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Murphy has been accepted towards fulfillment of the requirements for Ph.D degreein Food Science / I Major professor Date 06-11-79 0-7639 ’ OVERDUE FINES ARE 25¢ PER DAY PER ITEM Return to book drop to remove this checkout from your record. ENZYME ACTIVITIES OF HEAPTIC PEROXISOMES 0F LEAN AND OBESE MICE By Patricia A. Murphy A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science and Human Nutrition 1979 ABSTRACT ENZYME ACTIVITIES 0F HEPATIC PEROXISOMES 0F LEAN AND OBESE MICE By Patricia A. Murphy Hepatic peroxisomes and their enzyme activities have been examined in lean and obese C57BL/6J mice and in HA(ICR) mice. No difference in the numbers of peroxisomes per unit area was observed between lean and obese mice by electron microscopy. Clofibrate(p-chloro-phenoxyisobutyrate ethyl ester) feeding at 0.5% of the diet caused no increase in catalase activity in crude homogenates of liver or in peroxisome numbers, but did cause an increase in liver weight in lean and obese C57BL/6J male mice. Catalase activity did increase in HA(ICR) male mice fed the same clofibrate diet. Hepatic peroxisomes have been isolated on isopycnic sucrose gradients from white HA(ICR) mice and lean and obese CS7BL/6J mice. Nearly all the catalase activity was in the peroxisomal fraction. Matrix marker enzyme activities, catalase and urate oxidase of the peroxisomes and glutamate dehydrogenase of the mitochondria, were similar in amounts of activity in lean and obese C57BL/6J male and female mice. Membrane components, NADPHzcytochrome c reductase of the microsomes and B-hydroxybutyrate dehydrogenase of the mitochondria, had lower activity in the obese mice in inverse proportion to the Patricia A. Murphy larger liver size. The white HA(ICR) male mice had marker enzyme activities similar to the lean C57BL/6J male mice. Fed and fasted mice had similar marker enzyme activities on a per 9 liver basis. Activity for peroxisomal fatty acid B-oxidation was the same for obese and lean mice, fed and fasted mice or male and female mice per 9 liver, but peroxisomal B-oxidation was approximately 3 times higher in HA(ICR) male mice than in the C57BL/6J mice. Mitochondrial fatty acid B-oxidation was the same when comparing lean and obese mice or male and female mice but higher for fasted mice than fed mice. Mito- chondrial fatty acid B-oxidation was higher in HA(ICR) mice than in lean and obese CS7BL/6J mice. The ratio of mitochondrial to peroxisomal fatty acid B-oxidation activities were the same in all groups compared. Hepatic NAD:glycerol-3-P dehydrogenase was higher in obese male mice compared to lean male mice in both the peroxisomes and in total activity in the liver. Fasting reduced the cytosolic fraction of this enzyme activity in the lean mice but not in the obese mice. The peroxisomal enzyme remained unchanged during a fast in lean and obese mice. Female obese mice had higher cytosolic and peroxisomal NAD:glycerol-3-P dehydrogenase activity than the lean females. Obese females had higher total and cytosolic enzyme activities than obese males. Lean C57BL/6J mice and HA(ICR) mice had similar activities of NAD:glycerol-B-P dehydrogenase. On a per animal basis, there was more hepatic peroxisomal fatty acid B-oxidation and more peroxisomal NAD:glycerol-3-P dehydrogenase activity in obese mice. Thus, there does not appear to be a lowered Patricia A. Murphy amount of hepatic peroxisomal activity associated with increased weight. The evidence in this research does not support the hypothesis that peroxisomal metabolism wastes energy in animals. ACKNOWLEDGMENTS The author wishes to express a sincere thanks to Dr. Jim Kirk for his support, encouragement and constructive criticism during the writing of this dissertation and also during the entire course of this project. To D. N. E. Tolbert of the Department of Biochemistry, a sincere thanks for the research support, laboratory facilities and guidance involved in this project that would not have been possible without his support. To Bob Gee, Jeff Krahling and John Gauger, a very sincere thanks for the technical help and constructive dis- cussions. This project would not have been possible without their collective efforts. This project was supported by NIH Grant HD-0644l and 5 $07 RR07D49. To Drs. Brunner, Leveille and Romsos, a thanks for the reading of this manuscript. A special thanks to my parents for the many forms of support they have provided during this long road. And finally, a thanks to my many friends for their support and friendship, especially Mary, during the highs and lows of this project. 11' TABLE OF CONTENTS LIST OF TABLES .................... LIST OF FIGURES ................... INTRODUCTION ..................... LITERATURE REVIEW .................. W Cyciles.:::::::::::::::::: Peroxisomes ................... Peroxisomal Enzymes ............... Catalase ................... a-Hydroxy Acid Oxidase ............ D-Amino Acid Oxidase ............. Glyoxylate Aminotransferase ......... NADPzIsocitrate Dehydrogenase ........ Urate Oxidase ................ Carnitine Acyl Transferases ......... NADzGlycerol-B-P Dehydrogenase ........ Fatty Acid B-Oxidation ............ Hypolipidemic Drugs and Peroxisomes ....... STATEMENT OF THE RESEARCH PROBLEM .......... METHODS AND MATERIALS ................ Total Hepatic Catalase Content in Crude Homogenates Examination of Isolated Organelles from Lean and Obese Mice ................... Comparison of Peroxisomal and Mitochondrial Fatty Acid B-Oxidation ................ Statistics .................... RESULTS ....................... Total Hepatic Catalase Activity ......... Isolation of Hepatic Mouse Peroxisomes ...... Differences in Hepatic Peroxisomal Marker Enzymes iii Page vi Comparison of the Matrix and Membrane Associated Enzymes in the Lean and Obese C57BL/6J Mice ........... Comparison of B-Oxidation in the Lean and Obese C57BL/6J and HA(ICR) Mice ..................... Comparison of the Organelle Distribution of Fatty Acid B-Oxidation in the Mouse ................. NAD Reduction Assay for Fatty Acid B-Oxidation ..... Peroxisomal Oxygen Uptake Assay for Acid B—Oxidation ..................... Mitochondrial Oxygen Uptake Assay for Fatty Acid B-Oxidation ................... Mitochondrial/Peroxisomal Ratio of Fatty Acid B-Oxidation Activity ................. Comparison of NAD:Glycerol-3-P Dehydrogenase from Lean and Obese Mice ................... Comparison of NAD:Glycerol-3-P Dehydrogenase of Fasted Male Obese Versus Lean Mice ........ Comparison of NADzGlycerol-3-P Dehydrogenase of Fed Male Obese Versus Lean Mice .......... Comparison of NAD:Glycerol-3-P Dehydrogenase of Lean Male Fed Versus Fasted Mice . .. ....... Comparison of NADzGlycerol-B-P Dehydrogenase of Obese Male Fed Versus fasted Mice ......... Comparison of NAD:Glycerol-3-P Dehydrogenase of Fasted Lean Male Versus Female Mice ........ Comparison of NAD:Glycerol-3-P Dehydrogenase of Fasted Obese Male Versus Female Mice ....... Comparison of NAD:Glycerol-3-P Dehydrogenase of Fasted Female Obese Versus Lean Mice ....... Comparison of NAD:Glycerol-3-P Dehydrogenase of Fasted Male HA(ICR) and C57BL/6J Mice ....... DISCUSSION ........................... CONCLUSIONS .......................... APPENDIX ............................ BIBLIOGRAPHY .......................... iv Page 45 47 48 48 54 54 55 55 57 57 57 58 58 58 58 59 60 7O 72 79 Table LIST OF TABLES Page Liver weight and protein from livers of clofibrate pair-fed C57BL/6J lean and obese male mice ....... 27 Total hepatic catalase activity in clofibrate-fed C57BL/6J lean and obese male mice and HA(ICR) male mice .......................... 28 Equilibrium density of mouse liver organelles at 4° C . 36 Total hepatic enzyme activity of male lean and obese mice (C57BL/6J) and male HA(ICR) mice ......... 37 Liver and body weights of 2-3 month old mice ...... 38 Total hepatic marker enzyme activity of fasted male versus female C57BL/6J mice .............. 43 Total hepatic marker enzyme activity of fed versus fasted C57BL/6J male mice ............... 46 Hepatic B-Oxidation: Palmitoyl CoA dependent ..... 53 Hepatic NAD:Glycerol-3-P dehydrogenase ......... 56 Figure LIST OF FIGURES Liver tissue from lean CS7BL/6J male mouse. Peroxisomes (p) shown by dense staining with diaminobenzidine. Magnification = 38,000X ....... Liver tissue from obese C57BL/6J male mouse. Same treatment as Figure l. Magnification = l8,000X Liver tissue from lean CPIB-fed C57BL/6J male mouse. Same treatment as Figure l. Magnification = l8,000X . . Liver tissue from obese CPIB-fed C57BL/6J male mouse. Same treatment as Figure l. Magnification = 22,000X ................ Tube gradient profile for fasted HA(ICR) male mouse containing approximately 1.0 9 liver .......... Tube gradient profile for lean and obese C57BL/6J fasted male mice containing approximately 0.7 and 1.5 g of liver, respectively .............. Second catalase activity peak from lean C57BL/6J male mouse gradient showing microsomes and peroxisomes. Magnification = l2,000X ......... Tube gradient profile for lean and obese C57BL/6J fed male mice containing 0.82 and 0.94 9 liver, respectively ...................... Tube gradient profile for lean and obese C57BL/6J fasted female mice containing 0.35 and 0.69 9 liver, respectively ...................... vi Page 3T 3T 3T 3T 33 35 41 50 52 INTRODUCTION Obesity has been called the principal nutritional disease of humans living in most industrially developed nations (2l). Its cause is unknown (7). The complexities of obesity in man are summarized by Garrow (54). Experimentally, it is very difficult to make accurate measure- ments of daily energy balance in man in contrast to the rat. A net caloric imbalance of l-2% in man over a period of a few years has serious consequences in relation to weight gain or loss. The accuracy of techniques presently available to measure energy balance have a greater error than the imbalance they are supposed to measure. In man, energy regulation does not appear to be as accurate nor as rapid as in the rat. There is little evidence that the intake of energy in man is controlled by energy expenditure in the short or long term. In addition, the physiological signals that are effective in exper- imental animals are easily overridden in man. Garrow (54) suggests that there is good evidence for a metabolic adaptation in man to an energy imbalance. This is accomplished through a change in the meta- bolic rate in the same direction as the imbalance to maintain the equilibrium. The nature of this change is unknown but circumstantial evidence suggests that it could be a change in the rate of protein turnover (54). The concept that obesity may be genetically controlled in man finds favor with the close correlation between obesity and an endo- morphic somatotype during adolescence and with evidence from studies involving twins (l04). Because genetic tests are very expensive, time-consuming and inappropriate for man, the use of experimental animal models is mandated for studies involving energy regulation. LITERATURE REVIEW Obese Mouse There are many forms of obesity in rodents and they have been extensively reviewed by Bray and York (22), Hunt gt_al, (71) and Assimaopoulos-Jeannet and Jeanrenaud (6). The obese mouse (ob/ob) used in this study arose from the laboratory stock of the Jackson Memorial Laboratory, Bar Harbor, Maine, in 1950 (72). It has been used in many studies of obesity and extensively reviewed (31, 32, 56, 59-61, 128, 129, 153). The rationale for using this animal for this research is based on several parameters. Animals with the ob/ob gene can be detected at 10-15 days in age by lowered oxygen consumption (51, 78) or lowered body temperature (76, 147). There is a report that the ob/ob genetype can be detected successfully at even earlier ages (20). In the ob/ob animals, the body fat content, fat cell size, and serum insulin all increase prior to weaning (22, 46, 75, 79). Energy expenditure is decreased while hyperphagia and increased efficiency in food util- ization result in excess storage of calories as fat (22). Trayburn gt_gl, (147) have recently proposed that the obesity in these animals is due to a major defect in the energy consuming process. To support this concept, it is known that the obese is hypermetabolic prior to weaning (51, 78, 147); it cannot regulate its body temperature on cold exposure (76, 105, 115, 147, 157); and it remains in positive energy balance even when pair-fed to the lean littermates (5, 30, 65, 102). Even when the obese animal is placed on a restricted diet to reduce its weight to that of the leans, it still has a higher per- centage fat content (5, 23, 40, 160). Therefore, it has been con- cluded that an excess energy intake enhances but does not necessarily cause the obesity (5, 23, 40, 45, 120, 160). In ' ice, the obese animals contained 130% more fat than their lean littermates, but the animals were on1y_14% heavier ~c._‘ A __...._ _ f knim (93). In a five week period, these mice converted 3-4 times more food energy to body energy but only consumed 20-40% more energy. In contrast, only 70% of the dietary protein was converted to body protein (93). A difference in protein accumulation in preobese and obese mice has been reported (18), as well as a slower protein mass turnover in the obese animal during a fast (39). A significant difference in energy metabolism occurs at an early age in these animals (93). Bray gt_gl, (24, 158) have recently proposed that obesity in the ob/ob mouse is caused by an enzymatic defect through loss of the thyroid induced sodium- and potassium-adenosine triphosphatase. The difference in enzymatic activity cannot result from obesity in every animal model because mice made obese by gold-thioglucose treatment did not have a defective thyroid induced Na+-K+-ATPase. These results could be used to explain the lower body temperature, the inability to regulate body temperature and the increased food utilization efficiency in the obese mouse. Both liver and adipose tissue in the obese mouse have been studied extensively (22, 60). Histologically, obese mouse adipose tissue has larger (31, 32, 56) and more numerous fat cells (59). Although there is some controversy as to whether ob/ob mice have more fat cells (60), the number of cells has been reported to be dependent on the location of the fat depot (22). Hyperlipigenesis in adipose tissue of the obese mouse has been observed by several procedures. There is increased incorporation of acetate into fatty acids and lipids when epididymal adipose tissue is incubated in the absence of glucose (33, 66). Hellman and Nestman (62) observed increased esterification of palmitate in epididymal adipose tissue in the absence of glucose. The activity of an adipose glycerol kinase for the reesterification of fatty acids has been reported (94, 128). Obese mice appear to have an impaired ability to mobilize fat. Lochaya gt_gl, (94) reported lowered lipase activity. There are several reports of reduced fatty acid release from isolated adipose tissue after starvation (62, 101, 154) and after stimulation with epinephrin (90, 101). In hepatic tissue from the obese animal, there is a 5-10 times increase jg_yjyg_in glucose incorporation into liver fat (136) even after fasting (11, 159). Kornaker and Lowenstein (81) reported a greater capacity for fatty acid synthesis in the obese mouse liver due to higher levels of citrate cleavage enzyme. There have been numerous studies of hepatic and adipose tissue enzymes in lean and obese mice. Bray and York (22) reviewed these through 1971. The hepatic glycolytic, lipogenic, and gluconeogenic enzymes are increased in the obese animal with the exception of PEP carboxykinase. In adipose tissue, the lipolytic enzymes are decreased in the obese. Glycerol kinase activity has been reported to be high in obese adipose tissue, but these data are not supported by jg_yiyg_ data on fatty acid reesterification (62). Ig_yiyg, studies of hepatic lipogenesis showed 6-8 times higher activity in the obese mouse (73). This is confirmed enzymatically by the several fold higher activity of acetyl-CoA carboxylase activity in the obese mouse (29, 98). Maragoudakis gt_al, (98) also found a different response to a fasting-refeeding regime with the obese mouse. With a 48 hour fast followed by a 48 hour refeeding period, acetyl-CoA carboxylase increased 9 times in the lean animal while in the obese animal, the activity only increased two times. Therefore, the total activity in the two animals was equal. During a fast, acetyl-CoA carboxylase decreased by 1/3 in the obese mouse and by 1/5 in the lean animals. The workers concluded that there was a quantitative rather than a catlytic or regulatory difference in the enzyme levels in the two mice. Volpe and Marasa (152) reported an altered response in the obese animal on fasting-refeeding regime where the rate of degradation of fatty acid synthetase did not increase during fasting as it did in the lean animal. Fatty acid turnover and half-life has been determined by Mayer and colleagues (12). They concluded that significantly less fat was mobilized from stores in the obese mouse leading to a much slower turnover of fat stores and a longer half life. These reported findings indicate that the obese mouse has many factors in its enzymatic make-up that would contribute to a positive energy balance even under usually adverse conditions. However, little experimental work has explored differences in hepatic lipid degradation in the obese mouse. Futile Cycles The concept of futile or energy wasting cycles have been explored in several studies. Hue and Hers (70) reported futile cycling with the loss of ATP for g1ucose:glucose-6-P in mammalian liver and fructose-6-P:fructose-di-P in muscle but not liver. Rognstad gt_al, (131) have provided isotopic evidence for futile cycling between glucosezglucose-6-P and fructose-6-P:fructose-di-P in isolated liver parenchymal cells. Newsholme and Gevers (113) reported that the fructose-6-P:fructose-di-P recycling may benefit the system by making it more sensitive to various stimuli. In the bumble bee, Bgmbus affinis, the cycling is used to produce heat in the muscles (35). Evidence for futile cycling through pyruvate by hydrolysis of phosphoenolpyruvate has been given in perfused liver (52) and kidney cortex slices (132). The operation of futile cycles in glucose metabolism has been reviewed by Scrutton and Utter (131) and Newsholme and Gevers (113). Clark gt_g1, (36) have suggested that futile cycles may serve a regulatory function to balance ATP production and utilization. They also suggested that the recycling may be an imperfection in regulation or a leak. The wasted energy in futile cycles may occur at other sites in metabolism as well. The well-known site of energy wasting in plant peroxisomes through photorespiration suggest that animal peroxisomes could have a similar energy wasting function in animal tissues. Several reviewers have made this suggestion (44, 143, 144), so this aspect has been investigated further in my dissertation project. Peroxisomes Microbodies or peroxisomes are established as subcellular respiratory organelles (42, 43, 64, 67, 143). Microbodies have a single bounding membrane, dense matrix and, in general, contain enzymes associated with oxidative degradations involving flavin oxidases and catalase (144). The term microbody was introduced into the microscopy liter- ature by Rhodin in 1954 (130) to describe a special type of cytoplasmic body found in the convoluted tubule cells of mouse kidney having a single bounding membrane and a fine granular matrix. Rouiller and coworkers (53, 133) identified, in rat liver parenchymal cells, a similar particle which contained a dense core, as well as a single membrane and granular matrix. Microbody distribution as observed in numerous tissues and species, was summarized by Hruban (67). Microbodies were first recognized and called peroxisomes in rat liver through biochemical studies of the centrifugal behavior of urate oxidase, D-amino oxidase and catalase (l4, 16, 41). The term peroxisome comes from the initial use of an assay by de Duve's group for isolated microbodies through the peroxidic release of CO2 from formate as catalyzed by their catalase content. There is little evidence, however, for a peroxidative function of peroxisomes 1g_yjyg_ (144). The term peroxisome was used also by Tolbert gt 21, (143, 146) to describe microbodies in leaves which fulfilled de Duve's criteria of peroxisomes (42, 43). Studies with peroxisomes from Tetrahymena pyriformis showed the presence of enzymes of the glyoxylate cycle, as well as catalase (110, 111). Breidenbach and Beevers (17, 25, 38) introduced the term glyoxysome to describe microbodies in germinating fatty seeds that contained enzymes of the glyoxylate cycle and fatty acid B-oxidation. Catalase containing particles that were not mitochondria have been isolated from yeast (8). Certain algae have particles containing the glyoxylate enzymes (57). The peroxisomal system, in general, is made up of the asso- ciation of catalase with various H202 producing oxidases. This system catalyzes a two step reduction of molecular oxygen to water. The electron donors appear to vary with the cell type, but usually include glycolate, L-lactate and other L-amino acids (43). De Duve has com- pared the mitochondrial electron transport chain to the peroxisomal system in several important ways. First, peroxisomal respiration is not coupled to any mechanism for the retrieval of energy and, there- fore, catalyzes an essentially wasteful form of respiration. Photo- respiration in leaves of plants is the clearest example (143, 146). 10 Second, the rate of peroxisomal oxidation is almost directly proportional to oxygen tension, while mitochondrial respiration is essentially independent of oxygen tension, except at very low oxygen concentrations. In animals, there is no physiological data on the effect of oxygen on partitioning respiration between peroxisomes and mitochondria. However it has been assumed that mitochondria may have precedence over peroxisomes in utilization of limiting amounts of oxygen. Peroxisomes can respond to increases in oxygen content and, in plants, might have a function in protection from oxygen toxicity, although this has not been investigated in animals. Third, peroxisomes and mitochondria have different specificities to selected substrates. Urate and D-amino acids are metabolized preferentially by peroxisomes. Lazarow (88) and de Duve (44) propose that peroxisomes preferentially oxidize long chain fatty acids while mitochondria use shortened fatty acids. Peroxisomal Enzymes Peroxisomal enzyme content varies widely depending on the tissue from which they were isolated. By comparing the enzymes found in microbodies from different sources, an understanding of the origin of the hypothesis for their role in energy wasting therapy can be explained. In C3 plants, very active photorespiration occurs due to glycolate biosynthesis in the chloroplasts and its metabolism in the peroxisomes and mitochondria (l, 38, 116, 143). Oxygen uptake and C02 loss in these plants may be close to 50% of the gross photo- synthetic rate and drastically reduces net photosynthesis (114). 11 Several physiologically irreversible steps that are energy wasting occur in photorespiring plants in the conversion of ribulose- bisphosphate to glycine. In germinating fatty seeds such as peanut and castor bean, glyoxysomes, a micorbody, contain the enzymes of the glyoxylate cycle and fatty acid B-oxidation that are involved in the conversion of fatty acids to C4 acids that are then used for sugar synthesis in the germinating seeds (38). There is one energy wasting step in the first dehydrogenation in B-oxidation in these particles where the electrons are transferred to H202 which is then destroyed by catalase. No B—oxidation, apparently occurs in the mitochondria of these seeds. Glyoxysomes disappear from the germinating seed in about ten days, when the plant starts to photosynthetically produce its own sugars. During this changeover, the glyoxysomes are replaced by peroxisomes in the leaves of the plant. Until recently, peroxisomes in animals were not known to contain a defined cycle of respiratory or synthetic metabolism as do leaf peroxisomes or seed glyoxysomes. But many of the enzymes known to be contained in animal peroxisomes are associated with lipid degradation. Now the existence of fatty acid B-oxidation in liver peroxisomes establishes a metabolic sequence in the peroxisome. Catalase Catalase is found in all animal peroxisomes and is used as a marker for peroxisomes both cytologically (49, 114) and biochem- ically (145). The function of catalase in peroxisomes has been 12 suggested de Duve et_al, (42) to remove hydrogen peroxide produced by other enzymatic reactions in the peroxisomes, as well as extra- peroxisomally produced hydrogen peroxide. Catalase has been impli- cated in metabolic flux as measured by oxygen consumption that is peroxisomally dependent. In liver homogenates, Aebi and Suter reported that about 1/3 of the normal oxygen consumption by the liver was through the peroxisome (l). Oshino gt_gl, (116) demonstrated that approximately half the ethanol in liver cells was metabolized by catalase. They also showed that perfusing intact liver with urate and glycolate produced peroxisomal hydrogen peroxide and accounted for nearly half of the total liver respiration. Urate and glycolate metabolism have been shown to stimulate ethanol utilization by catalase several fold (142). Masters and Holmes (103) interpret this to mean that the peroxisomal system is capable of operating beyond its already high steady state levels. They also suggest that peroxisomal oxidation through catalase may occur without simultaneous increase in the pyridine nucleotide redox state in oxidizing ethanol. Jones and Masters (74) have compared catalase activities from various tissues and species. Catalase activity is always highest in the liver with substantial amounts in the kidney and blood. A much lower activity is found in other tissues. There is a marked species variation in peroxisomal and supernatant catalase activities as mea- sured in crude tissue homogenates. Rat and mouse livers have most of their catalase activity in the peroxisomal particles, while beef 13 and guinea pig liver catalase is found principally in the supernatant. Jones and Masters (74) attribute this to the need for protection of the less stable catalase of rat and mouse inside an organelle (per- oxisomes). No organelle separation was done, however, so peroxisomal breakage during tissue homogenation cannot be estimated. In rat liver, catalase accounts for as much as 40% of the peroxisomal protein (42). Catalase is used as a marker to identify peroxisomes for electron microsc0py using diaminobenzidine (49, 114). a-Hydroxy Acid Oxidase a-Hydroxy acid oxidase has been partially characterized from rat liver and kidney as well as pig liver and kidney peroxisomes (106). This enzyme catalyzes the aerobic oxidation of a-hydroxy acids with flavin mononucleotide as a cofactor to produce hydrogen peroxide (42, 43). The enzyme from rat liver peroxisomes is most active with glycolate as its substrate. Considerable activity