Protein-tyrosine Phosphatase-1B Negatively Regulates Insulin Signaling in L6 Myocytes and Fao Hepatoma Cells

Insulin signaling is regulated by tyrosine phosphorylation of the signaling molecules, such as the insulin receptor and insulin receptor substrates (IRSs). Therefore, the balance between protein-tyrosine kinases and protein-tyrosine phosphatase activities is thought to be important in the modulation of insulin signaling in insulin-resistant states. We thus employed the adenovirus-mediated gene transfer technique, and we analyzed the effect of overexpression of a wild-type protein-tyrosine phosphatase-1B (PTP1B) on insulin signaling in both L6 myocytes and Fao cells. In both cells, PTP1B overexpression blocked insulin-stimulated tyrosine phosphorylation of the insulin receptor and IRS-1 by more than 70% and resulted in a significant inhibition of the association between IRS-1 and the p85 subunit of phosphatidylinositol 3-kinase and Akt phosphorylation as well as mitogen-activated protein kinase phosphorylation. Moreover, insulin-stimulated glycogen synthesis was also inhibited by PTP1B overexpression in both cells. These effects were specific for insulin signaling, because platelet-derived growth factor (PDGF)-stimulated PDGF receptor tyrosine phosphorylation and Akt phosphorylation were not inhibited by PTP1B overexpression. The present findings demonstrate that PTP1B negatively regulates insulin signaling in L6 and Fao cells, suggesting that PTP1B plays an important role in insulin resistance in muscle and liver. Insulin signaling is regulated by tyrosine phosphorylation of the signaling molecules, such as the insulin receptor and insulin receptor substrates (IRSs). Therefore, the balance between protein-tyrosine kinases and protein-tyrosine phosphatase activities is thought to be important in the modulation of insulin signaling in insulin-resistant states. We thus employed the adenovirus-mediated gene transfer technique, and we analyzed the effect of overexpression of a wild-type protein-tyrosine phosphatase-1B (PTP1B) on insulin signaling in both L6 myocytes and Fao cells. In both cells, PTP1B overexpression blocked insulin-stimulated tyrosine phosphorylation of the insulin receptor and IRS-1 by more than 70% and resulted in a significant inhibition of the association between IRS-1 and the p85 subunit of phosphatidylinositol 3-kinase and Akt phosphorylation as well as mitogen-activated protein kinase phosphorylation. Moreover, insulin-stimulated glycogen synthesis was also inhibited by PTP1B overexpression in both cells. These effects were specific for insulin signaling, because platelet-derived growth factor (PDGF)-stimulated PDGF receptor tyrosine phosphorylation and Akt phosphorylation were not inhibited by PTP1B overexpression. The present findings demonstrate that PTP1B negatively regulates insulin signaling in L6 and Fao cells, suggesting that PTP1B plays an important role in insulin resistance in muscle and liver. insulin receptor substrate mitogen-activated protein kinase protein-tyrosine phosphatase polyacrylamide gel electrophoresis phosphatidylinositol 3′-kinase platelet-derived growth factor fetal calf serum Dulbecco's modified Eagle's multiplicity of infection bovine serum albumin After insulin binds to its own receptor, the insulin receptor is phosphorylated on its tyrosine residues, and tyrosine kinase activity is activated. The activated insulin receptor binds to insulin receptor substrates (IRSs)1 via the YMXM motif, and IRSs are also phosphorylated on tyrosine residues (1White M.F. Kahn C.R. J. Biol. Chem. 1994; 269: 1-4Abstract Full Text PDF PubMed Google Scholar). The tyrosine-phosphorylated IRSs activate their downstream signaling molecules, such as phosphatidylinositol (PI) 3-kinase and p21ras. Since tyrosine phosphorylation is essential for insulin signaling, the balance of activities between protein-tyrosine kinases and protein-tyrosine phosphatases (PTPase) appears to be very important for the effects of insulin. Several lines of evidence demonstrate that PTPase activity is increased in insulin-resistant states such as obesity and type 2 diabetes mellitus. It has been reported that obese human subjects have increased PTPase activity in skeletal muscle and adipose tissue (2Ahmad F. Azevedo J.L. Cortright R. Dohm G.L. Goldstein B.J. J. Clin. Invest. 