Cholecystokinin octapeptide: continuous picomole injections into the cerebral ventricles of sheep suppress feeding.

Cholecystokinin octapeptide decreased food intake in a dose-related manner when injected continuously into the lateral cerebral ventricles of sheep that had been deprived of food for 2, 4, 8, or 24 hours. In sheep deprived of food for 2 hours, as little as 0.01 picomole per minute suppressed feeding 35 percent 1 hour after beginning injection. Pentagastrin also decreased feeding in the 2-hour group, but only at a much higher dose range. Secretin had no effect. These findings support the hypothesis that cholecystokinin octapeptide acts on central nervous system structures that are involved in control of food intake.

Cholecystokinin antibody injected in cerebral ventricles stimulates feeding in sheep.

The role of brain cholecystokinin peptides in satiety was further assessed by using antibody to cholecystokinin to reduce cholecystokinin activity in the cerebrospinal fluid of sheep. Food intakes were increased approximately 100 percent during the 2-hour continuous injection of antibody into the cerebrospinal fluid. This supports the hypothesis that, during feeding, cholecystokinin is released into the cerebrospinal fluid, which transports it to the receptors that elicit satiety.

Novel treatments for obesity and osteoporosis: targeting apoptotic pathways in adipocytes.

Obesity and osteoporosis have grave consequences for human health, quality of life, and even the efficiency of the labor force and economy. However, these pathologies share a common cell progenitor, revealing a surprising target for drug research and development. Recent findings show that high adipocyte count in bone marrow is directly related to bone loss, as fat cells replace osteoblasts (or bone-forming cells). The objective of this review is to examine the importance of adipocyte apoptosis in the treatment of obesity and/or osteoporosis, with special emphasis on natural products as promising leads for drug development. We have induced in vivo adipocyte apoptosis, using leptin, ciliary neurotrophic factor (CNTF), beta adrenergic agonists and conjugated linoleic acid (CLA) in rodents. The results of leptin treatments on rats are suppressed food intake, reduced body weight, reduced body fat, adipocyte apoptosis, and elevated energy expenditure. Further, leptin treatment of leptin-deficient (ob/ob) mice increases endosteal bone formation and bone mineral density. Adipocyte apoptosis has also been induced in vitro using tumor necrosis factor-alpha (TNF-alpha), (-)-epigallocatechin gallate (EGCG) from Camellia sinensis and ajoene, from Allium sativum. Natural products have potential for inducing apoptosis of adipose tissue, inhibiting bone marrow adipogenesis and increasing the expression of osteogenic factors in bone, thereby yielding effective treatments for obesity and osteoporosis.

Central nervous system cholecystokinin and the control of feeding.

There is still much to learn of the sites and mechanisms of action of brain CCK peptides and their interaction with other brain peptides and neurotransmitters. The findings obtained from studies in sheep and other species provide evidence for brain CCK peptides functioning as important transmitters of hunger and satiety signals. We have hypothesized that signals, either neural or humoral, peripherally generated as a result of feeding induce secretion of CCK from specific (paraventricular) brain sites into the CSF (FIG. 1). Specialized ependymal cells, such as tanycytes, may take up CCK from the CSF for transport to receptor sites which mediate CCK's specific functions (such as changes in rumen motility, suppression of insulin secretion, and behavioral satiety). Evidence also exists to indicate the involvement of specific hypothalamic sites in either the release or the action of CCK.

CCK-octapeptide injected in CSF decreases meal size and daily food intake in sheep.

