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Chaperone-Mediated Regulation of Hepatic Protein Secretion by Caloric Restriction
Biochemical and Biophysical Research Communications 284, 335–339 (2001) doi:10.1006/bbrc.2001.4972, available online at http://www.idealibrary.com on
Chaperone-Mediated Regulation of Hepatic Protein Secretion by Caloric Restriction Joseph M. Dhahbi,* Shelley X. Cao,* John B. Tillman,† Patricia L. Mote,* Monica Madore,‡ Roy L. Walford,§ and Stephen R. Spindler* ,1 *Department of Biochemistry and ‡Department of Botany and Plant Sciences, University of California, Riverside, Riverside, California 92521; †Lockheed Martin Engineering & Sciences Company, Moffett Field, California 94035; and §Department of Pathology, School of Medicine, University of California, Los Angeles, Los Angeles, California 90024
Received May 8, 2001
Calorie restriction (CR) delays age-related physiological changes, reduces cancer incidence, and increases maximum life span in mammals. Here we show that CR decreased the expression of many hepatic molecular chaperones and concomitantly increased the rate and efficiency of serum protein secretion. Hepatocytes from calorie-restricted mice secreted twice as much albumin, 63% more ␣ 1-antitrypsin, and 250% more of the 31.5-kDa protein 2 h after their synthesis. A number of trivial explanations for these results, such as differential rates of protein synthesis and cell leakage during the assay, were eliminated. These novel results suggest that CR may promote the secretion of serum proteins, thereby promoting serum protein turnover. This may reduce the circulating level of damaging, glycoxidated serum proteins. © 2001 Academic Press
Key Words: mice; calorie restriction; chaperones; protein secretion.
Secretion efficiency is the fraction of a newly synthesized protein which is finally secreted by a cell. ER chaperone mRNA and protein abundance in the liver, kidney, and muscle are proportional to the amount of calories consumed (Ref. 10 and unpublished data). The fewer calories consumed, the lower the chaperone mRNA and protein levels. Of the eight ER chaperones studied, the mRNA abundance for all but one is negatively regulated in the liver of CR mice (11). We first found that these changes are initiated within weeks of a shift from consumption of a low calorie diet to a normal calorie diet (11). In more recent work, we found that chaperone mRNA abundance changes dynamically over the course of hours in response to calories consumed (unpublished data). This response is mirrored over the course of days in the level of chaperone protein. Here we show that the CR-related decrease in ER chaperone levels leads to increased efficiency and amount of hepatic serum protein secretion. MATERIALS AND METHODS
Molecular chaperones promote the degradation of proteins as well as their synthesis, maturation, and processing (1– 4). Misfolded, unassembled and a fraction of normal secreted proteins are retained in the ER and degraded via the ubiquitin–proteasome pathway, the major endoplasmic reticulum (ER) degradation pathway (5, 6). Molecular chaperones may facilitate the recognition of proteolytic substrates and serve as cofactors for the ubiquitinating enzymes. Thus, it is not surprising that the abundance of ER chaperones influences the secretion efficiency of many proteins (7–9). Abbreviations used: CR, dietary calorie restriction; CR mice, calorie-restricted mice; ER, endoplasmic reticulum; ERp57 and ERp72, endoplasmic reticulum protein 57 and 72; GRP75, GRP78, GRP94, and GRP170, glucose-regulated protein 75, 78, 94, and 170. 1 To whom correspondence and reprint requests should be addressed. Fax: 909-787-4434. E-mail: [email protected].
Mice and diets. Female, 28-month-old mice of the long-lived F 1 hybrid strain C3B10RF 1 have been described previously (11). Mice were weaned at 28 days, housed individually and subjected to either a control or a restricted diet. The composition of the defined diets and feeding regimen has been described in detail (10). Control and CR mice consumed ⬃95 and ⬃52 kcal per week, respectively. Mice were fasted for 24 h before use. RNA isolation and quantitation. Liver RNA was prepared and chaperone mRNA quantified as described (12). Specific mRNA levels were normalized to that of S-II mRNA. The murine albumin (13) and ␣ 1-antitrypsin (14) cDNA probes were radioactively labeled as described (15). Chaperone quantitation. Chaperone proteins were analyzed and quantified with western and protein dot blot as described (16). The antibodies used were: rabbit anti-GRP78 and anti-ERp57 (Affinity Bioreagents, Neshanic Station, NJ), anti-ERp72 (a gift from Michael Green, St. Louis University, School of Medicine, St. Louis, MO), anti-GRP170 (a gift from John Subjeck, Roswell Park Cancer Institute, Buffalo, NY) and anti-calreticulin (Stressgen Biotechnologies
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Corp., Victoria, BC, Canada). A goat anti-rabbit immunoglobulin conjugated with horseradish peroxidase (Accurate Chemical & Scientific Corp., San Diego, CA) was used as a secondary antibody. For GRP170, we used an alkaline phosphatase-linked secondary antibody. Serum and liver protein quantitation. Specific serum and liver proteins were quantified using Western blots (17). Ten microliters of a 1 to 100 dilution of serum or 10 g of total liver protein extract was subjected to 10% SDS–PAGE and transferred to nitrocellulose. ␣ 1Antitrypsin and albumin were detected with rabbit polyclonal antiserum, goat anti-rabbit immunoglobulins conjugated with horseradish peroxidase (Accurate Chemical & Scientific Corp.), then an ECL kit, used as described by the manufacturer (Amersham, Arlington Heights, IL). Quantitation was performed using phosphorimaging with ImageQuaNT (Molecular Dynamics, Sunnyvale, CA). Hepatocyte preparation. Hepatocytes were rapidly prepared using the in situ collagenase perfusion (18) and Percoll isodensity centrifugation methods (19, 20). Cells were resuspended in methionine-free Eagle minimum essential medium and counted. Only preparations of 90% or better viability as judged by trypan blue exclusion were utilized. Hepatocyte survival through the labeling and chase periods was evaluated by measuring lactate dehydrogenase release into the medium using Sigma kit No. 288 as described by the supplier (Sigma). Release was negligible. Protein secretion rates. Freshly isolated hepatocytes were cultured for 10 min at a density of 1.5 ⫻ 10 6 cells/ml in uncoated plastic dishes in methionine-free Eagle minimum essential medium at 32°C to deplete intracellular methionine. The medium was adjusted to 20 Ci/ml [ 35S]methionine (1000 Ci/mM) and the cells cultured for an additional 15 min. Unlabeled methionine was added to a final concentration of 1.1 mM and aliquots were collected at the times indicated in Fig. 2. Cells were separated from medium by centrifugation and stored at ⫺70°C. Proteins were quantified using SDS–PAGE and phosphorimaging. The fraction of a protein secreted was calculated by dividing the total amount of a specific radiolabeled protein secreted into the medium by the total amount of radiolabeled protein initially synthesized per million cells. Immunoidentification of secreted proteins. Hepatocytes were prepared and proteins radiolabeled for 135 min. Cells were removed by centrifugation and proteins immunoprecipitated with either rabbit polyclonal antiserum to mouse ␣ 1-antitrypsin or to mouse albumin (Accurate Chemical & Scientific Corp.) at 4°C for 4 h with gentle mixing and overnight incubation with protein A–Sepharose beads (Pharmacia). After washing and heating to 95°C for 5 min in 100 mM Tris–HCl (pH 7.2), 2.5% SDS, the beads were removed by centrifugation and proteins were resolved by 10% SDS–PAGE and quantitated using a phosphorimager. Rates of specific protein synthesis. After the 15-min labeling period, cells were harvested and lysed in ice-cold 50 mM Hepes (pH 7.2), 250 mM NaCl, 0.2% NP-40, 0.1% Triton X-100, 0.01% SDS, homogenized using five strokes with the B pestle of a Dounce homogenizer, and centrifuged for 5 min at 25,000g and 4°C. Proteins were immunoprecipitated from the extract with either anti-albumin or anti-␣ 1-antitrypsin antibodies as described. Radiolabeled proteins were resolved by 10% SDS–PAGE and quantified with ImageQuaNT. The rate of synthesis was expressed as the phosphorimager units (PI units) of each protein after 15 min of labeling per million cells. Specific activity of the tRNA-bound [ 35S]methionine pool. Hepatocytes were prepared and proteins radiolabeled as above using three animals from each dietary regimen. Hepatocytes were washed twice with phosphate-buffered saline using low speed centrifugation. RNA was isolated using TRI Reagent (12). After 3 ethanol precipitations, the specific activity of the tRNA-bound methionine pool was determined (21). Briefly, the RNA pellet was dissolved in 50 mM sodium carbonate (pH 10), and incubated at 37°C for 90 min to deacylate the aminoacyl-tRNAs. RNA was ethanol precipitated and the superna-
FIG. 1. Hepatic protein levels of ER chaperones in control and CR mice. (A) Hepatic ER chaperones in control (lanes 1–3) and CR (lanes 4 – 6) mice. Liver protein extract was analyzed by Western blotting, and the blots were probed with antibodies directed against the specific chaperones. (B) ER chaperones in control (open bars) and CR (filled bars) mice (n ⫽ 4). Liver proteins were quantified using protein dot blotting and specific antibodies. Data were quantified using a phosphorimager. Data represent means ⫾ SD. Significance is indicated: P ⬍ 0.01 (**) and P ⬍ 0.001 (***).
tant dried by vacuum centrifugation. Released amino acids were resuspended, converted to their phenylisothiocyanate derivatives, and separated by HPLC as described (21). Methionine was quantified using derivatized amino acid standards. The radioactivity in the methionine peak was quantified by liquid scintillation counting, and its specific activity calculated. Data analysis. Values are expressed as means ⫾ SD. Data were assessed with either Student’s t test (P ⬍ 0.05) or a one-way ANOVA followed by Fisher’s test (P ⬍ 0.01). Statistical analyses were performed with Minitab Statistical Software, Standard Version, 1992 (Minitab Inc., State College, PA).
RESULTS Previously we found that CR reduced the mRNA for 7 hepatic ER chaperones in mice (11). Here we investigated whether these changes in mRNA lead to similar changes in the levels of the chaperone proteins. The abundance of the 5 chaperones tested was reduced in CR mice, as judged by Western blotting (Fig. 1A). GRP78 was reduced by 65% (P ⬍ 0.001), endoplasmic reticulum protein 57 (ERp57) by 35% (P ⬍ 0.01), ERp72 and calreticulin by 60% (P ⬍ 0.01 and P ⬍ 0.001, respectively), and GRP170 by 40% (P ⬍ 0.001)
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FIG. 2. The rate of specific protein secretion by hepatocytes from control and CR mice. Freshly isolated hepatocytes from CR (open symbols) and control (filled symbols) mice were pulse-labeled with [ 35S]methionine. During a chase period in the presence of unlabeled methionine, samples of the medium and hepatocytes were collected and analyzed using SDS–PAGE. Shown are the results for proteins of 67,500, 49,000, and 31,500 apparent molecular weights (A, B, and C, respectively). The intensity of specific protein bands was determined using a phosphorimager. Data are means ⫾ SD of the percentage of each protein initially synthesized which had accumulated in the medium at the indicated times [n ⫽ 6; P ⬍ 0.001 (***)].
