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I. 1 0 II. 000'003'0100000011010:0IIV00 1.41. . ...10 000.0800IH0100..L0....101..0000 00015000010000 1010.000 0 ' [Ju01 00v .1091 . II. V I 00 I 1 L 0 0 7‘0.“- ‘D‘IP100CP.\00.&U.Q. l1t000w ”055.001.01.011.“- 9. i [00.1.1 -‘ aoffildlar'f.‘ [4’101‘1115‘ III‘I. .IJ‘f‘fflPsu..01 ”Ax-thréumg g‘u‘kug. .Et f’é. I I .(o I. 00 I. .IIO _ 1 1.001 1 1 . _l- I. 1': ..l'l THESI‘ 'u -H‘ i E. . A? .12. '7’." a? ’ initial» :17"? HUM; & 5U"? BUUK BINDERY INC. -lBRARY BINDERS "L: 92::sronr, MICHIGAN -‘ :n “i; A _ 51¢ a ”7 ' ABSTRACT Ct THE 115. £139. AND Bi mg LIPOLYTIC ACTIVITY OF Two PORCINE GROWTH HORMONE PREPARATIONS IN PIGS BY Brenda Lee Buzzell The lipolytic activity of commercially prepared porcine growth hormone (CPGH) and purified porcine growth hormone (PPGH) which had different biological activities, were studied in subcutaneous porcine adipose tissue in 21££2_and in finishing pigs in 3139. In experiment 1, the biological activity of CPGH and PPGH was deter- mined by the tibia test in two separate trials using hypophysectomized Long-Evans rats. The biological activity of CPGH and PPGH were deter- mined to be 1.1 IU/mg and 3.0 IU/ml, respectively. In experiment 2, subcutaneous adipose tissue from 3 meal fed and 3 §g_libitum fed pigs weighing between 69 and 77 kg were incubated with deionized distilled H20 (control), 50 ug/ml of CPGH, 10 ug/ml of PPGH (approximately equal in biological activity to CPGH) and 50 ug/ml of PPGH (equal in weight to CPGH) for 7 hours. The average rate of glycerol release from adipose tissue of ad libitum and meal fed pigs was greater (P < .05) in response to 50 ug/ml of CPGH than 10 ug/ml of PPGH, 50 ug/ml of PPGH or control. The time x treatment interaction was highly signifi- cant (P < .01). The accumulated medium glycerol was higher after 2 hr and rose linearly after the second hour in adipose tissue from both groups in response to CPGH. The rate of glycerol release was greater (nonsigni- ficant) than the control and 10 ug/ml of PPGH after the 3rd and 5th hour Brenda Lee Buzzell of incubation of adipose tissue from meal and ad libitum fed pigs, re- spectively, in response to 50 ug/ml of PPGH. The pattern of response to 10 ug/ml was essentially that of the control. The correlations between medium glycerol and FFA were .94 and .96 for meal and ad libitum fed pigs, respectively. In experiment 3, four Yorkshire x Hampshire crossbred barrows, ranging in weight from 69 to 77 kg, received either 0, .13 mg/kg PPGH, .34 mg/kg PPGH, or .34 mg/kg CPGH in saline on separate days with at least one day between treatments in a 4 by 4 Latin square experimental design. They were fed once per day for 4 hr; the feed was removed at least 12 hr before sampling. The treatments were infused over a 4 min interval and blood samples were collected at various intervals for the next 7 hours. Plasma FFA, glucose and serum insulin concentrations were determined on the samples. Plasma FFA levels were significantly greater (P < .05) at 30, 60, 90, 120 and 135 min in response to CPGH than.the other treatments at their re- spective times. At 150 min postinfusion, FFA levels were significantly greater (P < .05) in response to both CPGH and .34 mg/kg PPGH than the other treatments. The plasma FFA had returned to the level of the O-hr bleeding 180 min after infusion of CPGH. Plasma FFA varied significantly with time only in response to CPGH treatment. Plasma glucose levels varied significantly with time in response to .13 mg/kg and .34 mg/kg PPGH. Plasma glucose levels at the O-hr bleeding were significantly greater (P < .05) on the day of .13 mg/kg PPGH infusion Brenda Lee Buzzell than on the day .34 mg/kg PPGH was administered. Plasma glucose levels were higher (P < .05) in response to CPGH and both levels of PPGH than the control 210, 270 and 300 min postinfusion. 'At 330 min, glucose was significantly higher (P < .05) after infusion of .13 mg/kg PPGH than CPGH or saline. A depression (nonsignificant) in glucose was observed 15 min and 15 to 30 min after infusion of .13 mg/kg PPGH and .34 mg/kg PPGH, respectively. Serum insulin concentration varied significantly (P < .01) with time after administration of .13 PPGH, .34 PPGH, and .34 CPGH. Thirty min after infusion of both PPGH treatments, insulin levels were significantly (P < .05) less than the control. CPGH stimulated a greater (P < .05) release of insulin than the control 30 and 180 min postinfusion. At 330 min postinfusion, serum insulin levels were significantly greater (P < .05) in response to .13 mg/kg PPGH than the control or CPGH treatments. The high level of PPGH had higher values (P < .05) than the control at 330 min postinfusion. Both levels of PPGH elicited a higher serum insulin level than either the saline control or CPGH 390 min postinfusion. The correlation coefficients were .50 between insulin and glucose levels, .04 between FFA and glucose levels and .07 between FFA and insulin levels. Plasma glucose levels peaked at 135 min and 210 min in response to CPGH and both levels of PPGH, respectively, whereas plasma insulin levels peaked at 240, 330 and 210 min in response to CPGH, .13 mg/kg PPGH, and .34 mg/kg PPGH, respectively. After peak values were attained, both glucose and insulin values remained elevated in response to PPGH but re- turned to the control insulin and glucose levels in response to CPGH. THE I_N VITRO AND I_N VIVO LIPOLYTIC ACTIVITY OF TWO PORCINE GROWTH HORMONE PREPARATIONS IN PIGS By Brenda Lee Buzzell A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science and Human Nutrition 1975 ACKNOWLEDGEMENTS The author wishes to express her sincere appreciation to Dr. R. A. Merkel for his guidance and support throughout this study and for his advice in preparing this manuscript. The author gratefully appreciated the advice and the use of the facilities of Dr. D. R. Romsos. Apprecia- tion is also expressed to Dr. J. Fo Price and Dr. D. R. Romsos for serving on the guidance committee. The author wishes to thank Dr° E. R. Miller for his help with the pigs and the use of the swine research facilities. In addition the help of Mr. Roger Hale and Mr. Joe Strittmatter and their associates at the MSU swine barn was appreciated. The author wishes to thank Dr. R. Waterman for his patience in demon- strating the assay procedures used in this study. In addition, I would like to express sincere appreciation to Dr. W. T. Magee for his assistance with the statistical analyses of these data. Special thanks are extended to Dr. L. J. Machlin of Monsanto Chemical Company for providing the puri- fied porcine growth hormone used in this study. The author thanks Dr. G. A. Leveille for the continued financial support and the use of the facilities of the Department of Food Science and Human Nutrition. Appreciation is also extended to Dr. R. H. Nelson for the financial support of the project and the use of the animals and facilities of the Department of Animal Husbandry. Also thanks is expressed to Dr. H. D. Hafs for the use of the facilities in dairy physiology. ii Also, I would like to thank Mr. D. D. Crenwelge and Mr. J. S. Grigsby for their help in collecting the data and with analyzing the glucose. Sincere appreciation is extended to my fellow graduate students in the departments of Animal Husbandry, Food Science and Human Nutrition and Dairy Physiology for their help and advice. An additional special thanks go to my fellow graduate students in the meat laboratory for being such a jovial crew. Sincere appreciation is extended to Mrs. Kim Green for the typing of this manuscript. Special thanks and warm appreciation is expressed to Dr. J. P. Hitchcock for his advice, help, encouragement and sacrifices during this study. I also wish to thank my Parents Mr. and Mrs. Donald Buzzell for their encouragement and support during my educational pursuits. iii TABLE OF CONTENTS INTRODUCTION . . . . . . . . . . . . . . . . . . . . REVIEW OF LITERATURE . . . . . . . . . . . . . . . . . Lipolysis . . . . . . . . . . . . . . . . . . . . Activation of Hormone Sensitive Lipase . . Gluconeogenesis . . . . . . . . . . . . . . . . Growth Hormone . . . . . . . . . . . . . . . . . Purification of Porcine Growth Hormone . . . Control of GH Secretion . . . . . . . . . . Relationship Between the Pituitary and th Hypothalmlls O O O O O O O O O O 0 Growth Hormone Releasing and Inhibiting Peripheral Control of GH Secretion . . The Effect of GH on Lipolysis .. . . . . . . Effect of GH on Lipolysis In Vivo . . . Effect of GH on Lipolysis l§_Vitro . . The Effect of GH on Glucose Metabolism . . . Somatomedin . . . . . . . . . . . . . . . . Hormones The Relationship Between GH, Glucose and Lipolysis . . Serum Levels of GH in Pigs . . . . . . . . . Plasma FFA Levels in Pigs . . . . . . . . . Insulin . . . . . . . . . . . . . . . . . . . . . Synthesis and Release From the Pancreas . . The Effect of GH on Insulin Secretion . . The Effect of Insulin on Glucose Transport . Effect of Insulin on Lipolysis_in Vivo . . . iv Page 10 11 15 l6 16 17 20 20 23 27 29 29 30 32 32 33 35 36 37 Serum Insulin Levels in Swine . . . . . . . . . . Blood Glucose Levels in Swine . . . . . . . . . . The Effect of Insulin and Growth Hormone Interaction on Glucose and Lipolysis . . . . . . . . . . . . . . Effects of the Fed State . . . . . . . . . . . . . Inhibition of Insulin Action by GE . . . . . . . . Other Pituitary Lipolytic Hormones and Factors . . . . ACTH . . . . . . . . . . . . . . . . . . . . . . . TSH...................... MSH . . . . . . . . . . . . . . . . . . . . . . Anti-Insulin Peptide . . . . . . . . . . . . . . . Lipotrophic Factors . . . . . . . . . . . . . . . MTERIALS AND ETHODS O O O O O O O O O O O I O O O O O O 0 Experiment l-Tibia Test . . . . . . . . . . . . . . . . Experiment 2‘12 Vitro Trials . . . . . . . . . . . . . Adipose Tissue Biopsy . . . . . . . . . . . . . . Tissue Preparation in the Laboratory . . . . . . . Preliminary Trials . . . . . . . . . . . . . . . . lg Vitro Trial with PPGH and CPGH . . . . . . . . Glycerol Assay . . . . . . . . . . . . . . . . . . Free Fatty Acid Assay . . . . . . . . . . . . . . Preliminary Experiments . . . . . . . . . . . Experiment 3 . . . . . . . . . . . . . . . . . . . . . Experimental Animals . . . . . . . . . . . . . . . Catheterization . . . . . . . . . . . . . . . . . Experimental Design . . . . . . . . . . . . . . . Blood Collection . . . . . . . . . . . . . . . . . Blood Glucose . . . . . . . . . . . . . . . . . . Plasma Free Fatty Acid Assay . . . . . . . . . . . Insulin Radioimmunoassay . . . . . . . . . . . . . Statistical Analij-S O C O O O O C O C O O O O O O O O 40 40 41 42 42 44 45 45 47 48 48 50 50 50 51 52 52 53 53 54 54 55 55 56 57 57 58 60 RESULTS AND DISCUSSION . Experiment 1 Experiment 2 . Experiment 3 . Plasma FFA, Glucose and Serum Insulin Concentration . The Effect of PGH on Plasma FFA The Effect of PGH on Plasma Glucose Levels . . . . . . The Effect of PGH on Serum Insulin Levels . . . . . Combined Effects of PGH on FFA, Glucose and Insulin SUMMARY . . BIBLIOGRAPHY APPENDIX I . APPENDIX II. 0 O O O REAGENTS Figure 1. into the medium . APPENDIX II. APPENDIX II. APPENDIX II . APPENDIX 11. adipose APPENDIX III. APPENDIX III. APPENDIX III. APPENDIX III. Table 1. from adipose tissue of meal fed pigs Table 2. from adipose tissue of Ad libitum fed Table 3. adipose tissue of meal fed pigs . . . Table 4. tissue of Ag_libitum.fed pigs Table Table Table Table 1. 2. 3. 4. Time Study. Rate Accumulated medium Accumulated medium Accumulated medium Accumulated medium Raw data from pig Raw data from pig Raw data from pig Raw data from pig vi of glycerol released glycerol released glycerol released pigs 0 O O O C O O O FFA released from the 151-9 (Experiment 3) 146-6 (Experiment 3) 116-8 (Experiment 3) 205-7 (Experiment 3) Page 61 61 61 71 71 73 77 82 88 94 99 110 114 115 115 116 116 117 118 119 120 Page APPENDIX III. Table 5. Raw data - Glycerol Release lg_Vitro . . . 121 APPENDIX III. Table 6. Raw data - FFA Released In Vitro . . . . . 122 vii LIST OF TABLES Table 1 Experimental design for the ig_vivo experiment . . . . . 2 Effect of PGH preparations on lipolysis in porcine adipose tissue .11; Vitro O O O O O O O O O O O O O O O O O O O O D O 3 Plasma FFA values after infusion of saline, PPGH or CPGH 4 Plasma glucose values after infusion of saline, PPGH or CPGH C C C O O O C O O O O O O C C O C C O I I O O C O O 5 Serum insulin values after infusion of saline, PPGH or CEH O O O O O O O O O O O O O O O O O O O O O O O O O O 6 Simple correlation coefficients between plasma FFA, glucose and serum insulin concentration . . . . . . . . . APPENDIX II. 1 Accumulated medium.glycerol released from adipose tissue Of meal fed Pigs . O O O O O O O O O O O O C O O O O O O 2 Accumulated medium glycerol released from adipose tissue Of fl libitlml fed pigs 0 O O O C C O O C O O O O I. O O O 3 Accumulated medium FFA released from the adipose tissue Of meal fed pigs 0 O O O O O O O O O O C O O O O O O O O 4 Accumulated medium FFA released from the adipose tissue Of 93—1, libitm fed P185 0 o o o o o o o o o o o o o o 0 APPENDIX III. 1 2 Raw data from pig 151-9 (Experiment 3) . . . . . . . . Raw data from pig 146-6 (Experiment 3) . . . . . . . . Raw data from pig 116-8 (Experiment 3) . . . . . . . . Raw data from pig 205-7 (Experiment 3) . . . . . . . viii Page 56 62 75 79 83 88 115 115 116 116 117 118 119 120 LIST OF FIGURES Figure Page 1 The accumulated medium glycerol released from adipose tissue of ad libitum fed pigs . . . . . . . . . . . . . . . 64 2 The accumulated medium glycerol released from adipose tissue of meal fed pigs . . . . . . . . . . . . . . . . . . 65 3 The accumulated medium FFA released from adipose tissue 0f £9- libitunl fed pigs 0 O I O I O O O O O O O O O O O O O 69 4 The accumulated medium FFA released from adipose tissue Of meal fed pigs 0 0 O O O O O O C O O O O O O O O C O O I 70 5 Pattern of plasma FFA after infusion of saline, PPGH or cmH into Pigs O O O O O O O O O C O O O O O O O O O O O O 74 6 Pattern of plasma glucose and serum insulin after infusion of saline, PPGH or CPGH into pigs . . . . . . . . . . . . . 78 APPENDIX II. 1 Time Study. Rate of glycerol released into the medium . . 