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'r—Wv This is to certify that the thesis entitled HORMONAL AND DIETARY REGULATION OF ENZYME INDUCTION IN RAT LIVER presented by James Woodard Kurtz has been accepted towards fulfillment of the requirements for Ph.D. Biochemistry degree in W Major professor Date ifé/Xo 0-7639 OVERDUE FINES: 25¢ per day per item RETUMIIB LIBRARY MTERIALS: \— Place in book return to ve charge from circulation records © 1980 JAME S WOODARD KURTZ All Rights Reserved HORMONAL AND DIETARY REGULATION OF ENZYME INDUCTION IN RAT LIVER by James Woodard Kurtz A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1980 ABSTRACT HORMONAL AND DIETARY REGULATION OF ENZYME INDUCTION IN RAT LIVER by James whodard Kurtz To clarify the roles of glucose and insulin in the induction of hepa- tic lipogenic enzymes, the contributions of these two compounds to the induction of rat hepatic glucose 6-phosphate dehydrogenase (GGPDH), 6-ph05phogluconate dehydrogenase (6PGDH), and malic enzyme (ME) were examined. Normal and streptozotocin diabetic male rats were starved for three days, then refed a high carbohydrate diet for four days (hereafter abbre- viated S/R). In streptozotocin diabetic rats, the induction responses of G6PDH, 6PGDH, and ME proceeding from the three day starved to the four day high carbohydrate diet refed state, were 10 to 20% of those observed in similarly treated normal rats. This difference for diabetic rats was not due to inadequate glucose consumption since S/R diabetic rats consuned 59% more diet than S/R normal rats. Serum immunodetectable insulin concentra- tions of diabetic rats increased 2.6 fold in response to refeeding in both normal and diabetic rats. The latter insulin response did not lower serum glucose concentrations to normal levels. Supplementation of starved dia- betic rats with insulin during refeeding controlled serum glucose levels James woodard Kurtz and restored the induction of G6PDH, 6PGDH, and ME to levels above normal. Hepatocytes were isolated from 3-day starved male rats and incubated in serum-free Dulbecco's medium (DMEA). G6PDH specific activity increased 2.5-fold in 48 hour control incubations in DMEA and increased an addition- al 3.5-fold in the presence of 42 mU/ml insulin, 1 uM dexamethasone, and the absence of medium glucose. The effects of insulin and dexamethasone on GGPDH induction of G6PDH were dose dependent and additive. Increases in GGPDH specific activity by insulin and dexamethasone were independent of DNA synthesis. 6PGDH and ME specific activities decreased during the 48 hour control incubation in DMEA. Insulin, but not dexamethasone, pre- vented this decrease in activities and increased 6PGDH activity 20% above 0 hour levels. Cells incubated in DMEA with glucose in the absence of hormones showed no increase in G6PDH, 6PGDH, and ME activities. These results indicate that glucose alone is not sufficient to induce these liver enzymes and that insulin is required for the induction of GGPDH, 6PGDH and ME.ifl.lilQ and G6PDH and 6PGDH in 31332. The role of lysosomes as mediators of S/R, insulin, and glucocorti- coid stimulated enzyme induction in VlVO and in RH-35 cells was examin- ed. Although previously observed increases in lysosome fragility.during the first hours of refeeding were confirmed, further labilization or stab- ilization of lysosomes did not change the induction of G6PDH and 6PGDH compared with controls. Refeeding of a high glucose diet to starved rats resulted in no significant alteration in the association of lysosomal enzyme activities with purified liver nuclei compared with activities James Woodard Kurtz present in nuclei from 0 hour refed rats. Starved/refed streptozotocin diabetic rats, with or without insulin injection at doses sufficient to induce G6PDH and 6PGDH, showed no significant change in purified liver nuclear associated acid phosphatase activity throughout the experiment. The glucocorticoid responsive system, induction of tyrosine-a-keto- glutarate transaminase in RH-35 cells in culture and in adrenalectomized rats, was used to determine if glucocorticoids elicit a nuclear transloca- tion of lysosomes or lysosomal enzyme activity. Biochemical and electron microscopic examination revealed no change in the association of lysosomal hydrolases with nuclei of induced cells compared with those from uninduced cells. There was a 9-fold greater nuclear N-acetyl-efigfglucosaminidase activity in RH-35 cells than in hepatocytes despite the lower total activ- ity/DNA of RH-35 cells. The latter results do not support the extension of C.M. Szego's hypothesis for lysosomal mediation of hormone action to include S/R, insulin, and glucocorticoid action as described. Automated fluorometric methods developed for the analysis of DNA, protein, and selected enzyme activities: N-acetyl-B-D-glucosaminidase, G6PDH, and 6PGDH are described. ACKNOWLEDGEMENTS I wish to warmly thank Professor William W. Wells for his guidance and financial support during the course of this research. I would also like to thank my committee members, Drs. Jerry B. Hook, Allan J. Morris, John E. Wilson, and W. A. Wood for their very helpful suggestions. Finally, I would like to thank my wife Linda for her love and support throughout. ii TABLE OF CONTENTS Page LIST OF TABLES.......................................................vii LIST OF FIGURES......................................................ix ABBREVIATIONS........................................................xi INTRODUCTION Organization.................................................... 1 Literature Survey............................................... Induction of lipogenic enzymes............................ Induction of tyrosine aminotransferase by glucocorticoids. The lysosome as a mediator of hormone action.............. \JCWN N RationaleOOOOO0.0.0.0....OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO10 RaferenceSCOO0.0.0.0000...OOOOIOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 12 CHAPTER I. AUTOMATED FLUOROMETRIC ANALYSIS OF DNA, PROTEIN, AND ENZYME ACTIVITIES: APPLICATION TO METHODS IN CELL CULTURE ...... 16 AbstractOOOOOOOOOOOOOOOOOOOOOOOOOOOO0.0000000000000000000000 16 IntrOdUCtionoooooooooooooooooooooooooooooooooooooooooooooooo 16 Materials and mthOdSo O .0. O O O O O O O O O O. O O O. O O O O. O O 0 O O O O O O O O. O O 16 Sample preparations....................................... 16 Instrumentation........................................... 17 DNA analysis.............................................. 18 Protein analysis.......................................... 20 N-acetyl-e-D-glucosaminidase analysis..................... 21 Glucose 6-phosphate and 6-phosphogluconate dehydrogenase anallyslis.OOOOOOOOOOOOOOOOO...I...OOOOOOOOOOOOOOOOOOOOOOO 21 Resu1tSOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 22 DNA anaIYSiS.OOOOOOOOOOOOOOOOOO0.0...OOOOOOOOOOOOOOOOOOOOO 22 Protein analySiSOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 22 iii CHAPTER Page N-aCEtyT'8-D-gTUCOSBMTanaSE ana‘ySiSooooooooooooooooooooo 23 Glucose 6-phosphate and 6-phosphogluconate dehydrogenase anaTySTSoooooooooooooooooooooooooooooooooooooooooooooooo 23 DisCUSSion.OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO00...0.00.0000... 24 RaferenceSOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 25 II. INDUCTION OF TYROSINE AMINOTRANSFERASE IN RH-35 CELLS AND IN RAT LIVER. INVESTIGATION OF THE INVOLVEMENT 0F LYSOSOMES... 26 AbstractOOOI0.0.0000000000000000000000000000000000000000000. 26 IntrOductionOOOOOOOODOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 28 Materials and MethOdS.OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO. 29 C21] CUIture conditionSOOOOOOOOOOOOOOOOOOOOIOOOO0000...... 29 Analysis of enzyme activity, DNA, and protein concentrationOOOOOIOOOI.000......OOOOOOOOOOOOOOOOOOOOOOO 29 ETeCtron microscopyCOCOOOOOOO0.0...0.000000...0.0.0.000... 31 In Vivo stUdieSOCOOOOOOOOOOOOOOOOOOOOOOOO0......00.0.00... 32 ReSUIts.oooooooooooooooocooooooooooooooooooooooooooooooooooo 33 Development of the tyrosine aminotransferase induction system...0.0.00.00....0.0000000000000000000000 33 Lysosomal enzyme activity of nuclei during induction Of TAT.COOOOOOOOOOOOOOOOOOOOOO00.0.0...OOOOOOOOOOOOOOOOO 33 Discussion. ...... ........................................... 54 References... ......... ...................................... 57 III. INDUCTION OF LIPOGENESIS ENZYMES IN RAT LIVER DURING STARVATION—REFEEDING. INVESTIGATION OF THE INVOLVEMENT 0F LYSOSOMES................................................... 59 Abstract.................................................... 59 Introduction.................... ..... ....................... 61 Materials and Methods....................................... 62 Animal treatment.......................................... 62 samp1e preparation.C.OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO'..... 62 Enzyme analyseSOOOOOOOO00......I0.000000000000000000000000 63 iv CHAPTER Page Resu‘tSOOOOOOOOOOOO..0...0.000.000.0000000000000000000000000 65 G6PDH and 6PGDH induction and lysosome fragility.......... 65 Nuclear lysosomal enzyme activity during Starvation-refeeding.O..0.0.00000000000IOOOOOOOOOO0.00.. 74 DiSCUSSionCOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOCOOOOOOOO 89 References.OOOOOOOOOOOOOOOOOOOOOOO0.0.00.00.00.000.0.0.0.... 92 IV. INDUCTION 0F LIPOGENIC ENZYMES IN RAT LIVER AND IN PRIMARY CULTURES 0F ADULT RAT HEPATOCYTES........................... 94 summaryOOOOO0.00.00.00.00...0.0.0.000...OOOOOOOOOOOOOOOOOOOO 94 IntrOduction.O...0.0.000...OOOOOOOOOOOOOOO...OOOOOOOOOOOOOOO 96 Materials and MethOdSOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 98 MateriaISOCCOOOOO00.00.0000....000.00.000.000.0.0.0.000... 98 AnimalSOOOOOOOOOOOOOOOOOO0.0.0...OOOOOOOOOOOOOOOOOOOOOOOOO 98 Media preparation......................................... 99 Hepatocyte isolation......................................IOO Hepatocyte culture........................................101 Cell harvest and fractionation............................102 Analyses..................................................102 Resu1tSOOOOOOOOO0.000.000...OOOOOOOOOOOOOOO0.000000000000000104 Induction of lipogenic enzymes in normal and diabetic ratSOOOOOOO0.0000000000000000000000.0.000000000000000000104 C81] cu1ture.OOOOOOOOOOOOOOOOO...OOOOOOOOOOOOOOOOOOOOOOO..113 Induction of lipogenic enzymes in isolated hepatocytes....113 DiSCUSSion.OOOOOOOOOOOOOOOO ..... O ...... OOOOOOOOOOOOOOOOOOOOOI3O Raferences.....OOOOOOOOOOOOOOOOOOOOOO0.0...0.0.0.00000000000135 SUMMARY.OOOOOOOOOOOOOOOOOOOOOO0.00......OOOOOOOOOOOOOOOOOOOOO0.0.0.00138 ReferenCESOOOOOOOOOOOOOOOOOOOOOOOOOOO0.0.0.0.000000000000000140 APPENDIX.0.0...OOOOOOOOOOOOOOO00.0.0000...OOOOOOOOOOO0.00000000000000141 I. Comparison of Liver Total and Nuclear Cathepsin D and Liver Cytosolic Lipogenic Enzyme Activity between Genetically Obese Male Mice and their Lean Male Littermates...........................................I42 CHAPTER Page 11. Kinetics of Induction of Glucokinase in Rat Liver during Starvation-Refeeding..........................143 vi LIST OF TABLES Table Page Chapter I I. Assay Protocols........................................ 20 II. Fluorometer Filters and Settings....................... 21 III. Comparison of Reproducibility of Automated and Manual Methods Using the Coefficients of Variability of Regression Coefficients.............................. 22 IV. Productivity Comparison of the Manual and Automated MethOdS Described.0....O...OOOOOOOOOOOIOOOOOOOOOOO0.. 23 V. Enzyme Activities of Rat Liver Cells................... 23 Chapter II 1. Enzyme Activities of Rat Hepatocyte and RH-35 Cell FractionsOOOOOOO0.0000000000000000000000000000......O 50 Chapter III I. Liver and Body Heights of Normal and Hydrocortisone Injected Rats During Starvation-Refeeding............ 71 II. Effect of Corticosterone Injection on Liver Lysosome Fragility in Starved-Refed Rats...................... 72 III. Effect of Corticosterone Injection on Liver Glucose 6-Phosphate Dehydrogenase and 6-Ph05phogluconate Dehydrogenase in Starved-Refed Rats.................. 73 IV. Liver and Body Heights of Normal and Corticosterone Injected Rats During Starved-Refeeding............... 75 V. The Effect of Glucose Injection on Liver Lysosome Fragility and Induction of Dehydrogenases in Fed Rats.O....0...O0.00000000000000000000000.0.0.0.0....O 76 VI. Characteristics of Purified Rat Liver Nuclei........... 77 VII. Liver and Body Heights of Starved-Refed Rats........... 81 vii Table Page VIII. Effect of Starvation-Refeeding and Insulin Injection on Liver Total, Percent Free, and Nuclear Acid Phosphatase Activity in Normal and Streptozotocin Diabetic Rats........................................ 85 Chapter IV I. Liver Lipogenic Enzyme Activities of Normal and Streptozotocin-Diabetic Rats During Starvation-REfeedingOOOOOOOOOOOOOOOOOOOOOOOOOCOO...00105 II. Serum Glucose and Insulin Concentrations of Normal and Streptozotocin-Diabetic Rats During Starvation-RefeEding.00....0.0.0.0000....000000000000108 III. Diet Consumption and Liver Weight as Percent Body Weight of Normal and Streptozotocin-Diabetic Rats During Starvation-Refeeding..........................111 IV. Induction of Lipogenic Enzymes in Primary Cultures of Rat Hepatocytes.OOOOOOOOOOOOOOOOOOOOOOOO0.0.0.0000000124 V. Effect of Triiodothyronine on the Induction of Lipogenic Enzymes in Primary Cultures of Rat HepatocthSOOOOOOOOOOOOOOOOOOOOOOO0.0.0.0000...00.0.0126 VI. Effect of Aphidicolin on DNA Synthesis and Induction of Lipogenic Enzymes in Primary Cultures of Rat Hepatocytes..00...00.000.000.000...0.0.0.000000000000129 Appendix I. Comparison of Liver Total and Nuclear Cathepsin D and Liver Cytosolic Lipogenic Enzyme Activity between Genetically Obese Male Mice and their Lean Male LittermateSOOOOOOO0.00...OOOOOOOOOOOOO0.0.0.0.0000000141 viii LIST OF FIGURES Page Figure Chapter I 1. Gilford Model 3500 Computer-Directed Analyzer Interfaced with the Farrand Ratio Fluorometer-2.................... 2. Electrical and Vacuum Interconnections of the 3500 Fluorescence System..................................... 3. Thermostated Cuvette and Valve Connections of the Farrand Ratio Fluorometer-Z/Gilford 3500 Fluorescence System.... 4. Comparison of Fluorescence Obtained from the Assay of Standard Amounts of Calf Thymus DNA by the Ethidium Bromide Method Described when Performed Manually or Automatica]II‘YOOOOOOOOOO.OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO Chapter II 1. Growth Curve for RH-35 Cells.............................. 2. Effect of Dexamethasone Concentration on Tyrosine AminOtranSferase ACtiVit‘YOOOOOOOOOOOOOOOOOOOOOOOOO0.000. 3. Time Course of Induction of Tyrosine Aminotransferase in RH'35 ce115 by Dexamethasone.OOOOOOOOOOOOOOOOOOOOOOOO 4. Time Course of Tyrosine Aminotransferase and Nuclear Hexosaminidase Activity in RH-35 Cells Incubated With Dexamethasone.OOOOOOOOOOOOOOOOOOOO0.00.00.00.00..0. 5. Lysosomal Enzyme Activity of Nuclei from RH-35 Cells InCUbated With DexamethasoneOOOOOOOO0.0.0.0.0....00....O 6. Electron Microscopic Examination of Intracellular Lysosome Distribution after Treatment of RH-35 Cells With DexamEthasoneocoo.ooooooooooooooooooooooooooooooooo 7. Lysosomal Enzyme Activity of Liver Nuclei from Adrenalectomized Rats after Injection of Hydrocortisone 21-Sodium Succinate...................... ix 17 18 19 22 34 36 38 41 43 46 52 Figure Page Chapter III I. Effect of Hydrocortisone Injection on Liver Lysosome and Mitochondria Fragility of Starved-Refed Rats............ 66 2. Effect of Hydrocortisone Injection on Liver Glucose 6-Phosphate Dehydrogenase and 6-Phosphogluconate Dehydrogenase Activities of Starved-Refed Rats.......... 69 3. Lysosomal Enzyme Activity of Liver Nuclei from StarVEd-Refed RatSOOOOOIO...OOOOOOOOOOOOOOOOOIOO00...... 79 4. Effect of Insulin Injection on Lysosomal Enzyme Activity of Liver Nuclei from Fed Streptozotocin Diabetic Rats... 82 5. Characterization of Suspension Cultures of Hepatocytes ISO]ated from Starved Rats.OOOOOOIOOOOOOOOOOOOO0.0.0.0.. 87 Chapter IV 1. Time Course of Induction of Lipogenic Enzymes in Primary CUItureS 0f Rat HepatocyteSOOOO00.0.0.0.00.00.00.0000000114 2. G]ucose Dose-Response curve.OOOOOOOOOOOOOO00.00.00.0000000117 3. Insulin Dose-Response Curve...............................119 4o DEXBNEthaSOHE DOSE’ReSponSe Curve.00000000900coco-00000000121 Appendix 1. Time Course of Liver Glucokinase Activity during Starvation-RefeedinQOC0......0.0.0...OOOOOOOOOOOO0.0.0.0143 ABBREVIATIONS G6PDH glucose 6-phosphate dehydrogenase 6PGDH 6-phosphogluconate dehydrogenase ME = malic enzyme T3 = triiodothyronine TAT = tyrosine aminotransferase ACTH = adrenocorticotrOphic hormone PTH = parathyroid hormone TSH = thyroid stimulating hormone FSH = follicle stimulating hormone LH = leutinizing hormone RH-35 = Reuber hepatoma 35 MEN minimum essential medium (Eagle's) FBS fetal bovine serun 4-MU = 4-methylumbelliferone xi INTRODUCTION Organization This dissertation is divided into four chapters, each of which is in a form acceptable for publication in biochemical journals. Chapter I was authored by James N. Kurtz and William N. wells, published in Analytical Biochemistry 94:166-175 (1979), and is reproduced here by permission from Academic Press. Part of Chapter III (Table V) was my work and is pub- lished in the Journal of Nutrition 172:206-214 (1976) by Hartmut R. Schroeder, John A. Gauger, and William w. Nells. Chapter IV is in prep- aration to be submitted to the Journal of Biological Chemistry. Part I of the Appendix was presented as part of a poster entitled Lysosomal Enzymes and Liver Nuclei of the Genetically Obese Mouse authored by w.w. Wells, I.T. Mak, J.N. Kurtz, N.S. Henderson, C.A. Collins, and R.E. Ray (1979) at the XIth International Congress of Biochemistry, Toronto, Canada. LITERATURE SURVEY Induction of lipogenic enzymes. Lipogenesis is the successive link- age and reduction of the carbon units of acetate to form lipids. Several enzymes which are important in channeling carbons of various sources and reducing equivalents into fatty acids include: acetyl CoA carboxylase, ATP-citrate lyase, fatty acid synthetase, malic enzyme, glucose-6-phos- phate dehydrogenase, and 6-phosphogluconate dehydrogenase. The rate of lipogenesis is controlled largely through regulation of the activities of these enzymes. Control of lipid synthesis via regulation of acetyl CoA carboxylase and fatty acid synthetase has recently been reviewed (1). This survey will examine the dietary and hormonal factors which affect lipogenic enzymes with particular attention to the regulation of glucose- 6-phosphate dehydrogenase (GGPDH), 6-phosphogluconate dehydrogenase (6PGDH), and malic enzyme (ME). It has long been recognized that starvation lowers the activity and refeeding increases the activity of the lipogenic enzymes to levels above those of pre-starvation (termed "overshoot") (2,3,4). These early reports have been reviewed (5,6,7). The major dietary