is observed with lactate but very little with L-a—hydroxy butyrate. The enzyme cata- lyzes the oxidation of L-a-hydroxy isocaproate almost as well as glycolate and also has activity with a-hydroxy-caproate and a-hydroxy butyrate. The hepatic enzyme was not able to oxidize a-hydroxy acids of chain length greater than C The reason for these various 8' specificities is not known (106). a-Hydroxy acid oxidase shows very different substrate specificities between tissues and species. Rat liver a-hydroxy acid oxidase utilized glycolate, lactate and a-hydroxy isocaproate while 14 the enzyme isolated from rat kidney was inactive towards short chain a-hydroxyacids but was active for a-hydroxy isocaproate and a-hydroxypalmitate. The pig liver and kidney enzymes exhibit a principal specificity for glycolate (106). a-Hydroxy acid oxidase activity has been used to identify peroxisomes by microscopy by using a coupled reaction of nitro blue tetrazolium and a-hydroxy acid (3, 4, 137). Allen et a1. (3) reported positive reaction with D,L-a-hydroxyvalerate, D,L-a-hydroxybutyrate and L-lactate in rat kidney. D-Amino Acid Oxidase D-Amino acid oxidase has been identified in rat liver and kidney peroxisomes (15) using the oxidation of D-amino acids to keto acids and hydrogen peroxide production coupled to the peroxidation of formate. The enzyme has little activity towards L-amino acids. Farber gt_§l, (50) has used D-amino acid oxidase to visualize microbodies for microscopy in rat liver and kidney by using nitro blue tetrazolium coupled with various D-amino acids. They reported good staining with D-methionine, D-leucine, D-ethionine and D-isoleucine. Less intense staining was observed with D-tyrosine and D-phenylalanine. §1yoxylate Aminotransferase Glyoxylate aminotransferase is a characteristic enzyme of peroxisomes in which glyoxylate is transaminated to glycine with various amino acid donors (69, 80, 127, 143). In rat liver and kidney, the peroxisomal glyoxylate aminotransferase has been characterized (69) 15 and found to differ from those of spinach leaves in the specificity for the amino donor. The rat hepatic enzyme is specific for gly- oxylate and most active with leucine and phenylalanine and some activity with histidine as the amino donor. The rat hepatic glyoxylate aminotransferase fluctuates widely jg 1119 (69) as is seen with other peroxisomal enzymes (48, 83). Overnight fasting decreases the activity by 50%. The activity is increased moderately on high protein diets and in the terminal stages of starvation. No activity was detected at birth but increased thereafter, to a plateau at about 40 days of age in the rat. Female rats have been shown to have four times the activity of males. p-Chlorophenoxyisobutyrate (similar to clofibrate) increased the activity of the enzyme 2.5 times along with peroxisome proliferation (69). NADlesocitrate Dehydrogenase NADPzIsocitrate dehydrogenase has been reported to be associated with rat liver peroxisomes (92). Urate Oxidase Urate oxidase is always a peroxisomal enzyme if it is present in the tissues (42). Urate oxidase catalyzes an oxidation with oxygen of urate to allantoin and hydrogen peroxide (42). It is used as a peroxisomal marker enzyme (145). Urate oxidase, xanthine dehydrogenase and allantoinase have been reported in peroxisomes from avian and frog liver and avian kidney by Allen and coworkers (134, 151). Tolbert (144) 16 reported that active xanthine dehydrogenase is not found in peroxisomes from pig and rat kidney and liver. In rat liver, the core observed in peroxisomes is made up of urate oxidase (13, 68, 149, 150). This is probably true for all species with a peroxisomal core and urate oxidase activity (43). Birds, man and other primates lack cores in their peroxisomes and urate oxidase activity (2, 138). Carnitine Acyl Transferases Carnitine acetyl transferase and carnitine octanyl transferase have been identified as part of the peroxisomal enzyme complement in rat and pig liver peroxisomes but not in rat and pig kidney (100). These enzymes are also located in the mitochondria and the microsomes. These enzymes are not detected in microbodies, mitochondria or micro- somes of plants. The distribution of carnitine acetyl transferase was reported to be: 52% mitochondrial; 14% peroxisomal; and 34% microsomal. Peroxisomal and microsomal activities of carnitine octanyl transferase were approximately equal to carnitine acetyl transferase. The mitochondrial carnitine octanyl transferase exhibited an activity six times greater than the carnitine acetyl transferase (100). It has been suggested that the carnitine acetyl transferase in peroxisomes and microsomes acts to keep a reservoir pool of carni- tine acetyl residues and/or keep acetyl-CoA levels constant (100). The role of carnitine acyl transferases may be to transport acyl residues out across the peroxisomal membrane after fatty acid B-oxidation (89). l7 NAD:Glycerol-3-P Dehydrogenase NADzGlycerol-B-P dehydrogenase is located in peroxisomes isolated from livers of rat, chicken and dogs and from rat kidney (55). This enzyme is not found in spinach leaf peroxisomes. Con- versely, no malate dehydrogenase was found in animal peroxisomes but was very active in plant peroxisomes (156). It has been sug- gested that peroxisomal NAD:glycerol-3-P dehydrogenase acts in a membrane transport shuttle in a manner similar to the glycerol-B-P shuttle between the mitochondria and cytoplasm or to a malate dehydrogenase shuttle in plant peroxisomes (55). The peroxisomal enzyme is different kinetically and has a different mobility on polyacrylamide gels than the cytoplasmic NAD:glycerol-3-P dehydro- genase or the membrane FAD-linked mitochondrial glycerol-3-P dehydrogenase (R. Gee, J. B. Krahling 8 N. E. Tolbert, in preparation). Fatty Acid B-Oxidation Enzymes capable of B-oxidation of fatty acids have been reported in rat (88, 89) and mouse (112) liver peroxisomes. The first enzyme in the sequence, acyl CoA oxidase, has been isolated by Osumi and Hashimoto (117). This enzyme catalyzes a flavin-linked dehydrogenation of the substrate with oxygen uptake to produce hydrogen peroxide. Chain length specificities have been reported (88, 117). The long chain fatty acids (C14'C18) are oxidized best with little or no activity for short chain (C4-C8) acyl CoAs. B-Oxidation 18 activity in peroxisomes can be measured both by oxygen uptake through its catalase linkage in the first step or by substrate dependent NAD reduction in the third step catalyzed by B-hydroxy acyl CoA dehydro- genase (82, 89). Up to five cycles, but not the theoretical seven, have been observed with palmitoyl CoA oxidation by liver peroxisomes (88). This, in part, supports the chain specificity activity of the peroxisomal enzymes. Recently, Shindo and Hashimoto (139) have reported the activity of a fatty acyl CoA synthetase in rat liver peroxisomes, as well as good activity using palmitic acid as a substrate for B-oxidation. Therefore, the liver peroxisomes can activate and oxidize fatty acids. Hypolipidemic Drugs and Peroxisomes Feeding hypolipidemic drugs have been reported in numerous cases to proliferate peroxisomes (124-126, 140) and increase liver weight (19). The drugs have also been shown to change some total hepatic enzyme activities (26, 34, 84, 87, 96, 108, 121-123) in rat liver. The effects of clofibrate, a hypolipidemic drug, is selective in increasing enzyme activities. In the male rat liver, the specific activity of carnitine acetyl transferase and carnitine octanyl trans- ferase increased in isolated peroxisomes, mitochondria and microsomes from treated animals. There was also an increase in the mitochondrial carnitine palmitoyl transferase (77, 99). There was no increase in the specific activity of catalase and urate oxidase in the isolated organelle (47, 63, 99). Slight decreases in peroxisomal D-amino acid oxidase and a-hydroxy acid oxidase have been reported with clofibrate 19 treatment (91). The microsomal NADPH:cytochrome c reductase did not increase in specific activity (91). The flavin-linked glycerol-3-P dehydrogenase has also been reported to increase with hypolipidemic drugs (27, 28, 84). The decrease in serum triglycerides and neutral steroids has been a known function for hypolipidemic drugs for some time (141), but their mode of action is as yet unknown. A blockage of cholesterol synthesis at both premevalonate (9) and postmevalonate sites (10) has been suggested. Reports of inhibition of fatty acid synthesis at the level of acetyl-CoA carboxylase (97) and acyl-CoA-a-glycerolphosphate acyltransferase (28, 86) have been made. Coenzyme A and its deriva- tives have been reported to increase (107) with the administration of clofibrate. The effect of hypolipidemic drugs on the availability of acyl CoAs has also been suggested in regulating the hypolipidemic effect (99). More recently, there have been reports of increased fatty acid oxidation in livers of clofibrate fed rats (34, 58, 87, 96, 155). Peroxisomal proliferation does not seem to be necessary for hypolipidemic effects to be manifested. In female rats, no prolif- eration of peroxisomes occurs but there is a hypolipidemic response (85) and an increase in the total hepatic carnitine acetyl transferase (108). Phthalate esters have been reported to proliferate peroxisomes and increase some peroxisomal enzyme activities (109, 118, 119). 