(Fig. 1B). The decrease in chaperone protein levels parallels the decrease in the mRNA (11). The slightly faster migration of GRP170 from CR mice may result from differential posttranslational modification. We investigated the hypothesis that the altered abundance of ER chaperones may affect the rates of processing, and therefore the rates of secretion of hepatic proteins. Using freshly isolated hepatocytes, and pulse-chase studies, the secretion rate of the three most abundant secreted proteins was determined (Fig. 2). Shown are the rates of accumulation in the culture medium of proteins of 67,500 and 49,000 apparent molecular weight, which were subsequently identified as albumin and ␣ 1-antitrypsin (see below), and of an unidentified protein of 31,500 apparent molecular weight, here termed P31.5 (Figs. 2A, 2B, and 2C, respectively). By 60 min postlabeling, the amount of albumin, ␣ 1antitrypsin and P31.5 secreted by hepatocytes from CR mice was significantly greater than the amount secreted by hepatocytes from control mice (Fig. 2, P ⬍ 0.001). By 2 h post-labeling, the amount of albumin, ␣ 1-antitrypsin, and P31.5 secreted was 97, 63, and 250% higher for hepatocytes from CR mice. These results indicate that the secretion rate of these proteins is greatly enhanced by the CR diet. ␣ 1-Antitrypsin secretion appeared to reach a maximum 90 min after commencement of the chase with unlabeled methionine (Fig. 2B). Thus, hepatocytes from control mice appeared to secrete only about 50% of the ␣ 1-antitrypsin synthesized (Fig. 2B). In contrast, CR mice appeared to secrete approximately 85% of the
␣ 1-antitrypsin initially synthesized. These results suggest that CR led to an increase in both the rate and the final amount of initially synthesized protein secreted by hepatocytes. To allow quantitative comparison of the amounts of individual proteins secreted by hepatocytes from CR and control mice, we determined the specific activity of the 35S-methionyl-tRNA pools in the cells immediately after the pulse-labeling period. We found no difference in these specific activities (21.645 ⫾ 0.813 and 20.733 ⫾ 0.493 dpm/pmol from CR and control mice; P ⫽ 0.17). Thus, the relative rates of synthesis and secretion are directly comparable between hepatocytes isolated from control and CR mice. We investigated the possibility that the apparently higher secretion rates by hepatocytes from CR mice was due to greater rates of lysis of these cells during the chase phase of the studies. However, we were unable to detect any differential cell lysis. First, by SDS– PAGE we could detect no similarities between the profile of intracellular, radiolabeled proteins and those released into the medium during the chase period (data not shown). Second, no significant release of lactate dehydrogenase activity in the medium occurred during the chase period in cultures from either CR or control mice (data not shown). Third, there were no differences in viability of hepatocytes from CR and control mice as judged by cell count or trypan blue exclusion at the beginning or the conclusion of the chase periods. The apparent differences in the secretion rates of the proteins also are not artifacts of differential rates of synthesis of the proteins. There are no differences in the levels of albumin and ␣ 1-antitrypsin mRNA in control and CR mice (Fig. 3A). Further, the synthetic rates of ␣ 1-antitrypsin and albumin are the same in hepatocytes from CR and control mice (Fig. 3B). The amount of the proteins synthesized during the labeling periods is directly comparable because the total average incorporation of [ 35S]methionine into the proteins, and the specific activity of the t-RNA-bound methionine pool are the same in hepatocytes from control and CR mice. The proteins of 67,500 and 49,000 molecular weight were identified as albumin and ␣ 1-antitrypsin by several criteria. The radiolabeled proteins secreted following a pulse-labeling with [ 35S]methionine are shown in Fig. 4 lanes 2 and 6. As can be seen, the three most abundant secretory products are proteins of 67,500, 49,000, and 31,500 apparent molecular weight. Shown in lanes 3 and 7 are the proteins specifically precipitated using antibodies directed against mouse albumin and ␣ 1-antitrypsin, respectively. The immunoprecipitated albumin and ␣ 1-antitrypsin co-migrated with the proteins of 67,500 and 49,000 apparent molecular weight. Further, the 67,500 and 49,000 molecular weight proteins were completely removed from the medium by serial precipitations with the respective antibody (data not shown). P31.5 remains unidentified. It
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FIG. 3. The expression of albumin and ␣ 1-antitrypsin. (A) The abundance of hepatic albumin and ␣ 1-antitrypsin mRNA was determined by dot blotting of total RNA from control (open bars) and CR (filled bars) mice. The means ⫾ SD are shown (n ⫽ 6). (B) The rates of synthesis of albumin and ␣ 1-antitrypsin in hepatocytes of control (open bars) and CR (closed bars) mice (n ⫽ 6) were determined after a 15-min pulse labeling. Labeled proteins were immunoprecipitated, separated by SDS–PAGE, and quantified using a phosphorimager. The hepatic (C) and serum (D) levels of albumin and ␣ 1-antitrypsin from control (lanes 1–3) and CR mice (lanes 4 – 6) were determined by Western blotting.
is the prominent band just above the 30,000 molecular weight marker in Fig. 4, lanes 2 and 6. To determine whether enhanced rates of secretion of these proteins affected their steady-state levels, the hepatic and serum levels of ␣ 1-antitrypsin and albumin were determined by Western blotting (Figs. 3C and 3D). The hepatic levels of both proteins were reduced 30% in the CR mice, consistent with their increased rates of secretion and unchanged rate of synthesis in CR mice (P ⫽ 0.0013 and P ⫽ 0.0001 for ␣ 1antitrypsin and albumin, respectively; Figs. 2A and 2B and Fig. 3B). The serum levels of the proteins were also determined by Western blotting (Fig. 3D). No differences between control and CR mice were detected. These results indicate that the increased hepatic secretion of albumin and ␣ 1-antitrypsin in CR mice must be counterbalanced by increased plasma clearance.