components: carbohydrate, lipid, and protein have all been examined as affecting induction of these enzymes and will be briefly reviewed in that order. Sassoon gt 11. (8) reported that in normal starved/refed rats the degree of induction of G6PDH and 6PGDH was correlated with the amount of dietary glucose consumed. They also reported that a single injection of insulin (4-6 units) resulted in a 25% increase in G6PDH activity after 24 hours but concluded that dietary carbohydrate was the more important induction signal. In similar experiments from Holten's laboratory it was found that the rates of GGPDH (9) and 6PGDH (10) synthesis were signifi- cantly correlated with the amount of dietary carbohydrate consumed. In other experiments Rudak 35 31., (11) found that rats fed a 60% fructose containing diet induced GSPDH to the same extent as rats fed a 60% glucose containing diet. The authors stated that since insulin release occurred in one case (glucose fed) but not the other that dietary carbohydrate was the primary signal for induction of GGDPH (see Discussion of Chapter 4 of this dissertation for a more complete analysis of this point). Derr and Zeive (12) found that infusion of a glucose-amino acid solution into star- ved rats for 22 hours resulted in significant elevations in G6PDH and ME activity. Dietary lipid content has long been recognized to affect the starva- tion/refeeding stimulated increase in lipogenic enzymes (5). Induction of G6PDH was inversely related to the content of the fat and it was observed that unsaturated fatty acids were more effective in suppressing the induc- tion of G6PDH than saturated fatty acids. (13). Free fatty acids (14) and their CoA esters (15) inhibit the activities of most of the lipogenic enzymes in vitrg but it was suggested that, at physiological levels, this 'could not account for the lack of induction of dehydrogenase (14). Wolfe and Holten (16) found that fat fed rats fail to induce G6PDH because of an effect on the rate of G6PDH synthesis although effects on degradation rates were not entirely eliminated. Fatty acids may therefore regulate lipogenic enzyme activities by both short term effects on activity as well as long term effects on enzyme synthesis (17). Dietary protein, while required for the induction of lipogenic enzymes (6,18,19), in large amounts inhibited enzyme overshoot (6). This dietary protein requirement, although not ruled out as the result of a direct effect of specific amino acids on the control of enzyme induction, probably plays a supportive role to protein synthesis in general. Dietary effects cannot be isolated from subsequent hormonal actions when discussing the regulation of enzyme levels jg_vivo. Several hormones including insulin, glucocorticoids and thyroid hormones have effects on the induction of the lipogenic enzymes. Since the studies of Block and McLean (2) it has been recognized that insulin has an effect on the liver activities of G6PDH and 6PGDH. These workers found that the liver levels of G6PDH and 6PGDH dropped during alloxan diabetes. Others have reported that insulin injection into alloxan diabetic (20,21) or normal (22) rats increased the activities of these liver enzymes. Weber and Convery (23) have reported that starved/refed alloxan diabetic rats fail to induce liver G6PDH and 6PGDH unless supplemented with insulin (4U/day). Holten gt 91. (9,10,11) have critized the finding that insulin increases GGPDH and 6PGDH activity by stating that this is a secondary effect on diet consumption. Others have stated that insulin serves to induce these enzymes by enhancing glucose transport into liver cells and glucose is then the primary signal for the induction of G6PDH and 6PGDH (24). Very recently preliminary reports have appeared using the the isolated hepatocyte system to investigate the hormonal control of G6PDH and 6PGDH (25); however no full report has yet appeared. Glucocorticoids have been recognized as playing an important role in the starvation/refeeding stimulated induction of G6PDH and ME. Bedanier ‘gt.al. (26-29) found, in 60% of the experiments reported, that adrenalectomized rats were unable to show a full overshoot induction of G6PDH and ME unless supplemented with cortisol. The other 40% of the experiments showed some degree of overshoot induction in starved/refed adrenalectomized rats. Wilmer and Foster (30) found adrenalectomized rats deficient but not incapable of inducing G6PDH and 6PGDH during starva- tion/refeeding. When several different glucocorticoids were administered during starvation/refeeding (hydrocortisone was best), full induction was achieved. Other laboratories studying hormonal control of induction of gluco- kinase in primary cultures of rat hepatocytes have reported that glucocor- ticoids acted synergistically with insulin to induce this enzyme (31,32). The reports that fasting (33) and glucocorticoids (34,35) decrease insulin receptor affinity for insulin would appear to be in conflict with a syner- gistic action of the insulin and glucocorticoids. Thyroid hormones especially triiodothyronine, are known to regulate the activity of ME in rat liver (36,37) and in chick cells in culture (38). Very recently Towle gt 31. (39) demonstrated that starvation/re- feeding and thyroid hormone stimulated increases in the levels of rat liver malic enzyme activity and the rate of synthesis of ME were propor- tional to increases in levels of mRNA for ME. Likewise, 6PGDH activity increases during starvation/refeeding were found to be pr0portional to changes in the levels of 6PGDH mRNA (40). In contrast, G6PDH activity increases during starvation/refeeding, while proportional to increases in the rate of G6PDH synthesis and the level of enzyme protein, could not be attributed soley to increased mRNA (41). The recent findings that T3 is required for the insulin mediated induction of ATP-citrate lyase (42) and glucokinase (43) in primary cultures of isolated rat hepatocytes would suggest that T3 may be of importance to the induction of G6PDH and 6PGDH as well. Induction of tyrosine aminotransferase by glucocorticoids. Since the initial observations by Lin and Knox (44) that glucocorticoids increase the activity of tyrosine aminotransferase (TAT) in rat liver, much has been done to elucidate the mechanism. A recent comprehensive review of glucocorticoid hormone action is available (45). Kenney has shown by protein labeling and immunoprecipitation tech- niques that the increased activity of liver TAT after in vivg administra- tion of [14CJ-leucine and hydrocortisone to rats is due to a specific increase in TAT synthesis (46,47). This mechanism is also supported by the work of Goldstein, Stella, and Knox (48) who showed that in the isola- ted, perfused rat liver, addition of puromycin and hydrocortisone to the perfusate resulted in complete disappearance of glucocorticoid mediated TAT induction. Later Hager and Kenney found that similar rapid and speci- fic induction of TAT could be obtained in the perfused liver system by addition of glucagon or insulin to the perfusate (49). As with the estrogen responsive systems, it was found that specific cytoplasmic (50) and nuclear (51) receptor protein fractions for glucocor- ticoids existed in cultured liver cells. Using intact animals, Sekeris' group showed that upon entry of [3HJ-cortisol into the liver, approxi— mately 50% is associated, structurally unaltered, with cytosol, 12% is associated with both crude mitochondria and crude microsomes and 0.3% is associated with purified nuclei (52). The cytoplasmic cytosol receptor was found to have glycoprotein properties (neuraminidase and protease sensitive) with a sedimentation constant of 45 (52). In later reports, binding of [3HJ-cortisol to the cytoplasmic receptor glycoprotein(s) was shown to facilitate the amount of label associated with added purified 7 nuclei when compared with nuclei incubated with free [3HJ-cortisol or free [3HJ-cortisol plus bovine serum albumin (53). In these experiments [3HJ-cortisol-receptor complex formation not only enhanced nuclear bind- ing but also increased RNA polymerase activity of nuclei. Thus the cyto- plasnic receptor-steroid complex has been implicated as a functional intermediate in the induction process. Tomkins' group has shown that binding of the cytoplasmic receptor-dexamethasone complex with nuclear chromatin depends on activation. Activation can occur by incubation of the cytosol at 20°C, high (0.3 M NaCl) ionic strength, or low protein concentration (54,55) thus indicating, as in the estradiol system, that some type of structural modification must occur before binding and subse- quent hormone expression. Once in the nucleus the steroid-receptor com- plex binds to DNA (DNase sensitive binding) receptor sites (54). Binding is then followed by increased RNA synthesis (53). The mechanism by which transcription is regulated by the receptor-steroid complex has been studied primarily by Sekeris' group (56). To my knowledge no research has been done to elucidate the mechanism by which the glucocorticoid-receptor complex makes its way to the nucleus. Szego has proposed a mechanism for the transfer of the estrogen-receptor complex to the nucleus of extrogen sensitive target cells based on micro- sc0pic and biochemical experiments. A brief review of this evidence follows. The lysosome as a mediator of hormone action. Comprehensive reviews of the evidence for the lysosome as a mediator of hormone action are available (57,58). Within 1-2 minutes after adminstration in vivo of physiological amounts of estradiol-17B, the acridine orange stained lyso- somes of preputial gland and uterine cells were observed by fluorescence microscopy to localize in and about purified nuclei (59,60). In control experiments, injections of estradiol-17a or saline did not result in accu- mulation of lysosomes about the nuclei. Non-estradiol target organ lyso- somes were not responsive to estradiol treatment (59). These experiments demonstrate the specificity of hormone-lysosome interaction. In different studies (61) it was observed that the in vivo inter- action of physiological amounts of estradiol-17B with the preputial gland or uterine lysosome resulted in significant labilization of the lysosomal membrane compared with control (saline or estradiol-17a) injections. Membrane labilization was determined by a significant increase in the release of lysosomal enzymes from purified lysosomes to an isotonic or Triton X-100 incubation medium. Szego suggests (57) that the lysosomal enzymes released may in some way affect the observed structural changes of the cytoplasmic BS estradiol-receptor complex to the nuclear SS form (62). Pharmacologic doses of cortisol-21-acetate and propanolol just prior to estradiol-178 injections were able to reverse the previously observed: (a) metachromatic fluorescence pattern of crude and purified nuclear prep- arations, (b) lysosomal membrane labilization, and (c) intranuclear loca- tion of lysosomal hydrolases of preputial glands (63). Using adrenal demedullated rats, Szego £3 31. (64) have shown that within 5 minutes after injection of low doses of the polypeptide hormone ACTH, the adrenal cortical lysosomal membranes were labilized, i.e., more susceptible to autolysis. This response to ACTH did not occur in lyso- somes of non-target thyroid cells of hypophysectomized rats. Again this indicates specificity of lysosome hormone interaction. As with the observations with estrogen, ACTH treatment also resulted in the subsequent intranuclear location of lysosomal hydrolases (64). Other hormones including: PTH, TSH, FSH, LH, epinephrine, glucagon, and cyclic AMP are proposed to act by lysosomal mediation (57). Another report from Szego's laboratory (65) indicates the presence of an estradiol-178 receptor protein in the lysosol fraction of preputial glands (target organ for estrogens) of female rats. The lysosol fraction is the supernatant of a 105,000 x g - 1 hr centrifugation of a purified lysosome preparation which was previously incubated, with stirring, in a hypotonic buffer in 1 hour at 0-4°C. This lysosol protein (protease sen- sitive) posesses all of the criteria necessary for classification as a steroid receptor, i.e.: (a) target selectivity (nine-fold more receptors in preputial than liver cells), (b) stereospecificity (non-radioactive 17a-congener is unable to compete 3H-estradiol-17a from the protein except at very high concentrations), (c) protein nature (protease sensi- tive binding), and (d) high affinity (Ka = 1010 h-l) with low capacity (n = 10'13 moles/mg total lysosol protein) (65). In addi- tion, this lysosol receptor protein, like the cytosol estrogen and gluco- corticoid receptors, binds with greater affinity at 32°C than at 0°C. Szego speculates that a precursor-product relationship may exist. From this accumulation of morphological and biochemical evidence, Szego has hypothesized that "...the target-specific lysosome, on activa- tion by trophic hormone, serves as a mobile link for information transfer between the cell surface and the nucleoplasm" (57,58). RATIONALE The experiments performed here were designed to determine if Szego's hypothesis for lysosomal mediation of hormone action would apply to: (a) glucocorticoid hormone stimulated TAT induction in Reuber H-35 cells (Chapter II) and (b) starvation/refeeding stimulated induction of the lipogenic enzymes G6PDH, 6PGDH, and ME (Chapter III). The two major char- acteristics of lysosomal mediation of estradiol action reported by Szego i.e., lysosomal membrane labilization and relocalization of lysosomal enzyme activity to purified nuclei, were examined to determine if they were an integral part of glucocorticoid and starvation/refeeding stimula- ted enzyme induction in rat liver. The experimental approaches taken were similar to those of Szego and were an extension of work begun in Dr. Nells' laboratory (66-68). Prior to the examination of RH-35 cells, automated fluorometric techniques for analysis of DNA, protein, and enzyme activities in samples from cell cul- ture were developed. The presence of lysosomal hydrolases in nuclei of glucocorticoid treated RH-35 cells and adrenalectomized rats were examined biochemically and histologically. Likewise rat liver nuclei were examined for the presence of lysosomal enzyme activity during starvation/refeeding and after insulin supplementation to streptozotocin diabetic rats. Lyso- some fragility was also examined as a requirement for starvation/refeeding stimulated induction of the lipogenesis enzymes G6PDH and 6PGDH. In a final chapter, experiments using streptozotocin diabetic rats and isolated rat hepatocytes in primary culture were performed to deter- mine the relative contributions of glucose and insulin to the induction of the lipogenic enzymes G6PDH, 6PGDH, and ME. Hepatocyte isolation and 10 11 culture techniques were developed for this purpose. The role of glucocor- ticoids and triiodothyronine were also examined in the isolated hepatocyte system. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. LITERATURE REVIEW Volpe, J.J. and Vagelos, P.R. (1976) Physiol. Rev. 56, 339-417 G10Ck, 60E. and MCLean, PI (1955) BiOChemo J. 61, 390-397 Tepperman, H.M. and Tepperman, J. (1958) Diabetes 7, 478-485 Tepperman, J. and Teppenman, H.M. (1958) Am. J. Physiol. 193, 55-64 Tepperman, H.M. and Tepperman, J. (1963) Adv. in Enz. Reg. 1, 121-136 Potter, V.R. and Ono, T. (1961) Cold Spring Harbor Symposia on Quantitative Biology 26, 355-362 Gibson, D.M., Lyons, R.T., Scott, D.F. and Muto, Y. (1972) Adv. 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(1979) Endrocrinology 104, 205-211 Kahn, C.R., Goldfine, I.D., Neville, Jr., D.M. and DeMeyts, P. (1978) Endocrinology 103, 1054-1066 Olefsky, J.M., Johnson, J., Lin, F., Jen, P. and Reaven, G.M. (1975) Metabolism 24, 517-527 Wise, E.M. and Ball, E.G. (1964) Proc. Natl. Acad. Sci. USA 52, 1255-1263 Oppenheimer, J.H., Silva, E., Schwartz, H.L. and Surks, M.I. (1977):l;. Clin. Invest. 59, 517-527 Goodridge, A.G. and Adelman, T.G. (1976) J. Biol. Chem. 251, 3027-3032 Towle, H.C., Mariash, C.N. and Oppenheimer, J.H. (1980) Biochemistry 19, 579-585 Hutchison, J.S. and Holten, o. (1978) J. Biol. Chem. 253, 52-57 Sun, J.D. and Holten, o. (1978) J. Biol. Chem. 253, 6832-6836 Spence, J.T., Pitot, H.C., and Zalitis, G. (1979) J. Biol. Chem. 254, 12169-12173 Spence, J.T. and Pitot, H.C. (1979) J. Biol. Chem. 254, 12331-12336 Lin, E.C.C. and Knox, W.E. (1957) Biochim. Biophys. 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Chem. 248, 5866-5872 Sekeris, C.E. and Schmid, W. (1972) The Biochemistry of Gene Expression in Higher Organisms. Pollak, J.K. and Lee, J.W. (eds.) D. Reidel Publishing Co. Szego, C.M. (1974) in Recent Pro ress in Hormone Research, (R.0. Greep, ed), Vol. 30, pp. 171-23 , Academic Press, Inc., New York Szego, C.M. (1975) in Lysosomes in Biology and Pathology (Dingle, J.T. and Dean, R.T., eds.) Vol. 4, pp. 385-461, American Elsevier Publishing Co., Inc., New York Szego, C.M. and Seeler, B.J. (1973) J. Endocr. 56, 347-360 Verity, M.A., Munsat, T.L., Smith, R.E. and Szego, C.M. (1973) Annals of Int. Medicine 78, 725-738 Szego, C.M., Seeler, B.J., Steadman, R.A., Hill, D.F., Kimura, A.K. and Roberts, J.A. (1971) Biochem. J. 123,523-528 Jensen, E.V. and DeSombre, E.R. (1973) Science 182, 126-133 Szego, C.M. (1972) Gynec. Invest 3, 63-95 Szego, C.M., Rakich, D.R., Seeler, B.J. and Gross, R.S. (1974) Endocrinology 95, 863-874 65. 66. 67. 68. 15 Hirsch, P.C. and Szego, C.M. (1974) J. Steroid Biochem. 5, 533-542 Blosser, J.C. and Wells, W.W. (1972) J. Neurochem. 19, 1539-1547 Schroeder, H.R., Lawler, J.R. and Wells, W.W. (1974) J. Nutr. 104, 943-951 Schroeder, H.R., eauger, J.A. and Wells, W.W. (1976) Arch. Biochem. Biophys. 172, 206-214 Chapter ANALYTICAL BIOCHEMISTRY 94. 