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IIIII.|II‘ .IL'MIIII'IIIcA I... “MIMIflAI llllll ll llflll ll l l l l} l llillllll l 3123 This is to certify that the thesis entitled THE EFFECT OF SOMATOSTATIN 0N BODY WEIGHT AND FOOD INTAKE IN RATS presented by Barbara Baker Campbell has been accepted towards fulfillment of the requirements for M. S. degree inNutri tion -\ ‘ . ./; ’ ‘ - £1 ,d/gg WAY/V L/grLC-(L‘w {2/ Major professor Date 10/ 30/ 79 0—7 639 Linux? Defichigan Sum - Univaxity THE EFFECT OF SOMATOSTATIN 0N BODY WEIGHT AND FOOD INTAKE IN RATS By Barbara Baker Campbell 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 1979 ABSTRACT THE EFFECT OF SOMATOSTATIN 0N BODY WEIGHT AND FOOD INTAKE IN RATS By Barbara Baker Campbell Chronic protamine zinc somatostatin (PZ-SRIF) treatment will decrease body weight in rats. Whether this weight loss is due to decreased food intake or absorption is unknown. Male Sprague-Dawley rats (2509n treated with subcutaneous injections of PZ-SRIF (200 pg/kg) for seven days after pre-treatment with PZ-SRIF (lOO ug/kg) for sixteen days showed a significant decrease in body weight gain. This effect on weight gain was coupled with a sig- nificant decrease in food intake but no change in fecal lipids or xylose absorption. Following PZ-SRIF treatment, serum concentrations of glucose, immunoreactive insulin (IRI) and immunoreactive pancreatic glucagon (IRGa) were unchanged but serum immunoreactive total glucagon (IRGt) was decreased. Pancreatic islets showed diminished content and secretion of both IRI and IRGa. It is suggested that the observed weight loss in PZ-SRIF treated rats was due to a decrease in food intake rather than nutrient absorp- tion. Chronic PZ-SRIF treatment also appeared to selec- tively decrease circulating levels of enteroglucagon. ACKNOWLEDGMENTS I would like to express my sincere appreciation and gratitude to both Dr. N. Chenoweth and Dr. P. Foa for their guidance and assistance throughout this study. My acknowledgment and thanks are also extended to Dr. J. Gill and members of my committee, Dr. D. Romsos and Dr. R. Schemmel. I would also like to extend special thanks to Dr. J. Dunbar for his contributions to this project and Mrs. N. Foa for her valuable suggestions and encouragement. Finally, my deepest appreciation is extended to my husband, Bill, for his constant support and understanding. ii TABLE OF CONTENTS LIST OF TABLES. LIST OF FIGURES . INTRODUCTION. REVIEW OF LITERATURE. Regulation of Food Intake . Malabsorption . Somatostatin in Relation to Food Intake and . Malabsorption . MATERIALS AND METHODS . Animals and Treatments. Diet. . . Fecal Lipids. . . Xylose Tolerance Test . Blood Sampling. . . Islet Isolation and Incubation. Assays. . Statistical Analyses. RESULTS . Body Height, Food Intake and Fecal Lipids Xylose Tolerance Tests. . . . Serum Glucose, Insulin and Glucagon . Islet Studies . . DISCUSSION. SUMMARY AND CONCLUSIONS APPENDICES. BIBLIOGRAPHY. Page iv Table Al A2 A3 A4 LIST OF TABLES Design of experimental treatments. Composition of diet. Body weight gain, food intake and fecal lipids, treatment period I . . Body weight gain, food intake and fecal lipids, treatment period II, groups l and 2. Body weight gain, food intake and fecal lipids, treatment period II, groups 2 and 3. Twenty-four hour urinary excretion of xylose following treatment period II. Serum glucose, immunoreactive insulin (IRI) and pancreatic (IRGa) and total (IRGt) immuno- reactive glucagon values for protamine zinc (P2) and protamine zinc somatostatin (PZ- SRIF) treated rats . . . . . . Body weight and food intake. treatment periods I and II . . . . . . . . . . . Fecal lipids, treatment periods I and II Effect of chronic protamine zinc somatostatin (PZ-SRIF) treatment on immunoreactive insulin content of and secretion by isolated pancre- atic islets. . . . . . Effect of chronic protamine zinc somatostatin (Pl-SRIF) treatment on immunoreactive glucagon content of and secretion by isolated pancre- atic islets. . . . . . . . . . . . . . . iv Page 29 31 38 39 4O 42 43 65 67 69 7O Figure LIST OF FIGURES Immunoreactive insulin (IRI) content of pancreatic islets isolated from rats treated with 100 ug/kg protamine zinc somatostatin (PZ-SRIF) for sixteen days followed by 200 ug/kg PZ-SRIF for seven days. Immunoreactive insulin (IRI) secretion by pancreatic islets isolated from rats treated with lOO ug/kg protamine zinc somatostatin (PZ-SRIF) for sixteen days followed by 200 ug/kg PZ-SRIF for seven days. Immunoreactive glucagon (IRG) content of pancreatic islets isolated from rats treated with 100 ug/kg protamine zinc somatostatin (PZ-SRIF) for sixteen days followed by 200 ug/kg PZ-SRIF for seven days. Immunoreactive glucagon (IRG) secretion by pancreatic islets isolated from rats treated with lOO ug/kg protamine zinc somatostatin (PZ-SRIF) for sixteen days followed by 200 ug/kg PZ-SRIF for seven days. Page 46 48 51 53 INTRODUCTION Both intake and absorption of food must exist to pro- vide cellular energy. If a sufficient energy deficit occurs because of either decreased consumption and/or impaired absorption, the ultimate consequence may be loss of body weight. Regulation of food intake is a complex process. Many theories, both centrally and peripherally oriented, have been proposed to explain what signals hunger or satiety. After food is consumed, normal absorption of nutrients is dependent on the integrity of digestive processes in the gastrointestinal tract. Malabsorption of nutrients, especially fat, can occur if digestion is diminished. In addition to its primary function in decreasing growth hormone, the newly-discovered peptide somatostatin has been shown to have metabolic and central nervous system effects that may alter food intake. Somatostatin may be implicated in food intake control because of its central hypothalamic location, neurotransmitter actions or its effects on circulating metabolites and hormones that may act as peripheral hunger/satiety signals. Somatostatin may be involved in digestive events by inhibiting secretory processes of the stomach and pancreas as well as by altering gall bladder and gastrointestinal motility. In previous studies, chronic somatostatin treatment has been shown to produce a significant loss of body weight in growing and mature rats. Because of the potential effect of somatostatin on food intake and nutrient utilization it is not surprising to see a weight loss in somatostatin treated animals. Whether these reported weight losses are due to an effect of somatostatin on food intake or digestion and absorption of nutrients is presently unknown. REVIEW OF LITERATURE Regulation of Food Intake Classical studies of the role of the central nervous system have shown that the principal neural components which integrate the system for food intake regulation are located in the hypothalamus. The ventromedial hypothalamus (VMH) is the center for control of satiety (Hetherington, 1943) and the lateral hypothalamus (LH) the center for control of hunger (Anand and Brobeck, l95l). By lesion of the VMH it is possible to produce hyperphagia and by injury to the LH, aphagia. Conversely, stimulation of the VMH inhibits feeding and the LH initiates feeding even in satiated animals. Current neuroscience research has shifted the focus from the hypothalamus as an integrative center for hunger signals to the brain monoamines and their roles in hunger and satiety. Catecholamine pathways implicated in feeding are the dopamine nigrostriatal and the dorsal norepinephrine pathways. These central dopamine and norepinephrine neurons are required for initiation of ingestive behavior. Ungerstedt (l97l), Striker and Zigmond (l976) suggest that the syndrome of aphagia and adipsia following lesions of the lateral hypothalamus are caused by damage to the catecho- lamine pathways that project rostrally through this area and in particular, damage to the dopamine nigrostriatal pathway. Rowland and Antelman (1976) suggest that chronic activation of this dopaminergic system could underlie stress related hyperphagia. It has been further suggested that the activity of neurons containing the indoleamine, S-hydroxytryptamine, is reciprocally related to the activity of the catecholamine neurons so that serotonergic neurons might be expected to be involved in the cessation of inges- tive behavior (Saller and Striker, l976). Loss or destruc- tion of brain serotonin could then lead to hyperphagia (Breisch et al., 1976). However, this hyperphagia might be secondary to alteration in the secretion, metabolism or effectiveness of pituitary hormones. Knife cuts through areas where serotonergic neurons are expected to ascend have been reported not only to increase food intake but also to increase longitudinal growth and circulating levels of growth hormone (Palka et al., l97l; Mitchell et al., l973). Traversing the same pathway as part of the serotonin system is the ventral bundle of noradrenergic ascending fibers. The ventral bundle carries epinephrine as well as norepinephrine neurons as it travels from the midbrain to the lateral hypothalamus (Hoebel, 1977). Hyperphagia and increased body weight have also been reported to occur following selective damage to the ventral bundle (Hoebel, 1976; Gold, 1973). This hyperphagia following adrenergic depletion is also dependent on pituitary function (Ahlskog et al., 1975). Norepinephrine and epinephrine pathways ascending from the midbrain are, therefore, postulated to serve a satiety function (Ahlskog and Hoebel, 1973). On the contrary, Leibowitz (1976) using chemical stimulus data has theorized an alpha-adrenergic (norepineph- rine