1997; 100: 449-458Crossref PubMed Scopus (260) Google Scholar, 3Ahmad F. Considine R.V. Goldstein B.J. J. Clin. Invest. 1995; 95: 2806-2812Crossref PubMed Scopus (115) Google Scholar), and a 10% body weight reduction causes a decrease in overall adipose tissue PTPase activity with enhanced insulin sensitivity (4Ahmad F. Considine R.V. Bauer T.L. Ohannesian J.P. Marco C.C. Goldstein B.J. Metabolism. 1997; 46: 1140-1145Abstract Full Text PDF PubMed Scopus (111) Google Scholar). Furthermore, insulin infusion in vivo produces a rapid 25% suppression of soluble PTPase activity in muscles of insulin-sensitive subjects, but this response is severely impaired in subjects who are insulin-resistant (5McGuire M.C. Fields R.M. Nyomba B.L. Raz I. Bogardus C. Tonks N.K. Sommercorn J. Diabetes. 1991; 40: 939-942Crossref PubMed Scopus (87) Google Scholar). Moreover, increased PTPase activity is also observed in the liver (6Meyerovitch J. Backer J.M. Kahn C.R. J. Clin. Invest. 1989; 84: 976-983Crossref PubMed Scopus (88) Google Scholar) and skeletal muscle (7Ahmad F. Goldstein B.J. Metabolism. 1995; 44: 1175-1184Abstract Full Text PDF PubMed Scopus (116) Google Scholar) of diabetic rats. Taken together, abnormalities of PTPase activity are thought to be important to understand the molecular mechanism of insulin resistance. Previous studies showed that the tandem domain transmembrane enzymes, leukocyte antigen-related and leukocyte common antigen-related phosphatase/RPTPα, and the intracellular, single domain enzymes, protein-tyrosine phosphatase-1B (PTP1B) and SHP2 are candidate PTPases for the regulation of the insulin signaling pathway. In particular, PTP1B directly interacts with the activated insulin receptor (8Seely B.L. Staub P.A. Reichart D.R. Berhanu P. Milarski K.L. Raltiel A.R. Kusari J. Olefsky J.M. Diabetes. 1996; 45: 1379-1385Crossref PubMed Google Scholar) and exhibits the highest specific activity toward IRS-1 (9Goldstein B.J. Bittner-Kowalczyk A. White M.F. Harbeck M. J. Biol. Chem. 2000; 275: 4283-4289Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar). We have also reported that exposing Rat 1 fibroblasts expressing human insulin receptors to a high glucose condition impairs insulin-stimulated tyrosine phosphorylation of the insulin receptor and IRS-1 due to the increased PTP1B expression and activity (10Maegawa H. Ide R. Hasegawa M. Ugi S. Egawa K. Iwanishi M. Kikkawa R. Shigeta Y. Kashiwagi A. J. Biol. Chem. 1995; 270: 7724-7730Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Furthermore, overexpression of PTP1B by the electroporation method reduces the level of GLUT4 on the cell surface in primary cultured rat adipose cells (11Chen H. Wertheimer S.J. Lin C.H. Katz S.L. Amrein K.E. Burn P. Quon M.J. J. Biol. Chem. 1997; 272: 8026-8031Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Moreover, it is reported that mice lacking theptp1b gene show increased insulin sensitivity and resistance to high fat diet-induced obesity, which is supported by enhanced insulin-induced tyrosine phosphorylation of the insulin receptor and IRS-1 in muscle and liver (12Elchebly M. Payette P. Michaliszyn E. Cromlish W. Collins S. Loy A.L. Normandin D. Cheng A. Himms-Hagen J. Chan C. Ramachandran C. Gresser M.J. Tremblay M.L. Kennedy B.P. Science. 