amount of ␣ 1-antitrypsin increased from 53% of that initially synthesized in the control to 84% in the CR mice. The studies eliminated potential artifacts involving the specific activity of the intracellular methionine pool, the amount of each protein synthesized, and potential differential cell lysis. There is good evidence in the literature that ER chaperone levels can control the rate and extent of specific protein secretion (8, 9, 22). For example, a direct relationship has been found between the amount of protein trafficking through the ER and the level of GRP78 (23). GRP78 levels influence the secretion efficiency of many proteins that transiently associate with it in the ER (24). In general, the extent of association between a protein and GRP78 in the ER is inversely related to its secretion efficiency (24). For example, the secretion efficiencies of factor VIII, von Willebrand Factor variant, and tissue plasminogen activator are inversely correlated with the extent of their association with GRP78 (25). In this study, a significant proportion of Factor VIII was detected in a stable complex with GRP78 and not secreted. Overexpression of GRP78 by two- to threefold reduces secretion of von Willebrand factor and factor VIII (9). Overexpressing GRP78 several-fold delays the export of newly synthesized thyroglobulin (26). Conversely, reduction of GRP78 levels by 50 to 75% with antisense transcripts enhances secretion of a tissue plasminogen activator variant (8). Finally, induction of ER chaperones with calcium ionophores strongly reduces secretion of serum albumin, ␣ 1-antitrypsin, and ␣ 1-antichymotrypsin, although the loss of calcium from the ER may also have been involved in the loss of secretion efficiency (22).
DISCUSSION We report the novel result that the abundance of every ER chaperone tested was significantly reduced by CR and that these changes parallel previously reported reductions in the mRNA of these chaperones (11). The second novel result is that hepatocytes from calorie restricted mice secreted twice as much albumin, 63% more ␣ 1-antitrypsin, and 250% more P31.5 two hours after their synthesis. The maximum secreted
FIG. 4. Immunological identification of the 67,500 and 49,000 molecular weight proteins as albumin and ␣ 1-antitrypsin. Proteins in the medium from a pulse-chase study were precipitated with acetone (lanes 2 and 6), or immunoprecipitated with rabbit anti-mouse albumin antiserum (lane 3), or with rabbit anti-mouse ␣ 1-antitrypsin antiserum (lane 7), and analyzed by SDS–PAGE and phosphorimaging. An immunoprecipitation reaction identical to those shown in lanes 5 and 7, but lacking antiserum is shown in lane 5. The numbers to the left indicate the positions of radiolabeled molecular weight marker proteins in kiloDaltons (lanes 1 and 4).
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The ER proteins may be degraded at a higher rate in hepatocytes from control mice. ER chaperones, like their cytoplasmic counterparts, can facilitate protein degradation through the ubiquitin-proteasome pathway (1– 4). For example, in cell lines which synthesize IgG light chains in the absence of heavy chains, light chains are degraded in the ER with a half-life of 20 min (7, 27). GRP78 associates with the IgG light chains prior to their degradation, and there is a direct correspondence between the half-life of the protein’s association with GRP78, and the half-life of the protein. CR likely also promotes serum protein clearance, since the plasma levels of albumin and ␣ 1-antitrypsin did not change. Increased clearance should reduce the “dwell time” of these proteins in the circulation. Many lines of evidence support a role for glucose-modified and oxidized (glycoxidated) albumin and other serum proteins in the pathologies of diabetes and aging (28 – 30). CR produces relatively small reductions in serum glucose levels, but it effectively reduces the age-related accumulation of glycoxidation products (31–33). Thus, reduced serum protein dwell time may be a significant contributor to the reduced glycoxidation products and tissue damage found in CR animals. ACKNOWLEDGMENTS This work was supported by various donors to the research of S.R.S. J.M.D. and S.X.C. were recipients of fellowships from the Genetics Program of the University of California, Riverside.
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10. Spindler, S. R., Crew, M. D., Mote, P. L., Grizzle, J. M., and Walford, R. L. (1990) J. Nutr. 120, 1412–1417. 11. Dhahbi, J. M., Mote, P. L., Tillman, J. B., Walford, R. L., and Spindler, S. R. (1997) J. Nutr. 127, 1758 –1764. 12. Tillman, J. B., Dhahbi, J. M., Mote, P. L., Walford, R. L., and Spindler, S. R. (1996) J. Biol. Chem. 271, 3500 –3506. 13. Minghetti, P. P., Law, S. W., and Dugaiczyk, A. (1985) Mol. Biol. Evol. 2, 347–358. 14. Sifers, R. N., Ledley, F. D., Reed-Fourquet, L., Ledbetter, D. H., Ledbetter, S. A., and Woo, S. L. (1990) Genomics 6, 100 –104. 15. Tillman, J. B., Mote, P. L., Dhahbi, J. M., Walford, R. L., and Spindler, S. R. (1996) J. Nutr. 126, 416 – 423. 16. Fujita, T., Shirasawa, T., Uchida, K., and Maruyama, N. (1996) Mech. Ageing Dev. 87, 219 –229. 17. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76, 4350 – 4354. 