166-175 (I979) l L Automated Fluorometric Analysis of DNA, Protein, and Enzyme Activities: Application to Methods in Cell Culture‘ JAMES W. [(61172 AND WILLIAM W. WELLS2 Department nfBim'hwni.i!r_\'. Michigan State University. East Lansing. Michigan 48824 Received October 20. l978 Automated fluorometric methods for the analysis of DNA. protein. and selected enzyme activities for A-acetyl-B-D-glucosaminidase. glucose 6-phosphate dehydrogenase. and 6- phosphogluconate dehydrogenase are described. Instrumentation for these assays includes a Gilford 3500 computer-directed analyzer in conjunction wuh a Farrand Ratio Fluorometer-2 modified for flowthrough sampling. Comparisons were made between the automated fluorometric methods described and manual spectrophotometnc or fluorometric methods for reproducibility. speed of analysis. and quantitative correlation. Typical values of N- acetyl-B-D-glucosaminidase. glucose 6-phosphate dehydrogenase. and 6-phosphogluconate dehydrogenase activities obtained by these methods in isolated rat hepatocytes and Reuber H-35 hepatoma cells are reported. Studies using cell culture methodology often require the use of microassays. While manual fluorometric methods can provide the sensitivity needed. they are often ac- companied by reading fluctuation or back- ground noise which is a combination of a number of factors. including type and handling of sample. temperature. and fluorom- eter characteristics (1.2). This paper de- scribes the development of assay methods for representative cellular components using an automated fluorometer which eliminates much of the experimental error previously encountered. MATERIALS AND METHODS Sample preparation. Reuber H-35 hepatoma cells (RH-35 cells) were obtained from Dr. Richard Hanson. Temple Uni- versity. Philadelphia. Pennsylvania. Mono- layer cultures were grown in 75-cm2 plastic tissue culture flasks (Bioquest. Oxnard. Calif.) in a humidified atmosphere of 95% ‘ This work was supported by U. S. Public Health Service Grant AM l0209. -' To whom inquiries shouid be addressed. (m3-2697-‘79/050I66- IOSOZDO'O Copyrighi C 2979 by Acadermc Press. inc. All rights at reproduction in any :nrm rescrsga air/5% CO: at 37°C. Cells were grown in Eagle's minimum essential medium (MEM)3 with Earle‘s salts prepared from powder (Grand Island Biological Co.. Grand Island. N. Y.) supplemented with ID3 U/liter potas- sium penicillin G. 0.1 g’liter streptomycin (Chas. Pfizer and Co.. Inc.. New York. N. Y.). and 10 g/liter sodium bicarbonate and sterilized by filtration through a 0.45- pm filteriMillipore Corp.. Bedford. Mass). After filtration. the medium was tested for sterility for 1 week by innoculation into sterile aqueous solutions of 2.4% thiogly- colate and 3.7% brain-heart infusion (Difco Laboratories. Detroit. Mich) Before use. Eagle's MEM was further supplemented with nonessential amino acids and ID“? fetal calf serum (Grand Island Biological Co.). Each T-flask yielded approximately 4 x 107 cells at near confluency. ‘Abbreviations used: MEM. minimum essential medium (Eagle'Si: PBS. phosphate-buffered saline: EB. ethidium bromide: NAGase. N-acetyl-B-D-glu- cosaminidase: G6PDH. glucose 6-phosphate dehydro- genase: 6PGDH. 6-phosphogluconate dehydrogenase: J-MU. 4-methylumbelliferone. 16 17 AUTOMATED FLUOROMETRIC ANALYSIS Rat liver hepatocytes were obtained from ZOO-g male Holtzman rats by the two-step method of Seglen (3). Magnesium-free Krebs-Henseleit bicarbonate buffer (4) gassed with hydrated 95% 02/5‘7c CO, was used. Calcium was absent during preper- fusion and present during collagenase perfusion. Normal hepatocytes or RI-I-35 cells were suspended in 4 ml of ice-cold 0.32 M sucrose and 3 mM MgCl, to a density of approxi- mately 107 cells/m1. They were homogenized for 60 s in 18 x 104-mm glass test tubes using a Tekmar homogenizer (Tekmar Co.. Cincinnati. Ohio) equipped with a small probe (10 EN) and operated at 87 V. Three milliliters of this homogenate were centri- fuged at 800g for 10 min. To the 800g pellet was added 1.1 ml of 0.32 M sucrose and 3 FIG. I. Flurometer-l. mM MgCl,. and 1.1 ml of resuspended pellet was used to obtain purified nuclei as de- scribed by Szego and Seeler (5). The 800g supernatant (2 ml) was again centrifuged at 22.0003 for 15 min and this supernatant frac- tion was designated F. lnsrrumentation. Assays were performed using the Gilford System 3500 computer- directed analyzer (Gilford Instrument Co.. Inc., Oberlin. Ohio) modified so that the reading signal originated from a Farrand Ratio Fluorometer-2 (Farrand Optical Co.. Inc., Valhalla. N. Y.) instead of the standard Gilford 300-N spectrophotometer (Figs. 1 and 2). A thermostated. vacuum-operated. flow-through cuvette (70 [1.1). specially de- signed and available through the Gilford Instrument Company. was inserted in place of the standard Farrand cuvette holder FARIAND ‘LUOIOMETEI lNTIIFACE GIL-M431 Gilford Model 3500 computer-directed analyzer interfaced with the Farrand Ratio 18 KURTZ AND WELLS sauna/ounce r - - - - - - - I " - "' - I | ruse 70 I ' ' rowan I. g | acumen I I I '.. I. : cuvme I nun ' fl l I nuonoumn ) ‘ I mnnnce O I VACUUM ‘ J‘ C&- CUVI‘I'I’I 373nm” "WV“ 30 connecnou D J7 iico “L use cone 4 LINE cono Ina“ DATA nummn GlL-JSM- 1m soup use: an: ILICTIICAL Lines UN! CORD IIOKIN LINES AI! VACUUM UNIS F IG. 2. Electrical and vacuum interconnections of the 3500 fluorescence system. (Fig. 3). The Gilford Model 3500 computer- directed analyzer carries out programs directed by magnetic cards specified for standard methods in clinical chemistry. The most versatile cards for research are desig- nated as Kinetics 1 and 2 and Endpoints I and 2. For the general kinetics programs. a keyboard permits the operator to select any desired time interval. The utility of the sys- tem for the research laboratory can be ex- tended by the availability of special pro- grams obtained from the Gilford Instrument Laboratories. Inc. A series of integrated functions automatically conduct the meas- urements. store the data. and print out the results in appropriate units. For a fuller description of Model 3500 hardware. e.g.. transporter. pipetter/diluter. dispensers. and F arrand Ratio Fluorometer-2 to Gilford 3500 interface system. contact the Gilford Instru- ment Laboratories. Inc. These accessories to the standard Gilford 3500 and the Farrand Ratio Fluorometer-2 are available through the Gilford Instrument Laboratories. Inc. All assays were carried out in plastic strips (Gilford Instrument Co.. Inc.) containing four reaction cups per strip. In most cases standards and samples were pipetted man- ually into the reaction cups. and the strips were kept on ice until initiation of the re- action. For experimental programs, we have found that adequate reagent mixing is ob- tained when the volume of added reagent is equal to or greater than the volume of sample to which it is added. The calibra- tion sequence of standards (see Table 1) used in all analyses allows for the adjust- ment of the fiuororneter such that all read- ings are on scale at one range setting. Filters and fiuorometer settings used for all analyses are listed in Table 2. DNA analysis. DNA analysis was per- formed following the procedure of Karsten and Wollenberger (6.7). Reagents I and II were freshly made by mixing component stock solutions. Reagent I is 12.5 uyml RNase (Sigma Chemical Co.. St. Louis. Mo.: type l-A) and 4.13 IU/ml heparin (Sigma. type 1) dissolved in phosphate-buffered saline (PBS). PBS was made according to the method of Karsten and Wollenberger (6). Reagent II is 16.7 pig/m1 ethidium bromide (Sigma) in PBS. Calf thymus DNA (Sigma. type I), 0.1 mg/ml in PBS. was used as a’ standard. All stock solutions were stored at 4°C and were stable for at least 1 month. Purified nuclei are assayed for DNA by the Endpoint 1 and Bilirubin-direct pro- grams using the sequence of runs 1 and 2a (Table 1). Run 1 achieves the mixing of up to 19 AUTOMATED FLUOROMETRIC ANALYSIS ILIC‘I’IICAL CONNICTION TO INTERFACE GIL-m4” VACUUM CONNIC'UON Fla. 3. Thermostated cuvette and valve connections of the Farrand Ratio FIuorometer-l’Gilford 3500 fluorescence system. 0.2 ml of standard or sample with 1 m1 of a RNase/heparin mixture (reagent 1) in reac- tion cups. After run 1 the reaction strips are removed from the Gilford 3500 rack transporter and incubated 20 min at 37°C in a water bath and 15 min at room tempera- ture. Following incubations. run 2a accom- plishes the mixing of 1.5 m1 of the ethidium bromide solution (reagent II) with the samples and the reading of fluorescence. The final concentration of ethidium bro- mide is 10 ug/ml. The assay of whole homogenate fractions for DNA is similar to that of purified nuclei except that the high fluorescence due to whole homogenate alone necessitates the in- clusion of sample blanks (runs 1 and 2b). Run 1 is the same as in the assay of purified nuclei except that samples and standards are pipetted in two sets. The first set of samples serve as blanks and the second set of samples as complete reactions. After a 30-min in- cubation at 37°C and a 15-min incubation at room temperature. run 2b is started. In this run. 1.5 ml of PBS is mixed with the first set and reagent II is mixed with the second set of samples and standards. Fluorescence readings are automatically made in this run exactly 2.5 min after each mixing. Slopes. intercepts. and correlation co- efficients of standard curves are obtained by least-squares analysis. Calculation of the concentration of DNA in mg DNA) ml sample is as follows. Purified nuclei: (A - C )/(E)F . Whole homogenate: [(A - B) - (C - Dly (EiF. 20 KURTZ AND WELLS where A - fluorescence of sample + RNase «- hep- arin + PBS + ethidium bromide (EB) + EB-DNA complex, B = fluorescence of sample + RNase + hep- arin + PBS. C = fluorescence of RNase + heparin + PBS + EB. D = fluorescence of RNase + heparin + PBS E = SIOpe of standard curve (AF/15mg DNA), . F a undiluted sample volume (ml). For comparison. DNA was assayed man- ually by the diphenylamine method of Giles and Myers (8). Diphenylamine (Mal- linckrodt. St. Louis. Mo.) was twice re- crystallized from 100% ethanol. mp 52.8- 54°C. Calf thymus DNA, 0.1 mg/ml in PBS. was used as a standard. After the 18-h in- cubation. samples were centrifuged 15 min at 1146g at room temperature. and the ab- sorbance of the supernatant solutions was measured at 595 and 700 nm. Protein analysis. Protein analysis was performed by a modification of the method of BOhlen er al. (9). Reagent III is 0.2 M borate buffer. pH 9.25 (boric acid solution titrated with NaOH). and reagent IV is fluorescamine (Fluram Roche Diagnostics. Nutley, N. l.) at a concentration of 30 mg/ 100 ml in CaSO,-dried acetonitrile. Bovine serum albumin (Sigma) was used as a Stand- ard. Reagents III and IV can be stored in- definitely in the dark at room temperature. TABLE 1 ASSAY PROTOCOLS" Standards and samples Reagent addition ‘ Reac- Final Total Vol- tion sample assay ume cup volume volume Assay Program Run‘ Tower Reagent imli No. C oments‘ i ml l i ml l DNA Endposnt I l A I I I-4 Calibration sequence 0.2 2.7 (mode 1) 5-12 Standard curve i0- I5 pg DNAi Its-end Sample Bilirubin- Direet 2a A II I .5 Bilirubin-Direct 26 A PBS 1st set 1.5 II 2nd set 1.5 Protein Bilirubin-Di'rect l A III LS 1.41 Calibration sequence Di 2.! 8 IV 0.5 5- I2 Standard curve i0-I2 as protein) I3-end Samples NAGase Endpomt Ic I A V 0.25 l-J Calibration sequence 0.02 2.27 (mode I) 5 I” Standard curveio- (.5 nmol s—MUl Ill-end Samples Endpoint I: 2 A V1 2.0 (mode 3) GOPDH and Kinetics l l A VII 2.0 («1 Calibration sequence 0) ll 6PGDH‘ 5-12 Standard curve 10-20 ani NADPH) I3-end Samples Kinetics l 2 A VIII 2.0 ' See Materials and Methods for a description. ‘ A run is defined as the handling of samples and reagents in a programmed sequence. ' The calibration sequence is comprised of reaction cups I -A containing the lowest standard in cups I. 2. and 4. and the highest Standard in cup 3. ' Preineuhetion time - I s. incubation time - 20 s. and integration time - l0 s. 21 AUTOMATED FLUOROMETRIC ANALYSIS TABLE 2 FLUOROMETER FILTERS mo Serrmos" Pro- GOPDH. DNA tein NAGase 6PGDH Excitation wavelength mm 360 390 see no Filter number i7-ooi i7-Svli IT-OOI i7oooi Emission uavelength man 590 4'5 450 400 Filter number i3-69i iii-fit i3-’3i (3.7:) Excitation slit setting J I I 3 " In all assays. the following instrument settings were used: damping - Io. mode - sample range - I. temperature - 25'C. sample time I- S. \acuum t 10. and multiplication tactiir I :00. " Farrand Optical Co.. Inc.. Valhalla. .N Y ' Farrand Ratio Fluorometer-2. Whole homogenate. purified nuclei. and F fraCIions were assayed as indicated in Table I using the Gilford 3500 program for direct bilirubin analysis. The instrument pipetting system forcefully mixes standard or sample with 0.5 ml fluorescamine solu- tion and 1.5 ml borate buffer. pH 9.25, and the fluorescence is measured exactly 2.5 min after each mixing. The ordinate-inter- cept of the standard curve served as the blank since these samples do not contribute significantly to the fluorescence of the product. For comparison. protein was also de- termined manually by the procedure of Lowry et al. (10) using bovine serum al- bumin as standard. N-acetyl-B-D—glucosaminidase analysis. N-AcetyI-B-D—glucosamindase (NAGase) activity was determined by a method modi- fied from that of Robins et al. (11) using the Endpoint 1c program as summarized in Table 1. The aqueous 75 uMA-methylum- belliferone (Sigma) standard used for prep- aration of standards listed in Table I was stable at -80°C for at least 6 months. As- says of whole homogenate and F fractions were initiated (run 1) by the mixing of up to 0.05 ml sample or standard with 0.25 ml of the assay mix. reagent V. Reagent V, made on the day of assay from component stock solutions. was: 50 mM sodium citrate. pH 4.3. 0.2% lev) Triton X-IOO (Research Products International Corp.. Elk Grove Village. III.). and 1.03 mM 4-methylumbel- Iiferyl-N-acetyl-B-D-glucosaminide (Sigma). After a timed, room temperature incubation period (usually 15 to 20 min). reactions were stopped (run 2) by the addition of 2.0 ml of 20 mM 2-amino-2-methyl l-propanol (Sigma). pH 10.35, reagent V1. The fluorescence of each sample was measured in run 2. 9 s after addition of reagent V1. In the calculation of N-acetyI-B-D—glucosaminidase activity. the ordinate-intercept of the standard curve served as the blank since sample alone does not contribute significantly to the overall fluorescence. In addition. manual assays were performed by the same procedure as described for the automated method except that reactions and fluorescence readings were performed in IO x 75-mm test tubes. Glucose 6-phosphare and 6-phosphoglu- conate dehydrogenase analysis. The pro- cedure used for the determination of glu- cose 6-phosphate dehydrogenase (GGPDH) and 6-phosphog1uconate dehydrogenase (6PGDH) is a modification of that used by Rudaclt et al. (12) and is summarized in Table I. An appropriate volume of reagent VII is freshly prepared by mixing stock solutions to give: 115 mM Tris, pH 8.0, 10 ' mM MgC12, 50 uM NADP‘. 0.6 mM 6-phos- phogluconate. and 2 mM glucose 6-phos- phate (all organic chemicals from Sigma). Reagent VIII is identical to reagent VII ex- cept that glucose 6—phosphate is omitted. A 1 mM NADPH (Sigma) solution. pH 10. is pipetted manually into the reaction cups to give the various amounts of standard in- dicated in Table 1. For comparison. dehy- drogenase assays were also performed with the same assay mixture using a Gilford 2400-8 recording spectrophotometer (Gilford 1n- strument Co., Inc.). NADP’ and NADPH solutions were stored at -80°C: all other stock soluuons were stored at 4°C. Using the Kinetics 1 program. the com- bined glucose 6—phosphate and 6—phos- phogluconate dehydrogenase activity are measured during run I. and 6-phosphoglu- conate dehydrogenase activity alone is 22 KURTZ AND WELLS measured during run 2. Glucose 6-phos- phate dehydrogenase activity is calculated as the difference between the rates obtained in runs 1 and 2. Assays on F fractions are performed at 30°C. Statistical methods were employed ac- cording to Steel and Torrie (13). RESULTS DNA Analysis Figure 4 presents DNA standard curves for the ethidium bromide procedure per- formed either manually or by the automated method. To evaluate the reproducibility of the two procedures for analysis of DNA, the coefficients of variability (CV) of the regression coefficients (slopes) of either fluorescence or Ana-Am vs the concentra- tion of DNA were determined for a range of 10C 9Q- § i Q I m ‘6' o 1 l FLUMESCE:CE (arbitrary units) Q T a 6 O to a oi (oi. omi (’19) FIG. 4. Comparison of fluorescence obtained from the assay of standard amounts of calf thymus DNA by the ethidium bromide method described when performed manually or automatically. Manual assays I.) were performed by the same procedure as de- scribed for the automated procedure (I) except that reactions and fluorescence readings were carried out in I0 x 75-min test tubes. TABLE 3 Conrantson or Rarnooucmurv or AUTOMATED AND MANUAL METHODS Usmo THE Coerricmnrs or VARIABILITY or Reonesswn COEFFICIENTS Method'I Automated Manual Analysis CV 1%) n CV 1%) n DNA’ 1.02 44 1.47 48 Protein‘ 0.92 47 I . 14 33 NAGaser 0.71 2 1.55 12 6PGDH" 2.03 10 5.30 8 GéPDI-I' 13.4 8 13.6 6 “CV, coefficient of variability: n. number Of analyses. ° Regression analysis of standard curves. ' Regression analysis of enzyme velocity vs sample volume. 1 to'15 ug of DNA. These results, pre- sented in Table 3, indicate that the auto— mated method is more reproducible than the manual method. By increasing the fluorom- eter sensitivity IO—fold, as little as 0.1 ug of DNA could be detected: however, read- ing variability was :: 14%. Recovery of standard amounts of DNA added to samples of whole homogenate was 98%. A compari- son of the diphenylamine and ethidium bro- mide methods for the analysis of DNA in whole homogenate and purified nuclei frac- tions revealed no difference (analysis of variance) in quantities of DNA determined when fresh or once-frozen samples were analyzed (data not shown). A comparison of the time efficiency of performing the auto- mated and manual methods, Table 4, shows the automated method to be roughly 1.7 times more rapid. This comparison does not include the 18-h room temperature incuba- tion necessary in the manual method. Protein Analysis Determination of prOtein concentrations in four different preparations of rat hepato- cyte fraction F (1-4 mg protein/ml) by both 23 AUTOMATED FLUOROMETRIC ANALYSIS TABLE 4 PRODUCTIVITY COMPARISON or Tl-IE MANUAL AND AUTOMATED METHODS Descruaeo Method‘ Automated Manual DNA 44 26 Protein 56 30 NAGase 75 50 G6PDH and 6PGDH 67 48 ‘ Rates (analyseszhi include sample pipetting time. the manual Lowry and the automated fluorescamine methods gave similar results (no difference by analysis of variance). A comparison of the coefficients of vari- ability of regression coefficients for stand- ard curves of the manual Lowry protein assay with the automated fluorescamine as- say (Table 3) shows that the latter method was slightly more reproducible than the manual Lowry method. A comparison of the sensitivity of these methods in analyzing the same amount of protein as determined by the ratio of slopes of standard curves for each method. indicated that the fluo- rescamine method was 4.3 times more sensi- tive than the Lowry method. Table 4 shows that the speed of performing analyses by the automated method is nearly double that of the manual method. N-Acetyl-fi-D—Glucosaminidase Analysis Quantitative agreement between the manual and automated methods for this as- say was obtained (analysis of variance). Using the automated method described. the linearity of N-acetyI-B-D-glucosaminidase activity in isolated rat hepatocyte fraction WI-I with time and sample volume was veri- fied with samples having activities up to 1 nmol 4-methy1umbelliferone per minute. A comparison of the coefficients of variability of the regression coefficient for varying amounts of sample vs N-acetyI-B-D-glu- cosaminidase activity (Table 3), determined either automatically or manually, revealed the former method to be more than twice as reproducible as the latter. A comparison of time efficiency for the automated and manual methods (Table 4) indicated that the automated method was 1.5 times more ef- ficient than the manual method. Typical values of N-acetyI-B-Deglucosaminidase activity in WI! fractions of rat hepatocytes and RH-35 cells are shown in Table 5. Glucose 6-Phosphate and 6-Phosphogluconare Dehydrogenase Analysis Quantitative agreement between the manual and automated methods for these assays were obtained (analysis of variance). Using the automated method, dehydro- genase activity increased linearly with the volume of fraction F assayed up to an activ- ity of 15 nmol NADPI—I per minute. In this continuous assay, linearity of activity with time was determined for every sample using the Kinetics I program. This program dic- tates the storage of fluorescence readings TABLE 5 ENZYME ACTtvmes OF RAT Liven CELLs' Sample NAGase' G6PDH‘ 6PGDH' Rat hepatocytes 408.6 : 55.8 (4) 36.7 : 28.3 (4) 94.8 : 20.9 (4) RH-35 cells 139.0 : 59.0 (5) 48.0 : 15.0 (8) 28.1 e 5.2 (8) " N-AcetyI-B-O-glucosaminidase aetivities are from whole homogenate fractions and dehydrogenase activities are from 22.000g supernatant fractions (F). " nmoles 4-MU min‘I mg DNA". " nmoles NADPH min'l mg protein". 