'vwWw—‘fi THE PARTICIPATION 0F LYSOSOMES IN ENZYME INDUCTION IN RAT LIVER Thesis Ior Hm Degree of M. S. MICHIGAN STATE UNIVERSITY Ivan In Tong Mak I977 -r~ ‘W'flgfl‘fl a - . m -721 j 5&4 . i ‘ 1‘ r I LIST -, . RILILIJ‘II 5 in LC Univcmty "IFEIS *— ABSTRACT THE PARTICIPATION OF LYSOSOMES IN ENZYME INDUCTION ' IN RAT LIVER BY Ivan Iu Tong Mak Male rats of the Holtzman strain were fasted for three days and refed a diet high in carbohydrate (68.9%). The induc- tion of liver g1ucose-6-phosphate dehydrogenase and 6-phospho- gluconate dehydrogenase was monitored for up to 48 hours after refeeding. Induction occurred by 24 hours, and by 48 hours, 4.2 and 1.5 fold increases were observed for g1ucose-6-phos- phate dehydrogenase and 6-phosphogluconate dehydrogenase, respectively, compared with that of livers of pellet fed rats. After refeeding, lysosomes increased in fragility as judged by an increased release of acid phosphatase activity during standard homogenization. Fragility was greatest 3 hours after refeeding, but normal fragility was observed 24 hours after refeeding. Nuclei were isolated from the liver samples before and after refeeding. Those isolated just before refeeding revealed small latent acid phosphatase activity (4-6%). How- ever, after refeeding the carbohydrate-rich diet, a transient and significant (P<:0.01) increase in the latent activity occurred that was maximal (20%) at one hour, returning to nor- mal by 24 hours. Cross-mixing the 800xg nuclear pellet from livers of animals starved for 3 days with the 800xg supernatant Ivan Iu Tong Mak fraction from livers of animals refed the carbohydrate-contain- ing diet did not alter the nuclear lysosomal free (overt) or latent (detergent released) enzyme activity. Similarly, mixing the BOng nuclear pellet from levers of animals refed for 1 hour with the 800xg supernatant fraction from livers of ani- mals starved for 3 days, but not refed, did not change the nuclear lysosomal free or latent enzyme activity. Purified nuclei, further washed in hypotonic buffer, lost acid phospha- tase activity, but those isolated from livers of rats refed for one hour retained 10% of the enzyme latency, whereas all latency was lost from those isolated from uninduced rats. A second lysosomal enzyme, fB-galactosidase, became associated with the nuclei with the same temporal pattern as for acid " phosphatase. However, no variation in nuclear content of cytosolic lactate dehydrogenase occurred as a result of feed- ing the high carbohydrate diet to starved rats. When similarly starved rats were refed a diet high in *lipid and carbohydrate-free, no induction of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase was observed. Lysosomes were not temporarily fragile and purified nuclei did not exhibit increased latency of acid phosphatase activity. Evidence presented so far supports the hypothesis that liver lysosomal enzymes participate in early signals in the induc- tion of enzymes of lipogenesis. In another study, male rats were similarly fasted and refed the high carbohydrate diet. Twenty-four hours prior to refeeding, the experimental rats were injected with 85 mg/lOOg Ivan Iu Tong Mak body weight of Triton WR-l339 and the control rats with equal volumes of saline solution. The induction of the two lipogene- sis related enzymes in the liver of the Triton-treated rats was both delayed and depressed. Seventy-two hours after refeeding, 3.3 and 1.2 fold increases in specific activity were observed for G6PDH, and 6PGDH respectively, compared with that of livers of pellet fed rats, in the Triton-treated rats. Whereas the control rats exhibited increases in specific activity of 7.5 and 2 fold for the two enzymes. Nuclei from rats with similar star- vation and Triton treatments were also isolated before and after refeeding the high carbohydrate diet. .The previously observed transient and significant increase in nuclear acid phosphatase 1 hour after refeeding untreated rats was not observed after exposure to Triton WR-1339. The result suggests the possibi- lity that Triton WR-1339 impaired the function of liver lyso- somes in carrying early signals for the induction of enzymes lipogenesis. Additional work was carried out examining the dietary effect on RNA synthesis in liver nuclei of starved rats. Nuclei from liver of rats starved for 3 days and refed a diet high in carbohydrate demonstrated a significantly increased capacity to synthesize RNA 3 hours after the beginning of the refeeding. The increased RNA synthesis continued in nuclei of these rats and appeared to precede the induction of the enzymes of lipo- genesis. After 24 to 48 hours of refeeding,the isolated nuclei increased 2.2 fold in their capacity to synthesize RNA, compared with nuclei of livers of pellet-fed rats. When Ivan Iu Tong Mak similarly starved rats were refed a diet high in lipid and car- bohydrate-free, insignificant elevation in the capacity of RNA synthesis was observed in the isolated nuclei. In a further study, nuclei which were obtained from liver of starved rats and had been incubated for 30 min. at 25°C. with the post-nuclear fraction of liver homogenate from rats starved and refed the high carbohydrate diet for one hour exhibited a significantly (P<:0.0l) higher RNA synthesis acti- vity than that of the control nuclei which had been incubated with the post-nuclear fraction of liver from.similarly starved rats without refeeding. The lysosomes in the postnuclear frac- tion of the liver homogenate was suggested as the possible factor triggering the phenomenon. THE PARTICIPATION OF LYSOSOMES IN ENZYME INDUCTION IN RAT LIVER By Ivan Iu Tong Mak A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Biochemistry 1977 ACKNOWLEDGEMENTS I would like to express my deepest gratitude to Dr. William W. Wells for his guidance, encouragement, patience and continual financial support throughout the course of this research. I would also like to thank the members of my guidance committee, Dr. Loran L. Bieber and Allan J. Morris, for their honest criticisms and suggestions. All the members of Dr. Wells' laboratory, especially. James Kurtz, Jeffrey Nickerson and Rita Ray, have my acknowledgement for their supportive suggestions and enlight- ening discussions. In addition, I would like to extend my appreciation to Dr. Nannie Henderson for her performance of the scanning electron microscopy. ii TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES INTRODUCTION Organization . . Rationale and Objectives LITERATURE SURVEY Induction of Liver Hexose-Monophosphate Dehydrogenases The Lysosome as a Mediator of Hormone Action References Chapter I. THE ASSOCIATION OF LYSOSOMAL ENZYMES WITH NUCLEI DURING ENZYME INDUCTION IN RAT - LIVER . . . . . . . . . . . . . . Abstract Introduction Materials and Methods Animal Treatment Sample Preparation . Determination of Enzyme Activities by Computer Directed Spectrophotometry Acid Phosphatase . . LysosomalE- -Galactosidase . . Glucose 6 hosphate Dehydrogenase and 6- -Phosphogluconate Dehydrogenase DNA Determination . . Protein Determination . Electron Microscopy 111 Page vi vii 12 12 14 15 15 15 17 17 18 18 20 19 Chapter Page Results Induction of the Dehydrogenases . . . .20 Lysosomal Fragility and Transient Increase in Latent Nuclear Lysosomal Enzymes . . . 21 Studies of the Nuclear Lysosome Speci- ficity . . . . . . . . . 23 Scanning Electron Microscopy . . . . . . . 25 Discussion . . . . . . . . . . . . . . . . . 25 References . . . . . . . . . . . . . . . . . 30 II. EFFECT OF TRITON WR-1339 INJECTION ON INDUCTION OF LIVER HEXOSE-MONOPHOSPHATE SHUNT DEHYDROGENASES AND NUCLEAR LYSOSOMES IN THE FASTED-REFED RAT. . 41 Abstract . . . . . . . . . . . . . . . . . . 41 Introduction . . . . . . . . . . . . . . . . 43 Materials and Methods . . . . . J . . . . . . 43 Animal Treatment and Sample Preparation . . 43 Enzyme Assays and Metabolite Determina- tions . . . . . . . . . . . . . . . . . . 44 Hexosaminidase . . . . . . 44 Triglycerides and Glucose levels . . . . 45 Glucose- 6— —phosphate Dehydrogenase and 6-Phosphogluconate Dehydrogenase . . . 45 Results . . . . . . . . . . . . . . . . . . . 47 Effect of Triton WR-1339 on Induction of the Dehydrogenases . . . 47 Effect of Triton WR- 1339 on Plasma .Hexosamini- dase, Glucose and Triglyceride levels . 48 Effect of Triton WR-l339 on Nuclear Lysosomal Enzyme Activities and Liver Lysosomal Fragility . . . . . . . . . . . . . . . . 49 Discussion . . . . . . . . . . . . . . . . . 50 References . . . . . . . . . . . . . . . . . 54 III. DIETARY REGULATION OF RNA SYNTHESIS IN THE LIVER NUCLEI OF FASTED—REFED RATS . . . . . . . . . . 62 Abstract . . . . . . . . . . . . . . . . . . 62 Introduction . . . . . . . . . . . . . . . . 64 iv Chapter Page Materials and Methods . . . . . . . . . . . . 64 Chemicals . . . . . . . . . . . . . . . . 64 Animal Treatment . . . . . . . . . . . . . 65 Nuclear Samples . . . . . . . . . . . . . . 65 RNA Synthesis . . . . 65 In vitro Incubation of Liver Nuclei from —Starved Rats with 800xg Supernatant of Liver Homogenate from Rats Starved and Refed one hour the High Carbohydrate Diet . . . . . . . . . . . . . 66 DNA Content . . . . . . . . . . . . . . . . 67 Results and Discussion Conditions of RNA Synthesis . . . . 67 Dietary Effect on RNA Synthesis in Liver Nuclei of Starved Rats . . . 67 Effect of the Liver Post- nuclear Fraction from Starved Rats with or without one hour Refeeding of the High Carbohydrate Diet on RNA Synthesis Activity of Starved Rat Liver Nuclei . . . . . . . . . . . . 70 References . . . . . . . . . . . . . . . . . 74 CONCLUSION . . . . . . . . . . . . . . . . . . . . . . . 79 TABLE Chapter I. II. III. IV. Chapter I. II. LIST OF TABLES I Food consumption, body and liver weights of starved rats refed a high carbohydrate or high lipid diet. . . . . . Liver enzyme activities of rats starved 3 days and refed for 2 days a high carbohydrate or high lipid diet. . . . . . . . . . Acid phosphatase activity in rat liver nuclei after cross—mixing treatment Acid phosphatase activity of liver nuclei with or without hypotonic treatment in the starved rats refed a high carbohydrate diet" II Food consumption and body, and liver weights of starved rats injected with Triton WR-1339 or saline and refed a high carbohydrate diet Effect of Triton WR-l339 injection on plasma hexosaminidase, glucose and triglyceride levels of rats starved and refed a diet high in carbohydrate. vi Page . 32 . 34 . 35 . 36 . 55. . 57 FIGURE LIST OF FIGURES Page Chapter I 1. Chapter Chapter 1a. lb. A comparison of lysosomal membrane fragility with latent acid phosphatase activity of purified liver nuclei of rats starved for three days and refed a diet high in glucose. . . 37 Total -galactodidase and lactate dehydroge- nase activities of purified liver nuclei of rats starved for three days and refed a diet high in glucose. . . . . . . . . . . . . . . . . 38 A comparison of lysosomal membrane fragility with latent acid phophatase activity of puri- fied liver nuclei of rats starved for three days and refed a diet high in fat and carbohy- drate free . . . . . . . . . . . . . . . . . . . 39 Scanning electron micrograph of rat liver nuclei . . . . . . . . . . . . . . . . . . . . . 40 II Comparison of liver glucose- -6— —phosphate dehydro- genase and 6- -phosphogluconate dehydrogenase in the fasted- refed rat injected with saline or Triton WR-l339 . . . . . . . . . . . . . . . . . 60 A comparison of liver lysosomal fragility with acid phosphatase activity and the total -galac- tosidase of purified liver nuclei of rats starved for 3 days. The animals were treated with Triton WR- 1339 1 day before refeeding a diet rich in carbohydrate . . . . . . . . . 61 III Tgme course of incorporation of label from I H] UTP into RNA. . . . . . . . . . . . . . . . 