Cholecystokinin octapeptide (CCK-OP) is a potent and specific suppressor of feeding when administered as a continuous lateral cerebral ventricular injection in fasted sheep, and we have proposed that endogenous CCK-OP in the brain is released during meals and acts to terminate feeding. In previous studies, however, only relatively short-term effects of CCK-OP on feeding were examined. In the first experiment of the present series sheep were adapted to a 6-hr feeding period per day. CCK-OP injected continuously for 6 hr into the lateral ventricles reduced feeding during the entire feeding period (809 +/- 72 g, sham; 695 +/- 71 g, carrier; 505 +/- 69 g, CCK-OP; p less than 0.05). In addition mean feed intake for the two days (injection + first post injection day) was significantly reduced by CCK-OP; thus with CCK-OP, sheep did not compensate by the day after injection for the decreased feed intake on injection day. In a second experiment CCK-OP was injected into the lateral ventricles only during four consecutive 15 min meals 2 hours apart. With a dose of 0.159 pmoles/min CCK-OP, size of the second meal was reduced, but with 0.638 pmoles/min CCK-OP feeding during each of the first two meals was reduced and cumulative intake for the four meals was decreased. These results indicate that CCK-OP administered centrally can have long-term effects on feeding, and under appropriate conditions, could result in negative energy balance.

Satiety, hunger and regional brain content of cholecystokinin/gastrin and met-enkephalin immunoreactivity in sheep.

Cholecystokinin (CCK) and met-enkephalin (MEK) related peptides have been shown to alter feeding behavior subsequent to their injection into the peripheral circulation or directly into the brains of several species. To evaluate the potential role of endogenous brain pools of these peptides in feeding, groups of sheep were sacrificed either immediately following a meal (satiated) or after various intervals of food deprivation (hungry). Content of CCK-gastrin immunoreactivity in the anterior hypothalami of satiated sheep was elevated compared to 2, 4, or 24 hours of food deprivation. Content of MEK increased progressively with longer intervals of fasting (4 and 24 hours) in the amygdala and basomedial hypothalamus, whereas olfactory bulb content decreased with a similar time course. The results support a potential role for anterior hypothalamic CCK/gastrin in behaviors of satiety, whereas MEK neurons of limbic/rhinencephalic regions appear to form part of a separate circuit gradually activated by increasing hunger. Results are discussed in terms of potential target regions of the peptides, as well as the regional levels and feeding response of sheep as compared to available data from other species.

Cerebral ventricular transport and uptake: importance for CCK-mediated satiety.

Brain cholecystokinin (CCK) peptides have been proposed to be involved in the control of feed intake. We have examined the importance of the cerebral ventricular system in CCK-mediated satiety in sheep. Continuous injection of 0.64 pmol/min CCK-8 into the lateral ventricles (LV) decreased feeding, whereas injection of neither 0.64 nor 2.55 pmol/min CCK-8 into the cisterna magna (CM) significantly affected feeding. Thus, it is likely that the rostral, but not caudal, ventricular compartments and/or adjacent brain areas are involved in CCK-8 mediated satiety. The rate of injection of carrier solution (synthetic cerebrospinal fluid [CSF]) was found to affect feed intake during a continuous 75 min injection: feed intakes were greater during injection of sCSF at 0.10 ml/min than during either 0.03 ml/min sCSF or no injection (sham). Injection of 0.64 pmol/min CCK-8 in either 0.03 or 0.10 ml/min decreased feeding. The increased feeding during 0.10 ml/min sCSF injection may have been due to dilution of endogenous CCK released into CSF during the meal. To determine the percent recovery from CSF of exogenous CCK-8, CSF samples from CM were collected during 3 hr continuous LV injections of CCK-8 and inulin (for measurement of bulk absorption). Only 20 to 40 percent of administered CCK-8 was recovered in CM CSF. The loss of CCK-8 was probably not due to degradation in the CSF by proteolytic enzymes, since CCK-8 concentrations did not decrease during in vitro incubation at 37 degrees C for up to 24 hr. We propose that CCK-8 is released during feeding into the ventricular system, and subsequently taken up from CSF by specialized ependymal cells for transport to sites of action.

Differential distributions of cholecystokinin in hamster and rat forebrain.