114 ix INTRODUCTION Animal scientists are continually attempting to increase production efficiency, muscle mass and concomitantly decrease fat stores in meat animals. These animals tend to be less fat and more efficient. The im- provement of meat animals has been accomplished by improved selection techniques and nutrition. However, selection is a long, slow process. Furthermore, practically all of the improvements which can be made through nutrition have been realized with the present knowledge of bio- chemistry and physiology of the animal. The reduction in the amount of fat in meat animals is important for a variety of reasons. Production of fat is highly inefficient in terms of feed consumption per kilogram of gain since it occurs after bone and muscle growth have essentially been completed. Furthermore, the concern and implication of dietary fat is also an important issue currently. In order to decrease the amount of fat while increasing the amount of muscle, a thorough understanding of the physiology of the animal is required. Within the last few years, increasing attention has been directed to the role of the neuroendocrine system in muscle growth and fattening. Hormones, especially insulin, growth hormOne (GH), androgens and estrogens have been shown to increase muscle mass; GH and the steroid hormones have also been reported to decrease fat stores. GH was shown to diminish fat stores while simultaneously increasing the amount of lean mass in rat carcasses by Greenbaum (1953). Later, Turman and Andrews (1955) discovered that daily injections of GH increased feed efficiency, lean muscle mass and decreased backfat thickness in pigs. In 1972, Machlin confirmed the results of Turman and Andrews (1955) with daily injections of both commercially prepared porcine GH (CPGH) and PGH purified from CPGH. However, he discovered that purified PGH possessed less lipolytic activity than CPGH when rat and rabbit adipose tissues were incubated with each hormone preparation. Furthermore, other investigators have measured the lipolytic activity of GH from a number of species both in_yiyg_and in_yi££g. The results are inconsistent. In fact, the conflict has led a number of investigators to suggest that GB is contaminated with other lipolytic factors. Since the lipolytic activity of GH is still a controversial issue, further study is necessary to establish its unequivocal role in lipolysis. Thus, this study was undertaken to compare the lipolytic activity of two PGH compounds of different purities and biological activities on por- cine adipose tissue lfl.!i££2 and finishing pigs in 2139. The relationship between biological activity and lipolysis was also studied as well as the effects of an acute dose of PGH on plasma glucose and serum insulin levels. ’1 REVIEW OF LITERATURE Lipolysis Adipose tissue, the energy storage depot of the body, is very impor- tant in maintaining energy homeostasis of the animal. Adipose tissue stores excessive nutrients from ingested carbohydrates, fats and amino acids as triglycerides and releases free fatty acids (FFA) and glycerol when needed (Rizack, 1961). The formation of fat (lipogenesis) and the hydrolysis of fat (lipolysis) are controlled by the amount of nutrients and the primary and secondary effects of various hormones. Although lipogenesis depends upon the amount of nutrients in the bloodstream, lipolysis proceeds at all times but at different rates depending upon the stimuli affecting the adipose tissue at any given time. Therefore, the amount of adipose tissue is the net result of lipogenesis and lipolysis. The cleavage of triglycerides to form glycerol and FFA is catalyzed by hormone-sensitive lipase (HSL) as described in the reviews of Goodman (1970a) and Machlin (1972a). The glycerol is immediately released into the bloodstream.in_yiyg_or medium in zigrg since glycerol kinase which catalyzes glycerol to a-glycerol phosphate is present in very low activity in adipose tissue (Margolis and Vaughn, 1962). However, FFA are either reesterified or released into the bloodstream in_giyg or the medium in. 1132333 depending on the amount of nutrients in the cell. Reesterifica- tion occurs if sufficient glucose and/or glycogen are present to provide a—glycerol phosphate. If, glucose is not available, then FFA would be released into either the medium in yiggg or bloodstream in yiyg_where they are transported by albumin. According to Heindel _£‘_l. (1974), when the albumin binding sites are saturated with FFA, the FFA remain in the adipocyte although lipolysis continues as measured by glycerol re- lease. Consequently, triglycerides are cleaved into FFA which are utilized by muscles and glycerol which enters the gluconeogenic pathway.in the liver and kidney. Activation of Hormone Sensitive Lipase Although lipolysis has been shown to be continuous at a slow rate, the rate of lipolysis can be accelerated by the activation of HSL by hor- mones. Hormone action on lipolysis is mediated by the adenyl cyclase system which acts as a messenger between the cell membrane, to which the hormones bind, and the HSL in the cytoplasm. The first enzyme in the adenyl cyclase system is adenyl cyclase it- self which is found in the cell membrane. Furthermore, Birnbaumer and Rodbell (1969) have shown that a specific adenyl cyclase did not exist for each hormone but all hormones activated the same adenyl cyclase in rat adipocytes. Adenylcyclasecatalyzesthe formation of cyclic adenosine triphosphate (CATP) from ATP which formed cyclic adenosine 3', 5'-mono- phosphate (CAMP) (Ho and Sutherland, 1971; Butcher §£_al., 1968). In the next step, Corbin _£‘_l. (1970) using rat adipose tissue substantiated the previous discovery that CAMP activated protein kinase which in turn acti- vated HSL. Although CAMP and protein kinase had been implicated in the activation of HSL and/or lipolysis by Butcher t al. (1965, 1968) and Corbin _£‘_l. (1970), no definite proof was available until Huttunen and Steinberg (1971) determined the cofactors necessary for HSL activity from rat adipose tissue. Both Huttunen and Steinberg (1971) and Khoo and Steinberg (1974) using HSL purified from rat and chicken adipose tissue, respectively, discovered that protein kinase, CAMP, ATP and Mg2+ were necessary to activate HSL; however lipoprotein lipase (LPL) was not acti- vated with the same cofactors (Khoo and Steinberg, 1974). Furthermore, using a-32P ATP, Huttunen and Steinberg (1971) demonstrated that protein kinase activated HSL by catalyzing the transfer of the a-phosphate of ATP to HSL. Therefore, the adenyl cyclase system appears to activate only one lipase, HSL, which results in increased lipolysis. As mentioned previously, various hormones have been shown to acti- vate or inhibit the adenyl cyclase system by binding to the cell membrane. The method by which the hormones activate cAMP through adenyl cyclase varies with the hormone. Butcher _£__l, (1968) and Birnbaumer and Rodbell (1969) confirmed that the anterior pituitary hormones, adrenocorticotropin (ACTH), thyroid stimulating hormone (TSH) and lueteinizing hormone (LH) increased the accumulation of cAMP within 10 min when rat adipose tissue was incubated with caffeine and theophylline (both of which prevent the degradation of cAMP). In the same set of experiments, glucagon and the catecholamines, epinepherine and norepinepherine also stimulated the accu- mulation of CAMP. Furthermore when Birnbaumer and Rodbell (1969) added Propanol, a B-adrenergic blocker to rat adipocytes in vitro and Hertelendy ._£‘_l. (1970) infused propanol and epinepherine into sheep, lipolysis was inhibited. Consequently, ACTH and possibly TSH directly activated the adenyl cyclase system by binding to B-adrenergfl: receptors in the cell membrane. The anterior pituitary hormone, growth hormone (GH), has been shown to require a lag time for a significant increase in lipolysis to occur. Incubating isolated rat adipocytes in bicarbonate buffer containing albu- min with 1 ug/ml of bovine GH (BGH) and the glucocorticoid analog, dexa- methasone which has been shown to act synergistically with GH by Fain g3 31. (1965), Fain (1967) discovered lipolysis occurred after an hour lag period. In addition, the latter author showed the cause of lipolysis to be due to GH but not dexamethasone. Furthermore, when actinomycin-D and puromycin, which inhibit DNA dependent RNA synthesis and protein synthesis, respectively, were incubated with GH and dexamethasone for 4 hr, no in- crease in lipolytic activity was detected. Since Fain _£q_l. (1971) dis- covered that GH alone was responsible for the accumulation of cAMP in adipocytes, the apparent mode of action of GH on lipolysis was mediated through a protein whose exact function has not been determined. Conse- quently, HSL activating hormones may either act directly on the adenyl cyclase system such as ACTH and TSH do or indirectly as GH does. Although glucagon activated lipolysis, insulin has been shown to inhibit HSL by inhibiting the activation of adenyl cyclase and decreasing CAMP levels in adipocyctes. When Illiano and Cuatrecasas (1972) incubated adipocyte membranes with 5 uU/ml of insulin following incubation with ePinepherine and ACTH, the adenyl cyclase activity of the membranes decreased significantly. Also they reported finding some decrease in the baseline activity of adenyl cyclase when adipocyte membranes were incubated with 20 uU of insulin per milliliter. Consequently, insulin appeared to inhibit the effect of lipolytic hormones on adenyl cyclase. The action of insulin on CAMP has been documented for a long time although the exact mechanism of insulin action has been suggested only recently. Butcher _£._l- (1968), Manganiello _£ _1. (1971) and Khoo E; El' (1973) discovered that insulin significantly decreased the CAMP forma- tion caused by incubating rat adipocytes with epinepherine and ACTH; the decreased CAMP was also accompanied by decreased glycerol release. Loten and Sneyd (1970) discovered that high levels of insulin in the medium activated phosphodiesterase, which in turn inactivated CAMP in rat adipo- cytes. Zinman and Hollenberg (1974) discovered as little as 7.5 uU/ml of insulin, added to isolated adipocytes in yiggg, stimulated phosphodies- terase activity by 20%. Furthermore, lipolytic hormones such as epine- pherine and ACTH stimulated phosphodiesterase activity at the same time lipolysis occurred. The investigators suggested that insulin increased the rate of destruction of cAMP by activating phosphodiesterase, while decreasing the rate of CAMP formation by inhibiting adenyl cyclase. Gluconeogenesis Gluconeogenesis may be defined as the synthesis of glucose and usually occurs after depletion of glycogen stores in the liver during fasting (Exton gt al., 1970). Gluconeogenesis occurs in both the liver and kidney Sillce they are the only two tissues which have the necessary enzymes. The primary substrates of glyconeogenesis are lactate, amino acids and glycerol (Exton gt al., 1970). A number of hormones plus fasting have been shown to activate glu- coneogenesis. Garrison and Haynes (1973) discovered that isolated liver cells, incubated with glucagon and lactate, released glucose from the liver glycogen stores of fed rats but released newly synthesized glucose after a 20 min lag Period in the fasted rat. Furthermore, Exton _£._l- (1972) concluded that glucocorticoids were required for gluconeogenesis to occur in livers from fasted rats. When livers from adrenalectomized, adrenalectomized plus dexamethasone treated and normal rats were perfused in yi££g_the glucose production in liver from the adrenalectomized rat was much less than that from the normal rat, but dexamethasone injected into the adrenalectomized rat restored glucose synthesis. In addition, dexamethasone restored the depressed levels of gluconeogenesis to normal in adrenalectomized rats in response to glucagon and epinepherine. Exton _t _l. (1972) suggested that gluco- corticoids were required to maintain the responsiveness of cellular mechanisms to CAMP which were shown to be elevated in liver from fasted animals by Garrison and Haynes (1973). Therefore, gluconeogenesis occurred in response to fasting and certain hormones which require glucocorticoids to mediate their effect. Other hormones such as insulin and GH appear to inhibit or to have no effect on gluconeogenesis, respectively. Since insulin stimulated glucose transport and inhibited lipolysis, insulin blocked gluconeogenesis indirectly. Furthermore, insulin lowered the levels of CAMP which were shown to be required for gluconeogenesis (Exton g; 31., 1970). However, Exton.££ a}: (1972) after injecting .1 ug of GH twice daily for two days prior to perfusion of the liver, discovered GH had no effect on gluconeo- genesis. Although many compounds have been shown to be substrates for glucon- eogenesis, glycerol has been shown to be a substrate but FFA have been shown not to be a substrate for gluconeogenesis. In perfused liver, Ross ._§._l. (1967) and in isolated liver cells from fasted rats, Garrison and Haynes (1973) and Tolbert and Fain (1974) showed that glucose was synthe- sized from labeled glycerol. However, Ross _£__l, (1967) noted that the amount of glucose formed was small and suggested that the glycerol kinase Present in liver cells was the rate-limiting step. Exton _£“§l. (1970) calculated that glycerol would account for only a small percentage of glucose synthesis if all the glycerol were converted to glucose. Although plasma FFA do not serve as a substrate for gluconeogenesis, oxaloacetate has been shown to be incorporated into glucose in some experiments. Exton and Park (1968) and Tolbert and Fain (1974) reported an increase in labeled glucose from labeled oxaloacetate in both perfused livers and isolated liver cells from fasted rats, respectively. Both groups of investigators suggested that the oxaloacetate was converted to pyruvate extracellularly which accounted for the labeled glucose. Garrison and Haynes (1973) re- ported that oxaloacetate was not incorporated into glucose. Therefore, glycerol is incorporated into glucose in the liver but FFA do not appear to be. 10 Growth Hormone GH is secreted by the acidophils of the anterior pituitary gland and has been demonstrated to be necessary for the normal growth of mammals (Ganong, 1971). Although GH is known to be species specific due to dif- ferences in its amino acid composition and sequence, GH from one species will cross-react with another species in certain instances. GH from mammalian species which are lower on the phylogenetic scale than other mammalian species have been shown not to cause the typical GH response. However, GH from mammalian species higher on the phylogenetic will elicit the typical GH response in lower animals (Kostyo, 1974). Since the physiological response to human GH (HGH) has been shown to be the same in all species but since species specificity has been illus- trated, Kostyo (1974) reported that many investigators have postulated and searched for an "active core" which was responsible for CH actions in the GH molecule from all species. The physiological functions of GH has been illustrated to promd:e bone and muscle growth while decreasing fat stores (Ganong, 1971). GH has been shown to promote the growth of the epiphyseal plate through the action of somatomedin which was either synthesized in response to or altered from GH in the liver (MCConaghey and Sledge, 1970). Secondly, GH has been shown to increase protein synthesis by accelerating the rate of elongation (Kostyo and Rillemia, 1971) from the intracellular amino acid pool under normal physiological conditions (Turner, 1972). CH has been observed to decrease fat stores by mobilizing FFA in the rat and pig 11 by Greenbaum (1953) and Turman and Andrews (1955), respectively. Conse- quently, GH promoted "real growth" while depleting adipose tissue stores. Purification of Porcine Growth Hormone In order to have been able to ascertain the functions of GH described above, it was necessary to isolate and purify it from the pituitary gland. The other pituitary hormones must be removed from GE since some of them such as ACTH, thyroid stimulating hormone (TSH),melanocyte stimulating hormone (MSH), lipotrophic hormones and anti-insulin peptide (AIP) elicit responses similar to GH in the animal. Therefore, many procedures have been developed to isolate and purify GH from many mammalian species in- cluding the pig. Many procedures, most of which have been modified from HGH isolation and purification procedures have been developed for porcine GH (PGH). Although most researchers have begun with fresh pituitaries (Papkoff gt 31., 1962; Chappel and Carnegie, 1974), Chen _£._l- (1970) developed a procedure utilizing the Oxycel filtrate remaining after the adsorption of ACTH onto cellulose. In all cases, the isolated protein was shown to possess some GH activity by the tibia test (biological assay for GH). In the initial steps of most procedures, GH has been removed from the pituitary glands under either acidic or alkaline conditions. Since Chen t al. (1970) used Oxycel filtrate, the initial isolation procedure was developed by Payne t l. (1950). The hormones were extracted from acetone dried pituitary powder with glacial acetic acid which was heated to 70 C followed by another wash of glacial acetic acid and acetone. 12 To the acidic supernatant, Chen _£H_l. (1970) added 10M KOH to obtain a final concentration of .3M K and adjusted the pH to 8.5. After centri- fugation, the pH was adjusted to 4.0 with 4N HCl, and (NH4)2804 was added until the concentration was 1.25M. After centrifugation the pH of the redissolved precipitate was adjusted from 10.0 to 10.5 then to pH 8.5 with 4.0N HCl, and (NH4)2804 was added to obtain a final concentration of 1.2M. After centrifugation, the pH was adjusted to 7.0. The precipitate (FRACTION A) was redissolved, the pH adjusted to 4.0, then 4.9 from which a precipitate P1 was obtained. The solution remaining after centrifuga- tion was adjusted to pH 7.0 and .25M (NH After centrifugation, 4)2804. the concentration of (NH 804 was increased to 1.2M. The supernatant 4)2 waslyophilizedand designated fraction A1. The precipitate, P1,*was purified according to the procedure for A1 beginning with the pH adjust- ment of 10.0. The precipitate obtained following the addition of (NH4)2 4)2804 was called fraction A2. concentration of the supernatant was increased to 1.6M and SO to a final concentration of 1.2M (NH 4 The (NH4)2304 the precipitate remaining after centrifugation was called A3. 