Activation of Glycogen Phosphorylase with 5-Aminoimidazole-4-Carboxamide Riboside (AICAR)

The experimental evaluation of the contribution of glycogen phosphorylase (GP) to biochemical pathways is limited to methods that raise cAMP, activating the cAMP-dependent protein kinase/phosphorylase kinase/GP cascade. Such methods convert the unphosphorylated form, “GPb,” which catalyzes glycogenolysis only in the presence of appropriate allosteric activators such as AMP, to the phosphorylated, constitutively activated form, “GPa.” However, activation of GP in this way is indirect, requires a functional cAMP kinase cascade, and is complicated by other actions of cAMP. Here, we demonstrate a strategy for the experimental manipulation of GP in intact dermal fibroblasts, involving activation by the membrane-permeable adenosine analog 5-aminoimidazole-4-carboxamide riboside (AICAR) and inhibition by caffeine and Pfizer compound CP-91149, which bind to GP at distinct sites. Potential complications because of activation of AMP-activated protein kinase by AICAR were assessed with metformin, which activates this kinase but does not activate GP. Using this strategy, we show that glycogen can be a significant and regulatable precursor of mannosyl units in lipid-linked oligosaccharides and glycoproteins. The experimental evaluation of the contribution of glycogen phosphorylase (GP) to biochemical pathways is limited to methods that raise cAMP, activating the cAMP-dependent protein kinase/phosphorylase kinase/GP cascade. Such methods convert the unphosphorylated form, “GPb,” which catalyzes glycogenolysis only in the presence of appropriate allosteric activators such as AMP, to the phosphorylated, constitutively activated form, “GPa.” However, activation of GP in this way is indirect, requires a functional cAMP kinase cascade, and is complicated by other actions of cAMP. Here, we demonstrate a strategy for the experimental manipulation of GP in intact dermal fibroblasts, involving activation by the membrane-permeable adenosine analog 5-aminoimidazole-4-carboxamide riboside (AICAR) and inhibition by caffeine and Pfizer compound CP-91149, which bind to GP at distinct sites. Potential complications because of activation of AMP-activated protein kinase by AICAR were assessed with metformin, which activates this kinase but does not activate GP. Using this strategy, we show that glycogen can be a significant and regulatable precursor of mannosyl units in lipid-linked oligosaccharides and glycoproteins. Glycogen, a glucose polymer, is a key store of hexose in mammalian cells. Its formation from UDP-Glc and breakdown to Glc-1-P 1The abbreviations used are: Glc-1-P, glucose-1 phosphate; Glc-6-P, glucose-6 phosphate; Fru-6-P, fructose-6 phosphate; Man-1-P, mannose-1 phosphate; Man-6-P, mannose-6 phosphate; AICAR, 5-aminoimidazole-4-carboxamide riboside; AMPK, AMP-activated protein kinase; GP, glycogen phosphorylase; LLO, lipid-linked oligosaccharide; HPLC, high pressure liquid chromatography. are catalyzed, respectively, by glycogen synthase and glycogen phosphorylase (GP). GP exists in two forms, depending upon its phosphorylation state (1Johnson L.N. FASEB J. 1992; 6: 2274-2282Crossref PubMed Scopus (264) Google Scholar). “GPb,” the unphosphorylated form, can be stimulated by allosteric activators such as AMP. Alternatively, phosphorylase kinase can convert the enzyme into its phosphorylated, covalently activated form “GPa,” which no longer requires allosteric activation. The regulation of GP may be complicated further by phosphatases and phosphatase inhibitors and the abilities of enzymes such as AMP-activated protein kinase (AMPK) (2Polekhina G. Gupta A. Michell B.J. van Dendersen B. Murthy S. Feil S.C. Jennings I.G. Campbell D.J. Witters L.A. Parker M.W. Kemp B.E. Stapleton D. Curr. Biol. 2003; 13: 867-871Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar) and protein phosphatase-1 (3Newgard C.B. Brady M.J. O'Doherty R.M. Saltiel A.R. Diabetes. 