20 In summary, it is known that animal peroxisomes contain various enzymes linked to flavin oxidases and catalase. No defined cycles of futile respiration have been observed in animal peroxisomes as found in plant peroxisomes. There is much inter- and intra-species variation in peroxisomal enzyme content and their response to various agents. STATEMENT OF THE RESEARCH PROBLEM This research was designed to examine the involvement of peroxisomes in a proposed energy wasting scheme in the control of calorie utilization by mammals. The animal model chosen was the obese-hyperglycemic mouse (C57BL/6J) and its lean littermate. They were used to determine possible differences in the activities of peroxisomal enzymes for the obese compared to its lean littermate. This animal was chosen because it becomes obese without using cal- orically dense diets and can be identified by lowered oxygen con- sumption from its lean littermates before phenotypic expression of obesity. The protocol for this study was to examine the activities of catalase, urate oxidase, palmitoyl-CoA dependent NAD reduction (B-oxidation) and NAD:glycerol-3-P dehydrogenase of peroxisomes isolated from livers of lean and obese mice by isopycnic sucrose density gradient centrifugation. The data obtained from these experiments will aid in the determination of whether there is a basis for an energy wasting differential between lean and obese animals. A further under- standing of peroxisomal involvement in lipid metabolism as well as a connection with obesity has been achieved. 21 METHODS AND MATERIALS Male and female lean (C57BL/6J) mice and their genetically obese and hyperglycemic littermates were from Jackson Memorial Laboratory, Bar Harbor, Maine. For comparison and development of procedures, six week old white [HA(ICR)] male mice from Spartan Research, Haslett, Michigan, were used. Total Hepatic Catalase Content in Crude Homogenates Total hepatic catalase content in crude homogenates of liver in these mice was examined initially in this research. The effect of clofibrate (CPIB or p:chloro-phenoxyisobutyrate ethyl ester) on these animals was also explored. Two month old male mice were separated into four groups: lean animals fed the control diet; obese animals fed the control diet; CPIB- fed lean animals; and CPIB-fed obese animals. The groups were pair-fed to the obese mice fed the CPIB diet. The control diet consisted of ground Wayne Lablox (Allied Mills, Chicago, Illinois). The CPIB diet consisted of ground Wayne Lablox and 0.5% (w/w) clofibrate (Ayerst Laboratories, Inc., New York). An additional two groups of mice, lean and obese, were fed the CPIB diet gg_libitum. For comparison, the male HA (ICR) mice of 6-8 weeks of age were used to examine the effect of CPIB feeding. These mice were divided into two groups, control and CPIB-fed, ag_libitum. These animals were fed the same 22 23 batch preparation as the lean and obese mice. All animals were fed this diet for two weeks prior to sacrifice. The mice were fasted overnight to reduce liver glycogen. The animals were killed by decapitation, the liver rapidly exposed and perfused with cold homogenizing buffer (8.5% sucrose, 0.01 M phosphate, pH 7.5). Portions of the livers from individual animals were processed for peroxisome visualization by electron microscopy according to the procedure of Fahimi (49) and Novikoff gt_gl, (114) using diaminobenzidine. The weighed livers were minced individually with scissors and homogenized in a loosely-fitting mechanically driven Potter- Elvehjem homogenizer. The homogenate was filtered through miracloth and the volume measured. This material was taken to determine the catalase activity and protein concentration. Catalase activity was measured spectrophotometrically at 240 nm by following the rate of loss of a standardized solution of H202 (145). Protein was determined according to the method of Lowry et_al, (95). Examination of Isolated Organelles from Lean and'Obese Mice Obese and lean littermate control mice, 8-12 weeks of age, and HA (ICR) male mice, 6-7 weeks of age, were used to study enzyme activities in isolated organelles as described by Murphy §t_al, (112, Appendix A). In addition, a second catalase peak of activity from zonal gradients of lean male mouse liver were examined by electron 24 microscopy. The fraction was fixed in 12.5% glutaraldehyde, 50% sucrose, 0.5 mM EDTA, pH 7.5, for 30 minutes at 4° C. The fixed solution was diluted 1:1 with washing buffer (12.5% glutaraldehyde, 0.5 mM EDTA, pH 7.5) and centrifuged at 40,000 rpm in a Ti 60 rotor for the Beckman L-2 centrifuge for 30 minutes at 4° C. The pellet was washed and collected in 25% sucrose, 0.5 mM EDTA, pH 7.5. The isolated peak was then postfixed with 1% 0504 in 0.10 M cacdylate, pH 7.5, overnight. The fixed fraction was processed for electron microscopy. Ultrathin sectioning was done on a LKB 4801A ultra- microtome. The sections were stained with saturated uranyl acetate for 30 minutes and counter stained with lead citrate for 10 minutes. The sections were examined in a Zeiss 100 or a Philips 200 electron microscope. Comparison of Peroxisomal and Mitochondrial Fatty Acid B-Oxidation To compare the distribution and magnitude of fatty acid B-oxidation in lean and obese mice as well as the HA(ICR) mice, similar procedures were used for organelle separation as previously described (112, Appendix A) with the following modifications. (1) For obese mice, one liver was used per experiment and distributed in three gradient centrifuge tubes. For lean C57BL/6J and HA(ICR) mice, because of the smaller liver size, two livers were pooled per experiment. (2) For better estimation of the efficiency of tissue homogenation, a fraction of the total homogenate was saved for further thorough homogenation in a Ten-Broeck homogenizer. The remainder of the homogenate was filtered through Miracloth (Chicopee Mills, Inc., 25 Milltown, New Jersey), and the organelle separation was carried out as described (112, Appendix A). The total homogenate was used to determine protein and enzyme activities to compare with the limited homogenation of the organelle preparation. (3) An initial centrigu- gation at 5,000 rpm for 15 minutes was included in the fractionation procedure followed by the 25,000 rpm centrifugation for 3 hours as previously described (112, Appendix A). (4) After development in the centrifuge, the gradients were collected in 1 ml fractions from the top of the tube using a modified ICR tube gradient pump and a Gilson fraction collector. Marker enzyme activities were determined as previously described (112, Appendix A). Fatty acid B-oxidatidn was measured spectrophotometrically by NAD reduction as previously described (112, Appendix A) and poloragraphically by oxygen uptake according to Krahling gt_gl, (82). NADzGlycerol-3-P dehydrogenase activity was measured spectrophotometrically as used previously (112, Appendix A). Lean and obese male and female mice were used in this portion of the research. All animals were deprived of food for 18 hours prior to sacrifice (from 3 p.m. the preceding afternoon to 9 a.m. the fol- lowing morning) in all fasted animals. Fed animals were allowed free access to food up until time of sacrifice. Statistics Experimental means were compared using Student's t test except for fatty acid B-oxidation data (Table 9) where factorial analysis of variance was employed. RESULTS Total Hepatic Catalase Activity The liver weights and protein contents of the livers are given in Table l. Obese animals had obviously larger and more fatty livers by visual inspection, as well as larger liver weights. The obese animals fed the control diet had higher protein content than the lean mice fed the control diet. The mice fed the CPIB diet had increased liver weights compared to their gg_libitum fed control mice, but there was no increase in liver protein. Thus, in the C57BL/6J strain of mice, clofibrate did induce the reported increase in liver weight but not in protein concentration. Catalase activity did not increase in pair-fed C57BL/6J mice or these gg_1ibitum fed mice in contrast to data reported for other mice (123, 124). The data is presented in Table 2. Obese C57BL/6J mice fed the control diet had significantly less catalase activity per 9 liver but significantly higher specific activity than the lean C57BL/6J mice. Clofibrate feeding did not increase catalase content either on a pair-fed basis or an ag_libitum basis. In fact, clofibrate seemed to enhance the small difference in activity observed between lean and obese mice. When HA(ICR) mice were fed the same diet from the same batch preparation used for the lean and obese CS7BL/6J mice, the 26 27 Table 1. Liver weight and protein from livers of clofibrate pair-fed C57BL/6J lean and obese male mice N Liver weight (g) Protein mg/g liver Control Lean 7 1.14:0.14a’b mezoa Obese 7 2.13:0.283’d 1741186 Clofibrate-fed Lean 5 1.60: 0.19!”C 132 e 37“3 Obese 7 2.93:0.35‘“d 1631179 Note: Values sharing common superscripts are significantly different at level tested: a c d p< .001 ’ ’ p< .005b p< .059. 