Rapid Activation of Glycogen Phosphorylase by the Endoplasmic Reticulum Unfolded Protein Response
2002; Elsevier BV; Volume: 277; Issue: 47 Linguagem: Inglês
10.1074/jbc.m205001200
ISSN1083-351X
AutoresArvind Gill, Ningguo Gao, Mark A. Lehrman,
Tópico(s)Transgenic Plants and Applications
ResumoEndoplasmic reticulum (ER) stress is associated with misfolding of ER proteins and triggers the unfolded protein response (UPR). The UPR, in turn, helps restore normal ER function. Since fastidious N-linked glycosylation is critical for folding of most ER proteins, this study examined whether metabolic interconversions of precursors used for glycan assembly were controlled by the UPR. Thus, eight enzymes and factors with key roles in hexose phosphate metabolism were assayed in cytoplasmic extracts from primary dermal fibroblasts treated with UPR inducers. Stimulation of only one activity by the UPR was detected, AMP-independent glycogen phosphorylase (GP). GP activation required only 20 min of ER stress, with concurrent decreases in cellular glycogen and elevations of its metabolites Glc-1-P and Glc-6-P. Addition of phosphatase inhibitors to enzyme extracts from unstressed cells mimicked the effect of ER stress on GP activity, suggesting that phosphorylation of GP or a regulatory factor was involved. These data show that the UPR can modulate hexose metabolism in a manner beneficial for protein glycosylation. Since activation of GP appears to occur by a rapid post-translational process, it may be part of a general strategy of ER damage control, preceding the well-known transcription-dependent processes of the UPR that are manifested hours after the occurrence of ER stress. Endoplasmic reticulum (ER) stress is associated with misfolding of ER proteins and triggers the unfolded protein response (UPR). The UPR, in turn, helps restore normal ER function. Since fastidious N-linked glycosylation is critical for folding of most ER proteins, this study examined whether metabolic interconversions of precursors used for glycan assembly were controlled by the UPR. Thus, eight enzymes and factors with key roles in hexose phosphate metabolism were assayed in cytoplasmic extracts from primary dermal fibroblasts treated with UPR inducers. Stimulation of only one activity by the UPR was detected, AMP-independent glycogen phosphorylase (GP). GP activation required only 20 min of ER stress, with concurrent decreases in cellular glycogen and elevations of its metabolites Glc-1-P and Glc-6-P. Addition of phosphatase inhibitors to enzyme extracts from unstressed cells mimicked the effect of ER stress on GP activity, suggesting that phosphorylation of GP or a regulatory factor was involved. These data show that the UPR can modulate hexose metabolism in a manner beneficial for protein glycosylation. Since activation of GP appears to occur by a rapid post-translational process, it may be part of a general strategy of ER damage control, preceding the well-known transcription-dependent processes of the UPR that are manifested hours after the occurrence of ER stress. Endoplasmic reticulum (ER) 1The abbreviations used are: ER, endoplasmic reticulum; AMAC, 2-aminoacridone; AZC, l-azetidine-2-carboxylic acid; CDG, congenital disorder of glycosylation; CSN, castanospermine; DTT, dithiothreitol; eIF, eukaryotic initiation factor; FACE, fluorophore-assisted carbohydrate electrophoresis; GP, glycogen phosphorylase; LLO, lipid-linked oligosaccharide; PERK, PKR-like ER kinase; SLO, streptolysin-O; TN, tunicamycin; UPR, unfolded protein response. stress initiates signals that emerge from the ER lumen, and activate cytoplasmic and/or nuclear responses, which in turn alter ER function. The general paradigm of ER stress signaling is termed the unfolded protein response (UPR) (1Patil C. Walter P. Curr. Opin. Cell Biol. 2001; 13: 349-356Google Scholar, 2Mori K. Cell. 2000; 101: 451-454Google Scholar, 3Ma J. Hendershot L.M. Cell. 2001; 107: 827-830Google Scholar), which can be triggered by agents that cause misfolded proteins to accumulate within the ER lumen, such as dithiothreitol (DTT), tunicamycin (TN), thapsigargin, castanospermine (CSN), and azetidine-2-carboxylic acid (AZC) (4Shang J. Koerner C. Freeze H.H. Lehrman M.A. Glycobiology. 