or epinephrine) receptor mechanism located in the medial hypothalamus with a role in hunger stimulation, and beta-adrenergic and dopaminergic receptor mechanisms located in the lateral hypothalamus with a role in hunger suppression. This new theory can be correlated with the classical VMH/LH theory of satiety and feeding if the alpha-adrenergic system excites feeding by inhibiting part of the classical VMH satiety system and the beta-adrenergic system causes satiety by inhibiting part of the LH feeding system (Hoebel, 1977). Regardless of proposed pathways, if mechanisms in the brain centrally control food intake, they must respond to nutrient related chemical changes in the extracellular environment, neural inputs or external recep- tors which monitor environmental events such as temperature fluctuations of the periphery. Metabolic factors such as glucose, fat breakdown pro- ducts, amino acids or peptide hormones such as insulin, glucagon, growth hormone or cholecystokinin-pancreozymin (CCK-PZ) are postulated to be peripheral sensors (Bray, 1976). The blood levels of these substances fluctuate according to the metabolic state and alteration in their concentrations could evoke changes in feeding behavior. The hypothesis that blood glucose controls feeding has maintained interest for two decades. Mayer (1953) postu— 1ated that the VMH might respond to changes in the rate of glucose utilization and modify food intake. This hypo- thesis has been supported by studies demonstrating that injection of glucose into the ventricles reduces food intake (Herberg, 1960) and injection of phloridzin, a drug which blocks glucose uptake, increases food intake (Glick and Mayer, 1968). Injections of the cytotoxin gold thio- glucose (GTG) in mice selectively destroys the VMH. This lesion will cause hyperphagia and obesity. The glucose moiety is necessary for the production of the lesion which suggests that gold is accumulated by cells that can trans- port glucose (Debons et al., 1962). Using electrophysio- logical recording techniques, glucosensitive neurons have been identified in the LH as well as the VMH. These neurons are believed to be the functional units of the hypothalamic control system (Oomura et al., 1969). These neurons change their rate of firing in response to modifications in blood glucose. When glucose utilization is enhanced, neurons of the LH have a decreased discharge frequency whereas VMH neuron firing is increased above control levels (Oomura, 1976). This increase is followed by a post-excitatory depression below resting levels. The biphasic nature of the VMH neuronal response suggests that perhaps there is one type of glucoreceptor with a range of thresholds (Marrazzi, 1976). Exactly how these VMH neurons are sub- sequently involved in the regulation of metabolic homeo- stasis, including food intake, is presently unknown. In addition to central glucoreceptors, liver and intestinal receptors which produce afferent discharges on changes in blood glucose are proposed to exist (Bell, 1976). The lipostatic hypothesis of Kennedy (1953) proposed that some metabolite of fat, plasma free fatty acids or glycerol, serves as a feedback signal for food intake regu- lation. Glycerol, released into the circulation in propor- tion to the rate of hydrolysis of triglycerides, was viewed as the most likely candidate. Glycerol then may be an indirect messenger through glucose formation in the liver or a direct messenger via possible glycerol receptors in the hypothalamus. The rapid change in circulating concentrations of free fatty acids and their response to variations in glucose and insulin concentrations make them an unlikely feedback element. Fatty acids and glycerol are elevated in states of both deprivation and obesity; therefore it is difficult to imagine how these substrates could serve, by themselves, as reliable metabolic indices. However, hunger seems to be correlated with fatty acid release and satiety correlated with fatty acid uptake by adipose tissue (Brobeck, 1974). Studies of hypothalamic neurons demonstrate that electro- osmotic application of free fatty acids to the glucosensitive neurons increases the activity of the feeding center (LH) and decreases activity of the satiety center (VMH). The neuronal monitoring of increased free fatty acids causes activation of the LH and disinhibition of LH suppression by VMH and these changes may motivate eating (Oomura, 1976). Although free fatty acids and glycerol have received the most attention as possible lipostatic regulators of food intake, other possible regulatory substances coming from adipose tissue are the steroids (Hervey, 1969) or prosta- glandins (Baile et al., 1973)- Alterations in blood amino acid concentration seem to decrease food intake when the protein content of the diet is very low (Peng et al., 1974) or high (Mellinkoff et al., 1956); the proportions of amino acids in the diet deviate from requirements (Rogers and Leung, 1973); or when the diet is deficient in an essential amino acid (Harper, 1975). The receptor system for detecting amino acid deficiency appears to be in the brain although not localized in the VMH (Bray, 1976). Besides nutrient metabolites, hormonal factors play a role in modifying food intake. Although insulin is secreted continuously in basal amounts, a major stimulus for increases from basal secretions is the presence of the products of digestion. One of the major actions of insulin is to enable the tissues of the body to utilize and/or store products of digestion. If rats are regularly injected with insulin, food intake increases and body weight rises (MacKay et al., 1940; Hoebel and Teitelbaum, 1966). This increased food intake could result from peripheral hypo- glycemia. However, there is increasing evidence for a direct involvement of insulin with food intake mechanisms. Several researchers (Bagdade, 1968; Decker and Hagerup, 1967; Bernstein et al., 1975) have shown that basal insulin levels positively correlate with body weight in humans. VMH lesioned hyperphagic animals show increased insulin levels independent of overeating (Woods and Porte, 1976). In experiments in which animals are given large alimentary doses of glucose, there is a paradoxical stimulation of food intake. Prolonged and exaggerated insulin response is believed to be responsible for the increased food intake (Rezek et al., 1979). Insulin-sensitive receptors in the central nervous system have also been demonstrated. Debons et al. (1977) showed that insulin is necessary for gold thioglucose destruction of the VMH glucoreceptors. Szabo and Szabo (1972), by carotid artery insulin injections and jugular vein anti-insulin serum injections,demonstrated a decrease in systemic blood glucose. This reduction in glucose, evidence for insulin-sensitive receptors in the 10 brain, was later shown to be due to a direct neural effect on hepatic metabolism and not mediated by pituitary (Szabo and Szabo, 1975b) or pancreatic hormones (Szabo and Szabo, 1975a). Injection of insulin directly into the VMH followed by the same decrease in systemic blood glucose has localized these central nervous system insulin-sensitive receptors in the VMH (Storlein et al., 1975). Electroosmotic appli- cation of insulin to neurons of the VMH will decrease firing frequency and LH insulin exposure will cause increased neuronal firing; whereas if glucose is applied with the insulin, VMH neurons will increase firing (Oomura, 1976). More recently, Havrankova et a1. (1978) have demonstrated insulin receptor binding throughout the nervous system. However, Goodner and Berrie (1977) showed only median eminence and not ventral medial or lateral hypothalamic tissue insulin receptor binding. These authors suggest that insulin communicates with deeper brain centers by neuronal transmission. deCastro et a1. (1978) have suggested that glucagon together with insulin are important in maintaining body weight. Manipulation of glucagon together with insulin, affecting glucose availability and utilization, will alter intake in a predictable manner according to the glucostatic mechanism of food intake. Injections of glucagon into humans (Shulman et al., 1957) and rats (Sudsaneh and Mayer, 1959) will reduce food intake. As in the case of insulin 11 it is not known whether this effect is strictly related to the glycemic state or also to the central nervous system. Growth hormone has also been implicated in regulation of food intake. In humans and experimental animals, injec- tions of growth hormone are accompanied by an increase in food intake and growth. Growth hormone is not essential for obesity to develop because rats without pituitaries will still become obese after VMH injury (Bray, 1974). However, in hypophysectomized, intact VMH animals a decrease in food intake has been observed (Kennedy and Parrott, 1958). Gastrointestinal hormones, by acting as satiety signals, may have a role in regulating food intake. Smith et a1. (1974) have suggested that cholecystokinin-Pancreozymln (CCK-PZ) may be one of these hormones. In rats with open gastric fistulas, satiety did not occur if food failed to enter the small intestine. Intake of food was inhibited by injections of physiological doses of CCK-PZ or by closing the gastric cannula and allowing entry of food into the intestines. A more direct role for CCK-PZ as a neuro- regulator of food intake has been suggested by findings that CCK peptides are found in the brain and appear to be localized in cortical neurons (Muller et al., 1977). Extracts of the cerebral cortex of genetically obese mice with hyperphagia contain diminished brain immunoreactive CCK compared