76 Effect of nuclei concentration on RNA synthesis. . . . . . . . . . . . . . . . . . . . 76 vii FIGURE Page RNA synthesis activities in liver nuclei ob- tained from rats starved for 3 days and refed for various time a diet high in carbohydrate or a diet high in lipid . . . . . . . . . . . . 77 RNA synthesis activities in liver nuclei which were from starved rats and had been incubated for various time with the 800xg supernatant of liver homogenate from either the starved rats with 1 hr. refeeding of the high carbohydrate diet, or from starved rats without refeeding. . 78 viii INTRODUCTION Organization The text of this thesis is divided into three chapters, each following a format used in most biochemical journals. Chapter I is presented as it will appear in Archives of Biochemistry and Biophysics 183 in September, 1977, under the title of "The Association of Lysosomal Enzymes with Nuclei During Enzyme Induction in Rat Liver" by Iu T. Mak and William 'W. Wells. Chapter II is in preparation to be submitted for" publication. Chapter III represents additional results from a couple of recent preliminary experiments probing the mole- cular mechanism of the investigations relating lysosomes to enzyme induction in this laboratory. Rationale and Objectives Enzyme levels in intact animals and in a variety of animal cells in culture can be altered by physiological, nutri- tional and hormonal changes and by administration of various pharmacological agents (l-5). Any of these changes would be able to regulate enzyme amount by affecting enzyme synthesis or degradation (1). It is generally recognized that the surround- ing membranes of cells and their peripherally dispursed organel- lar components receive the first impact of the specific and 1 “Mb—P I __- ._.— non-specific physical and chemical agonist or hormones which are capable of interacting with one or another receptor site on the cell surface (6-9). The specific agonist then under- goes physical transfer into the cytoplasm where it forms a complex with a cytoplasmic receptor, which is then transmitted through the cytoplasm to the nucleus, resulting in the subse- quent modulated transcription and ultimately specific protein synthesis (8, 10-15). What is less evident are the means through which these remote cellular stimuli are communicated to the intranuclear environment which is the control core of the growth response. C. M. Szego has proposed that the primary lysosomal population of certain hormonal target cells (endoé crine glands) participates in the reception of the agonist . and serves as the mobile link in its transcytoplasmic migra- tion toward and invasion of the nuclear envelope (16). Con- commitantly, lysosomal membrane destabilization through hor- monal action may result in enhanced fusion with or uptake of various macromolecules and thus for potential reduction of template restrictions in the cellular target. Dr. Szego re- cently extended her model to a generalized pattern of lysosome— mediated enhancement of nucleocytoplasmic communication for a variety of circumstances which likewise lead to cellular acti- vation (17). It is to the investigation of the possible role parti- cipated by lysosomes in the process of enzyme induction in mammalian liver that the research reported herein is directed. For this purpose, I have selected as a model rat liver 6-phos- phogluconate dehydrogenase and glucose-6-P dehydrogenase which catalyze the oxidative steps of the pentose phosphate shunt and are associated with liver lipogenesis. LITERATURE SURVEY A. Induction of Liver Hexose-Monophosphate Dehydrogenases. A well known example of liver enzyme induction in re- sponse to dietary manipulation is the marked stimulation of the pentose phosphate shunt dehydrogenases in fasted animals refed a diet rich in carbohydrate (2, 4, 5, 18). Other lipo- genic enzymes respond in a similar manner as the pentose phos- phate dehydrogenases to nutritional manipulation, suggesting that this group of functionally related enzymes is regulated in a coordinate fashion (4). The changes in the levels of the dehydrogenases have been generally agreed to involved in- creased protein synthesis (4, 19-22). Hormonal involvement in the regulation is indicated by the fact that thyroxine, estra- diol-l7- , dihydroepiandrosterone, cortisone, and insulin have all been showed to alter the level of the enzymes in liver (23-25). Finally, interactions between the dietary and hor- monal factors were suggested by the ability of insulin to aug- ment the effect of high carbohydrate diets on the level of the enzyme in liver (26, 27). In most studies, a delay of 24-48 hours was required for the full effect of the refeeding to be observed (18-20). Significantly, the rates of 6PGDH and G6PDH synthesis were found to be directly proportional to dietary carbohydrate intake (19, 21, 22). It was suggested that the effect of insulin was only indirect by affecting the amount of diet consumed (21, 22). Szepesi and Berdanier also concluded that the signal for their induction was carbohydrate (20). However, the release of insulin that accompanies refeeding starved rats a carbohydrate-rich diet may trigger early events in the liver that precede the first measurable increase in lipogenic related enzyme activity by several hours after re- feeding (20). In any case considerable delay in the induction of lipogenic enzymes strongly suggests that either dietary carbohydrate or responding hormones trigger some intermediary systems that carry the signal to the nuclei and initiate the induction process. B. The Lysosome as a Mediator of Hormone Action. Other than the sole function of indiscriminating destruc- tion as first visualized by de Duve and Wattiaux (28), several properties of lysosomes have been revealed to recommend them- selves for consideration in the participation of hormone action. In two reports Allison summerized the capacity of lysosomes for selective accumulation of a large variety of chemical sub- stances, organic and inorganic, soluble and particulate (29, 30). Lysosomes were also reported to be characterized by extra- ordinary propensity for rapid and reversible fusion with other cellular membrane, notably the plasmalemma (31, 32) and pino- cytotic vacuoles (33). Henning g£_al. observed that the mem- brane of lysosomes exhibited a degree of similarity to the plasmalemma of the homologous tissue in lipid composition and in certain carbohydrate components (34, 35). This degree of similarity would be essential to the predisposition toward fusion. In another report (11), Allison and Mallucci indi- cated that lysosomes were often deployed at cytoplasmic peri- phery in cells under basal condition suggesting that they are in'a strategic position to intercept agonist which have entered the intracellular environment by means of receptors at the cell surface. It was also reported that lysosomes were capable of saltatory translation at speeds well beyond those which characterize Brownian movement (36). In addition, it was discovered that lysosomes may release their sequestered hydrolases in graded concentration and without membrane rup- ture to a degree proportional to magnitude of the provocative stimulus (31, 37). The observation suggested the potential” of lysosomes, by limited attack up on existing macromolecules in the environment, of being able to trigger certain metabolic processes in the affected cell. Furthermore, lysosomes were observed concentrated ina conspicuously perinuclear position in cells activated by a variety of circumstances (11, 38, 39). Since 1970, in several related reports, Szego and her colleagues first proposed that protein synthesis in estrogen- stimulated uterine cells and preputial glands of rat involved the participation of lysosomes. It was first observed that at short intervals after the in vivo injection of physiological amounts of estradiol-l7-F3 to gonadectomized rats, lysosomes of the preputial gland or uterus were significantly labilized compared with those from rats with saline or estradiol-l7-‘f administration (40). Such observation was not found for lysosomes of non-target tissues. Membrane labilization was determined by a significant increase in the release of lysosomal enzymes from purified lysosomes to an isotonic or Triton X-100 containing incubation medium. In addition, the structural latency of mitochondrial marker enzymes from the same tissue sourse under identical conditions was unaffected by steroid hormone pretreatment. Concurrently, another report showed that intense and rapid accumulation, in vitro, of“ 3H-estradiol-l773 occurred A in macromolecular fractions extracted by hypotonic saline from highly purified lysosomes following isolation of those organelles in structually intact state from preputial glands of ovariec— tomized rats (41). These results were supported by their counterpart in vivo studies (16). In a latter report (42), Hirsch and Szego indicated the presence of an estradiol-l779 receptor protein in the lysosol fraction of preputial glands of female rats. The lysosol fraction was the supernatant of a 105,000xg 1 hr centrifugation of a purified lysosomes pre- paration which was previously incubated with stirring in hypo- tonic buffer for 1 hr. at 00 ~4OC. This lysosol protein possessed high affinity, low capacity characteristics of bind- ing the hormone. This protein was also found to be target tissue specific and stereospecific for the hormone, and pro- tein in nature. Moreover, like the cytosol estrogen receptors, the lysosol receptor protein bound with greater affinity at 32°C than at 0°C.. Szego thus speculated that lysosomal pro~ teins may serve as the source of the cytosolic receptor mole- cules for steroid hormones. More biochemical and morphological evidence was then reported in another study (43). Within 1-2 min. after in vivo administration of physiological amounts of estradiol—1776 , the lysosomes of preputial gland and uterine cells were observed by fluorescence microscopy to localize in and about purified nuclei. In control experiments, injections of estradiol-17-6‘ or saline did not result in accumulation of lysosomes about the nuclei. Non-estradiol target organ lysosomes were not responsive to the estradiol treatment. Evidence of nuclear invasion by lysosomes in estrogen-stimulated uterine cells was later provided by the study in which inclusion bodies staining positive for acid phosphatase were observed in the' nuclei (44, 45). ' From another approach (48), Szego demonstrated that pretreatment with glucocorticord, a stabilizer of lysosomal membrane (46, 47), strikingly curtailed the stimulatory influence of estrogen upon target-specific lysosomal labilization and transcytoplasmic migration in the rats. Using adrenal demedullated rats (49), Szego gt_al. showed that within 5 min. after injection of low doses of the polypeptide hormone ACTH, lysosomal labilization and centri- petal mobilization of lysosomes in adrenal cortical cells. Similar observations had been studied for testosterone and diethylstilboestrol (40). Other hormones studied including thyroid-stimulating hormone, parathyroid hormone, luteinizing hormone, epinephrine and glucagon were thought of as acting by lysosomal mediation (l6, l7). From all these morphological and biochemical evidences, Szego has hypothesized that target-specific lysosomes, on acti- vation by trophic hormone, serves as a mobile link for infor- mation transfer between the cell surface and the nucleoplasm. Finally, a recent report (50) from this laboratory indi- cated that dietary galactose evoked labilization of liver lyso- somal membranes, as judged by an increase in the soluble frac- tion of several lysosomal enzymes and greater sensitivity to osmotic shock of the lysosomes from galactose—fed chicks. In another report (51), refeeding the rats starved for three days a high carbohydrate diet resulted in the labilization of liver lysosomes within 3 hr. and prior to the subsequent induction of the first two enzymes of the pentose pathway. Both findings indirectly support Szego's lysosomal mediation hypothesis. 