2002; 12: 307-317Google Scholar). Typically, ER stress activates the stress sensors Ire1p and ATF6 (3Ma J. Hendershot L.M. Cell. 2001; 107: 827-830Google Scholar) to cause transcription of genes encoding chaperones, folding enzymes, and other proteins that enhance ER function. Appearance of the respective gene products occurs several hours after initiation of ER stress. For such ER proteins, there is no evidence that regulation by the UPR involves post-translational import or activation. Additionally, ER stress can temporarily inhibit cellular protein synthesis, lessening the load of misfolded protein entering the lumenal space. This occurs within 10–20 min of the application of ER stress by rapid post-translational phosphorylation of eIF2α by the PKR-like ER kinase or PERK (5Harding H.P. Zhang Y. Ron D. Nature. 1999; 397: 271-274Google Scholar). Translation arrest is reversible (for example, lasting 2 h in fibroblasts, Ref. 4Shang J. Koerner C. Freeze H.H. Lehrman M.A. Glycobiology. 2002; 12: 307-317Google Scholar), allowing the subsequent translation of mRNAs encoded by UPR-responsive genes. Previous studies from this laboratory with primary dermal fibroblasts identified another rapid effect of the UPR, involvingN-linked glycosylation of ER proteins (4Shang J. Koerner C. Freeze H.H. Lehrman M.A. Glycobiology. 2002; 12: 307-317Google Scholar, 6Doerrler W.T. Lehrman M.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13050-13055Google Scholar). ER quality control is highly dependent upon covalent attachment of the oligosaccharide Glc3Man9GlcNAc2 to specific asparaginyl residues of nascent ER proteins. This requires the synthesis of a lipid-linked oligosaccharide (LLO), Glc3Man9GlcNAc2-P-P-dolichol. TN is a specific inhibitor of the synthesis of this LLO and, as a result, its application with cells causes ER protein misfolding and ER stress. Conditions that cause accumulation of premature LLO intermediates also result in ER stress (7Lehrman M.A. J. Biol. Chem. 2001; 276: 8623-8626Google Scholar). However, it was found that the UPR can compensate by promoting extension of such premature LLOs to Glc3Man9GlcNAc2-P-P-dolichol (6Doerrler W.T. Lehrman M.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13050-13055Google Scholar). Of particular interest is DTT-induced stress, which stimulates LLO synthesis within 20 min, and is attenuated by adaptation of cells to ER stress (4Shang J. Koerner C. Freeze H.H. Lehrman M.A. Glycobiology. 2002; 12: 307-317Google Scholar). This suggests a form of stress relief in which one or more components of the pre-existing pathway for generation of Glc3Man9GlcNAc2-P-P-dolichol is activated post-translationally, rather than being synthesized de novo. No evidence was obtained for UPR activation of hexose transport or transferases involved in synthesis of Glc3Man9GlcNAc2-P-P-dolichol (6Doerrler W.T. Lehrman M.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13050-13055Google Scholar). Since multiple undermannosylated intermediates (Man2–5GlcNAc2-P-P-dolichol) were all extended by the UPR, it is feasible that the UPR acts by increasing synthesis of one or more precursors of the mannosyl residues. In this study we present evidence that AMP-independent glycogen phosphorylase (GP) can mediate enhanced LLO extension by the UPR. Activation of GP is rapid, and may be part of a general program of ER damage control that precedes transcriptional events regulated by the UPR. Human dermal fibroblasts, obtained from various sources (see below, Table II), were cultured and subjected to ER stress with DTT, TN, CSN, or AZC as described (6Doerrler W.T. Lehrman M.