1999; 283: 1544-1548Crossref PubMed Scopus (1905) Google Scholar). Thus, PTP1B appears to play an important role in the regulation of insulin signaling. In the present study, we employed the adenovirus-mediating gene transfer technique, and we analyzed the effect of PTP1B overexpression on insulin signaling in model cells of insulin target tissues, such as L6 myocytes and Fao cells. In both cells, PTP1B overexpression markedly inhibited insulin-stimulated tyrosine phosphorylation of the insulin receptor and IRS-1 and resulted in a significant inhibition of the association between IRS-1 and the p85 subunit of PI 3-kinase and Akt phosphorylation as well as MAP kinase phosphorylation. Moreover, glycogen synthesis was also inhibited under both basal and insulin-stimulated conditions. The present findings demonstrate that PTP1B negatively regulates insulin signaling in muscle and liver cells. Human insulin was provided by Lilly. Anti-PTP1B antibody, anti-IRS-1 antibody, and anti-p85 N-SH2 antibody were purchased from Upstate Biotechnology Inc. (Lake Placid, NY). Anti-phospho-Akt antibody and anti-phospho-MAP kinase antibody were from New England Biolabs (Beverly, MA). Horseradish peroxidase-conjugated phosphotyrosine antibody (RC20H) and insulin receptor antibody were from Transduction Laboratories (Lexington, KY). Horseradish peroxidase-linked anti-rabbit and anti-mouse antibodies, anti-ERK2 antibody, anti-Akt1 antibody, and anti-PDGF receptor antibody were from Santa Cruz Biotechnology (Santa Cruz, CA). Dulbecco's modified Eagle's (DME) medium and fetal calf serum (FCS) were obtained from Life Technologies, Inc. All radioisotopes were obtained from PerkinElmer Life Sciences. XAR-5 film was obtained from Eastman Kodak Co. All other reagents and chemicals were purchased from Sigma. L6 cells, which were provided by Dr. A. Klip (The Hospital for Sick Children, Toronto, Canada), were grown and maintained in minimum Eagle's medium-α containing 50 units/ml penicillin, 50 μg/ml streptomycin, and 10% FCS in a 5% CO2 environment. The cells were reseeded in the appropriate culture dishes and, after reaching subconfluency, the medium was changed to minimum Eagle's medium-α containing 2% FCS. The medium was then changed every 2 days until the cells were fully differentiated, typically after 5 days. Fao cells, which were from Dr. C. R. Kahn (Joslin Diabetes Center, Boston, MA), were grown and maintained in DME medium containing 50 units/ml penicillin, 50 μg/ml streptomycin, and 10% FCS in a 5% CO2 environment. Prior to experimentation, the cells were trypsinized and reseeded in the appropriate culture dishes. The Ad-E1A-transformed human embryonic kidney cell line 293 was cultured in DME high glucose medium containing 50 units/ml penicillin, 50 μg/ml streptomycin, and 10% FCS in a 5% CO2 environment. The recombinant adenovirus containing PTP1B cDNA was generated as described previously (13Wang J. Cheung A.T. Kolls J.K. Bryer-Ash M. Diabetes. 1999; 48 Suppl. 1: 328Google Scholar). 2J. Wang, A. T. Cheung, J. K. Kolls, W. W. Starks, A. Martinez, D. Dietzen, and M. Bryer-Ash, manuscript in preparation. PTP1B wild-type cDNA was subcloned into pACCMVpLpASR(+) plasmid (14Gluzman Y. Reichl H. Solnick D. Gluzman Y. Eucaryotic Viral Vectors. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1982: 187-192Google Scholar). This plasmid contains 1.3 map units of adenovirus 5 (Ad5) left end, cytomegalovirus early promoter, PUC19 polylinker site, and SV40 poly(A) signal sequences, followed by map units 9–18 of the Ad5 genome. The resulting recombinant plasmid was then cotransfected into 293 packaging cells with pJM17 plasmid (15McGrory W.J. Bautista D.S. Graham F.L. Virology. 