24 KURTZ AND WELLS (7 per second) for three operator-specified intervals of time and then calculates AF per minute and percent deviation from linearity for each analysis. The described assays seldom accelerated or decelerated more than 1% from a linear rate. Comparison of the coefficients of variability of the regres- sion coefficients of varying amounts of sample vs enzyme activity, determined by the automated and manual methods de- scribed (Table 3), indicate that the auto- mated methods are equal to or more re- producible than the manual methods for glucose 6-phosphate and 6-phosphoglu- conate dehydrogenase activity determina- tions respectively. As shown in Table 4, use of the automated method resulted in the proc- essing of samples 1.4 times more rapidly than spectrophotometric analysis using a Gilford 2400-5. Typical values of the dehy- drogenase activities of fraction F from iso- lated rat hepatocytes and RH-35 cells are shown in Table 5. DISCUSSION While we have found the diphenylamine method of Giles and Myers (8) for analysis of DNA entirely adequate for rat liver. iso- lated rat hepatocytes. and RH-35 cells. the automated ethidium bromide method de- scribed gives comparable results and is more rapid and slightly more reproducible. We therefore agree with Karsten and Wollen- berger (7) that the ethidium bromide assay is the method of choice for tissue culture work. In the assay of whole homogenate, samples were somewhat turbid; however. adequate correction for this could be made by the inclusion of apprOpriate blanks. Therefore. fluorometry by surface illumina- tion is not necessary and right-angle i1- lumination can be used. This is in agreement with the findings of others (7,14,15). It was also found that the extensive heating (50°C. 1 h) recommended by van Dyke and Szustkie- wicz(16) was not needed and that incubation at 37°C for 20 min as indicated by Karsten and Wollenberger (6.7) was sufficient. The lower fluorescence per microgram DNA in the automated method compared with read- ings obtained with the manual method (Fig. 4) is due to the smaller size of the flow-through cuvette. This does not present a problem in routine assay since gain ad- justments are made to give a standard to blank ratio of 30 for 10 pg DNA. While quantities less than 1 pg of DNA can be measured, they are accompanied by a cor- responding increase in reading variability. Although unnecessary for these analyses. greater sensitivity could be achieved with this system by use of a time-averaging program. Analysis of isolated rat hepatocyte frac- tion F for protein by the automated fluoresc- amine method described gave results which wereisimilar to those obtained by the Lowry . method. In addition, the method described is much simpler, faster, and as reproducible as the Lowry method over a range of 0.5 to 50 pg of protein. The limit of protein de- tected by the automated fluorescamine method described is 0.5 pg. This sensitivity has proved adequate for analyses of isolated rat hepatocyte and RH-35 cell fractions. The ability to precisely control the timing of multiple reagent additions to large num- bers of samples made the described sys tem particularly useful for endpoint assays of enzyme activities. This was illustrated by the highly reproducible measurement of N-acetyl-fi-D-glucosaminidase activity (Table 3). The values of N-acetyl-B—D-glu- cosaminidase in isolated rat hepatocytes reported in Table 5 (408.6 : 55.8 nmol4-MU min"1 mg DNA“) are slightly lower than those previously reported for whole liver [500-3000 nmol mm" mg DNA" (17,18), assuming 2.4 mg DNA/g fresh tissue (19)] owing to differences in substrates and cell types. Automated methods for the measure- ment of several other lysosomal enzymes are available (20). The values obtained for isolated rat hepa- tocyte G6PDH and 6PGDH (Table 5) using 25 AUTOMATED FLUOROMETRIC ANALYSIS the fluorometric method compare well with results obtained in rat liver by this and other laboratories (12.21.22). The elevation of G6PDH activity in the RH-35 tumor cell line compared with levels in isolated rat hepato- cyte (Table 4) is consistent with the find- ings of Selmeci and Weber (23). For the analysis of DNA. protein, and the activities of the representative en- zymes, N-acetyl-B—D-glucosaminidase, glu- cose 6—phosphate. and 6-phosphogluconate dehydrogenase in isolated rat hepatocytes or Rl-I-35 cells, the methods described are as simple and reliable as the more conven- tional assay methods. The combined auto- mated and fluorometric aspects of the methods described provide for the rapid and reproducible handling of increased numbers of samples derived from relatively small amounts of tissue or cultured cells. ACKNOWLEDGMENT We wish to acknowledge the generous cooperation of the Gilford Instrument Co.. Oberlin. Ohio. for mak- ing advanced-design interface hardware and program assistance available for this study. REFERENCES I. Lowry. O. R.. and Passonneau. J. V. (1972) A Flexible System of Enzymatic Analysis. Aca- demic Press. New York. 2. Udenfriend. S. (1962) Fluorescence Assay in Biol- ogy and Medicine. Vol. I. pp. 96- 124. Academic Press. New York. 3. Seglen. P. O. (1976) in Methods of Cell Biology (Prescott. D. M.. ed.). Vol. X111. pp. 29-83. Academic Press. New York. 4. Ross. 8. D. (1972) Perfusion Techniques of Bio- . Selmeci. L. E., and Weber. G. chemistry. p. 23. Oxford Univ. Press (Claren- don). London. . Szego. C. M.. and Seeler. B. .1. (1973) J. Endu- crinol. 56. 347- 360. . Karsten. U.. and Wollenberger. A. (1972) Anal. Biochem. 46, 135-148. . Karsten. U.. and Wollenberger. A. (1977) Anal. Biochem. 77. 464-470. . Giles. K. W.. and Myers. A. (1965) Nature iL'un- don) 206. 93. . BOhlen. P.. Stein. 8.. Dairman. W.. and Uden- friend. S. (1973) Arch. Biochem. Biophys. 155. 213-220. . Lowry. O. H.. Rosebrough. N. 1.. Farr. A. L.. and Randall. R. J. (19511 J. Biol. Chem. 193, 265- 275. . Robins. E.. Hirsch. H. E.. and Emmons. S. S. (1968) J. Biol. Chem. 243. 4246-4252. . Rudack. D.. Chisholm. E. M.. and Holten. D. (1971)]. Biol. Chem. 246, 1249-1254. . Steel. R. G. D.. and Torrie. .1. H. (1960) Principles and Procedures of Statistics. McGraw-l-lill. New York. - . Boer. G. J. (1975) Anal. Biochem. 65, 225-231. . El-Hamalawi. A. A.. Thompson. 1. S.. and Barker, G. R. (19751.4nal. Biochem. 67, 384—391. . van Dyke. R.. and Szustkiewicz. C. (1968) Anal. Biochem. 23, 109-116. . Conchie. J., Findlay. 1.. and Levvy. G. A. (1959) Biochem. J. 71, 318-325. . Sellinger. O. 2.. Beaufay. H.. Jacques. P.. Doyen. A.. and de Duve. C. ( 1960) Biochem. J. 74, 450- 456. . Schneider. W. C. (I945)J. Biol. Chem. 161. 293- 303 . . Tappel. A. I... (1972) in Lysosomes: A Laboratory Handbook (Dingle. J. T.. ed.). pp. 136-149. American Elsevier. New York. . Schroeder. H. R.. Gauger. .1. A.. and Wells. W. W. (1976) Arch. Biochem. Biophys. 172. 206-214. . Clock. G. 5.. and McLean. P. (1954) Biochem. J. 56, l7l-l75. (1976) FEBS Lett. 61, 63-67. Chapter 11 Induction of Tyrosine Aminotransferase in RH-35 Cells and in Rat Liver. Investigation of the Involvement of Lysosomes. ABSTRACT Lysosomes have been implicated in the process of translocation of the estradiol-receptor complex to nuclei of target tissues shortly after exposure to this hormone (Szego, C.M. (1974), see reference 2). The glucocorticoid responsive system, induction of tyrosine aminotransferase (TAT) in RH-35 cells in culture and in adrenalectomized rats, was used to determine if glucocorticoids elicit a similar nuclear translocation of lysosomes or lysosomal enzyme activity. TAT activity of confluent monolayer cultures of RH-35 cells incubat- ed with 1 uM dexamethasone (optimal dose) increased to a maximum of 8-fold above control after 8 hours of incubation. Hydrocortisone (2 uM) produced a slightly lower (3-fold) increase in activity in a 5 hour incu- bation. Isolated nuclei were examined for the lysosomal hydrolase acti- vities: acid phosphatase, N-acetyl-B-Q-glucosaminidase, cathepsin D, and a-glucoronidase at various times after addition of 1 uM dexamethasone to cells. In separate studies, RH-35 cells were stained for acid phsopha- tase and examined by electron microscopy at various times after addition of 1 uM dexamethasone. These experiments revealed no consistent change in the association of lysosomal hydrolases with nuclei of induced cells 26 27 compared with those from control cells. An analogous j__vivo experiment was performed by injecting an inducing dose of hydrocortisone (10 mg/100 g body weight) into adrenalectomized rats. Nuclear acid phosphatase activities were lower after adrenalectomy and were further decreased or unchanged at early times after hydrocortisone injection. Comparison of RH-35 cell and normal isolated hepatocyte nuclear N-acetyl-81Q-glucosa- mindase activities revealed a 9-fold greater association of activity with RH-35 cell nuclei despite lower RH-35 cell activity per cellular DNA. The results presented here do not support the extension of Szego's hypothesis to include glucocorticoid action. The possibility remains that such a phenomenon occurred but at a magnitude not detectable by the present methods. INTRODUCTION The current understanding of glucocorticoid hormone action has recently been thoroughly reviewed (1). One aspect of glucocorticoid action about which little is known is the means by which the hormone-re- ceptor complex migrates through the cytosol to the nucleus. Szego has proposed a mechanism for the transfer of the estrogen-receptor complex to the nucleus of extrogen sensitive-target cells (2). Within 1 to 2 minu- tes after administration 1 vivo of physiological amounts of 17B-estra- diol, the acridine orange stained lysosomes of preputial gland and uter- ine cells were observed, by fluorescence microscopy, to localize in and about nuclei (3, 4). Control injections of 17a-estradiol were ineffec- tive in eliciting this response and non-target tissues were unresponsive to 17e-estradiol (3). Nuclei purified from these tissues by conventional procedures showed 15-fold increases in the lysosomal: acid phosphatase, B-glucuronidase, and acid ribonuclease II within 15 min of 175-estradiol injection (4). Examination of lysosome enriched fractions from preputial gland cells of ovariectomized rats revealed the presence of estradiol receptor proteins (5). The present investigation was designed to examine the possibility that lysosomes are involved in the translocation of the glucocorticoid- receptor complex to the nuclei. Induction of TAT in RH-35 cells was the hormone responsive system chosen because translocation of the hormone-re- ceptor complex to the nucleus is well documented as an integral part of the induction of TAT (1). 28 MATERIALS AND METHODS Cell culture conditions. Reuber H-35 hepatoma cells obtained from Dr. Richard N. Hanson were grown in MEM containing 10% FBS and normal amounts of non-essential amino acids as previously described (6). During culture, medium was changed every four days. Eighteen hours prior to experiments, FBS-containing medium was removed, the cells were rinsed once with PBS-deficient medium then replenished with 10 ml FBS-free medium. Experiments were, as indicated, begun either by changing medium to one containing the experimental conditions or by addition of a concentrated stock solution of the experimental compounds to incubation medium. In initial studies, cells were detached from the surface of the T-flasks by a 6 min trypsinization at 37°C in an atmosphere of 95% air/5% C02 using a 0.25% trypsin in a solution containing (per liter): 1 9 glucose, 8.9 NaCl, and 0.4 9 KCl. The cell suspension was then decanted into a graduated conical centrifuge tube and centrifuged 4 min at 3/4 full speed in a table top centrifuge (IEC, Model HN), washed once with 6 ml 0.32 M sucrose containing 3 mM MgCl2. The cell pellet was then brought to 3 ml with 0.32 M sucrose, 3 mM M9012 and homogenized and centrifuged as described previously (6) to obtain 800 x g-10 min and 22,000 x g-15 min supernatants and purified nuclei. In later experiments, cells were washed twice with 0.32 M sucrose, 3 mM MgClz while attached to the flasks and harvested in 0.32 M sucrose, 3 mM MgClz with a rubber policeman in a final volume of 3 ml. Homogenization and fractionation were then performed as indicated above. Analysis of enzyme activity, DNA, and protein concentration. Tyrosine aminotransferase (EC 2.6.1.5) activity was determined 29 30 according to the procedure of Granner and Tomkins (7) in the presence of 2 mM diethyl-dithiocarbamate which is used to inhibit breakdown of the endproduct, p-hydroxyphenylpyruvate, by p-hydroxyphenylpyruvate oxidase. Due to a slight drift in the absorbance of p-hydroxyphenylpyruvate (A331nm) with time, readings were taken at 60 min after stopping reactions. Assays were begun by the addition of sample to complete reaction mixtures pre-incubated to 37°C, then stopped by addition of KOH as indicated (7). The pH optimum for RH-35 cell TAT was determined to be 7.6. One unit (U) of activity is 1 umole p-hydroxyphenyl pyruvate formed per minute. Cathepsin 0 (EC 3.4.23.5) was determined by the [3HJ-acetyl hemo- globin method of Barrett (8) with slight modification. The substrate, [3HJ-acetyl hemoglobin, was prepared according to the procedure of Barrett (8) and had a specific activity of 0.02 uCi/mg soluble hemo- globin. The reaction mixture contained 3.6 mg of [3H]-hemoglobin, 0.24 M formate buffer, pH 3.0, 0.1% triton X-100 and sample and water to a final volume of 210 pl. Assays were started by the addition of sample (86 ul) to the assay mixture which has been pre-incubated 5 min at 45°C. Incubations were 90 min at 45°C and were stopped by the addition of 1 ml 3% (w/v) TCA at 4°C. The sanmles were centrifuged at 1100 x g for 15 min. Aliquots (0.5 ml) of the supernatant were counted in Bray's solu- tion (9) in a Beckman CPM-100 liquid scintillation spectrometer.. The pH optimum for this enzyme in RH-35 cells was determined to be 3.0. Activity is expressed as TCA soluble tritium cpm per mg DNA in a 90 min. incubation. 31 a-glucuronidase (EC 3.2.1.31) activity was determined at 25°C using the fluorometric method of Robins £3 21, (10). The pH optimum of this enzyme in RH-35 cells was detennined to be 3.6. N-acetyl-s-D-glucosaminidase (EC 3.2.1.30) (hexosaminidase) activity and protein and DNA concentrations were determined fluorometrically as previously described (6). For both B-glucuronidase and hexosaminidase activities, one unit (U) is 1 umole 4-methylumbelliferone released per minute under the assay conditions. Electron microscopy. At varibus times after addition of dexamethasone to confluent T-flasks of RH-35 cells, medium was decanted and cells were fixed with 5 ml of 2% glutaraldehyde (10% stock glutaraldehyde from Electron Microscopy Sciences diluted 1/5 with 0.1 M Na cacodylate, pH 7.2, just before use). While cells were being fixed they were scraped from T-flasks into 10 x 1 cm test tubes and incubated at room temperature a total of 20 min. Cells suspensions were centrifuged as described above for cell harvesting and washed once with 0.15 M Na acetate buffer, pH 5.0. Cells were then resuspended in Gomori acid phosphatase stain (11) and incubated for 40 min at 37°C in the absence and presence of 0.01 M NaF which inhibits acid phosphatase and therefore served as a control for acid phosphatase staining. Following this incubation, cells were washed twice with 0.4 M sodium acetate buffer, pH 5.0 to remove non-specific staining. Cells were dehydrated by washing consecutively in 25, 50, 75, and 95% ethanol solutions, 10 min in each and finally for 30 min in 100% ethanol. Samples were then given to June Mack of the electron microscopy facility of the Pesticide Research Center, Michigan State University. Cells were embedded in Epon-Araldite and ultrathin sectioned with a Sorvall MT—2 ultramicrotome. Sections were stained with saturated uranyl 32 acetate and post-stained in a saturated lead citrate solution. Cells were then examined using a Philips 300 transmission electron microscope. In vivo studies. Male Holtzman rats 180-200 g were adrenalectomized or sham operated according to the procedure of Zarrow_gt_al. (12) and given lab chow and 1% NaCl as a drinking solution, ad libitum. Four days after adrenalectomy, rats were starved for 19 h then injected with hydrocorti- sone 21-Na succinate (10 mg/100 body weight) or saline and sacrificed by decapitation at various times after injection. Livers were rapidly removed and placed in 0.32 M Sucrose 3mM MgClz at 0-4°C. Four grams of liver were minced with scissors then combined with 23 ml 0.32 M sucrose 3 mM MgCl2 and homogenized 305 at 65V with a Tehmar homogenizer. The whole homogenate was filtered through 4-ply cheese-cloth and centrifuged at 800 x.g for 10 min. The 800 x g supernatant was then centrifuged for 15 min at 22,000 x g to obtain a supernatant containing free lysosomal enzyme activity. The entire 800 x g pellet was mixed with 20 ml of 2.4 M sucrose and 1 mM MgC12 and centrifuged for 45 min at 30,000 rpm in a Beckman L-2 ultracentrifuge with a 30K rotor. The purified nuclear pel- let was carefully resuspended in 1.5 ml 0.32 M sucrose 3 mM MgCl2. DNA analysis was performed by the method of Giles and Meyers (13) as describ- ed previously (6). RESULTS Development of the tyrosine aminotransferase induction system. Reuber H-35 cells grew in MEM containing 10% FBS at a logarithmic rate as shown in Figure I. Doubling time for the cells during the logarithmic phase of growth was 24 hours. Cells were routinely innoculated at a density of 106 cells per flask and used in experiments 1 week later a density of 4 x 107 cells per flask. At this stage, cells were in a confluent monolayer and, as indicated in Figure 1, not rapidly replicat- ing. The induction of TAT in RH-35 cells incubated in the presence of dexamethasone is demonstrated in Figures 2 and 3. As shown in Figure 2, the optimum dose of desamethasone for the induction of TAT was approxi- mately 1 uM. TAT activity increased rapidly during the first five hours after addition of dexamethasone (Figure 3) then leveled by 8 hours at an 8-fold elevation above control activity and began to decline slightly after 10 hours of incubation. During the same time period, the activity of TAT remained constant in cells incubated in the absence of dexametha- sone. RH-35 cells were also responsive to hydrocortisone. In experi- ments not shown here, TAT specific activity increased 3-fold in RH-35 cells incubated 5 hours with 2 uM hydrocortisone 21-succinate as compared with activities of control cells incubated 5 hours in the absence of steroid. The time course and extent of induction of TAT by dexamethasone in RH-35 cells are in general agreement with the findings of others (21). Lysosomal enzyme activity of nuclei during induction of TAT. In order to determine if glucocorticoids stimulated an increase in liver nuclear lysosomal enzyme activity in a manner similar to that reported by 33 34 Figure 1. Growth Curve for RH-35 Cells. 