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13050-13055Google Scholar). Cells were cultured continuously in medium containing 5 mm glucose in all experiments presented. To verify the effects of UPR inducing treatments (except TN) on LLO synthesis, controls (not shown) were performed with cells labeled with 40 μCi/ml [2-3H]mannose, in which case a 20-min incubation with medium containing 0.5 mm glucose was used. [3H]LLOs were extracted with chloroform/methanol/water (10:10:3), and [3H]oligosaccharides were released from dolichol-P-P and analyzed by HPLC as described (6Doerrler W.T. Lehrman M.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13050-13055Google Scholar).Table IIEffects of DTT- and TN-induced ER stress on 5′-AMP-dependent and -independent glycogen phosphorylase activities in primary dermal fibroblastsFibroblast cultures:−AMP+AMPControlDTTTNControlDTTTNunits/min/mg prot. ± S.E.% control% controlunits/min/mg prot. ± S.E.% control% controlAdultCRL-19040.080 ± 0.005188 ± 27204 ± 230.299 ± 0.015110 ± 9100 ± 3(n = 8)(n = 6)(n = 4)bTN-treated CRL-1904 cultures had a set of controls (n = 6) different than those for DTT, with average activities of 0.082 ± 0.006 (−AMP) and 0.278 ± 0.006 (+AMP).(n = 8)(n = 6)(n = 4)bTN-treated CRL-1904 cultures had a set of controls (n = 6) different than those for DTT, with average activities of 0.082 ± 0.006 (−AMP) and 0.278 ± 0.006 (+AMP).CRL-18920.075 ± 0.003140 ± 9188 ± 200.200 ± 0.011113 ± 7137 ± 9(n = 7)(n = 5)(n = 5)(n = 7)(n = 5)(n = 5)CRL-19870.068 ± 0.0071391700.192 ± 0.006122135(n = 2)(n = 1)(n = 1)(n = 2)(n = 1)(n = 1)F-120.066 ± 0.008134 ± 6NDcNot determined.0.170 ± 0.01390 ± 4ND(n = 2)(n = 2)(n = 2)(n = 2)F21–30.068 ± 0.003152 ± 8ND0.178 ± 0.002109 ± 3ND(n = 2)(n = 2)(n = 2)(n = 2)PediatricaCRL-7514: 4-year-old Caucasian male, neuroblastoma. CRL-2114: 2-year-old Black male, no clinical abnormalities recorded. CRL-1513: 4-year-old male, ethnicity unrecorded, multiple congenital defects (LLO profiles were normal and responsive to ER stress (4), inconsistent with CDG-I). CRL-1474: 7-year-old male, ethnicity unrecorded, glioma.CRL-75140.027 ± 0.0002194 ± 18171 ± 160.085 ± 0.001102 ± 495 ± 1(n = 2)(n = 2)(n = 2)(n = 2)(n = 2)(n = 2)CRL-21140.026 ± 0.001205 ± 9180 ± 710.068 ± 006101 ± 686 ± 17(n = 2)(n = 2)(n = 2)(n = 2)(n = 2)(n = 2)CRL-15130.037 ± 0.002161 ± 51770.106 ± 0.00497 ± 1294(n = 2)(n = 2)(n = 1)(n = 2)(n = 2)(n = 1)CRL-14740.044 ± 0.00182 ± 31020.120 ± 0.00473 ± 667(n = 2)(n = 2)(n = 1)(n = 2)(n = 2)(n = 1)Glycogen phosphorylase was assayed without or with 5 mm5′-AMP in cytoplasmic extracts from cells treated in the absence (control) or presence of 2 mm DTT for 15 min, or 5 μg/ml TN for 60 min, and normalized to protein content, as described under “Experimental Procedures.” Cultures with CRL designations were from the American Type Culture Collection. F-12 was a gift of Dr. H. Freeze, Burnham Institute, and F21–3 was obtained from National Psoriasis Foundation Tissue Bank. Adult donors were clinically normal. Pediatric donors were normal or had clinical abnormalities not expected to affect the parameters measured here. For reasons that are unclear, the average GP activities among the 5 adult cultures (−AMP, 0.072; +AMP; 0.21) were higher than those for four pediatric cultures (−AMP, 0.034; +AMP, 0.095). GP activity in only one of 9 cultures, pediatric CRL-1474, did not respond to ER stress. Since this culture also had the highest GP activity among the four pediatric cultures, there is a possibility of constitutive activation of GP.a CRL-7514: 4-year-old Caucasian male, neuroblastoma. CRL-2114: 2-year-old Black male, no clinical abnormalities recorded. CRL-1513: 4-year-old male, ethnicity unrecorded, multiple congenital defects (LLO profiles were normal and responsive to ER stress (4Shang J. Koerner C. Freeze H.H. Lehrman M.A. Glycobiology. 