Propensity to form conditioned taste aversions augments anorexia in obese (ob/ob) mice with B16 melanoma.

Ob/ob mice (OB) with B16 melanoma become anorectic, but lean mice (LN) do not. Present studies suggest that this difference reflects a greater bent for OB to form conditioned taste aversions (CTA). In Exp 1, healthy OB formed stronger CTAs than LN to a saccharin taste paired with lithium chloride (LiCl, 3 mEq/kg ip). In Exp 2, the OB-LN difference of Exp 1 was decreased by giving naltrexone (10 mg/kg sc) before LiCl, which suggested opiate involvement. Exp 3 showed that OB tumor anorexia vanishes if foods dissociated from tumor growth are given: OB fed a constant diet became anorectic 16 days after B16 inoculation; giving a new diet on Day 16 delayed anorexia onset for 8 days; a second new diet on Day 32 abolished anorexia for 24 hr. LN with tumors ate all diets at nontumor control levels. OB survived melanoma longer than LN regardless of diet, but OB fed a varied diet died first; thus, anorexia may enhance OB survival.

Reduction of learned helplessness by central administration of quaternary naltrexone.

Prior research has established that escape impairment resulting from prior inescapable shock (IS) could be reversed by the peripheral administration of the opiate antagonist naltrexone (NTX), but not the quaternary form of naltrexone (QNTX), which when systemically administered, does not readily pass the blood-brain barrier. As it was unclear whether the failure of systemically administered QNTX to reduce shuttle escape deficits following exposure to IS could be attributed to reasons other than the restricted access of QNTX to receptor sites in the brain, rats were affixed with chronic indwelling ventricular cannulae to allow direct brain administration of QNTX. The present experiment found a significant attenuation of the escape deficit produced by prior inescapable shock following the intracerebroventricular (ICV) administration of QNTX (10 micrograms/rat). These data provide further evidence of a mediational role for central opiate receptors in the expression of escape interference following inescapable shock.

Central mediation of naloxone-induced anorexia in the ventral tegmental area.

Central naloxone injections were used to show that endogenous opioids in the ventral tegmental area (VTA) regulate consumption of palatable foods. Peripheral injections of naloxone were more effective in reducing the consumption of a sweet solution in normally fed rats than in animals maintained at 85% of their free-feeding body weight. A dose of 10 micrograms/side naloxone injected into the VTA reduced consumption in normally fed rats, whereas a dose of 25 micrograms/side did the same in food-restricted animals. The inactive isomer, (+) naloxone, did not reduce consumption; the effect has chemical specificity. Naloxone injected 0.5, 1.0, or 2.0 mm above the VTA did not reduce consumption; the effect has anatomical specificity. Naloxone effectively decreases the eating of palatable foods, but not eating for survival. This has important implications for the use of opioid antagonists in weight-loss programs.

Opioid-induced linear running in obese (ob/ob) and lean mice.

Earlier research has shown that opioids stimulate behavioral activation in mice whereas opioid antagonists attenuate this activation. We conducted an experiment to determine the dose-response curve of FK33824, a potent Met-enkephalin analogue. FK33824 produced an unusual form of behavioral activation we called "linear running" in which the mice ran continuously in one direction and were nearly oblivious to environmental stimuli. This may be the kind of running that occurs naturally during migration. Wheel running activity of genetically obese (ob/ob) and lean (C57BL/6J ?/+) mice was measured following the intracerebroventricular infusion of 0.1, 1.0, 10.0 and 100.0 ng of FK33824. The lowest dose did not alter baseline running, whereas the 1.0 and 10.0 ng doses significantly increased running in both genotypes. We found a genotype difference with the highest dose tested, the lean mice ran at baseline levles and displayed ataxia whereas the obese mice continued to show increased running without ataxia. We hypothesize that genetic differences in the enkephalin mechanisms of C57 lean and obese mice are responsible for linear running.

Decreased glucocorticoid binding and receptor activation in brain of genetically diabetic (MDB/MDB) mice.