3.4 x 105 cells were innocu- lated into tissue culture flasks containing 15 ml MEM with 10% FBS and incubated as described in Materials and Methods section. Medium change is indicated by arrows. Each point is the mean i 5.0. for three or four flasks. TOO .4 O (106) 1.0 CELL NUMBER 0.1 35 ‘ d —( —l I Z —( - Illl ‘ -l c- — d .1 - fl - d .1 q - 1 d "‘ 1 d ‘1 J t 1 l 1 I J l J j J J 3‘ O 5 15 18 Figure 1. 10 DAYS Growth Curve for RH-35 Cells. 36 Figure 2. Effect of Dexamethasone Concentration on Tyrosine Aminotrans- ferase Activity. RH-35 cells grown to confluency were incubated in FBS- deficient medium 19 hours prior to initiation of induction. At 0 hour, 1 ml of concentrated stock solutions of dexamethasone were added to the incubation medium resulting in the final concentrations indicated. Cells were incubated 12 hours with dexamethasone then harvested by trypsiniza- tion, fractionated, and analyzed as described in Materials and Methods. Values are means i 5.0. for three flasks. TAT ACTIVITY (M U/MG PROTEIN) 50 43 C) (H C) 37 '41 ‘ T I l l T T l {P l l L l 1 NONE 9 8 7 6 5 -LOG [DEXAMETHASONE](M) Figure 2. Effect of Dexamethasone Concentration on Tyrosine Aminotransferase Activity. 38 Figure 3. Time Course of Induction of Tyrosine Aminotransferase in RH-35 Cells by Dexamethasone. Conditions were as described in Figure 2. Dexamethasone concentration was 1 uM. Values are means 1 5.0. for three flasks. (MU/MG PROTEIN) TAT ACTIVITY I00 ‘1 C” 50 25 39 +DEXAMETHASONE \ ---.. CONTROL\ ................ 1C) I l 1 l J l I o 2 4‘6 8 IO l2 HOURS Figure 3. Time Course of Induction of Tyrosine Aminotransferase in RH-35 Cells by Dexamethasone. 4O Szego gt 31. (2) for estradiol action in rat uterus and preputial glands, the following experiments were performed. Figure 4A shows the time course of nuclear hexosaminidase activity in RH-35 cells incubated in the presence and absence of 2 uM dexamethasone. Nuclear hexosaminidase acti- vity of control and dexamethasone treated cells did not differ except at the 3.5 hour time point where control nuclear activity was nearly twice that of dexamethasone treated cells. Hexosaminidase activities determin- ed in the absence of 0.2% triton X-100 were 50-70% of the activity in the presence of triton X-100 and paralleled activities determined with deter- gent. This constant amount of structural latency is consistent with the presence of lysosomes in the preparations of nuclei. TAT activity of the dexamethasone treated cells increased 4 to 5-fold during this time course (Figure 4B). In this experiment the induction was begun by a change of median. In order to minimize non-hormone effect of medium change, in the experiment shown in Figure 5, a small amount of concentrated dexametha- sone was added to cultures without changing the medium. Also, earlier time points were examined. Nuclear a-glucuronidase, cathepsin D, and hexosaminidase activities changed in parallel during the course of incu- bation of RH-35 cells with 1 uM dexamethasone. This observation is con- sistent with the presence of a single class of lysosomes in nuclear frac- tions. Hexosaminidase activity of nuclear fractions, but not that of e-glucuronidase or cathepsin D, was significantly elevated at 15‘min com- pared with 0 min. However this increase proved to be irreproducible. In order to determine if the association of lysosomes with nuclei was a loose one which was perhaps disrupted by homogenization and purifi- cation of nuclei, RH-35 cells were examined microscopically after the 41 Figure 4. Time Course of Tyrosine Aminotransferase and Nuclear Hexosa- minidase Activity in RH-35 Cells Incubated with Dexamethasone. Confluent cultures of RH-35 cells were incubated in FBS-deficient EMEM for 19 hours prior to the start of the experiment. At 0 time, medium was changed to PBS-deficient EMEM with (circle) and without (squares) added dexametha- sone at a concentration of 2 x 10 uM. At the times indicated, cells were harvested by trypsinization then fractionated and analyzed as described in Materials and Methods. Purified nuclei (panel A) were assayed for hexosaminidase activity with (closed symbols) and without (open symbols) a final concentration of 0.2% triton X-100. TAT activity (panel B) was determined in 22,000 x g supernatants as described in Materials and Methods. Values are means i 3.0. for three flasks. 42 O) O SE(NIU/Mo DNA) :s O TAT ACTIVITY(MU/NIG erect) NUCLEAR HEXOSAMINIDA Figure 4. Time Course of Tyrosine Aminotransferase and Nuclear Hexosaminidase Activity in RH-35 Cells Incubated with Dexamethasone. 43 .mxmmpwuh snow cow .o.m w mcmms mcm mmzpm> .waum o cpmgmgumu use Puum mmmvvcoczospmim use mmmww Icvsmmoxm: .cwvuumm muosumz ms» :. vmnpcummu mm umumcopuumce new Npumz 25 m .mmoeuam z mm.o _E m.¢ :* :mEmuwpoa emanac a new: umamm>eun mcm: mppmu .umumopvcw mmswu ms» u< .2: H mo comuocucmucou pm=_$ m on mcommgpmsmxmu xuoum umpmeucmucou mo coppwuum mg» »n gamma mm: acmewcmaxu .uemswcqum mg» mo acmum mzu on cowca mesa; Na 2“: acmwuwmmcimmm P5 o“ cw cmumnaucp mem: mF—mu mmuzm mo mmcsupzu ucmspw Icou .mcommsummemo new; umpmnsucH m~_mu mmizm some Pm—uaz we apw>wuo< mea~cw .meomomam .m mczmwm 44 .mcommcumsmxmo saw: umumaaucm mp_mo mmnzm some _m_u:z mo xu_>_uu< msa~cm Pmsomomxg .m mcam'm 2.3 ms. : .om. _ .wmr . mu m.nv A A J/ $32332 m: 58.28388 i . n All/H.LOV 45 addition of 1 uM dexamethasone. At various times after hormone addi- tions, cells were fixed, stained for lysosomes by the Gomori's lead medi- um (11), then further prepared and examined under the electron micro- scope. The results of this experiment are shown in Figure 6A-F. Lyso- somes are identifiable by the halo of electron opaque lead phosphate which precipitates around the lysosome as the result of a reaction of lead in the medium with the phOSphate liberated from B-glycerophosphate by the action of acid phosphatase. Golgi and endoplasmic reticulun are known to stain by this method (11) as is evident in Figure 6. There was no evidence for a fusion or peri-nuclear localization of lysosomes in the cells examined. Figure 6E, 10 min after exposure of cells to 1 uM dexa- methasone, showed a close association of lysosomes with nuclei but this was not a common observation. . In order to determine whether changes in nuclear lysosomal enzyme activities were small in relation to endogenous activity and therefore difficult to demonstrate, nuclear hexosaminidase and acid phosphatase activities were determined in normal and RH-35 cells. Results shown in Table I indicated that RH-35 cells had 3-times more hexosaminidase acti- vity per mg nuclear DNA than normal hepatocytes. As a percentage of total cellular hexosaminidase, RH-35 cell nuclei had 9-fold more hexosa- minidase activity than present in hepatocyte nuclei. Preliminary examin-' ation of acid phOSphatase activities indicates the reverse pattern of association. In order to examine the possibility that glucocorticoids in 1119 might therefore stimulate an observable increase in nuclear lysosomal enzyme activity in a manner similar to estradiol action reported by Szego, the following experiment was performed. Rats were 46 Figure 6. Electron Microscopic Examination of Intracellular Lysosome Distribution after Treatment of RH-35 Cells with Dexamethasone. Conflu- ent cultures of RH-35 cells containing FBS-deficient MEM were exposed to dexamethasone (1 uM). At (A) 0 time, (B) 0.5 min, (C) 2 min, (0) 4 min, (E) 10 min, and (F) 30 min, medium was decanted and cells were fixed for 20 min with 2% glutaraldehyde then stained 40 min with Gomori acid phos- phatase stain. Cells were then dehydrated with ethanol, embedded in Epon-Araldite, thin sectioned and stained with saturated uranyl acetate then post-stained in saturated lead citrate. Magnifications are 10,000 and 40,000. 47 B 0.5 minutes, 10,000X Figure 6 A and 8. Electron Microscopic Examination of Intracellular Lysosome Distribution after Treatment of RH-35 Cells Ni th Dexamethasone. 48 C 2 minutes, 10,000X J}: J; D 4 minutes, 10,000X Figure 6 C and 0. Electron Microscopic Examination of Intracellular Lysosome Distribution after Treatment of RH-35 Cells With Dexamethasone. 49 F 30 minutes, I0,000X Figure 6 E and F. Electron Microscopic Examination of Intracellular Lysosome Distribution after Treatment of RH-35 Cells With Dexamethasone. 50 Table I. Enzyme Activities of Rat Hepatocyte and RH-35 Cell Fractions.1 ' Activity Fraction Enzyme Rat hepatocytes RH-35 cells WH hexosaminidase 408.6 1 55.8 139.0 1 59.0 acid phosphatase 66.1 1 2.3 9.20 1 4.40 PN hexosaminidase 6.31 1 3.68 19.2 1 14.7 acid phOSphatase 4.71 1 0.82 2.99 1 1.08 (PN/WH)100 hexosaminidase 1.39 1 0.72 12.6 1 5.9 acid phosphatase 7.2 1 1.4 41 1 26 1Whole homogenate (WH) and purified nuclear (PN) fractions were pre- pared as described under Methods. Hexosaminidase activity is mU 4-MU/mg DNA. Rat hepatocyte values are from 3 rats with 11 observations and RH-35 cell values are from 2 different passages of cells with 5 observa- tions. Acid phosphatase activity is nmoles Pi/hr/mg DNA. Rat hepato- cyte values are from 1 rat with 3 observations and from 1 passage of RH-35 cells with 3 observations. Rat hepatocyte values are from 4 rats with 14 observations and RH-35 cell values are from 3 different passage of cells with 8 ovservations. Values are means 1 5.0. with 4 x 10 cells per observation. adrenalectomized and maintained for five days to remove endogenous gluco- corticoids. Rats were then starved for 18 hours in order to put them in a physiological state which would require glucocorticoids. Animals then received 10 mg/100 g body weight hydrocortisone 21-succinate and were sacrificed at various times after injection. This dose of hydrocortisone is optimal for the in lilQ induction of TAT in adrenalectomized rats (14). Purified liver nuclei were isolated and analyzed for lysososmal enzyme activity at various times after injection. The results of two such experiments are shown in Figure 7A and 7B. In one experiment (Figure 7A) nuclear cathepsin D and acid phosphatase activities declined steadily after injection of hydrocortisone to 60 and 40% respectively of initial activities at 45 min after injection. In a second experiment (Figure 78) it was noted that adrenalectomy alone lowered the nuclear acid phosphatase activity to values 50% of those in unoperated or sham operated rat liver nuclei. Injection of hydrocortisone into adrenalec- tomized rats had no effect on nuclear acid phosphatase during the early period of TAT induction. Injection of unoperated or sham operated rats with saline decreased nuclear acid phosphatase activity to values 70% of initial activities at 60 min after injection. Saline injection into adrenalectomized rats slightly increased nuclear acid phosphatase activity by 60 min after injection. 52 .ucvoa 1mm mace m to» .o.m H mamma mcm mmspm> .cmuomwcw mcom_ucooocuxs .vaPEopomp -mcmccm mvmaumncw mew—mm .um~FEouom_m:meum mumuommcwca .umNVEouumpmcmeum mumpumncw mcwpwm .umumcmao Emzm mumuomncwca .vmumcmao Emcm mumuumncw m=_Pcm .vmumcmaocz mumuumncwcz .vmumcmgocz ”mponexm .mwm»_m=m »u_>_uom msx~cm FmEOmoma— to» mac—punt» campus: umwwwesa :Pmuao op muocumz can mpmwcmmmz cw vmnpeummv mm cmmmmuoca mcmz mcm>wp vcm cowaumwcw cmuem m—m>emu:_ cme_u um umupmpcumm mcmz m—mswc< .mcppmm co azmwmz mean a oofi\mpmcvooam Eswvomnam mcomvucooocu»; me OH new: :oPpommcw cu cowea ; ma um>cmam cmza .macu m cow umcwmucwme .umumcmao Emsm Lo umNWEouumpmcmccm mcmz Am mmfiicmav mum; mpwz .cmwmmu mamm mg» mo mpcmswcmaxm muacmamm o3» mew m was < mpmcmm .mumcvuuam E=_comIHN m:Om_ucouoevx: mo cowuumhcu cmuwm mama cm~_soaumpmcmcc< soc» wmpozz cm>p4 we apw>_gu< machm PmEOmomam .N mesmwm .mumzwuuam e=_uom-- mcomwucouoccax eo :owuumncm empmm mpmm vm~_sopumpmcmec< some Pmpusz em>m4 mo pr>w¢u< mstcm Fmeomomxg .u mezmwm 53 wmzbzi mm...32_§ N on oe on om 0. cm 6 n . . T . . o m mzom_Eooomo>:. W a co. . V com 0 .. com 0 M r com A O . . \ fi mmfiéemoze nun” ooe W .. 9% 00¢ u «a a q q q q 0“ com I oo. . mm. I on. o 263120 .. m: ALIAILOV EWAZNB ENE—ICON DISCUSSION Following diffusion into the cell (15, 16 ), glucocorticoids bind to glucocorticoid binding protein G (17) which is involved in TAT induction by stimulating RNA synthesis (18, 19 ). The mechanism by which the glucocorticoid-receptor G complex moves to the nucleus is unknown. Most authors suggest that translocation of steroid-receptor complexes from the cytoplasm into the nucleus is dependent upon the binding of steroid-re- ceptor complexes to high affinity binding sites of chromatin (20). Move- ment to the nucleus would occur by diffusion in order to maintain a homo- genous intracellular distribution of free receptor-hormone complexes. Gannon gt 31. (20) suggest that the hormone-receptor complex is "excluded" from the cytoplasm and preferentially accumulated in the nucleus due to the high intranuclear water content (1.6:1 = nuclear Hgozcytosol H20). Szego has evidence (2) that lysosomes move to and associate with nuclei in response to estradiol stimulation of target cells and that in this way the estradiol-receptor complex is transported into the nucleus. Of primary importance is the demonstration that the RH-35 cells in culture respond in a typical fashion to added glucocorticoids. The results presented in Figures 1, 2, and 3 confirm the well documented growth characteristics and inducibility of TAT in RH-35 cells in culture in response to added dexamethasone (21). The value of total rat hepato- cyte acid phosphatase reported in Table I (66.1 1 2.3 nmoles Pi/hr/mg DNA) is in agreement with that reported by Munthe-Kaas st 21. (22) (approximately 100 nmoles Pi/hr/mg DNA). The hexosaminidase value reported in Table I (408.6 1 55.8 nmoles 4 MU/min/mg DNA) is lower 54 55 than that reported by Munthe-Kaas 23431. (approximately 900 nmoles p-nitrophenol/min/mg DNA) (22). This discrepancy could be explained by differences in temperature of assay (37°C for Munthe-Kaas gt a1. (22) versus 25°C for results presented here) as well as the known variations of hexosaninidase activity towards different substrates (23-25). The lysosomal enzyme activities of RH-35 cells and nuclear fractions of nor- mal hepatocytes have not been previously reported. The remaining experiments conducted here (Figures 4 to 7) were per- formed to examine the possibility that the translocation concept of Szego, proposed originally for estradiol action (2), could be extended to include glucocorticoids. This seemed likely because of the many similar- ities of the early events of estradiol (26) and glucocorticoid (1, 27, 28) action i.e., steroid hormones, cytosol receptors of 200,000 MW, tem- perature dependent conformational change of receptor, translocation to the nucleus, binding to DNA, and specific stimulations of transcription. The biochemical and microscopic results presented here (Figures 4-7) do not support the extension of Szego's model for estradiol action to include glucocorticoid action in RH-35 cells or rat liver. Since the method of nuclei preparation was according to Szego and Seeler (3) and the enzyme analyses used were sufficiently sensitive to detect changes of the magnitude reported (10-15 fold) in “purified nuclei" of preputial glands stimulated with estradiol (4) it is difficult to suggest that such a phenomenon occurred but was not detected. An alternate possibility is that liver lysosomes do move to the nuclei in response to glucocorticoid stimulation but not to the same extent as observed in preputial glands or uteri under the influence of estradiol. Examination of this possibility 56 would require the development of more sensitive techniques. Of interest is the finding (Table I) that nuclei of RH-35 cells have higher activi- ties of hexosaminidase than normal rat hepatocytes despite lower activity per total cellular DNA. A preliminary comparison of RH-35 and normal rat hepatocyte nuclear acid phosphatase activities revealed the opposite trend (Table I). An intriguing possibility is that this is a general characteristic of tunor cells. 11. 12. 13. 14. 15. 16. 17. 18. REFERENCES Rousseau, G.G. and Baxter, J.D. (eds.) Glucocorticoid Hormone Action (1979) Springer-Verlag, Berlin Szego, C.M. (1974) in Recent Progress in Hormone Research, (R.0. Greep, ed), Vol. 30, pp. 171-233 Academic Press, Inc., New York Szego, C.M. and Seeler, B.J. (1973) J. Endocr. 56, 347-360 Szego, C.M., Steadman, R.A., and Seeler, B.J. (1974) Eur. J. Biochem 46, 377-386 - Hirsch, P.C. and Szego, C.M. (1974) J. Steroid Biochem. 5, 533-542 Kurtz, J.W. and Wells, W.W. (1979) Anal. Biochem. 94, 166-175 . Granner, D.K. and Tomkins, G.M. (1970) in Methods in Enzymology (Tabor, H. and Tabor, C.N., eds.) Vol. XVIIA, p. 633-637, Academic Press, New York ‘ Barrett, A.J. (1972) in Lysosomes. A Laboratory Handbook (J.T. Dingle, ed.), p. 104, North-Holland Publishing Co., London Bray, G.A. (1960) Anal. Biochem. 1, 279-285 . Robins, E., Hirsch, H.E., and Emmons, 8.5. (1968) J. Biol. Chem. 243, 4246-4252 Essner, E. (1973) in Electron Microscopy of Enzymes. Principles and Methods (Hayat, M.A., ed.) Vol. 1, pp. 44-76, Van Nostrand Reinhold Co., New York Zarrow, M.X., Yochim, J.M., McCarthy, J.L. and Sanborn, R.C. (1964) Experimental Endocrinology, pp. 194-196, Academic Press, New York Giles, K.W. and Myers, A. (1965) Nature 206, 93 Kenney, F.T. and Flora, R.M. (1961) J. Biol. Chem. 236, 2699-2702 Rao, H.L., Rao, G.S., Holler, M., Brener, H., Schattenberg, P.J., and Stein, W.D. (1976) Hoppe-Seyler's Z. Physiol. Chem. 357, 578-584 Plagemann, P.G.W. and Erbe, J. (1976) Biochem. Pharmacol. 