2002; 12: 307-317Google Scholar), inconsistent with CDG-I). CRL-1474: 7-year-old male, ethnicity unrecorded, glioma.b TN-treated CRL-1904 cultures had a set of controls (n = 6) different than those for DTT, with average activities of 0.082 ± 0.006 (−AMP) and 0.278 ± 0.006 (+AMP).c Not determined. Open table in a new tab Table IER stress does not appreciably stimulate the activities of seven key enzymes and factors involved in hexose phosphate metabolismEnzyme or factor assayedTreatmentActivityPercent of controlmean ± S.E.Phosphomannose isomerase (PMI)Control (6)8.7 ± 0.6–DTT (5)9.1 ± 0.6104CSN (2)10.2 ± 0.3117TN (3)8.9 ± 0.2102Phosphomannomutase (PMM)Control (6)3.0 ± 0.3–DTT (5)2.9 ± 0.395CSN (2)3.7 ± 0.4121TN (3)2.7 ± 0.388Fructose-1,6-bisphosphatase (F-1,6-BPase)Control (5)1.9 ± 0.3–DTT (5)1.7 ± 0.390CSN (5)1.6 ± 0.382TN (5)1.3 ± 0.466Phosphoglucomutase (PGM)Control (2)13.5 ± 1.7–DTT (2)13.8 ± 1.5103Phosphofructokinase (PFK)Control (5)5.3 ± 0.5–DTT (5)5.7 ± 0.6107CSN (5)6.0 ± 0.4114TN (5)5.8 ± 0.2111Hexokinase (HK)Control (4)1.2 ± 0.2–DTT (4)1.2 ± 0.193TN (2)1.0 ± 0.177Fructose-2,6-bisphosphate-like PFK activator(s)Control (6)16.4 ± 1.4–DTT (6)14.6 ± 1.889CSN (6)20.6 ± 2.7126TN (6)16.0 ± 5.798All assays are described under “Experimental Procedures.” Activities are calculated per mg of protein in SLO extracts. PMI, PMM, PGM, HK, and F1,6BPase activities were all measured in enzyme-linked assays and are reported as μmol of NADPH formed per min. PFK is measured as μmol of Fru-1,6-bisP formed per min. Fru-2,6-BP-like activity per 106 cells is reported as the equivalent pmol of authentic Fru-2,6-BP giving the same enhancement of PFK activity. Parentheses indicate the number of times each stress treatment was tested. Open table in a new tab Glycogen phosphorylase was assayed without or with 5 mm5′-AMP in cytoplasmic extracts from cells treated in the absence (control) or presence of 2 mm DTT for 15 min, or 5 μg/ml TN for 60 min, and normalized to protein content, as described under “Experimental Procedures.” Cultures with CRL designations were from the American Type Culture Collection. F-12 was a gift of Dr. H. Freeze, Burnham Institute, and F21–3 was obtained from National Psoriasis Foundation Tissue Bank. Adult donors were clinically normal. Pediatric donors were normal or had clinical abnormalities not expected to affect the parameters measured here. For reasons that are unclear, the average GP activities among the 5 adult cultures (−AMP, 0.072; +AMP; 0.21) were higher than those for four pediatric cultures (−AMP, 0.034; +AMP, 0.095). GP activity in only one of 9 cultures, pediatric CRL-1474, did not respond to ER stress. Since this culture also had the highest GP activity among the four pediatric cultures, there is a possibility of constitutive activation of GP. All assays are described under “Experimental Procedures.” Activities are calculated per mg of protein in SLO extracts. PMI, PMM, PGM, HK, and F1,6BPase activities were all measured in enzyme-linked assays and are reported as μmol of NADPH formed per min. PFK is measured as μmol of Fru-1,6-bisP formed per min. Fru-2,6-BP-like activity per 106 cells is reported as the equivalent pmol of authentic Fru-2,6-BP giving the same enhancement of PFK activity. Parentheses indicate the number of times each stress treatment