with non-obese littermates. This finding 12 suggests that a lower amount of CCK in the brain may be causally related to the unrestrained appetite of these mice (Straus and Yalow, 1979). Enterogastrone, a preparation now known to be rich in CCK, has also been reported to inhibit intake in mice (Schally et al., 1967). In contrast, the gastrointestinal hormones secretin and gastrin will not induce satiety in rats (Smith et al., 1974). Rather than signals coming from the duodenum or jejunum, Deutsch et a1. (1978) believe that the stomach signals satiety. They showed that when the stomach is isolated from the duodenum by means of an inflatable cuff, compen- satory feeding occurs when liquid nutrient is withdrawn from the stomach. This phenomena could be related to neural inputs to the hypothalamus as Opposed to hormonal regula- tion of food intake. Gastric distension is known to diminish food intake and gastric fistulas which allow food to leave the stomach immediately prevent satiety. When food enters into and distends the stomach, stretch recep— tors in the stomach wall are activated and stimulate the vagus nerve (Paintal, 1954). This stimulation of vagal afferents from gastric distention increases neuronal activity of the VMH (Sharma et al., 1961). These findings suggest that gastric distention stimulates the vagus nerve which in turn affects the satiety center. Another type of signal that might influence hypOthalamic feeding systems is heat production as reflected in changes 13 of body temperature (Brobeck, 1960). Body temperature is regulated in the preoptic region of the anterior hypothala- mus. Interaction within the hypothalamus between the temperature regulation and food intake system may cause an animal to overeat when exposed to the cold and undereat when exposed to heat. Animals with lesions in the anterior hypothalamus overeat in the heat and eat too little in the cold (Hamilton and Brobeck, 1966). Warming or cooling the preoptic region leads to suppression or stimulation of feeding (Andersson and Larsson, 1961). There is also a rapid increase in heat production follow- ing food ingestion. This added heat could be utilized as a signal that feeding has occured (Brobeck, 1974). Thyroid hormones can modulate the basal metabolic rate and cause an increase in heat production by accelerated catabolism of fats, proteins and carbohydrates (Bray and Campfield, 1975). In response to a thyroxine induced increase in thermogene- sis, lean mice will adjust their food intake so that increased heat production will not result in body weight loss whereas obese mice cannot adjust and lose weight (Vanderlmig et al., 1979). From the previous discussion it is apparent that regu- lation of food intake is a complex physiological process involving the recognition and integration of many different types of signals. Much effort has been devoted to identi- fying signals and receptors that initiate hunger and satiety 14 and hypothalamic integrative mechanisms for these signals. No single physiological mechanism can fully explain appe- tite or satiety. Food intake is suppressed when glucose supply is abundant, fat stores become large, protein intake is imbalanced, insulin or growth hormone levels are low or glucagon and cholecystokinin are high. A variety of chemical signals exist with one common result on food intake. In addition to or perhaps interrelated with these signals are more generalized factors influencing food intake - gastric distention or environmental temperature and body heat content. Although each mechanism is proposed to exist, how they all interact to exert a common control is not fully understood. Malabsorption After food intake has been initiated in response to the signals discussed previously, the ingestion of foodstuffs does not necessarily ensure a sufficient supply of energy to maintain body weight. Nutrients from the food must be delivered by processes of digestion and absorption to the cells where they can be utilized as energy substrates. The gastrointestinal tract serves to digest and absorb foods and nutrients supplied to it exogenously by the diet as well as endogenously from luminal secretions and sloughed cells. Failure to absorb ingested nutrients or to reabsorb endo- genous biologically useful substrates results in malabsorption 15 (Holt, 1977). The cardinal feature of malabsorption is steatorrhea followed by weight loss (Borgstrom, 1969). In humans ingesting a typical diet supplying 50-100 9 fat per day, steatorrhea is defined as an excretion of greater than 5 g of fat as fatty acids in the feces per day (Frazer, 1969). This quantity signifies fecal fat in excess of 10-20% of ingested dietary fat. Excessive fat excretion is believed to be the most sensitive index of intestinal tract malfunc- tion. The most important cause of faulty fat digestion and absorption can be ascribed to lack of pancreatic enzymes or effective bile salts activity. Disturbances in various aspects of motility including contraction, segmentation, mixing and rate of transit previously have been considered to be important in the etiology of steatorrhea. However,the effects of alterations in motility on intestinal absorp- tion are little understood. Increases in the forward propulsion of intestinal contents and possibly decreased time for absorption can be compensated for by increased mixing of the food with digestive secretions and exposure to absorptive surfaces. Absorption could possibly increase, decrease or remain the same (Losowsky et al., 1974). Pancreatic digestive enzymes play an essential role in normal gastrointestinal functioning. Deficiency of pan- creatic enzymes may be brought about by atrophy of the 16 pancreatic alveolar cells with consequent lack of produc- tion of the enzymes, by obstruction of the pancreatic ducts or by ineffective stimulation for release of the enzymes into the lumen of the intestine. The gastrointestinal hormones, ch01ecystokinin-pancreozymin and secretin are major stimuli to the pancreas for release of enzymes, bi- carbonate and water. If these hormones are deficient, the pancreatic digestive enzymes, amylase, lipase and a variety of proteases may be insufficient for complete digestion of the energy nutrients. Except in cases of very severe pancreatic insufficiency, a significant malabsorp- tion of carbohydrates is not a severe problem. Pancreatic secretions normally contain excess amylase and in addition, a large portion of carbohydrates are mainly dependent on intestinal brush border enzymes. As in the case of carbo- hydrates, malabsorption of nitrogen often fails to develop in spite of diminished secretion of pancreatic enzymes. According to Crane (1969), increased proteolytic activity by pepsin may compensate for lack of pancreatic secretions. The decrease in bicarbonate leads to a lowered upper intes- tinal pH and subsequent continuation of peptic activity beyond the stomach. In contrast to carbohydrate and pro- tein digestive enzymes, human and rat pancreatic juice contain three distinct lipolytic enzymes. Therefore, a pancreatic enzyme deficiency is most effective in decreasing fat digestion and absorption. 17 Bile salts released from the gall bladder by CCK-PZ stimulation also play an important part in the digestion and absorption of fats. If a bile deficiency exists or bile is completely lacking, fat absorption declines but does not disappear completely. In rats with external biliary fistulas, fat digestion was still 40-70% of normal levels (Krondl et al., 1971). However, in rats deficient in both pancreatic juice amibile, the fat level after absorp- tion in the lymph was 8 mg whereas in normal rats on the same diet the level was 120 mg of fat (Wiseman, 1964). These results would seem to emphasize the importance of pancreatic lipase for normal fat digestion. The entire digestive and absorptive processes follow a cascade of events: food signals the gastrointestinal hormones, these hormones then signal the pancreas and the pancreas then releases digestive secretions. A block at any point in this chain of events could compromise the ability to handle nutrients and malnutrition and weight loss could result. Somatostatin in Relation to Food Intake and Malabsorption Somatostatin, or somatotrophin release inhibiting factor (SRIF), is a small peptide composed of fourteen amino acids. It is produced in the lateral hypothalamus which, as discussed previously, is considered the feeding center in the hypothalamic theory of food intake regulation. Somatostatin is carried through neurons to the median 18 eminence of the hypothalamus where it is stored. From the nerve endings, it is delivered into the hypopheseal portal system and subsequently carried to the anterior pituitary where it exerts one of its functions, inhibition of growth hormone (GH) or thyroid stimulating hormone (TSH) (Brazeau et al., 1974; Guillemin and Gerich, 1976; Hansen and Lund- baek, 1976). In addition to being considered a hypothalamic hormone, a neuromodulator or neurotransmitter role for somatostatin has been suggested. Because of its presence in numerous neurons, its release by depolarizing stimuli (Lee et al., 1978), its direct effects on central and peripheral nerves, and its behavioral effects in animals, somatostatin may have a significant role in the regulation of nervous system functions, possibly including neural regulation of food intake. By light and electron microscope studies and immuno- fluorescent techniques, somatostatin has been demonstrated in many areas of the nervous system: nerve cell bodies in the thalamus; neo and limbic cortical areas, the area of the brain in charge of emotions; the