25, 1489-1494 Beato, M., Schmid, W., and Sekeris, C.E. (1972) Biochim Biophys. Acta 263, 764-774 Peterkofsky, B. and Tomkins, G.M. (1968) Proc. Nat. Acad. Sci. 60, 222-228 57 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 58 REFERENCES Sekeris, C.E. and van der Meulen, N. (1974) Acta Endocronologica Suppl 191, 173-190 Gannon, F., Katzenellenbogen, B., Stancel, G., and Gorski, J. (1976) The Molecular Biology of Hormone Action, pp. 137-149, Academic Press, Inc., New York, NY‘ Reel, J.R., Lee, K.L., and Kenney, F.T. (1970) J. Biol. Chem. 245, 5800-5805 Munthe-Kaas, A.C., Berg, T., and Seljelid, R. (1976) Expt. Cell Res. 99, 146-154 Conchie, J., Findlay, J., and Levvy, C.A. (1959) Biochem. J. 71, 318-325 Weissmann, B., Hadjiioannou, S., and Tornheim, J. (1964) J. Biol. Chem. 239, 59-63 Findlay, J., Levvy, C.A., and Marsh, C.A. (1958) Biochem. J. 69, 467-476 Jensen, E.V. and De Sombre, E.R. (1973) Science 182, 126-134 Thompson, E.B. and Lippman, M.E. (1974) Metabolism 23, 159-202 Litwak, G. (1979) T185 4, 217-220 Chapter III Induction of Lipogenesis Enzymes in Rat Liver During Starvation-Refeeding. Investigation of the Involvement of Lysosomes. ABSTRACT The intracellular distribution of lysosomal enzymes was examined during the course of G6PDH and 6PGDH induction by a regimen of starva- tion/refeeding. Lysosomal fragility was determined by the non-particu- late activity as a percentage of total activity (i.e., percent free acti- vity) under standard homogenization procedures. For acid phosphatase, hexosaminidase, and B-galactosidase, percentage free activity increased significantly (42, 120, and 42% respectively) in comparison with normal rat liver levels at 9 hours after feeding a high carbohydrate diet to 3 day starved rats. Mitochondrial fragility, as measured by the percentage free fumarase activity, was not significantly altered from a value of approximately 50% during the course of starvation/refeeding. Labiliza- tion or stabilization of lysosomes by a single glucose injection (750 mg/100 g body weight) into fed rats or corticosterone injection (2.5 mg/100 g body weight) during a starvation/refeeding regimen, respective- ly, was not correlated with changes in the capacity for induction of G6PDH and 6PGDH when compared with respective controls. Nuclear hexosa- minidase specific activity but not that of cathepsin D or acid phospha- tase, increased significantly above normal levels during a 3 day starva- tion period. Refeeding of a high glucose diet to 3 day starved rats resulted in no significant alteration in the association of lysosomal 59 6O enzyme activities with purified nuclei compared with activities present in nuclei from 0 hour refed rats. Starved/refed streptozotocin diabetic rats, with or without insulin injection at doses sufficient to induce GGPDH and 6PGDH, showed no significant change in purified nuclear asso- ciated acid phosphatase activity throughout the experiment. The results presented here do not support the role for lysosomes as a mediator of enzyme induction in the starved-refed rat as suggested by Szego (see references 1 and 2) for estradiol and ACTH action. INTRODUCTION Starvation followed by refeeding results in elevations of liver lipogenic enzymes above normal levels (3). The mechanism by which diet and insulin (see Chapter 4) serve to induce G6PDH and 6PGDH is not clear. Szego gt 31. (for review see 1 and 2) have presented evidence that, in estradiol responsive systems, estradiol administration resulted in the rapid (within 2 min) association of lysosomes with nuclei (4,5) as well as lysosome fragility (4,6). 'Previous work from this laboratory (7,8) has indicated that the starvation-refeeding stimulated induction of GGPDH and 6PGDH was associated with lysosome changes observed by Szego gt_al. in estradiol responsive tissues. The purpose of the present investiga- tion was to further examine the involvement of lysosomes, both fragility and nuclear association, in the process of dietary and hormonal stimula- tion of G6PDH and 6PGDH induction. _ 61 MATERIALS AND METHODS Animal treatment. Male rats weighing initially 150-200 g were pur- chased from the Holtzman Co., Madison, WI and housed and fed rat chow (Wayne Lab-Blox, Allied Mills, Inc.) as described (7). The high glucose diet was 68.9% glucose, 20% casein, 5% corn oil, 5% salt mix (Wesson, Osborn modified), 1% vitamin mix (Nutritional Biochemicals Inc.) and 0.1% choline chloride. The Wesson (Osborn modified) salt mix was supplemented to 10 ppm Cu2+ using CuSO4, 50 ppm Mn2+ using MnSO4, and 50 ppm Zn2+ using Zn0 as recommended by Greenfield and Briggs (9). Streptozotocin, a gift from Dr. W.E. Dulin of Upjohn Co., Kalamazoo, MI, was dissolved in 0.1 M sodiun citrate buffer, pH 4.5 and injected into the femoral vein of ether anesthetized rats within 10 min of solution preparation. Hepatocytes were isolated from rats by the collagenase method of Seglen (10) as described in detail in the Materials and Methods section of Chapter 4 of this dissertation. Insulin, Regular and Lente Iletin, 100 U/ml, was from Eli Lilly and Co. Sample preparation. Rats were sacrificed by decapitation and livers quickly removed and placed in 0.25 M sucrose, 2 mM MgClz. Homogeniza- tion and fractionation by differential centrifugation were as described previously (7) to obtain 800 x g - 15 min and 22,000 x g - 15 min super- natant fractions for analysis of lysosomal enzyme activities and a 105,000 x g - 60 min fraction for analysis of dehydrogenase activity. Hepatocytes were fractionated as described previously (11). Purified nuclei were obtained using the procedure of Szego and Seeler (4) except 'high-speed centrifugation was with a fixed-angle rotor (30K) for liver samples and a swinging bucket rotor (SW 27.1) for isolated hepatocytes. 62 63 Enzyme analyses. Acid phosphatase activity was determined at 25°C, pH 5.0 by the rate of release of phosphate from a-glycerophosphate by two different methods. Method 1 was described by Schroeder gt 31. (7) and method 2 was described by Mak and Wells (8). Both methods have the same initial assay conditions which are a modification of those described by Vaes and Jacques (12). The methods differ in the detection of phosphate. Method 1 utilizes an isobutanolzbenzene extraction of TCA soluble phos- phate and method 2 measures free phosphate in the reaction mixture after clarification of samples with sodium dodecyl sulfate. Hexosaminidase activity was determined at 37°C, pH 3.8 by the rate of release of p-nitrophenol from p-nitrophenyl-N-acetyl-B-Q-glucosaminide as described by Schroeder gt_gl, (7). a-Galactosidase activity was determined at 37°C, pH 5.0 by the rate of release of p-nitrophenol from p-nitrophenyl- a-Q-galactopyranoside as described by Schroeder et 31. (7) except that the final substrate concentration was raised to 1.5 mM. B-Glucuronidase activity was determined at 37°C, pH 5.0 by the rate of release of p-nitrophenol from p-nitrophenyl-5-Q-glucuronide as described by Schroeder gt 31. (7). Cathepsin D activity (method 1) was determined at 45°C, pH 3.4 by the rate of release of TCA soluble peptides as tyrosine equivalents from hemoglobin substrate as described by Barrett (13) or (method 2) by the rate of release of TCA soluble [3HJ-acetyl-peptides from [3HJ-acetyl-hemoglobin at pH 3.0, 45°C as described by Barrett (13). Method 2 is described in detail in the Materials and Methods sec- tion of Chapter 2 of this dissertation. Tyrosine equivalents were deter- mined by the method of Lowry gt 31. (14) using L-tyrosine as a standard. All lysosomal enzyme activities were determined in the presence of 0.2% (w/v) Triton X-100 (Rohm and Haas). Fumarase activity was determined at 64 25°C, pH 7.4 by the increase in C-C double bond formation from L-malate as described by Hill and Bradshaw (15). GGPDH and 6PGDH activities were determined by the spectrophotometric method described previously (11). Lactate dehydrogenase activities were determined at 25°C by measuring the rate of reduction of NAD+ in the presence of L-lactate using the lac- tate dehydrogenase kit (LD-P14, Gilford Diagnostics) in conjunction with a Gilford 3500 spectrophotometer. Protein was determined according to Lowry gt El! (14) with bovine serum albumin as the standard and DNA was determined by the diphenylamine method of Giles and Myers (16) as des- cribed previously (11). All biochemicals were purchased from Sigma Chemical Co., St. Louis, MO except where indicated otherwise. Statisti- cal methods were according to Steel and Torrie (17) with a = 0.05. Means not having a common superscript are significatly different as determined by analysis of variance, least significant difference test. RESULTS GGPDH and 6PGDH induction and liver lysosome fragility. Figure 1A and 18 shows the effects of hydrocortisone injection on the fragility of rat liver lysosomes and mitochondria during a starvation/refeeding regimen. The percentage free activity i.e., the activity/ml of the post-mitochon- drial-lysosomal supernatant (22,000 x g - 15 min) as a percent of the activity/ml of the post nuclear supernatant (800 x g - 15 min), is used as an index of lysosome fragility. The percent free activity of liver acid phOSphatase, hexosaminidase, and s-galactosidase of normal control rats were 17.2 1 2.1, 4.1 1 1.0, and 11.8 1 1.6% respectively. Feeding rats a high glucose diet for 8 days led to a slight but not significant elevation in the percentage free activity of acid phoSphatase, hexosamin- idase, and e-galactosidase compared with the percentage free activity of these enzymes in livers of rats fed a chow diet (Figure 1A and 1B). Starvation for 3 days (0 hour refed) led to a significant increase only in percentage free hexosaminidase activity compared with percent free hexosaminidase activities in livers of rats fed a chow diet. Refeeding 3 day starved rats a high glucose diet for 9 hours resulted in significant elevations in the percent free activity of all three liver lysosomal enzymes above the respective percent free activities present in livers of chow diet fed control rats. The percent free activities of acid phos- phatase, hexosaminidase, and e-galactosidase were 24.4 1 2.4, 9.0 1 2.7, and 16.7 1 1.9, respectively. This peak in percent free activity was followed, after 2 days of refeeding, by a return to normal levels of percent free activity. The percent funarase which was free in normal chow fed rats was 49.9 1 6.2%. Starvation-refeeding had no significant 65 66 .mumc a mo .o.m « mew mmapo> .mcao; a van .N .m .m.o .m.ou mcmz mms_u :owpomncm .ugmvmz auon m ooH\mcom_ucouocuxc as m.~ saw: umpumncw memz mum; mumo -Pucw mm_uewu ummopu can umuomncwcz mcmz mum; mamopncm mmFULFu :ch .m chma cw czozm mew mmmcmssm use mmmuwmopumpmmru new < pmcma cw cxozm mcm mmmuwcwsmmoxm; vac AH vozums xn umNAPmcmv mmmumsamosg uwum mo mmwu_>puum mmem ucmuema .ucmuoccmqam Acpe mH I m x comv campuacuumoa ms» to _E\>uw>wuum mg» mo acmucmg a mo accumccmasm Acpe ma I m x ooo.-v Pmsomomprumoa mg» mo FE\>aV>Puom mzu m? xuw> Iwuum emmcw ucmucmme .mvozumz can m—mpcmpmz cm cmnweummc mm cm~x_mcm was cmemamea mcmz mcm>wm .vm -umo_vcw mmswu mzu um umuwmwcomm man .126; c an um_c mmoozpm sow; mew ummmc .mzmu m com vm>empm mcmz m_aewea .emeem. .msae 1 Loc Ameeeuez new mpeweaaaz mamv Amy Same amee=_m new; we“ to ADV ease pee umm memz mpocmcou .umm: mcmz m omNIoom >__mwp_:w mcvugmwmz mum; :mENHFo: mpmz .mumz umwmmivm>cmpm mo zappromcm o_cvco;uoaw: vcm msomong Lm>p4 co cowuumncn mcompucouocvx: mo mommwm .H mezmwm 67 .mmmm umwm¢Ium>emum mo xuwpwmmcm m_1u:o;uopwz ucm msomomz4 cm>wm :o compumncH mcomwucouoeu»: mo uummmm .H mgaapm omumm manor Nb. mwv m n; my o 11 - m - q q u on .. m I 9. -T a... II .I. om I I.u~ I ”1941522... 1 om I 1.9m l I w I . lav. mm_ t 400- . 4 _>. I— SOOF 1 0 < 200- e .L_1 Ioo- é .- 0 . O C G O I 2 3 DAYS REFED Figure 2. Effect of Hydrocortisone Injection on Liver Glucose 6-Phosphate Dehydrogenase and 6-Phosphogluconate Dehydrogenase Activities of Starved-Refed Rats. 71 .mgm.ez seen a ocfi\me m.~ me emameoe .mpmg e we .o.m N mcmme mew mm=Fu> .H mezo,u ow ucmmm. mg» :- umnvcummu acmEFngxm ms» mo :o_um::pu:ou m m, mpghm mm.o a m~.m o~.o « m~.m m a wmm m « mmu can ummmg «um um>cmum mm.o A mm.m -.o « Ne.m Hg « NNN n a new :we cmmmc .cm um>cmum -.o « oH.¢ -.o m ¢~.m m N m- N m m- gm ummmc .um um>cmum o~.o a mm.m m~.o « m~.m o~ « “mm A N HNN cm ummmc .vm um>eoum N~.o a m~.m eH.o N ~¢.m mu a mom a A mfiu sm.~ umwmc .um um>cmum I mfiwo « am.m I ~H u HHN um um>cmum - efi.o a oe.e - m « NAN gape emeeepm ewe I m~.o H ww.v I a a Raw 2656 um; um» + I + I avmuumncp meow—ucouogua; avmuumncp mcompacouocua; acmEpmmc» ARV a “game: seem em_e= ea>13 Amy Seamus xeem .ae_e m.m:_umm»mxI:o_mm>cmum mcpczo mama vmuumncm mcom_ugouogu»: ucu pascoz mo musmpmz xvom can Lm>wm .H m—amh 72 .muee m mo .o.m H mcems mes mmepe> .eso; c we aceemmeme op m>euepme mesa; m.e ecu .m.~ .m.HI memz mms_u copuomncu .pgmemz xvoa m ooH\m:oemumoueueou me m.~ cue: :oeuomncp we mucmmne eo mucmmmea cu emmme mcmpm I new + ms» .ucmaecemazm Aces mHIm x oowv eemposcIumoa mga mo _E\»ue>.uue ms» eo acmuema e we aceaecemqem Aces mHIm x ooo.-v pesomomszumoa mg» we —E\»u.>_uoe mgu me x».>_uoe mmew ucmoemm .meogumz ecu mpeeemuez c. emn_eumme me emeeameq mem: mem>we .emueoeece mme_u me» we emupwpeuem ecu .eao; c we umee mmooapm sm.; ms» emwme .mhee m eow em>eeum mem: apespce eemmmme .mxee a“ eow Amvozpmz ecu m—eeemue: mmmv amen mmouapm saw; may eo 26:6 pee em» memz mpoeucoo .emma mem: m oomIoma appeeurce ocegmpmz mane =eE~u_o= mpeze e~.m a m.e e.em.m e m.eH eH.~ « e.m e.e~.~ « e.e gee eeeee .em ee>eeem e.ee.~ e m.- em.~ 4 e.em e.ee.e e e.e e.e_.o « m.m ee eeeee .em ee>eeem e.e~.e a e.mfi e.ee.HH e m.~m e.ee.e e e.m e.ee.e e e.m em eeeee .em eeeeeem I a.e~.~ e m.m~ - e.ee.~ e e.m em ee>eeem I ee.~ n e.- - em.“ H ~.e ae.e emee=_e ewe I e.ee.~ e e.~H - e.e~.~ « e.e :eee pee eee + I + I . acmEueme» emuumncp mcoempmoo_ueou emuumncp mcoemumomeaeou Ammem av mmeeemouoepecIu Ammem am mmeeezwsemoxmz .meee eeeee-ee>eeem :e App—Fame; msomomxm em>pm co coeuomncm mcoempmooeueou mo pumeeu .Hfi mpne» 73 .peepe: eeee a cep\ee m.~ ee eeeeeee .cpmaoea me\:e mee mmppe>epue mmecmmoeezgmo .HH mpae» o» ecmmmp mgp :p umaweumme pamEeemaxm mg» mo zopuechpcou e we meghm e.e e ~.mep e.em e e.~ep e.pep e e.eem e.oe e e.ep~ ewe eeeeeIIem ee>eepe e.~p p e.ee m.~ e N.me e.~ e e.~p e.p e p.ep ee eeeee .ee eeeeepe e.e e e.ee e.p p e.pe ~.~ e e.ep e.o e e.pp em eeeee .em ee>eepe I .e.m e e.ee I e.e e e.e~ em eeeeepe I ~.p~ e e.eep I e.~ e e.eep peee eeepepe eee I e.ep p o.ee I e.e e e.e~ eeep pee eee + I + I acmEpeme» aumuomwce mcoemamoueueou nempumnce mcoempmouepeou Iowan magma Domflflm eeeeeIee>eepm ep xeeee eee :eeee eespp ee eepppeeep eeeeepeOpepeep ee peace“ .ppp epeee 74 B-galactosidase activity (Table II). Percent free hexosaminidase acti- vity was unaltered by this treatment. Liver percent free a-galactosidase. activity of rats injected with corticosterone was not altered during starvation-refeeding. The ability of rats to induce GGDPH and 6PGDH in response to starvation and refeeding for 2 days was unaltered by injec- tion of corticosterone during the refeeding period. This result is shown in Table III. Liver and body weights for this experiment are shown in Table IV. The effect of glucose injection into fed rats on liver percentage free lysosomal enzyme activity was examined to determine whether glucose was the mediator of the fragility increases observed during refeeding. These results are shown in Table V. Three hours after a single injection of glucose at (750 mg/100 g body weight), there was a significant 3.5, 2.7, and 1.5-fold increase in the percentage free activity of hexosamini- dase, B-galactosidase, and e-glucuronidase respectively above water in- jected control activities at 3 hours after injection. Water injection did not affect the percentage of free lysosomal enzyme activity. Fragil- ity returned to normal one day after glucose injection and remained at basal levels for the remaining two days of the experiment. A single in- jection of glucose into fed rats had no significant effect on the liver activities of G6PDH and 6PGDH (Table V). Liver and body weights for this experiment are alsoshown in Table V. Nuclear lysosomal enzyme activity during starvation-refeeding. Some characteristics of the nuclei isolated by the method described are shown in Table VI. The protein/DNA ratio of purified nuclei of starved-refed rats showed a slight but significant increase (6.8%) in the first 1.5 hours after refeeding (Table VI). Control fed rats had higher nuclear 75 mee mmsHe> .peeeez seen a eop\ee e.~ ee eeeeese .maee m es Io.m « msems .HH mHne» up usmamH msp sH umuHeummu usmsHemsxm ms» mo soHHessHHsou e we mHsHe mo.o H mm.¢ ¢¢.o « ew.e e u mHN uH mH.o « mo.e o~.o a mw.m mH H HRH u ¢~.o a om.m m¢.o a Hm.m mm « NoH mH NH.o « mm.m I u Hope « wH.¢ I H me.c « mm.¢ I mm H 44 NHN me me mom moH mum ewe eeeee «em ee>eepe so umwme .um um>eepm sm umwme .um um>eepm um um>eepm pmHu mmoosHm um» :oso pee umw + I + numuomnsp msoemumouHueou aumuumnsp msoemumouHHLou HsmEuemeH Hay a peepes eeee Hep pempe: gape: easep seem peepe e.asHummemsIsoHpe>eepm msHeso mung umpomnsH msoemumoupueoo use Heseoz es mpsmpm: Huom use em>Hp .>H mHneH '76 .mpee e be .o.m « usems mee mmsHe> .sHmuees asxza mee mm.u.>HHue zscse use :sswc .usepesemssm HsHE mH I m x ocwv eem-uasIumos mgu no He\»u.s.uoe mgp be «smegma e we useaesemaam HsHE mH I a x cco.-V HeeomomaHIHmcs mgp be Hs\»HH>.Hue mg» m. :uHsr mmee psmuems mee mm=He> mmeuHsueausHuIe use .mmeuHmoaue—eoIe .mmeuHsHEemoxm: .muosemz use mHeHemue: s. umnHeummu me umeaHese use umeesms mum: meme—H .umpeUHus. meHu use pe eeppe.epee eee eees e pe psepex eeee a espepe e.p pe A.. ee.p=_em eeep=.e use e ee HI. eepex ea...pe.e ep.1 .s.. umpumns. mem: apes .Haosmsoesa pmHu :csu gee e um; mum: a cemIccm a_—e_p.sH as.;u_m: muee seENHHoz mHe:e eo.e e m.HH em.“ « e.w~ em.e e e.m~ ew.m « m.m~ + eo.e e m.H~ eo.m « a.w~ I ec.e e o.cm I :sosu evIN « ~.wH eHIe e c.mH ea.m a c.- ea.m « c.m~ + em.m e o.mH ew.~ « e.c~ I eH.H « m.m~ I :sscc ec.e « mINH em.. a H.mH em.~ « m.mH u~.m « o.w~ + em.o e e.mH ee.n « n.~H I ec.m « H.oH I mmeupsoesuaHoIe ea~.o « muIa eom.c « ee.a e~u.¢ « KNIm ac.o e m.m~ + emw.c « a~.n emm.o « cm.c I eme.c « em.m I mmeuHmouueHewIe ewm.c « as." e-.o H Hm.m eo~.s « as.~ a~:.~ e H.cH + eaH.c « mm.~ eNm.o a Ho.m I eee.o « cm.~ I mmeuHs H.533: mmHHH>Huu< means“ em>HH cm.c « w~.e mH.o e ms.m m~.c e N~.m nH.c « en.e + mc.c e wa.n mH.o e oo.e I mc.o H oc.m I . . He. pee_e2 eeeeepseee: ee>.p n e men NH « cum «H e HHm we « ccn + H e own nH « on I xH « man I Has peeees eneem Heeee mHeu m wheu w New H K m emmouaa 3.3.8.... eeepppHee eepee eepe e.mees ums s. memesmaceussmo be :u—HuausH use HHHHHaees mesmemxp em>HH so soHHommsH mmou=H¢ be aumeeu msp .> mHee» 77 .muee m seem we: suHs: :umHu mmousHm um»= usmoxm muee e we .o.m n msems mee mmsHe> .2; so» Hs\ms o.e on m.H use :3 so; Hs\me mm.o o» om.o Eoew ummsee msOHHeeusmosoo HH umpmeess ms» use umuesoepueewsz mem: szv mmuesmmosos em>HH mHos: .m mesmeu cu usmmmH msu sH umneeummu usmEHemsxm msu mo soHuessHHsoo e we meshe HéH neoé e we eH.