10. 11. 12. 13. 14. 15. 16. REFERENCES Shimke, R. and Doyle, D. (70) in Ann. Rev. Biochem. (Snell, E. 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Szego, C. M., Rakich, D. R., Seeler, B. J. and Gross, R. S. (74) Endocr. 26, 863. Schroeder, H. R., Lawler, J. R. and Wells, W. W. J. Nutr. 104, 934. Schroeder, H. R., Gauger, J. A. and Wells, W..W. Arch. Biochem. Biophys. 172, 206. , 179. (74) (76) CHAPTER I THE ASSOCIATION OF LYSOSOMAL ENZYMES WITH NUCLEI DURING ENZYME INDUCTION IN RAT LIVER ABSTRACT Male rats of the Holtzman strain were fasted for three days and refed a diet high in carbohydrate (68.9%). The induction of liver glucose-6-phosphate dehydrogenase and 6-phosphog1uconate dehydrogenase was monitored for up to 48 hours after refeeding. Induction occurred by 24 hours, and by 48 hours, 4.2 and 1.5 fold increases were observed for glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, respectively,.com- pared with that of livers of pellet fed rats. After re- feeding, lysosomes increased in fragility as judged by an increased release of acid phosphatase activity during standard homogenization. Fragility was greatest 3 hours after refeeding, but normal fragility was observed 24 hours after refeeding. Nuclei were isolated from the liver sam- ples before and after refeeding. Those isolated just before refeeding revealed small latent acid phosphatase activity (4-6%). However, after refeeding the carbohydrate-rich diet, a transient and significant (P<<0.01) increase in 12 13 the latent activity occurred that was maximal (20%) at one hour, returning to normal by 24 hours. Cross-mixing the 800xg nuclear pellet from livers of animals starved for 3 days with the 800xg supernatant fraction from livers of animals refed the carbohydrate-containing diet did not alter the nuclear lysosomal free (overt) or latent (deterj gent released) enzyme activity. Similarly, mixing the 800xg nuclear pellet from livers of animals refed for 1 hour with the 800xg supernatant fraction from livers of animals starved for 3 days, but not refed, did not change the nuclear lysosomal free or latent enzyme activity. Purified nuclei, further washed in hypotonic buffer, lost acid phosphatase activity, but those isolated from livers of rats refed for one hour retained 10% of the enzyme latency, whereas all latency was lost from those isolated from unin- duced rats. A second lysosomal enzyme,/3-galact’osidase, ‘became associated with the nuclei with the same temporal pattern as for acid phosphatase. However, no variation in nuclear content of cytosolic lactate dehydrogenase occurred as a result of feeding the high carbohydrate diet to starved rats. When similarly starved rats were refed a diet high in lipid and carbohydrate-free, no induction of glucose—6-phos- phate dehydrogenase and 6-phosphogluconate dehydrogenase was observed. Lysosomes were not temporarily fragile and puri- fied nuclei did not exhibit increased latency of acid 14 phosphatase activity. Though the evidence presented does not establish a direct correlation between lysosome migra- tion to nuclei as a required function in enzyme induction, the temporal and specific nature of the phenomenon support the hypothesis that liver lysosomal enzymes participate in early signals in the induction of enzymes of lipogenesis. INTRODUCTION It is well recognized that the enzymes of liver lipo- genesis are induced after refeeding fasted rats a diet rich in carbohydrate (1-6). To elicit the induction of pentose phosphate shunt dehydrogenases, the administration of an excess of glucose for an extended period of time is required (1. 7, 8). The mechanism by which nuclei receive an induc; tion signal is relatively unknown. Since induction occurs many hours after the return of insulin to normal levels, it has been suggested that insulin may not be direCtly involved (5, 8, 9). However, the release of insulin that accompanies refeeding starved rats a carbohydrate-rich diet (10) may trig- ger early events in the liver that precede the first measure- able increase in lipogenesis related enzyme activity by several hours. Recently, we reported that a transient fragility of liver lysosomes occurred before the induction of pentose phos- phate shunt dehydrogenases by a carbohydrate-rich diet (11). To support the hypothesis that lysosomes carry the induction signal from cell membrane to nuclei, lysosomal translocation would have to occur prior to induction, be transient and be specific for refeeding diets that promote induction of lipogenesis 15 enzymes. The present studies provide evidence for the early translocation of lysosomes to nuclei after refeeding fasted rats diets rich in carbohydrate. MATERIALS AND METHODS Animal Treatment. Male rats of the Holtzman strain weighing approximately 150 g were housed in groups of 4-5 in plastic cages containing wood chips. A control group was fed a com- mercial chow (Lab-Blox, Allied Miles, Inc.) and water, 66 libitum, throughout the experiments. Animals were fasted with free access to water for three days and refed a diet containing 20% casein, 68.9% glucose monohydrate, 5% corn oil, 5% Wesson salt mixture, 0.1% choline chloride and 1% vitamin mixture (11, 12). In another experimental series, the simi- larly fasted rats were refed the high fat diet reported by Sassoon,_g£_62; (12), consisting of 33% casein, 53% lard, 5% corn oil, 5% Wesson salt mixture, 0.1% choline chloride, 2.9% alphacell and 1% vitamin mixture. Sample Preparation. Rats were killed by decapitation. Livers were quickly excised and chilled in ice cold 0.25 M sucrose and 2 mM MgC12. All subsequent operations were performed at 0-4t. Samples of liver (4 g) were homogenized in a 15% (w/v) 0.25 M sucrose and 2 mM MgCl2 solution with a Tekmar Model SDT tissue disruptor (65 volts for 30 sec.) followed by fil- tration through six layers of cheese cloth. Aliquots were set aside as whole homogenate samples. The resulting homogenates 16 were processed by differential centrifugation to obtain the 800xg, 22,000xg and 105,000xg supernatant fractions as de- scribed by Schroeder, g§_§22 (11). Nuclear preparations were purified by high speed centrifugation in 2.4 M sucrose as described by Szego, 62_ EE: (13) except that a fixed angle rotor was used. The crude nuclear pellet from the 800xg centrifugation was resuspended in the homogenizing medium to a total volume of 2 ml and di- luted with 20 ml of 2.4 sucrose containing 1 mM MgCl The 2. suspension was centrifuged at 50,000xg for 45 min. at 0°C in a 30K rotor. The purified nuclear pellet was resuspended in 0.25 M sucrose for biochemical analysis or electron microscopy. In a study of possible nuclear contamination by CYtO? plasmic lysosomes, crude nuclei (800xg pellet) isolated from rats starved for 3 days (zero time) and those starved for 3 days and refed the high carbohydrate diet for one hour were cross-mixed with the supernatants of one hour and zero time, respectively. The resuspended mixtures were incubated at 00C for 45 min. and were recentrifuged at 800xg to obtain crude nuclei which were then purified by centrifugation in 2.4 M sucrose as previously described. In another experiment, the highly purified nuclear samples from the livers of rats fed the chow diet, the starved animals and those refed for one hour were resuspended in a hypotonic solution containing 10 mM NaCl, 3 mM MgCl and 10 mM Tris-HCl, pH 7.4, at 00C 2 for 45 min. The hypotonically treated nuclei were then reiso- lated by 800xg centrifugation and resuspended in 0.25 M sucrose for analysis. To further assess the specificity of lysosomal 17 enzyme migration to nuclei, nuclei were assayed for the cyto- solic enzyme, lactic dehydrogenase (EC 1.1.1.27) by the method of Hacker,.gg_§2; (14). The enzyme was not inhibited by Triton X-100 (0.2% w/v). Reaction rates at 30°C were measured in a Gilford 3500 computer directed spectrophotometer at 340 nm. Determination of Enzyme Activities by Computer Directed §pectrophotomet§y. Acid Phosphatase (EC 3.1.3.2). The enzyme activity was assayed by a modified method of Vaes and Jacques (15). Inorganic phos- phate measurements were adapted from the methods of Ames (l6) and Tashima (17). The reaction mixture contained in final concentration: 0.05 M lg-glycerophosphate, 0.2% (w/v) Triton X-100, 0.1 M Na Acetate, pH 5.0 and an appropriate amount of enzyme. Triton X-100 was not included when the overt (free) nuclear acid phosphatase activity was measured. .The difference in total and overt activity was taken to represent latent (lysosomal) acid phosphatase. The reaction was incubated at room temperature in cups mounted on a Gilford 3500 computer directed spectrophotometer and was stopped by the addition of twice the assay volume of a reagent consisting of 1.86% (w/v) ascorbic acid, 0.46% (w/v) ammonium molybdate and 0.75% (w/v) sodium dodecylsulfate (SDS) in 1.0 N H2804. For each individual reaction, a corresponding blank was prepared in which the stop- per reagent was added with the enzyme. The absorbance was measured at 700 nm after at least 1.5 hr. incubation at room temperature or 20 min. incubation at 45°C for color development. Deproteinization was eliminated as all the proteins in the 18 reaction mixture were sufficiently solubilized by the SDS. By using mode 1 of the End Point-2 Program, Gilford Model 3500, the desired enzyme volume, reaction mixture and stopper reagent were pipetted at the proper time intervals automati- cally.‘ A standard curve of inorganic phosphate (KH2P04) was analyzed with each assay. The instrument was then programmed by mode 3 to read the absorbance of each reaction and automa- tically print out the net absorbance change. Lysosomal fl-Galactosidase (EC 3.2.1.23). The total enzyme activity was assayed according to Blosser and Wells (18) using p-nitrophenyl-fi-Q-galactopyranoside as substrate. The 500 }1 reaction mixture contained in final concentration: 3 mM p-nitrophenyl-fi -D-galactopyranoside, 0.2% (w/v) Triton X-100, 50 mM sodium phosphate and 50 mM sodium citrate buffer, pH 5.0, and enzyme. After incubation at 37°C for 60 min., the reaction was stopped with 1 m1 of 2.7% (w/v) trichloroacetic acid. Denatured protein was removed by centrifugation and an aliquot (1 ml) of the supernatant solution was treated with an equal volume of a sodium borate solution (0.108 M NaZB407'H20 + 0.133 N NaOH) and the resulting p-nitrophenol released was measured at 410 nm in a Gilford 3500 spectrophotometer. Glucose-6-Phosphate Dehydrogenase (EC 1.1.1.49) and 6-Phospho— gluconate Dehydrogenase (EC 1.1.1.43). The dehydrogenases from the 105,000xg supernatant fraction were assayed at 30°C according to a modification by Rudack, et a1. (19) of proce- dure 2 of Bottomley, et a1. (20). The assay was performed 19 with the Gilford 3500 computer directed spectrophotometer using the General Kinetics-l program. All the above reactions were routinely run at two to three enzyme concentrations to verify linearity and in all cases, the reaction rates were shown to increase linearly with time and enzyme concentration. DNA Determination. DNA content in the nuclear preparations and in the whole tissue homogenates was measured by a method modified after Hill and Whatly (21). To 10—20 lul of tissue homogenate or 2-5 ,ul of the nuclear preparation were added 30 ‘,ul of a 1% sodium deoxycholate solution. After 5-10 min. at room temperature the volume was brought to 2 ml with an aqueous solution containing 50 mM Tris, pH 7.4, followed” by