Binding of [3H]triamcinolone acetonide (TA) to cytosolic receptors and subsequent in vitro activation of glucocorticoid-receptor complexes were studied in whole brain and liver from misty diabetic mice (mdb/mdb) and their control littermates (??/++). Binding was specific for glucocorticoid receptor (GcR) since the specific glucocorticoid, RU26988, was used to compete with [3H]TA for binding. Reduced [3H]TA binding was observed in whole brain and liver in diabetic animals when compared to control animals. Within the brain, binding was significantly (P less than 0.05) decreased in cortex, hippocampus, and hypothalamus. No significant differences in binding were found in the striatum or "midbrain". GcR binding was similar in diabetic and control animals until 2 months of age when overt diabetic symptoms appeared and the GcR binding was lower in diabetic animals. Though GcR from mdb/mdb brain cytosol could be thermally activated, the extent of activation was significantly (P less than 0.05) less than that for controls. These data indicate that GcR in liver and brain cytosol are decreased in mdb/mdb mice and that the GcR available for binding in mdb/mdb brain cytosol appears less capable of undergoing activation and binding to DNA-cellulose than GcR from control brain cytosol. Decreased GcR activation in brain cytosol from mdb/mdb mice was associated with increased dissociation of [3H]TA from the GcR. These results suggest that the decreased negative feedback previously observed in diabetic animals may be due to decreased binding of hormones and a decreased level of activation of hormone bound receptor complexes.

Shock controllability and opioid substrates of escape performance and nociception: differential effects of peripherally and centrally acting naltrexone.

Rats exposed to inescapable shock exhibited analgesia and a significant impairment of shock-escape learning in a shuttle box situation 24 hr later. In contrast, rats exposed to escapable shock or to no shock displayed neither effect. Naltrexone (10 mg/kg) significantly reduced the analgesia and completely eliminated the escape deficit in inescapably shocked rats but induced hyperalgesia, coupled with a marked deterioration of escape performance, in escapably shocked and nonshocked rats. The same dose of quaternary naltrexone, which has low ability to cross the blood-brain barrier, had no effect on either the antinociception or the escape deficit produced by inescapable shock, although it also induced escape impairment and hyperalgesia in rats preexposed to escapable shock or to no shock. A second experiment demonstrated that both the escape interference and the antinociceptive consequences of prior inescapable shock could be reduced partially by a much lower dose (1 mg/kg) of naltrexone but 50 times this amount of quaternary naltrexone was still without effect. These results imply that the consequences of exposure to inescapable shock are mediated by activation of central opioid processes whereas naltrexone-induced effects in escapably shocked and nonshocked animals may be peripherally mediated. The relevance of these findings to the possible role of nociception in escape performance is discussed.

Opposite effects of opiate agonists on metabolic weight loss in mice.

Morphine (M) produced a dose-related decrease in metabolic weight loss (MWL) over a three-hour period, despite the fact that it significantly enhanced locomotor activity in male mice. This conservation of energy was specific to opioid receptors because naltrexone HCl (N) blocked it. These receptors were localized mainly to the periphery because a form of N that does not enter the brain, MR2663 BR (QN) eliminated the conservation response and instead increased MWL. The increase in MWL induced by M plus QN was blocked by MR2266 (MR), a specific kappa receptor antagonist. We suggest that this increase is due to the unmasking of a kappa opioid system located in the brain that acts to enhance energy expenditure. In support of this idea, administration of the kappa receptor agonist U-50,488H (U) produced a dose-related increase in MWL to levels nearly twice that of saline controls, while markedly reducing locomotor activity. These actions were blocked by MR but not by N. They were not blocked by QN and this suggests they originate mainly in the brain. Increasing amounts of M caused increasing inhibition of metabolic weight loss induced by a constant amount of U. This supports the idea of an antagonistic relationship between the two opioid systems.

Food intake during tumor growth: anorexia in genetically obese ob/ob mice and hyperphagia in lean mice.

Recent findings indicate that obese (ob/ob) mice suffer a low incidence of lung metastasis and survive longer than lean (+/?) littermates following injection with B16 melanoma cells [34]. The present study examined the food intake of obese and lean mice during the growth of this tumor. Mice from both groups increased their food intake by small and approximately equal amounts during the first three quarters of the survival period following injection with 10(6) cells, and body weights remained fairly stable. During the final quarter, however, obese mice became anorexic whereas lean mice became intensely hyperphagic; body weights changed accordingly. Thus, food intake is differentially affected by tumor growth in this form of genetic obesity.

Serum calcitonin, calcium and thyroxine in young and old Zucker fatty rats (fa/fa).

