Activation of Nesfatin-1-Containing Neurones in the Hypothalamus and Brainstem by Peripheral Administration of Anorectic Hormones and Suppression of Feeding via Central Nesfatin-1 in Rats.

Peripheral anorectic hormones, such as glucagon-like peptide (GLP)-1, cholecystokinin (CCK)-8 and leptin, suppress food intake. The newly-identified anorectic neuropeptide, nesfatin-1, is synthesised in both peripheral tissues and the central nervous system, particularly by various nuclei in the hypothalamus and brainstem. In the present study, we examined the effects of i.p. administration of GLP-1 and CCK-8 and co-administrations of GLP-1 and leptin at subthreshold doses as confirmed by measurement of food intake, on nesfatin-1-immunoreactive (-IR) neurones in the hypothalamus and brainstem of rats by Fos immunohistochemistry. Intraperitoneal administration of GLP-1 (100 µg/kg) caused significant increases in the number of nesfatin-1-IR neurones expressing Fos-immunoreactivity in the supraoptic nucleus (SON), the area postrema (AP) and the nucleus tractus solitarii (NTS) but not in the paraventricular nucleus (PVN), the arcuate nucleus (ARC) or the lateral hypothalamic area (LHA). On the other hand, i.p. administration of CCK-8 (50 µg/kg) resulted in marked increases in the number of nesfatin-1-IR neurones expressing Fos-immunoreactivity in the SON, PVN, AP and NTS but not in the ARC or LHA. No differences in the percentage of nesfatin-1-IR neurones expressing Fos-immunoreactivity in the nuclei of the hypothalamus and brainstem were observed between rats treated with saline, GLP-1 (33 µg/kg) or leptin. However, co-administration of GLP-1 (33 µg/kg) and leptin resulted in significant increases in the number of nesfatin-1-IR neurones expressing Fos-immunoreactivity in the AP and the NTS. Furthermore, decreased food intake induced by GLP-1, CCK-8 and leptin was attenuated significantly by pretreatment with i.c.v. administration of antisense nesfatin-1. These results indicate that nesfatin-1-expressing neurones in the brainstem may play an important role in sensing peripheral levels of GLP-1 and leptin in addition to CCK-8, and also suppress food intake in rats.

Electroacupuncture modulation of reflex hypertension in rats: role of cholecystokinin octapeptide.

Acupuncture or electroacupuncture (EA) potentially offers a nonpharmacological approach to reduce high blood pressure (BP). However, ~70% of the patients and animal subjects respond to EA, while 30% do not. EA acts, in part, through an opioid mechanism in the rostral ventrolateral medulla (rVLM) to inhibit sympathoexcitatory reflexes induced by gastric distention. CCK-8 opposes the action of opioids during analgesia. Therefore, we hypothesized that CCK-8 in the rVLM antagonizes EA modulation of sympathoexcitatory cardiovascular reflex responses. Male rats anesthetized with ketamine and α-chloralose subjected to repeated gastric distension every 10 min were examined for their responsiveness to EA (2 Hz, 0.5 ms, 1-4 mA) at P5-P6 acupoints overlying median nerve. Repeated gastric distension every 10 min evoked consistent sympathoexcitatory responses. EA at P5-P6 modulated gastric distension-induced responses. Microinjection of CCK-8 in the rVLM reversed the EA effect in seven responders. The CCK1 receptor antagonist devazepide microinjected into the rVLM converted six nonresponders to responders by lowering the reflex response from 21 ± 2.2 to 10 ± 2.9 mmHg (first vs. second application of EA). The EA modulatory action in rats converted to responders with devazepide was reversed with rVLM microinjection of naloxone (n = 6). Microinjection of devazepide in the absence of a second application of EA did not influence the primary pressor reflexes of nonresponders. These data suggest that CCK-8 antagonizes EA modulation of sympathoexcitatory cardiovascular responses through an opioid mechanism and that inhibition of CCK-8 can convert animals that initially are unresponsive to EA to become responsive.

CCK-8 and PGE1: central effects on circadian body temperature and activity rhythms in rats.