the addition of 50 ,ul of 200 lug/ml Mithramycin (Pfizer Co.) in 300 mM MgC12. The mixture was agitated ‘and the fluorescence was measured at 540 nm with excitation at 435 nm using a Farrand Ratio Fluorometer-2 interfaced and directed by mode 3 of the End Point-1 program of the Gilford 3500 computer. This more sensitive fluorometric ana— lysis of DNA gave values that agreed favorably with that ob- tained by the dephenylamine reaction (22). Electron Microscgpy. Nuclei were prepared for examination by scanning electron microscopy by a modification of the prece- dure described by Kirschner and Rusli (23). Nuclear prepara- tions suspended in 0.25 M sucrose with 2 mM MgCl2 were fixed for 4-18 hours at 4°C by mixing with an equal volume of 3% 20 glutaraldehyde in 0.2 M cacodylate buffer, pH 7.4. After fixation, nuclei were rinsed three times with 0.1 M cacody- late buffer, pH 7.4, containing 7.5% sucrose and dehydrated through a graded series of ethanol solutions (5 minutes each) to 100% ethanol. They were then dried on glass cover slips in a Sorvall Critical Point Drier using C02 as the carrier gas and coated with approximately 200 X of gold in a Film-Vac model EMS-41 sputter coater. Specimens were examined in an ISI Super Mini I Scanning Electron Microscope operating at an accelerating voltage of 15KV. Protein Determination. Protein concentration was determined by the method of Lowry, et a1. (24), and modified for auto- mated analysis by the Gilford 3500 computer directed End . Point-l program with bovine serum albumin as the standard. RESULTS Induction of the Dehydrogenases. A minimum of two complete experiments were performed for each series with rats of the same size, sex and age. The dietary intake of the groups refed a high carbohydrate diet and the groups refed a high lipid diet were comparable. In addition to diet consumption, Table I contains data for body and liver weights. The response of liver glucose-6-phosphate dehydrogenase and 6-phosphogluco- nate dehydrogenase activities to the refeeding of either the high carbohydrate diet or the high lipid diet is represented in Table II. Refeeding starved rats the high carbohydrate diet resulted in an increase, over that of the liver of 21 pellet-fed rats, in the specific activities of 4.2 and 1.5 fold for G6PDH and 6PGDH, respectively after 48 hours. When the high lipid diet was refed to starved rats, the specific activities of these two lipogenic enzymes in the liver remained virtually unchanged for 48 hours. Lysosomal Fragility and Transient Increase in Latent Nuclear Lysosomal Enzymes. In previous reports from this laboratory (11), there was an increase in rat liver lysosomal fragility as judged by an increased release of latent enzymes after refeeding starved rats a carbohydrate-rich diet. In the present study, the free acid phosphatase activity (as % of total) in the 22,000xg supernatants was followed from zero " to 48 hours after refeeding a high carbohydrate diet or a high lipid diet. The degrees of liver lysosomal fragility in starved rats in response to the refeeding of a high carbo- hydrate diet are shown in Fig. l. The percent of acid phos- phatase that was free significantly increased from 20.9 f 0.85% at zero time to 27.9 I 1.30% (P-<:0.0l) 3 hours after refeed- ing. The total nuclear acid phosphatase and the overt nuclear acid phosphatase activities were assayed immediately after the highly purified nuclear fractions were prepared. Refeed- ing the high carbohydrate diet to the starved rats caused a transient and significant (p-<:0.01) increase in both overt and latent acid phosphatase activities in liver nuclei that reached a maximum 1 hr after refeeding the diet. The total acid phosphatase activity in the nuclear samples from the rats 22 receiving a diet high in carbohydrate for one hour was signi- cantly higher than that from zero time (725 f 41.5 vs. 484 f 47.3 nmoles Pi/mg DNA/hr, (P <30.01). Prior to refeeding, 6.0% of the total nuclear acid phosphatase was latent. In comparison, the latency rose significantly (P<< 0.01) to 20% of total 1 hr. after refeeding the high carbohydrate diet. The total nuclear acid phosphatase activity in the rat liver declined rapidly 3 hr. after refeeding to values not signifi- cantly different from those observed before refeeding. The latency of the nuclear enzyme activity returned to 4.9% 3 hours after refeeding the diet. I The observed increases in both overt and latent acid' phosphatase activities in the nuclear preparations were not, accompanied by detectable alteration in total acid phosphatase activities in the whole homogenates based either on tissue weight or DNA content. However, using the nuclear and homo- genate enzyme activities per unit DNA, we calculate that no more than 0.3-0.5% of the total activity resides in nuclei. Total lysosomal fi?-galactosidase activity was measured in the nuclear samples which had been frozen and thawed (Fig. 2). The activity rose significantly (P <10.0l) from a mean of 50.9 I 3.1 nmoles p-nitrophenol/hr/mg DNA in the nuclear samples at zero time to a mean of 73.8 I 3.5 nmoles p-nitro- phenol/hr/mg DNA 1 hr. after refeeding, and decreased to a mean of 57.6 f 8.9 nmoles p-nitrophenol/hr/mg DNA 24 hrs after refeeding. As a control for nonspecific cytosolic enzyme binding to liver nuclei, we measured lactate 23 dehydrogenase in typically purified nuclei during the course of a starvation-high carbohydrate diet refeeding study (Fig. 2). During the period that lysosomal enzymes associated with nuclei, nuclear lactate dehydrogenase activities did not vary significantly. A significant decrease in the amount of lactate dehydrogenase did occur 24 hrs. after refeeding. The results presented in Fig. 3 demonstrate that there was no significant change in liver lysosomal fragility over the 3 hour period after refeeding the high fat diet as judged by the percent of total acid phosphatase activity that was overt, that is, in the 22,000xg supernatants. However, a significantly (P‘<'0.01) increased lysosomal fragility was observed 24 hours after refeeding the diet. The results shown in Fig. 3 also indicated no detectable change in either overt or latent acid phosphatase activity in the liver nuclei from rats at various time points after refeeding the high lipid diet. Again, the total nuclear ’8 -ga1actosidase activity reflected a similar pattern with that of acid phosphatase; that is, no detectable alteration in total nuclear activity was found. Studies of the Nuclear Lysosome Specificity. A study was con- ducted to evaluate the possibility of an artifactual pheno- menon in which the refeeding of a high carbohydrate diet for one hour to starved rats might facilitate the sticking of liver lysosomes to liposome-free nuclei during the tissue homogenizing procedure. Crude nuclei isolated from rats killed at zero time and 1 hour after refeeding were cross-mixed 24 and incubated with the 800xg supernatants of the samples of one hour and zero time, respectively, as described in the Materials and Methods section. The latent and overt acid phosphatase activities in the cross—mixed nuclei are presented in Table III. After treatment, the nuclei from the 1 hr. rat liver still retained increased overt and latent acid phos- phatase activity as compared with that of zero time nuclei. Moreover, lysosomes in the 800xg supernatants of the one hour rat liver samples failed to alter the low latent activity of the enzyme in the nuclei from zero time rat livers. Further attempts were made to evaluate the extent of lysosomal binding to nuclei partially purified by sucrose density centrifugation by resuspending nuclei from the com-. mercial chow-fed control, 3 day starved (zero time) and 1 hour refed rat groups in the hypotonic solution described in Methods and Materials. The overt and latent acid phospha— tase activities in the treated and corresponding untreated nuclear samples are shown in Table IV. After the hypotonic treatment, acid phosphatase activity was reduced in all the nuclear preparations. A decrease in the protein to DNA ratio in all samples after the wash indicated that an appreciable amount of protein was removed. At least some of the nuclei had been disrupted during the process of mechanical washing and resuspending. After the treatment in a hypotonic solution, there was virtually no latent acid phosphatase activity exhi— bited in the nuclei from the chow-fed or starved groups. Latent activity (11%) of the enzyme could still be observed in the 25 nuclei from the starved rats refed for 1 hr. Lysosomal acti- vity of nuclei from different groups of experimental rats varied with the starting weight of the animals. However, the agreement within experimental studies is excellent as shown by the small standard deviations from the mean. Scanning Electron Microscopy. Typical nuclei isolated from the 800xg pellets of rat liver whole homogenates and washed by sedimentation through 2.4 M sucrose and 1 mM MgCl2 are visualized in Fig. 4 by scanning electron microscopy. The nuclei appear relatively uncontaminated by unbroken cells and cellular debris. DISCUSSION In agreement with numerous previous studies (1-8), the activity of the hexose monophosphate shunt dehydrogenases in the liver of the starved rat rebounded 48 hours after male rats were refed a high carbohydrate diet (Table 11). Further- more, a carbohydrate requirement for the induction of the dehydrogenases was confirmed (10) since refeeding a high lipid containing diet evoked no change in their activity. Szepezi and Berdanier (10) concluded from similar studies of the dehy- drogenases that the signal for their induction was carbohy- drate, though insulin could not be ruled out as a factor. The overshoot of dehydrogenase activity also required ade- quate dietary protein. The hypothesis that insulin may serve as a derepressor of genetic information in the liver of diabetic 26 rats has been tested by comparing the template activity for RNA synthesis of chromatin from liver of insulin treated diabetic rats to that of chromatin from liver of insulin- deficient diabetic rats (25). The time course study showed that the template activity reached a peak at two hours and was found to be 28% greater than that of chromatin from liver of diabetic rats not treated with insulin. Though serum glucose and insulin levels were not determined in the present study, we may assume by analogy with studies of others (5, 10) that insulin mobilization after refeeding the high carbohy- drate diet reached an initial peak at a time (approximately 2 hr.) when changes in lysosomal status also were apparent.' In contrast, no stimulation of serum insulin levels is observed in starved rats refed a high lipid diet (10). The stability of the lysosomal membrane under various metabolic conditions has been reviewed extensively (26—28). We previously reported that liver lysosomal fragility in chickens varied diurnally in an apparent response to dietary and hormonal stimuli (29). This observation was extended to the starved-refed rat model where manipulation of dietary and hormonal factors could be conducted (11). In these studies a labilization of liver lysosomes was apparent 3 hours after refeeding a high carbohydrate diet to rats previously starved for 72 hours. Hyperglycemia, induced in rats fed a commercial chow diet by Streptozotocin toxicity, or hypoglycemia induced in normally fed rats by insulin injection, failed to elicit an enhanced liver lysosomal fragility. In neither instance 27 were the dehydrogenases induced to overshoot.. When insulin or glucose was administered to normal fasted rats, a signi- ficant lysosomal fragility occurred within 3 hr. In the present study, we again observed liver lysosomal fragility that reaches a peak 3 hours after refeeding the