We determined the serum levels of calcitonin (CT), calcium (Ca), and thyroxine (Ti) in lean (?/+) and fatty (fa/fa) male Zucker rats 10 weeks and 10-12 months of age. The most dramatic finding was a high level of serum CT (3.24 +/- 1.18 ng/ml) in young fatties whereas sera from young leans were all below the limit of assay detection (less than 0.120 ng/ml, p less than 0.01). Young fat rats also had elevated levels of both Ca (11.2 +/- 0.2 vs. 9.7 +/- 0.2 mg/dl, p less than 0.001) and Ti (6.7 +/- 0.48 vs. 4.72 +/- 0.28 micrograms/dl, p less than 0.01). In older animals the mean serum level of CT increased further in the fatties and became readily measurable in leans (5.67 +/- 1.94 vs. 1.49 +/- 0.55, p less than 0.01). Thyroid C-cells, identified immunohistochemically, were abundant in both leans and fatties at this age but were substantially more numerous in the fat rats (p less than 0.001). Calcium levels increased somewhat in the older leans, but still remained higher in the fat rats (p less than 0.05). Thyroxine values were essentially the same for old animals of both genotypes (5.07 +/- 0.61 vs. 5.54 +/- 0.88). Age effects were not significant for any measure in the fat animals, but in the leans there were significant age-related increases in CT (p less than 0.02) and serum Ca (p less than 0.05).

Beta-endorphin immunoreactivity in the plasma of patients with the Prader-Labhart-Willi syndrome and their normal siblings.

No significant difference was found in the range or mean values of ir-beta-endorphin in the plasma of 6 patients with the Prader-Labhart-Willi syndrome compared to 7 of their normal siblings. The hypothesis that some of the symptoms of the P-L-W syndrome are due to excessive opioid activity is not supported by measurement of peripheral levels of ir-beta-endorphin.

Genetically obese mice: resistance to metastasis of B16 melanoma and enhanced T-lymphocyte mitogenic responses.

The metastasis of B16 melanoma cells differed significantly in obese (ob/ob) and lean (+/?) female mice of strain C57BL/6J. When the mice were inoculated subcutaneously with melanoma cells at 10 to 11 months of age, the primary tumor grew more slowly in obese than in lean littermates and the frequency of lung metastasis was greatly reduced. When the mice were injected with the cells at 4 to 7 months, the primary tumor grew at the same rate in obese and lean mice, but the obese mice again showed a significantly reduced frequency of lung metastasis. That this effect was related to an enhanced immunocompetence in obese mice was supported by the finding that splenic lymphocytes of ob/ob mice showed three times the proliferative response to the T-cell mitogen concanavalin A compared with the proliferative response of lean control mice. The ob/ob mouse may provide a model for the study of enhanced immunocompetence in obese individuals.

Secretion of calcitonin in the genetically obese Zucker rat (fa/fa).

Previously we found that adult Zucker fatty rats have C-cell hyperplasia and increased thyroidal calcitonin (CT) compared to lean controls. In this study we have evaluated both secretion of CT and responsiveness to CT in order to see whether they, too, were altered. Fat rats and lean littermates, 13-15 months old, were used. CT secretion was provoked by (1) feeding for 2 hr after an 18-hr fast, (2) giving pentagastrin iv, and (3) injecting CaCl2 iv. CT was measured by radioimmunoassay. Responsiveness to CT was examined by giving porcine or salmon CT iv and measuring serum Ca 1-3 hr later. For CT secretion, compared to leans the fat rats showed (1) higher fasting serum Ca and CT and a greater rise in CT after feeding, (2) a similar 5- to 10-fold increase in CT after iv pentagastrin, and (3) a greater rise in both serum Ca and CT at various times between 5 min and 3 hr after iv CaCl2. For CT responsiveness, fat and lean rats were equally responsive to iv CT in terms of the fall in plasma Ca 1-3 hr later. The results show that fat rats can secrete as much or more CT in response to provocative stimuli as lean rats and that they appear normally responsive to injected CT. Therefore, inability to release CT and insensitivity to CT do not underly the C-cell hyperplasia, increased thyroidal CT, and increased circulating CT in the fat rat.

Opiate antagonists overcome the learned helplessness effect but impair competent escape performance.

Rats exposed to inescapable shocks exhibited deficiencies in learning to escape shock in a novel situation 24 hours later (learned helplessness). Opiate antagonists (naloxone or naltrexone) blocked the learned helplessness effect, allowing efficient escape performance on the subsequent test. In contrast, these drugs impaired the performance of rats pretrained with escapable shocks and animals with no previous exposure to shock. Both effects occurred at small doses and increased substantially with higher doses. The results suggest a significant role for endogenous opiates in the induction of learned helplessness as well as in the acquisition of efficient escape behavior.