subcommissural organ; and the periventricular region in the anterior hypothalamus (Hokfelt et al., 1975; Pelletier, l976). Somatostatin is also located in secretory granules of neuronal fibers localized in the arcuate, suprachiasmatic, ventromedial and ventral premammillary nuclei, organum vasculosum of the 19 lamina terminalis, external zone of the median eminence, spinal ganglia and dorsal horns of the spinal cord, the lamina propria of the gut and Auerbach's plexus (Luft et al., 1978). In the same SRIF positive neurons of the spinal ganglia, the presence of dopamine-B-hydroxylase, the enzyme catalyzing the conversion of dopamine to norepinephrine, has been demonstrated (Elde et al., 1978). This finding suggests the presence of somatostatin and a known neuro- transmitter, norepinephrine, in a single neuron. Somatostatin also exerts a direct depressant activity on central and peripheral neurons. It has been shown to depress spike discharge frequency and amplitude when applied microiontophoretically to neurons of the cortex, brain stem and hypothalamus (Renaud et al., 1975). Given intravenously to rats, somatostatin increased pentobarbital sedation and the lethal dose for strychnine (Kastin et al., 1978). In addition somatostatin will inhibit the electrically induced release of acetylcholine in guinea pig myenteric plexus (Guillemin, 1976). In contrast to these central depressant effects already referred to, evidence for stimulatory activities of somatostatin is provided by behavioral obser- vations. Somatostatin will potentiate the motor effects of L-dopa (Kastin et al., 1978). After direct cerebral adminis- tration to rats, it will reduce REM sleep, cause paraplegia in extension, and produce circular running evolving into catatonia (Rezeck et al., 1976; Havlicek et al., 1976). 20 Opposing effects of somatostatin on the nervous system - depression or stimulation - are believed to be dose related. Another hypothalamic peptide, melanocyte stimulating hormone release inhibiting factor, has also been shown to have biphasic dose-related effects (Kastin et al., 1978). In addition to its presence in the nervous system, somatostatin has been found in similar concentrations in a discrete population of cells, the 0 cells (Orci et al., 1975). These cells are located in the pancreatic islets and in the mucosa of the gastrointestinal tract, mainly in the pylorus and fundus of the stomach and in smaller amounts in the duodenum, jejunum and ileum (Arimura et al., 1975). Somatostatin is also located in a small number of parafol- licular cells in the thyroid (Hokfelt et al., 1975). Insulin and glucagon, also produced by pancreatic islet cells, are important hormones for nutrient utilization and may be involved in food intake regulation. In humans, fasting insulin and glucagon plasma levels as well as insulin and glucagon responses to various stimuli are diminished by somatostatin infusion (Christensen et al., 1974; Efendic and Lins, 1978). Virtually all known stimuli of insulin secretion are blocked by somatostatin - glucose, isoproterenol, tolbutamide, glucagon, arginine and secretin (Gerich, 1976). Similarly, glucagon responses to meals, intravenous arginine, insulin induced hypoglycemia, epine- phrine and insulin deprivation are suppressed by somatostatin 21 (Guillemin and Gerich, 1976). Since the inhibition of insulin and glucagon produced by somatostatin can be reduced by phentolamine, an alpha-adrenergic blocker, it is believed that somatostatin inhibits both these hormones by interac- tion with alpha-adrenergic pathways (Taborsky et al., 1978). Despite concomitant lowering of both plasma insulin and glucagon levels, short term infusion of somatostatin will induce hypoglycemia in rats due to indirect suppression of glycogenolysis and gluconeogenesis (Byrne et al., 1977). Prolonged somatostatin infusion, however, has been reported to cause hyperglycemia due to decreased glucose utilization (Lins and Efendic, l976). Somatostatin will also cause a decrease in plasma free fatty acids in normal (Byrne et al., 1977) or diabetic rats (Micossi et al., 1976). This effect results from either increased tissue utilization of these lipid moieties (Byrne et al., 1977) or, more likely, decreased lipolysis due to glucagon inhibition (Micossi et al., 1976). The presence of somatostatin in the 0 cells, which are situated in the pancreas between the glucagon secreting alpha cells and the central mass of insulin secreting beta cells, raises the possibility that it may function as a local regulator of insulin and glucagon release (Unger, 1977). The islet cells would receive information from one another via the interstitial spaces and exert a paracrine influence in which insulin inhibits glucagon and may 22 decrease somatostatin, glucagon stimulates insulin and somatostatin and somatostatin inhibits insulin and glu- cagon (Unger et al., 1978). In addition to these actions within the islets, there is evidence that somatostatin may function in nutrient homeostasis. By inhibition of various digestive events in response to signals from enteric hormones and rising nutrient concentrations, somatostatin may restrain nutrient entry from the gut. With somatostatin functioning in this capacity, the pancreas would have control over exogenous nutrient flux as well as control, by means of insulin and glucagon, over endogenous nutrient flux (Unger et al., 1978). ’ Somatostatin has been shown to cause a circulation- dependent delay in carbohydrate absorption. During somato- statin infusion, Wahren and Felig observed a 30% reduction in splanchnic blood flow coupled with reduced reactive hyperglycemia in diabetic patients after oral but not intravenous glucose tolerance tests (Wahren and Felig, 1976). Somatostatin infusion will also inhibit galactose absorption in humans and rats. The decrease in blood galactose is proposed to result from delayed gastric emptying, diminished gastrointestinal blood flow or direct inhibition of galactose uptake by somatostatin (Wagner et al., 1978). In rhesus monkeys, it was shown that somato- statin inhibited triglyceride absorption after ingestion of 23 a mixed meal (Koerker et al., 1978). Sakurai et a1. (1975) also reported a failure of blood triglycerides to rise during a fat load in somatostatin treated dogs. Somatostatin has been shown to suppress various gastro- intestinal hormones: secretin, cholecystokinin-pancreozymin (CCK-P2), gut glucagon, vasoactive intestinal peptide (VIP), gastric inhibitory polypeptide (GIP), gastrin, and motilin. Many of the digestive functions signaled by these hormones are consequently reduced. Alcohol or HCl-induced release of secretin and olive oil induced secretion of CCK-PZ from the duodenal mucosa in humans was inhibited by somatostatin (Raptis et al., 1978). Basal, non-stimulated pancreatic exocrine secretion (Boden et al., 1975) and release of pan- creatic fluid and bicarbonate in response to CCK-P2 and secretin in dogs (Boden et al., 1975; Konturek et al., 1976b) and man (Dollinger et al., 1976; Konturek, 1976) were inhibited by somatostatin. Chariot et a1. (1978) also showed that infusions of somatostatin resulted in a decrease in basal flow and vagal and acetylcholine stimu- lated bicarbonate and protein secretion from the rat pancreas. In a study by Folsch et a1. (1978), somatostatin inhibited basal and CCK-P2 stimulated enzyme and volume secretion in the rat. However, it did not influence secretin stimulated bicarbonate concentration or rate of secretion at the dose used for CCK-PZ-stimulated enzyme inhibition. It has been suggested that somatostatin 24 infusion inhibits pancreatic secretion by a decrease of acetylcholine release at nerve endings and a direct inhi- bition at pancreatic effector cells (Chariot et al., 1978). In addition to decreasing CCK-P2 and pancreatic exocrine secretions, somatostatin reduces gall bladder emptying elicited by exogenous CCK-PZ or intraluminal digestive products. This inhibition could lead to bile stasis (Holtermuller et al., 1977). Gut glucagon, or glucagon-1ike-immunoreactivity (GLI), released during glucose, long chain triglycerides or amino acid absorption has been demonstrated to be inhibited by somatostatin infusions (Sakurai et al., 1975). Somatostatin will suppress both VIP production from vipomas, with a reduc- tion in small intestinal juice production (Lennon et al., 1975), and endogenous GIP release with a reduction in insulinotropic action (Pederson et al., 1975). Somatostatin has also been shown to reduce the endo- crine and exocrine activity of the stomach. In humans, it inhibits gastrin release (Konturek, 1976; Konturek et al., l976a; Raptis et al., 1975; Vatn et al., 1977) both after a test meal and during insulin hypoglycemia (Barros D'Sa et al., 1978). In cats, vagally-induced gastrin release is suppressed by somatostatin (Uvnas-Wallensten et al., 1977). Somatostatin has a direct effect on the parietal and peptic cells to suppress the secretion of gastric acid and pepsin in cats (Albinus et al., 1976; Gomez-Pan et al., 25 1975), dogs (Barros D'Sa et al., 1975; Hummelt et al., 1977) and humans(Raptis et al., 1975; Vatn et al., 1977). The inhibitory potency of somatostatin is most effective on gastric acid secretion induced by pentagastrin, urecholin or a peptone meal and less pronounced on histamine stimU* lated secretion (Creutzfeldt and Arnold, 1978). Vagal integrity is not mandatory for inhibition by somatostatin. Reduction of acid and pepsin was observed in vagally inner- vated and denervated stomach pouches (Konturek et al., l976c). The gastric blood flow also does not change during somatostatin infusion (Konturek et al., 