was tested. Cell monolayers were treated with streptolysin-O (SLO (Murex brand, distributed by Corgenix, UK)) to selectively permeabilize the plasma membranes as described (8Martys J.L. Shevell T. McGraw T.E. J. Biol. Chem. 1995; 270: 25976-25984Google Scholar, 9Anand 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: 487-501Google Scholar), allowing collection of diffusable cytoplasmic components with minimal physical perturbation or organelle breakage. Each 100-mm dish contained ∼1 × 106 cells. After treatment with SLO on ice for 4 min, unbound SLO was removed by washing the monolayers with ice-cold phosphate-buffered saline. 2 ml of modified transport buffer (containing 78 mm KCl, 4 mm MgCl2, and 50 mm Na-HEPES, pH 7.4, but lacking DTT as originally formulated, Ref. 8Martys J.L. Shevell T. McGraw T.E. J. Biol. Chem. 1995; 270: 25976-25984Google Scholar) prewarmed to 37 °C were added. After 15 min at 37 degrees, the dishes were placed on ice for an additional 5 min. The buffer was collected and used for measurements of hexose-metabolizing enzymes. Protein was measured with a dye binding assay (Bio-Rad) with bovine serum albumin as a standard. Comparisons of extracts made this way with extracts made with 1% Triton X-100 showed that Triton X-100 recovered 5–6 times as much protein, but a similar amount of total GP (not shown). The various stress treatments described in this study did not influence the amount of total protein recovered in cytoplasmic extracts. Assays were linear over the incubation periods indicated. Except for PFK, all enzymes in 0.2 ml of cytoplasmic extract were assayed in 1 ml of buffer containing 50 mm K-HEPES, pH 7.2, 25 mm KCl, and 5 mm MgCl2, reactions were performed in the presence of 1.5 units of glucose-6-P dehydrogenase (Sigma, catalog number G8529) and 0.2 mm NADP+ (Sigma, catalog number N0505), and product was assessed spectrophotometrically at 340 nm for the reduction of NADP+ to NADPH, as described (10Van Schaftingen E. Jaeken J. FEBS Lett. 1995; 377: 318-320Google Scholar). Except for PFK, the incubation times at 37 °C, and other reaction components, are indicated below for each enzyme. In all cases, assay values with blank reactions lacking the enzyme substrate were subtracted out. All data were normalized to protein content. Hexokinase: reactions were incubated for 15 min with 1 mm d-glucose and 1 mm Mg-ATP. Phosphoglucose isomerase (PGI): reactions were incubated for 15 min with 1 mm fructose 6-phosphate. Phosphomannose isomerase (PMI): reactions were incubated for 2 h with 1 mm mannose 6-phosphate and 7.13 units PGI (Sigma, catalog number P5381). Phosphomannomutase (PMM): reactions were incubated for 2 h with 0.8 mm mannose 1-phosphate, 0.1 mm glucose 1,6-bisphosphate, 0.35 units PMI (Sigma P5153), and 7.13 units of PGI. Fructose-1,6-bisphosphatase: reactions were incubated for 15 min with 10 μm fructose 1,6-bisphosphate and 7.13 units of PGI. Phosphofructokinase (PFK): 0.2 ml of extract was mixed with 1 ml of buffer containing 1 mm fructose 6-phosphate, 0 or 1 mm Mg-ATP, 50 mm Tris-Cl, pH 8.0, 0.1 mm Na3EDTA, 6 mm MgCl2,0.16 mm NADH, 0.4 units of aldolase (Roche Diagnostics cat. 102652), 2.4 units of triose-phosphate isomerase (Roche Diagnostics cat. 109762), and 0.4 units of α-glycerophosphate dehydrogenase (Roche Diagnostics cat. 127752), and incubated for 15 min at 37 °C. The ATP-dependent formation of fructose 1,6-bisphosphate was determined with an enzyme-linked method (11Furuya E. Uyeda K. J. Biol. Chem. 1981; 256: 7109-7112Google Scholar). Fructose 2,6-bisphosphate (or similar activators) was measured by activation of PFK as described (12Van Schaftingen E. Lederer B. Bartrons R. Hers H.-G. Eur J Biochem. 