Rats and golden hamsters show a differential feeding response to intracranial injections of cholecystokinin (CCK). Rats, but not hamsters reduce food intake after CCK injections into the hypothalamic paraventricular nucleus. In view of this species difference, we undertook an immunohistochemical study of the distribution of CCK-immunoreactivity in the hamster hypothalamus and remaining forebrain. CCK-immunoreactive perikarya were abundant in the neocortex, claustrum, hippocampal formation, amygdaloid complex, bed nucleus of the stria terminalis, nucleus of the lateral olfactory tract and in the magnocellular basal nucleus. CCK-immunoreactive neurons had a more restricted distribution in the diencephalon and were relatively rare in the preoptic area-hypothalamus. The only exception was the suprachiasmatic nucleus and adjacent medial anterior hypothalamus, in which CCK-immunoreactive neurons were numerous. CCK-containing perikarya were not observed in the hamster hypothalamic paraventricular and supraoptic nuclei, where they have been reported to occur in the rat. Groups of CCK-positive perikarya were also noted in the hamster thalamic paratenial and parafascicular nuclei. CCK-immunoreactive fibers/terminals were localized in the caudate and putamen, periventricular zones, dorsolateral geniculate, thalamic reticular nucleus and the superficial layer of the optic tectum. Fiber/terminal labeling was also present in those regions associated with CCK-immunoreactive perikarya. Our results indicate that the telencephalic distribution of CCK-containing neurons in the hamster appears to be similar to that reported in the rat. However, several differences occur in the diencephalon. Perhaps the most striking is that the hamster differs from the rat in having a large group of CCK-containing neurons in the suprachiasmatic nucleus, and in lacking the CCK-containing perikarya observed in the rat paraventricular and supraoptic nuclei. These differences may underly species differences in feeding responses to intracranial CCK injections and gonadal responses to short photoperiods. Our data further suggest that the distribution of neuropeptides and other neuroactive substances may not always be conserved during evolution.

Immunohistochemical distribution of cholecystokinin, dynorphin A and Met-enkephalin neurons in sheep hypothalamus.

The distribution of cholecystokinin (CCK)-, Met-enkephalin (M-ENK)- and dynorphin (DYN)-like immunoreactive perikarya were examined in the sheep hypothalamus using the peroxidase-anti-peroxidase technique. CCK- and DYN-containing neurons were found primarily in the suprachiasmatic nucleus (SCH) and supraoptic nucleus (SO). No CCK- or DYN-containing neurons were found in the paraventricular nucleus (PVN). M-ENK-containing neurons were found mainly in the PVN of the hypothalamus. In addition, M-ENK neurons were found in the dorsomedial (DMH), lateral (LH), anterior (AH) and periventricular hypothalamic areas. The distribution of these neuropeptides may provide a basis for understanding differences in responsiveness to centrally administered peptides.

CCK-octapeptide injected into cerebral ventricles of sheep decreases plasma insulin level.

Cholecystokinin-8 (CCK-8) was injected into the lateral cerebral ventricles (LV) of conscious sheep to determine its effect on secretion of three hormones important in regulation of peripheral metabolism: growth hormone (GH), glucagon, and insulin. Three hr continuous LV injection of 0.64 pmol/min CCK-8 decreased plasma insulin concentration approximately 30% throughout the three hr period, compared to control injection, and this effect occurred independently of whether or not the sheep were permitted to eat during injection. Only high doses of desulfated CCK-8 affected plasma insulin levels, thus suggesting the involvement of specific CCK receptors. Plasma glucose and GH concentrations were unaffected by LV injections, but plasma glucagon concentration was also decreased by CCK-8. LV injection of 0.64 pmol/min CCK-8 in sheep given an IV bolus injection of glucose (1.0 g/kg body mass) resulted in significantly lower plasma insulin and higher plasma glucose concentrations than during control LV injections; thus CCK-8 is capable of suppressing insulin secretion even under stimulated conditions. Since cisterna magna injections of CCK-8 were not effective in suppressing insulin secretion, more rostral brain sites are probably responsible for mediating this effect.

Cerebral ventricular injections of CCK-octapeptide and feed intake: the importance of continuous injection.