Cholecystokinin-octapeptide (CCK-8) has been shown to possess an acute thermogenic and hyperthermic action when given intracerebroventricularly in slightly restrained rats. To substantiate the febrile nature of that hyperthermia freely moving animals should be used and together with body core temperature, at least one behavioral parameter, such as general activity, should also be recorded. In the present studies, Wistar rats (N=34) exposed to thermoneutral (26-28 degrees C) or cold (4 degrees C) ambient temperature and to a 12:12-h light/darkness schedule were infused intracerebroventricularly with CCK-8 or prostaglandin E1 (PGE1) for several days using ALZET minipump and changes in body core temperature and general activity were recorded by biotelemetry (Minimitter). In rats exposed to a thermoneutral ambient temperature, low doses of CCK-8 induced slight but significant rises of day minima of circadian body temperature rhythm (CBTR) and with a high dose (1 microg/h) of the peptide--infused either at thermoneutrality or during cold exposure--an increase of acrometron could also be recorded. All of these changes were observed only during the first 2-4 days of 7-day-long infusions. Intracerebroventricular infusion of PGE1 administered at thermoneutrality in a dose of 1 microg/h for 7 days induced a marked rise in body core temperature with a disappearance of CBTR in some rats for 2-3 days or with rises of day minima/acrometron in others. General activity--running parallel with CBTR in periods without infusions--tended to be decreased when core temperature rose during the first couple of days of intracerebroventricular infusion of higher doses of CCK-8 or of PGE1. The decreased general activity--one component of sickness behavior--together with an increased body core temperature found in the present study, supports the view that they are components of a genuine fever induced by the central effect of the two mediators used.

Differential startle reactivity following central CCK-8S and systemic Boc CCK-4 administration in mice: antecedent stressor history and testing condition.

The influence of intraventricular cholecystokinin-8S (CCK-8S) and systemic N-t-Boc-Trp-Met-Asp-Phe-amide (Boc CCK-4) was evaluated in the acoustic and fear-potentiated startle paradigms in CD-1 mice. In the light + tone startle condition. CCK-8S increased startle 168 hr after administration, compared with saline. In the tone startle condition, CCK-8S decreased startle immediately and 24 hr after administration, compared with saline. Among nonshocked mice, CCK-8S increased startle at 48 and 168 hr, compared with saline. In the light + tone condition, 5 microg Boc-CCK-4 did not influence startle, whereas 15 microg Boc CCK-4 decreased startle immediately, 24 hr, and 48 hr following administration. Results demonstrate that antecedent environmental experiences interact with subsequent pharmacological challenges in provoking the temporal expression of alterations in startle magnitude.

A possible interaction between CCKergic and GABAergic systems in the rat brain.

Cholecystokinin sulfated octapeptide (CCK-8S) was given to rats i.p. at single doses of 10 and 100 nmol/kg, respectively. It produced a modification in GABA levels in several areas of the rat brain. After 30 min of injection, the lower dose (10 nmol/kg) increased GABA levels in striatum by 31% (P<0.05). The higher dose (100 nmol/kg) enhanced GABA levels either in hippocampus by 78% (P<0.05) or in frontal cerebral cortex by 81% (P<0.05) and decreased in olfactory bulbs by 57% (P<0.01). Thus, these results show that systemic injection of CCK-8S, produced regional specific changes on GABA levels in brain, and these effects were dose-dependent. Systemic pretreatment with the CCK(B) receptor antagonist, PD 135,158, 1 mg/kg i.p., on the endogenous levels of GABA in certain regions was also studied. The selective CCK(B) receptor antagonist, PD 135,158, did not have an effect per se on the endogenous levels of GABA but prevents the action induced by the neuropeptide. We suggest that the action of CCK may be mediated via a selective action on the CCK(B) receptor subtypes.

Brain regions where cholecystokinin suppresses feeding in rats.

