Glycogen Phosphorylase mRNA Research Articles

Long-Term Administration of Dehydroepiandrosterone Accelerates Glucose Catabolism via Activation of PI3K/Akt-PFK-2 Signaling Pathway in Rats Fed a High-Fat Diet.

Dehydroepiandrosterone (DHEA) has a fat-reducing effect, while little information is available on whether DHEA regulates glucose metabolism, which would in turn affect fat deposition. To investigate the effects of DHEA on glucose metabolism, rats were administered a high-fat diet containing either 0 (HCG), 25 (HLG), 50 (HMG), or 100 (HHG) mg·kg-1 DHEA per day via gavage for 8 weeks. Results showed that long-term administration of DHEA inhibited body weight gain in rats on a high-fat diet. No statistical differences in serum glucose levels were observed, whereas hepatic glycogen content in HMG and HHG groups and muscle glycogen content in HLG and HMG groups were higher than those in HCG group. Glucokinase, malate dehydrogenase and phosphofructokinase-2 activities in HMG and HHG groups, pyruvate kinase and succinate dehydrogenase activities in HMG group, and pyruvate dehydrogenase activity in all DHEA treatment groups were increased compared with those in HCG group. Phosphoenolpyruvate carboxykinase and glycogen phosphorylase mRNA levels were decreased in HMG and HHG groups, whereas glycogen synthase-2 mRNA level was increased in HMG group compared with those in HCG. The abundance of Glut2 mRNA in HMG and HHG groups and Glut4 mRNA in HMG group was higher than that in HCG group. DHEA treatment increased serum leptin content in HMG and HHG groups compared with that in HCG group. Serum insulin content and insulin receptor mRNA level in HMG group and insulin receptor substrate-2 mRNA level in HMG and HHG group were increased compared with those in HCG group. Furthermore, Pi3k mRNA level in HMG and Akt mRNA level in HMG and HHG groups were significantly increased than those in HCG group. These data showed that DHEA treatment could enhance glycogen storage and accelerate glucose catabolism in rats fed a high-fat diet, and this effect may be associated with the activation of PI3K/Akt-PFK-2 signaling pathway.

Polysaccharides from Liriopes Radix ameliorates hyperglycemia via various potential mechanisms in diabetic rats

Liriopes Radix, which is regarded as both drug and healthy diet, is drunk as tea and used in traditional Chinese medicine to treat diabetes. Based on our previous studies, investigated the hypoglycemic effects and explored the mechanisms of total polysaccharides from Liriope spicata var. prolifera (Liriopes Radix) in a diabetic rat model. TLSP reduced hyperglycemia in diabetic rats. The oral glucose tolerance test showed that TLSP could improve the glucose tolerance of diabetic rats. Damage to liver and pancreas tissue was inhibited after treatment with TLSP. Moreover, TLSP increased glycogen content, glucokinase (GK) and glycogen synthetase (GS) activities, and suppressed the elevation of glucose-6-phosphatase (G6Pase) and glycogen phosphorylase (GP) activities in liver. Compared with the diabetic control group, GK and GS mRNA expression were significantly elevated, while G6Pase and GP mRNA expression were decreased in TLSP groups. In addition, TLSP could inhibit glycogen synthase kinase-3β expression and increase insulin receptor, insulin receptor substrate-1, phosphoinositide 3-kinase, protein kinase B and glucose transport protein-4 expression in liver. TLSP showed hypoglycemic function. Improvement of glucose metabolism and insulin-signaling transduction were possible mechanisms.

Differential expression of genes and changes in glucose metabolism in the liver of liver-specific glucokinase gene knockout mice