CCK-octapeptide (CCK-OP) is a potent suppressor of feed intake when administered as a continuous injection into the lateral cerebral ventricles of sheep. We compared the effect on feeding of CCK-OP given as bolus and continuous injections to assess the importance of the continuous injection method. A total dose of 30.6 pmoles of CCK-OP given as one, four, or weight bolus injections had no effect on feed intake; whereas the same total dose given as a 45 min continuous injection reduced feed intake 80 percent. We also compared the effects of beginning the continuous injection immediately before, 5 min before, and 15 min before the sheep began a meal. Feed intakes were significantly lower when CCK-OP injection was begun 15 min before the meal, than whe n injection was begun immediately before or 5 min before the meal, suggesting that the site of action of CCK-OP for its effect on feeding is not the immediate vicinity of the lateral ventricles. The complete lack of effect of bolus injections of CCK-OP is unexpected; the explanation probably awaits further information on kinetics of CSF formation and CCK-OP degradation, site of action of CCK-OP, and the role of the CSF in transport of the peptide.

Circadian rhythm of feeding induced changes in hypothalamic Met-enkephalin concentrations.

Accumulating evidence suggests that opioid peptides play an important role in the hunger component of the control of food intake. The enkephalins, one of the opioid peptide families, stimulate feeding when injected into specific hypothalamic areas and endogenous concentrations change with the fed/fasted condition of rats and sheep and with phase of circadian cycle. To demonstrate a possible circadian rhythm in feeding-induced changes in Met-enkephalin (MEK), 54 male rats initially weighing 255 +/- 3 g were adapted to a 12-hr fast during the light (light-fasted) or dark (dark-fasted) phase of the circadian cycle, then sacrificed before (non-fed) or after (fed) being allowed to eat a meal. In non-fed compared with fed rats, MEK concentrations were higher in the paraventricular nucleus (PVN, 170 vs. 109 pg/mg tissue, p less than 0.05) and ventromedial hypothalamus (VMH, 209 vs. 161 pg/mg tissue, p less than 0.05) in the dark (light-fasted) but not light (dark-fasted), even though rats ate a larger meal in the light (8.6 vs. 5.0 g, p less than 0.01). In rats fed the same amount of food in the light (dark-fasted) as ad lib fed rats in the dark (light-fasted), MEK concentrations did not differ in the PVN or VMH, suggesting that circadian rhythm is more important than meal size. Rats gavaged with an amount of milk equal in calories to dark ad lib-fed rats (light-fasted) had MEK concentrations not different from light-fasted non-fed rats (216 vs. 209 pg/mg tissue, NS) suggesting that feeding behavior, pregastric stimuli and/or form of diet is important for influencing MEK concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in brain CCK concentrations with peripheral CCK injections in Zucker rats.

Evidence suggests that the satiety responses to peripherally administered CCK are mediated by a CNS component(s). Since CCK concentrations in the hypothalamus can change with degree of hunger, they may also be involved in the feeding response to peripherally administered CCK. Six-hr fasted rats were administered saline or 2 micrograms/kg CCK-8 and half were allowed to eat a meal. They were sacrificed after a meal or after the fast and hypothalamic content of CCK was measured by RIA. In rats injected with CCK, compared with those injected with saline, CCK concentrations were decreased in the ventromedial hypothalamus (VMH, 39 vs. 47 pg/mg tissue, p less than 0.004) and dorsomedial hypothalamus (17 vs. 21 pg/mg, p less than 0.009) and increased in the lateral hypothalamus (28 vs. 19 pg/mg, p less than 0.01). CCK concentrations in fed compared with fasted rats were higher in the VMH (47 vs. 39 pg/mg, p less than 0.002) and in obese compared with lean rats CCK concentrations were higher in the paraventricular nucleus (48 vs. 38 pg/mg, p less than 0.05), suprachiasmatic nucleus (46 vs. 34 pg/mg, p less than 0.008) and VMH (52 vs. 34 pg/mg, p less than 0.001). Since peripheral injections of CCK influenced concentrations of CCK in hypothalamic areas associated with feeding, these results provide evidence that the feeding response to peripherally injected CCK may be mediated by changes in CCK content of specific brain areas.

Changes in brain met-enkephalin concentrations with peripheral CCK injections in Zucker rats.

