + insulin group was lower than that in the control on day 2 and in RA group on day 6, respectively. GSK3β expression levels in insulin and RA + insulin groups were higher than that in the control on day two. The total p-GSK3β normalized to β-actin showed an increase in the insulin group compared to the RA group on day 6. The p-GSK3β/GSK3β ratio in the insulin group was higher than that in RA and RA + insulin groups on day 6. Conclusions Treatments with RA and insulin increase the glycogen content in L6 cells via upregulation of glycogenesis. A synergy of RA and insulin can be seen. The increases of total GS and decreases of p-GS/GS ratio contribute to the elevation of GS activity. RA likely exerts its glycogenic effects through changes of the total GS level and phosphorylation by GSK3β. Funding Sources Internal fund at the Univenrsity of Tennessee.

Heterogeneity in Insulin‐Stimulated Glucose Uptake Among Different Muscle Groups in People

Insulin‐stimulated glucose uptake in skeletal muscles is the major mechanism for postprandial glucose disposal. Insulin‐stimulated glucose uptake is greater in type I than in type II muscles in rats. The cellular capacity for glucose uptake and disposal (insulin receptor, GLUT4, hexokinase, and glycogen synthase expression) is also greater in isolated human type I compared with type II fibers. The goal of the present study was to test the hypothesis that insulin‐stimulated glucose uptake rate varies among different muscle groups and is greater in trunk (erector spinae, rectus abdominis, obliques) than leg (quadriceps, hamstrings) muscles and greater in the hamstrings than the quadriceps because trunk muscles generally have a greater percentage of type I fibers than leg muscles and the hamstrings have a greater percentage of type I fibers than quadriceps muscles. We determined abdominal and thigh muscle glucose uptake rates in 10 healthy lean women (age: 43±13 years, BMI: 23±2 kg/m2; mean±SD) by using a hyperinsulinemic‐euglycemic clamp procedure in conjunction with dynamic positron emission tomography imaging after bolus injection of 18F‐fluorodeoxyglucose. In addition, [6,6‐2H2]glucose was infused to determine whole‐body glucose disposal rate per kg fat‐free mass, which is commonly used as an index of muscle glucose uptake, and a muscle biopsy from the vastus lateralis was obtained to determine myofiber composition. Glucose uptake rate in the erector spinae (128±30 μmol/kg/min) was nearly twice as great as in the rectus abdominis (66±14 μmol/kg/min; P&lt;0.01) and approximately 50% greater than in the obliques (88±23 μmol/kg/min; P&lt;0.05). Quadriceps and hamstring glucose uptake rates were not different from each other (85±42 and 91±37 μmol/kg/min, respectively; P&gt;0.05) and not different from glucose uptake rates in the rectus abdominis and obliques but less than in the erector spinae (P&lt;0.05). No significant correlation between fiber type composition and muscle glucose uptake rate in the quadriceps was detected. Whole body insulin stimulated glucose disposal rate was 93±28 μmol/kg fat‐free mass/min. We conclude that: i) insulin‐stimulated muscle glucose uptake is heterogeneous among muscle groups, ii) it is unlikely that differences in fiber type composition among muscles are responsible for the heterogeneity in glucose uptake rates, and iii) whole‐body glucose disposal rate per kg fat‐free mass does not accurately reflect glucose uptake rates in individual muscles.Support or Funding InformationNIH DK115400, DK56341 (Nutrition Obesity Research Center), DK020579 (Diabetes Research Center), and UL1TR000448 (Clinical Translational Science Award), and ADA 1‐18‐ICTS‐119.

Conjugated linoleic acids diminish glycogen synthase and glycogen synthase kinase-3 expression in muscle cells of C57BL/6J mice - in vitro and in vivo study.

Conjugated linoleic acids (CLA) have been extensively advertised as dietary supplements to reduce fat and increase muscle mass. However, the role of CLA in glycogen metabolism is still largely unknown. The aim of this study was to assess the effect of CLA on glycogen synthesis in vitro (CCL 136 cell line human) and CLA in vivo (C57BL/6J mice). The materials used were the CCL 136 muscle cell line and muscles of female C57BL/6J mice (n = 52), housed at animal laboratory facility and feed with "MURIGRAN", a standard feed prepared for rodents (Agropol, Poland). Chemically pure fatty acids were added to soybean oil. CLA isomers (c9,t11 CLA, t10,c12 CLA, and as a mixture (1:1)) were administered with feed. Supplementation in mice started at week 6 of age and lasted for 4 weeks. Methods used in the study were real time- PCR - quantification of gene expression, Western blot glycogen synthase kinase-3 (GSK3α 9) and glycogen synthase (GS) protein, glycogen staining by PAS. Quantitative determination of glycogen by spectrophotometry and intracellular reactive oxygen species was measured the intracellular oxidation of dichloro-dihydro-fluorescein diacetate (DCFH-DA). In vitro data showed that GS and GSK3 expression was lower in cells cultured with different CLAs and a mixture of CLAs. GS gene expression was significantly decreased in cells cultured with c9, t11 CLA (P < 0.04) and t10, c12 CLA (P < 0.05) as well as the mixture of both isomers. The GSK3α gene expression was reduced in cells cultured with a mixture of CLA (P < 0.02), whereas phosphorylation of GSK3α increased in cells cultured with c9, t11 CLA GSK3α (P < 0.05). In vivo data showed a reduction in the glycogen concentration among mice fed a diet containing t10, c 12 CLA and a mixture of CLA isomers. We conclude that both CLA isomers can affect the synthesis of glycogen in muscle cells through the regulation of GS and GSK3α gene expression.