18. Klaunig, J. E., Goldblatt, P. J., Hinton, D. E., Lipsky, M. M., Chacko, J., and Trump, B. F. (1981) In Vitro 17, 913–925. 19. Nemoto, N., and Sakurai, J. (1993) Jpn. J. Cancer Res. 84, 272–278. 20. Nemoto, N., Sakurai, J., Tazawa, A., and Ishikawa, T. (1989) Cancer Res. 49, 5863–5869. 21. Mitchell, D. E., Gadus, M. V., and Madore, M. A. (1992) Plant Physiol. (Rockville) 99, 959 –965. 22. Lodish, H. F., and Kong, N. (1990) J. Biol. Chem. 265, 10893– 10899. 23. Satoh, M., Nakai, A., Sokawa, Y., Hirayoshi, K., and Nagata, K. (1993) Exp. Cell Res. 205, 76 – 83. 24. Little, E., Ramakrishnan, M., Roy, B., Gazit, G., and Lee, A. S. (1994) Crit. Rev. Eukaryotic Gene Expr. 4, 1–18. 25. Dorner, A. J., Bole, D. G., and Kaufman, R. J. (1987) J. Cell Biol. 105, 2665–2674. 26. Muresan, Z., and Arvan, P. (1998) Mol. Endocrinol. 12, 458 – 467. 27. Knittler, M. R., Dirks, S., and Haas, I. G. (1995) Proc. Natl. Acad. Sci. USA 92, 1764 –1768. 28. Masoro, E. J., Katz, M. S., and McMahan, C. A. (1989) J. Gerontol. 44, B20 –B22. 29. Vlassara, H., and Bucala, R. (1996) Diabetes 45(Suppl. 3), S65– S66. 30. Beisswenger, P. J., Makita, Z., Curphey, T. J., Moore, L. L., Jean, S., Brinck-Johnsen, T., Bucala, R., and Vlassara, H. (1995) Diabetes 44, 824 – 829. 31. Cefalu, W. T., Bell-Farrow, A. D., Wang, Z. Q., Sonntag, W. E., Fu, M. X., Baynes, J. W., and Thorpe, S. R. (1995) J. Gerontol. A Biol. Sci. Med. Sci. 50, B337–B341. 32. Dyer, D. G., Dunn, J. A., Thorpe, S. R., Lyons, T. J., McCance, D. R., and Baynes, J. W. (1992) Ann. N.Y. Acad. Sci. 663, 421– 422. 33. Sell, D. R., Lane, M. A., Johnson, W. A., Masoro, E. J., Mock, O. B., Reiser, K. M., Fogarty, J. F., Cutler, R. G., Ingram, D. K., Roth, G. S., and Monnier, V. M. (1996) Proc. Natl. Acad. Sci. USA 93, 485– 490.
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Chaperone-Mediated Regulation of Hepatic Protein Secretion by Caloric Restriction Joseph M. Dhahbi,* Shelley X. Cao,* John B. Tillman,† Patricia L. Mote,* Monica Madore,‡ Roy L. Walford,§ and Stephen R. Spindler* ,1 *Department of Biochemistry and ‡Department of Botany and Plant Sciences, University of California, Riverside, Riverside, California 92521; †Lockheed Martin Engineering & Sciences Company, Moffett Field, California 94035; and §Department of Pathology, School of Medicine, University of California, Los Angeles, Los Angeles, California 90024
Received May 8, 2001
Calorie restriction (CR) delays age-related physiological changes, reduces cancer incidence, and increases maximum life span in mammals. Here we show that CR decreased the expression of many hepatic molecular chaperones and concomitantly increased the rate and efficiency of serum protein secretion. Hepatocytes from calorie-restricted mice secreted twice as much albumin, 63% more ␣ 1-antitrypsin, and 250% more of the 31.5-kDa protein 2 h after their synthesis. A number of trivial explanations for these results, such as differential rates of protein synthesis and cell leakage during the assay, were eliminated. These novel results suggest that CR may promote the secretion of serum proteins, thereby promoting serum protein turnover. This may reduce the circulating level of damaging, glycoxidated serum proteins. © 2001 Academic Press
Key Words: mice; calorie restriction; chaperones; protein secretion.
Secretion efficiency is the fraction of a newly synthesized protein which is finally secreted by a cell. ER chaperone mRNA and protein abundance in the liver, kidney, and muscle are proportional to the amount of calories consumed (Ref. 10 and unpublished data). The fewer calories consumed, the lower the chaperone mRNA and protein levels. Of the eight ER chaperones studied, the mRNA abundance for all but one is negatively regulated in the liver of CR mice (11). We first found that these changes are initiated within weeks of a shift from consumption of a low calorie diet to a normal calorie diet (11). In more recent work, we found that chaperone mRNA abundance changes dynamically over the course of hours in response to calories consumed (unpublished data). This response is mirrored over the course of days in the level of chaperone protein. Here we show that the CR-related decrease in ER chaperone levels leads to increased efficiency and amount of hepatic serum protein secretion. MATERIALS AND METHODS
Molecular chaperones promote the degradation of proteins as well as their synthesis, maturation, and processing (1– 4). Misfolded, unassembled and a fraction of normal secreted proteins are retained in the ER and degraded via the ubiquitin–proteasome pathway, the major endoplasmic reticulum (ER) degradation pathway (5, 6). Molecular chaperones may facilitate the recognition of proteolytic substrates and serve as cofactors for the ubiquitinating enzymes. Thus, it is not surprising that the abundance of ER chaperones influences the secretion efficiency of many proteins (7–9). Abbreviations used: CR, dietary calorie restriction; CR mice, calorie-restricted mice; ER, endoplasmic reticulum; ERp57 and ERp72, endoplasmic reticulum protein 57 and 72; GRP75, GRP78, GRP94, and GRP170, glucose-regulated protein 75, 78, 94, and 170. 1 To whom correspondence and reprint requests should be addressed. Fax: 909-787-4434. E-mail: [email protected].