There is increasing evidence that peptides in the brain are important in the control of food intake. Administration of opioid and CCK peptides have elicited hunger and satiety, respectively. To evaluate the interaction of these peptides and their role in the central nervous system, concentrations of met-enkephalin were measured in the hypothalamus of rats following peripheral administration of CCK; in addition, effects of feeding and fasting and obesity were studied. In CCK- vs. saline-injected rats met-enkephalin concentrations were decreased in the paraventricular nucleus (PVN), suprachiasmatic nucleus (SC), supraoptic nucleus (SON), dorsomedial hypothalamus (DMH) and ventromedial hypothalamus (VMH). In fed compared with fasted rats met-enkephalin concentrations were higher in the anterior hypothalamus (AH) and lower in the SC; in obese compared with lean rats, concentrations were higher in the AH, PVN, SC, SON, DMH, lateral hypothalamus and VMH. These results show that peripheral injections of CCK can decrease concentrations of met-enkephalin in the brain and suggest a mechanism by which these peptides may interact to influence behavior. In addition, the findings support the hypothesis that the hyperphagia which is typical of obese rats may be due to increased concentrations of met-enkephalin.

Meal-stimulated increased concentrations of beta-endorphin in the hypothalamus of Zucker obese and lean rats.

Increased opiate peptide concentrations in brain and plasma have been associated with increased feeding. The role of beta-endorphin in the control of food intake and obesity was examined by measuring concentrations in hypothalamus, pituitary and plasma of hungry (6-hr fasted) and satiated (5 min after a meal) Zucker obese and lean rats. beta-Endorphin concentrations (1) in satiated vs. hungry rats were increased in the VMH (90 vs. 79 pg/mg tissue, p less than 0.05) and decreased in the supraoptic nucleus (65 vs. 78 pg/mg tissue, p less than 0.05), (2) in obese vs. lean rats were decreased in the VMH (79 vs. 90 pg/mg tissue, p less than 0.05) and (3) in female vs. male rats were increased in the anterior hypothalamus (123 vs. 59 pg/mg tissue, p less than 0.01) and VMH (90 vs. 79 pg/mg tissue, p less than 0.05). Analysis of a phenotype by feeding condition interaction revealed that obese but not lean rats had higher beta-endorphin concentrations in the satiated vs. hunger condition. However, plasma beta-endorphin concentrations did not differ with feeding condition, phenotype or sex. Intermediate and posterior but not anterior pituitary beta-endorphin concentrations were lower in obese than lean rats. Thus, there is some evidence for a relationship between beta-endorphin concentration and feeding in the hypothalamus, but beta-endorphin concentrations in plasma do not appear to be influenced by feeding condition or obesity.

Meal-stimulated increased concentrations of CCK in the hypothalamus of Zucker obese and lean rats.

CCK is a putative satiety peptide found to be active when administered peripherally and centrally. Concentrations of CCK have been measured in the brains of fed and fasted animals, but as yet no clear correlation with feeding has been found. In the present experiment rats were sacrificed after a 6-hr fast or 5 min after a meal. Areas of the hypothalamus were removed from these rats and assayed for CCK content. The relationship between obesity and CCK content in specific areas of the brain was also investigated by using Zucker obese and lean rats. In fed rats the CCK concentrations were higher than in fasted rats in the ventromedial hypothalamus (VMH) (56 vs. 42 pg/mg tissue, p less than 0.005), lateral hypothalamus (38 vs. 27 pg/mg, p less than 0.01) and supraoptic nucleus (48 vs. 39 pg/mg, p less than 0.01). In obese rats the concentrations were higher than in lean rats in the VMH (56 vs. 41 pg/mg, p less than 0.003), dorsal medial hypothalamus (37 vs. 30 pg/mg, p less than 0.04) and anterior hypothalamus (61 vs. 37 pg/mg, p less than 0.001). Average concentrations of CCK in all hypothalamic areas were higher in females than males (50 vs. 40 pg/mg, p less than 0.001). Thus, CCK concentrations in specific areas of the hypothalamus increased with feeding, supporting the potential role of CCK in the central nervous system as a satiety peptide. Further, although the concentrations of CCK in obese rats were higher than those in lean rats, the changes in CCK concentration with feeding were the same, showing that obesity is not a consequence of decreased concentrations or concentration changes of CCK in brain.