0n the other hand, Papkoff t l. (1962) using the method of Li (1954) in the initial stages of purification, extracted the hormones from fresh pig pituitaries by adding calcium hydroxide, pH 10.3, to the ground pitui- taries. Varying amounts of saturated (NH4)ZSO were added to the super- 4 natant at pH 6.8 and after the fifth addition of (NH the precipitate 4)2S°4 was dialyzed until it was free of salt. The precipitate remaining after dialysis was redissolved in water and adjusted to pH 5.3. After each 13 centrifugation, the pH of either the supernatant or precipitate was ad- justed to 6.8, 10.5 and 7.0. After centrifugation following the last pH adjustment, the supernatant was lyophilized, redissolved in water at pH 4.0 and various amounts of NaCl were added. After treatment with NaCl, 3 series of pH adjustments from 10.0, 8.7, and 6.8 followed by centrifu- gation after each pH change, were performed on the precipitate. The precipitate, remaining after centrifugation of the pH 6.8 supernatant, was redissolved in water at pH 9.0 and lyophilized. This fraction was purified using the procedure of Papkoff _£_§l. (1962). Despite the dif- ferences in reagents, both methods described above employed wide ranges in pH to extract PGH. Furthermore, the end product of the first stage of purification, using the procedures of Chen t al. (1970) was assayed and found to contain ACTH, TSH and prolactin. However, Li (1954) discovered growth promoting activity but no contamination by other pituitary hormones when he isolated HGH using the same procedure. The final steps of purification and isolation of PGH were achieved by column chromatography. Papkoff _£M_l. (1962) purified the precipitate they isolated using the method of Li (1954) by countercurrent distribution with 2-butanol-.4% dichloroacetic acid followed by elution with .1M acetic acid on a Sephadex-SO column. The eluate, after lyophilization was assayed for GH, TSH, ACTH and MSH. GH activity was present, and contamination by TSH, ACTH and MSH were less than .1 mU/mg and 1 U/mg and .005%, respect- ively. Furthermore, when electrophoresis was employed, a single band was observed. When Chen t 1. (1970) developed the final steps of the proce- dure only Chromatography was used to purify PGH. 14 After redissolving fractions Al, A2 and A3 in .02M sodium borate, pH 8.0, Chen _£_§l. (1970) placed the dissolved fractions on a DEAE-cellu- lose column equilibrated with .02M sodium borate and eluted them with a linear gradient of NaCl ranging from .033M to .3M. PGH, obtained by this method, had GH activity of 1.6 IU/mg and was contaminated with .00025 USP units/mg of ACTH. When PGH was subjected to polyacrylalide gel electro- phoresis, major slow and minor fast bands of PGH were observed. After additional column chromatography, the fast band was removed and Chen 35 31 (1970) postulated the fast band might be caused by deamidation of PGH. Furthermore, the isoelectric point of both hormones was ascertained to be 6.3. Although PGH had been isolated and purified as indicated by elec- trophoretic methods and assayed for other pituitary hormones as described above, a third method of PGH isolation, which was developed by Machlin (personal communications), yielded PGH which had greater growth promoting activity than the other purification procedures as measured by the tibia test. Starting with PGH with an activity between .5 and l IU/mg, Machlin used a method similar to Chen _£‘al. (1970) to purify the hormone. The PGH was dissolved in .03M tris buffer, pH 8.8, applied to a DEAF-cellulose column equilibrated with .03M tris buffer, pH 8.8, applied to a DEAE- cellulose Column equilibrated with .03M tris, pH 8.8 and eluted with the same buffer. After lyophilization, PGH was redissolved in .03M tris buffer, pH 8.8, and eluted through the same columns with .04M NaCl .03M tris, pH 8.8. Fractions 33 to 40 were combined, and (NH 504 was added 4)2 15 to make a 60% solution. After centrifugation the supernatant was dialyzed against water and lyophilized. The growth promoting activity was ascer- tained to be 3.0 USP units/milligram. However, no assays for other pituitary hormones were performed on PGH. Consequently, by subjecting the GH molecule to column Chromatography, PGH with greater growth-promoting activity and purity was obtained. To summarize the purification procedures of PGH, the initial phase was the removal of the hormone from the pituitaries followed by the removal of small amounts of contaminants. Chappel and Carnegie (1974) questioned the effect of prolonged exposure to highly alkaline or highly acidic com- pounds on the integrity of the PGH molecule. Although many functions of PGH have not been shown to be decreased by the harsh treatment involved in the isolation and purification procedure, the real effects of the treatment on these functions are unknown. Control of GH Secretion GH is synthesized and released from the acidophils of the anterior pituitary under the Control of the hypothalmus (Schally §£.fll" 1973). The hypothalmus has been shown to secrete two hormones which either stimulate or suppress GH synthesis in and release from the anterior pituitary. The hypothalmus itself has receptor sites for stimuli from both the nervous and Circulatory systems. Consequently, stimuli from the periphery control the secretion of GH. 16 Relationship Between the Pituitaryiand the Hypothalmms. In a review of hypothalamic regulatory hormones by Schally $3111. (1973), they described the anatomical relationship between the hypothalmus and the anterior pi- tuitary gland. The hypothalmus itself is located in the diencephalon at the base of the brain where it forms the floor and part of the walls of the third ventricle. The median eminence, which is a part of the floor of the third ventricle, Connects the hypothalmus with the anterior pitui- tary gland. Furthermore, the median eminence and the anterior pituitary gland contain a common portal blood system while nerve fibers connect the hypothalmus and the median eminence. Consequently, Chemical substances such as hormones can be transmitted through the nerves of the hypothalmus to the portal blood vessels in the median eminence to the anterior pitui- tary gland. Growth Hormone Releasingiapd InhibitingiHormones. The existence of two hypothalmic GH releasing hormones, GH releasing factor or hormone (GHRH) and somatostatin or CH inhibiting hormone (GHIH) have been known for some time in sheep, cattle, pigs and rats. Schally t al. (1968) and Mittler t 1. (1970) incubated purified GHRH from porcine hypothalmi with rat pituitary cells ig_vitro and discovered that bioassayable GH was re- leased into the medium. Furthermore, Mittler t al. (1970) discovered that GHRH increased the synthesis of GH in rat pituitaries in vitro. The response of immunoreactive GH to purified sheep GHRH in_vivo in rats re- ported by Frohman t al. (1971), paralleled the results obtained ig vitro. Although the purified or synthesized GHRH did not stimulate immunoreactive 17 GH from pituitaries lg vitro, Schally _£‘_l. (1973), isolated another pep- tide from the hypothalmus which stimulated release from rat pituitaries. t al., isolated, purified and sequenced somatosta- In 1973, Brazeau tin from ovine hypothalmi and then synthesized the peptide. When rat pituitaries were incubated with 1.0M or greater concentration of synthe- sized or native somatostatin in_yi££g, radioreactive GH released into the medium was significantly decreased. When Brazeau _£.al, (1974) injected as little as 2.0 ug of somatostatin subcutaneously into rats and stimulated GH release with either sodium pentobaritol or stress, they discovered the increase in plasma levels of GH which occurred in the control was either inhibited or decreased in the treated animals. Martin (1974) and Peracchi t al. (1974) confirmed that somatostatin reduced immunoreactive plasma GH in rats and man, respectively. Furthermore, Peracchi t l. (1974) and Koerker _£._l. (1974) discovered that somatostatin caused a large decrease in immunoreactive insulin in humans and baboons, respectively. In an attempt to discover if the effect of somatostatin on insulin was direct or indirect, Johnson _£__l. (1975) perfused rat pancreata with arginine both with and without somatostatin ig_yiyg and discovered that somatostatin blocked insulin release. Therefore, somatostatin not only has been shown to inhibit GH secretion but also to prevent insulin release from the pan- creas provided it enters the peripheral bloodstream in large enough quan- tities. Peripheral Control of GH Secretion. Many different Chemical compounds from the periphery of the body affect the secretion of GH. Although effects 18 of some compounds such as FFA remain controversial, the effects of GH and glucose on GH secretion are fairly well defined in some mammals although not necessarily in pigs. Other hormones such as insulin appear to exert their effects through glucose rather than directly on the hypothalmus or pituitary itself. Although little work has been done with pigs, researchers using other mammals have proven that exogenous GH inhibited endogenous secretion of GH. When Sawano _£__l. (1967) injected either saline or GH into rats be- fore injecting GHRH, GHRH did not deplete pituitary stores in the GH treated rats but did in control rats. Not only was there a lack of GH depletion in the pituitary gland after GHRH injection, but Sakuma and Knobil (1970) demonstrated that other substances such as Pitressin and insulin which increased plasma GH levels in saline-infused monkeys did not increase GH levels after GH infusion. In addition, Abrams _£‘_l. (1971) discovered that the inhibitory effect of exogenous GH reduced the normal GH response in humans for as long as 12 hr after the cessation of three daily injections of GH for six days. Consequently, the evidence presented above strongly supported the existence of a negative feedback of plasma GH on the hypothalmus in order to inhibit GH secretion. The peripheral substrates such as glucose and insulin have been shown to affect GH release while plasma FFA levels appeared to have no effect on plasma GH. Machlin _£‘_l. (1968a) and Swiatek _£‘al. (1968) reported that plasma GH levels increased in the pig in response to hypoglycemia induced by insulin, a rapid decline in plasma glucose or fasting. Moreover, 19 the increase in CH secretion in response to the stimuli was much lower in pigs than in normal weight humans, but comparable to obese humans. Yet the sluggish response in plasma GH was not due.to obesity since Swiatek _£._l- (1968) discovered the same sluggish response in 4 day old pigs. Consequently, plasma levels of GH are increased by glucose and insulin. The effect of FFA on plasma GH levels appear to be minimal in pigs. Machlin _£._l- (1968a) concluded that FFA were not directly related to GH levels specifically but were inversely related to insulin levels while the animal was being fasted. In a review article, Reichlin (1973), re- ported that FFA in primates do not exert an effect on plasma GH except infused FFA were found to decrease nocturnal plasma GH levels by Blackard _£‘_l. (1971). However, Quabbe _g _l. (1971) showed that a decrease in plasma FFA, induced by nicotinic acid, caused an increase in GH levels in humans within 2 hr with or without hypoglycemia. Furthermore, when FFA returned to normal, the plasma GH levels decreased. Consequently, Quabbe g; _l. (1971) suggested that FFA did not have a direct controlling effect but rather induced other Changes which increased GH levels. When Bfiber _£‘_l. (1971) measured GH levels after plasma FFA were lowered, they dis- covered that GH levels rose immediately and returned to resting levels after plasma FFA reached their lowest level and were elevated, respectively. Consequently, plasma FFA levels appeared to have no effect on GH secretion in pigs but did in man although experiments similar to those in humans have not been performed with pigs. Stress has been reported to increase plasma GH levels in primates (Reichlin, 1973), sheep (Machlin 3; al., 1968b) and pigs (Machlin g; 31., 20 l968a,b). Machlin _E._l. (l968a,b) observed an increase in plasma GH levels in pigs 30 min after either saline or glucose was injected. Fur- thermore, exercise has been shown to elicit GH secretion in man by Hagen t 31. (1972). Therefore, stress by either injection or exercise has been shown to increase plasma GH levels. The Effect of GH on Lipolysis GH was suggested to have lipolytic activity by Greenbaum (1953) when rats, injected daily with GH, showed increased protein and decreased fat stores. Later, Turman and Andrews (1955) and Machlin (l972a,b) dis- covered daily injections of PGH decreased average backfat thickness while increasing the amount of body protein. In order to ascertain whether GH had lipolytic activity, numerous investigators have injected GH 13.3129 and/or incubated adipose tissue with GH ig.yi££g. Although some progress has been made, the effects of GH on lipolysis remain controversial. Effect of GH on Lipolysis IE Vivo. The ig_vivo lipolytic effect of GH obtained from many species has been demonstrated to be biphasic. The first phase has been Characterized by a decrease in FFA followed by an increase in the second phase. In the early studies only lipolysis was observed. Raben and Hollenberg (1959) observed increased plasma FFA 18 hr after partially purified PGH, BGH, simian GH (SGH) or HGH was injected into dogs. In 1964, Winkler t al. noted that the plasma FFA levels were increased in dogs within 1 hr and stabilized between 3 and 6 hr after injection of 21 BGH. By infusing labeled palmitate, they discovered the initial rise was due to an increased rate of FFA release which was paralleled by increased tissue uptake although uptake was not as great as the release since plasma FFA plateaued above control levels. Consequently the rise of plasma FFA in response to GH has been documented for sometime. The biphasic effect of GH on lipolysis has been demonstrated in pigs by Machlin gt _1. (l968a), dogs (Rathgeb gg'al., 1970) and humans (Fine- berg and Merimee, 1974; Berle §£_§l., 1974). When Machlin _£‘§l. (l968a) injected PGH intravenously into fasted 27 kg pigs, a 40 to 60% decrease in plasma FFA was observed within 30 min, followed by a 3 to 4 fold in- crease in plasma FFA Concentration within 60 minutes. The plasma FFA level remained elevated for over 4 hours. Likewise, Vezinhet._5_;l. (1971) demonstrated an insulin-like effect, which was followed by an in- crease in glucose levels and elevated FFA levels which peaked l to 2 hr but returned to control levels 9 hr after PGH administration into hypo- physectomized rabbits. Rathgeb _E‘_l. (1970) reported a decrease in plasma FFA, although not statistically significant, at 30 min, followed by a continual increase through 4 hr after injection of canine (CGH) or BGH into dogs. Likewise Berle _£‘_l. (1974) discovered a significant decrease in plasma FFA and glycerol levels, followed by a significant increase between 90 and 150 min after injection of HGH into humans. These findings were Confirmed by Fineberg and Merimee (1974). Berle _£‘_l. (1974) postulated that GH initially accelerated FFA and glycerol metabo- lism which was later followed by accelerated lipolysis. 