Enhanced tumor resistance and immunocompetence in obese (ob/ob) mice.

Genetically obese mice (C57BL6/J-ob/ob) have enhanced resistance to metastasis of the B16 melanoma in comparison with lean littermate controls. Here we show that this difference is not due to differences in the health status of these mice. We show also that the obese mice have enhanced immunocompetence as indicated by enhanced proliferative responses of their splenic lymphocytes to T-cell mitogens but not to a B-cell mitogen. The obese mice also produced twice as many antibody-secreting cells in their spleens in response to immunization in vivo with sheep erythrocytes as did lean mice. There were no differences between the two genotypes in cytolytic T lymphocyte activity after immunization in vivo with allogeneic cells. An opioid theory of enhanced immunocompetence could account for our results.

Increased immunoreactive dynorphin and leu-enkephalin in posterior pituitary of obese mice (ob/ob) and super-sensitivity to drugs that act at kappa receptors.

Posterior pituitaries of obese mice (ob/ob) contained significantly more immunoreactive dynorphin (P less than .01) and leu-enkephalin (P less than .01) than their lean littermates. Drinking in obese mice was stimulated by 0.3%, and feeding by 10%, of the dose of ethylketocyclazocine, a kappa receptor agonist, needed to produce extra feeding and drinking in lean mice. Obese mice also showed greater and longer lasting suppression of ingestion after MR-2266, a kappa antagonist, than did lean mice. MR-2266 was much more effective than naloxone in suppressing schedule-induced polydipsia in rats. These results indicate that kappa receptors are involved in feeding and drinking and that obesity is associated with changes in these receptors and their ligands.

Presence of immunoreactive calcitonin in the hypothalamus and pituitary lobes of rats.

We have found calcitonin-like immunoreactive material in extracts of hypothalami from six-month old, male rats. The level of this immunoreactivity, 0.21 ng/hypothalamus, is substantial considering out lower limit of detection of 0.006 ng of rat calcitonin. However, the hypothalamus contains less calcitonin-like immunoreactive material than either the anterior lobe (1.16 ng) or neurointermediate lobe (0.81 ng) of the pituitary. Taken together these three sources of calcitonin-like immunoreactive material contain less than one thousandth the immunoreactivity found in the thyroid. The exact nature of the calcitonin-like immunoreactive material found in these extrathyroidal sites and its physiological role, if any, remain to be discovered. The recent report of the occurrence of calcitonin receptors in the hypothalamus and other brain regions in conjunction with our finding of calcitonin-like immunoreactivity in the hypothalamus suggests that calcitonin-like molecules may be active within the brain.

Suckling: developmental indicator of genetic obesity in mice.

The Bar Harbor obese-hyperglycemic mouse (ob/ob) characteristically develops its obese phenotype during the postweaning period, when access to food is unrestricted and its behavioral phenotype of hyperphagia appears. In the present study suckling behavior (viz., nipple attachment latencies and total suckling time) of lean and preobese mouse pups, derived from matings of heterozygous lean patients, was measured in either fed or fasted conditions from 6 to 21 days postpartum on anesthetized dams. Twenty-hour milk deprivation selectively channeled pup activity into suckling for both preobese and lean mice, although, in general, preobese mice nipple-attached sooner and suckled longer than leans. In particular, fed preobese pups suckled longer than fed lean pups from 18 days on, providing a preweaning behavioral indicant of a genetic tendency to obesity when environmental constraints on overeating are minimized.

Obesity and the development of the diffuse neuroendocrine system.

The diffuse neuroendocrine system may play a critical role in the events of both adaptation for impending famine and of arousal from the opioid-induced conservation state. This may occur by means of the opioid and anti-opioid peptides of APUD cells acting in conjunction with shifts in biogenic amine uptake and release.

Hibernation: an opioid-dependent state?

Hibernation reduces substantially the heart rate of hamsters as well as the respiratory rate, the body temperature and the arousal level. The heart rate is reversed dramatically by the injection of low doses of Naloxone and in some cases the hamster arouses prematurely from hibernation. The effect is not due to the pain of the injection because saline injections do not produce such changes. The effect requires a pre-existing state of hibernation because Naloxone has no cardioacceleratory or arousal effect in non-hibernating hamsters that have had their heart rate and body temperature decreased substantially during hypothermia. These results suggest that endogenous opioids may contribute specifically to the state of hibernation. Moreover, a physiological role may exist for an anti-opioid system in the promotion of arousal from hibernation.