The gut-brain peptide, cholecystokinin (CCK), inhibits food intake when injected either systemically or within the brain. To determine whether CCK's effect in the brain is anatomically specific, CCK-8 (0. 8, 4, 20, 100, 500 pmol) was microinjected into one of 14 different brain sites of rats, and its impact on subsequent food intake was measured. CCK-8 at 500 pmol significantly suppressed intake during the first hour post-injection following administration into six hypothalamic sites (anterior hypothalamus, dorsomedial hypothalamus, lateral hypothalamus, paraventricular nucleus, supraoptic nucleus, ventromedial hypothalamus) and two hindbrain sites (nucleus tractus solitarius, fourth ventricle). Although lower doses were sometimes effective (anterior hypothalamus, dorsomedial hypothalamus, nucleus tractus solitarius), there appeared to be no significant difference in potency among sites. Injections into the medial amygdala, nucleus accumbens, posterior hypothalamus, dorsal raphe, and ventral tegmental area were either ineffective or produced a delayed response. The higher doses required for most sites, as well as the widespread effectiveness of CCK-8 within the hypothalamus, suggest that spread of CCK-8 to adjacent brain sites, and (or) to the periphery, may have been required for anorexia to occur. Findings reported in an accompanying paper provide strong evidence that paraventricular nucleus injection of CCK-8 (500 pmol) did not increase plasma CCK-levels sufficiently to suppress feeding by a peripheral mechanism. Together, these results suggest that CCK may be acting as a neurotransmitter or neuromodulator within two different brain regions to produce satiety - one region which includes the nucleus tractus solitarius in the hindbrain, and another more distributed region within the medial-basal hypothalamus.

Decelerating gastric emptying: therapeutic possibilities in type 2 diabetes.

There is clear evidence of a positive correlation between carbohydrate absorption, plasma concentration of glucose, and the rate of gastric emptying. This suggests that clinical manipulation of gastric emptying rates may have therapeutic potential in glycaemic control. Cholecystokinin (CCK-8) has been shown to delay gastric emptying in individuals with Type 2 diabetes, but its potential as a therapy is limited by the need to administer it intravenously. The preferred routes of administration would be intramuscular injections, an intranasal spray or the use of orally ingested CCK analogues. Alternatively, the oral administration of an agent that enhances endogenous release of CCK could represent an important approach to the treatment of Type 2 diabetes. Agents such as POT II may have a therapeutic indication in patients with recently diagnosed Type 2 diabetes.

Treatment of parenteral nutrition-associated cholestasis with cholecystokinin-octapeptide.

The authors investigated whether parenteral nutrition-associated cholestasis (PNAC) in surgical neonates could be alleviated by the administration of cholecystokinin-octapeptide (CCK). Two groups of infants were studied, after major abdominal or cardiac surgery in the newborn period. The low-dose group consisted of three infants with PNAC who received cholecystokinin-octapeptide (Sincalide) at a dose of 0.02 micrograms/kg intravenously (IV), twice daily. The high-dose group comprised eight infants with PNAC who received an initial dose of 0.02 micrograms/kg IV or intramuscularly, three times daily on the first day, followed by a daily doubling of the dose up to as high as 0.32 micrograms/kg. In the low-dose group, direct bilirubin levels declined a mean of 50.2 +/- 14.5%. In the high-dose group, direct bilirubin levels declined a mean of 23.4 +/- 14.3%. In three patients in the high-dose group, no decline occurred. All three had clinical signs of overt liver failure and died of liver failure within 2 months after treatment with CCK. By excluding these patients from the high-dose group, the decline in bilirubin levels increased to 49.6 +/- 10.9%. Side effects from CCK occurred in two patients and consisted of abdominal pain and feeding intolerance. Treatment with CCK appears to be associated with a decline in direct bilirubin levels, provided overt liver failure has not developed.

Inhibition of serotonin synthesis attenuates inhibition of ingestive behavior by CCK-8.