22 The lipolytic effect of GH‘ig‘yiyg_has been challenged by Trygstad (1967). After purifying GH by gel chromatography with .OSM tris-hydro- chloride buffer, the purified and unpurified GH and the lipid mobilizing fraction (LMF) were injected subcutaneously into 39 libitum fed rabbits. The partially purified GH caused an increase in plasma FFA levels within 1 hr and FFA remained elevated through 7 hr; but the purified GH did not increase plasma FFA levels at any time during the sampling period. The LMF which was removed during purification also increased plasma FFA levels. Consequently, Trygstad (1967) suggested that the lipolytic activity of GH was due to contamination by other lipolytic hormones from the pituitary gland. Elevated plasma FFA levels obtained after GH administration have been shown to decrease after a daily injection for 7 to 10 days in limited-fed rats (Cheng and Kalant, 1968) and dogs (Winkler £5 31,, 1964; Rathgeb 25. t l. (1964) observed elevated plasma FFA .11., 1970). Although Winkler 4 hr after the first few days of daily GH administration, FFA had returned to control levels after the seventh day. Rathgeb _E‘_l. (1970) confirmed these findings. Cheng and Kalant (1968) observed normal plasma FFA levels after 10 days of GH administration in limited-fed rats even though pro- tein synthesis continued to occur. However, elevated plasma FFA levels Continued to be elevated in ad libitum fed rats. Even though plasma FFA were decreased glycerol remained elevated in dogs after 10 days of daily GH administration according to Winkler t l. (1969) and Rathgeb t 1. (1970). Winkler t al. (1969) suggested that either increased FFA reester- ification and/or oxidation in the adipose tissue accounted for the decrease 23 in plasma FFA. Consequently, GH has been shown to increase lipolysis even though serum FFA were not elevated. Effect of GH on Lipolysis In_Vitro. The controversy which surrounded the lipolytic activity of GH in_yiyg has also been reported for adipose tissue ig_yi££g. In the Classical study by Fain _£‘al. (1965), BGH with and without dexamethasone, was shown to increase lipolysis both in intact adipose tissue or isolated adipocytes from normal, fasted rats after in- cubation for 4 hours. Both:.01 pg/ml and 1.0 pg of BGH/ml of medium with and without dexamethasone, respectively, accelerated lipolysis as measured by FFA release. Since neither dexamethasone nor GH alone activated lipo- lysis significantly, Fain _£__l. (1965) concluded that GH and dexametha- sone act synergistically to accelerate lipolysis. Lipolysis could not be induced in intact adipose tissue incubated for 4 hr with or without dexa- methasone unless glucose was added to the medium; however isolated adipo- cytes did not require glucose for lipolysis. Furthermore, at least 2 hr were required before the lipolytic effects of GH on either adipocytes or tissue were observed because protein synthesis was required before lipoly- sis occurred. In addition to BGH, Fain _£H_l. (1965) reported that .01 ug/ml HGH and SGH and .l ug/ml PGH significantly accelerated lipolysis in isolated rat adipocytes incubated in the presence of dexamethasone for 4 hours. The lipolytic response to BGH has been confirmed by Hamid _£Hal. (1965) in an experiment in which adipose tissue was incubated with 5 “g of BGH. 24 Swislocki _£_al. (1971) purified NIH BGH, which was shown to be heterogenous on polyacrylamide gel electrophoresis, and then incubated .01 Imyml BGH fragments obtained from trypsin digests with adipose tissue from hypophysectomized rats. All of the fractions accelerated lipolysis. However, only one which corresponded to the major band of the NIH BGH preparation on gels induced the typical lipolytic response to GH in yiyg as well as in 31539. Furthermore, results of Wieser t al. (1974) sup- ported the observations of Fain t l. (1965) and Swislocki t l. (1971) that GH stimulated lipolysis. Wieser _£‘_l. (1974) compared the lipolytic effect of several BGH preparations derived from different purification procedures and varying in biological activity as measured by the tibia test, on adipocytes, which were isolated from fed female rats and incubated with dexamethasone and theophylline. As the biological activity of the BGH, which ranged from .93 to 2.3 IU/mg increased, the lipolytic activity of BGH increased. Although contaminants were observed in the gels of SDS- polyacrylamide electrophoretograms, Wieser _§._l. (1974) suggested that the contaminants observed on the SDS gels were not responsible for the accelerated lipolysis produced 4 hr after GH administration. Although purification has been suggested to increase the lipolytic effect of BGH, Trygstad (1967) and Machlin (l972b) have proposed that the lipolytic activity of HGH and PGH, respectively, decrease with purification. When Trygstad (1967) purified HGH and incubated both the partially puri- fied and purified GH with rabbit fat pads, he discovered that 100 H8 of purified GH/1.1 ml were required to elict the same release of FFA into the medium as .1 pg of partially purified GH. Although a faint band 25 remained, the purified GH was shown to be more homogenous by disc gel electrophoresis than the unpurified GH. Furthermore, a fraction, isolated during the purification procedure, was found to release a significant amount of FFA when .1 ug/ml was incubated with fat tissue. Consequently, Trygstad (1967) suggested that the lipolytic activity of GH may be caused by the light band which was still observed after the purification of GH following separation by disc gel electrophoresis. This suggestion of Trygstad (1967) was supported by Machlin (l972a,b) who incubated PGH with adipose tissue from normal, fed rats and rabbits. Prior to purification, commercially available PGH was found by Machlin (l972b) to separate into two major bands by disc gel electrophoresis. Following purification, the biological activity of the PGH increased from .9 to 2.2 USP units/milligram. Commercial, purified and NIH PGH preparations (the latter, purified according to Cheng _£'_l, (1970), had a tibia activity of 1.0 USP unit/mg) were incubated with both rat and rabbit adipose tissue for 4 hours. The amount of glycerol released from rat adipose tissue by NIH and purified PGH was 3% and less than 1%, respectively, of the Commercial PGH. Simi- larly, glycerol release by rabbit adipose tissue was 9% and less than 1%, respectively, of commercial PGH. Since the lipolytic activity of GH de- creased as its biological activity increased, Machlin (1972b) suggested that lipolytic activity was not an "intrinsic part of the PGH molecule and was not necessary for tibia activity." Consequently, until a homogen- ous GH preparation is isolated and purified, the lipolytic activity of GH will continue to be debated. 26 Although.the debate Concerning the lipolytic activity of GH Continues, other investigators using hypophysectomized rats may have provided insights for possible explanations. The response of adipose tissue from hypophy- sectomized rats to lipolytic agents in 21553 has been shown to be decreased with the length of time after hypophysectomy (Schillinger and Gerhards, 1974). When Goodman (1968a) incubated adipose tissue from hypophysectom- ized rats in the presence of GB for 3 hr, it did not increase the basal production of glycerol or FFA. However, when .5 mg/ml theophylline were added to the medium following a 3 hr preincubation with .01 ug/ml GH, gly- cerol production was significantly increased above the theophylline or GH controls. These results were suggested to imply that GH conditions the lipolytic "machinery" of the cell without initiating lipolysis itself. Further evidence was provided by Schillinger and Gerhards (1974) who de- monstrated that 50 Mg of GH and 500 pg of corticosterone injected into hypophysectomized rats per day significantly increased lipolysis in iso- lated adipocytes in_yi££g in response to either .15 ug of epinepherine or .5 ug/ml of ACTH per m1 of incubation medium, whereas neither 500 pg of corticosterone nor 50 pg of GH injected into the hypophysectomized rats induced a lipolytic response. Consequently, the lipolysis observed after a large dose of GH alone may be due to contaminants. However, lipolysis observed after GH and glucocorticoid administration is probably due to the potentiation of the lipolytic machinery by CH and initiation of lipo- lysis by glucocorticoids. Therefore, if GH does possess lipolytic activity, it is probably indirect. Id 27 The Effect of GH on Glucose Metabolism The "insulin-like” effect or decrease in plasma glucose and FFA which was observed within 1 hr following GH administration in pigs by Machlin t l. (1968a), in dogs by Rathgeb t l. (1970), in humans by Fineberg and Merimee (1974) and Berle t al. (1974) has been studied quite extensively in rats by Goodman. Goodman (1965) reported that 30 min after 12 vivo administration of 50 ug or 1 mg/rat BGH, the uptake and incorpora- tion of labeled glucose into fatty acids and CO was accelerated in vitro. 2 However, by 3 1/2 hr after GH administration in 2139, the uptake and in- corporation of labeled glucose by rat adipose tissue was depressed 39 3133. In another experiment, Goodman (1965) discovered that glucose uptake in adipose tissue from rats injected with BGH 3 1/2 hr prior to incubation with GH in XIEEQ did not occur. Yet glucose uptake did occur 24 hr after the ig.yiyg injection under the same conditions. Furthermore, when Goodman (1970b) studied the antilipolytic effect of GH on epinepher- ine-stimulated lipolysis in fat pads from hypophysectomized rats, in yitgg, he discovered that .l ug/ml BGH decreased the elevated glycerol production due to epinepherine. However, when the fat pads were preincu- bated with GH for 3 hr prior to exposure to epinepherine, GH did not decrease epinepherine-stimulated lipolysis. Goldman and Bressler (1967) discovered that the activity of the en- zyme involved in the transport of long Chain fatty acids across the mitochondrial membrane, long Chain acyl CoA-carnitine acyltransferase (LCAT) was increased in hypophysectomized rats by five daily GH 28 administrations 20 hr prior to the time that LCAT was assayed in adipose tissue. Adipose tissues from the same rats, incubated with labeled glu- cose were observed to increase glucose incorporation into FFA and C02. Batchelor and Mahler (1972) using ad libitum fed normal rats, observed a significant increase in incorporation of labeled glucose into C02 and triglycerides 90 min after GH administration only if the lipid content of the adipocytes was being depleted. If the lipid content was not being depleted, no significant increase was observed above controls. Therefore, GH-stimulated lipolysis has been shown to be somewhat dependent on in- creased glucose metabolism. The mechanism of the uptake of glucose from the medium has been suggested to be an increased rate of entry into the cell. When 50 or 100 Ag of BGH were injected in yiyg into hypophysectomized rats 30 min prior to incubation of the adipose tissue with L-arabinose, Goodman (1966) ob- served a significant increase in the intracellular accumulation of L- arabinose. However, the increase in intracellular L-arabinose was not demonstrated 3 hr after in yiyg injection of GH. Consequently, the uptake of glucose in response to GH observed by Goodman (1966) was mediated by the activation of the glucose transport system. The accelerated transport of glucose into the cell has been shown to last no more than 3 hr after GH administration both in yigg_and in 21353. Three hours after GH administration, lipolysis has been detected and the utilization of glucose has been shown to decrease. Goodman (l968b) re- ported that the reduction of glucose uptake by adipose tissue observed 4 hr after GH administration was blocked by cycloheximide, an inhibitor of 29 protein synthesis. Based on the work of Fain _£__l, (1971) and the stu- dies reviewed above, the initial "insulin-like" effect of GH has been shown to be diminished by protein synthesis which in turn stimulated lipolysis. Somatomedin Somatomedin has been shown to have "insulin-like" activity both in ,yi££2_and in_yiyg_by Underwood _£‘§l. (1972). The authors discovered that 21Jsomatomedin/ml inhibited epinepherine-induced lipolysis as much as did 150 ”U of insulin/ml after incubation of rat adipose tissue in yiggg. Furthermore, the slopes of the dose response curve for insulin and somatomedin gs. epinepherine-stimulated glycerol release i2_yi££g_ were almost identical. In addition, 50 U of somatomedin injected in_yiyg 4 hr prior to in 31553 incubation of adipose tissue from hypophysectom- ized rausdecreased glycerol release. Although the actiOn of somatomedin resembled the early action of GH on lipolysis, Underwood _£‘_l. (1972) doubted that the early "insulin-like" effect of GH was attributable to somatomedin since somatomedin required 3 hr for plasma levels to increase. Furthermore, they suggested that the lipolytic activity of GH might be due to a lipotrophic factor such as that reported by Trystad (1967). The Relationship Between GH, Glucose and Lipolysis Different polypeptides of the GH molecule have been shown to be re- sponsible for the lipolytic and "insulin-like" activity of GH. 30 Bornstein _£._l- (1968) discovered that IN-G or somantin and AC-G or cataglykin, fragments of ovine GH (OGH), inhibited and stimulated, respectively, the synthesis of FFA from acetate in rat liver slices. After further experiments, Bornstein (1972) concluded that somantin in- hibited glucose uptake and oxidation and accelerated lipolysis by inhibi- ting certain enzymes in the glycolytic pathway. Cataglykin had "insulin- like" activity and reversed somantin effects by activating the same glycolytic enzymes which somantin inactivated. According to Bornstein's (1972) hypothesis, GH is secreted from the pituitary and is transported to the tissue which hydrolizes the GH molecule to release the appropriate peptide depending on the glucose levels. Somantin and cataglykin are released when glucose levels are low and high, respectively. Although somantin has been isolated from the media which contained either muscle, adipose tissue or liver slices, the hypothesis still does not explain the "insulin-like" effect of GH in fasted animals or on the adipose tissue both 13 vivo and in vitro. Serum Levels of GH in Pigs Many studies have been undertaken to determine the serum levels of GH in pigs at different ages. Swiatek _£‘_l. (1968) reported that perinatal pigs had plasma GH levels of 81 ng/ml which decreased to 17 ng/ml follow- ing feeding. When 1 and 3 week old pigs were fasted for 72 hr, plasma GH values remained at approximately 20 ng/ml and did not increase signifi- cantly due to fasting. As pigs grow older, Siers and Hazel (1970) reported a decline in plasma GH values from 5.41 to 3.33 to 2.82 ng/ml for 17.2, 51.6 and 92.6 kg pigs, respectively. Machlin t l. (l968a), measured 31 plasma GH in overnight fasted pigs and reported values of 10.7 and 5.9 ng/ml for pigs weighing 46 and 75 kg, respectively. Therefore, Siers and Hazel (1970) concluded that GH decreased with age. The plasma GH levels of pigs weighing 70 to 90 kg have been reported to have considerable diurnal variation. The plasma GH values for 67 to 90 kg pigs have been reported to be 3.68 ng/ml with a range of 1.82 to 6.90 ng/ml by Siers and Trenkle (1973), 9.44 to 13.10 ng/ml by Topel 2E El- (1973) and 5.9 ng/ml in fasted pigs by Machlin t 1. (l968a), approxi- mately 3.0 ng/ml by Bidner _£_§l, (1973). Furthermore, Siers and Trenkle (1973), after taking blood samples every half hour from 9:00 am to 3:00 pm from ad libitum fed 67 to 84 kg crossbred gilts, reported that GH values doubled or tripled around 9:30 and 10 am and again around 1:30 to 2:00 pm. Consequently, Siers and Trenkle (1973) suggested that administration of exogenous GH which does not increase the average daily plasma level by more than 200 to 300% would remain within normal physiological limits. However, exogenous GH can increase GH levels dramatically and in pro- portion to the amount injected. Machlin _£H_l. (1968a) reported that plasma GH levels decreased from 2000 to 900 ng/ml and 200 to 100 ng/ml in 50 min following injection of 30.0 and 3.0 mg of PGH, respectively, into 27 kg pigs which had been fasted overnight. Furthermore, based on the rate of decrease in plasma GH levels, the half-life was calculated to be between 20 and 30 minutes. However, after infusing either 30.3 and 60.6 ng/min/kg GH into 15 week old pigs, Althen and Gerrits (1974) re- ported the half-life of GH to be approximately 12.0 min in 90 kg pigs. 32 Plasma FFA Levels in Pigs Plasma FFA levels in swine after feeding and fasting have been re- ported by a number of investigators. In neonatal pigs, Swiatek _£._l. (1968) reported plasma FFA levels of 320 qu/l (microequivalents/liter) in the fed animal which rose to 640 qu/l in the fasted pig. In 20 to 24 kg barrows, FFA levels rose from 150 qu/l to 1000 qu/l during fasting (Machlin 2; al., l968a). Similarly Grigsby g£_al, (1972) reported 314 qu/l and 607 qu/l of FFA in the 81 kg fed and fasted pig, respectively. However, Siers and Trenkle (1973) observed 134 qu/l which ranged from 70 to 250 qu/l in fed 67 to 84 kg gilts. Furthermore, the FFA values fluc- tuated from about 50 qu/l to 250 qu/l over a 7 hr period within the same pig - Insulin In a review article, Fritz (1971) described insulin as an anabolic hormone since it had been shown to increase lipid, protein and glycogen synthesis. Along with these anabolic processes, insulin has also been shown to reduce catabolic processes such as lipolysis, gluconeogenesis and ketogenesis. More specifically, insulin has been reported to be necessary for the transport of glucose and amino acids into cells, for the conversion of inactive glycogen synthetase to active glycogen synthe- tase, and for RNA synthesis (Friden and Lipner, 1971). Furthermore, by inducing the influx of glucose into the adipocyte and by activating certain glycolytic enzymes, insulin decreases FFA and glycerol release. Consequently insulin has a wide variety of actions in mammalian species. 