high carbo- hydrate diet, but not after refeeding the high lipid diet. 4 Because lysosomes have been proposed as normal media- tors of hormone action (30, 31), and since lysosomal fragility and redistribution of lysosomal enzymes from normal intra- cellular locations to perinuclear or intranuclear locations were early and specific responses to hormones, we considered the time course examination of liver nuclei for lysosomal enzyme activity (latent activity) to be crucial to determine whether a diet-initiated hormone-communicated signal for enzyme induction was a valid hypothesis. It was important to assess the possible nonspecific attachment of a typical cytosolic enzyme, lactate dehydrogenase, to nuclei of the livers of starved rats fed the high carbohydrate diet. No significant difference in lactate dehydrogenase was observed during the critical early time course of the study. A signi- ficant increase in the latency of nuclear acid phosphatase occurred and reached a peak 1 hr. after refeeding starved rats a high carbohydrate diet (Fig. 1). No similar translo- cation of a small proportion of the total lysosomes (0.3- 0.5%) to the liver nuclei occurred when starved rats were refed a high lipid diet. The lysosomes associated with the nuclei 1 hr. after refeeding a high carbohydrate diet were not 28 attached by spurious stickiness since nuclei from livers of starved rats failed to acquire latent lysosomal activity as a result of cross-incubation with the 800xg supernatant frac- tion from liver homogenates obtained from rats refed a high carbohydrate diet for 1 hr. Washing of purified nuclei de- rived from starved rat liver in hypotonic solution reduced the latency altogether, but liver nuclei from rats refed a high carbohydrate diet for 1 hr. retained a significant amount of latency after the same treatment. These results suggest that some if not most of the intact lysosomes were exposed to the surface of the nuclei or loosely bound to the nuclei from both starved and starved-refed rats, but that for the latter nuclei, a significantly greater prOportion were either inter- nalized or more tightly bound to the nuclear membrane. Scan- ning electron microscopy, while useful in monitoring the purity of the nuclear preparations, was not able to discern the presence of intact lysosomes on the nuclear membrane in preparations from either starved or starved-refed rats. The translocation of lysosomal acid phosphatase and fig-galactosi- dase from the extranuclear to the nuclear fraction as well as the transient fragility of lysosomal membranes to mechani- cal disruption are compatible with a mechanism that involves the participation of lysosomal enzymes in derepression of lipogenesis enzymes in starved rats. Though the evidence gained so far for this function of lysosomes is only circum- stantial, it is of interest that in a variety of instances such as liver regeneration (32), hormone target cell 29 interaction (30) or exogenous mitogenic influences (33), a lysosome-mediated nucleocytoplasmic network of communication has been independently suggested. Further consideration of this hypothesis will require a more direct demonstration of the function of lysosomes as a mediating agent for the recep- tion, transduction and propagation of the action of a meta- bolic signal triggering enzyme induction. ACKNOWLEDGMENTS Scanning electron microscopy was kindly performed by Dr. Nanine S. Henderson, Department of Biochemistry, at the. Center for Electron Optics, Michigan State University. 10. 11. 12. 13. 14. 15. l6. 17. REFERENCES Tepperman, J. and Tepperman, H. M. (1958) Am. J. Physiol. 193, 55-64. Lin, E. C. C. and Knox, W. E. (1956) Biochem. Biophys. Acta 26, 85-88. Conney, A. H. and Burns, J. S. (1959) Nature (London) 184, 363-364. Numa, S., Matsuhashi, M. and Lynen, F. (1961) Biochem. 2. 334, 203-217. Gibson, D. M., Lyons, R. T., Scott, D. F. and Muto, Y. (1972) Adv. Enz. Reg. 26, 187-204. McDonald, B. E. and Johnson, B. C. (1966) J. Nutr. 62, 161-167. ' Bottomley, R. H., Pitot, H. C., Potter, V. R., and Morris, H. P. (1963) Cancer Res. 22, 400-409. Derr, R. F. and Zieve, L. (1974) J. Nutr. 104, 65-68. Stark, M. J., and Frenkel, R. (1974) Life Sci. 26, 1563-1574. Szepesi, B. and Berdanier, C. D. (1971) J. Nutr. 101, 1963-1574. Schroeder, H. R., Gauger, J. A. and Wells, W. W. 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(1976) Scanning Electron Microscopnyart V, in "Identification and Characterization of Isolated Cell Organelles by High Resolution Scanning Electron Microscopy," pp. 153-162. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R. C. (1951) J. Biol. Chem. 193, 265-275. Morgan, C. R. and Bonner, J. (1970) Proc. Nat. Acad. Sci. 66, 1077-1080. . Deter, R. L. and de Duve, C. (1967) J. Cell Biol. 223 437-449. Weissmann, G. and Thoma, L. (1964) Recent Progr. Hormone Res. 26, 215-245. Ignarro, L. J. (1975) in Lysosomes in Biology and Pathology, (Dingle, J. T. and Dean, R. T. eds.), Vol. 4, pp.48l-523. North-Holland Publishing Company, Amsterdam. Schroeder, H. R., Lawler, J. R., and Wells, W. W. (1974) J. Nutr. 104, 943-951. Szego, C. M. (1974), in Recent Progress in Hormone Research (Creep, R. 0., ed.), Vol. 26, pp. 171- 33, Academlc Press, New York. Szego, C. M. (1975) in Lysosomes in Biology and Pathology (Dingle, J. T. and Dean, R. T., eds.) Vol. 6, pp. 385- 477, North-Holland Publishing Company, Amsterdam. Adams, R. L. P. (1963) Biochem. J. 62, 532-536. Allison, A. C. and Mallucci, L. (1964) Lancet 2, 1371- 1373. 32 nva pom pom ow.m me.N o - . a eessmcou peso eweue>< ea.o H mm.m 3N.o.u se.m ea.o H mm.m. e~.o H me.e m A.ez swam w ooa\wv MON? -3 w. W... EM? a...“ w. 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Group II were liver nuclear samples from starved rats refed a high carbohydrate diet for 1 hr. mixed with the 800xg supernatants of liver homogenates from rats starved for 3 days. bExpressed as nmoles P- released per mg DNA per hour at room temperature (n=4 f S. D.). 36 .Am I av .oudumummfiou Boon um .H£ Mom <29 we pom vommmamu «m mmHoEG mm pmmmmumxmn .ems me new eo ee .e.s me .HomIeass as om eee mmumz_zs m .aomz SE OH mo wowumsmaoo coaunaom owcouomkn m as poumndoaw 6963 monEMm mnu mo muodwwa< .cosuomm mamwumumz pom whosumz won as omnsuommp mm Uwumaoms mHmB Hmaoszm .m .m H m I a em.o _H mm.m mm.e _H em.m . em.o H me.m mzm\emeeeem I e.mm I m.m- I em.m Ase seeeeem emeeese ee.H emm mm 4. com em 4. 3mm semsmeee meees mm + mmm so + mom es + mom sem>eeee use>o mwoummue saamosoouomsm mm.o _H os.m em.e H mm.m mm.e H me.m mzm\emeeeem I m.mm I e.e I em.e Ase seeeeem emeee>< em 4. 3mm em.H mos mm_H smm sem>eeee meeee mm + see 3m + eem mm + sme ses>meee eue>o cosumummmum pumvsmum .HS H Ummmm GOHumerMUm Cmm 3050 HQHUDZ cosum>umum mmmn m mmmm m memumnmmosm Uso¢ .muowp oumuvmsonumo swan m mommy mums vm>H6um ozu CH unmaummuu oscouomhn udotuHB Ho nuwB Hmaonc Hm>fia mo huw>wuom mmmumzmmosm vwo< >H emeee 37 2% O 300 Total C: e. r 2 E “a _ oten’r e 600- fl 0 E . m ,, a: 5 /L 3? m 2 6 ' Free P; g 2400- peIIeI- fed -30 3 Q. 0 U) .C O O. ._ I . :2 M‘NFQ < 3:" 2 200— ~20 a, Q) 3 I: 2 U E . O l l I, l JL J :| | IO 1: o l 2 3 T24 48 TIME AFTER REFEEDI NG (h r.) Figure l. A comparison of lysosomal membrane fragility (free acid phosphatase, % of total) with latent acid phosphatase activity of purified liver nuclei of rats starved for three days and refed (heavy arrow) a diet high in glucose as described under "Methods." Each point is the mean + standard deviation of 5 animals. The symboIs are described in the figure. 38 2 A s 3 a so- 0 E I a f S :3 I 7.: '5 6C“' ' m E I g c m v -I.4 cm 2.: pellet—fed 8 a I? 2. f; 40- —I.2 g a C)- o '5 fill) 22 .2 ‘//£2 0, ° 73 (D q'LZO*- «0.23::l 8 -O.6 E.» <7 ‘e’ 3 z Z O I I I I II I Am I 0.4 o I 2 3 D' 24 T453 3 TIME AFTER REFEEDINGIhrs) Figure 2. Total fi-galactosidase and lactate dehydro- genase activities of purified liver nuclei of rats starved for three days and refed (heavy arrow) a diet high in glucose as described under ”Methods." Each point is the mean 2 standard deviation of 5 animals. The symbols are described in the figure. 39 (D O O "I 600- ‘ (Total Free W31” W7 g4 pellet-f ed 200-; ~20 4C“)— Nuclear Acid PhOSphalase(n moles/hr/ngNA) o—o Free Acid Phosphatase ( %of Total) 1 l I I IL 1 l o I 2 3T"24"4s TIME AFTER REFEEDING (hrs) 0 (5 Figure 3. A comparison of lysosomal membrane fragility (free acid phosphatase, % of total) with latent acid phosphatase activity of purified liver nuclei of rats starved for three days and refed (heavy arrow) a diet high in fat and carbohydrate free as described under "Methods." Each point is the mean + stan- dard deviation of 5 animals. The symbols are described in the figure. 40 A Figure 4. Scanning electron micrograph of rat liver nuclei purified as described under "Methods." The magnification is 3200x. CHAPTER II EFFECT OF TRITON WR-l339 INJECTION ON INDUCTION OF LIVER HEXOSE-MONOPHOSPHATE SHUNT DEHYDROGENASES AND NUCLEAR LYSOSOMES IN THE FASTED-REFED RAT1 ABSTRACT The effect of Triton WR-1339 on induction of liver glu- cose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) was investigated. Male rats of the Holtzman strain were fasted for 3 days and refed a diet high in carbohydrate (68.9%). Twenty-four hours.prior to refeed- ing, the experimental rats were injected with 85 mg/lOOg body weight of Triton WR-1339 and the control rats with equal volumes of saline solution. The induction of the two lipogenesis related dehydrogenases in the liver of the Triton-treated rats was both delayed and depressed. In the Triton-treated rats, seventy-two hours after refeeding, 3.3 and 1.2 fold increases in specific activity were observed for G6PDH and 6PGDH respectively, compared with that of livers of pellet fed rats. In contrast, the control rats exhibited increases in specific activity of 7.5 and 2 fold respectively for the two enzymes. Hexosaminidase activities, glucose and triglyceride levels from plasma of both rat groups were compared. Very high triglyceride concentration and relatively higher hexosaminidase levels were observed in the plasma of the 41 42 Triton-treated rats, compared with that of plasma of the control rats for most of the experimental period. How- ever, 3 hours after refeeding the diet, significant elevations in both plasma glucose and hexoaminidase levels were demonstrated by the control rats but not by the Triton-treated rats. In another experimental series, liver nuclei from I rats with similar starvation and Triton treatments were isolated before and after refeeding the high carbohydrate diet. The previously observed transient and significant increase in nuclear acid phosphate 1 hour after refeeding untreated rats was not observed after eXpoSUre to Triton WR-l339. The evidence presented supports the possibility I that Triton WR-1339 impaired the function of liver lysosomes in carrying early signals for the induction of enzymes of lipogenesis. INTRODUCTION The non-ionic detergent, Triton WR-l339, is selectively taken up by and accummulated within lysosomes (l). The finding by Davies that Triton WR-1339 loaded lysosomes are not accessible to endocytosed proteins suggests that engorged lysosomes may cease to participate in normal functions (2). In our study of the induction of lipogenesis enzymes by refeeding fasted rats a diet rich in carbohydrate (3), we provided evidence for the early translocation of lysosomes to nuclei after refeeding. The purpose of the present investigation was to determine whether Triton interference with rat liver lysosomes would' alter the nature or temporal pattern of the translocation , phenomenon and the subsequent lipogenic enzyme induction after refeeding starved rats a carbohydrate rich diet. MATERIALS AND METHODS Animal Treatment and Sample Preparation. In Part A of the experiment, male