28 Table 2. Total hepatic catalase activity in clofibrate—fed C57BL/6J lean and obese male mice and HA(ICR) male mice Catalase Animal n mnol min'1g liver'] Innolmin'1 mg protein"1 Pair-fed Control lean 7 6.80: 1.53b 79.4: 3.6b Control obese 7 5.23: 0.50b 90.4: 11.6b’d CPIB leanT 6 5.35: 1.59 71.8: 15.1 CPIB obese 7 4.61: 0.99 75.7 : 20.2d Ad lib--fed CPIB Lean 7 2.34: 0.37al 21.2: 2.8 Obese 6 1.81 : 0.14al 17.8: 1.1 Ad lib-~fed HA(ICR) Control 7 2.75: 0.51c 25.1 : 5.5a CPIB 7 4.11: 0.98c 45.9: 14.8al TCPIB = clofibrate (chlorO-prphenoxyosobutyrate ethyl ester). Note: Values sharing common superscripts are significantly different at level tested: p< .Ola’c p< .05b’d. 29 increase in catalase activity was observed as previously reported (123, 124) relative to the control. It was concluded that clofibrate does not affect peroxisomal catalase in the C57BL/6J male mice as it does in other strains Of mice (123, 124). It does, however, increase liver weight in the C57BL/6J mice. Electron microscope examination of thin sections from control lean and Obese C57BL/6J mice showed no gross difference in the numbers of peroxisomes, i.e., no higher or lower number in the Obese animal, as shown in Figures 1 and 2. Pictures Of livers Of clofibrate-fed animals showed no increase in peroxisome numbers compared to the respective controls (Figures 3 and 4). These pictures indicate there was no apparent difference in the numbers of peroxisomes between lean and Obese C57BL/6J mice and no increase in peroxisome numbers on clofibrate feeding at 0.5% Of the diet as reported for other rodents (84, 108, 121-126, 140). Isolation Of Hepatic Mouse Peroxisomes Cell organelles in liver homogenates from fasted male HA(ICR) mice (Figure 5) and fasted lean and Obese C57BL/6J mice (Figure 6) were separated on isopycnic sucrose density gradients. The peroxisomes, identified by catalase and urate oxidase, were well separated from the mitochondria, identified by glutamate dehydrogenase and B- hydroxybutyrate dehydrogenase. The lysozomal marker, acid phosphate, was primarily located in the mitochondrial peak with a small amount Of tailing into the peroxisomal area (data not shown). The microsomes, 30 Figure 1. Liver tissue from lean C57BL/6J male mouse. Peroxisomes (p) shown by dense staining with diaminobenzidine. Magnification = 38,000X. Figure 2. Liver tissue from Obese C57BL/6J male mouse. Same treatment as Figure l. Magnification = 18,000X. Figure 3. Liver tissue from lean CPIB—fed C57BL/6J male mouse. Same treatment as Figure 1. Magnification = 18,000X. Figure 4. Liver tissue from Obese CPIB-fed C57BL/6J male mouse. Same treatment as Figure l. Magnification = 22,000X. 31 Figure l Figure 3 Figure 4 Figure 5. 32 Tube gradient profile for fasted HA(ICR) male mouse containing approximately 1.0 9 liver. H Catalase (mmol/min) HNADH (nmol/min) I—u N A 0 reduction (umol/min) O 05 33 White HA (ICR) K Cytochrome c I-\\ reductase Glutamate \L Cotolose dehydrogenase} \ l e - *Glutomate dehydregenose (nmol/min) o 8 o o ‘0 F500 "400 -300 "200 L100 \, "Palmitoyl CoA /\ dependent NAD reduction IOO Glycerol-3- P dehydrogenase Dihyroxyocetone-P I" \ 5.0 2.5 reductase «f \b _ II \‘ ,4 » “kw”- , ‘1 e.»— ,A “j 1‘ 0v ‘90-. v," ‘IW o: l 110 20 3O 4O 50 Volume (ml) °—-° NADPH=Cyt c reductase (nmoI/min) o-oUrate oxidase (umOl/min) 0--0 NADH oxidation (umoI/min) 34 Figure 6. Tube gradient profile for lean and Obese C57BL/6J fasted male mice containing approximately 0.7 and 1.5 g Of liver, respectively. 35 EE\_oE588838. u ;udo h b b L F m 2 2.52653 E.E\_oEEV $295 1.. EEQBES 88.518651 EEBEsSQBL om_ um ucmcmwm_c zpucmommwcmpm wee mpawcomcmnzm coeeoo mcwcmsm mm=~m> ”muoz mowpm hemp“ some 882 o.ooomnflopmp am. he m Amumvw_ coo cm>wp m can cowuomem xmma cw : mEXNcm a co. ewe , cwouocg me can wows Amquwuom oex~cm owuaao; Page» .v opnmh 38 Table 5. Liver and body weights Of 2-3 month Old mice Animal n Liver (9) Body (9) Nels Lean 5 1.56: .15“ 26.56: 2.32b Obese 5 4.36: .64b’c 53.19: 5.20 HA(ICR) 3 1.54: .25 26.25: 1.85 Eenele Lean 5 1.23: .345"d 21.30: 1.48b b,d Obese 3 2.73: .50 49.13: 2.31 Note: Values sharing common superscripts are significantly different at level tested: a p<:.05 p< .Olb’c’d. With both the HA(ICR) and the C57BL/6J mice, less than 5% of the catalase or urate oxidase activity was present in the supernatant fraction. Almost all of the activity was in or tailed into the peroxi- somal peak indicating that there may have been little peroxisomal breakage during homogenation. These results differ from those of the rat where about 50-75% of the catalase is in the supernatant and is attributed to peroxisomal fragility or to two subcellular locations of catalase (74). Inclusion of 0.01% ethanol in the gradient, which has been reported to prevent its inactivation by the formation of an inactive peroxide complex (37), did not increase the catalase activity. Data from mouse gradients, particularly the lean mice, indicate some of the peroxisomal material tailed into the mitochondrial area with 39 a minor peak slightly less dense than the mitochondria (Figure 6). The identity Of this material has been examined by electron microscopy (Figure 7). In gradients from both the rat and the mouse liver, the presence of some of the peroxisomal marker enzyme activity in less dense sucrose than the main peroxisomal band appears to be due to an area containing a mixture of microsomes and peroxisomes. The profile of the mitochondrial matrix enzyme, glutamate dehydrogenase, and the mitochondrial membrane enzyme, B-hydroxybutryrate dehydrogenase, coincide. This indicated that no significant amount of single membrane mitochondrial fragments were present. Differences in Hepatic Peroxisomal Marker Enzymes The obese C57BL/6J mice at 3 months of age averaged 53 g body weight and 4.4 9 liver weight. On the average, they were two times heavier than their lean littermates and their livers were about three times larger. The HA(ICR) and the lean C57BL/6J mice had the same liver weight and body weight on the average. Under these circum- stances, the enzymatic data from the HA(ICR) mice and the 057BL/6J lean and Obese mice have been reported as specific activity in the isolated subcellular organelle from the sucrose gradient and as total activity per 9 liver, per liver and 100 g body weight. The specific activity of the marker enzymes in the isolated peroxisomes (catalase and urate oxidase) and the mitochondria (gluta- mate dehydrogenase) and the total activity per 9 liver were the same in the obese and lean C57BL/6J and the HA(ICR) mice (Table 4). These Figure 7. 40 Second catalase activity peak from lean C57BL/6J male mouse gradient showing microsomes and peroxisomes. Magnification = 12,000X. 41 Figure 7 42 similarities were true even though the obese mice had larger and more fatty livers at three months Of age. Consequently, the total peroxi- somal and mitochondrial activity increased per liver from the Obese mice in proportion to the liver size compared with the lean C57BL/6J but not the HA(ICR). Approximately the same fraction of the liver, about one-third, was used for each gradient so the obvious increase in Figure 6 in the total activity in the Obese liver was due to the increased liver size. An interesting comparison was made between the lean C57BL/6J mice and the HA(ICR) which showed a higher total catalase activity per liver in the HA(ICR) mice even though the liver size is the same in the two animals (Table 5). This increased activity was even more apparent when expressed on a per 100 g body weight basis. The obese CS7BL/6J mice still had higher activity per liver for catalase and glutamate dehydrogenase than the HA(ICR) mice but on a per 9 liver basis, the reverse was found for catalase activity. This indicates that the HA(ICR) mice have a different enzymatic makeup than the lean C57BL/6J even though both animals are lean. Total hepatic marker enzyme activities for male and female C57BL/6J lean and obese mice are compared in Table 6. No differences were found between the male and female animals with the marker enzyme activities on a per 9 liver basis. The differences observed between lean and Obese male mice are seen again in a comparison of the lean and Obese female mice. This is believed to be due to the large size of the liver of the obese animal. 43 . 6. v9.5. .mo. vae 93.6 m.o..no.m ~52; m 32.3 .330 «3.7332 eeméhod 868m; 8 2e... .33.. N.” 85m To .+.m.o m6 :6 m 38.3 .53 Afigpoeé m.m 836 ad 85. 5o 8 F; e 22.. .53 333835.. 38.335 P... 853 m; ANN P. IN. N m 22.3 .325 N.m:.¢N 58.1.0.3 8. :N. m 8 3m... .3an m; 35.2 edemN m. o 8m. N m 32.6... .53 A5535... mg $13 K; “Na N. pom. m e 3e... .53 633.8 385 S. .8 SN N .8 m2 m 8 m... m 32.3 .388 m3 8. Se eom heNN S 8 S m .29.. .388 R: ANNN MN .43 2 .u. 