33 Synthesis and Release from the Pancreas Although some species differences in the amino acid sequence have been reported, insulin is composed of two fragments A and B, which con- tain 21 and 30 amino acid residues, respectively, connected by disulfide bridges (Friden and Lipner, 1971). In a review article, Lacy (1974) presented the current theory about insulin synthesis. Elevated blood glucose stimulates the synthesis of proinsulin in the endoplasmic reticu- lum of the islets of Langerhans. Proinsulin, which is the A and B Chains connected by a "C" Chain of amino acids, is conveyed to the Golgi complex where the "C" Chain, is removed. The A and B Chains (insulin) are sur- rounded by a smooth membrane. The smooth membrane encased granules (beta granules) are pinched off from the Golgi sacs. The beta granules are stored between the microtubules which trans- locate them to the plasma membrane upon glucose stimulation. Grodsky g; .11. (1974) have observed that newly synthesized insulin is not released immediately but stored until the next glucose stimulation. The release of insulin into the bloodstream has been established to be initiated by hyperglycemia, the most potent stimulant, gastrointestinal hormones and glucagon. The release of insulin in response to high blood glucose levels has been shown to be biphasic by investigators both in 21559 (Grodsky, 1972) and in_yiyg (Porte and Pupo, 1969). When isolated islets of Langerhans were perfused with 1 mg/ml of glucose in an in yitrg system, Grodsky (1972) observed a sharp spike in plasma insulin levels within the first 5 min but a second increase was 34 not observed. When 1.5 to 5.0 mg/ml of glucose were perfused through the system, a rapid increase followed by a rapid decrease in medium insulin was observed within 5 minutes. Fifteen to 20_min after glucose adminis- tration, insulin levels were elevated for another 30 to 45 minutes. Furthermore, Grodsky (1972) demonstrated that insulin levels con- tinued to spike even though insulin had been released within 5 min.when higher levels of glucose were perfused through the system. These obser- vations prompted Grodsky (1972) to postulate that insulin was stored in packets in the islets of Langerhans and each packet released insulin when its threshold for glucose concentration had been reached. Furthermore, Porte and Pupa (1969) demonstrated the same biphasic response in humans in_yiyg after a prolonged infusion of 300 mg of glucose/min followed by an injection of 5 g of glucose. A sharp rise and fall in plasma insulin levels occurred within the first 2 to 5 min followed by another rise, although not as great as the initial spike. Consequently, the biphasic release of insulin in response to hyperglycemia is directly dependent on the plasma or medium glucose Concentration. Although the biphasic release of insulin in response to hyperglycemia has not been reported in pigs, many investigators have demonstrated the release of insulin in response to high plasma glucose levels. When Mach- 1in _£‘_l. (l968a) injected .75 g glucose/kg of body weight into fasted 40 to 50 kg pigs, plasma insulin concentration increased 5- to 6-fold initially and then dropped to 3-fold after 15 min where it remained for 2 hr postinjection. Likewise, after infusing 100 ml/kg hr of glucose for 1 hr, or after an oral glucose load, a 3- to 5-fold increase in plasma 35 insulin was noted at approximately the same time plasma glucose peaked and insulin remained elevated for at least 1.5 hr after blood glucose peaked. Furthermore, Machlin _£‘_l. (l968a) reported that the insulin response to hyperglycemia in pigs was much lower than in normal humans. The reason the biphasic elevation of plasma insulin was not observed may have been due to the time sequence of sampling since samples were removed every 15 min during the first hour and every half hour for the subsequent 2.5 hours. Siers and Trenkle (1973) designed an experiment whereby blood samples from 67 to 84 kg crossbred gilts, which were housed in separate stalls, and fed ad libitum were collected every half hour. The investigators discovered that plasma insulin increased or decreased suddenly and without "any observable environmental force". Since the correlation Coefficient between glucose and insulin was found to be .54, Siers and Trenkle (1973) concluded that plasma glucose and insulin "fluctuated in the same direction at the same time". Therefore, pigs respond to elevated plasma glucose by secreting insulin, although the pattern of the response has not been described in detail. The Effect of OH on Insulin Secretion GH has been shown to stimulate insulin secretion in dogs (Altszuler _£‘al., 1968) and in rats (Van Lan g; 31., 1974) but not in pigs (Machlin .££"§1., 1968a). Altszuler 2E.§l- (1968) reported a significant increase in plasma insulin concentration in fasted dogs following 3 to 8 days of 36 injection of 1 mg/kg/day of BGH. Furthermore, the plasma insulin concen- tration tended to be elevated after one day. Van Lan _£._l- (1974) using hypophysectomized rats, also found that the decreased plasma insulin values due to hypophysectomy returned to that of normal fasted rats after BGH had been administered every other day for 3 weeks. However, when Machlin ‘_£._l. (1968a) injected either .11 or 1.11 mg of PGH/kg of body weight into fasted 27 kg pigs, they observed a 62% increase (nonsignificant) in plasma insulin levels 4 hr after injection in the two pigs for which the 4 hr-values were presented. Consequently, GH whether endogenous or exo- genous increased insulin secretion. The Effect of Insulin on Glucose Transport Insulin has been proven to increase the transport of glucose into many different cells including adipocytes both in_yi£rg, Crofford and Renold (1965a) incubated epidymal fat pads with glucosewith and without insulin and found that insulin was responsible for the transport of glu- cose into the cells. Furthermore, Crofford and Renold (1965b) demonstrated that insulin stimulated the transport of some sugars, primarily D-glucose and to a lesser extent mannose and L-glucose into rat adipocytes. The decrease in plasma glucose levels in response to insulin has been demonstrated in many species including the pig. Machlin _£._l. l968a) obtained a decrease in blood glucose concentration from 91 mg/100 ml to 36 mg/100 ml 30 min after the intravenous injection of .3 U of insulin/kg of body weight into fasted 45 to 75 kg pigs. Although the 37 plasma glucose levels began to increase 1 hr after infusion, the values remained below control levels for at least 3 hours. Conversely, when glucose was either fed or injected intravenously into pigs, the elevated plasma insulin concentration caused a decrease in plasma glucose values over a 3 hr period. Machlin _£._l° (l968a) reported that both the rate of insulin secretion and the rate of glucose Clearance in response to either an oral or intravenous glucose load was less than that reported for normal human subjects. Furthermore, the rate of glucose clearance observed in pigs corresponded to the rate observed in humans with diabetes mellitus. Consequently, insulin has been shown to decrease high blood glucose levels in pigs. Effect of Insulin on Lipolysis in Vivo As discussed earlier, insulin has been shown to inhibit lipolysis by inactivating adenyl cyclase and activating phosphodiesterase which de- creased CAMP levels in yiggg. The antilipolytic effect also has been shown in 3129 in some species of mammals. Hollenberg and Raben (1959) showed that GH-elevated FFA levels Could be decreased by the infusion of insulin and glucose in dogs. Similarly, Cheng and Kalant (1970) discovered that .015 U of insulin/kg of body weight reduced plasma FFA levels to 45% of the control level in normal, fasted humans within 30 minutes. Abrams gtnal. (1971) obtained similar results following .1 U of insulin/kg body weight injection into normal humans. In these studies, the blood glucose levels were also depressed which indicated that glucose was transported 38 into the cell. Once in the cell, glucose provided the substrate for a- glycerol phosphate necessary for the reesterification of FFA. Consequently, the FFA would not be released but reesterified (Goodman, 1970a). Furthermore, a number of investigators have found that endogenous plasma insulin levels were inversely related to plasma FFA. During a 3 day fast, the changes in plasma FFA levels were inversely related to the Change in insulin in 20 to 24 kg pigs (Machlin g£_§l,, l968a). In 3 week old pigs, Swiatek 2; al- (1968) noted the same inverse relationship be- tween plasma insulin and FFA levels. However, Siers and Trenkle (1973) using nglibitum fed gilts which were submitted to little environmental stress did not obtain any relationship between plasma FFA and insulin concentration. Consequently, insulin apparently controlled FFA levels during fasting when blood glucose levels are decreasing, but insulin does not exert a fine control over FFA levels. Serum Insulin Levels in Swine Serum insulin levels in swine fluctuate with the fed-state of the pig as it does in other species. In 64 to 87 kg crossbred gilts which were §g_ libitum fed and bled via a catheter every half-hour for 6 hr, Siers and Trenkle (1973) discovered that the average insulin value for all pigs and all time periods was .60 ng/ml with a range of .25 to 1.07 ng/milliters. The insulin levels in each pig were "subject to large and sudden increases and decreases” which were not attributable to any environmental stimulus. Swiatek t l. (1968) reported plasma insulin values ranging 39 between 10 and 30 uU/ml in the young, fed pig to less than 4 uU/ml in the fasted pigs. In 84 kg pigs, Grigsby §£_§l, (1972) reported an average of 6 uU/ml of insulin during fasting and sham.feeding which rose to 104 uU/ml after feeding. Similarly, Machlin _£__l, (l968a) reported an average serum insulin value of 5.8 uU/ml in 20 to 24 kg fasted pigs. Other in- vestigators have reported a wide range of values from 26 to 27 uU/ml by Romsos t al. (1971) to 132 uU/ml by Wood a a1; (1971). Blood Glucose Levels in Swine Like insulin, blood glucose levels do not appear to exhibit any diur- nal variation but do respond to feeding and fasting. The following plasma glucose levels have been reported in §g_libitum fed pigs: 90 mg/100 ml by Romsos t 1. (1971), 87 mg/100 ml in 67 to 84 kg pigs by Siers and Trenkle (1973), Topel t al. (1973) and Grigsby EENQL- (1972). (Blood glucose levels of pigs in the fasted state have been reported to be lower than the values of pigs in the fed state as one would expect. t al. (1971) reported that fasted 84 kg Grigsby gghal. (1972) and Romsos pigs had plasma glucose values around 70 mg/100 ml, while Machlin g§_gl, (l968a) and Swiatek t El: (1968) reported glucose levels of 85 mg/100 ml and 80 mg/100 ml, respectively, from younger pigs. Therefore, reported average plasma glucose values were about 15 mg/100 ml lower among fasted compared to fed pigs. 40 The Effect of Insulin and Growth Hormone Interaction on Blood Glucose and Lipolysis Effects of the Fed State Since some of the early experiments, investigators have shown that the lipolytic response of GH could be blocked with either a meal, glucose load or insulin. Goodman and Knobil (1959) studied the effect of GH on lipolysis in monkeys which were either fasted, fed once per day or ad libitum fed. GH injected into fasted monkeys increased plasma FFA 1.5 fold over the fasted control at 4 and 8 hours. However, 30 to 60 times the dose of SGH necessary to increase plasma FFA in fasted monkeys had no effect on lipolysis in 3Q libitum fed animals. Furthermore, the monkeys which were fed just once per day did not respond to GH when it was injected immediately after feeding but did respond when GH administration followed an overnight fast. Raben and Hollenberg (1959) discovered that insulin and glucose, infused after GH administration, decreased the elevated plasma FFA levels in dogs. Trueheart and Herrera (1971) showed the same phenomena in rats and offered a partial explanation. When fed and fasted hypophysectomized rats were injected with GH iguyiyg the incorporation of labeled glucose into CO and total lipids into the adipose tissue which 2 was incubated ig vitro with insulin was significantly less among fasted than fed rats. Furthermore, the incorporation of labeled glucose into CO2 and FFA was not statistically different between the fed GH injected and the saline injected hypophysectomized rats. Since insulin appears to 41 block the lipolytic activity of GH; fasting which has been shown to be decreased in plasma insulin levels in pigs (Machlin g2 31., l968a) as well as other mammals appeared necessary for the lipolytic effect of GH to be observed. During prolonged fasting, all food may be excreted from the digestive tract but at different rates depending on the species. The rate of food passage through a pig has been shown to be very slow by a number of in- vestigators. When Castle and Castle (1956) fed pigs grain marked with a dye, they discovered that the mean retention time of the grain, which was mixed with water to form a mash, was 33.1 and 35.2 hr for the morning and night feeding, respectively. In a later experiment, Castle and Castle (1957) fed the recommended daily allowance based on the pig's weight, and either .5, 1.5 or 2.0 times the RDA and observed a mean retention time of 28.7, 32.3, 25.8 and 25.9 hr, respectively. The investigators concluded that as the amount of feed increased, the retention time decreased. Fur- thermore, O'Hea and Leveille (1969), after adapting 87 kg pigs to meal feeding, reported that food residue remained in the digestive tract 18 hr after the last meal. Based on this observation, they suggested that the pigs were not in the postabsorptive state until 18 hr after feeding. Inhibition of Insulin Action by GH After either fasting in which plasma levels of GH were elevated or Chronic GH administration, the effect of insulin on plasma glucose has been reported to be reduced. Yalow t l. (1969) showed that humans whose 42 plasma GH levels were elevated after the first glucose tolerance test secreted more insulin but blood glucose levels remained elevated after the second glucose tolerance test. However, if the GH response to the first glucose test was abolished, glucose tolerance remained the same. Lostroh (1974) confirmed the observation of Yalow _£‘_l. (1969) in hypo- physectomized rats which were injected with OGH or saline 9 days prior to insulin injection. The GH treated rats maintained higher blood glucose levels than the saline treated rats. Consequently, GH appears to decrease insulin sensitivity which might serve as a homeostatic mechanism to main- tain energy during fasting. Other Pituitary Lipolytic Hormones and Factors Besides GH, the pituitary gland secretes other lipolytic hormones, notably MSH, from the intermediate lobes of the pituitary TSH, ACTH, anti- insulin peptide and lipotrophic factors. Some of these hormones, namely TSH, MSH and ACTH have been listed as possible contaminants of GH by Hollenberg g£_§l, (1961) and Freinkel (1961). Anti-insulin peptide has recently been isolated from porcine pituitary glands and has been shown to have about the same molecular weight as GH although the isoelectric point is much more acidic than that of GH. ACTH ACTH has been shown to stimulate the growth of the zona fasciculata and zona reticularis of the adrenal Cortex and the synthesis and secretion 43 of glucocorticoids from both of these zones, besides the activation of lipolysis (Friden and Lipner, 1971). The lipolytic activity of ACTH both ig_yiyg and in yi££g_has been demonstrated in a number of species. Furthermore, the lipolytic activity of ACTH has been demonstrated when the animals were in both a fed or fasted state. When Hollenberg _E _l. (1961) compared the amount of ACTH necessary to stimulate lipolysis in adipose tissue from fed and fasted rats in_yi£rg, they discovered that .001 ug/ml and .003 ug/ml of porcine ACTH, respectively, significantly increased the release of FFA into the t al. (1972) noted that isolated adipocytes medium. Furthermore, Exton from adrenalectomized rats released only 66% of the glycerol of those from normal rats incubated under the same conditions. When ACTH was added to the medium, glycerol release was elevated in adipocytes from both the normal and adrenalectomized rats above their respective control levels but not to the same absolute amount. Since the lipolytic response to cAMP was reduced in adipocytes from adrenalectomized rats, Exton _E.