Mice and diets. Female, 28-month-old mice of the long-lived F 1 hybrid strain C3B10RF 1 have been described previously (11). Mice were weaned at 28 days, housed individually and subjected to either a control or a restricted diet. The composition of the defined diets and feeding regimen has been described in detail (10). Control and CR mice consumed ⬃95 and ⬃52 kcal per week, respectively. Mice were fasted for 24 h before use. RNA isolation and quantitation. Liver RNA was prepared and chaperone mRNA quantified as described (12). Specific mRNA levels were normalized to that of S-II mRNA. The murine albumin (13) and ␣ 1-antitrypsin (14) cDNA probes were radioactively labeled as described (15). Chaperone quantitation. Chaperone proteins were analyzed and quantified with western and protein dot blot as described (16). The antibodies used were: rabbit anti-GRP78 and anti-ERp57 (Affinity Bioreagents, Neshanic Station, NJ), anti-ERp72 (a gift from Michael Green, St. Louis University, School of Medicine, St. Louis, MO), anti-GRP170 (a gift from John Subjeck, Roswell Park Cancer Institute, Buffalo, NY) and anti-calreticulin (Stressgen Biotechnologies
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Corp., Victoria, BC, Canada). A goat anti-rabbit immunoglobulin conjugated with horseradish peroxidase (Accurate Chemical & Scientific Corp., San Diego, CA) was used as a secondary antibody. For GRP170, we used an alkaline phosphatase-linked secondary antibody. Serum and liver protein quantitation. Specific serum and liver proteins were quantified using Western blots (17). Ten microliters of a 1 to 100 dilution of serum or 10 g of total liver protein extract was subjected to 10% SDS–PAGE and transferred to nitrocellulose. ␣ 1Antitrypsin and albumin were detected with rabbit polyclonal antiserum, goat anti-rabbit immunoglobulins conjugated with horseradish peroxidase (Accurate Chemical & Scientific Corp.), then an ECL kit, used as described by the manufacturer (Amersham, Arlington Heights, IL). Quantitation was performed using phosphorimaging with ImageQuaNT (Molecular Dynamics, Sunnyvale, CA). Hepatocyte preparation. Hepatocytes were rapidly prepared using the in situ collagenase perfusion (18) and Percoll isodensity centrifugation methods (19, 20). Cells were resuspended in methionine-free Eagle minimum essential medium and counted. Only preparations of 90% or better viability as judged by trypan blue exclusion were utilized. Hepatocyte survival through the labeling and chase periods was evaluated by measuring lactate dehydrogenase release into the medium using Sigma kit No. 288 as described by the supplier (Sigma). Release was negligible. Protein secretion rates. Freshly isolated hepatocytes were cultured for 10 min at a density of 1.5 ⫻ 10 6 cells/ml in uncoated plastic dishes in methionine-free Eagle minimum essential medium at 32°C to deplete intracellular methionine. The medium was adjusted to 20 Ci/ml [ 35S]methionine (1000 Ci/mM) and the cells cultured for an additional 15 min. Unlabeled methionine was added to a final concentration of 1.1 mM and aliquots were collected at the times indicated in Fig. 2. Cells were separated from medium by centrifugation and stored at ⫺70°C. Proteins were quantified using SDS–PAGE and phosphorimaging. The fraction of a protein secreted was calculated by dividing the total amount of a specific radiolabeled protein secreted into the medium by the total amount of radiolabeled protein initially synthesized per million cells. Immunoidentification of secreted proteins. Hepatocytes were prepared and proteins radiolabeled for 135 min. Cells were removed by centrifugation and proteins immunoprecipitated with either rabbit polyclonal antiserum to mouse ␣ 1-antitrypsin or to mouse albumin (Accurate Chemical & Scientific Corp.) at 4°C for 4 h with gentle mixing and overnight incubation with protein A–Sepharose beads (Pharmacia). After washing and heating to 95°C for 5 min in 100 mM Tris–HCl (pH 7.2), 2.5% SDS, the beads were removed by centrifugation and proteins were resolved by 10% SDS–PAGE and quantitated using a phosphorimager. Rates of specific protein synthesis. After the 15-min labeling period, cells were harvested and lysed in ice-cold 50 mM Hepes (pH 7.2), 250 mM NaCl, 0.2% NP-40, 0.1% Triton X-100, 0.01% SDS, homogenized using five strokes with the B pestle of a Dounce homogenizer, and centrifuged for 5 min at 25,000g and 4°C. Proteins were immunoprecipitated from the extract with either anti-albumin or anti-␣ 1-antitrypsin antibodies as described. Radiolabeled proteins were resolved by 10% SDS–PAGE and quantified with ImageQuaNT. The rate of synthesis was expressed as the phosphorimager units (PI units) of each protein after 15 min of labeling per million cells. Specific activity of the tRNA-bound [ 35S]methionine pool. Hepatocytes were prepared and proteins radiolabeled as above using three animals from each dietary regimen. Hepatocytes were washed twice with phosphate-buffered saline using low speed centrifugation. RNA was isolated using TRI Reagent (12). After 3 ethanol precipitations, the specific activity of the tRNA-bound methionine pool was determined (21). Briefly, the RNA pellet was dissolved in 50 mM sodium carbonate (pH 10), and incubated at 37°C for 90 min to deacylate the aminoacyl-tRNAs. RNA was ethanol precipitated and the superna-
FIG. 1. Hepatic protein levels of ER chaperones in control and CR mice. (A) Hepatic ER chaperones in control (lanes 1–3) and CR (lanes 4 – 6) mice. Liver protein extract was analyzed by Western blotting, and the blots were probed with antibodies directed against the specific chaperones. (B) ER chaperones in control (open bars) and CR (filled bars) mice (n ⫽ 4). Liver proteins were quantified using protein dot blotting and specific antibodies. Data were quantified using a phosphorimager. Data represent means ⫾ SD. Significance is indicated: P ⬍ 0.01 (**) and P ⬍ 0.001 (***).
tant dried by vacuum centrifugation. Released amino acids were resuspended, converted to their phenylisothiocyanate derivatives, and separated by HPLC as described (21). Methionine was quantified using derivatized amino acid standards. The radioactivity in the methionine peak was quantified by liquid scintillation counting, and its specific activity calculated. Data analysis. Values are expressed as means ⫾ SD. Data were assessed with either Student’s t test (P ⬍ 0.05) or a one-way ANOVA followed by Fisher’s test (P ⬍ 0.01). Statistical analyses were performed with Minitab Statistical Software, Standard Version, 1992 (Minitab Inc., State College, PA).