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In the obese animals, catalase increased in the male animal due to the larger liver size, however, no difference was measured on a per 100 g body weight basis. Urate oxidase activity was different between the male and female lean mice on a per liver basis but not between the obese male and female mice. In neither comparison was there a difference on a per 100 g body weight basis. The activity of glutamate dehydrogenase, the mitochondrial marker enzyme, changed similarly to catalase. There was no apparent difference in activity between the lean male and female mice. On a per liver basis, glutamate dehydrogenase was higher in the male obese animals than in the obese female mice. This difference was large enough to effect a significant difference to the body weight comparison. The male and female mice have a similar marker enzyme distri- bution on a per 9 liver basis. The lean and obese female mice differ 45 in the same way as the lean and obese male mice. The differences in body and liver size seem to cause some, but not all, of the differences observed between the male and female mice on a per liver basis and on a per 100 9 liver basis. The marker enzyme activities between fed and fasted male mice are shown by the data in Table 7. No differences were observed in the activity levels due to fed versus the fasted state. The same differ- ences in activity based on a per liver basis were seen in comparing the fed lean and obese males as seen in comparing the fasted lean and obese males. Comparison of the Matrix and Membrane Associated Enzymes in the Lean and Obese C57BL/6J Mice Catalase and urate oxidase of the peroxisomes and glutamate dehydrogenase of the mitochondria are located in the matrix of these organelles and were not different in specific activity between the lean and obese animals. In contrast, the membranous enzymes, NADPHzcyto- chrome c reductase of the microsomes and B-hydroxybutyrate dehydrogenase of the mitochondria, decreased two to threefold per 9 liver in the obese mice. For these membranous enzymes, there was no difference per liver as shown by the data in Figure 6, even though the livers from the obese mice were about three times larger. Thus, there appears to be a difference of hepatic matrix enzymes of the organelles but no difference in the membrane components with obesity. 46 o c Q nmo v m2. X. "umpmmu Fm>mp an ucmcmmwwu xppcmuwmwcmwm use mgawgumgmqam coseou mcwgmsm mmapm> "muoz N.w.flN.¢N N.v.flo.mp e.P.fiN.m e umummw .mmwno w.o.flN.mp am.onflm.o m.ouflm.N N am» .mmmno m.~.nm.mp N.F “N.m N._.flm.m e nmummw .cmm3 Acws\~osnv _.e.flm.e_ n_.FAH¢.e m.o.flm.N N awe .cmm3 mmmo_xo mane: m._.fl¢.o_ m._nfio.m ¢.o.flm.— w teammm .mmmno 90:4. umNnm...” mdnfip m um... 638 man: 3:: 2.2.. e .5ng .53 2.5.2.3 a... 3... mm... 3.. N... S... m 8.. .58.. mmacmmocifiu 32.33... cup “FNe om fleNN up “Pm m umummw .mmwao Nanflo—F mmopnfleoN F “Nm m we» .mmmao . mm womN mNuflmN 0N “Fm m umpmmm .cmm3 Acws\PoEEV em “mmm mm_ “mm N “mm m um» .cmm3 mmmpmpmu agape: xuon Lm>wp can gm>wp a Log : Fmswc< mea~cm a cop .wa wows m—me wo\3mumu umpmmw mamgw> vow we xuw>muum weaned .mxems orange; Punch .N m_nmh 47 Comparison of B-Oxidation in the Lean and Obese C57BL/6J and HA(ICR) Mice The assay procedure for peroxisomal palmitoyl-GOA dependent reduction of NAD did not measure the mitochondrial B-oxidation (82, 88, 89). The palmitoyl—GOA dependent NAD reduction shown by the data in Figures 5 and 6 coincided with the distribution of the peroxisomal catalase. These results with the mouse extend the observation made by Lazarow and de Duve (89) in the rat that liver peroxisomes catalyze B-oxidation. The peroxisomal B-oxidation was 5-6 times more active per 9 liver, per liver and per loo 9 body weight in the HA(ICR) mice compared to the lean C57BL/6J mice (Figures 5 and 6, Table 4). The specific activity did not exhibit a significant difference. Total fatty acid B-oxidation in the peroxisomes for the obese mouse livers was about twice as much per g of liver as in the lean mice. This resulted in a fivefold difference in total B-oxidation on a per liver basis. This increase was pronounced in specific activity of the iso- lated peroxisomes (Table 4). These results indicated a higher activity for B-oxidation in liver peroxisomes of the obese mice even though the other peroxisomal enzymes were not higher per 9 liver or per mg protein. Comparison of the HA(ICR) mice to the obese C57BL/6J mice indicated a higher specific activity of fatty acid B-oxidation in peroxisomes from obese mice, but threefold higher total activity per 9 liver in the HA(ICR) mice. On a per animal basis, expressed as either per liver or per loo 9 body weight, the HA(ICR) mice and the obese C57BL/6J mice have an equal capacity to oxidize fatty acids in the peroxisomes. 48 Comparison of the Organelle Distribution of Fatty Acid B-Oxidation in the Mouse The gradient profiles for fed male mice and fasted female mice are shown in Figures 8 and 9. The marker enzymes, catalase and urate oxidase for the peroxisomes and glutamate dehydrogenase for the mito- chondria, are fairly well separated for each other. There was sig- nificant mitochondrial contamination of the peroxisomal peak in the lean male and female mice. In comparing mitochondrial and peroxisomal B-oxidation, the assays used measured only the activity of the enzyme from the organelle indicated (82). In the mouse, there is approximately an equal distribution of B-oxidation between the two organelles. This is apparent in Figures 8 and 9 and in Table 8. NAD Reduction Assay for Fatty Acid B-Oxidation When B-oxidation was measured in the peroxisomal fraction by the rate of NAD reduction, the only significant differences observed were between the HA(ICR) mice and the lean and obese C57BL/6J mice. In both cases, the HA(ICR) mice had higher activity than the lean or obese. There was no significant difference in the activity between the lean and obese mice whether fed or fasted in this experiment. This is in contrast to data already reported. When the data from both experiments is combined, no significant difference was found due to the large variance in the data. No apparent difference was observed in peroxisomal activity between male and female mice. 49 Figure 8. 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To nméN m 5... .53 a $ a u a m m: weomwxogma a meompxogma ucmpmccqum Pouch c Pmswc< Pugm>wp m p-22 o300 2 ~ § noo« 200 l ’ l , )lOO k x .,J = - A- - E E A \ S .6 E e E, Urate 3 E \1' ‘, F’Dlmnoyl CoA ‘8 (133 ,’ s" dependent 3 <1 ,NAD reduction 2 Z O I 5 f 1“- A—A “ ‘‘‘‘‘ {5‘ O '0 < so E GlycerOI - 3 - P E .6 dehydrogenase E ’5 \ '5 E e 3 .3 9. _ 9 é 05 F / ' < 2 5 13 8 , a .. Duherxyacetone-P / K :3 reductase " ~ 7 ‘g‘ . g) z /'\ ‘I ‘0 Z 7 I J \— IIIIII ’4’ ‘ . 4.".‘0‘c )4] ‘0‘ 5" " ‘ A A .6 20 so :0 so Volume (ml) FIG. 1. Isopycnic sucrose density gradient frac- tionation of the homogenate containing 1.0 g of liver from a white mouse HA(ICR). All the assays were run on the same preparation except for glycerol-3-P dehydrogenase and dihydroxyacetone-P reductase which were from another preparation containing 0.77 g of liver. white mice as compared with the CS7BL/6J mice. This was repeatedly observed and no explanation can be offered for this other than biological variation. Data in Figs. 1 and 2 also Show that the B-oxidation activity in the hepatic peroxisomal fraction from the white mouse was two- to threefold more ac- tive than in comparable fractions from the lean C57 BL/6J mice. In this study, how- ever, the Bar Harbor lean C57BL/6J mice have been used so that comparisons could be made with the obese mutant and its lean littermate. 181 With both C57BL/6J and the HA(ICR) mice, less than 5% of the catalase or urate oxidase activity was present in the super- natant fraction. Almost all the activity was in or tailed from the peroxisomal peak, as if there had been little breakage of the per- oxisomes during homogenization. These re- sults differ from those reported for the rat, where about 50 to 75% of the catalase is in the supernatant and attributed either to peroxisomal fragility or to two subcellular locations of catalase (9). Inclusion of 0.01% ethanol in the gradient, which has been used to prevent the formation of an inactive per- oxide complex (15), did not increase the cat- alase activity. Data from mouse gradients, particularly the lean mouse, indicate some of the peroxisomal catalase tailed into the mitochondrial area (Fig. 2). The identity of this tailing material in the mouse gradients has been examined by electron microscopy. For both the mouse and the rat, the per- oxisomal marker enzyme activity above the main band appears to be due to an area con- taining a mixture of peroxisomes and mi- crosomes. The profile for the mitochondrial matrix enzyme, glutamate dehydrogenase, and the mitochondrial membrane enzyme, B-hy- droxybutyrate dehydrogenase, coincide. This indicates that no Significant amount of single membrane mitochondrial fragments were present. Differences in Hepatic Peroxisomal Enzymes from Lean and Obese Mice The obese Bar Harbor CS7BL/6J mice at 3 months of age averaged 53 g body wt and 4.4 g liver wt. On the average they were twofold