§l. (1972) suggested that the glucocorticoids have a permissive role in lipo- lysis. However, ACTH appeared to act directly on lipolysis in_yi££g. The lipolytic response to ACTH in both adrenalectomized and normal rats has also been shown in 3133. Hollenberg _£‘_l. (1961) injected be- tween .1 and 1.0 mg of porcine ACTH into adrenalectomized rats and observed a 2 fold increase in plasma FFA 40 min after injection. In addition, when adipose tissue from the same rats were incubated for 3 hr an increase of 8 “moles/g of FFA was reported for the treated rats above the untreated controls. Similarly Spriovski t l. (1975) discovered that .5 pg of ACTH 44 (.052 uU) injected into normal and adrenalectomized rats increased gly- cerol release 4 fold within the first 30 min after injection. However, plasma FFA were depressed since only 17% was released by the adrenalec- tomized rats, compared to 84% by the normal rat. Since adrenalectomy caused lower circulating levels of glucocorticoids, ACTH must stimulate lipolysis directly while glucocorticoids inhibit glucose utilization as well as stimulate cAMP activity (Exton g£_gl,, 1972) in adipose tissue. Not only has ACTH caused lipolysis in rats, it also has a similar effect in rabbits. When Romans gghal, (1974) infused rabbits with 1.5 m U of ACTH/min/kg of body weight, they observed a 6 to 18 fold increase in plasma FFA after 1 hr which gradually declined even though infusion con- tinued. Therefore ACTH directly stimulated lipolysis in both rats and rabbits. TSH TSH has been shown to increase the biosynthesis and release of thy- roid hormone from the thyroid gland and induce the release of FFA from adipose tissue (Friden and Lipner, 1971). The lipolytic activity of TSH has been shown in yiggg.in a number of species. When fat pads from ii libitum fed rats were incubated for 1.5 and 3 hr with .25 to .5 U of TSH/m1, an increase in FFA released into the medium was observed by Freinkel (1961). Later Rudman §£.§l, (1963) observed that 1.0, .l and 10 pg TSH/m1 stimulated lipolysis in adipose tissue from guinea pigs, rats and dogs, respectively, but not in adipose tissue from rabbits, hamsters and one pig i§_vitro. After incubating 45 bovine TSH with isolated rat adipocytes inhyiggg_for 4 hr, Fain _£“_1, (1965) found that 280 uU/ml of incubation medium stimulated lipolysis with or without dexamethasone demonstrating that glucocorticoids were not required for lipolysis to occur. Furthermore, the authors concluded that TSH was not present in large enough quantities to account for the lipolytic activity of GH since more BGH containing 400 uU/ml of TSH was necessary to stimulate lipolysis than one containing 4 uU/ml of TSH. Therefore, TSH does possess lipolytic activity although it does not appear to be a contaminant of GH. MSH MSH has been isolated in two forms a and B from the intermediate lobe of the pituitary gland. Alpha and B-MSH have been shown to stimulate the synthesis and distribution of melanin, which is the skin pigment. In addition, MSH has also been shown to stimulate lipolysis in adipose tissue in yiggg,(Friden and Lipner, 1971). In 1963, Rudman _£‘_1. (1963) demon- strated the lipolytic activity of a- and B-MSH in yiggg in adipose tissue from rabbits, guinea pigs, and dogs. However, since the lipolytic activity of MSH has not been studied in vivo, the true physiological effect of MSH has been questioned by Fain (1970a) and Friden and Lipner (1971). Anti-Insulin Peptide Recently an anti-insulin peptide (AIP) with an isoelectric point of 4.1 and a molecular weight of 22,000 (Louis, unpublished data) has been 46 isolated from bovine, porcine, ovine and human anterior pituitaries under acidic conditions (Louis E; al., 1966; Louis and Conn, 1968). Since AIP was first discovered in patients with lipotrophic diabetes, its actions have been reported to mobilize FFA from fat depots while causing diabetes even though plasma insulin levels were elevated (Louis and Conn, 1968). AIP has been shown to be a less potent lipolytic agent than GH. In the early work, Tutwiler and Chiefet (1973) noted increased lipolysis when rat epididymal fat pads were incubated with .1 to 10 ug/ml AIP in the presence or absence of glucose. Furthermore, cycloheximide, added to the medium at the same time as AIP inhibited lipolysis. Wieser _£ _1. (1974) discovered that AIP caused a slight release of glycerol when 2 ug/ml were incubated ingi££2_with adipocytes from fed rats. Cycloheximide blocked glycerol release completely. When Wieser _£__1, (1974) incubated rat adipocytes with GH under the same conditions, they found that .01 ug/ml of BGH caused a significant increase in lipolysis which cyclohexi- mide also blocked. Since GH and AIP respond to cycloheximide in the same manner and since more AIP was required to stimulate lipolysis, Wieser g5 ‘11. (1974) suggested that AIP might contain 2 to 4% GH. The lipolytic effect of AIP has been studied in 2132 also. Tutwiler (1974a) discovered that AIP decreased fasting blood glucose levels while increasing FFA 6 hr after injecting 4 mg/kg into fasted rats. In another study, Tutwiler (1974b) reported that FFA were significantly elevated 3 hr after a glu- cose tolerance test was administered 6 hr after AIP administration to fasted rats. The plasma FFA concentration after a glucose tolerance test 47 6 hr following a saline injection served as a control. Despite the resem- blance of the lipolytic activity of AIP to GH, Tutwiler (1974a,b) reported only .048 IU/mg of GH activity when the rate of gain test was used. Con- sequently GH and AIP, have similar effects on FFA mobilization in 2133 and $3 xitgg which Wieser t l. (1974) suggested might be due to GH contam- ination of AIP. Lipotrophic Factors A series of lipotrophic factors have been isolated from the pituitary gland. One of the first such factors was the LMF which Trygstad (1967) separated from HGH by column chromatography or isolated directly from human pituitary glands. The IMF increased plasma FFA, which peaked after 2 hr, in ad libitum fed rabbits ig_yiyg. The rabbit fat pads showed a significant increase in lipolysis after 3 hr incubation with either .1 pg or 1.0 pg of LMF per 1.1 ml of medium in yigrg. In addition, Schleyer 23 a1, (1974) have isolated two lipotrophic hormones, P-LFIe and P-LFIId which were relatively free of ACTH contamination as measured by the biolo- gical assay for ACTH or in the radioimmunoassay. Since the lipolytic activity of P-LFIe and P-LFIId in rat adipose tissue ig_yigg were compar- able to ACTH but were not significantly contaminated with ACTH, Schleyer t 1. (1974) suggested that P-LFIe and P-LFIId were lipolytic hormones. MATERIALS AND METHODS In order to ascertain the acute lipolytic activity of two PGH pre- parations of differing biological activities both 13 yiggg and in yiyg, this study was divided into three experiments. In the first experiment, the tibia test was performed to determine the biological activities of both purified (PPGH) and commercially prepared PGH (CPGH). In the second experiment of the study, saline, CPGH, or one of two levels of PPGH were incubated with porcine adipose tissue ig_yi££g. In experiment 3, pigs were infused with either saline, CPGH, or one of two levels of PPGH. The lower level of PPGH was comparable to the biological activity of CPGH while the higher level was equal in weight to the CPGH administered both in vitro and in vivo. Experiment 1 - Tibia Test The biological activity of CPGH (Somatotropin, STH, Growth Hormone Lot 3785, Nutritional Biochemicals Inc., Cleveland, Ohio) and PPGH (lot 614276, supplied courtesy of Dr. L. J. Machlin, St. Louis, Missouri) was determined by the tibia test developed by Greenspan _£‘_l. (1949) as modified by Papkoff and Li (1962). The biological activity of each was determined in two separate trials. In the first trial, the standard, NIH PGH, lot P52CP (supplied courtesy of Dr. L. E. Reichert of the National Institute of Arthritis and Metabolic Diseases) had a biological activity of 1.47 IU/milligram. PPGH, assayed in the first trial, served as the standard for the second trial. 48 49 In both the trials, Long-Evans rats, hypophysectomized between 26 and 28 days of age (Charles River Laboratory, Wilmington, MA), were weighed on three consecutive days after arrival to obtain an initial weight and reweighed two weeks later. Any rat gaining more than 7 g or less than 4 g was eliminated from the study. In the first trial, eight of the remaining rats were randomly assigned to be injected with saline and four rats were assigned to each group which received 29 pg of PPGH/rat, 58 pg of PPGH/rat, 29 ug/rat of NIH PGH or 58 pg/rat of NIH PGH. The hormones were dissolved in isotonic saline and .5 m1 of the solution were injected twice per day over a four day period beginning the day following the final weighing. In the second trial, five rats were assigned to the saline group and six rats were assigned to each of four groups. These groups received either 25 pg of CPGH/rat, 50 Mg of CPGH/rat, 25 pg of PPGH/rat or 50 pg of PPGH/rat. Each rat was injected with .5 m1 of saline or CH dissolved in saline twice per day over a four day period beginning two weeks after the final weighing. Twenty-four hours following the last injection, the rats from both trials were killed and both tibiae were removed, split and fixed accord- ing to Papkoff and Li (1962). The tibiae were split at the proximal end in the midsaggital plane, washed in water for 10 min and transferred to acetone for 6 minutes. Following the acetone wash, the tibiae were re- washed in water for 3 min and then fixed in 2% silver nitrate for 2 min- utes° Following fixation, the tibiae were placed in water under a strong 50 light until the calcified tissues turned brown. The tibiae were stored in 70% ethanol until the epiphyseal plate width was measured. The tibia widths were measured with an eyepiece micrometer with an A.O. Smith dissecting microscope. Prior to measurement, the eyepiece micrometer was calibrated against a stage micrometer. Eight width mea- surements of the epiphyseal cartilage of each tibia were recorded and then averaged. The tibiae widths from each treatment group were averaged and the activity ascertained by the method of Papkoff and Li (1962). Experiment 2 -‘ln Vitro Trials Adipose Tissue Biopsy According to the procedure of O'Hea and Leveille (1969), adipose tissue was removed from the subcutaneous fat layer in the shoulder region of unanesthetized pigs weighing more than 60 kilograms. The hair was clipped with a pair of scissors and an incision, varying in length from 2.5 to 7.5 cm was made in the skin with a razor blade. A rectangular section of subcutaneous alipose tissue was surgically removed and placed in .9% saline at 37 C. The tissue was returned to the laboratory imme- diately after excision. Tissue Preparation in the Laboratory The tissue was prepared for incubation according to O'Hea and Leveille (1969). Briefly, slices, weighing approximately 90 to 150 mg, were made with a Stadie-Riggs hand microtome and weighed on a Cahn Electrobalance. 51 Then, the tissue was placed in 25 m1 flasks containing the appropriate hormone and 3 m1 of Krebs Ringer bicarbonate buffer (1/2 Ca concentration) pH 7.4 (DeLuca, 1972, Appendix I.A.l) containing 4% FFA poor bovine serum albumin (BSA, Sigma Chemical Company) and 1 mM dextrose. The flasks were gassed with 95% 02:5% CO2 atmosphere, and were agi- tated (90 cycles/min) in a metabolic shaker (Dubnoff Instrument Co.) at 37 C. Three flasks containing adipose tissue were incubated for each treatment and adipose tissue samples from.the same pig were exposed to all treatments. The medium was changed every hour after the second hour for 5 addi- tional hours. The medium was changed by removing the flask from the shaker and transferring the tissue to the new flask containing fresh medium. After the tissue was removed, the medium was divided into two parts, frozen and stored at -20 C. Preliminary Trials In order to ascertain the amount of CPGH necessary to obtain a lipo- lytic response, .06 ml distilled deionized water (DDHZO) containing 0, 30, 60, 90, 120, 150, 180, 210, 240, 270 or 300 pg of CPGH were added to the flasks which were incubated with adipose tissue from an ad libitum fed pig as described above. Although there was little difference in the amount of FFA and glycerol released between 60 and 270 pg of CPGH, 150 pg of CPGH elicited the great- est amount of glycerol and was used for subsequent experiments. 52 Adipose tissue was removed from three fasted pigs, incubated with either .05 m1 of DDH20 or 150 pg of CPGH dissolved in 50 p1 of DDHZO. The procedure used for excising and incubating the adipose tissue was the same as that described above. The amount of both glycerol and FFA released into the medium peaked between 4 and 6 hr and began to decline during the seventh hour. Thus, the length of time the tissue was incubated with GH was chosen to be 7 hours. 