1976a). The mecha- nism for this somatostatin induced inhibition of gastric secretion has not been fully elucidated. By interfering with normal gastroduodenal motility and serum motilin levels, somatostatin will decrease gastric emptying. Bloom et a1. (1975) described retardation of gastric emptying and motilin levels during somatostatin infusion in humans. In dogs, it was found that somatostatin has a distinct motor effect on the duodenum but not on gastric antral contractions. The differential effect could diminish the antral-duodenal pressure gradient and inter- fere with gastric motility (Boden et al., 1976). Tansy et al. (1978) also showed the anatomical dependence of the motor effects of somatostatin. Intravenous somatostatin depressed the stomach tonus and contractile activity and increased the excitation of the small intestine segmental 26 motor activity. It was suggested that relaxation of the stomach could be the result of either endogenous catecho- lamine release, direct action of somatostatin on adrenergic receptors, or inhibition of acetylcholine release in the myenteric plexus. Since somatostatin-induced small intes- tine contractile events were antagonized by atropine, it was believed that these events were mediated by effects of somatostatin on acetylcholine -like receptors (Tansy et al., 1978). In summary, from the brain to the pancreas, somato- statin has ubiquitous domains with numerous and varied effects. Many of the actions of somatostatin are related to the biological processes of food intake, digestion and absorption. Impairment of any of these processes could lead to a decreased energy supply and weight loss. In two previous studies, a decrease in body weight was reported in rats treated with somatostatin. Brazeau and coworkers (1974) demonstrated a decreased rate of body weight gain (P<0.01) in young (125 9) male rats receiving three daily subcutaneous injections of somatostatin for eight days. Micossi and collaborators (1976) reported a significant (P<0.05) weight loss in normal and diabetic adult (470-570 9) rats treated with somatostatin for two weeks. Both of these studies, however, focused on other results of chronic somatostatin treatment such as growth hormone inhibition or decrease in serum glucose and lipid 27 levels. Weight loss was merely observed as a secondary result. The present experiment was designed to determine whether these reported weight losses were due to an effect of somatostatin on food intake, digestion and absorption of ingested nutrients, or a combination of these parameters. MATERIALS AND METHODS Animals and Treatments Male Sprague-Dawley rats weighing approximately 250 g were randomly divided into three groups of eight each and individually housed in metabolic cages in a temperature controlled room (22°C). Linear somatostatin1 to be used for treatment was attached to protamine zinc according to the method of Brazeau et a1. (1974) to increase its biological half life from two to four minutes to six hours. In the first sixteen day treatment period, rats were injected subcutaneously twice daily at 9:00 a.m. and 3:00 p.m. with equal volumes of the following: Group 1, saline; Group 2, protamine zinc; Group 3, 100 ug/kg body weight protamine zinc somatostatin (Table 1). Both saline and protamine zinc were used as controls to negate the possibility of prota- mine zinc affecting the parameters studied. After sixteen days of Treatment period I it appeared somatostatin had exerted no effects at the dosage level used. In the previous studies of Brazeau et a1. (1974) and Micossi et a1. (1976), effects on body weight had been 1Biodata, Rome, Italy; Ayerst Laboratories, Montreal, Canada (Treatment II, Group I rats only). 29 Table 1. Design of experimental treatments1 Treatment period2 Group I 11 l saline PZ-SRIF (100 ug/kg) 2 P2 P2 3 PZ—SRIF PZ-SRIF (100 ug/kQ) (200 u9/k9) 1 Treatments of saline, protamine zinc (P2) and protamine zinc somatostatin (PZ-SRIF) were administered as two daily subcutaneous injections. 2Treatment period I lasted sixteen days; treatment period 11 lasted fourteen days for groups 1 and 2 and seven days for group 3. 30 observed after seven and fourteen days, respectively, of protamine zinc somatostatin therapy. In Treatment period II,therefore, animals from Group 1 were begun on protamine zinc somatostatin injections and animals of Group 2 were maintained as a protamine zinc control for fourteen addi- tional days of treatment. Animals of Group 3 were continued on protamine zinc somatostatin injections at an increased dosage of 200 ug/kg body weight for seven days. Diet All rats were fed a high fat,semi-purified diet (Table 2) and allowed water ad libitum. Rats were given a nine day acclimation period to the diet. Throughout the study, light/dark cycles were adjusted to match treatment with feeding time. Food cups were presented after the 9:00 a.m. injection and animals left undisturbed in a darkened room. At 4:00 p.m. food was taken away, weighed and daily consumption recorded. Animal body weights were measured and recorded every third day. Fecal Lipids A three-day accumulation of feces was collected for each rat throughout both treatment periods and maintained as a separate specimen for analysis. Following the experi— mental periods, these fecal collections were dried to a constant weight in a 60°C oven and then finely ground with a mortar and pestle. A 0.5 g aliquot of each specimen was 31 Table 2. Composition of diet Ingredient Percentage Sodium caseinate 20.0 DL—methionine . 0.3 Cornstarch 37.1 Sucrose 10.0 Corn oil/lard (1:1) 25.0 Vitamin mix1 0.4 Mineral mix2 4.0 Choline chloride 0.2 Cellulose3 3.0 1Composed of: (in mg/kg diet): thiamin HCl, 22; pyridoxine, 22; riboflavin, 22; Ca pantothenate, 66; P-amino benzoic acid, 110; menadione, 50; inositol, 100; ascorbic acid, 200; niacin, 100; vitamin 812, 0.3; biotin, 0.6; folic acid, 4; (in IU/kg diet) vitamin A acetate, 20,000; alpha tocopherol acetate, 100; vitamin D3, 2,200 IU and cerelose to 4.0 9. 2Salt mixture, Draper 4164, United States Biochemical, Cleveland, Ohio 44128 (Draper et al., 1964). 3Avicel Food Prototype 174-2, FMC Corporation, Marcus Hook, Pennsylvania 19061. 32 acidified with concentrated hydrochloric acid, extracted with petroleum ether and analyzed by a gravimetric method for total lipid content (Henry et al., 1974). Xylose Tolerance Test Xylose tolerance tests are commonly used clinically as tests for intestinal malabsorption. Since xylose is a non-metabolizable pentose, a high percentage recovered in the urine after a test dose is usually indicative of normal intestinal absorption. To test for normal intes- tinal absorption in experimental animals a xylose toler- ance test was administered to all animals. Prior to using this test in experimental rats, a dose of 0.5 g/kg body weight xylose, the amount usually used clinically for children, was tried on normal, untreated rats. No vomi- ting was observed and approximately 50% was routinely recovered in a twenty-four urine specimen. 0n the basis of these results, at the end of treatment period II animals were fasted overnight and a 0.5 g/kg body weight 1 was delivered into the dose of a 15% solution of xylose stomach by means of a stainless steel cannula. Rats were allowed only water throughout the test period, and a 24-hour urine specimen was collected, volume measured and frozen for analysis the following week. Injections continued as scheduled during the xylose test period and 1Pfanstiehl Laboratories, Waukegan, Illinois. flew-f— was as: ~ \ . “‘1 in“: ' 33 for two subsequent days. Xylose concentration is the urine samples was analyzed by the spectrophotometric method of Roe and Rice (1948). This method is based on the principle that xylose in the presence of heat and acid will form furfurals which react with the reagent p-bromoaniline to form a pink color. Blood Sampling Prior to Treatment period I each rat was fasted over- night, anesthetized with ether and a blood sample was collected from the intra-orbital sinus by means of a heparinized capillary pipet. At the end of Treatment period II, on the morning of the third day following the xylose tolerance tests and after an overnight fast, the animals were injected according to treatment group, anes- thetized with ether and a post-treatment blood sample was collected. After both pre- and post-treatment collections, blood was placed in a chilled tube containing proteolytic I allowed to clot and then centrifuged. enzyme inhibitor The serum samples were frozen for later determinations of glucose, insulin and glucagon. Animals from Groups 1 and 2 were then killed by ether inhalation. Islet Isolation and Incubation After collection of post-treatment blood samples, the pancreas was removed from one half of the rats in Group 3 1Trasylol; FBA Pharmaceuticals, Inc., New York, New York. 34 and the islet cells were isolated by the collagenase diges- tion technique of Lacy and Kostianovsky (1967). Duplicate samples of five islets, selected for uniform size with the aid of a dissecting microscope, were incubated in 2 m1 of Krebs bicarbonate buffer containing 0.2% bovine serum plus either glucose (50 or 300 mg/100 ml) or glucose (50 or 300 mg/100 ml) and arginine (200 mg/100 ml). The islets were incubated for 2 hours in a Dubnoff metabolic shaker at 37°C in an atmosphere of 95% oxygen and 5% carbon dioxide. Following incubation, the islets were removed from the media by centrifugation (500 r.p.m. for 2 minutes at 4°C) and the buffer frozen for later hormone analysis. Triplicate samples of five islets were also homogenized in 3 m1 of Krebs buffer with added bovine serum albumin (0.2%). This homogenate was also stored for later glucagon and insulin analyses. After a 24 hour period following Treatment period II during which no somatostatin was given, the remaining half of the rats of Group 3 were anesthetized, their pancreas removed and then these animals were killed. The islet cells were removed, incubated, and homogenized using the same procedures as described previously. Pancreatic islets from normal untreated rats weighing approximately 250-350 g were isolated, homogenized and incubated for purposes of comparison with islets of PZ-SRIF treated rats. 35 Assays Serum samples were analyzed for glucose content on a YSI Model 23A Glucose Analyzer.l using the glucose oxidase method. Insulin was determined according to the radioimmu- noassay of Hales and Randle (1963). Serum glucagon concen- tration was determined using the method of Foa et a1. (1977) except that a dextran coated slurry was used to separate free from bound hormone. Pancreatic glucagon was determined using AGS (antiglucagon sera) 18, an antibody which binds specifically to pancreatic glucagon. AGS 10, which binds to pancreatic as well as intestinal glucagon-like materials, was used to determine total immunoreactive glucagon. Islet incubation media and homogenized islets were measured for glucagon content as described above but only AGS 18 was used. Insulin was assayed by the method of Malaisse et a1. (1967). Statistical Analyses All calculations were performed on a Hewlett-Packard Model 65 programmable calculator. Analysis of variance, Dunnett's t and Student's t (Gill, 1978) were used to detect mean differences (P<0.05) in body weights, food intake, fecal lipids and xylose absorption among treatment groups. Pre- and post-treatment serum levels of glucose, insulin and glucagon were analyzed by paired t tests. 1Yellow Springs Instrument Co., Inc., Yellow Springs, Ohio 45387. 36 Post treatment serum levels of gut glucagon for somato- statin-treated animals were compared to levels for prota- mine zinc treated animals using Student's t test. Differen- ces in insulin and glucagon content of islets and islet response to stimuli for somatostatin treated versus normal animals were analyzed by analysis of variance and Dunnett's t test. RESULTS Bodpreight, Food Intake and Fecal Lipids Rats treated with saline, protamine zinc (P2) or pro- tamine zinc somatostatin (PZ-SRIF) showed no significant differences in body weight gain, food intake or fecal lipids after sixteen days of treatment (Table 3; refer also to Appendix Tables Al and A2 for additional data). In Treatment period II, after fourteen days of PZ-SRIF or PZ injections, rats showed a slight but statistically non-significant decrease in body weight after PZ-SRIF treatment. There were no significant changes in food intake or fecal lipids (Table 4; Appendix Tables Al and A2). After an additional seven days in which they received an increased dose of PZ-SRIF (200 ug/kg body weight) Treatment II, Group 3 rats showed a significant decrease (P<0.05) in body weight gain. This was accompanied by a significant decrease (P<0.05) in food intake but no changes in fecal lipids when compared to controls (Table 5; Appen- dix Tables A1 and A2). 37 38 .vowgwa ucmEummgu on» mo new on» an mmapm> Go momso>m Amt moss» mo mmosm>m awn omen» < .m aaogm cw mums ugmvo ecu N ucm _ masogm cm mums cm>wm Low mmspm> mucmmmgamg cams gumm mews: .zmm H memos, Am¥\mn oopv no.0 H wn.o no.0 H n©.o N.o H P.¢ ¢.o H m.m ~.w H ¢.©w mammuNa m No.0 H mw.o wo.o H mo.o N.o H ¢.¢ ¢.o H F.m m.w H m.mw Na N no.0 H m©.o mo.o H om.o N.o H o.v ¢.o H m.m ~.w H m.~m wcwpmm F Pacwa _acuwcu ¢_a=ma mpawuwcn m wxmpcm coom m oop\xmu\m zmc\p;mww3 seen a oop\m .cwmm pcmsummgh qsoso mn2a_g Pagan oxapcw coca agave: scam ~.PH nomgma newspmmgp .mnwgmp qumw ucm mxmacm woo» .cwmm uzmwmz atom .m «Pack 39 .coPLmn acmsumwgu on» mo new mzp um mm=~m> we mumgm>m amt moss» mo mmmgm>m zen mossy om com mmapm> mucmmogamg cums sumo mews: 2mm H mcuwzp op.o H wN.o Pp.o H mm.o F.o H m.e N.o H m.e o.m H 0.0m Na N Amx\mn oo_v F_.o H mm.o No.o H om.o N.o H m.¢ N.o H m.¢ ~.m H m.e¢ ufimm1Na P guard Fawuwc~ Hpmcwm mpmwumcm m mxmpcw nooN m oop\adc\m hon\u;mww3 Anon a oop\m .cpmm pcmEummg» asogw macaw_ Paooc oxaucc coca “coco: Huom .NUCM F masogm .HH vowgma “cospmmcu .mnma__ qumm new wxmucw coo» .cwmm usumm} zoom .e «Fame 4O .mumg umummgu Na cusp Amo.ovav cmzop zpucmuvmpcmpmm .HH vomgma ucmspmwgu Ho use on» an mmzpm> mo mmmcm>m xmu mags» <¢ .HH cowgma acmEummgu mcwccwmmn op Lowga mmspm> mo wmmgm>m amu mmgcu oam mgu Lo» umszmcm mew: N aaogm LoH HH cowgma Hawsumwgh mo mxmu cm>mm amng one .N qzogm Low mama :mmugon new m aaogm so» mamu cw>mm uwammp HH nomgma ucmspwmgpN .mpmg “capo LoH mmapm> mpcmmmgamg some :omm mgmgz 2mm H mcmm:P mo.o H FN.F No.0 H mh.o m~.o H m.m N.o H P.H mo.m H c.0N “m3\mn oomv mmmm1Na m mo.o H mP.F No.0 H mm.o N.o H m.e N.o H m.¢ o.m H m.Pm Na N PHCHL FHHchH YHPH:_L m_HHHH=H m mxmpcw voow d oop\xmv\m xwu\u;mwmzlxvon w oop\m .cwmm pcmsumogh asoso mcHaH_ Hague oxapcw Hood Hgmwmz atom . N van N masocm .HH nowgmq pamEpmwcu .mupapp qumw ucm mxmpcw,uoow .cpmm usmmmy xuom .m mpnmk 41 Xylose Tolerance Test There was no significant difference in the percent of ingested xylose excreted in the collected 24-hour urine specimen for any animals (Table 6). The mean urine volume excreted for the 24-hour period was 14.8 i 1.0 ml. Serum Glucose,_Insulin and Glucagon The mean fasting serum values for glucose, immuno- reactive insulin (IRI), immunoreactive pancreatic or alpha cell glucagon (IRGa) and total glucagon (IRGt) are listed in Table 7. All pre-treatment values were within normal limits. Serum glucose, insulin and pancreatic glucagon showed no change for P2 control and PZ-SRIF treated animals. Animals of Group 3, treated with 100 ug/kg PZ-SRIF for fourteen days followed by 200 ug/kg PZ-SRIF for an addi- tional seven days showed a significant decrease (P<0.05) in immunoreactive total glucagon using paired t analysis. The animals of Group 1 treated with 100 ug/kg PZ-SRIF for fourteen days also show a decrease from pre-treatment total glucagon levels. These values were not significantly dif- ferent using paired t analysis due to the large standard error in initial pre-treatment levels. Because it is not known if the effects of somatostatin are dose related, values for all animals treated with PZ-SRIF (Groups 1 and 3) were combined for statistical analyses. Using the paired t test, post treatment total glucagon concentration was 42 Table 6. Twenty-four hour urinary excrptgon of xylose following treatment period II 9 Group Treatment Percent excretion3 1 PZ-SRIF 55.6 1 3.2 (100 ug/kg) 2 Pl 56.7 i 3.0 3 PZ-SRIF 52.5 i 3.0 (200 ug/kg) 1Xylose administered as a 0.5 g/kg body weight dose. 2Treatment period II lasted fourteen days for groups 1 and 2 and seven days for group 3. 3Means i SEM where each mean represents values for seven rats in group 1 and eight rats for groups 2 and 3. 43 .Hmma a m.pcmu=um mcwms mpmswcm AN azoguv uwummgp Na Low m=_m> Pmcmm cusp AmNo.ovmV gmzop apucmo -Hmwcmwm ucm mpmxpmcm H umcwma mcha mapm> Hmwuwcw cusp AmNo.ovav Lmzop xppcmowmmcmwm N .mwmapmcm u uogwma mcwm: mzpm> Hmwpwcw 2mg» Amo.ovav gmzop xppcmuwmwcmwmm .mwmapmcm Fmowumwpmum Low ummmgm>m ucm umcwneoo crumumouwsom ucwn m:HEmpoLg new: cmummgu masocm Low mmzpm>m .HH nowsma ucmspmmgu meHm :mxmu mquEcm voopme .H uoHLma “coaummgu Ho cowummumc_ op gowgg coxmu mmFQEmm noopmm .mmcwazogm HH vowcma ucmsummghN . .m azogm cw mam; Hgmwm ecu N was F masocm :H mum; =m>mm Lo» mmapm> mucmmmgamg cums sumo mgmgz sum H mcmmz. HP H mH H mp H m_ H N H m H H H NmHH wmm HH_ oHF Hm HH o_. ,__ HHNm-Na mm + _ NN H Hm H ON H mp H H H m H H H m H AHH\HH HHNV HHmH mom mm_ Hm_ Hm NH H__ mop LHNm-Na m an H NN H FF H mN H N H H H m H H H HNm HHm Hm, Hm, mm HH m_H mp_ Na N N_ H HN H HN H HN H m H m H N H H H AHH\H: copy HHH HHm mNF mH. NH NH Ho_ m_P LHmm-Na F _H=HH HHHHHHH _H=HH _HHHH:H PHHHH FHHHHHH H_H:HH mFHHHHHH He\ma _E\mql Fe\:11 FH\HE HHHEHHHLH Nasozw HHHH HHHH HHH HHHHHHH qumg nmummgu AmHmmuNav swampmoumsom ocw~ mcHEmpoga ucm ANNV ocw~ mcHEmpoLa L0H mmzpm> commuzpm m>wuumogoczssw ApumHv Page» use Ammev ovummgocma ucm AHNHV cwpamcw m>Hpummgocsssw .mmoospm Eacmm .N mpam» 44 significantly lower (P<0.025) than pre-treatment levels. This post-treatment value was also significantly lower (P<0.025) than post-treatment levels for P2 treated rats using Student's t test. Islet Studies Chronic protamine zinc somatostatin treatment signifi- cantly diminished (P<0.01) the insulin content of isolated pancreatic islets to less than half of normal levels (Figure 1; individual values given in Appendix Table A3). Twenty- four hours after somatostatin treatment, islet insulin content increased slightly but still was significantly lower (P<0.05) than normal amounts. Insulin secretion by isolated pancreatic islets (Figure 2; Appendix Table A3) was also suppressed by chronic somato- statin therapy. In response to a non-stimulatory concen- tration of glucose (50 mg/100 ml), islets of somatostatin treated rats showed decreased response when compared to islets of untreated animals (P<0.01). This same dose of glucose plus added arginine (200 mg/100 ml) served as a stimulation for insulin release in normal islets. However in PZ-SRIF treated islets there was little response (P<0.01). Twenty-four hours after PZ-SRIF treatment there was still negligible insulin secretion by the treated islets when compared to controls (P<0.01). In response to an insulin stimulatory concentration of glucose (300 mg/100 ml) either Figure 1. 