1982; 129: 191-195Google Scholar,13Uyeda K. Furuya E. Luby L.J. J. Biol. Chem. 1981; 256: 8394-8399Google Scholar). Rabbit muscle PFK (Sigma cat. F2129) and PFK in extracts from control and stressed cells were tested. Activators were obtained by alkaline extraction as follows. 100-mm dishes were placed on ice and washed with ice-cold phosphate-buffered saline twice. 1 ml of 0.1m NaOH (ice-cold) was added, and cells were scraped, transferred to a glass tube, and sonicated for 20 s. The mixture was heated at 80 degrees for 5 min. Samples were cooled on ice and centrifuged for 20 min at 14,900 rpm in a microcentrifuge. The supernatants were neutralized using 1 m HEPES (free acid) and added to PFK assays. In our hands, PFK was activated 2–3-fold by extracts from control cells. Extracts prepared by SLO permeabilization (see above) were assayed for GP activity (14Robson R.L. Morris J.G. Biochem. J. 1974; 144: 513-517Google Scholar). A 0.2-ml extract was diluted into 1 ml of assay buffer containing 20 mm sodium phosphate (pH 7.2), 2 mm MgSO4, 1–2 mmNADP+, 2 μg/ml glucose-6-phosphate dehydrogenase, 3 units/ml rabbit muscle PGM, and 3 μm glucose 1,6-bisphosphate. When indicated, 5 mm 5′-AMP was included. The reaction was initiated by adding 100 μl of 10 mg/ml glycogen (Sigma cat. G1508). GP activity was determined spectrophotometrically at 340 nm over a period of 5 h at 37 °C, and values for blank reactions lacking extract were routinely subtracted out. Over this period assays were linear for both time and amount of extract. The reaction was strictly dependent upon the presence of glycogen, PGM, and orthophosphate (not shown). Data presented for GP activity in the presence of AMP represent the total activity measured, without subtraction of activity obtained in the absence of AMP. Cellular alkaline extracts containing hexose phosphates (see above) were used. Since some hexose phosphates can be modified by alkali, key results were corroborated (not shown) with 70% ethanol extracts. Assays (1 ml) included 50 mm Tris-Cl (pH 7.5), 10 mm MgCl2, 0.25 mmNADP+, and 0.2 ml of extract. 2 μg/ml glucose-6-phosphate dehydrogenase (Sigma G7877) was added, and absorbance at 340 nm was measured after 15 min. Controls performed in the absence of glucose-6-phosphate dehydrogenase were subtracted out. Cell monolayers were extracted by scraping into 70% ethanol. The extract was clarified by centrifugation, dried under N2(g), dissolved in water, and applied to a 1-ml column of Dowex AG1-X2 (formate form). After washing with 20 ml of water, which removed essentially all neutral hexose, hexose phosphates were eluted with 15 ml of 4 m formic acid (15Spiro M.J. Diabetologia. 1984; 26: 70-75Google Scholar) and dried under N2(g). Recovery of a Glc-6-P standard was greater than 90% (not shown). However, hexose 1-phosphates were highly sensitive to hydrolysis, and under these conditions more than 95% of Glc-1-P standard was recovered as glucose. Thus, glucose appearing in the 4 m formic acid eluate represented Glc-1-P. Hexoses and hexose phosphates were modified with 2-aminoacridone (AMAC) and separated with a modified monosaccharide composition gel (16Gao N. Lehrman M.A. Glycobiol. 2002; 12: 353-360Google Scholar) composed of a 20% acrylamide separating gel with 125 mm Tris borate, pH 8.3 and a 6% acrylamide stacking gel with 63 mm Tris borate, pH 6.8. The running buffer was 0.1 m Tris borate (pH 8.3). All other aspects of the electrophoresis were similar to those described for commercial monosaccharide composition gels (Glyko). AMAC-modified sugars were quantified with a BioScan fluorescence scanner as described (16Gao N. Lehrman M.A. Glycobiol. 2002; 12: 353-360Google Scholar). Glycogen was extracted from cell monolayers in 100-mm dishes, pre-chilled on ice, and washed with ice-cold PBS, by scraping with 1 ml of ice-cold 70% perchloric acid followed by sonication at 0 degrees (17Keppler D. Decker K. Bergmeyer H.U. Methods Of Enzymatic Analysis. 