1988; 163: 614-617Crossref PubMed Scopus (550) Google Scholar), which carriers Ad5 genomic DNA and propagated as described previously (16Kolls J. Peppel K. Silva M. Beutler B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 215-219Crossref PubMed Scopus (212) Google Scholar). Mature recombinant Ad5 encoding PTP1B wild type was thus generated after in vivo homologous recombination between these two plasmids. Since 293 cells were originally derived from adenovirus transformation, the missing E1 gene function of pJM17 was provided in trans. The resulting recombinant virus containing the PTP1B was denoted as Ad5-PTP1B and was replication-defective (at least in cells lacking the E1 region of the adenovirus) but fully infectious. L6 myocytes and Fao cells were infected at a multiplicity of infection (m.o.i.) of 10–50 plaque formation units/cell for 1 or 2 h with stocks of either a control recombinant adenovirus (Ad5-ctrl) containing the cytomegalovirus promoter, pUC 18 polylinker, a fragment of the SV40 genome, or the recombinant adenovirus containing PTP1B (Ad5-PTP1B). Transduced cells were incubated for 56 h at 37 °C in 5% CO2 and appropriate medium with 2% heat-inactivated serum, followed by incubation in the starvation media required for the assay. The efficiency of adenovirus-mediated gene transfer was ∼90% as measured by immunocytochemistry. Ad5-ctrl- or Ad5-PTP1B-infected cells were starved for 16 h in DME regular glucose medium with 0.05% FCS. The cells were stimulated with 100 ng/ml insulin for 5–10 min at 37 °C and lysed in a solubilizing buffer containing 20 mm Tris, 1 mm EDTA, 140 mm NaCl, 1% Nonidet P-40, 50 units/ml aprotinin, 1 mmNa3VO4, 1 mm phenylmethylsulfonyl fluoride, 50 mm NaF, pH 7.5, for 30 min at 4 °C. The cell lysates were centrifuged to remove insoluble materials. For Western blot analysis, whole cell lysates (20 μg of protein per lane) were denatured by boiling in Laemmli sample buffer containing 100 mm dithiothreitol and resolved by SDS-PAGE. Gels were transferred to nitrocellulose by electroblotting in Towbin buffer containing 20% methanol. For immunoblotting, membranes were blocked and probed with specified antibodies. Blots were then incubated with horseradish peroxidase-linked second antibody followed by chemiluminescence detection, according to the manufacturer's instructions (Amersham Pharmacia Biotech). Glycogen synthase activity was determined as described previously (17Egawa K. Sharma P.M. Nakashima N. Huang Y. Huver E. Boss G.R. Olefsky J.M. J. Biol. Chem. 1999; 274: 14306-14314Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Differentiated L6 myocytes were infected with Ad5-PTP1B or Ad5-ctrl at 50 m.o.i. for 1 h and grown in medium containing heat-inactivated serum (2%) for 72 h. The cells were serum- and glucose-starved in DME with no glucose, 0.1% BSA, 2 mm pyruvate medium for 3 h and then stimulated with or without 200 ng/ml insulin for 30 min in 5 mm glucose containing medium. Cells were washed with ice-cold phosphate-buffered saline three times, scraped in the buffer containing 50 mm Tris-HCl, 10 mm EDTA, 100 mm KF, pH 7.4, and sonicated. After centrifugation, the protein concentration was measured. 10 μg of protein was used to determine the ability to stimulate incorporation of [14C]UDP-glucose into glycogen in the presence or absence of glucose 6-phosphate. Glycogen synthesis was measured as described previously (18Maegawa H. Kobayashi M. Ishibashi O. Takata Y. Shigeta Y. Am. J. Physiol. 1986; 251: E616-E623PubMed Google Scholar) with some modification. Fao cells were infected with Ad5-PTP1B or Ad5-ctrl at 10 m.o.i. for 2 h and grown in medium containing 2% heat-inactivated serum for 56 h. The cells were serum-starved for 16 h, and the medium was then replaced with DME medium containing 1% BSA. The cells were incubated with [14C]glucose (0.4 μCi/well) and 100 ng/ml insulin for 2 h in a CO2 incubator, washed with phosphate-buffered saline 3 times, and lysed with 2 n NaOH at 55 °C. The synthesized [14C]glycogen was precipitated with cold glycogen in 66% ethanol and washed, and the radioactivity was measured. The values are expressed as mean ± S.E., unless otherwise stated. Scheffe's multiple comparison test was used to determine the significance of any differences among more than three groups. p < 0.05 was considered significant. Differentiated L6 myocytes and Fao cells were infected with recombinant adenovirus expressing wild-type PTP1B as described under “Experimental Procedures”. Following 72 h of incubation, the cells were lysed and analyzed by SDS-PAGE followed by Western blotting with anti-PTP1B antibody (Fig. 1). Small amounts of endogenous PTP1B were observed in both cell lines. PTP1B was expressed in a dose-dependent manner in each cell line. Total phosphatase activity also increased dose-dependently in PTP1B overexpressing cells as reported previously2 (13Wang J. Cheung A.T. Kolls J.K. Bryer-Ash M. Diabetes. 