13 Vitro Trial with PPGH and CPGH Adipose tissue was removed from three ad libitum fed Hampshire x Yorkshire crossbred barrows weighing between 72.0 and 74.0 kg in the first trial. Three Yorkshire x Hampshire crossbred barrows weighing between 69 and 77 kg 17 days prior to experimentation were included in the second trial. These pigs were adapted to meal feeding as described in experi- ment 3. They were biopsied between 13 and 14 hr following the removal of feed. The adipose tissue from both groups of pigs was excised and sliced as described above. The tissue was incubated with either DDHZO, 10 pg/ml PPGH, 50 pg/ml PPGH or 50 pg/ml CPGH. Again, the medium was changed every hour after the second hour for 7 hr and transferred to disposable test tubes and stored as described above. Glycerol Assay Glycerol was assayed according to the enzymatic method of Wieland (1963). A .2 ml aliquot of the sample, blank (KRB) or standards (ranging 53 from .05 to .5 pmoles/ml) were placed into 12 x 75 mm disposable culture tubes. After pipetting the samples, .05M.ATP and .02M diphosopyridine nucleotide (NAD)(Sigma Chemical Co.) were dissolved in DDHZO and added to the hydrazine buffer containing 1M hydrazine, .02M glycine and .002M 2+ . Mg , pH 9.8 (Appendix I.B.l). One and four-tenths ml of a-glycerol phosphate dehydrogenase (Sigma Chemical Co.) diluted with .6 ml of DDH 0 followed by the addition of 2 2.5 mg (.450 m1) of glycerol kinase (Sigma Chemical Co.) diluted to 2.0 ml with DDHZO, were added to the hydrazine buffer containing NAD and ATP (Appendix I.B.2). Within 10 min after addition of glycerol kinase, 1.6 ml of buffer were added to the sample, mixed gently, and incubated at 25 C for 50 minutes. The absorbance of NADH was read at 340 nm on a Gilford Micro-Sample Spectrophotometer 300N and recorded on a Data Lister, Model 4008. The amount of glycerol in the samples were determined by the equation of Weiland (1963). Free Fatty Acid Assay Preliminary Experiments. The FFA assay was done according to Dun- combe (1963) as modified by Itaya and U1 (1965). Five ml of Cu-reagent (Appendix I.C.l) were added to 16 x 125 mm screw cap test tubes followed by 5 m1 of redistilled chloroform. A .5 ml aliquot of sample, standards or the blank were added to the test tubes. The test tubes were capped and shaken on a metabolic shaker for 30 min followed by centrifugation at 1600 rpm. The copper layer was aspirated and the chloroform layer was filtered through No. 1 Whatman filter paper. After filtration, .5 m1 54 of .1% sodium diethyldithiocarbamic acid, dissolved in redistilled n- butanol, were added to the chloroform filtrate, mixed and read immediately at 440 nanometers. The standard curve was drawn from all values of the standards run with each experiment. The FFA assay of experiment 2 followed basically the same procedure as that described above, but several modifications were incorporated. The Cu-reagent was modified as described in Appendix I.C.2. In addition, a 2 ml aliquot of the chloroform layer was mixed with .5 ml of the .1% sodium diethyldithiocarbamic acid solution and read immediately at 440 nm on a Beckman DU Spectrophotometer (Model 2400). Sample FFA values were read from the standard curve which was determined each day. In addition, all glassware was acid washed prior to use. Eyeriment 3 Experimental Animals Four Hampshire x Yorkshire crossbred barrows from the MSU swine herd and weighing between 69 to 77 kg were adapted to meal feeding and metabo- lism cages at least three days prior to catheterization and 8 days prior to treatment. The pigs were maintained on a 13% protein MSU finisher ration mixed with enough water to form a wet mash. They were fed at approximately 5 pm daily and the feed was withdrawn around 9 pm. After catheterization, the pigs returned to the metabolism cages in a room in which the temperature was maintained between 22 and 25 C. The pigs were each fed 2.7 kg of MSU finisher ration per day as described above. 55 Catheterization Five days prior to treatment, the four pigs were surgically catheter- ized at the MSU Veterinary Clinic. They were anesthetized with halothane gas, and placed in dorsal recumbency. An incision was made anterior to and either to the left or right side of the sternum, to expose the jugular vein. Approximately 3 cm of Silastic medical grade tubing (.016 cm inside diameter and .033 cm outside diameter, Dow Chemical Co., Midland, MI) was inserted into the jugular vein. The catheter was loosely tied around the vein and sutured to the subcutaneous adipose tissue. A loop was left in the catheter just under the skin and the catheter was threaded through a trocar. The trocar was passed subcutaneously to the dorsal midline and exteriorized through an incision anterior to the scapula. The external portion of the catheter was coiled in an adhesive tape patch which was both sutured and cemented to the skin. A blunted 18 gauge needle was inserted into the catheter. The catheter was filled with heparin (480 units/ml) and sealed with a 1 ml tuberculin syringe. Experimental Design The experimental design, presented in Table l was a 4 x 4 Latin square. The pigs received either saline, .13 mg/kg of PPGH, .34 mg/kg of PPGH or .34 mg/kg of CPGH dissolved in .OlN NaOH saline, pH 10.8 and enough .OlN HCl was added to adjust the pH to between 8.0 and 9.0. The amount of CH each pig received was based on his weight 8 days prior to the first treatment. At least one day separated treatments. 56 TABLE 1. EXPERIMENTAL DESIGN FOR THE INDVIVO EXPERIMENT Pig Day No. l 2 3 4 Treatments 1 .13 PPGH: .34 PPGH .34 CPGH Saline 2 .34 CPGH .13 PPGH Saline .34 PPGH 3 Salinecd .34 CPGH .34 PPGH .13 PPGH 4 .34 PPGH Saline .13 PPGH .34 CPGH 3.13 mg PPGH/kg body weight was infused. b.34 mg CPGH/kg body weight was infused. cSaline was infused. d.34 mg PPGH/kg body weight was infused. Approximately 1.5 hr before the O-hr bleedings were collected, the heparin solution in the catheter was replaced with 3.5% sodium citrate. Two O-hr bleedings were taken 1.25 and 1 hr prior to infusion. The GH or saline was infused over a 4 min period, and the time at the end of infus- ion was designated as O-time. The catheter was flushed with 1.5 m1 of sterilized physiological saline immediately after infusion. Blood Collection Blood was collected with a 10 ml syringe at the following times after injection: 15, 30, 60, 90, 105, 120, 135, 150, 180, 210, 240, 270, 300, 330, 360, 390 and 420 minutes. Approximately 5.5 ml of blood were mixed with .75 ml of .1M sodium oxalate while the remainder was allowed to clot. The blood samples which contained sodium oxalate were centrifuged at approximately 2000 rpm for 10 min in a refrigerated Sorvall Medium Angle centrifuge. The samples were transferred to plastic vials, frozen in dry 57 ice and ethanol, and transferred to a 0 C freezer. After all bleedings were completed, the samples were transferred to and stored in a -20 C freezer until assayed. The clotted samples were transferred to a refri- gerator. Five hr after the last samples were collected, the blood was centrifuged at 2000 rpm in a Sorvall RC-3 refrigerated centrifuge for 10 minutes. The serum was frozen in plastic vials at -20 C until insulin was determined. Blood Glucose Plasma blood glucose was determined by the COD-PERID method as described in the Boehringer Mannheim Co. Bulletin 7453 (1974). The enzyme (lots 617486 and 623880) was dissolved in 1000 m1 of distilled H20 and stored between 0 and 4 C. Five ml of the enzyme solution (room tempera- ture) were added to either .02 ml of the H 0 blank, standards (100, 200, 2 300, 400 mg of glucose/100 ml) or samples. The enzyme was gently mixed with the sample and incubated for 50 min at room temperature. Absorbance was read at 600 nm with a Gilford Micro-Sample Spectrophotometer 300N. A standard curve was drawn and the extinction coefficient determined for each assay. The plasma glucose values were divided by .785 to adjust for the added sodium oxalate. Plasma Free Fatty Acid Assay The plasma FFA were determined by the method of Duncombe (1963) as modified by Itaya and Ui (1965), Weenick (1969) and Bidner (1970). Two 58 ml of phosphate buffer, pH 6.1 and 5 m1 of chloroform followed by .3 ml of blank, plasma or standard were added to 16 x 100 mm screw cap test tubes. The tubes were shaken at least 30 times and then allowed to stand for at least 15 minutes. The PO4 buffer (upper layer) was aspirated and 2.5 ml of Cu reagent (Appendix I.C.2) were added immediately. The tubes were shaken 30 times and centrifuged at 1600 rpm (Sorvall HL-8 head) for 20 minutes. The Cu layer (upper layer) was aspirated and the chloroform layer, which contained the FFA, was filtered through Whatman No. 1 filter paper. A 2 ml aliquot of chloroform was thoroughly mixed with .5 ml of .1% sodium diethyldithiocarbamic acid dissolved in n-butanol (prepared fresh each day) and the absorbance of the mixture was read immediately at 440 nm on a Beckman DU Spectrophotometer. Chloroform served as the machine blank and chloroform which was carried through the procedure served as the assay blank. The concentration of plasma FFA was deter- mined by adding palmitate, dissolved in absolute ethanol, to pig standard serum. A new standard curve was drawn for each assay. If no FFA were detected in the serum, the standard in which FFA could not be detected was used for that value. In addition, if all values were detected, a French curve was used to approximate the FFA value at which the optical density was zero. The plasma FFA values were divided by .785 to account for the added sodium oxalate. Insulinggadioimmunoassay Serum insulin concentration was measured by the double antibody assay described by Meiburg (1973). The following is a description of the assay. 59 1. Either 250 or 150 pl of buffer B1 (.05M phosphate buffered saline - 1% BSA, pH 7.4; Appendix I.D.2) followed by either 250 or 350 pl of serum (enough to make 500 pl) were added with a Hamilton syringe to 12 x 75 mm disposable culture tubes. Four hundred p1 of B1 were added to tubes used for standards. Porcine insulin standards (100 pl; Eli Lily; Appendix I.D.3) which were prepared in buffer B1 and ranged from 1.435 to 239.0 pU /tube, were pipetted to tubes as described above. 2. On day zero, 200 pl of guinea pig anti-porcine insulin serum (GPAPI, Miles Laboratory), diluted l:100,000 with guinea pig control serum (1:400; Appendix I.D.S), were pipetted into each tube as described above. The tubes were stirred gently and incubated at 4 C for 24 hours. 3. On day one (24 hr later), 100 p1 of 125I-insulin (IM-38, specific activity - 50 p CI/pg, Amersham/Searle; Appendix I.D.6) which contained 18,000 cpm, were added to each tube. The tubes were gently stirred and incubated at 4 C for 24 hours. 4. On day two (48 hr later), 200 pl of sheep anti-guinea pig gamma globulin (SAGPGG), diluted to 1:31 with PBS-EDTA (Appendix I.D.7), were added to all tubes which were then gently stirred and incubated at 4 C for 96 hours. 5. On day six (96 hr after the addition of second antibody), 2.5 ml of PBS (Appendix I.D.8) were added to the tubes. The tubes were centri- fuged at 2800 rpm in a Sorvall RC-3 refrigerated centrifuge for 30 minutes. The supernant was carefully decanted; the tubes were inverted on absorbant paper to dry for 30 min and then were wiped dry. The tubes were counted 60 for 4,000 counts or 4 min in a Nuclear-Chicago Model 4320 autogamma scin- tillation counter. Tube number, time and counts were printed by a tele- type (Teletype Corp). The average counting time for each standard in the three sets of standards was used to calculate a regression equation with linear, quad- ratic and cubic components. Regression coefficients, calculated on a CDC 6500 computer, were entered into an Olivetti computer (Programma 101, Olivetti Underwood, New York) which corrected for dilution. Tube number and counting time of the sample were entered and value for unknowns were automatically calculated. Statistical Analysis The statistical analysis was done by a CDC 6500 computer at the MSU computer center. Data from experiment 2 were analyzed by two-way analysis of variance. Data from.experiment 3 were analyzed by three-way analysis of variance and least squares analysis of variance. Where significance was indicated, Duncan's New Multiple Range Test was performed to determine which means were significantly different in both experiments (Steel and Torrie, 1960). RESULTS AND DISCUSSION Experiment 1 The amounts of the PPGH and CPGH administered both Ea yiggg and ia_ yiyg_were based on the "biological activity" of both preparations. The biological activity or "tibia activity" of PPGH (lot 614276) was found to be 3.0 IU/mg which agreed with results reported earlier by Machlin (personal communication). The biological activity of CPGH (lot 3486) used in this study was determined also to be 1.1 IU/mg by the tibia test. According to the specifications given by Nutritional Biochemicals Inc. the biological activity of CPGH was reported to be between .5 and 1.0 IU/milligram. Experiment 2 The basal rate (control) of glycerol release which is a measure of lipolysis from adipose tissue of meal fed pigs was approximately twice that of ad libitum fed pigs as shown in Table 2. Also, the rate of gly- cerol release from adipose tissue of meal fed pigs in response to CPGH and both levels of PPGH was 200% and 30% greater, respectively, than the response of adipose tissue from.ag_libitum fed pigs. The length of time between biopsy and incubation was 1 hr longer for the adipose tissues of .fli libitum fed pigs than that from the meal fed pigs. The influence of time lapse prior to incubation has not been reported; hence, the effect upon the data cannot be evaluated. On the other hand, since pigs are nibblers, the ag libitum fed pigs may have eaten shortly before biopsy, 61 62 TABLE 2. EFFECT OF PGH PREPARATIONS ON LIPOLYSIS IN PORCINE ADIPOSE TISSUE IN VITRO Glycerol release Concentration pmoles/hr g wet wt Treatment in medium Meal fed 5g libitum fed Control (DDHZO) 50 p1/m1 .41a .25a PPGH 10 pg/ml .76a .28a PPGH 50 pg/ml 1.268 .468 CPGH so LJJg/ml 3.08b 2.27b SE = .49 SE = .16 SE = Standard error of the mean. Different superscripts in the same column indicate significance at P<305 in column 1 and P<.01 levels in column 2. whereas the meal fed pigs had not eaten within 12 hr of biopsy. Grigsby ._£‘_1. (1972) reported that plasma insulin levels were elevated between 15 min and 7 hr after feeding; thus, the adipose tissue from ag libitum fed pigs may have still contained insulin. The residual insulin, may have been responsible for decreased lipolysis in adipose tissue from ag libitum fed pigs. Furthermore, Goodman and Knobil (1959) and Goodman (1970) both observed that either fasting or one meal per day was necessary for the lipolytic response to GH to occur ianyiyg. Although adipose tissue from ag_libitum fed rats (Fain a£_a1,, 1965) responded to GH 12_ yiggg, the effect of fasting on adipose tissue in this study may have po- tentiated the lipolytic activity of PGH. The average rate of glycerol release from adipose tissue from both meal and ag libitum fed pigs in response to CPGH administration was 63 significantly greater than either the control or PPGH, while the latter two groups did not differ significantly. The significantly greater re- sponse of porcine adipose tissue to CPGH supports the reports of Machlin (1972). When he incubated rat and rabbit adipose tissue with CPGH (dif- ferent lot) before and after purification (similar to that procedure used to purify PPGH), he reported that the purified PGH accumulated only 1% as much medium glycerol as CPGH did. Furthermore, Trygstad (1967) observed a reduced lipolytic response to HGH following its purification. The latter author removed a lipolytic mobilizing factor (LMF) which was shown to be lipolytic la 33332, In addition Goodman (1968) was unable to stim- ulate lipolysis with BGH ighgiggg'without the addition of dexamethasone to the incubation medium. On the other hand, Swislocki _£._l. (1971) stimulated lipolysis with purified BGH in adipose tissue from hypophysec- tomized rats. The pattern of accumulated medium glycerol, released from adipose tissue of ag libitum and meal fed pigs in response to the control and GH treatments, is presented in figures 1 and 2, respectively. The means and standard errors of the means are presented in Appendix 11, tables 1 and 2 for meal and aa libitum fed pigs, respectively. The 7 hr period was chosen as the length of time for incubation since the average rate of glycerol release tended to decrease in adipose tissue from 3 fasted pigs during the 7th hour (Appendix II, figure 1). A significant time x treatment interaction was observed in medium glycerol released from adipose tissue of both ad libitum and meal fed pigs. Figure 1. 61)- 1&04 AtO' 110- 110- 'L0- 64 C) Control ‘ D 10 pg/ml PPGH 50 pg/ml PPGH 50 pg/ml CPGH A A A A O O O ‘ A’. T’:/- ‘ ¢I‘--!=:-.--llll:'---l ' /‘~'/:' . r l l l I l 1 I 2 3 4 5 6 7 Hr The accumulated medium glycerol released from adipose tissue of ag libitum fed pigs. 210:1 I 19.0- 18.0- 17.0 - 16.0- 15.0- 14.0- 13.0- 12.0- H .0 a 10.0-4 9.0—J 8,0" pmolesfg 7.0% 6,0- 5,0- 4,0- 3.0-1 2.0-l 1.0- Figure 2. 