RESULTS Previously we found that CR reduced the mRNA for 7 hepatic ER chaperones in mice (11). Here we investigated whether these changes in mRNA lead to similar changes in the levels of the chaperone proteins. The abundance of the 5 chaperones tested was reduced in CR mice, as judged by Western blotting (Fig. 1A). GRP78 was reduced by 65% (P ⬍ 0.001), endoplasmic reticulum protein 57 (ERp57) by 35% (P ⬍ 0.01), ERp72 and calreticulin by 60% (P ⬍ 0.01 and P ⬍ 0.001, respectively), and GRP170 by 40% (P ⬍ 0.001)
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FIG. 2. The rate of specific protein secretion by hepatocytes from control and CR mice. Freshly isolated hepatocytes from CR (open symbols) and control (filled symbols) mice were pulse-labeled with [ 35S]methionine. During a chase period in the presence of unlabeled methionine, samples of the medium and hepatocytes were collected and analyzed using SDS–PAGE. Shown are the results for proteins of 67,500, 49,000, and 31,500 apparent molecular weights (A, B, and C, respectively). The intensity of specific protein bands was determined using a phosphorimager. Data are means ⫾ SD of the percentage of each protein initially synthesized which had accumulated in the medium at the indicated times [n ⫽ 6; P ⬍ 0.001 (***)].
(Fig. 1B). The decrease in chaperone protein levels parallels the decrease in the mRNA (11). The slightly faster migration of GRP170 from CR mice may result from differential posttranslational modification. We investigated the hypothesis that the altered abundance of ER chaperones may affect the rates of processing, and therefore the rates of secretion of hepatic proteins. Using freshly isolated hepatocytes, and pulse-chase studies, the secretion rate of the three most abundant secreted proteins was determined (Fig. 2). Shown are the rates of accumulation in the culture medium of proteins of 67,500 and 49,000 apparent molecular weight, which were subsequently identified as albumin and ␣ 1-antitrypsin (see below), and of an unidentified protein of 31,500 apparent molecular weight, here termed P31.5 (Figs. 2A, 2B, and 2C, respectively). By 60 min postlabeling, the amount of albumin, ␣ 1antitrypsin and P31.5 secreted by hepatocytes from CR mice was significantly greater than the amount secreted by hepatocytes from control mice (Fig. 2, P ⬍ 0.001). By 2 h post-labeling, the amount of albumin, ␣ 1-antitrypsin, and P31.5 secreted was 97, 63, and 250% higher for hepatocytes from CR mice. These results indicate that the secretion rate of these proteins is greatly enhanced by the CR diet. ␣ 1-Antitrypsin secretion appeared to reach a maximum 90 min after commencement of the chase with unlabeled methionine (Fig. 2B). Thus, hepatocytes from control mice appeared to secrete only about 50% of the ␣ 1-antitrypsin synthesized (Fig. 2B). In contrast, CR mice appeared to secrete approximately 85% of the
␣ 1-antitrypsin initially synthesized. These results suggest that CR led to an increase in both the rate and the final amount of initially synthesized protein secreted by hepatocytes. To allow quantitative comparison of the amounts of individual proteins secreted by hepatocytes from CR and control mice, we determined the specific activity of the 35S-methionyl-tRNA pools in the cells immediately after the pulse-labeling period. We found no difference in these specific activities (21.645 ⫾ 0.813 and 20.733 ⫾ 0.493 dpm/pmol from CR and control mice; P ⫽ 0.17). Thus, the relative rates of synthesis and secretion are directly comparable between hepatocytes isolated from control and CR mice. We investigated the possibility that the apparently higher secretion rates by hepatocytes from CR mice was due to greater rates of lysis of these cells during the chase phase of the studies. However, we were unable to detect any differential cell lysis. First, by SDS– PAGE we could detect no similarities between the profile of intracellular, radiolabeled proteins and those released into the medium during the chase period (data not shown). Second, no significant release of lactate dehydrogenase activity in the medium occurred during the chase period in cultures from either CR or control mice (data not shown). Third, there were no differences in viability of hepatocytes from CR and control mice as judged by cell count or trypan blue exclusion at the beginning or the conclusion of the chase periods. The apparent differences in the secretion rates of the proteins also are not artifacts of differential rates of synthesis of the proteins. There are no differences in the levels of albumin and ␣ 1-antitrypsin mRNA in control and CR mice (Fig. 3A). Further, the synthetic rates of ␣ 1-antitrypsin and albumin are the same in hepatocytes from CR and control mice (Fig. 3B). The amount of the proteins synthesized during the labeling periods is directly comparable because the total average incorporation of [ 35S]methionine into the proteins, and the specific activity of the t-RNA-bound methionine pool are the same in hepatocytes from control and CR mice. The proteins of 67,500 and 49,000 molecular weight were identified as albumin and ␣ 1-antitrypsin by several criteria. The radiolabeled proteins secreted following a pulse-labeling with [ 35S]methionine are shown in Fig. 4 lanes 2 and 6. As can be seen, the three most abundant secretory products are proteins of 67,500, 49,000, and 31,500 apparent molecular weight. Shown in lanes 3 and 7 are the proteins specifically precipitated using antibodies directed against mouse albumin and ␣ 1-antitrypsin, respectively. The immunoprecipitated albumin and ␣ 1-antitrypsin co-migrated with the proteins of 67,500 and 49,000 apparent molecular weight. Further, the 67,500 and 49,000 molecular weight proteins were completely removed from the medium by serial precipitations with the respective antibody (data not shown). P31.5 remains unidentified. It
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FIG. 3. The expression of albumin and ␣ 1-antitrypsin. (A) The abundance of hepatic albumin and ␣ 1-antitrypsin mRNA was determined by dot blotting of total RNA from control (open bars) and CR (filled bars) mice. The means ⫾ SD are shown (n ⫽ 6). (B) The rates of synthesis of albumin and ␣ 1-antitrypsin in hepatocytes of control (open bars) and CR (closed bars) mice (n ⫽ 6) were determined after a 15-min pulse labeling. Labeled proteins were immunoprecipitated, separated by SDS–PAGE, and quantified using a phosphorimager. The hepatic (C) and serum (D) levels of albumin and ␣ 1-antitrypsin from control (lanes 1–3) and CR mice (lanes 4 – 6) were determined by Western blotting.