Ingestive behavior was activated in male rats by intraoral infusion of a 1-M solution of sucrose. Injection of cholecystokinin octapeptide (CCK-8; 1.6 or 5.0 micrograms) inhibited ingestion of the sucrose solution and increased the concentration of 5-hydroxytryptamine (5-HT) in the paraventricular hypothalamic nuclei. The inhibitory effect of the low, but not the high, dose of CCK-8 was attenuated by depleting 5-HT in the brain with p-chlorophenylalanine (PCPA; 100 mg/kg for 3 days). Treatment with 5-hydroxytryptophan (20 mg/kg) increased the concentration of 5-HT in the brain of rats pretreated with either NaCl or PCPA and enhanced the inhibitory effect of CCK-8 on ingestive behavior in the PCPA-, but not NaCl-, treated rats. 5-HT may play a role in the mechanism of action of CCK-8 but additional factors must be involved.

Role of hypothalamic cholecystokinin octapeptide in the colonic motor response to a meal in rats.

The effects of hypothalamic microinfusions of cholecystokinin octapeptide and its antagonist L364,718 on cecocolonic myoelectrical activity were evaluated by electromyography in fasted and fed rats. The rats were chronically fitted with electrodes implanted on the cecum and proximal colon and cannulas placed bilaterally in either the ventromedial or lateral hypothalamus. In fasted rats, microinfusion of cholecystokinin octapeptide (10 ng/kg) into the ventromedial hypothalamus increased the spike-burst frequency of the cecum and the colon by 45.6% and 43.7%, respectively, during the 30-minute period after treatment. The injection of cholecystokinin octapeptide (10 ng/kg) into the lateral hypothalamus had no effect on either cecal or colonic motility. Feeding increased the frequency of cecal and colonic spike bursts by 52.1% and 50.1% for 30 minutes postprandially. When infused bilaterally into the ventromedial hypothalamus 10 minutes before feeding, L364,718 (1 or 5 micrograms/kg) abolished the increase of the frequency of cecal and colonic contractions induced by the meal. Infused into the lateral hypothalamus at similar dosages, L364,718 had no effect on the postprandial enhancement of cecocolonic motility. Increase of cecocolonic spike-burst frequency induced by feeding or by cholecystokinin octapeptide injected into the ventromedial hypothalamus was abolished by previous intracerebroventricular but not intraperitoneal administration of atropine (1 microgram) and 4-diphenylacetoxy-N-methylpiperidine (1 microgram), a selective muscarinic M2-receptor antagonist. In contrast, pirenzepine (1 microgram, intracerebroventricularly) did not significantly reduce the meal- or cholecystokin octapeptide-induced increase in cecal and colonic motility. These results suggest that, in rats, (a) cholecystokinin octapeptide is involved in the generation of the cecocolonic motor response to a meal and these effects are mediated through cholecystokinin octapeptide receptors located in the ventromedial hypothalamic nuclei, and (b) these postprandial colonic motor changes involve central cholinergic activation through muscarinic M2 receptors.

Comparative activity of two cholecystokinin analogues with partial agonist activity: effects on food intake and brain monoamines.

Two analogues of the C-terminal heptapeptide of cholecystokinin have been synthesized, in which the C-terminal phenylalanine residue has been replaced by a phenylethylester (JMV 180) or a phenylethylamide (JMV 170) group. They have been shown to present partial agonist CCK activity on pancreatic amylase release. In this study, the effects of the two peptides were investigated on food intake and brain monoamine metabolism after intraperitoneal (IP) and intracerebroventricular (ICV) administration. Neither peptide was active on feeding after IP administration but both decreased food intake after ICV injection, with a slightly higher potency for JMV 170. JMV 180 induced no change in monoamine metabolism whatever the route of administration. JMV 170 IP decreased cortical levels of dopamine and its metabolites. This effect was stronger after ICV injection and was accompanied by changes in serotonergic metabolism in the hypothalamus and cortex. Contrary to CCK8 S, which is more active on feeding after peripheral injection, the feeding effects of the analogues obtained by modification of the C-terminal phenylalanine residue appear to involve a central site of action. Furthermore, phenylethylamide substitution (JMV 170) gives rise to greater potency on monoaminergic variations than replacement with a phenylethylester (JMV 180) and the effect is enhanced following central administration.

Cholecystokinin does not stimulate prosomatostatin-derived peptides in man.