2000; 49: 1967-1977Crossref PubMed Scopus (158) Google Scholar) to bind to glycogen. Recently, interest has been generated in glycogen as a source of hexose units in N-linked glycoproteins (4McMahon R.J. Frost S.C. Am. J. Physiol. 1996; 270: E640-E645PubMed Google Scholar, 5Gill A. Gao N. Lehrman M.A. J. Biol. Chem. 2002; 277: 44747-44753Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). Protein N-linked glycosylation requires the lipid-linked oligosaccharide (LLO) Glc3Man9GlcNAc2-P-P-dolichol. The oligosaccharide unit is transferred by oligosaccharyl transferase to asparaginyl residues on nascent proteins (6Kornfeld R. Kornfeld S. Annu. Rev. Biochem. 1985; 54: 631-664Crossref PubMed Scopus (4078) Google Scholar). LLO synthesis requires contributions of enzymes in both the cytoplasm and the endoplasmic reticulum. In the cytoplasm, precursor nucleotide sugars are synthesized. In contrast, the endoplasmic reticulum is the site of assembly of the LLO, requiring UDP-GlcNAc, GDP-mannose, and UDP-glucose as well as the lipids mannose-P-dolichol and glucose-P-dolichol, which are synthesized from GDP-mannose and UDP-glucose, respectively. Two independent approaches have suggested that phosphorolysis of glycogen to form Glc-1-P can be stimulated under particular forms of cellular stress and that the Glc-1-P can be converted sequentially to Glc-6-P, Fru-6-P, Man-6-P, Man-1-P, and GDP-mannose in sufficient quantities to substantially enhance glycoconjugate synthesis. Impaired LLO mannosylation in cultured 3T3-L1 and Chinese hamster ovary-K1 cells correlated with glycogen depletion resulting from glucose starvation (4McMahon R.J. Frost S.C. Am. J. Physiol. 1996; 270: E640-E645PubMed Google Scholar). In addition, increased glucose phosphate production and glycogenolysis were coincident with endoplasmic reticulum stress and improved LLO mannosylation (5Gill A. Gao N. Lehrman M.A. J. Biol. Chem. 2002; 277: 44747-44753Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). Although such experiments suggest that glycogen may be a significant precursor of mannosyl units for glycoconjugate synthesis, in each case glycogenolysis was activated indirectly. This problem could be solved by deliberate activation of GP. However, current approaches for experimental stimulation of GP in intact cells require activating the cAMP-dependent protein kinase/phosphorylase kinase cascade by adding an appropriate receptor agonist, an activator of adenylyl cyclase, or a membrane-permeable form of cAMP. Activation of phosphorylase kinase by these methods results in the conversion of GPb to GPa, but other pathways also respond to cAMP, so it would be difficult to attribute any effects on LLO synthesis solely to activation of GP. Here, we examined 5-aminoimidazole-4-carboxamide riboside (AICAR), which like metformin (7Zhou G. Myers R. Li Y. Chen Y. Shen X. Fenyk-Melody J. Wu M. Ventre J. Doebber T. Fujii N. Musi N. Hirshman M. Goodyear L.J. Moller D.E. J. Clin. Investig. 2001; 108: 1167-1174Crossref PubMed Scopus (4576) Google Scholar) is widely reported to cause activation of AMPK in cells (8Winder W.W. Hardie D.G. Am. J. Physiol. 1999; 277: E1-E10PubMed Google Scholar). AICAR enhanced LLO extension as anticipated, but surprisingly its mechanism was inconsistent with that of metformin (9Shang J. Lehrman M.A. J. Biol. Chem. 2004; 279: 9703-9712Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Through a pharmacological approach, we show that AICAR enhanced LLO synthesis primarily by stimulating GP not AMPK. Reagents—Compound CP-91149 (10Martin W.H. Hoover D.J. Armento S.J. Stock I.A. McPherson R.K. Danley D.E. Stevenson R.W. Barrett E.J. Treadway J.