65 A (J Control I! 10 ug/ml PPGH 2 50 pg/ml PPGH 50 pg/ml CPGH A A o A o 0’ I o A I I o I . ' - I ‘ O 9 O -/0 // l U I I l I 1 2 3 4 5 6 7 Hr The accumulated medium glycerol released from adipose tissue of meal fed pigs. 66 The rate of glycerol release in successive hours either remained constant or declined for both groups of pigs in the control treatment. Secondly after the first 2 hr of incubation, the accumulated glycerol in response to CPGH was approximately twice that of any of the other treatments and the two levels of PPGH were essentially the same as the control (figures 1 and 2). The large accumulation of glycerol during the first 2 hr of incubation in response to CPGH was in opposition to the results reported by Fain and Saperstein (1970) who found a nonsignificant response after a 2 hr incubation. However, no increase in lipolysis was observed in response to PPGH administration during the first 2 hr of incubation. In fact, the initial tendency of 50 pg/ml PPGH to increase lipolysis was not observed until the 3rd and 5th hr of incubation of adipose tissue from meal fed and 2a, libitum fed pigs, respectively. Fain and Saperstein (1970) also reported a 3 hr delay in GH stimulated lipolysis. During the delay, protein syn- thesis occurred and they showed it was essential for glycerol release. They also suggested that the rapid onset of GH stimulated lipolysis, similar to that of CPGH, was due to contamination of other pituitary hor- mones such as IMF, ACTH or TSH. Furthermore, Goodman (1965) observed in- creased glucose uptake and incorporation into C0 ‘13 vitro for 1 hr 2 following ia'vivo GH administration which he called "insulin-like" activity. However, the lipolytic effect was observed 3.5 hr following the GH injection. Therefore, the initial lipolytic activity of CPGH observed in this study may be due to other lipolytic pituitary hormones since CPGH stimulated lipolysis earlier than that shown for more highly purified GH. 67 After lipolysis was stimulated, as measured by the increased rate of glycerol accumulation, CPGH tended to stimulate the release of glycerol at a constant rate, whereas PPGH tended to fluctuate and increased it at a significantly slower rate than CPGH. The CPGH probably contained con- taminants which have been reported to stimulate lipolysis. Furthermore, the elevated lipolytic activity of CPGH in comparison to the lipolytic activity of PPGH supported the observation of Machlin (1972) that as lip- olytic activity increased the "tibia activity" decreased. A dose response relationship, although not significant, appeared to exist between the two levels of PPGH. As shown in table 2, the average hourly release of glycerol from adipose tissue of both groups of pigs in response to 50 pg/ml PPGH was almost twice the response to 10 pg/ml PPGH. Furthermore, the pattern of response was much different as shown in figures 1 and 2. Fifty pg/ml PPGH stimulated the rate of glycerol release above that of the control rate and 10 pg/ml PPGH following the 3rd and 5th hr of incubation of adipose tissue from meal and aa libitum fed pigs, respectively. The rate of glycerol release in response to 10 pg/ml PPGH was similar to the control value except during the last hour of incubation of adipose tissue from meal fed pigs when the rate tended to increase. The delayed response and the stimulatory effect of fasting on GH-stimulated lipolysis, which have been attributed to (Fain and Saperstein, 1970) and found necessary for CH activity, (Goodman, 1970a; Goodman and Knobil, 1959) tended to suggest that the lipolytic activity of 50 pg/ml might be due to GH itself. 68 Machlin (1972) established a dose response relationship with 3 levels of a preparation similar to the PPGH used in this study when he observed that PPGH had approximately 1% of the lipolytic activity of CPGH. Trygstad (1967) also observed some remaining, ia_yi££g lipolytic activity with HGH after the removal of LMF, which significantly increased lipolysis during 3 hr of incubation. However, Trygstad (1967) also observed a dis- tinct, faint band in the same-area as the HGH band on disc gels following the purification of GH and suggested that the faint band might account for the lipolytic activity of the purified HGH. On the other hand, Wieser ._£‘_1. (1974) noted a large molecular weight band (not GH) on SDS gel electrophoretograms obtained from a variety of relatively pure BGH pre- parations, but suggested that the band was not responsible for lipolysis. Since the higher level of PPGH elicited a greater release of glycerol than the low level used in this study, contamination of PPGH with other lipolytic factors cannot be ruled out. Consequently, the exact cause of the dose response relationship to PPGH cannot be determined based on these experiments. The pattern of FFA release is presented in figures 3 and 4 for ag libitum and meal fed pigs, respectively. The correlations between glycerol and FFA were .96 and .94 for the ag_libitum and meal fed groups, respect- ively. Although the FFA data verified the effect of the GH treatments on lipolysis, glycerol was deemed a more reliable measure of lipolysis. Glycerol cannot be phosphorylated in adipose tissue due to the almost complete lack of glycerol kinase (Margolis and Vaughn, 1962) whereas FFA are subject to reesterification. pmoles/g Figure 3. 430p 4e0- 44.01 3894 36.0- 340- 30.01 zeal 2co- 20.03 180- 169. 14g— 120. 100- so. so. 40. Zilq If If 69 (J Control D 10 pg/ml PPGH 050 pg/ml PPGH 50 pg/ml CPGH A ‘0 0’ , 3 /e/"/ A say:/- ,.;..-—-!;,,,4/ - ' “_ I r I I I I I 2 3 4 5 6 7 The accumulated medium FFA released from adipose tissue of ag libitum fed pigs. FFA released in response to 10 pg/ml PPGH was the same as that released by the control. 70 50.04 Control 10 pg/ml PPGH 50 Isa/m1 PPGH 50 pg/ml CPGH 48.0- >000 46.0- 44.01L J 33.0-1 36117 34.0 1 0' 30.0-1 28.0- 26.0~ J II 20.0.. 18.04 pmoles/g 16.0- 14.0 d 12.01 0 10.0- 8.0- I 6.0 "‘ A 4.0- O \ D H 2.0- :/ J T r I 2 3 4 5 6 7. Hr Figure 4. The accumulated medium FFA released from adipose tissue of meal fed pigs. 71 Summarization of the ia_yi££2_results suggest that l) CPGH probably contains contaminants which stimulate lipolysis, 2) the biological activ- ity, when used as a measure of purification of GH, was inversely related to the direct lipolytic activity of GH, 3) PPGH tended to have lipolytic activity but whether the activity was due to contamination or CH itself was not determined and 4) fasting increased the lipolytic response to all treatments and especially the PPGH treatments. Experiment 3 Plasma FFA, Glucose and Serum Insulin Concentration The plasma FFA values obtained in this study were lower than those reported in the literature. In this study, the plasma FFA values for the saline group averaged 105.6 qu/l and ranged between 88 and 160 qu/liter. Allee et a1. (1972) reported that 22 kg, meal fed pigs averaged 355 qu/l prior to feeding and 219 qu/l 7 hr after fasting. (However, Siers and Trenkle (1973) reported that aa_libitum fed gilts, weighing 67 to 84 kg, averaged 134 qu/l over a 6 hr period and some of the gilts averaged less than 100 qu/liter. Although the pigs in this experiment were meal fed, the plasma FFA more closely resembled those of ag_libitum fed pigs than of those pigs in the postabsorptive state. These pigs may not have been in the postabsorptive state due to the slow rate of food passage through the digestive tract of the pig. Castle and Castle (1956) reported that the average retention time for grain fed pigs was between 33 and 35 hours. Later, O'Hea and Leveille (1969) 72 reported that 87 kg pigs were in the postabsorptive state 18 hr after feeding since food was observed in the digestive tract prior to that time. The effect of the amount of feed each pig ate on plasma insulin, FFA and glucose levels also suggested that some of the pigs remained in the absorptive state. Although the feed was not reweighed after feeding, a visual appraisal of how much each pig ate was recorded. Any pig that ate less than the other pigs, had higher average plasma FFA levels (188 qu/l) than the other pigs (95 qu/l). Consequently, all pigs may not have been in the postabsorptive state which would also account for some of the variability seen among plasma FFA values. Thus, the plasma FFA values should be compared among the treatments rather than to the litera- ture values. The plasma glucose levels of the pigs in this experiment averaged 96 mg/100 ml for the saline control 81‘0“}? and 94 mg/100 ml for the 0-hr bleedings. The values were higher than the plasma glucose levels reported by Siers and Trenkle (1973) who detected 87 mg/100 ml in the plasma of 87 kg pigs. Grigsby _£‘_1. (1972) reported values ranging from 71 mg/100 ml in the fasted state to 117 mg/100 ml after feeding. Likewise Machlin a; a1. (l968a) reported plasma glucose values ranging from.epproximately 150 tug/100 ml in the fed state to 90 mg/100 ml in the fasted state. Con- sequently, the plasma glucose levels observed in this experiment were comparable to those reported in the literature. Serum insulin values ranged from 19 pU/ml to 222 pU/ml. The average control (saline) values were 59.2 pU/ml and the O-hr bleedings averaged 73 59.5 pU/ml. The values reported in the literature range between 6 uU/ml (Machlin a; al., 1968; Grigsby agual., 1972) in the fasted state to 132.0 pU/ml (Wood a; al., 1971). Therefore, the plasma insulin values reported in this study were within the range reported in the literature. The Effect of PGH on Plasma FFA The FFA response to the four treatments employed in this study are presented in figure 5. The FFA levels in response to the control (saline) .13 mg/kg PPGH and .34 mg/kg PPGH treatments did not vary significantly with time but the FFA levels of the pigs that received the .34 mg/kg CPGH treatment varied significantly (P<.0005) with time. As can be seen in table 3, plasma FFA concentration of the CPGH treated pigs was signifi- cantly greater than the other treatments at 30, 60, 90, 120 and 135 min postinfusion. Although FFA were high at 105 min, they did not differ significantly between treatments probably due to the large pig to pig variation. At 150 min postinfusion, plasma FFA levels were higher in response to both CPGH and .34 mg/kg PPGH than to the other two treatments. The increase in plasma FFA in response to CPGH was 6 times the O-hr and lO-times the levels of the other treatments during the peak period which occurred 60 to 90 min postinfusion. After 90 min the plasma FFA decreased continuously and returned to control levels by 180 min postinfusion (figure 5). The plasma FFA levels were significantly (P<.05) elevated in response to .34 mg/kg PPGH at 150 min postinfusion. Furthermore, a similar spike zoo—l l00{ FA FA I I I I 60 120 180 240 300 360 420 FFA 0 control 4 D .13 mg/kg PPGH O .34Ing/Itg PPGH A .34mg/kg CPGH lOO. I T ° ' ob ' I50 I 150 ' 2'40 31:0 360 420 min FFA I I T r l l 1 I T ‘7 oo :20 no no zoo zoo 420 min FIGURE 5. Pattern of plasma FFA chow infusion of saline PPGH o: CPGH into pigs. 75 TABLE 3. PLASMA FFA VALUES AFTER INFUSION OF SALINE, PPGH OR CPGH Treatment Bleeding Control PPGH PPGH CPGH period Saline .13 mg/kg ' .34 mg/kg .34 mg/kg_ min qu/l qu/l qu/l qu/l 0 93 1 25(1) 94 1 36 140 1 81 146 + 93 15 94 1 48 106 1 30 94 1 34 258 1 142b 30 92 1 383 160 1 92a 98 1 32a 334 1 188b 60 116 1 338 114 1 48a 100 1 348 962 1 798b 90 88 1 18a 88 1 28 94 1 44a 969 1 635 105 98 1 28 99 1 28 94 1 28 625 1 265b 120 149 1 84a 92 1 38a 106 1 348 571 1 239b 135 86 1 35a 120 1 98a 78 1 143b 316 1 13g 150 96 1 42a 88 1 198 220 1 126 301 1 79 180 94 1 3o 67 1 12 90 1 30 145 1 52 210 102 1 42 101 1 3 144 1 61 96 1 12 240 93 1 22 74 1 10 176 1 158 82 1 22 270 97 1 46 74 1 18 118 1 66 168 1 112 300 114 1 62 104 1 14 134 1 73 162 1 113 330 120 1 54 142 1 34 145 1 128 122 1 61 360 161 1 122 126 1 48 162 1 124 110 1 66 390 104 1 14 103 1 14 266 1 276 110 1 28 420 99 1 54 110 1 15 204 1 229 102 1 58 Ileean 1 standard deviation. Different superscripts on the same line indicate the data differed signi- ficantly (P<.05). (although nonsignificant) was observed at 120 and 135 min postinfusion.in the control and .13 mg/kg PPGH treatments, respectively (figure 5); Con- sequently, it was impossible to determine whether the response was due to the treatment or a normal phenomenon which occurred each morning at appro- ximately the same time. Neither the control nor .13 mg/kg PPGH treatment differed significantly from each other at any of the time periods. The dramatic rise and fall of plasma FFA levels in response to CPGH within 3 hr suggest that CPGH contains a lipolytic factor. Although 76 Vezinhet _£‘_1. (1971) observed a peak in plasma FFA levels between 1 and 2 hr after injection of PGH into hypophysectomized rabbits, the values did not return to normal levels until 9 hr after injection. Furthermore, a significant increase in plasma FFA was not observed until 2 hr after infusion of HGH into humans as reported by Fineberg and Merimee (1974). Similarly, Rathgeb _E‘_1. (1970) observed a significant increase in plasma FFA but the increase did not occur until 4 hr postinfusion of CGH into dogs. Machlin _£.al. (1968a) did not observe an increase in plasma FFA until 60 min after infusion of PGH into 27 kg fasted pigs and the levels peaked between 120 and 180 minutes. The delayed response has been shown to be necessary for lipolysis to occur ;a_y;££g by Fain and Saperstein (1970). However, in this study, an increase (P=.l to .05) was observed 15 min after infusion of CPGH. Trygstad (1967) observed a similar increase in plasma FFA in response to HGH in ag_libitum fed rabbits within the first 2 hr which was also observed in this study. However, after purifi- cation of the same HGH during which LMF was removed, the lipolytic activity was markedly reduced although still detectable. Since a faint band of protein was still visible in the region of the GH molecule, on disc gels, Trygstad (1967) suggested that the slight lipolytic activity obtained with the purified HGH was due to contamination of the GH preparation. Trygstad (1967) also suggested that the lipolytic activity of GH was caused by con- tamination of the GH preparation rather than the GH molecule itself. Although a slight increase in lipolysis was observed after .34 mg/kg PPGH, the results obtained in this experiment would tend to support the obser- vations of Trygstad (1967). 77 The Effect of PGH on Plasma Glucose Levels Plasma glucose levels for each treatment at each time period are presented in figure 6. Plasma glucose levels did not differ significantly with time over the bleeding period for either control or CPGH treatments. However, plasma glucose levels changed significantly (P<.01) with time in response to both levels of PPGH. The average O-hr plasma glucose levels were significantly lower on the days when .34 mg/kg PPGH was scheduled for administration than when the .13 mg/kg PPGH treatment was administered. The other two treatments had average O-hr glucose levels between the two PPGH levels. However, there is no apparent explanation for the significant differences between treatments at the O-hr bleedings. Insulin also was lower and FFA were higher (although nonsignificant in both instances) on days when .34 mg/kg PPGH were administered than on days when .13 mg/kg PPGH were administered. Since insulin and glucose have been shown to be correlated, r=.54, (Siers and Trenkle, 1973) and FFA levels are inversely related to plasma glucose and insulin, the glucose, FFA and insulin data tend to support each other. As can be seen in figure 6, plasma glucose levels tended to decline within the first 15 min following infusion of either .13 or .34 mg/kg PPGH in relation to both the respective 0-hr bleeding values and the con- trol value at 15 minutes. Furthermore, plasma glucose values reached their nadir at 30 min postinfusion in response to .34 mg/kg PPGH although the response to .13 mg/kg PPGH had begun to increase again. The tendency for glucose to decrease within the 1st hr after infusion or injection of Insulin __ Glucose ......... ~120 E '0 A“‘k‘-A~ _.|° a A. FA A-vA-MA“ \ \.b‘d \\ p'm : A Auk A O I‘o-I ~k’ .90 3 ~80 a a 1204 lm E INT 5 . \ a = 00- o ' c -1 o 60 3. 40-4 20- .L ‘ j 0 I j I T I T I I I I 7 V 60 120 180 240 300 360 420 min Insulin __ Glucose ......... ~120 E -....3. \ ”&~‘°Q‘J"os“o- O ~IW 3 ‘ o. §~‘ --o.-- _A. o 140- 0 o— o' ‘0""° ~90 3 Ta ~80 1 a 1204 ~70 E 1004 '5 . \ e‘ ... 3 S .5 3 60-1 1 40.. ‘ L 20- l T I I Y ° 6'0 1 1'20 ' 150 2'40 ' 300 360 420 min FIGURE 6. Panern of plasma glucose and serum insulin or CPGH into pigs. 78 ,uu insulin/ml 20 0 control | l' a .I3mg/lrg PPGH 6.122. ________ o .34Ing/Itg PPGH A .34Ing/llg CPGH 49.0..O\ Q. o--Q o" \o " 0‘0 ; o’oo‘cr' «d \ I, dd -120 / 00ml °'Jo'1'20 180'2'40I3'00'3'60'4'20 Inin Insulin Glucose ..-—--..-. 420-5 .11. 4:. a' f “ [7,. u“~q mo_ 1:1. 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