heavier than their lean littermates and their livers were about threefold larger. Under these circumstances enzymatic data in Fig. 2 were evaluated and reported in Table I as Specific activity in the isolated subcellular organelle from the sucrose gra- dient and as total activity per gram of liver, per liver, or per 100 grams body weight. The specific activities of the marker en- zymes in the isolated peroxisomes (catalase, and urate oxidase) and the mitochondria (glutamate dehydrogenase) and the total ac- tivity per gram of liver were the same from 75 182 MURPHY ET AL. Bar Harbor C57BL/6J 5+ LEAN i ‘ Cyoichiorne c I l reducmse Y ._. Catalase (mmol/mm) a IOO "Glplomote dehydrogenase (nmol "J ‘ ~—- ‘NADP cyl c reduCIose (nmol/rnm) .. ' 1.9- Hydvolybulyrale dehydrogenase g 3 a >——-o NAD 8- HydtOxbUlyrole ‘ IOO dehydrogenase (nmcl/mln) N O 5 Palmitoyl CoA Dependent NAD reductioMrrml/mnl HUrale Oidase (LImOI/mm) PulmImyl Col: .6 oxldmlon NAD Glycerol- P dehydrogense (umol Imlnl V -—' NAD= Glycerol-3P Dehytroganse N DlhydrOIy acetone-P ' reductase v-w NADH Dihydroxyocetone - P Reductose (umol/mnn) AAAAAA so ‘éou‘ao‘ 40 so Volume (ml) FIG. 2. Isopycnic sucrose density gradient fractionation of the homogenate containing 0.75 and 1.71 g of liver from a lean or obese C57BL/6J mouse, respectively. The B-oxidation activity from the lean mouse has been multiplied by 10 for comparison. the lean and obese mouse (i.e. , ratio of about 1 in Table I). This similarity was true even though the obese mouse at 3 months of age had a larger and more fatty liver. Conse- quently, the total peroxisomal and mito- chondrial activity increases per liver from the obese mouse in proportion to the liver size. In Fig. 2 approximately the same 76 ENZYME ACTIVITIES OF PEROXISOMES FROM LEAN AND OBESE MICE 183 TABLE I TOTAL HEPATIC ENZYME ACTIVITY or LEAN AND OBESE MICE (C57BL/6J)a Per mg protein in peak activity fraction Per g liver Per liver Per 100 g body wt Obese] Obese! Obese! Obese/ Enzyme N Units lean Units lean Units lean Units lean Catalase Lean 5 4.3 2 1.2 1 4 1 20 10 78 t 29 2 9 290 1 96 1 5 (mmol/min) Obese 5 5.9 x 1.7 ' l 14 ' 224 z 80‘ ' 421 1 146 ° Glutamate dehydrogenase Lean 4 0.038 I 0.007 1 2 0.7 l 1 1.7 I 0.9 3 2 6.4 t 3.5 1 6 (umol/min) Obese 4 0.047 I 0.010 ' _ 0.4 i 5.6 1 1.5‘ ‘ 10.4 t 1.9 ' Urate oxidase Lean 4 0.28 t 0.08 1 2 3.5 0 9 5.2 : 1.7 2 5 19.5 t 7.5 l 2 (umoUmin) Obese 4 0.34 r 0.11 ' 3.2 _ ° 13.0 t 4.7‘ ' 24.7 t 8.2 ' Palmitoyl-CoA oxidation Lean 5 9 t 3 3 1 263 z 143 1 8 309 z 199 5 l 1536 t 851 2 5 (nmol/min) ()bese 4 28 z 12" ' 468 : 59“ ' 2027 t 461‘ ' 3800 x 734” ' " The units are cited in column one for each enzyme and the specific activity is calculated on the basis of the protein content of the isolated peak activity fraction. peroxisomes for catalase. urate oxidase. and palmitoyloCoA oxidation and mitochondria for glutamate dehydrogenase. ' l’ < 0.01. “ P < 0.05. fraction (about 1/3) of the total liver homog- enate had been used for each gradient, so the increase in total activity in the obese liver was due to an increase in the liver Size. Since the liver in the obese mouse was larger on a body weight basis than in the lean mouse, the change in peroxisomal and mitochondrial marker enzymes increased per 100 gram of body weight. Comparison of Matrix and Membrane-Associated Enzymes Catalase and urate oxidase of the per- oxisomes and glutamate dehydrogenase of the mitochondria are located in the matrix of these organelles and were not different in specific activity between the lean and obese mouse. In contrast the membrane en- zymes, N ADPHzcytochrome c reductase of the microsomes and B-hydroxybutyrate de- hydrogenase of the inner mitochondrial membrane, decreased two- to threefold per gram of liver in the obese mouse. For these membranous enzymes there was no differ- ence per liver, as seen in Fig. 2 even though the liver from the obese mouse was about threefold larger. Thus there seems to be a difference of hepatic matrix enzymes of the organelles but no difference in membrane components with obesity. This difference should be examined for other enzymes and , on a larger scale with zonal sucrose gradients. Comparison of fi-Oxidation in the Lean and Obese C5 7BL/6J Mouse and the White Mouse The assay procedure for peroxisomal pal- mitoyl-CoA-dependent reduction of NAD did not measure mitochondrial B—oxidation (6, 8). The palmitoyl-CoA-dependent N AD reduction shown in Figs. 1 and 2 coincided with the distribution of the peroxisomal cat- alase. The results with the mouse extend the observation of Lazarow and De Duve (6) with the rat that liver peroxisomes cata- lyze B—oxidation. The peroxisomal B-oxida- tion was two— to threefold more active per gram of liver from the white mouse (Fig. 1) than from the black Bar Harbor lean mouse (Fig. 2). To compare the distribution of B-oxidation between peroxisomes and mito- chondria, it will be necessary to extend these assays using procedures (8) that will quantitatively measure B-oxidation in mito- chondria from the sucrose gradient used to separate the two organelles. Preliminary results indicate about equal distribution of B-oxidation between the two particles from the mouse liver. Total B-oxidation in the peroxisomes from the obese mouse liver was about twofold as much per gram of liver as in the lean mouse, which resulted in a difference of over fivefold per liver. This increase was pronounced in the enzymatic specific activ- 77 184 ity with the isolated peroxisomes (Fig. 2 and Table I). The results indicate a higher activity for B-oxidation in the liver peroxi- somes of the obese mouse, even though the other peroxisomal enzymes, catalase and urate oxidase, were not higher per gram liver or per milligram protein. Differences in Hepatic NAD:glycerol-3-phosphate Dehydrogenase in the Obese Mouse The amount of this enzyme in the per- oxisomal fraction of the liver was small, be- ing about one-tenth of that in the cytosol (Figs. 1 and 2, Table II). This limited ob- servation with mice indicates that NAD: glycerol-P dehydrogenase previously re- ported for rat liver peroxisomes (7) may also be a constituent of mouse peroxisomes. The glycerol-3-P dehydrogenase activities in the peroxisomal and supernatant frac- tions from the mouse liver migrated sim- ilarly during thin layer isoelectric focusing but have different mobilities during electro- phoresis on 7% polyacrylamide gels. The TABLE II DISTRIBUTION AND TOTAL ACTIVITIES or N ADIGLYCEROL-3-PHOSPHATE DEHYDROGENASE IN THE LIVERS or FIVE LEAN AND OBESE MICE Supernatant fraction Peroxisomal fraction“ Percentage distribution Lean 10 t 3.5 76 t 6 Obese 14 t 2.5* 76 :t 2 p.mol/min g liver Lean 0.9 1- 0.3 10 t 5 Obese 2.8 :- 2.1"‘ 18 t 12 p.mol/min mg protein Lean 0.2 t 0.1 0.5 t 0.5 Obese 0.8 I 0.3" 0.9 t 0.4 " Peroxisomal fraction includes volumes 8—24 ml on gthe gradients; supernatant fraction includes volumes 144-55 ml on the gradient. * P < 0.01. ** P < 0.05. ._——.___ __ —— MURPHY ET AL. activities from the two compartments also differ kinetically (R. Gee, unpublished). The activity of glycerol-3-P dehydrogenase in the peroxisomal fraction from the obese mouse liver was higher than that in the lean mouse when expressed on either a protein or gram of liver basis (Table II). The cy- toplasmic pool of glycerol-3-P dehydro- genase was higher in the obese mouse by about the same magnitude as in the peroxi- somal fraction. DISCUSSION The mouse has been found to be a par- ticularly good animal for studying liver per- oxisomes. The peroxisomal enzymes are very active per gram of tissue and the per- oxisomes appear to be much less fragile than in the rat liver as judged by little catalase activity in the supernatant fraction. Better peroxisomal isolation was experienced with the white HA(ICR) mouse than with the lean C57 BL/6J mouse. In addition the white mouse provided several fold higher specific activity for B-oxidation in the isolated per- oxisomes. Three comparisons can be made for the enzyme activities of subcellular particles from the livers of obese with those from the lean littermates of the C57 BL/6J mouse at 3 months of age. (A) The matrix enzymes, catalase and urate oxidase of the peroxi- somes and glutamate dehydrogenase of the mitochondria, remained about constant per gram of liver from either lean or obese ani- mal. Since the obese animal had a larger liver, the total activity per liver increased proportionately. (B) Two membrane en- zymes that were examined, NADPH:cyto- chrome 0 reductase of the microsomes and B-hydroxybutyrate dehydrogenase of the mitochondria, were lower in the obese mouse in inverse proportion to the larger size of the liver. Therefore, the amount of these two enzymes per liver was not dif- ferent. (C) A higher specific activity and total activity of peroxisomal fatty acid B- oxidation, as well as some increase in NAD:glycerol-3-P dehydrogenase, was ob- served in the obese mouse. Peroxisomal fatty acid B-oxidation in the obese mouse was about threefold higher in 78 ENZYME ACTIVITIES OF PEROXISOMES FROM LEAN AND. 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