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1176-1181Crossref Scopus (221) Google Scholar) was a generous gift of Pfizer. AICAR (number A611700) was from Toronto Research Chemicals. AICAR-P (ZMP, number A1393), metformin (number D5035), and caffeine (number C0750) were from Sigma. All of the above reagents were prepared as stock solutions in water. Cell culture media were from Invitrogen, and sera were from Atlanta Biologicals. [2-3H]d-Mannose (10–20 Ci/mmol) was from Amersham Biosciences, and 2-deoxy-[G-3H]d-glucose (10 Ci/mmol) was from American Radiolabeled Chemicals. Culture of Normal and CDG Type I Dermal Fibroblasts—Dermal fibroblasts were cultured as described earlier (11Doerrler W.T. Lehrman M.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13050-13055Crossref PubMed Scopus (46) Google Scholar, 12Shang J. Korner C. Freeze H. Lehrman M.A. Glycobiology. 2002; 12: 307-317Crossref PubMed Scopus (47) Google Scholar) in RPMI 1640 medium with 10% fetal bovine serum. Cells were generally grown to 80–90% of confluence for experiments. As described earlier (12Shang J. Korner C. Freeze H. Lehrman M.A. Glycobiology. 2002; 12: 307-317Crossref PubMed Scopus (47) Google Scholar) the fibroblasts used were normal CRL-1904 (American Type Culture Collection). Analysis of [3H]Mannose-labeled LLOs and N-Linked Glycans by HPLC—Glycans were analyzed as described (11Doerrler W.T. Lehrman M.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13050-13055Crossref PubMed Scopus (46) Google Scholar, 12Shang J. Korner C. Freeze H. Lehrman M.A. Glycobiology. 2002; 12: 307-317Crossref PubMed Scopus (47) Google Scholar) by incubating cells for 20 min in RPMI 1640 medium containing 0.5 mm glucose, 10% dialyzed fetal bovine serum, and 40 μCi/ml (2.5 μm) [3H]mannose. [3H]-labeled LLOs and N-glycans were recovered and fractionated by HPLC as described previously (11Doerrler W.T. Lehrman M.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13050-13055Crossref PubMed Scopus (46) Google Scholar, 12Shang J. Korner C. Freeze H. Lehrman M.A. Glycobiology. 2002; 12: 307-317Crossref PubMed Scopus (47) Google Scholar, 13Zeng Y. Lehrman M.A. Anal. Biochem. 1991; 193: 266-271Crossref PubMed Scopus (23) Google Scholar). In some experiments 0.1 mm unlabeled d-mannose was included during the 20-min labeling period. When indicated, the peak heights of [3H]mannose-labeled glycans detected by HPLC were normalized to their mannose contents to determine their percentages in glycan pools (11Doerrler W.T. Lehrman M.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13050-13055Crossref PubMed Scopus (46) Google Scholar). Hexose Uptake Measurements—Uptake of tritium-labeled mannose and 2-deoxyglucose was done over a 10-min period as described (9Shang J. Lehrman M.A. J. Biol. Chem. 2004; 279: 9703-9712Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Assay of Glycogen Phosphorylase in Fibroblast Extracts—Assays were performed essentially as described (5Gill A. Gao N. Lehrman M.A. J. Biol. Chem. 2002; 277: 44747-44753Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). Extracts were prepared by streptolysin O permeabilization (14Anand M. Rush J.S. Ray S. Doucey M.A. Weik J. Ware F.E. Hofsteenge J. Waechter C.J. Lehrman M.A. Mol. Biol. Cell. 2001; 12: 487-501Crossref PubMed Scopus (75) Google Scholar). A solution was prepared (0.56 ml) with 31.3 mm sodium phosphate (pH 7.4), 3.13 mm MgSO4, 3.57 mm NADP+, and 1.79 mg/ml glycogen (Sigma catalog number G1508) and allowed to stand overnight at room temperature (empirically it was noticed that assay reproducibility was greatly enhanced by keeping the NADP+ solution at room temperature for at least 2 h). Water, enzymes (glucose-6-phosphate dehydrogenase to 5.7 μg/ml, and rabbit muscle phosphoglucomutase to 4.3 units/ml), glucose 1,6-bisphosphate (to 4.3 μm), and any activators or inhibitors were added to a final volume of 0.7 ml. The reaction was initiated by adding 0.3 ml of extract containing 0.02 mg of cytoplasmic protein or the corresponding buffer. GP activity was determined spectrophotometrically at 340 nm. Multiple data points were collected over a period of 4 h at room