To investigate the role of liver-specific expression of glucokinase (GCK) in the pathogenesis of hyperglycemia and to identify candidate genes involved in mechanisms of the onset and progression of maturity onset diabetes of the young, type 2 (MODY-2), we examined changes in biochemical parameters and gene expression in GCK knockout (gckw/–) and wild-type (gckw/w) mice as they aged. Fasting blood glucose levels were found to be significantly higher in the gckw/– mice, compared to age-matched gckw/w mice, at all ages (P<0.05), except at 2weeks. GCK activity of gckw/– mice was about 50% of that of wild type (gckw/w) mice (P<0.05). Glycogen content at 4 and 40weeks of age was lower in gckw/– mice compared to gckw/w mice. Differentially expressed genes in the livers of 2 and 26week-old liver-specific GCK knockout (gckw/–) mice were identified by suppression subtractive hybridization (SSH), which resulted in the identification of phosphoenolpyruvatecarboxykinase (PEPCK, also called PCK1) and Sterol O-acyltransferase 2 (SOAT2) as candidate genes involved in pathogenesis. The expressions of PEPCK and SOAT2 along with glycogen phosphorylase (GP) and glycogen synthase (GS) were then examined in GCK knockout (gckw/–) and wild-type (gckw/w) mice at different ages. Changes in PEPCK mRNA levels were confirmed by real-time RT-PCR, while no differences in the levels of expression of SOAT2 or GS were observed in age-matched GCK knockout (gckw/–) and wild-type (gckw/w) mice. GP mRNA levels were decreased in 40-week old gckw/– mice compared to age-matched gckw/w mice. Changes in gluconeogenesis, delayed development of GCK and impaired hepatic glycogen synthesis in the liver potentially lead to the onset and progression of MODY2.

The Effects of Estradiol and Catecholestrogens on Glycogen Catabolism and Glucose Mobilization in the Uterus of the Mink (Neovison vison).

Embryonic development prior to formation of the placenta depends in part on uterine glandular secretions (histotroph) that are rich in carbohydrates, including glycogen. In mink, total uterine glycogen concentrations decrease 55% between estrus and the peri-implantation period, presumably to meet the metabolic demands of developing embryos. Estradiol-17beta (E2) stimulates uterine glycogen production and circulating E2 levels peak during proestrous and decline thereafter. We have shown that the effects of E2 are mediated in part through the catecholestrogens: 2-hydroxycatecholestradiol (2-OHE2) and 4-hydroxycatecholestradiol (4-OHE2) produced by the endometrium. It is however, unknown what is responsible for glycogen catabolism as the time of implantation draws near. While progesterone concentrations are increasing at this time, exogenous progesterone stimulated uterine glycogen accumulation in mink with intact ovaries. Although E2 levels are significantly reduced during most of the peri-implantation period, a transitory peak in E2 secretion occurs near the expected time of implantation. It is therefore possible that E2 and/or catecholestrogens facilitate both glycogen synthesis and breakdown. Therefore, to investigate the effects of these steroids on glycogen catabolism and glucose mobilization, our objectives were to determine the effects of E2, 2-OHE2 and 4-OHE2 on (1): uterine glycogen content and (2): gene expression levels for glycogen-synthase, glycogen-phosphorylase, and glucose-6-phosphatase. Mink (N=24) were ovariectomized and assigned at random to one of four groups: 1 = Controls, 2 = E2, 3 = 2-OHE2, and 4 = 4-OHE2. Animals were injected sc once daily with hormone (40ug/kg BW) in sesame seed oil, or oil only (controls) for 3 consecutive days. On day four, mink were sacrificed and uteri collected. Total uterine glycogen concentrations (mg/g dry wt) were increased 8-fold by E2 and 4-OHE2 and 4-fold by 2-OHE2, compared to controls (P<0.05). Interestingly, the percent glycogen content of the glandular epithelium was increased 4 to 5 fold by 4-OHE2 and E2 (P<0.05), but was not different between 2-OHE2 and controls. Expression of glycogen-synthase mRNA did not differ between E2, 4-OHE2 and controls, but was reduced 5-fold in response to 2-OHE2 (P<0.01). Uterine glycogen-phosphorylase mRNA expression was increased 3-fold by E2 and 4-OHE2, and 10-fold by 2-OHE2 (P<0.01). The expression of glucose-6-phosphatase mRNA was greatest in response to 2-OHE2, followed by 4-OHE2 and then E2 (P<0.05). We conclude that E2 and/or catecholestrogens influence both glycogen synthesis and breakdown, perhaps as a result of different concentrations or exposure time. Although 4-OHE2 is considered to be a more potent estrogen than 2-OHE2, our data show that 2-OHE2 had a greater effect than E2 and 4-OHE2 on the expression of all glycogen metabolizing genes investigated here. It is possible that as the time of implantation approaches, the production and/or sensitivity of the uterus to 2-OHE2 predominates over both 4-OHE2 and E2, promoting glycogen catabolism and glucose mobilization. Such a mechanism, could serve to regulate the release of glucose from uterine glycogen reserves, providing a source of nutrition to the developing embryos, which may be essential for implantation and reproductive success. (Funded by Fur Commission USA and NIH INBRE P20RR016454) (poster)