6. VCH, Weinheim, Germany1974: 1127-1131Google Scholar). After removal of insoluble material by centrifugation, aliquots were used for determination of glucose with or without prior enzymatic degradation of glycogen. Glycogen degradation was achieved by combining 0.4 ml of extract with 0.2 ml of 1 m KHCO3 and 2 ml of 1 mg/ml amyloglucosidase (Sigma A3514) solution, followed by shaking at 40 °C for 2 h. The incubation was stopped by adding 1 ml of 70% perchloric acid, neutralized with 2–3 mg solid KHCO3to a pH of 6.9–7.3, and centrifuged at 4000 rpm for 15 min. Glucose was assayed by mixing up to 0.1 ml of the supernatant with 1 ml of buffer containing 0.3 m triethanolamine chloride, pH 7.5, 1 mm ATP, 0.1 mm NADP+, 4 mm MgSO4, and 5 μg/ml glucose-6-phosphate dehydrogenase. After incubation at 37 °C for 5 min, absorbance at 340 nm was determined. Hexokinase (Sigma H5625) was added to a final concentration of 1.4 units/ml. After an additional 5 min at 37 °C, absorbance at 340 nm was again determined, from which the first reading was subtracted to determine the glucose-specific change in absorbance. After subtraction of values obtained without amyloglucosidase treatment and comparison with standard glucose samples, the quantity of glycogen-derived glucose was determined. Compared with tissues such as liver, brain, and muscle, little is known about regulation of glycolysis, gluconeogenesis, or glycogenolysis in dermal fibroblasts. A number of regulatory points in hexose metabolism with the potential to modulate LLO mannosylation were therefore evaluated to determine whether they might be regulated by the UPR (Fig. 1). ER stress conditions shown previously to stimulate LLO extension were chosen. These conditions did not cause cytoplasmic stress, itself a potential regulator of hexose metabolism. 2J. Shang and M. A. Lehrman, unpublished observations. DTT was particularly useful because it acted within 20 min of addition to cells, and its effects on LLO extension were due to an ER stress response rather than a direct chemical reduction of a regulatory enzyme (4Shang J. Koerner C. Freeze H.H. Lehrman M.A. Glycobiology. 2002; 12: 307-317Google Scholar). However, for the current study two significant modifications were made. First, cells were maintained continuously in medium with 5 mm glucose. In prior studies, a 20-min period in 0.5 mm glucose medium was necessary to provide a pool of truncated LLOs. Second, TN could not be used in prior studies since it directly inhibits LLO synthesis. TN was used in the present study since any UPR effectors should be activated by it. Cytoplasmic extracts were prepared by gentle permeabilization of the plasma membrane with streptolysin-O (SLO) to minimize release of material from damaged intracellular organelles (8Martys J.L. Shevell T. McGraw T.E. J. Biol. Chem. 1995; 270: 25976-25984Google Scholar). Thus, our assays were limited to readily diffusable components. No changes were observed for seven key enzymes and co-factors (TableI). There were no apparent stress effects on enzymes (phosphomannose isomerase, phosphomannomutase) that might divert Fru-6-P, a key intermediate in glycolysis, to mannosyl phosphates. No changes were detected in the enzymes (phosphofructokinase, fructose-1,6-bisphosphatase) or cytoplasmic activators of phosphofructokinase (such as Fru-2,6-bisP) that regulate interconversion of Fru-6-P and Fru-1,6-bisP, and thus possibly shift hexose units from glycolysis to glycoprotein synthesis. Consistent with the prior lack of evidence for enhanced uptake glucose and mannose from culture medium (6Doerrler W.T. Lehrman M.