~ e e.mm .3...” umepme .um um>eepm e.ep eeo.e e Ne.~ ee.p e e.em ee.p eeeee .ee ee>eepm e.ep epp.e e ee.~ ee.p p p.ee em ee>eepe m.ep p.eee.o e ee.e ee.p e e.ee pope eeepe_e eee. e.ep pes.c e ee.e em.~ e e.ee seep pee eee ze\=: ze =3 OPHML (ZD\: wwHOLn— acmH—BMOLF e._eppez eeeep pee eeeeeeee eo epppepeeppeeeee .p> e_eee 78 protein/DNA ratios than nuclei of starved rats. This was a reflection of the higher protein content of livers from well fed rats (Table VI) com- pared with the protein content of starved rat liver. Nuclei were 8-10 um in diameter with nucleoli plainly visible. Nuclear lysosomal enzyme activities of rat liver during starvation- refeeding are shown in Figure 3. Liver nuclei from eleven day glucose diet fed rats showed no differences in specific activities of cathepsin D, hexosaninidase, and acid phosphatase compared with the same lysosomal enzyme activities of nuclei from control rats fed a chow diet. Starva- tion for 3 days resulted in a significant increase above control fed levels for liver nuclear hexosaminidase specific activity but not for cathepsin D or acid phosphatase specific activity. Refeeding starved rats a high carbohydrate diet lowered liver nuclear acid phosphatase activity to levels significantly below those of chow diet fed rats but not below those of 0 hour refed rats. Nuclear cathepsin D and hexosamin- idase activities were not significantly altered from starvation levels by refeeding the high carbohydrate diet. Liver and body weights during this experiment are shown in Table VII. Nuclear lysosomal activity was measured after injection of insulin into fed streptozotocin diabetic rats (Figure 4). Acid phosphatase acti- vity decreased significantly 10 minutes after insulin injection then returned to normal by 60 minutes after injection. Nuclear cathepsin D activity did not vary significantly throughout the injection period. Diabetic rats which were starved and refed the high carbohydrate diet, with or without insulin supplementation at doses sufficient to induce G6PDH, 6PGDH, and ME (see Chapter 4 of this dissertation), showed no significant variation in nuclear acid phosphatase or percent free acid 79 Figure 3. Lysosomal Enzyme Activity of Liver Nuclei from Starved-Refed Rats. This is a continuation of the experiment shown in Table VI. Male Holtzman rats weighing initially 150-170 g were used. Controls were fed rat chow (C) or the high glucose diet (G) (see Materials and Methods) for 12 and 11 days respectively. "Refed" animals were starved for 3 days, refed the high glucose diet at 0 hour, and sacrificed at the times indi- cated. Purified liver nuclei were prepared and analyzed for lysosomal hydrolase activities, DNA, and protein content as described in Materials and Methods. Units: cathepsin 0 (method 1), ug tyrosine equivalents/hr/ mg DNA; hexosaminidase, nmoles p-nitrophenol/hr/mg DNA; acid phosphatase (method 1) nmoles Pi/hr/mg DNA. Values are mean 1 S.D. for 4 rats except G which was from 3 rats. 35 8O CATHEPSI N D jEk‘TTTT“-i:} __§§ 25 ‘ I5 1 ‘ I ' >_ 700- " LT. > F3 600i HEXOSAMINIDASE . " 2 500- % ‘ o: < . 33' 40c:1 ‘ J J ' o 7 - " 3 oo 2 60 _ ACID ‘ O PHOSPHATASE I D 400- ‘ 20 I4 ' L——I— ' J—tJ 0o I 2 3 4 6 Figure 3. HOURS REFED Lysosomal Enzyme Activity of Liver Nuclei from Starved-Refed Rats. 81 .mpee m see» me: sues: eumHu mmouaHm um»: psmuxm wees e we .s.m « msems mee mmzHe> .m meaaHe ou usmmmH ms» sH umeeeummu psmEHemsxm msu mo scepessHHsou e mH mHsHe NHIO H ¢HIM ma H NON :mIM Dmmmg .Um vm>gmum w~.o H oo.m eH H ecu :mIH uwwwg .vm U¢>mem mOIO H wwIN mg H oma um U¢>Lmam mH.o e m~.m m e wmm pmHu mmouaHo um» OMIO H NQIM cw fl Hum 30:0 Hog twv Hay enumwnzewwmm wwwmpdmhwn usmspemeh e.epee eeeeeIeeseepm ee mpeeeez eeee eee easep .pp> epeee 82 .mHesHse e ecu .o.m u msems mee mmsHe> .Hsmseemsxm ms» psosmsoese :osu pee um» mem: mpee HH< .mpee umeomuses: .ueumneeuIsos .Heseos See 23: E Steppe e832 eeetees 2: me .9. .33 2.2.2. 8:3 .s 532:8 N55 eerie . mmHoEs .mmepessmoss ueue "wees: apH>Heu< .smemmu maem msp mo mesmseemsxm mpeeesmm 93H sH umses Iemumu memz mmHeH>Hpoe o.spsmmsueu use mmeuessmoss ueu< .muosemz use mHeHempez sH umnHeummu we mmee IH>Hpue AN uosemsv s spmsmspeo eo AN uospmsv mmepessmoss uHue eoe umeaHese use umeeames memz emHuss e9: emcee: .eepepeeee 8e: 2: pe eepceepe... eee Hoe eSee... ee 3 239. see. e 8:: m pe e: Ismse sue: .s.H umpumnse mem: mpee .esos o pe .emHeH exeu esoe .psmem: Huon mx\seuopo~oesmepm as me es soeuomusH .>._ »n oHumneHu mues memz m ooonmH msHsmHmz muee seseuHo: mHe: .meex ueemneeo seuopoe Iousmepm ume Eoee HmHuaz em>ep mo xue>euu< msaesm Hegemomap so soHeumnsH sHHsmsH eo uumeem .s mesmee 83 .meem oeumaeeo sHuoHoNopsmeem ums Eoee emHusz em>ep eo zue>euu< maxesm Hesomomxp so soeeomusH seHsmsH eo Homeeu manor mwhnzzz o «M e om on 8 o. o u d HTd “h - I J I- II. 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Z mefiéemoze 92 13 A mw eo mHmaHese an umsHEemHmu me usmemeeeu 2Huseoeeesmem mee useeomemssm soEEoo e mse>e2 Hos msem2 .ssoem ems muee s we .o.m « mseme mee mmsHe> .peom He 2HH>Huoe m2 use 2owsu .zosuu eoe umeaHese mem: msoeuoee» usepesemszm use eoos H eom m x coo.moH He ummseeepsmo mem: .NHumz 25 N .mmoeusm 2 mm.o se H>\:v mmH .mmuesmmosos em>ep .soeuemseeeusmo emuee umeomHHoo me: asemm .mue so NHumz 25 N .mmoeosm 2 m~.o sH umoeHs use ummeoxm aHuHsee mem: mem>HH use .mmsou mmseeepsmu umeeseeesmsss se um IuomHHou me: uooHn xszeu .soeueueseomu 22 umopmeeoem mem: mHeEHs< .N usmeHemsxm sH esos mso se umszm Isoo assoEe Heeop use H HsmEHemoxm sH psmHm: xuos HesHe a ooH ems ussose 2HHeu mmeem>e msp mH um53m Isoo pmeo .msHummeme eo soHHeHHHsH emeee sHE om Hemem: Auon m ooH\= m we sm>Hm me: sHHsmsH eeHemme eo soepomnse HemsoueemseepsH mHmsem e .N Hsmeeemsxm sH .psmem: zuos m ooH\= mH He aHHeu msomsepeunsm umpomnse me: HseHsmsH mesmpv sonsmsmsm osHN sHHsmsH H Hsmaeemsxm sH .umueoeusH me soeuepsmsmHsssm seHomsH usosee: eo see: AN psmseemsxmv esos H eo AH esmeeemsxmv maeu e eoe Hmuosum2 use mHeHemHe2 mmmv umHu mmousHm sues e umeme smsu .mxeu m eoe um>eeum mem: muee .soepomnse empee 2mm: H msessemmm .Aesmem: xuos m2\me muv sHooHoNoHsmeum mo soeeomuse msosm>eeHsH 2n oeumneeu muee mem: m omNIoom ase Ismem: muee seENHHoI mHe2 .HHH use HH mHseH on mmHHose omHe soeuseeummu mes» .mseummemsIsoHpe>eepm mseeso wees oepmseeoIsHuouoeousmeum use Heseoz eo mmHHH>HHo< mazes“ oesmmosep em>ep .H mHneH 1116 I III! t9.m~ « n<.m 4 VI ec.c « UH.2 +3 eN.H 2H.H . mI®Hm 0.:— 'h. 0 N N 22 ue.m~ « m.mum uH.~o « m.~e~ m s__:ms. + ue umeme .um uo>eeam .UHHeseHu ue.o_ e o.~m se.cH e m.- e u: umeoe .un ue>eeum .uppmeeHu eH.~ H o.~m en.H « m.HH m um um>eepm .uHueeeHu u=.mm e c.xe~ oH.eeum .Heseos e~.u e ~.nm eo.~ « v.2H m um um>eeum .HeEeos sm.~ e 2.20 s~.m e a.s~ e Heseos .H I-I seeee :28 s - peeepee: pad .IéI IveIeapIeeIImquQOeiIDIe 3 eu< mega IIII .aseummemmIsoeue>eepm as.e=: apes oepmseeoIs.uopo~opsmesm us< Hes.o2 eo mmHHH>Hpu< maaesu oHsmaooHp em>HH .H o—aeh 107 7.0, and 17.6 fold elevated above activities determined in non-insulin treated, starved-refed diabetic rats and were 2.0, 1.5, and 1.7 fold elevated respectively above activities of starved-refed normal rats. Diabetic rats were unable to properly dispose of incoming dietary glucose whereas nornal rats had this capacity. Seven days after strepto- zotocin injection, serum glucose of rats rose from the normal level of 8.8 1 0.4 (n = 4) to 30.8 1 4.0 mM (n 8 61). As indicated in Table II, three days of starvation of diabetic rats resulted in significantly reduced serum glucose levels.' In normal rats, serum glucose levels drop- ped significantly from 8.8 to 6.1 mM after three days of starvation. Serum glucose levels of 3 day-starved normal and 3 day-starved diabetic rats then rose 2.0 and 3.3 fold, respectively, after refeeding the high carbohydrate diet for 1 hour. After 4 days of feeding the high carbohy- drate diet, the serum concentration of glucose of normal rats returned to pre-starvation levels while the serum glucose concentration of diabetic rats remained elevated at 31.0 mM. The mean concentrations of serum insulin in streptozotocin diabetic rats were in all cases lower than those of normal rats although these differences were not always statistically significant (Table II). In response to starvation, the serum insulin concentration of both normal and diabetic rats was lowered to 8 and 3 uU/ml, respectively, in experi- ment 1 and to 24 and 19 uU/ml, respectively, in experiment 2. Both normal and streptozotocin diabetic rats which had been starved for 3 days, then refed a high-carbohydrate diet for 1 hour, responded with a 2.6 fold elevation in serum insulin concentration above levels found prior to refeeding. Although the immunochemically detectable insulin response to refeeding in diabetic rats was proportional in amount to that 108 .H mHneH o» usmomH mmm .asHummemmxsoHHe>eeem msHeso wees upumneHaIsHoouoNousmeum use Heseoz mo msoHueeHsmusou sHHsmsH use mucus—w sseem .HH mHneh 109 epeemep + 2p eeeee .em ee>eepm .pepeeeee uoouHA um.~ u o.o m usooHA em.o « a.~ w s—HsmsH + .um um>eeum .UHHmeeHu uNH u om uH.~ « m.e~ m 2H umwme .um um>eeum .uHumueHu em « mH s~.H u m.~ e um um>eeum .qumneHu neH e No uu.H a H.~H m 2H umwme .um um>eeum .Heseos eeH a em sm.o a H.u m um um>eeum .Heseos .N um « NH uo.eH « o.Hm .e ue umeme .um um>eeum .qumneHu em e m u.ee.H a m.w m um um>eeem .oHumueHu mus e HNH s~.o u e.w m ue umwme .um um>eeum .HeEeos aw u w ew.o a m.o m um um>eeum .HeEeos umw e um ee.o « w.w e Heeeos .H HHE\::M sHpsmsH stu mmouus s usmsueme» .psxm .mseummemmIsoHee>eeum msHeso wees oeumeeHoIsHuopoeousmeum us< Heseoz mo msoHueepsmusou sHHsmsH us< meson—m soemm .HH mHne» 110 of normal rats, the former was ineffective in controlling serum glucose (Table II). Insulin, when injected into streptozotocin diabetic rats during refeeding, was effective in lowering serum glucose. This indicates that peripheral tissues of diabetic rats were responsive to exogenous insulin and that the immunodetectable insulin of diabetic rats was either quantitatively inadequate or abnormal in hypoglycemic activ- ity. After four days of feeding, normal and diabetic rat serum insulin levels were elevated above starvation levels (Table II, experiment 1), however refed normal rat serum insulin levels were 7-fold higher than those in refed diabetic rats. During the four day refeeding period, diabetic rats consumed 60% more diet daily-per 100 9 final body weight than normal rats starved and refed in the same way (Table III). Daily insulin administration to starved-diabetic rats during the refeeding period lowered the average food consumed daily per 100 g body weight to a level which was still 23% above that of starved-refed intact rats (Table III). Liver weight per 100 g body weight was reduced by starvation and returned to normal values after refeeding in both normal and diabetic rats. In diabetic refed rats, supplementary insulin dramatically increased liver weights per 100 g body weight as compared with other groups. Livers of this group were fatty in appearance. Despite an intake of dietary glucose which would normally promote an overshoot of lipogenic enzyme induction in the intact rat, starved-refed diabetic rats were incapable of overshoot induction unless supplemented with insulin. Insulin was not required for the return of enzyme activi- ties from starvation to normal levels. However, since insulin was detec- table immunochemically in diabetic rats and because it was uncertain 111 .H mHne» op usmmmH mmm .asHummwmmIsoHHe>eepm msHeso muem oepmseeo IsHooHoNoHsmepm use Heseoz mo psmemz xuom usmoems m< Hememz em>HH us< soHpsEsmsou amen .HHH mHneh 112 nem.o « Hm.m em.H « ~.~ m sepsmsH + 2H umeme .um um>eeem .oepmeeeu ee~.e e e~.e e e eeeeee. I .ee ee>eepe .pepeeepe eu~.c « mm.m e~.~ a ~.m m 2H umeme .um um>eeem .oHemeeHu emH.o « e~.m o e um um>eeem .oeumeeeu emH.o u mm.~ ee.H e ~.m m 2H ummme .um um>eeum .Heseos emH.o « Nm.~ o e um um>eepm .Heeeos .N ueoIs « om.e ee.o a H.~H m sHHsmsH + ue umeme .um ue>eepm .oHemeeHu omm.o « ae.¢ ue.m « e.mH e ue umeme .um um>eepm .oHumeeHu e.emH.o « w~.m o m um um>eeem .uHemseHu ue~.c « ~e.e em.H e e.m m ue umeme .um um>eepm .HeEeos euH.o « mw.~ o m um um>eeum .Heseos sHH.o « m~.m III e HeEeos .H Husmem: muse av quumssmsou s psmseeme» ..enxm peeee: easep pees .oseummemmIsoHue>eeem msHeso wees uHumeeHaIseoouoNopsmeum e5 Heeaz .5 222: see. peepeee e< p.33: e2: 22 539528 pas a: ezee 113 whether insulin was acting directly on the liver cell alone or in concert with glucose or other signals, we selected the relatively less complicat- ed isolated rat hepatocyte system in culture for further investigation of lipogenic enzyme induction. Cell culture. Hepatocytes isolated by the methods described were rou- tinely 90% viable as determined by exclusion of trypan blue with cell yields of 2 to 5 x 108 per rat. Hepatocytes maintained in culture as described attached to the FBS-coated dishes within the first 40 min after plating. After 24 hours of culture, 1/3 to 1/2 of the cells which were plated remained attached. After rinsing the cells gently as described, approximately 95% of the cells which remained on the plate excluded trypan blue and, of the cells removed from the tissue culture plate, 10% excluded trypan blue. There was very little cell death beyond the first day in culture. The presence of dexamethasone (1 pH) in the culture medium improved plating efficiency and increased cell aggregation into "trabecular aggregates“ (36). Induction of lipogenic enzymes in isolated hepatocytes. As shown in Figure 1A, incubation of hepatocytes isolated from 3 day starved rats with inducing medium (serum-free DMEA containing 27 mM glucose, 42 mU/ml insulin, and 1 uM dexamethasone) resulted in a 13-fold increase in G6PDH activity over three days in culture, a 4-fold increase above activity of cells incubated for three days in control medium. Control medium was serum-free DMEA which contained 5.6 mM glucose. 6PGDH activity of "induced" cells dropped 30% in the first and second days of culture then returned to the level present at the start of the culture by the third day (Figure 1B). Malic enzyme activity of “induced“ cells remained con- stant or decreased slightly for two days, then increased 2-fold by the 114 .mmsmeu mmese eoe .o.m e mseme mee mmsHe> .msomespmsexmu 2: H use .sHHsmsH Hs\=s Ne .mmoosHa 25 n.- yo msoHeeeusmusou Hes.» sup: umusmemHsssm sm eHe mmm\~ou am .umHeHuHess e se noun He umpensuse use msoep Ieuue msoEeos use mpeHoneme msoHee> msHseepsou HH use .umeepmspmmse emspm .maeu m eoe um>eepm mem: m cNNIooN msHsmHm: muee seENpHoz mHez .mmuauopesmz Hem eo mmeauHsu heeeees sH mmsaesm uHsmmooHH we soeuususH mo mmesou mee» .H mesmee 115 eteeemzt 222.952. oo :JOKFZOU ox\\AnHmmooo2. o. mee m . . . o posezoo. W Mu .Ioe H \«I smoooz. ow rouse . Hm (UIaIOJd fiw/nw) Ail/010V .mmpxuouesmz pea eo mmespHsu 2eeEHes sH mmsxesm uHsmmosHp we so_eu:usH mo mmeoou meek .28... ”.2: 225332. N . .H mesmee \. _ . osezoo \\\\\\x w I 2.. * .II smoooz. Iommo IIHyw (UIGIOJd fiw/nw) KIIAIIOV 116 third day (Figure 1B). For both 6PGDH and ME, activities of control and induced cells were parallel, the induced state having slightly higher activities. The responsiveness of the selected hepatocyte lipogenesis enzymes to varying amounts of glucose, insulin, and dexamethasone is shown in Figures 2-4. The specific activities of GGPDH, 6PGDH, and ME in hepato- cytes incubated for 0 and 48 hours in control medium lacking glucose, insulin, and dexamethasone and 48 hours in medium containing 42 mU/ml insulin and luM dexamethasone with varying amounts of glucose are shown in Figure 2. In control medium, hepatocyte G6PDH activity increased 3.3-fold during 48 hours in culture. Cells incubated with insulin and dexamethasone, in the absence of glucose, had 11.4 and 3.5-fold elevated GGPDH activity above cells incubated 0 and 48 hours in control mediun. Addition of glucose up to 22 mM had no further stimulating effect on the induction of GGPDH by insulin and dexamethasone. 6PGDH and ME activities of hepatocytes incubated in control medium dropped during 48 hours in culture and for 6PGDH but not ME, could be restored to 0 hr levels by addition of insulin and dexamethasone. As in the case for G6PDH activi- ty, addition of glucose had no effect on 6PGDH and ME activities in hepa- tocytes incubated with insulin and dexamethasone. Optimal concentrations of insulin and dexamethasone at a glucose concentration of 27.3 mM for the induction of GGPDH in hepatocytes from 3 day starved rats were 40 to 160 mU/ml and 1 to 10 pH respectively as shown in Figures 3 and 4. The degree to which glucose and hormones in- teract to affect lipogenesis enzyme induction in hepatocytes from starved rats was examined using optimal inducing concentrations of insulin (42 mU/ml) and dexamethasone (1 pH); 27.3 mM glucose and 15 uM T3 in 117 Figure 2. Glucose Dose-Response Curve. Hepatocytes were prepared, cul- tured, and analyzed as described in Figure 1. In this experiment, incu- bation medium was a modified DMEA which was glucose-free. "No additions“ refers to the absence of added insulin and dexamethasone. Glucose con- centration varied as indicated and insulin and dexamethasone concentra- tions, where added, were 42 mU/ml and 1 uM respectively (final concentra- tions). Values are means 1 5.0. for 3 dishes. 118 e GSPDH . 6 o 80_§~ /§\ E i K E EH/lefl-I: ~53 eo- Insulinaoexomeihosone is; 4or 4 c, 20 _§*“4e In. No Add. _ E QR OIII. No Add. _ B 0 l J Jfij‘ | 1‘ l 560- 6PGDH d 3;:4‘4<)__ ‘_——lI---filt—T. 73—1t7'37711'EZ/91Ie4 ,_ InsulInaoexomeihosone ._ 20 __ 0 hr. No Add. . 2 48M. No Add. 5 O I 1 1 [#15 | M | 4 ME 20 70 hr. 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A See aeeeeepeeeae . 38:3 . e. 3 .HI. ease... H mm... .H.-. ee_.: H Bee .2... p.13: 3.... 5.3.... + 382.. 4 H. .2. 3. e3... A .e... :7. e8... p 2.2.x .emI. esee A See .2; .. esaeepeaxee . H. e... .N. ea... H 2... .HI. e2... e 3.8 .H.-. ease... H 3...... :52... e... 5.3... . He. .2. 2.. edema H Re .3. e.p .3 H 3.3 .8... .636 A 2.3 .2... Hi. 382.. + H... 3 .2. e538... H ea... .3 piece H 8.3 .2. 3.6.2... p 8.8 .2: 2. me e. e... e... .2... 28% e3. :28 See... 3 .22.... E33... 2F Hspmpoem 252.... 2pH>Hpu< mssesu psoszemeH .mmpxuopeam: pe2 mo mmeopHou res—ea sH amazes. 0.529.... .o 5325:. 9... so msHsoeaspouoIe» .o post. .> m2: 128 Although cell growth is reported by most authors not to occur in primary cultures of adult rat hepatocytes, there are reports that, espe- cially during prolonged incubation, cell division can occur (see 36). Microscopic examination of cells in culture revealed a progressive increase in confluence of cells suggesting the possibility of cell divi- sion. This possibility was particularly important since the induction of GGPDH could be linked to a demand for ribose needed for DNA synthesis. Also because insulin is known to have growth promoting characteristics in other types of mammalian cells (46) it was essential to examine whether insulin was mitogenic in these cells. Aphidicolin, at 5 ug/ml, a concen- tration sufficient to block DNA synthesis in dividing cultures of Hela (47) and CHO cells (personal communication from Dr. John A. Boezi), did not block the insulin and dexamethasone mediated induction of G6PDH nor was there an effect on total DNA content (Table VI). Thus, induction occurred in these cultures of hepatocytes in a,manner which was indepen- dent of DNA synthesis. 