45 Immunoreactive insulin (IRI) content of pancreatic islets isolated from rats treated with 100 ug/kg protamine zinc somatostatin (PZ-SRIF) for sixteen days followed by 200 pg/kg PZ-SRIF for seven days. PZ-SRIF indicates islets homogenized immediately after treatment; 24 hrs. post PZ-SRIF indicates islets homogenized 24 hours after last treatment. Values represent mean 1 SEM. Asterisks denote significant difference from normal values: *P<0.05, **P<0.01. IRI Content (uU/islet) 46 2000 " A I 1500 _a O O O n V I» 500‘" lllllllllllllllll-i. Normal PZ-SRIF 24 hrs. post PZ-SRIF Figure 2. 47 Immunoreactive insulin (IRI) secretion by pancreatic islets isolated from rats treated with 100 ug/kg protamine zinc somatostatin (PZ-SRIF) for sixteen days followed by 200 ug/kg PZ-SRIF for seven days. Islet cells were incubated for 2 hours in 1) low glucose, 50 mg/100 ml (50 Glu); 2) low glucose plus 200 mg/100 ml arginine (Glu 50 + Arg); 3) high glucose, 300 mg/ml (300 Glu); and 4) high glucose plus 200 mg/100 ml arginine, (Glu 300 + Arg). Values represent mean 1 SEM. Asterisks denote significant difference from normal values: **P<0.01. IRI secretion (uU/islet/Z hrs.) 600 400 N O O Glu 50 48 Glu 50 + Arg Glu 300 m Normal PZ-SRIF "ll ,5: 24 hrs . I PZ-SRIF Glu 300 + Arg post 49 alone or in combination with added arginine (200 mg/100 ml), untreated islets showed increased response and maximal secretion. Islets from PZ-SRIF treated animals in response to these stimuli secreted a considerably higher level of insulin than at non-stimulatory concentrations of glucose, but did not reach normal levels (p<0.01) or return to normal levels after 24-hours of non-treatment. These results, therefore, show that isolated pancreatic islets of normal rats responded to media stimuli as shown by increased insulin release. Islets of the PZ-SRIF treated rats responded to the presence of media stimuli but show an inhibited secretion of insulin. Immunoreactive glucagon content of isolated pancreatic islets of PZ-SRIF treated rats also showed a significant depression (P%0.05) from levels for normal islets (Figure 3; Appendix Table A4). Twenty-four hours after PZ-SRIF treatment, islet glucagon content had increased to levels lower but not significantly different than normal. Secretion of glucagon by isolated pancreatic cells in response to a stimulus waslowered by chronic somatostatin treatment (Figure 4; Appendix Table A4). Arginine or low glucose levels (50 mg/100 ml) serve as stimuli for pancre- atic glucagon release. Normal islets responded to these stimuli in the media by an increased glucagon secretion. PZ-SRIF treated islets showed a very low response when com- pared to normal levels and did not recover normal secretion Figure 3. 50 Immunoreactive glucagon (IRG) content of pancreatic islets isolated from rats treated with 100 ug/kg protamine zinc somatostatin (PZ-SRIF) for sixteen days followed by 200 ug/kg PZ-SRIF for seven days. PZ-SRIF indicates islets homogenized immediately after treatment; 24 hrs. post PZ-SRIF indicates islet homogenized 24 hrs. after last treatment. Values represent mean i SEM. Asterisk denotes significant difference from normal values: *P<0.05. 51 0 0 0 4 ..l._________________________ 3000 2000 1000 AHH_HH\HHV Hcaucoo HNH PZ-SRIF 24 hrs. Normal post PZ-SRIF Figure 4. 52 Immunoreactive glucagon (IRG) secretion by pancreatic islets isolated from rats treated with 100 ug/kg protamine zinc somatostatin (PZ-SRIF) for sixteen days followed by 200 ug/kg PZ-SRIF for seven days. Islet cells were incubated for 2 hours in 1) low glucose, 50 mg/100 ml (50 Glu); 2) low glucose plus 200 mg/100 ml arginine (Glu 50 + Arg); 3) high glucose, 300 mg/ml (300 Glu); and 4) high glucose plus 200 mg/100 ml arginine (Glu 300 + Arg). Values represent mean 1 SEM. Asterisks denote significant difference from normal values: **P<0.01. IRG secretion (pg/islet/Z hrs.) 3000 2500 2000 1500 1000 500 ** T Glu 50 *‘k 53 Glu 50 + Arg Glu 300 Glu 300 + Arg w Normal 5 PZ-SRIF :' 24 hrs.post PZ-SRIF 54 within a 24-hour period. In a high glucose media (300 mg/ 100 ml), glucagon secretion by untreated islets responded similarly to a 30 mg/100 ml glucose stimulus; PZ-SRIF treated islets showed a suppressed response (P<0.01). At this same glucose concentration the presence of added arginine acts as a further stimulus for glucagon secretion and somewhat overrides the suppression by an increased glucose concentration. Thus, in normal islets, there was an increased response to stimuli, but in PZ-SRIF treated islets there was a diminished glucagon response with minimal recovery after 24 hours. DISCUSSION The incubated pancreatic islets showed that chronic somatostatin treatment suppressed both the production, as measured by content, and release of insulin and glucagon. These results provide evidence that the PZ-SRIF which was administered to these rats was biologically active. The changes in islet cell hormone production and release are most likely due to a direct effect of somatostatin on the alpha and beta cells, possibly due to its presence in the pancreatic 0 cells and its paracrine function. It is well known that somatostatin will suppress both basal and stimu- lated serum insulin and glucagon levels (Christensen et al., 1974; Gerich, 1976; Guillemin and Gerich, 1976; Efendic et al., 1978). Insulin suppression could also be due to the actions of somatostatin on the gastrointestinal tract causing diminished gastrin, secretin and CCK-PZ release (Konturek, 1976). These hormones have been hypothesized to be gastrointestinal factors that are released in response to glucose and stimulate the pancreas to release insulin (McIntyre et al., 1965). Lack of these hormones could decrease pancreatic hormone secretion. Since electrical stimulation of the vagus nerve tends to increase insulin secretion (Frohman et al., 1967) somatostatin could decrease 55 56 insulin by inhibiting cholinergic activity (Chariot et al., 1978). Although there was a definite decrease in the pancreatic islet hormones, their concentrations in the serum did not show the same decreases in response to PZ-SRIF treatment. Similarly, there was no difference in serum glucose concen- tration between PZ-SRIF treated or PZ treated animals. This result could be due to a concomitant suppression of both insulin and glucagon. With opposing actions on glucose metabolism, the end result of insulin and glucagon suppres- sion by PZ-SRIF could be no change in fasted serum glucose values. Serum immunoreactive insulin and pancreatic gluca- gon also showed no alteration after somatostatin treatment. Since the serum values represent a fasted state, perhaps with chronic somatostatin treatment, altered hormone function may become evident only in response to a metabolic challenge. Also, with diminished islet cell hormone pro- duction a compensatory mechanism, decreasing metabolic clearance as a function of receptor-site catabolism, might exist. The serum total immunoreactive glucagon of 200 ug/kg PZ-SRIF treated rats did show a significant decrease (P<0.05) from normal fasting levels. In addition, if values for all PZ-SRIF treated rats are combined and Student's t as opposed to Dunnett's t test is used, there wasa significant decrease (P<0.025) from normal rats. Whether this analysis represents a legitimate as opposed to a statistically advantageous 57 manipulation is debatable. However, by separating treatment effects into dose levels, a dose-response relationship is attributed to somatostatin. Whether the effects of somato- statin can be considered dose dependent is presently not known. In this study, different results for body weight gain and food intake were observed for PZ-SRIF treatment at 100 ug/kg for fourteen days followed to 200 ug/kg for seven days compared to treatment at 100 pg/kg for fourteen days. If length of treatment does not account for differences, effectiveness of somatostatin treatment could be dose-depen- dent, and then all results should be separated according to dosage. However, if values for all PZ-SRIF treated animals are not combined, gut glucagon was still significantly lowered by 100 pg/kg followed by 200 pg/kg PZ-SRIF treatment. Decreased serum total immunoreactive glucagon levels by somatostatin were also reported by Matsuyama et a1. (1979). Circulating levels of enteroglucagon have been shown to be elevated in hyperphagia and very low in states of starvation (Pearse et al., 1977), which is opposite to the responses observed for pancreatic glucagon. Thus, the lowered gut glucagon levels of PZ-SRIF treated rats might reflect merely a state of food deprivation as seen by decreased food intake in these animals. According to Unger et a1. (1978), extra pancreatic alpha cells are more sensi- tive to insulin and small quantities instantly turn off the secretion of extra-pancreatic glucagon. Perhaps these 58 cells are also more sensitive to PZ-SRIF. In this study, there was no significant change in body weight gain due to chronic somatostatin therapy (100 ug/kg) for sixteen or fourteen days in 250 g or 330 9 rats, respec- tively. This dose was identical to that used by Micossi et a1; (1976) in 470 9 rats. These investigators reported a significant decrease in body weight within fourteen days. However, in young rats (125 g) Brazeau et a1. (1974) using a dose of 100 ug, (equivalent to 800 pg/kg body weight) three times daily, showed a significant decrease in body weight gain after seven days. Perhaps in an animal that is growing rapidly, as opposed to a mature rat, an increased dosage is necessary to produce results. In view of similar weight gains for control and animals treated with 100 ug/kg PZ-SRIF for fourteen or sixteen days, it was not surprising that there was no change in food intake or fecal lipids