Characterization of Hepatic Glucose Metabolism Disorder with the Progress of Diabetes in Male Spontaneously Diabetic Torii Rats

The Spontaneously Diabetic Torii (SDT) rat has recently been established as a new model of non-obese type 2 diabetes. In this study, we examined changes in hepatic glucose metabolism in prediabetic and diabetic SDT rats compared with age-matched control rats. The prediabetic state was confirmed at 16 weeks of age, and the diabetic state was confirmed at 24 and 32 weeks of age. Decreases in glucokinase mRNA levels and activity were observed in the prediabetic state. In this state, glycogen synthase activity and glycogen content were also decreased in the SDT rat. In addition to the above changes, glycogen phosphorylase mRNA and activity were decreased and gluconeogenetic enzyme mRNA levels were significantly increased in the diabetic state. These results indicate there is a great potential that abnormalities in hepatic glucose metabolism play a role in the progression to onset of diabetes. We suggest that the SDT rat is a valuable diabetic model for investigations into mechanisms or causes of progression to diabetes.

Inhibition of glycogen phosphorylase (GP) by CP-91,149 induces growth inhibition correlating with brain GP expression

The role of glycogenolysis in normal and cancer cells was investigated by inhibiting glycogen phosphorylase (GP) with the synthetic inhibitor CP-91,149. A549 non-small cell lung carcinoma (NSCLC) cells express solely the brain isozyme of GP, which was inhibited by CP-91,149 with an IC 50 of 0.5 μM. When treated with CP-91,149, A549 cells accumulated glycogen with associated growth retardation. Treated normal skin fibroblasts also accumulated glycogen with G1-cell cycle arrest that was associated with inhibition of cyclin E-CDK2 activity. Overall, cells expressing high levels of brain GP were growth inhibited by CP-91,149 correlating with glycogen accumulation whereas cells expressing low levels of brain GP were not affected by the drug. Analyses of 59 tumor cell lines represented in the NCI drug screen identified that every cell line expressed brain GP but the profile was dominated by a few highly GP expressing cell lines with lower than mean GP-a enzymatic activities. The correlation program, COMPARE, identified that the brain GP protein measured in the NCI cell lines corresponded with brain GP mRNA expression, ADP-ribosyltransferase 3, and colony stimulating factor 2 receptor α in the 10,000 gene microarray database with similar correlation coefficients. These results suggest that brain GP is present in proliferating cells and that high protein levels correspond with the ability of CP-91,149 to inhibit cell growth.

Hepatic Alanine-glyoxylate Aminotransferase Activity and Oxalate Metabolism in Vitamin B6 Deficient Rats

Hepatic Alanine-glyoxylate Aminotransferase Activity and Oxalate Metabolism in Vitamin B6 Deficient Rats

Glycogen phosphorylase is activated in response to glucose deprivation but is not responsible for enhanced glucose transport activity in 3T3-L1 adipocytes

We have previously shown that glucose deprivation activates glucose transport in a time- and protein synthesis-dependent fashion in 3T3-L1 adipocytes, a mouse cell line. Coincident with this is loss of glycogen. Because glycogen phosphorylase (GP) is responsible for glycogen degradation, we have examined its regulation to determine the relationship between transport activation and glycogen turnover. We first cloned the adipose GP cDNA and found sequence similarity to rat and human liver GP. Because the mouse liver GP cDNA sequence was unavailable, we cloned this cDNA as well and showed 100% identity between mouse adipose and liver sequences. A 3.1 kb transcript was readily observed in total RNA isolated from mouse liver or adipose by Northern blot analysis but, surprisingly, not in either total or poly(A) selected RNA from 3T3-L1 adipocytes. To evaluate regulation in 3T3-L1 adipocytes, we amplified GP mRNA from total RNA using multiplex, semi-quantitative PCR but found that expression did not change in response to deprivation. GP protein levels did not change either. However, endogenous GP activity from glucose-deprived cells was significantly elevated relative to controls, due to an increase in the phosphorylated form of GP (GP a). Finally, we overexpressed GP to determine its direct influence on the glucose transport system. These results were negative, which suggests that the nutrient control of glucose transport and GP occurs independently despite kinetic similarities in transport activation and glycogen turnover.