129 .mo.o v a .ummu mucmgm$m.u u:mo.».=a.m ammo. .mu:m..m> vo m.mz.a:m xn umc.semumu mm u:m.mmw.c apacmo_».:m.m use ua—gomgmqam :oeeoo m mcw>mg uo: menu: .mmsm.c mugs» Low .o.m H mcome mew mm=.m> .vmumo.c=. mm :..ou.u.=qm we mocmmnm Lo mocwmwen on. c. mco.u.u=oo m:.o=oc. smug: umuunaoc. mew: m..mo Hewswemnxm m.;u c. .. m.=m.. :. umaPLUmwu mm um~x.o:m ccm .umeau.=o .cm.mgm.a mem: amazooucgmzm nm~.o n mm.¢ mm~.. « mo.m~ ue..m A -.~¢ m.m. + we nem.o a m..¢ mc¢.~ a cm.¢~ om~.mH u mo..¢ m.m. . we m~¢.o n mo.~ mmm.. a om.- neo.. « mo.mH w.«. + cw n.mmw.o A mm.m mmo.. n o..m~ n-.. H ow.w. m.m. . em u.~.o A o~.m aw¢.e a mm.me we... n mm.m ¢.o~ . o m: In... Imago .a.~.a\m=. ..s\m= m. .m.=o;. we.. puo< mex~cm pcmspmmgh .mmuxuoucng aux we mm.=u.=u xgmswea :. mmexncw u.=maog.. $o co.uo=u:. use m.mmzp:>w m.nm. DISCUSSION The amount of dietary carbohydrate consumed during refeeding was considered by some authors (2-7) to be the primary signal for the induc- tion of hepatic lipogenesis enzymes. Gozukara et 31. (4) have shown a significant linear correlation between the kcal of dietary carbohydrate consumed/day/IDD g body weight and the immunochemically detected rate of synthesis of GGPDH in starved-refed rats. In another study from the same laboratory (2), rats were starved then refed a 60% fructose containing diet. Following refeeding, it was observed that the induction of GEPDH was esentially the same as that achieved by refeeding a 60% glucose diet where an insulin response is known to occur. The authors concluded that insulin must be ruled out as an intermediary signal between diet consump- tion and induction of GGPDH based on a report that fructose failed to elicit an insulin release from rabbit pancreas (48). However, Sugawa-Katayama and Morita (49) found that rats starved and refed a 69% fructose diet responded with a significant increase in serum immunodetec- table insulin. If dietary glucose is the primary signal for the induc- tion of lipogenic enzymes, then one would expect rats depleted of insulin to be as capable of inducing G6PDH as intact rats. The results presented here (Table I) do not support this view. Streptozotocin-diabetic rats given the same starvation-refeeding regimen as normal rats are signifi- cantly impaired in their ability to induce GGPDH, 6PGDH and ME. The induction of G6PDH, 6PGDH, and ME in diabetic rats was reduced to 10, 22, and 13% respectively of that in intact starved-refed rats. These results confirm those of Weber and Convery (16) and extend them to include ME. In addition, the results presented here demonstrate that this impaired 130 131 ability of a diabetic starved/refed rat to induce lipogenic enzymes to the same extent as a normal starved/refed rat is not due to a dimimished diet consumption by the refed diabetic rat (Table III). In fact, diabetic refed rats consume more diet/100 g body weight than normal refed rats (Table III) and should, if dietary glucose were the major stimulus, show enhanced induction of the lipogenic enzymes. The observation pre- sented here that isolated hepatocytes incubated with insulin and dexa- methasone in the absence of glucose are able to induce G6PDH (Figure 2) also supports the conclusion that glucose is not required for the induc- tion of GGPDH. Another opinion (lb-18,21) is that insulin plays an important role in the induction of lipogenic enzymes by starvation-refeeding. In the present study, injection of starved diabetic rats with insulin during the refeeding period resulted in the significant induction of the three hepatic lipogenic enzymes examined (Table I), thus extending the work of Weber and Convery (16) to include ME. As shown in Table III, insulin supplementation to starved diabetic rats during refeeding lowered the diet consumption/100 g body weight compared with non-insulin treated starved/refed diabetic rats. Thus the observed induction of the lipogen- ic enzymes in insulin supplemented starved-refed diabetic rats cannot be attributed to an increased diet consumption. The simplest interpretation of these results is that the injected insulin acted directly on the liver to increase the lipogenic enzyme activities, however it was still pos- sible that insulin was acting either in concert with some other in vivo factors or indirectly by stimulation of another organ to release an inducing factor(s). In cultured hepatocytes from 3 day starved rats, it was possible to demonstrate the induction of G6PDH by added insulin in 132 the absence of glucose (Figure 2). The addition of up to 22 mM glucose did not alter the induction process. The consistent 2-fold increase in G6PDH activity in 48 hour cultures of hepatocytes from 3 day starved rats to levels found in hepatocytes from normal unstarved rats could be explained as the result of the translation of a pool of stable GGPDH mRNA. Szepesi gt 31. (50) have found an 8-azaguanine resistant increase in GGPDH activity in starved-refed rats and suggest that this is due to the translation of a pool of stable GGPDH mRNA which is not degraded dur- ing starvation. If such a pool existed in hepatocytes from 3-day starved rats, then replenishment of amino acids, in the absence of further induc- ing signals, could lead to a return of G6PDH activity to pre-starvation levels. The stimulatory effect of dexamethasone on the induction of G6PDH in primary cultures of rat hepatocytes (Table IV) is consistent with the observations, j__vivo, of Berdanier gt Eli (23-26). Optimal amounts of insulin and dexamethasone, in combination induced GSDPH to a greater extent than either alone. The additive nature suggests that these agents are acting by different mechanisms. Recently, Holten gt 31. (28,30) reported that the dietary and hormonal stimulated increase in GGPDH mRNA was not sufficient to account for the observed magnitude of increase in G6PDH synthesis (29). In this regard, Peraino (51) has suggested that glucocorticoids may exert a permissive role in the induction of G6PDH and glucokinase, j__vivo, by making more intracellular amino acids available for translation. Unlike the in_vivo response to injected insulin (Table I), 6PGDH does not appear to be induced to a great extent by insulin jn_vitro (Figure 3 and Table IV). In dose-dependent fashion, insulin appears to 133 prevent a decrease in enzyme activity during culture. ME also does not respond to insulin addition in vitrg (Figure 3 and Table IV) to the extent observed after insulin administration in 1119 (Table 1). Recent results of Nakayama and Holten indicate that a greater induction of 6PGDH by insulin occurred after incubation of hepatocytes for 7 days (27). Dexamethasone did not improve the response of 6PGDH or ME to insulin in the studies reported here. Thyroid hormone participation in the induction of GBPDH, 6PGDH, and ME was examined because of the well docunented effects of T3 on the induction of ME in the rat in vivg (33) and in chick embryo liver cells in culture (52). Also, the recent reports of Spence gt 11. which showed that hepatocytes obtained from thyroidectomized rats did not induce ATP-citrate lyase (53) or glucokinase (54) in response to insulin and dexamethasone unless pre-treated with T3 indicated the possible impor- tance of thyroid hormones for the induction of other lipogenic enzymes. Addition of T3 to cultures of hepatocytes incubated as described in Table V indicated that this hormone was slightly inhibitory but does not prevent the insulin and dexamethasone stimulated induction of GGPDH. The activity of 6PGDH was also lower in cells incubated 48 hours in the pres- ence of T3 and the various combinations of glucose, insulin, and dexa- methasone shown in Table V while cellular ME activity was elevated only slightly by such treatment. The lack of a significant T3 effect on ME activity in hepatocytes from 3 day starved rats could be explained by the relatively long tI/Z of ME in response to T3 (4 days) (55) and the decrease in the nuclear T3 binding capacity during starvation (56). 134 In conclusion, the in vivo and jn_vitro results presented here indi- cate that glucose, either consumed by starved diabetic rats or added to the culture medium of hepatocytes obtained from 3 day-starved rats alone is not sufficient to elicit an induction of GGPDH, 6PGDH, or ME. Insu- lin, when injected into starved diabetic rats during a period of refeed- ing, resulted in an induction of GGPDH, 6PGDH, and ME in a manner inde- pendent of dietary glucose consumption. In isolated hepatocytes, insu- lin, and dexamethasone, in the absence of medium glucose, acted separate- ly or together to induce G6PDH. 6PGDH and ME activities were not greatly affected by these treatments. The mechanism by which insulin and dexa- methasone induce rat liver G6PDH remains to be elucidated. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. REFERENCES Gibson, D.M, Lyons, R.T., Scott, D.F. and Muto, Y. (1972) Adv. Enz. Reg. 10, 187-204 Rudak, D., Chisholm, E.M. and Holten, D. (1971) J. Biol. Chem. 246, 1249-1254 Rudak, 0., Gozukara, E.M., Chisholm, E.M. and Holten, D. (1971) Biochem. Biophys. Acta 252, 305-313 Gozukara, E.M., Frolich, M. and Holten, D. (1972) Biochem. Biophys. Acta 286, 153-163 Sassoon, H.F., Watson, J. and Johnson, B.C. (1968) J. Nutr. 94, 52-56 - Sassoon, H.F., Dror, Y., Watson, J.J. and Johnson, B.C. (1973)§LL_ Nutr. 103, 321-335 Derr, R.F. and Zieve, Lo (1974) Jo ”utro 104, 65-68 Szepesi, B. and Freedland, R.A. (1968) Can. J. Biochem. 46, I459-1470 Wolfe, R.G. and Holten, D. (1978) J. Nutr. 108, 1708-1717 Nace, C.S. and Szepesi, B. (1976) J. Nutr. 106, 285-291 Muto, Y. and Gibson, D.M. (1970) Biochem. Biophys. Res. Com. 38, 9-15 Yugari, Y. and Matsuda, T. (1967) J. Biochem. 61, 541-549 MCDOflald, BOEO and JOhnSOn, B.C. (1965) Jo Nutr. 87, 161-167 Potter, V.R. and Ono, T. (1961) Cold Spring Harbor Symposia on Quantitative Biology 26, 355-362‘ Szepesi, B. and Berdanier, C.D. (1971) J. Nutr. 101, 1563-1574 Weber, G. and Convery, H.J.H. (1966) Life Sciences 5, 1139-1146 Freedland, R.A., Cunliffe, T.L. and Zinkl, J.G. (1966) J. Biol. Chem. 241, 5448-5451 Steiner, D.F. and King, J. (1964) J. Biol. Chem. 239, 1292-1298 Glock, G.E. and McLean, P. (1955) Biochem. J. 61, 390-397 Goodridge, A.G. and Adelman, T.G. (1976) J. Biol. Chem. 251, 3027-3032 135 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 136 Steiner, D.F. (1964) Nature 204, 1171-1173 Nakamura, T., Aoyama, K., Kato, S., Tomita, Y., Tanaka, K. and Ichihara, A. (1979) Carcino-Embryonic Proteins Vol. II, p. 723-728, Elsevier, Amsterdam Berdanier, C.D., Wurdeman, R. and Tobin, R.B. (1976) J. Nutr. 106, 1791-1800 Wurdeman, R., Berdanier, C.D. and Tobin, R.B. (1978) J. Nutr. 108, 1457-1461 Berdanier, C.D. and Shubeck, D. (1979) J. Nutr. 108, 1766-1771 Bouillon, D.J. and Berdanier, C.D. (1980) J. Nutr. 110, 286-297 Nakayama, R. and Holten, D. (1980) Abstract No. 1689, A580 Meeting, New Orleans, LA Holten, 0., Taylor, M.A., Wolf, R.G., Hutchison, J.S. and Sun, J.D. (1979) Federation Proc. 38, 612 Winberry, L. and Holten, D. (1977) J. Biol. Chem. 252, 7796-7801 Sun, J.D. and Holten, D. (1978) J. Biol. Chem. 253, 6832-6836 Procsal, D., Winberry, L. and Holten, D. (1976) J. Biol. Chem. 251, 3539-3544 Hutchison, J.S. and Holten, D. (1978) J. Biol. Chem. 253, 52-57 Towle, H.C., Mariash, C.N. and Oppenheimer, J.H. (1980) Biochemistry 19, 579-585 Mak, I.T. and Wells, W.W. (1977) Arch. Biochem. Biophys. 183, 38-47 Schroeder, H.R., Gauger, J.A. and Wells, W.W. (1976) Arch. Biochem. Biophys. 172, 206-214 Seglen, P.O. (1976) jn_Methods in Cell Biology (Prescott, D.M., ed.), Vol. 13, pp. 29-83, Academic Press, New York Seglen, P.0. and Fossa, J. (1978) Expt. Cell Res. 116, 199-206 Seglen, P. (1977) Biochim. Biophys. Acta 496, 182-191 Seglen, P. (1976) Biochim. Biophys. Acta 442, 391-404 Seglen, P. (1978) Biochem. J. 174, 469-474 Tanaka, K., Kishi, K., and Ichihara, A. (1979) J. Biochem. 86, 863-870 Kurtz, J.W. and Wells, W.W. (1979) Anal. Biochem. 94, 166-175 43. 44. 45. 46. 47. 48. 490 50. 51. 52. 53. 54. 55. 56. 137 Hsu, R.Y. and Lardy, H.A. (1969) in Methods in Enzymology (Lowenstein, J.M., ed.), Vol. 13: pp. 230-235, Academic Press, New York Hales, C.N. and Randle, P.J. (1963) Biochem. J. 88, 137-146 Steel, R.G.D. and Torrie, J.H. (1960) Principles and Procedures of Statistics, McGraw-Hill Book Co., Inc., New York Holley, R.W. (1975) Nature 258, 487-490 ' Pedrali-Noy, G., Spadari, 5., Miller-Faures, A., Miller, A.0.A., Kruppa, J., and Koch, G. (1980) Nucleic Acids Research 8, 377-387 Grodsky, G.M., Eatts, A.A., Bennett, L.L., Vcella, C., McWilliams, H.B., and Smith, D.F. (1963) Am. J. Physiol. 205, 638-644. Sugawa-Katayama, Y. and Morita, N. (1977) J. Nutr. 107, 534-538 Szepsesi, B., Berdanier, C.D., Diachenko, S.K. and Moser, P.B. (1971) g; Nutr. 101, 1147-1152 Peraino, C. (1967) J. Biol. Chem. 242, 3860-3867 Goodridge, A.G. and Adelman, T.G. (1976) J. Biol. Chem. 251, 3027-3032 Spence, J.T., Pitot, H.C., and Zalitis, G. (1979) J. Biol. Chem. 254, 12169-12173 Spence, J.T. and Pitot, H.C. (1979) J. Biol. Chem. 254, 12331-12336 Schimke, R.T. and Doyle, D. (1970) Ann Rev Biochem 39, 929-976 Dillmann, W.H. and Oppenheimer, J.H. (1979) Endocrinology 105, 74-79 SUMMARY The development of automated fluorometric analyses for common biochemical determinations (Chapter I) may prove helpful to persons doing large numbers of samples from tissue culture materials. The experiments performed in Chapters II and III were designed to determine if the model for lysosomes proposed by Szego (1,2) for estradiol action could be extended to include glucocorticoid stimulated induction of tyrosine aminotransferase in RH-35 cells and starvation-refeeding stimulated induction of lipogenesis enzymes in rat liver. The results presented here do not support this extension. One could argue that the disruptive nature of the cellular fractionation techniques used may have disturbed the physiological association of lysosomes with nuclei, however it should be noted that the nuclear purification procedure used was that of Szego and Seeler (3). Also the electron microscopic evidence failed to show any redistribution of lysosomes within the RH-35 cell after glucocorticoid stimulation at time points comparable to those chosen by Szego. The observations of Schroeder £3 31. (4), that there is a transient liver lysosome fragility coincident with a shift from the starved to the refed state, was confirmed by the results of Chapter 111. However, the failure of lysosome stabilization and labilization to alter expected changes in the induction of lipogenic enzymes would suggest that, while a transient fragility is occurring simultaneously with induction, it is not an integral part of the induction signal pg: 32. It is possible that these fragility changes could reflect recently observed association of internalized insulin-receptor complexes with lysosomes (5,6). This 138 139 represents an exciting role for lysosomes as agents of either destruction or further processing of hormonal stimuli. In the final Chapter, evidence is presented that insulin is required for the overshoot induction of GGPDH jg_yjtgg_and 12 £119 and that glucose alone is neither sufficient nor necessary for induction of GGPDH. This is in direct disagreement with the previous results of Holten gt 21. (7,8). This discrepancy is discussed in detail in Chapter IV. REFERENCES Szego, C.M. (1975) in Lysosomes in Biology and Pathology (Dingle, J.T. and Dean, R.T., eds.) Vol. 4, pp. 385-461, American Elsevier Publishing Co., Inc., New York Szego, C.M. (19740 in Recent Progress in Hormone Research (Greep, R.0., ed.) Vol. 30, pp. 171-233, Academica Press, New York Szego, C.M. and Seeler, B.J. (1973) J. Endocr. 56, 347-360 Schroeder, H.R., Ganger, J.A., and Wells, W.W. (1976) Arch. Biochem. Bioghys. 172, 206-214 Carpentier, J-L., Gorden, P., Barazzone, P., Freychet, P., LeCam, A. and Orci, L. (1979) Proc. Natl. Acad. Sci. 76, 2803-2807 Goldfine, I.D., Jones, A.L., Hradek, G.T., Wong, K.Y., and Mooney, J.S. (1978) Science 202, 760-763 Rudak, D., Chisholm, E.M. and Holten, D. (1971) J. Biol. Chem. 246, 1249-1254 Gozukara, E.M., Frolich, M. and Holten, D. (1972) Biochim. Biophys. Acta 286, 153-163 140 APPENDIX 141 142 .mxuo.n mo mmmno use com. :u.: :m_mmc xoo.n mum—aeoo cm~.sovcm. .mo:o..m> we m.ma.o=m mem: mo.um..mum .mwumagmua.. cam. mo was» so.» .mo.o v a. a.ucmo.w.=m.m memww.v cmmza .mwmmgucmgwa :. :m>.m m.ms.:m Ho gangs: ms. Lo. .o.m H memos mew mm=.m> .p.om pm :.E\vmo:uoen zao_uom Iowan ecu :oaom u.c= ..pom o :qumsumu Lam—02: so. ...\:.e om\mo. x Emu u .m>..\= .x..>..om a :.mqm;umu .m>.. .mpou Lo. .. . .muamgu mo co.uumm muoguoz on. :. umn..omwu mm emc.semumu mew: mm.u.>.uom mmmcmmoeu53mc new .. Luggage Ho =o.uumm muoguoz ms» :. umn..ummu mm um=.semamu mem: mm.u.>u.om o =.mqw;ucu .wz .Loagmz 2mm .xgoumeonm. :omxowa us. so.» to:.ouno moans.waa.. mzomx~oeoumz .o msomaNoEo; .msgo: ..m;» can :.w..m no\no . ao\.m~mu mg» mem: «0.2m (anHHH..m. H w.mo .m..,a.m H o.~. .m.. 0mm H Noun .m.. .e.. H H... .nw..mm.~ H .m.m mm.-~m. amano .m.. M.. H e.em .m.. 0.. H o.~. .m.. o.m H .Hom .m.. ma.o H om.H .m.. Hm.o H mo.~ mm.-~m. cma. .m...m.~. H m... .N...m.~ H M.. .m...¢~.m H Hm.m .m.. mH.N H a... .N....m.o H mM.. a.-.e amana .m.. m.m H m..~ .m.. N.. H 3.. .m.. mm.~ H .awm .m., wo.~ H .o.o .N..Y.H.o H .~.mi m.-.. cam. zow.o zoaau ...\=. .maac. H.as.=< ~c.muo.a ms\=s. auw>Puu< Law—uaz zpwwwuu< .m>.. rmHOP ma< »u_>.uu< mea~cu o..omouau o :.mao;umu m.mwumaemaap. m.mz cum. L.mgu can mo.z m.mz mmmno a..mu.um=mw cmmzumn au.>.uo< mex~cm o.:mmoa.. UPFOmouxu .m>.. can a cpmamsumu Law.o:z can .muo» Lm>.. we comwemaaou .. m.nw~ 143 .m:.uwm.m. newest Humane mm.. u x ..\N e. u N... "co.umzcm mg. m:.m: .e.a.o.a me\=.s\ma.os= mm.m u How. we. m..m H mm”. .HHHH ea=.HHgo H..aH=as..aaxa a.a HH=.a. .eomiuw. .o. .mmm .Ncm .>u< .mnafi. ..m um .z.o .comn.w op m:.ueouom .ms.. vcm .xuw>.pum :. omcwco mo accumcoo mum. .x..m.u.c. au.>.uom mex~cm ..m>m. macaw xucmpm 2m: on» an xaw>.uum mex~cm .mE.» cw>wm a pm xu.>.uom mechm II II II II II +3400 LLJLIJLIJXH .mewcz . Hx-a .mam. - mm... - .H.. n m... .co.um=cm on. So.» vmum.:o.mo mm: w:.. am..ov mg. .... empamgo Ho :o..omm meozumz ms» :. non..ommu mm mm; cws.am. m:.umw»meico.um>.mum we. .oom. .Nmminm .a x. .o> amo.oex~cm :. mvozumz c. umn..ummu mm um:.5.m.mu mem: mm.p.>..om mmm:.xoos.m .omouau .m>.. .m:.cmmwmmico.um>.mum ac..=v xp.>.uo< mmmcpxoo:.u .m>.. Ho mmeaoo we.» .. m.:a.. 144 .m=.vmmmmxi=o.um>emum mcpeau Au.>.uo< mmmcpxoo=.o .m>.. .o mmeaoo we.. 62... ms. 7. 1.... H _ _ 7 '9 S: .. m.=m.u (3 CU CD