1999; 48 Suppl. 1: 328Google Scholar) (data not shown). The infected cells were stimulated with 100 ng/ml insulin for 5 min and lysed. The cell lysates (200–500 μg of protein) were immunoprecipitated with receptor antibody or anti-IRS-1 The were analyzed by Western blotting with phosphorylation of the insulin receptor and IRS-1 were more than 70% in the Ad5-PTP1B infected cells in both cell lines The membranes were and with anti-p85 The association between IRS-1 and the p85 subunit of PI 3-kinase also in PTP1B overexpressing cells (Fig. and and the reduction of IRS-1 tyrosine phosphorylation (Fig. and we the downstream signaling of PI PTP1B expressing L6 myocytes or Fao cells were stimulated with insulin for 10 and analyzed by Western blotting with Akt antibody (Fig. of PTP1B inhibited insulin-induced Akt phosphorylation in both cell lines. of which is downstream of PI was also by PTP1B overexpression (data not shown). Differentiated L6 myocytes were infected with PTP1B and control at 50 m.o.i. for 1 h. Following 72 h of incubation, the cells were serum- and glucose-starved for 3 h and stimulated with 200 ng/ml of insulin for 30 of PTP1B inhibited insulin-stimulated glycogen synthase activity ± PTP1B ± p < we [14C]glucose incorporation into glycogen in Fao cells. The Fao cells were incubated with insulin and [14C]glucose for 2 h, and glycogen synthesis was measured as described under “Experimental in overexpression of PTP1B inhibited glycogen synthesis at both basal ± PTP1B ± p < and insulin-stimulated ± PTP1B ± < The PTP1B expressing cells were stimulated with insulin for 10 and to SDS-PAGE followed by Western blotting with MAP kinase antibody PTP1B expression inhibited insulin-stimulated MAP kinase phosphorylation in both cell lines. we the effects of PTP1B on platelet-derived growth factor signaling. Ad5-ctrl- or Ad5-PTP1B-infected L6 myocytes were stimulated with 30 ng/ml PDGF for 5 and then analyzed by SDS-PAGE followed by Western blotting with antibody or Akt antibody (Fig. PTP1B expression not either PDGF receptor tyrosine phosphorylation or Akt phosphorylation. Thus, the PTP1B overexpression appears to be specific for insulin signaling. We previously reported that exposing Rat 1 fibroblasts expressing human insulin receptors to high glucose impaired the insulin-stimulated tyrosine phosphorylation of the insulin receptor and IRS-1 due to the increased expression and activity of PTP1B (10Maegawa H. Ide R. Hasegawa M. Ugi S. Egawa K. Iwanishi M. Kikkawa R. Shigeta Y. Kashiwagi A. J. Biol. Chem. 1995; 270: 7724-7730Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). evidence has that overexpression of PTP1B the level of GLUT4 on the cell surface in primary cultured rat adipose cells (11Chen H. Wertheimer S.J. Lin C.H. Katz S.L. Amrein K.E. Burn P. Quon M.J. J. Biol. Chem. 1997; 272: 8026-8031Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). it was reported (12Elchebly M. Payette P. Michaliszyn E. Cromlish W. Collins S. Loy A.L. Normandin D. Cheng A. Himms-Hagen J. Chan C. Ramachandran C. Gresser M.J. Tremblay M.L. Kennedy B.P. Science. 