rats of the Holtzman strain weighing about 200g were housed in groups of 4 in plastic cages containing wood chips. A control group was fed a commer- cial diet (LabBlox, Allied Mills, Inc.) and water, 66 libitum, throughout the experiment. Animals were fasted with free access to water for 3 days and refed a diet high in car- bohydrate as previously described (3). The diet consisted of 20% casein, 68.9% glucose monohydrate, 5% corn oil, 5% Wesson salt mixture, 0.1% choline chloride and 1% Vitamin mixture. Twenty-four hours before refeeding, the 43 44 rats were injected intraperitoneally with either 85 mg per 100g body weight Triton WR-l339 (Ruger Chemical Co. Inc. Irvington, N. J.) or 0.9%, w/v, sodium chloride in equal volumes. lAnimals were anesthetized with diethylether and blood was collected in chilled heparinized tubes by heart puncture. Sample were centrifuged at 1000xg for 10 min. and the plasma was removed. In part B of the experi- ment, male rats of the Holtzman strain weighing about 150g were housed in groups of 5. They were starved, injected with Triton, and refed the high carbohydrate diet as in part A. These rats were killed by decapitation. The livers of either intracardially bled or decapitated rats were excised and immediately placed in ice cold 0.25 M sucrose " containing 2 mM MgCl . Samples of liver (4g) were homo- genized and processeg by differential centrifugation to obtain the 800xg, 22,000xg and 105,000xg supernatant frac- tions as previously reported (3). Liver nuclei purified by high speed centrifugation in 2.4 M sucrose as previously described (3) were also obtained from animals of Part B of the experiment. Enzyme Assays and Metabolite Determinations. Hexosaminidase (EC 3.2.1.30). The enzyme from the plasma samples was assayed by modification of the method employed by Blosser and Wells (4) and adapted to a program develOped for the Gilford 3500 computer directed SpectrOphotometer in this laboratory. The reaction mixture contained in final concentration: SmM p-nitrOphenyl-N-acetyl:P-glucos- 45 aminide, 0.2% (w/v) Triton X-100, 50 mM sodium citrate buffer, pH 4.2, and enzyme. After incubation at room temperature the reaction was stOpped with twice the assay volume of 150 mM sodium glycinate, ij>10. Reactions stOpped immediately after enzyme addition served as blanks. The amount of p-nitrophenol released was measured auto- matically by the Gilford 3500 spectrophotometer at 410 nm. Triglycerides and Glucose levels. To determine glucose levels in the plasma samples, the samples were first deproteinized by the method of Somogyi (5). An aliquot (50‘nl) of the plasma was mixed with 0.5 ml of titrated 5% ZnSO and 0.5 ml of 0.3 N Ba(0H) . The mixture was shaken,4centrifuged, and the upper Elear supernatant was used for glucose determination. The glucose content of the deproteinized plasma and triglycerides of the whole plasma were determined by using the Gilford System 3500 Computer Directed Analyzer and the Worthington/Gilford Automated Glucose and Triglyceride Kits. G1ucose-6-phosphate Dehydrogenase G6PDH (EC 1.1.1.49) and 6-Phosphogluconate Dehydrogenase 6PGDH (EC 1.1.143). The dehydrogenases were assayed in the 105,000xg supernatant fraction with the Gilford 3500 computer directed Spectro- photometer as previously described (3). Acid phOSphatase (EC 3.1.3.2) and P-Galactosidase (EC 3.2.1.23) assays, DNA and protein levels were determined by methods previously described (3). All the reactions were routinely run at two to three enzyme concentration and for various lengths 46 of time to verify linearity with time and enzyme concen- tration. Total enzyme activity refers to that detected in the 800g supernatant fraction in the presence of 0.2% (w/v) Triton X100 (Rohm.& Haas). Free activity signifies that measured in the 22,000g supernatant in the presence of 0.2% (w/v) Triton X-100. The free activity is also expressed as a percentage of the total units without regard to quantity of protein. RESULTS Effect of Triton WR-1339 on Induction of the Dehydrogenases. Table I contains data for body and liver weights and diet consumption of animals used for part A of the experiment. The dietary intake of the groups receiving either the Triton or saline injections are comparable. The response of liver G6PDH and 6-PCDH activities to refeeding the high carbo- hydrate diet up to 72 h in these two rat groups is repre- sented in Fig. 1. At the start of refeeding (zero time) the activity levels for the two dehydrogenases in livers of rats with Triton treatment were essentially the same as that of the saline control rats. Upon refeeding the saline control rats, the expected increase in both dehydrogenase activities was observed by 24 h; and by 72 h the Specific activities of C6PDH and 6PGDH had risen, reSpectively to 7.5 and 2 times the levels in the livers of pellet-fed rats. When the high carbohydrate diet was refed to the starved rats which had received the Triton injections, the response of both enzymes was substantially depressed or delayed. By 24 h no significant induction had occurred for either enzymes. By 72 h the specific activities of G6PDH and 6PGDH in the livers of this group had increased only 3.3 and 1.2 fold respectively. These increases were significantly smaller than those in saline control rats (P <:0.01). 47 48 Effect of Triton WR-1339 on Plasma Hexosaminidase,yglucose and Triglyceride levels. By twenty-four hours after the injection of Triton, the concentration of triglyceride in the plasma of the experimental rats had risen 28 fold greater than that in the plasma of pellet-fed rats (Table II). We attribute this, in part, to the known capacity of detergents to associate with triglycerides in the plasma in such a way as to reduce their rate of reuptake by the liver (6,7). The triglyceride concentration remained high up to 48 h, but has drapped 88% by 72 h after refeeding. Three days of fasting decreased plasma triglyceride levels in saline injected rats to one-fourth those in the pellet-fed rats. On refeeding, the plasma triglyceride levels rose gradually and by 72 h had risen to 60% of the normal level. After the resumption of feeding the high carbohydrate diet, plasma glucose levels in rats of the saline control group rose significantly by 3 h (p <:0.05) and then returned to normal by 24 h (Table II). On the other hand, only an insignificant increase in plasma glucose levels was observed by 3 h for the Triton-treated rats. At 24 to 72 h after refeeding the diet, the plasma glucose levels from neither group were significantly different than those observed just before refeeding. The plasma hexoaminidase activities at different times after refeeding are presented in Table II. At 3 hours after refeeding a significant (p <:0.01) elevation in plasma hexosaminidase levels was observed in the saline injected 49 rats. These levels had returned to normal by 24 h. Triton- injected rats had significantly higher plasma hexosaminidase activities than saline-injected rats just before refeeding (368 2,31 vs. 295 i 3.4 nmoles/ml/hr). Further elevation in this activity had occurred by 3 hours after refeeding for this Triton-treated group and the levels remained elevated relative to saline-treated controls throughout the experiment. Effect of Triton WR-1339 on Nuclear Lysosomal Enzyme Activities and Liver Lysosomal Frggility. Our previous study (3) found increased lysosomal membrane fragility in the liver of fasted rats refed a high carbohydrate diet: Evidence for increased fragility was, first, the increased" release of latent (lysosomal) enzymes after standard mechan- ical cell disruption and, secondly, a transient increase in total and latent nuclear lysosomal enzyme activities after refeeding starved rats the high carbohydrate diet. In the present study (part B of the eXperiment) rats of similar size (about 150g) were used to determine whether the Triton treatment would alter these observed phenomena. After 3 days of fasting the liver-lysosomes of Triton- treated rats were more fragile than those only starved for 3 days, as judged by the higher free acid phosphatase activities (as % of that activity in 800xg supernatants) in the 22,000xg supernatants (Fig 2) 30.3 i 4.7 vs. 20.9 i 0.8 . When the diet was refed the rats pretreated with Triton, the fraction of total acid phosphatase activity that was in the 22,000xg SUpernatant increased in the first 50 hour but not significantly. The lysosmmal fragility of this rat group remained high throughout the experiment. The total and free nuclear acid phOSphatase activities were assayed immediately after the highly purified nuclear fractions were prepared. The results in Fig. 2 reveal a slight increase over the starved controls in the mean total. nuclear acid phosphatase activity during the first hour after refeeding. However, this difference was not statis- tically (P:< 0.05) significant. Neither did the total activities differ significantly from.the correSponding free activities at any time after refeeding. Total lysosomal e-galactosidase activity was measured in the nuclear samples which had been frozen and thawed (Fig. 2). 'The total nuclear B-galactosidase activity reflected a pattern similar with that of total nuclear acid phosphatase. No significant change in total nuclear activity occurred in the 48 hours after refeeding. I DISCUSSION Wattiux,.66_§2, (1) found that injection of Triton WR-l339 caused a very marked decrease in the density of hepatic lysosomes. The subsequent analysis of this pheno- menon showed it to be the consequence of massive intra- lysosomal storage of the detergent. After uptake of the detergent, the lysosomes swell and become more fragile (8) as indicated in this study by the increased % free acid phOSphatase released in the 22,000xg supernatants (Fig. 2). Such lysosomes can not be considered normal and may not 51 function physiologically. Evidence supporting this assumption is found in Davies' report (2) which suggested that Triton WR-l339 filled lysosomes represent inert residual bodies and do not participate in one of the main functions of lysosomes, the digestion of protein taken into the cell by phagocytosis. Therefore, it is a reasonable assumption that Triton WR-1339 would also impair the pos- sible function for lysosomes as carriers of information from the plasmalemma to the nuclear envelope and the nucleoplasm presumably due to the inability of "tritosomes" to fuse with phagosomes (9). Szego has recently discussed the possible role of the lysosomes as agents mediating the action of hormones and other cellular messengers in specific target tissues (10). Evidence supporting this hypothesis has been reported for both steroid hormones (11) and peptide hormone (12). In previous studies, the lability of liver lysosomes was increased 3 hours after refeeding a high carbohydrate diet to rats previously starved for 72 hours (13). Furthermore, a significant increase in latent acid phosphatase occurred in the nuclei and reached a peak 1 h after refeeding the diet (3). Because lysosomal fragility and redistribution of lysosomal enzymes from normal intracellular locations were early and specific responses to hormones (10,11), we considered the interference with the normal functions of lysosomes to be a useful approach to determine whether. the speculated role of lysosomes in enzyme induction would 52 be impaired; and whether normal induction of the enzyme would be altered. This study demonstrated that the induction of two enzymes of lipogenesis, g1ucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase after refeeding fasted rats a carbohydrate rich diet was significantly suppressed and delayed by a prior Triton WR-1339 injection. It further demonstrates that no significant translocation of lysosomal enzymes to the liver nuclei occurred when the diet is refed the Triton-treated rats. Nevertheless, 72 h after refeeding the diet, 3.3 and 1.2 fold increases in Specific activity for G6PDH and 6PGDH, respectively, are observed in the Triton-administered rats. It is likely that not all liver lysosomes are incapacitated by Triton WR-l339. In his extensive review of lysosomes heterogeneity (14), Davies concluded that there are two or more lysosomal papulations in rat liver, and that not all lysosomes of rat liver are affected by Triton WR-1339. Thus, the small population of liver lysosomes which retain functional activity might account for the partial induction of the lipogenic enzymes. This study also shows that the rise in plasma glucose levels in the first 3 hours after refeeding is significantly greater for Triton-injected rats than for saline-treated controls. This suggests the possible effect of Triton WR-1339 on stimulation of insulin release or alternatively on glucose transport from the intestine to the blood 53 resulting in the delayed induction of the dehydrogenases. In addition, Triton WR-l339 is known to induce hyperlipemia in the blood of animals (15,16); the presence of high lipid levels in blood might affect the induction of the dehydro- genases independent of the participation of lysosomes. Our present study revealed a direct alteration of the physical properties of liver lysosomes in rats and a simultaneous interference with the inductin of the hexose monophOSphate shunt lipogenesis related dehydrogenases. Whether the interference with the enzyme induction is directly or indirectly affected by the impaired function of lysosomes remains to be further clarified. KomVGLfl 10. ll. 12. 