temperature. Over this period assays were linear, and the resulting slopes were used to determine activity. Values for blank reactions lacking extract were routinely subtracted out. Stimulation of LLO Extension by AICAR—Potential enzyme activators and inhibitors were tested with primary dermal fibroblasts refed daily with RPMI 1640 medium containing normal glucose (11 mm). To evaluate the effects on synthesis of LLOs and glycoproteins, cells were then labeled for 20 min with 2.5 μm [2-3H]mannose in medium adjusted to 0.5 mm glucose. In our hands this glucose concentration shifts the distribution of LLOs from mostly Glc3Man9GlcNAc2-P-P-dolichol (i.e. completed LLO) to Man2-5GlcNAc2-P-P-dolichol intermediates as the predominant species (9Shang J. Lehrman M.A. J. Biol. Chem. 2004; 279: 9703-9712Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 11Doerrler W.T. Lehrman M.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13050-13055Crossref PubMed Scopus (46) Google Scholar). The effects of 0.5 mm glucose on LLO synthesis are prevented if the labeling reaction also includes 0.1 mm mannose, which efficiently drives LLO mannosylation (11Doerrler W.T. Lehrman M.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13050-13055Crossref PubMed Scopus (46) Google Scholar). Thus, enzyme modulators could be evaluated for stimulatory effects on LLO synthesis by labeling in medium with 0.5 mm glucose, whereas inhibitory effects could be discerned with medium containing 0.5 mm glucose and 0.1 mm mannose. Because metformin is a positive modulator of LLO synthesis (9Shang J. Lehrman M.A. J. Biol. Chem. 2004; 279: 9703-9712Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar) we examined AICAR, as it has been widely reported that treatment of cells with AICAR, like metformin, activates AMPK (8Winder W.W. Hardie D.G. Am. J. Physiol. 1999; 277: E1-E10PubMed Google Scholar). This requires intracellular conversion of AICAR to the phosphorylated nucleotide 5-aminoimidazole-4-carboxamide ribotide (designated AICAR-P for simplicity) by adenosine kinase (15Sabina R.L. Kernstine K.H. Boyd R.L. Holmes E.W. Swain J.L. J. Biol. Chem. 1982; 257: 10178-10183Abstract Full Text PDF PubMed Google Scholar, 16Sabina R.L. Patterson D. Holmes E.W. J. Biol. Chem. 1985; 260: 6107-6114Abstract Full Text PDF PubMed Google Scholar). AICAR-P allosterically activates purified AMPK, although with a substantially lower affinity than AMP (17Corton J.M. Gillespie J.G. Hawley S.A. Hardie D.G. Eur. J. Biochem. 1995; 229: 558-565Crossref PubMed Scopus (1051) Google Scholar, 18Henin N. Vincent M.F. Van den Berghe G. Biochim. Biophys. Acta. 1996; 1290: 197-203Crossref PubMed Scopus (94) Google Scholar). Depending upon the cell type and the experimental system, AICAR-P in intact cells appears to act primarily either by allosteric activation of AMPK (19Longnus S.L. Wambolt R.B. Parsons H.L. Brownsey R.W. Allard M.F. Am. J. Physiol. 2003; 284: R936-R944Crossref PubMed Scopus (114) Google Scholar) or by making AMPK a better substrate for the upstream-activating kinase complex containing LKB1 (20Hawley S.A. Boudeau J. Reid J.L. Mustard K.J. Udd L. Mäkelä T.P. Alessi D.R. Hardie D.G. J. Biol. 2003; 2: 28.1-28.15Crossref Google Scholar). In any case, the mechanism of AICAR-P differs from that of metformin, which does not activate the isolated enzyme but can activate AMPK in intact cells (7Zhou G. Myers R. Li Y. Chen Y. Shen X. Fenyk-Melody J. Wu M. Ventre J. Doebber T. Fujii N. Musi N. Hirshman M. Goodyear L.J. Moller D.E. J. Clin. Investig. 2001; 108: 1167-1174Crossref PubMed Scopus (4576) Google Scholar, 21Hawley S.A. Gadalla A.E. Olsen Hardie D.G. Diabetes. 