Intestinal infusion of a liquid diet alters CCK and NPY concentrations in specific brain areas of rats.

Cholecystokinin (CCK) and neuropeptide Y (NPY) have been implicated in the control of food intake in a number of species. This study was carried out to determine 1) whether nutrient-related stimulation of the upper small intestine could activate central CCK and NPY neuronal systems, resulting in changes in concentration of these peptides in specific brain areas, and 2) the influence of the circadian cycle on nutrient-related effects. Four groups of rats received treatments of either 1.0 ml saline (S) or Ensure liquid diet (E) infused into the duodenum either during the dark (D) or light (L) phase of the circadian cycle. CCK and NPY concentrations in extracts of specific brain areas were measured by RIAs. CCK concentration in the supraoptic n. (SON) was higher in D than in L, regardless of infusion treatment, and in the dorsal parabrachial n. area (DPN), CCK concentration was higher in E than S infused rats, regardless of circadian phase. CCK concentration in the dorsal motor vagal n. area (DMV) was higher in E, but only during L. NPY concentration was higher in DPN and paraventricular n. areas (PVN) and lower in the suprachiasmatic n. area (SC) after E, regardless of circadian phase. The changes in concentration of CCK and NPY in specific brain areas in response to food in the upper intestine suggest that nutrient-related signals from the intestine can activate specific CNS CCK and NPY-containing neural pathways.

Alpha 2-adrenoceptor blockade does not block feeding induced by neuropeptide Y in sheep.

We tested the hypothesis that blockade of central alpha 2-adrenergic receptors would prevent neuropeptide Y (NPY)-induced feeding. Nine young female sheep were fitted with lateral ventricula cannulas. Bolus intracerebroventricular (ICV) injection of 3 nmol of NPY increased feed intake after 30 min between 45 and 153% in three experiments. A bolus ICV injection of 400 or 100 nmol of the alpha 2-antagonist, yohimbine, either 5 or 30 min before NPY injection, did not attenuate this response. Instead, yohimbine increased feed intake over NPY-induced feeding by 52 to 55%. We interpret these data as evidence that the putative NPY feeding pathway in feed-sated sheep is not dependent on the type of alpha 2-adrenergic mechanism which can be blocked by ICV injection of yohimbine.

Clonidine has a bidirectional effect on operant responding for food.

Clonidine, an alpha-noradrenergic agonist, has had inconsistent effects when administered peripherally in previous studies of feeding behavior. The present experiment was undertaken to evaluate clonidine using an operant feeding paradigm (continuous reinforcement schedule) to provide detailed data on the time course of its effects. Over an entire four-hour session, all doses of clonidine tested (25, 50, or 100 micrograms/kg) increased bar-pressing. The 50 micrograms/kg dose was most effective. An examination of the time course of responding revealed that the initial effect of clonidine was to decrease responses with the duration and magnitude of the decrease directly proportional to dose. However, clonidine also prolonged a phase of steady responding for food once the animals resumed bar-pressing, resulting in a net increase of food intake. Future investigations of clonidine should take into account the effects of increasing dose on delayed onset of feeding.

Medial hypothalamic lesions in Syrian hamsters: characterization of hyperphagia and weight gain.

Syrian golden hamsters (Mesocricetus auratus) received bilateral lesions aimed at the ventromedial hypothalamus (VMH) or a sham lesion. In the first study, some of the animals in each surgical group were housed in standard sedentary conditions while others had free access to running wheels. The lesions produced a 30% increase in the daily intake of chow, and this was accomplished exclusively by increased meal sizes. As a result, lesioned hamsters gained body weight relative to controls both on the chow diet and in a subsequent high fat diet phase. The effects were comparable in both sedentary and exercising groups. The lesions produced increases in body length and fat content. In the second study, lesions were made in the VMH or in adjacent nuclei and, after an initial period on chow, the hamsters were then given a choice between chow and high fat diet. The lesioned hamsters showed no unusual preference for the high fat diet but, as before, those with damage to the VMH or paraventricular nucleus (PVN) showed exaggerated body weight gain. Hamsters with these lesions were hyperinsulinemic in both fed and fasted conditions at the end of the study.