13. 14. 15. 16. REFERENCES Wattiux, Rl, Wibo, M. and Baudhuin, P. (1963) in CIBA Foundation Symposium on Lysosomes (De Reuck, A. V. S. and Cameron, M. P. eds.): pp. I76-200 J. and A. Churchil, London. Davies, M. (1973) Biochem J. 135, 57. Mak, I. T. and Wells, W. W. (1977) Arch. Biochem, Biophys. 183, (in press). Blosser, J. C. and Wells, W. W. (1972) J. Neurochem. 22, 1539. Somogyi, M. (1945) J. Biol. Chem. 160, 69. Robinson, D. S. (1963) Adv. L2p. Res. 2, 133. Scanu, A. M. (1965) Adv. Lip. Res. 2, 63. De Duve and Wattiaux (1966) Ann. Rev. Physiol. 26, 435. Davies, M., Lloyd, J. B. and Beck, F. (1971) Biochem. J. 121, 21. Szego, C. M. (1975) in Lysosomes in Biology and Patho- logy_ (Dingle, J. T. and Dean, R. T., eds.)- 6, pp. 385- 477, North-Holland Publishing Co., Amsterdam. Szego, C. M. (1974) in Recent Progress in Hormone Research (GREEP, R. 0. ed.7726, pp. 171-233, Academic Press, New York. Szego, C. M., Rakich, E. R. Seeler, B. J. and Gross, R. S. (1974) Endocrinology 95, 863. Schroeder, H. R., Cauger, J. A. and Wells, W. W. (1976) Arch. Biochem. Biophys. 172, 206. Davies, M. (1975) in Lysosomes in Biology and Pathology (Dingle, J. T. and Dean, R. T., eds.) 6, pp. 301-348, North-Holland Publishing Co., Amsterdam. Kellner, A., Correll, J. A. and Ladd, A. T. (1951) J. Exp. Med. 22, 373. Friedman, M. and Byers, S. O. (1953) J. Exp. Med. 62, 117. 54 55 em.m e - - - I m esmv eem Hem OH.¢ o I I I I H toadmcoo uosa 0wmum>< sm.e H me.m mm.e H me.m - - - - m A.e3 seem m oem\mv mm.e “.ms.m em.e H mm.m em.e H ee.e s emmmez Oesmm e H msm mm.“ mmH - I I - m mm H mam mm H ems es “.mem s Ame eemmez seem musom m Case 0 Houucou anouu muuomoum com 3030 umm seem emu memeeemem Neemm ease mmmHImz ceases tees eeeeemeH eeem .MDOHQ oumnpmsonumu Cwsm m pmwmm tam mosamm H0 pm>umum mo muanOB H0>HA pom .mpom paw aowuaasmcou pooh H emees 56 .poHHmQ HmucmfiHhmmxo woumOHch 0:0 How was H0O UmEflmcoo ume mwmnm>m OCH oum3 moDHm>n .m .m H e I e Ame eeseHe> Hmnwo mo CoHusHom ocHHmm NO.O H0 AHV .uB mvon w OOH H0O OmmHIMB couHHH mo we mm HmnuHo nuHs .O .H UmuomnaH ouoz mumu Ono waHpommmu ouommn muson em .HOHO mumupssonnmo stn a woman vow muson us now vmummm mno3 Aw OON HDQHOV chHumIamENuHom mnu mo muwu mHmzm e.ms m.ee m.sH m msmv eem Hem m.es e.ee m.em s eeeeeeeo seem emeeesm mm.e H NH.m mm.e H mm.e me.e H mm.e m s.e3 seem m eem\mv se.e H em.e mm.e H ee.e me.e H ee.e s eemmes Reese em.H mam s.H emm mm H mmH m m _H omm m H sam mm_H Hem s Ame eemeez seem meson Nu muoom we mudom em Odouo huummonm uem seem emu mameeemem seemm ease .s.e.eeeev H emeee emm H emem eem_H eeem . 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O—-——I TN; M\ >~ O O LIJ “E E F.\'-C:\Q_4 '— .1. O _ -0 m 3.\I-<-I I I l ' (uIaIOJd bw/Sllunw') H090 9 H O—-I “NT; \ 3; O LIJ O—l -—2 \ P- 'O .23 2."? -O < ET“ A ' ' ° ' O 8 ‘ o Q‘ N (uIaIOJd Ow/suun W) H0d9 9 61 800 g- ‘ Total fl “Galactosidaso Nucleor Hydrolose Activity (:1 moles/hr/mg DNA) Free Acid Phosphatase (% of Total) 400- ~40 zoo~ 9 . I 9 ~30 g‘pellet-fed -20 0 l I l I II | H l , o a 2 3 j 24 48 '0 TIME AFTER REFEEDINGMrs). Figure 2. A comparison of liver lysosomal fragility (free acid phosphatase, Z of total) with acid phosphatase activit (expressed as nmoles/hr/mg DNA at 25°C? and the total -galactosidase (expressed as 10X nmoles/ hr/mg DNA at 37°C) of purified liver nuclei of rats starved for 3 days. The animals were treated with Triton WR-1339 1 day before re- feeding (heavy arrow) a diet rich in carbohy- drate as described under Materials and Methods. Each point is the mean i_standard deviation of 5 animals. CHAPTER III DIETARY REGULATION OF RNA SYNTHESIS IN THE LIVER NUCLEI OF FASTED-REFED RATS ABSTRACT Nuclei from liver of rats starved for 3 days and refed a diet high in carbohydrate (68.9%) demonstrated a significantly increased capacity to synthesize RNA 3 hours after the beginning of the refeeding. The increased RNA synthesis continued in nuclei of these rats and appeared to precede the induction of the enzymes of lipogenesis. After 24 to 48 hr. of refeeding, the isolated nuclei increased 2.2 fold in their capacity to synthesize RNA, compared with nuclei of livers of pellet-fed rats. When similarly starved rats were refed a diet high in lipid and carbohydrate free, insignificant elevation in the capacity of RNA synthesis was observed in the isolated nuclei. In another study, nuclei which were obtained from liver of starved rats and had been incubated for 30 minutes at 25°C. with the post-nuclear fraction of liver homogenate from rats starved and refed the high carbohydrate diet for 1 hr., exhitibed a significantly ( p<<0.01) higher RNA synthesis activity than that of the control nuclei which had been incubated with the post-nuclear fraction of liver from similarly starved rats without refeeding. The 62 63 lysosomes in the postnuclear fraction of the liver homo- genate was suggested as the possible factor triggering this phenomenon. INTRODUCTION In agreement with the literature (1-4), our previous study showed that the enzymes of liver lipogenesis were induced by refeeding fasted rats a diet rich in carbohydrate, but not by refeeding a diet rich in lipid and carbohydrate free ( Ch. I). Our study also demonstrated that the refeed- ing of the high carbohydrate diet, but not the high lipid diet, to the starved rats evoked an early increase in liver lysosomal fragility and a transient increase in the nuclear lysosomal enzyme activity. In the present study, we have . examined the time course effect of the high carbohydrate diet and high lipid diet refeeding on RNA synthesis in the rat liver and correlated the time course of the effect after refeeding the diets to the induction responses of the enzymes of lipogenesis. A preliminary attempt was also undertaken here to demonstrate the stimulatory effect of the post-nuclear fraction of liver homogenates from rats starved and refed the high carbohydrate diet for 1 hr. on the RNA synthesis capacity in liver nuclei of starved rats. MATERIALS AND METHODS Chemicals. [5,6-3HJ-UTP (sp. radioactivity 41.6 Ci/m Mole) was obtained from New England Nuclear Corp. Boston, Mass.; unlabelled nucleotide triphOSphates and dithiothreitol were 64 65 from Sigma Chemical Co. St. Louis, Mo.. Animal Treatment. Male rats of the Holtzman strain (Madison, Wi.) weighing 150-180g were used. The rats in groups of four were starved with free access to water for 72 hours and were then refed a high carbohydrate diet or a high lipid diet. The compositions of the two diets had been detailed in Chapter I. A control group was fed a com- mercial chow and water ad libitum.throughout the experiment. Nuclear Samples. At various times up to two days after refeeding, the rats were killed by decapitation, the livers were quickly removed and homogenized in 0.25 M sucrose con; taining 2 mM.MgC12. Highly purified nuclei were prepared ” essentially by the method described in Ch. I. The final nuclear pellet was resuSpended in a solution containing 25% glycerol, 5 mM Magnesium.acetate, 50 mM HEPES buffer, pH 7.6, and 5 mM dithiothreitol to give a concentration of 2-4 mg/ml DNA. The resuspended nuclei were divided intoaliquots which were either freshly assayed for RNA synthesis or immediately stored at -80°C.. No loss of RNA synthesis ability was found in nuclei stored for up to one month. RNA Synthesis. The conditions used to assay RNA synthesis were modified after the method of Sarma, gg‘al. (5). Each incubation mixture of 80‘pl contained in final concentration: 12.57. glycerol, 5-10 pC of [5,6-3HJ-UTP, 0.05 mM cold UTP, 0.4 mM of the other three nucleotide triphosphates, 5 mM magnesium acetate, 2.5 mM dithiothreitol, 25 mM HEPES buffer 66 pH 7.6, 0.3 M ammonium sulfate and the nuclear sample (20-40,ug of DNA). The incubation was started by briefly vortexing and transferring a tube to a water bath at 25°C.. Ten,pl aliquots in duplicate were removed before starting the incubation and after 10 min. and 20 min. of incubation. To the lO‘pl aliquots, 0.1 ml of 1% sodium dodecyl sulphate, 10 mM EDTA.were added. After standing for 20 min. at room temperature, 1 ml of ice cold 10% trichloracetic acid was added. The precipitate was collected on a 2.4 cm Whatman GF/C filter, washed twice with cold 10% TCA and twice with 95% alcohol. The filters were dried in an oven at 110°C. for 20 min. and counted by liquid scintillation in a toluene fluor. In vitro Incubation of Liver Nuclei from Starved Rats with 800 xggSupernatant of Liver Homogenate from.Rats Starved and Refed 1 Hr. the High Carbohydrate Diet. To study the possible stimulatory effect of the post- nuclear liver cytosol from starved rats refed a high carbo- hydrate diet for 1 hr. on RNA polymerase activity, male rats of the same size were used. After 3 days of starvation, some of the rats were refed the high carbohydrate diet for 1 hr. The rats were sacrificed and the livers were homo- genized in 15% (w/v) 0.25 M sucrose containing 2 mMngClz as previously described (Ch. 1). After centrifugation at 800xg for 10 min., crude nuclei (isolated from about 1.5g of liver tissue) from the zero time rats (starved without refeeding) were resuspended with either 6 ml of the 800xg 67 supernatant from the rats refed the high carbohydrate diet for 1 hr. or with 6 ml of the 800xg supernatant from starved rats as the control. The resuSpended mixtures were incu- bated at 25°C. for various time up to 2 hr. in this study. The incubation was stopped by transferring the samples to ice. The samples were then immediately recentrifuged at 800xg for 10 min. to obtain the crude nuclei which were again purified by centrifugation in 2.4 M sucrose as pre- viously described. Nuclear samples were assayed for RNA synthesis. DNA Content. DNA was determined by the method develOped in this laboratory as described in Chapter I. RESULTS AND DISCUSSION Conditions of RNA Synthesis. The Optimal concentration of 0.3 M ammonium sulphate was used to give maximal activity of RNA polymerase for this system” The temperature of the incubation was chosen to be 25°C. at which temperature RNA synthesis was found to be linear for