2002; PubMed Scopus Google Scholar). As in AICAR enhanced extension of LLO intermediates and and increased the of transferred to protein and However, if LLO extension was by the of 0.1 mm mannose and AICAR no the of AICAR was than that of metformin for was in cells and in which LLO synthesis by mannose (9Shang J. Lehrman M.A. J. Biol. Chem. 2004; 279: 9703-9712Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). However, the of metformin on mannose (9Shang J. Lehrman M.A. J. Biol. Chem. 2004; 279: 9703-9712Abstract Full Text Full Text PDF PubMed Scopus (22) Google was than that of AICAR of metformin (9Shang J. Lehrman M.A. J. Biol. Chem. 2004; 279: 9703-9712Abstract Full Text Full Text PDF PubMed Scopus (22) Google AICAR not of 2-deoxyglucose in fibroblasts of mannose to the of AICAR on LLO of or was with cells in the or presence of 2 mm AICAR of cells were in the or presence of 2 mm AICAR as were with 40 μCi/ml 40 μCi/ml with AICAR, or μCi/ml [3H]mannose. was as in of LLOs were analyzed from of and HPLC were the corresponding in To that AICAR was than metformin upon mannose for its on LLO synthesis, were done with 40 μCi/ml 40 μCi/ml with AICAR, or μCi/ml [3H]mannose. As in with 40 μCi/ml treatment of cells with AICAR has of an on mannose than μCi/ml [3H]mannose. However, the of Glc3Man9GlcNAc2-P-P-dolichol were for 40 μCi/ml for 40 μCi/ml with AICAR, and for μCi/ml that mannose was not a significant for AICAR treatment of AICAR-P Glycogen Phosphorylase in Fibroblast has been reported to activate the form of glycogen in B. 1996; PubMed Scopus Google Scholar) and (19Longnus S.L. Wambolt R.B. Parsons H.L. Brownsey R.W. Allard M.F. Am. J. Physiol. 2003; 284: R936-R944Crossref PubMed Scopus (114) Google Scholar) muscle In both AICAR-P activated GP in muscle but distinct were that phosphorylase kinase been reported to be a substrate of AMPK D. Hardie D.G. Biochim. Biophys. Acta. PubMed Scopus Google Scholar) a to a A. A. B. Kemp B.E. Witters L.A. Biol. Chem. 2000; PubMed Scopus Google in muscle GPb activation was to the of AICAR-P to AMPK with a phosphorylation of phosphorylase kinase and phosphorylation of GPb B. 1996; PubMed Scopus Google Scholar) than to the of AICAR-P to the AMP activation site on However, the experiments not to or an and were done under in which enzymes in the cascade were with to the In contrast, in activation by the effects of AICAR was to allosteric activation of GP by AICAR-P than activation of a kinase cascade. This was by the that treatment of muscle with AICAR to increased glycogen no in the phosphorylation state of AMPK, and no of activation of AMPK or GP. However, phosphorylation of a substrate of AMPK suggested that AICAR activate AMPK that a of muscle GPb could be activated by AICAR-P as well as AMP in the of not which is with allosteric activation of GPb by mm AMP and mm and in stimulated GP activity in and whereas AICAR and metformin not not In these experiments cytoplasmic were and reactions were done in the of the results are with allosteric activation and in with a enzyme cascade LLO Extension by of Glycogen determine AICAR enhanced LLO extension by activating GP in intact fibroblasts, two inhibitors of GP were which to the site of GP (1Johnson L.N. FASEB J. 1992; 6: 2274-2282Crossref PubMed Scopus (264) Google no on GP activity by not However, it the stimulation of GP in by AMP and AICAR-P and Using the for LLO synthesis in caffeine by not mannosylation of LLOs lower and However, as in and caffeine the stimulatory of AICAR on LLO extension are of independent not the of 0.1 mm mannose on LLO synthesis and that it not the Although both GP and AMPK are for metformin is only to activate the GP in was not activated by metformin not and the of metformin to LLO extension was not by caffeine and Pfizer compound CP-91149 is a of GP, both in and in (10Martin W.H. Hoover D.J. Armento S.J. Stock I.A. McPherson R.K. Danley D.E. Stevenson R.W. Barrett E.J. Treadway J.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1176-1181Crossref Scopus (221) Google Scholar). CP-91149 to a site on GP M. Danley D.E. J.L. McPherson R.K. Treadway J.L. Hoover D.J. Chem. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) distinct from the caffeine CP-91149 was used to AICAR by stimulating GP. Although CP-91149 was as an of GPa, it also of muscle in the of AMP muscle GPb activated by AMP not As in and CP-91149 both and GP in fibroblasts CP-91149 was then tested as an of the extension of LLOs in intact cells 20 CP-91149 by not LLO extension a and but it the stimulatory of AICAR and This that AICAR by stimulating GP. The inhibitory effects of CP-91149 were with inhibition by was noticed that the of the AICAR treatment for LLO synthesis was and the of CP-91149 was as an the of AICAR was lower lower When AICAR increased Glc3Man9GlcNAc2-P-P-dolichol above of the LLO CP-91149 no with the effects of metformin on LLO extension were not by CP-91149 AICAR from metformin in in of the that the primarily of AICAR and metformin in fibroblasts are GP and AMPK, respectively. The of AICAR but not metformin on LLO extension was by caffeine and The of metformin but not AICAR on LLO extension was upon its to mannose but not metformin, stimulated GPb in results were because AICAR has been reported to activate AMPK in a number of and the fibroblasts were to with other these cells may have a of GP to AMPK. Thus, AICAR may be activating both but GP activation may be detected and have a is that mannose in fibroblasts is by an AMPK cascade that is to metformin but not to is that cells functional LKB1 (20Hawley S.A. Boudeau J. Reid J.L. Mustard K.J. Udd L. Mäkelä T.P. Alessi D.R. Hardie D.G. J. Biol. 2003; 2: 28.1-28.15Crossref Google but mannose in cells is to metformin (9Shang J. Lehrman M.A. J. Biol. Chem. 2004; 279: 9703-9712Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). is that AICAR was converted to AICAR-P in fibroblasts, as it is in mammalian (8Winder W.W. Hardie D.G. Am. J. Physiol. 1999; 277: E1-E10PubMed Google because AICAR not activate GP in upon formation of AICAR-P from AICAR in Chinese hamster cells R.L. Patterson D. Holmes E.W. J. Biol. Chem. 1985; 260: 6107-6114Abstract Full Text PDF PubMed Google Scholar) and muscle (15Sabina R.L. Kernstine K.H. Boyd R.L. Holmes E.W. Swain J.L. J. Biol. Chem. 1982; 257: 10178-10183Abstract Full Text PDF PubMed Google Scholar) are with intracellular in the sufficient to activate GP. A AICAR concentration of mm in AICAR-P of mm in muscle and mm in muscle (15Sabina R.L. Kernstine K.H. Boyd R.L. Holmes E.W. Swain J.L. J. Biol. Chem. 1982; 257: 10178-10183Abstract Full Text PDF PubMed Google to 2 mm AICAR used in this intracellular AICAR-P of Chinese hamster ovary-K1 cells for h with 0.7 mm AICAR of of cellular protein R.L. Patterson D. Holmes E.W. J. Biol. Chem. 1985; 260: 6107-6114Abstract Full Text PDF PubMed Google Scholar). In our fibroblasts a volume of ml would 0.5 of that of fibroblasts with 2 mm AICAR for h done in mm intracellular In any case, these results show that the of an activator with inhibitors that act by distinct and can be used for the experimental evaluation of GP. This strategy be AICAR-P activates GP allosterically or AMPK to phosphorylase because the two inhibitors are for GPb and results also demonstrate that the Glc-1-P resulting from glycogenolysis can be a significant source of hexose units for glycoconjugate assembly and a for activation of glycogenolysis by the protein as the cause of enhanced LLO extension (11Doerrler W.T. Lehrman M.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13050-13055Crossref PubMed Scopus (46) Google Scholar, 12Shang J. Korner C. Freeze H. Lehrman M.A. Glycobiology. 2002; 12: 307-317Crossref PubMed Scopus (47) Google Scholar). Thus, the GP modulators used with a for glycoconjugate synthesis. GP and AMPK respond to in which can be by in cytoplasmic hexose Such are to LLO synthesis and in GP and AMPK may have in a of hexose for LLOs and for other such as that require for glycogen phosphorylase assays and for with cell are for the gift of CP-91149 from and Treadway for

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