Expression of the gene encoding glycogen phosphorylase is elevated in diabetic rat skeletal muscle and is regulated by insulin and cyclic AMP.

Glycogen phosphorylase regulates the breakdown of glycogen into glucose, but as previous studies have demonstrated, the control of glycogen metabolism becomes deregulated in diabetes mellitus. Messenger RNA levels encoding several different proteins are altered in skeletal muscle biopsies of patients with insulin-dependent and non-insulin-dependent diabetes. The possible alteration of expression of the gene encoding the skeletal muscle isoform of glycogen phosphorylase during diabetes has not previously been investigated. We examined the effect of streptozotocin-induced diabetes and insulin treatment on glycogen phosphorylase mRNA in rat skeletal muscle; glycogen phosphorylase mRNA levels were elevated in diabetic rat muscle tissue, but were partially suppressed in diabetic rat muscle following insulin treatment. To distinguish between the effects of insulin and counter-regulatory hormones on glycogen phosphorylase mRNA levels, we employed differentiating rat L6 myoblasts in culture. Insulin stimulated the accumulation of glycogen phosphorylase mRNA as determined by Northern blot analysis. Moreover, insulin and dibutyryl cAMP stimulated expression of a transiently transfected chloramphenicol acetyl transferase reporter gene under the control of the muscle glycogen phosphorylase promoter in differentiating myotubes in culture, suggesting that the effects of insulin and counter-regulatory hormones on glycogen phosphorylase mRNA are at the level of transcription. These results suggest that insulin and epinephrine may participate in the induction of the glycogen phosphorylase gene during myogenesis; moreover, activation of this gene in muscle tissue may be a contributing factor in impaired glycogen storage during uncontrolled diabetes.

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

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

Effect of vitamin B6 deficiency on the expression of glycogen phosphorylase mRNA in rat liver and skeletal muscle.

The effect of vitamin B6 deficiency on the expression of glycogen phosphorylase mRNA in rat liver and skeletal muscle was investigated. The level of phosphorylase mRNA in the muscle of vitamin B6-deficient rats was reduced to 40% of that in the control rats. By contrast, the phosphorylase mRNA level was increased 5-fold in the liver of the deficient animals. It was also found that the expression of the beta-actin gene, generally regarded as a 'housekeeping' gene, was unaffected by B6 deficiency in the muscle but was enhanced in the liver of the deficient animals. These observations suggest that vitamin B6 may modulate the transcriptional activation of the phosphorylase gene in a tissue-specific manner.

Complete cDNA sequence for rabbit muscle glycogen phosphorylase

The cDNA for the nearly full-length rabbit muscle glycogen phosphorylase mRNA has been isolated and sequenced. The cDNA is rich in G and C nucleotides. This feature is especially striking at the 3rd position of codons, where 86% of the 843 amino acid codons terminate with G or C. Methionine, presumably the initiation residue, is found at position—1, suggesting that the removal of only a single methionine residue precedes the amino-terminal acetylation at serine. Eight differences between the deduced amino acid sequence and the previously determined protein sequence are discussed.

Quantitation of muscle glycogen phosphorylase mRNA and enzyme amounts in adult rat tissues

Mammalian glycogen phosphorylases comprise a family of isozymes that are expressed selectively in a variety of cell types. As an initial step towards understanding the molecular processes that regulate the differential expression of the phosphorylase family, we have begun a quantiative examination of isozyme expression in vivo. In this paper, we report quantitative estimates of the amounts of the muscle (M) isozyme and its mRNA in adult rat tissues. Quantitative estimates of the amount of M-phosphorylase were obtained by an analysis involving electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose filters and sequential treatment with M-isozyme specific antibody and radioactively-labelled protein A. M-phosphorylase mRNA amounts were determined by an analysis involving transfer of RNA from agarose gels to nitrocellulose filters and subsequent hybridization with radioactively labelled rat M-phosphorylase cDNA. These studies indicate that M-phosphorylase is present in all tissues tested with the possible exception of liver. These are skeletal muscle, heart, brain, stomach, lung, kidney, spleen and testis. Quantitation of M-phosphorylase amounts indicate that there is a wide spectrum of variation (over 1000-fold range) in the relative amounts of the M-isozymes in these tissues. Relative mRNA levels parallel isozyme levels indicating that the major control of expression of this isozyme is governed by mRNA accumulation.