up to 45 min. (Fig. la). At 37°C incorporation of labeled UTP into RNA was linear for only 15 min. Under the chosen conditions, the rate of RNA synthesis was also linearly dependent on the concentration of nuclei (Fig. lb). With the high concentration of ammo— nium sulphate selected, the RNA synthesis was presumed to be chiefly mRNA (6,7). Dietary Effect on RNA Synthesis in Liver Nuclei of Starved Rats. The dietary intake of the rats refed the high carbo- 68 hydrate diet and the high lipid diet were comparable and similar to that observed in our previous study (Ch. I). The pellet control rats were sacrificed at the same time of the day (9:00 a.m.) as that for rats of zero time, 24 hr. and 48 hr. groups. RNA synthesis activities were assayed immediately after the highly purified nuclear samples were prepared. Fig. 2 reports the time course of RNA synthesis activities in liver nuclei isolated from the fasted rats after refeeding a high carbohydrate diet or a high lipid diet. By the end of the starvation period, the RNA synthesis capacity in the liver nuclei was reduced to approximately 60% of that of pellet fed control. Upon refeeding the starved rats the high carbohydrate diet, elevation in RNA synthesis activity was observed by 3 hr. By 6 hr. the activity was already 2 fold that of the zero time rats or about the same activity level of those of the pellet control group. The RNA synthesis capacity in liver nuclei of these groups of rats continued to increase after 24 to 48 hr. of refeeding the diet. By 48 hr., a 2.2 fold enhancement for the RNA synthesis activity in the nuclei over that of the liver nuclei of pellet fed rats was ob- served in this study. In our previous study of the induction of the liver enzymes of lipogenesis (Ch. I), the increase in the hexose 'monphosphate shunt dehydrogenases was not seen until 24 hr. after refeeding the starved rats the high carbohydrate diet. The mechanisms evoking the response of the lipogenic enzymes have been generally agreed to involve increased 69 protein synthesis (3,8). However how the induction signal is transmitted to the nuclei is relatively unknown. Insulin has been suggested to be involved in the regulation of the induction of the enzymes as it was known that when diabetic rats were supplied with insulin, the activities of several liver enzymes increased and that the increase was dependent on both RNA and protein synthesis (9). Among the hepatic enzymes whose activities were found to be controlled by insulin level were G6PDH and 6PGDH. It was also demon- it" strated that insulin mobilization after refeeding the high carbohydrate diet reached an initial peak at 2 hr. To test the hypothesis that insulin may serve as a stimulator of genetic information, Morgan and Bonner (10) showed that insulin treatment to diabetic rats increased the prOportion of the diabetic genome which was available for transcription. Furthermore, the ability of insulin to mediate long term effects through its direct action on intracellular structure was demonstrated by the recent report that 125I-labeled insulin could enter the intact cell and bind to the nucleus (11). The results of the present investigation indicated that the transcriptional activation in the induced rat liver nuclei was observed after the transient increase in nuclear lysosomal enzyme activity (1 hr. after refeeding) and the increased fragility of liver lysosomes was apparent. The results reflected the transcriptional activation preceded the induction of the enzymes; and the continual increase in 70 the induction of the dehydrogenases was also preceded by a continual elevation of the RNA synthesis capacity in the nuclei. When the high lipid diet was refed to the starved rats (Fig. 2), the RNA synthesis activity from the liver nuclei remained virtually unchanged up to 6 hr. after re- feeding. By 48 hr. the activity was still just about 90% of that of the pellet fed rats. It is of interest that the results observed here correlated well with the fact that when the high lipid diet was refed to the starved rats, no stimulation of serum.insulin levels was observed (8), no early translocation of lysosomal enzymes to the nuclei, no. early increase in lysosomal fragility in liver and no subsequent induction of the dehydrogenases were demonstrated. Effect of the Liver Post-nuclear Fraction from Starved Rats with or without 1 Hr. Refeeding of the High Carbohydrate Diet on RNA Synthesis Activity of Starved Rat Liver Nuclei. In our previous study (Ch. I), a transient increase in nuclear lysosomal acid phOSphatase occurred and reached a peak 1 hr. after refeeding starved rats a high carbohydrate diet. As lysosomes have been prOposed as normal mediator of the action of hormones and other cellular biocatalytic sub- stances (12); our experimental evidence supported the possibility that by 1 hr. refeeding the diet to the starved rats, the liver lysosomes might be activated by the diet initiated hormone-communicated stimulus and were translocated to the nucleus in preparation for gene derepression. The 7l preliminary report herein.was an ig_yit£g study under arbitrarily chosen conditions, of the possible stimulatory effect of the post-nuclear fraction of liver homogenate from the rats starved and-refed 1 hr. of the high carbohydrate diet on the transcriptional activity in the nuclei. Pre- sumably the post-nuclear fraction.would contain the activated lysosomes. The RNA synthesis activities of the highly purified liver nuclei isolated after the incubation period for various time at 25°C. were represented by Fig. 3. Nuclei which had been incubated with the zero time rat (the starved rats without refeeding) liver 800xg supernatant exhibited the expected decrease in activity with the time they had been incubated at 25°C., presumably due to gradual degradation of the RNA polymerase. But nuclei which had been incubated with the 800xg supernatant of the 1 hr. refed rats demonstrated a temporal higher RNA synthesis activities which reached the peak for the nuclei which had been incubated for 30 min. Normally, the RNA polymerase in these nuclei should degrade at the same rate as those incubated with the liver 800xg supernatant of the zero time rats at 25°C. Thus the shaded area between the upper and lower curves in Fig. 3 could be attributed to the stimulatory effect of the post-nuclear fraction of the 1 hr. refed rats. The RNA synthesis capacity of the nuclei which had been incubated for 30 min. with the 800xg supernatant of the 1 hr. refed rats was higher than those nuclei without any incubation and was significantly (p70.01) higher than those control nuclei 72 which had been incubated with the 800xg supernatant of the zero time rats under the same conditions. However nuclei had been incubated longer than one hour exhibited no significant difference in the activity between these two groups. The mechanism of the observed stimulatory effect on the nuclei RNA synthesis capacity is unknown. It is possible that the activated lysosomes or lysosomal constituents triggered the activation. However, an alternative activator generated in the liver cytosol after refeeding the high carbohydrate cannot be excluded. Our next approach will be to examine whether purified liver lysosomes or lysosomal constituents would have any direct effect on the transcriptional activity in nuclei. Although this strategy is more unphysiological, a direct examination of the possible function of lysosomes as a 'mediating agent for the reception, transduction and prOpa- gation of the action of a metabolic signal triggering enzyme induction will ultimately be required. In conclusion, the study here has showed that the nature of the diet refed to the starved rat has a direct effect on transcriptional activity in the rat liver. But it is not known whether this regulation is achieved by a larger template availability for RNA polymerase and/or by an increased activity and/or availability of the enzymes itself. Further studies on the template availability of . chromatin and the total amount of DNA-dependent RNA polymerase 73 and the synthesized RNA species analysis would certainly give rise to a better understanding of the mechanism in the molecular level of our model of enzyme induction. 10. ll. 12. REFERENCES Tepperman, J. and Tepperman, H. M. (58) Amer. J. Physiol. 193, 55. Numa, S., Matsuhashi, M. and Lynen, F. (61) Biochem. Z. 334, 203. Gibson, D. M., Lyons, R. T., Scott, D. F. and Muto, Y. (72) Adv. Enz. Reg. l8, 187. . McDonald, B. E. and Johnson, B. C. (66) J. Nutr. 81, 161. Sarma, M. H., Reman, E. R. and Baglioni, C. (76) Bioch. Biophy. Acta 418, 29. Widnell, C. C. and Tata, J. R. (64) Biochem. Biophys. $222 81, 513. ' Novello, F. and Stirpe, F. (70) Febs. Letters 8, no. 1, 47. " Szepesi, B. and Berdanier, C. D. (71) J. Nutr. 101, 1563. Steiner, D. F. (66) Vitamins Hormones 84, l. Morgan, C. R. and Bonner, J. (70) Proc. Nat. Acad. Sci. 88, 1077. Goldfine, I. D., Smith, G. J., Wong, K. Y. and Jones, A. L. (77) Pro. Nat. Acad. Sci. lg, 1368. Szego, C. M. (75) in Lysosomes in Biology and Pathology (Dingle, J. T. and Dean, R. T., eds.) 4, pp. 385-477, North-Holland Co., Amsterdam. 74 Figure l a. Figure l b. 75 Time-course of incorporation of label from [3H] -UTP into RNA. At various time aliquots in duplicate were removed and TCA-precipitable counts determined as described under "Methods". Effect of nuclei concentration on RNA synthesis. Aliquots in duplicate were removed at 0 time, 10 min. and 20 min. after incubation at 25°C and the TCA- precipitable counts determined as described under "Methods". 3H-UMP Incorporated (pmoles/mg DNA) 3H~UMP Incorporated (pmoles/IO min.) 76 0i 0 O O T 2000f O IOOO- 0 l I I I 0 IO 20 30 4O TIM E (MinufeS) 6r 4- 2— O 1 1 L J 0 20 4o .60 , 80 Nuclear DNA Content in the Incuboflon Mixture (,ug) 77 l500" I000"- pe/llet-fed /1/ LAVi/H/ 3H-UMPlncorporated (pmoles/mg DNA/lo min.) or 8 I i—G—i I6—a\.——' I" Ci I 1 l I II I IL I O I 3 6 IV 24 j! TIM E AFTER REFEEDING (hrs) Figure 2. RNA synthesis activities in liver nuclei obtained from rats starved for 3 days and refed for various time a diet high in carbohydrate (H) or a diet high in lipid (C>——-<)). Experimental condi- tions were as described under "Methods". n = 4 $.30 D. 400— k 300- §\\\ 200- L I W l l l l o 30 so 90 :20 INCUBATI ON Tl M E AT 25°C(min.) Figure 3. RNA synthesis activities in liver nuclei which were from starved rats and had been incubated for various time with the 800g supernatant of liver homogenate from either the starved rats with 1 hr. refeeding of the high carbohydrate diet (H), or from starved rats without refeeding (C>—-C>), Experimental conditions were as described under "Methods". n = 3': S. D. CONCLUSION The preceding sections have examined the possible involvement of liver lysosomes in the induction of liver enzymes of lipogenesis. Evidence presently available does not prove whether the induction of the enzymes of lipo- genesis in rat liver is dependent on the function of the lysosome. However the temporal and specific nature of the phenomena observed strongly suggest that the partici- pation of lysosomes is required for the deve10pment of the full effect of the diet-initiated hormone-communicated” signal for enzyme induction. Since there is a growing body of evidence to indicate a contribution of cytOplasmic structures to the control of gene expression; and since lysosomal constituents including nucleases and proteases as well as acidic matricular proteins have been postulated to play specific roles in gene activation processes leading to anabolic pathways, evidence presented here has provided certain novel aspects for the study of the general role of lysosomes in nucleocytOplasmic communication. 79 M'TITIilfil7I1fljfllfljfi!Lilitflfflfil'liflilfljfl'flfilT'55