1999; 283: 1544-1548Crossref PubMed Scopus (1905) Google Scholar) that the PTP1B mice showed increased insulin sensitivity and resistance to high fat diet-induced obesity with enhanced insulin-induced tyrosine phosphorylation of the insulin receptor and IRS-1 in muscle and liver. in insulin-induced tyrosine phosphorylation of the insulin receptor and IRS-1 in adipose tissue was not also showed that increased insulin sensitivity in mice was as insulin-stimulated glucose was in skeletal adipose tissue was Boss O. J.K. J.L. J.M. N. M. Y. A. Kahn Biol. 2000; PubMed Scopus Google Scholar). Furthermore, it has been reported that overexpression of PTP1B insulin-induced tyrosine phosphorylation of the insulin receptor and IRS-1 by but not the glucose in Y. S. Kahn J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). Therefore, the role of PTP1B for insulin effects appears to be In the we PTP1B wild-type in L6 myocytes and Fao cells adenovirus-mediated gene transfer to the effect of PTP1B on insulin signaling. PTP1B overexpression markedly inhibited insulin-stimulated MAP kinase Akt and glycogen synthesis in both L6 myocytes and Fao cells with inhibition of tyrosine phosphorylation of the insulin receptor and These effects were specific for insulin signaling, because PTP1B overexpression not phosphorylation of PDGF receptor and Akt (Fig. to derived from the effects of PTP1B on insulin signaling appears cell In PTP1B overexpression inhibited tyrosine phosphorylation of the insulin receptor and IRS-1 by but Akt phosphorylation and glucose were Y. S. Kahn J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). The inhibition of tyrosine phosphorylation of the insulin receptor and IRS-1 were in L6 myocytes and Fao cells, this be for the effects of PTP1B among these cells. It has been reported that PTP1B to the and is activated after into the by of its Full Text PDF PubMed Scopus Google Scholar, A. M. J. PubMed Scopus Google Scholar). of PTP1B be in each cell line. for these findings be the expression of endogenous In PTP1B not play an important role because its expression level is and other phosphatases be Thus, overexpression of PTP1B show effects than in L6 and Fao cells. In L6 myocytes and Fao cells, both insulin-stimulated MAP kinase phosphorylation and PI 3-kinase were inhibited by PTP1B overexpression. insulin-stimulated MAP kinase phosphorylation was blocked by PTP1B overexpression in Y. S. Kahn J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). This be to the cell type of PTP1B PTP1B overexpression in not insulin resistance in the by The for this between in vivo and in is is the of the expression PTP1B overexpression was increased by in the liver of the in this study, it was increased by in cells. PTP1B overexpression showed effects among L6 Fao cells, and PTP1B insulin resistance in muscle and liver cells but effects in Y. S. Kahn J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar) in the culture to the of the PTP1B mice (12Elchebly M. Payette P. Michaliszyn E. Cromlish W. Collins S. Loy A.L. Normandin D. Cheng A. Himms-Hagen J. Chan C. Ramachandran C. Gresser M.J. Tremblay M.L. Kennedy B.P. Science. 1999; 283: 1544-1548Crossref PubMed Scopus (1905) Google Scholar, Boss O. J.K. J.L. J.M. N. M. Y. A. Kahn Biol. 2000; PubMed Scopus Google Scholar). It was reported that insulin signaling was impaired in muscle but was not in liver of IRS-1 mice by expression T. K. H. K. Y. R. Y. F. S. Y. N. Y. T. Biol. 1996; PubMed Scopus Google Scholar), but the to the gene in mice insulin resistance in the liver H. J.M. S. Y. D. S. S. White M.F. PubMed Scopus Google Scholar). Furthermore, it was reported that plays an important role in adipose tissue of IRS-1 mice Y. S. H. R. K. K. T. E. H. S. Y. Y. T. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). These findings demonstrate that specific insulin signaling in or phosphatase be important in adipose In the present findings that PTP1B negatively regulates insulin signaling in muscle and liver cells. the activity of PTP1B be important in the molecular mechanism of insulin resistance in the type 2 diabetic We Dr. A. Klip (The Hospital for Sick Children, Toronto, and Dr. C. R. Kahn (Joslin Diabetes Center, Boston, for L6 cells and Fao cells,

Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.