is the prominent band just above the 30,000 molecular weight marker in Fig. 4, lanes 2 and 6. To determine whether enhanced rates of secretion of these proteins affected their steady-state levels, the hepatic and serum levels of ␣ 1-antitrypsin and albumin were determined by Western blotting (Figs. 3C and 3D). The hepatic levels of both proteins were reduced 30% in the CR mice, consistent with their increased rates of secretion and unchanged rate of synthesis in CR mice (P ⫽ 0.0013 and P ⫽ 0.0001 for ␣ 1antitrypsin and albumin, respectively; Figs. 2A and 2B and Fig. 3B). The serum levels of the proteins were also determined by Western blotting (Fig. 3D). No differences between control and CR mice were detected. These results indicate that the increased hepatic secretion of albumin and ␣ 1-antitrypsin in CR mice must be counterbalanced by increased plasma clearance.
amount of ␣ 1-antitrypsin increased from 53% of that initially synthesized in the control to 84% in the CR mice. The studies eliminated potential artifacts involving the specific activity of the intracellular methionine pool, the amount of each protein synthesized, and potential differential cell lysis. There is good evidence in the literature that ER chaperone levels can control the rate and extent of specific protein secretion (8, 9, 22). For example, a direct relationship has been found between the amount of protein trafficking through the ER and the level of GRP78 (23). GRP78 levels influence the secretion efficiency of many proteins that transiently associate with it in the ER (24). In general, the extent of association between a protein and GRP78 in the ER is inversely related to its secretion efficiency (24). For example, the secretion efficiencies of factor VIII, von Willebrand Factor variant, and tissue plasminogen activator are inversely correlated with the extent of their association with GRP78 (25). In this study, a significant proportion of Factor VIII was detected in a stable complex with GRP78 and not secreted. Overexpression of GRP78 by two- to threefold reduces secretion of von Willebrand factor and factor VIII (9). Overexpressing GRP78 several-fold delays the export of newly synthesized thyroglobulin (26). Conversely, reduction of GRP78 levels by 50 to 75% with antisense transcripts enhances secretion of a tissue plasminogen activator variant (8). Finally, induction of ER chaperones with calcium ionophores strongly reduces secretion of serum albumin, ␣ 1-antitrypsin, and ␣ 1-antichymotrypsin, although the loss of calcium from the ER may also have been involved in the loss of secretion efficiency (22).
DISCUSSION We report the novel result that the abundance of every ER chaperone tested was significantly reduced by CR and that these changes parallel previously reported reductions in the mRNA of these chaperones (11). The second novel result is that hepatocytes from calorie restricted mice secreted twice as much albumin, 63% more ␣ 1-antitrypsin, and 250% more P31.5 two hours after their synthesis. The maximum secreted
FIG. 4. Immunological identification of the 67,500 and 49,000 molecular weight proteins as albumin and ␣ 1-antitrypsin. Proteins in the medium from a pulse-chase study were precipitated with acetone (lanes 2 and 6), or immunoprecipitated with rabbit anti-mouse albumin antiserum (lane 3), or with rabbit anti-mouse ␣ 1-antitrypsin antiserum (lane 7), and analyzed by SDS–PAGE and phosphorimaging. An immunoprecipitation reaction identical to those shown in lanes 5 and 7, but lacking antiserum is shown in lane 5. The numbers to the left indicate the positions of radiolabeled molecular weight marker proteins in kiloDaltons (lanes 1 and 4).
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The ER proteins may be degraded at a higher rate in hepatocytes from control mice. ER chaperones, like their cytoplasmic counterparts, can facilitate protein degradation through the ubiquitin-proteasome pathway (1– 4). For example, in cell lines which synthesize IgG light chains in the absence of heavy chains, light chains are degraded in the ER with a half-life of 20 min (7, 27). GRP78 associates with the IgG light chains prior to their degradation, and there is a direct correspondence between the half-life of the protein’s association with GRP78, and the half-life of the protein. CR likely also promotes serum protein clearance, since the plasma levels of albumin and ␣ 1-antitrypsin did not change. Increased clearance should reduce the “dwell time” of these proteins in the circulation. Many lines of evidence support a role for glucose-modified and oxidized (glycoxidated) albumin and other serum proteins in the pathologies of diabetes and aging (28 – 30). CR produces relatively small reductions in serum glucose levels, but it effectively reduces the age-related accumulation of glycoxidation products (31–33). Thus, reduced serum protein dwell time may be a significant contributor to the reduced glycoxidation products and tissue damage found in CR animals. ACKNOWLEDGMENTS This work was supported by various donors to the research of S.R.S. J.M.D. and S.X.C. were recipients of fellowships from the Genetics Program of the University of California, Riverside.
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