for any of these groups. The decrease in body weight seen in PZ-SRIF (200 ug/kg) treated rats appeared to be related to decreased food intake as opposed to malabsorption since there was no significant difference in fecal lipids or xylose tolerance from PZ treated rats. Decreased food intake was apparent after one day of 200 ug/kg PZ-SRIF treatment. Therefore, higher dosage of PZ-SRIF instead of an additional seven days of treatment may have been responsible for the decline in food intake and consequent decreased body weight gain. 59 This effect on food intake agrees with a study done by Rezek et a1. (1978) in which repeated three-hour hepatic portal infusions of somatostatin were reported to reduce daily food intake by 29% causing a loss of body weight. In further studies, (Rezek et al., 1979) intraperitoneal administration of 100 ug/rat SRIF caused a two-hour complete anorexia followed by a decreased food intake during the following 24 hours. In a study by Lotter and lloods (1977), intraperitoneal injections of 1000 ng/kg SRIF caused a 50% decrease in food consumed in a 30-minute period. Since somatostatin is produced in the lateral hypo- thalamus (Guillemin and Gerich, 1976), and may act as a neurotransmitter (Pimstone and Berelowitz, 1978) it con- ceivably could be involved in the complex process of food intake. Somatostatin has been shown to have a depressant effect on the neurons of the cortex and hypothalamus (Renaud et al., 1975). It could, therefore, depress the dopaminergic neurons of the lateral hypothalamus postulated to be involved in the initiation of feeding (Ungerstedt, 1971). Inhibition of these neurons by SRIF could result in apagia and adipsia. Because somatostatin is also located in the gastro- intestinal tract (Elde, 1978), it could act as peripheral hunger/satiety signal and communicate via neurotransmission directly with the central nervous system to control food intake. Somatostatin could affect food intake indirectly 60 by modulating peripheral signals that also may influence food intake. Gastric distention has been shown to decrease food intake (Paintal, 1954). By delaying gastric emptying (Bloom et al., 1975) and thereby promoting prolonged gastric distention somatostatin could decrease food intake. Somatostatin has been shown to depress the gastrointestinal hormones: motilin (Bloom et al., 1975), GIP (Pederson et al., 1975), VIP (Lennon et al., 1975), gut glucagon (Matsuyama et al., 1979), secretin (Raptis et al., 1978), and gastrin (Raptis et al., 1975; Abbinus et al., 1976; Konturek et al., 1976a; Vatn et al., 1977). By causing a shutdown of the gastrointestinal endocrine system, it is conceivable that food intake may be depressed. If the circulating concentra- tions of insulin or growth hormone are viewed as plausible peripheral signals for food intake, then somatostatin by inhibiting these hormones (Lundbaek et al., 1977; Brazeau et al., 1974) could decrease food intake. Byrne et a1. (1977) have shown that the net effect of somatostatin infusion on blood glucose is hyperglycemia due to decreased glucose utilization. This state of glycemia according to the glucostatic mechanism of food intake could serve to limit food intake. However, since somatostatin will inhibit glucagon (Lundbaek et al., 1977), hepatic glu- cose production by decreased glucagon-dependent glyco- genolysis and consequent decreased availability of glucose may cause an increase in food intake according to the 61 glucostatic mechanism. Furthermore, increased food intake could be expected on the basis that SRIF inhibits chole- cystokinin-pancreozymin (Raptis et al., 1978) or thyroid stimulating hormone (Hansen and Lundback, 1976). A decrease in either of these hormones could signal an increased food intake. Since somatostatin has been shown to reduce digestive functions (Raptis et al., 1975, 1978; Albinus et al., 1976; Konturek, 1976; Holtermuller et al., 1977; Chariot et al., 1978) and cause malabsorption of carbohydrate (Wahren and Felig, 1976; Wagner et al., 1978), protein (Sakurai et al., 1975), and fats (Koerker et al., 1978; Sakurai et al., 1975) an increase in fecal lipids might be expected in somatostatin treated animals; however, increased fat excretion was not observed in this experiment. The lack of influence of PZ-SRIF on fecal lipids may have been caused by the increased fat content of the diet. It was originally believed that since PZ-SRIF is proposed to decrease pancreatic exocrine secretion (Folsch et al., 1978; Chariot et al., 1978) and consequently pancreatic lipase, any possible malabsorptive effects would be intensi- fied on a diet containing 25% rather than the usual 4% fat. However, an increased fat content could have served as an inducer for increased lipase output since pancreatic enzymes 62 adapt to the type of food normally ingested (Felber et al., 1974). Hence, the dose of somatostatin may not suppress secretin, CCK-PZ and pancreatic exocrine secretion suffi- ciently to offset an increase in lipase caused by the high fat diet. According to Losowsky et a1. (1974), over 80% of pancreatic exocrine function must be lost or enzymes in the duodenal juice must be less than 10% of normal levels before steatorrhea will result. In the previous positive tests for malabsorptive effects of somatostatin, blood levels of the ingested or infused nutrient were monitored for 2-3 hours during somatostatin infusion. Blood levels of glucose, xylose (Wahren and Felig, 1976), galactose (Wagner et al., 1978), amino-nitro- gen (Sakurai et al., 1975) and triglycerides (Sakurai et al., 1975; Koerker et al., 1978) were significantly lower than controls. By possible concomitant delay in gastrointestinal motility (Tansy et al., 1978), gastric emptying (Bloom et al., 1975), and splanchnic blood flow (Wahren and Felig, 1976) absorption could be merely delayed but not diminished. Thus, if absorption were delayed, increased fecal lipids would not necessarily be expected. Results of the xylose tolerance test showed no impaired intestinal absorption for PZ-SRIF treated rats. However, the length of collection of excreted xylose, 24-hours, would also not differentiate the possibility of delayed absorption. SUMMARY AND CONCLUSIONS Male Sprague-Dawley (250 9) rats treated with PZ-SRIF (200 ug/kg body weight) for seven days after an initial pre-treat- ment for sixteen days of PZ-SRIF (100 ug/kg body weight) showed a significant decrease in body weight gain. The level of PZ-SRIF used for this treatment was also suffi- cient to suppress the immunoreactive insulin and glucagon content and secretion of pancreatic islets isolated from these animals. The decreased gain in body weight in these rats was coupled with a significant decrease in food intake but no change in fecal lipids or xylose absorption. Somato- statin did not alter fasting serum levels of glucose, insulin or pancreatic glucagon in experimental rats but selectively and significantly decreased total glucagon which includes gastrointestinal as well as pancreatic glucagon. It is suggested, therefore, that the observed weight loss in PZ-SRIF treated rats was due to a decrease in food intake rather than a decrease in nutrient absorption. Enteroglucagon, which may be involved in nutrient homeo- stasis, was also decreased by somatostatin. Whether lowered gut glucagon is causally' related to, or merely an effect of, decreased food intake is unknown. 63 64 Although not the central focus of this study, it is suggested that somatostatin could influence food intake by its 1) central hypothalamic location and neural influence 2) peripheral gastrointestinal location and/or 3) inhibition of metabolites and hormones. 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HHmm1NN NwN + mHm Nmm + NmN NmN + HN NmH + Nm com + mom mumoo mgoo; HN NmN H mmH Nam H mom NmN H mm NmH H HN Hoe H HNm HHHmm1No wN H mHo cm H mHo mN H omH mH H mmH mHH H HmHH mHoELoz HLH + com HHH com HHH HLH + OH HHo om HHo mgao; NNHonH\:: uonHND: NcoHHoLoom cHHamcH Hcoucou cHHomcH muonH oHaooLocoo oonHomH Na :oHHoLoom too No acmucoo :HHomcH o>HuuoohocossH co ucosuomgp HHHmmuNov :Huoumouosom ucHN ocHEouogo uHcoggu Ho uumHmN .m< mHaop xHucooo< 70 .muonH HoeLo: cog» HHo.ovov ooon aHucooHHHcmHmN .muonH Hosoo: cusp Hmo.ovov Loon NHpcooHHHcmHm .pcosuoogu umoo mgao; HN oouonzocH oco oopoHomH mNoo =o>om HH\H: HON No HoonHoH HNHH coonHH LHH HHNm-Na Hx\mz ooH HHH: HHHHHHH Hum; .pcmEHomgp goumo NHouoHvoEeH oouonoocH oco oouoHomH mama co>om HHHH: HON Na HoonHoH HHHH coonHH LoH HHHm-Na HJHH: ooH HHHz HHHHHLH mom; 0 Low HHmmuNa zoom muonHm LOH HHmmnNm EoLH muonH H .muog usmHoz oHnooooEou ompoogucz Eosm muonHm .ng< + com :Huv .ochwmgo HE ooHNmE OQN moHo omooon smH; HH new mH35 oomv HENmE oom .omouon smH; Hm "ng< + om =Huv ochHmoo HE ooHNmE oON maHo mmoooHu 3oH HN “HaHm omv HE ooH\me om .omooon :oH HH cH mgao; N Low wouoaaocm mponHN .muonH uanm Low mooHo> mucomogomg :oHumLoom comooaHm Ho zoos zoom moms: HuHmmuNo Hmoo mono; HN coo HHNmuNa Lom :ooo muonH o>Hozp oco mHmHmH Hmsgo: HgmHo go» moaHo> mpcomogoog acoucoo :omooon uonH goH come on» woos; sz H mcomzH mHHmmuNo NomN H mmm NOHH H HmN NNwm H mmH NNoH H NmN HNH H mmmN Hmoo mgoo; HN NomN H HHm NOHH H HmH NNwm H NmN NNoH H NoH oHNH H NmmH HHHNmuNo omN H momH OHH H mom Nmm H mmmN NoH H NHN mHm H Nmmm mHoELoz HHH + com HHo oom HHo HLH + OH HHo om HHo HHHHH\HH mgoo; NNHonHNmo acoucoo comooon NcoHuogoom comouoHu HmuonH oHpoooocmo copoHomH An :oHuoLuom oco Ho ucmucoo comooon o>HuooogocaeeH :o acoeumogu HHHNmuNoV cHuopmouosom ocHN mcHEouoLa uHcoggu Ho HooNNm .H< m HHHH xHHHHHHH BIBLIOGRAPHY BIBLIOGRAPHY Ahlskog, E. and Hoebel, B.G. 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