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13050-13055Google Scholar), no measurable changes were detected for hexokinase. Phosphoglucomutatase, inhibition of which could prevent storage of imported hexose as glycogen, was also unaffected. Considering these and earlier (6Doerrler W.T. Lehrman M.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13050-13055Google Scholar) results, it is clear that ER stress does not cause widespread changes in activities involved in hexose metabolism. As shown in Fig.2, panel A, GP activity was detectable in fibroblasts in both its more active (AMP-independent) and less active (AMP-dependent) forms. Although detailed information about this enzyme in fibroblasts is lacking, by extension from tissues such as liver and muscle it is possible that phosphorylation converts the less active form, GPb, to the more active form, GPa (18Johnson L.N. FASEB J. 1992; 6: 2274-2282Google Scholar). Caffeine, which blocks AMP binding to GPb, inhibited the AMP- dependent activity but not the AMP-independent activity (Fig. 2, panel B). When cells were treated with DTT, TN (Fig. 2, panel C), or AZC (not shown) at concentrations known to cause ER stress (4Shang J. Koerner C. Freeze H.H. Lehrman M.A. Glycobiology. 2002; 12: 307-317Google Scholar), AMP-independent GP activity in extracts increased, but AMP-dependent activity was unaffected. Activation of GP occurred within 20 min of DTT addition. Direct addition of DTT to assays, or its inclusion during extract preparation, did not affect GP activity (not shown). Thus, stimulation by DTT was not due to a direct chemical reduction of GP or a modulator of GP, as expected since the cytoplasmic environment in which GP exists is highly reducing. In addition to the fibroblast culture used in the experiments displayed in the figures, ER stress with DTT or TN increased GPa but not GPb activities in seven other adult and pediatric dermal fibroblast cultures (Table II). The stimulation of GP by ER stress did not exceed 2-fold, but the following sections provide evidence that this stimulation was significant. Dermal fibroblasts contained, on average, 230 nmol of glycogen-derived glucose per 106 cells. In the presence of orthophosphate, GP converts glycogen into Glc-1-P. Small but consistent losses of glycogen were observed with DTT (23 ± 2%, n = 13) and TN (12 ± 2%, n = 3). For DTT, the decrease occurred after 15 min of addition. Since cellular glycogen loss may be counterbalanced by its synthesis, the apparently weaker effect of TN may be related to the fact that it acted more slowly (within 1 h). Thus, the resulting glycogenolysis would be more easily compensated by glycogen synthesis. These losses of glycogen, though small, were more than adequate to account for downstream effects on hexose phosphates and LLOs (Table III).Table IIIStoichiometry of hexose flux resulting from ER stressForm of hexoseType of ER stressnmolGlycogen-derived Glc-1-P produced2 mm DTT525 μg/ml TN18Glc-1-P detected2 mm DTT (FACE)2.4Glc-6-P detected2 mm DTT (FACE)4.52 mm DTT (assay)3.05 μg/ml TN (assay)3.8200 μg/ml CSN (assay)1.3Neutral hexose in Glc3Man9GlcNAc2-P-P-dolichol required0.02Estimates are based upon 106 cells. Quantities of glycogen-derived Glc-1-P were calculated from the amount of glycogen consumed, as indicated in the text. Cytoplasmic hexose phosphates due to ER stress were determined by FACE and enzyme assay as described under “Experimental Procedures,” with values from unstressed cells subtracted out. The total amount of glucose plus mannose in Glc3Man9GlcNAc2-P-P-dolichol detected by FACE in dermal fibroblasts grown in 5 mm glucose (12Van Schaftingen E. Lederer B. Bartrons R. Hers H.-G. Eur J Biochem. 19
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