In man, plasma cholecystokinin (CCK) and somatostatin-28 (S-28) levels increase after ingestion of a mixed meal. Both peptides originate from the gastrointestinal tract. In supra- and periphysiological doses, CCK stimulates the release of somatostatin-14 from in vitro pancreatic islets and gastric cells and increases circulating somatostatin-like immunoreactivity in dogs, leading to the conjecture that CCK regulates somatostatin-like immunoreactivity secretion. Nonetheless, whether CCK is responsible in part for the meal-induced rise in S-28 in man has not been established. Therefore, the present study was designed to determine if CCK, at both physiological and supraphysiological concentrations, increases the circulating levels of prosomatostatin (proS)-derived peptides in humans. On 3 separate days, five healthy men ate a mixed liquid meal or received iv infusions of CCK at rates of 18 or 38 pmol/kg.h. Plasma levels of pro-S-derived peptides, including pro-S, S-14, S-13, S-28, and CCK, were measured. Basal CCK levels averaged 0.9 +/- 0.1 pmol/L and increased after the meal to a peak level of 5.4 +/- 1.5 pmol/L and averaged 3.1 +/- 1.2 pmol/L over 90 min. The mean basal levels of pro-S, S-14, and S-13, measured collectively, was 6.1 +/- 0.4 pmol/L eq S14 and was unaltered by food intake. The S-28 level was 6.7 +/- 0.6 pmol/L and rose to a zenith of 13.1 +/- 3.3 pmol/L by 90 min. Infusion of CCK at 18 and 38 pmol/kg.h produced steady state plasma CCK levels of 4.1 +/- 1.1 and 9.9 +/- 1.5 pmol/L, respectively. Basal levels of pro-S-derived peptides were unaltered during the infusion of either the low or high dose of CCK. We conclude that CCK by itself is not a physiological signal to the release of pro-S-derived peptides in man.

Acute and long-lasting effects of peripheral injection of caerulein and CCK-8 on the central GABAergic system in mice.

Acute and long-lasting effects of peripheral injection of caerulein (CLN) and cholecystokinin octapeptide (CCK-8) on the gamma-aminobutylic acid (GABA) content and the GABA accumulation by aminooxyacetic acid (AOAA) in the discrete brain regions of mice were examined. The content and accumulation of GABA in the striatum, hypothalamus, and frontal cortex was measured with high performance liquid chromatography with electrochemical detection (HPLC-ECD). The GABA content slightly decreased in the striatum 60 min after CLN and CCK-8 were administered, whereas it slightly increased in the hypothalamus and frontal cortex. Moreover, with CLN and CCK-8, the GABA accumulation after AOAA treatment decreased in the striatum and hypothalamus 30 min after injection. Meanwhile, when administering CLN, the GABA content as well as the GABA accumulation after AOAA treatment increased in the striatum and frontal cortex 1 day after injection, and continued to increase the second and third day in the striatum. These results showed that peripheral injection of CLN and CCK-8 had effects on the central GABAergic system with local specific actions, and also the long-lasting and time-dependent biphasic effects of CLN.

[Antagonism of morphine analgesia and electroacupuncture analgesia by cholecystokinin octapeptide (CCK-8) administered in periaqueductal gray (PAG) of the rabbits].

We have reported that intracerebroventricular (i. c. v.) injection of 1-4 ng of CCK-8 to the rat produced a remarkable antagonistic effect on morphine analgesia. In order to study the species specificity and the site of action, CCK-8 was microinjected into the PAG of the rabbit, and its influence on morphine analgesia and electroacupuncture analgesia was observed. The latency of the escape response (ERL) to radiant heat focused on the snout was measured as an index of the pain threshold. Microinjections were made via cannulae chronically implanted into the PAG. The drug solutions were delivered in a volume of 1 microliter, at a speed of 0.125 microliter/min. The ERL was measured for a period of 60 or 70 minutes at 10 min intervals. 1. CCK-8 administered unilaterally to the PAG of the rabbit at a dose of 3 ng antagonized the analgesia induced by morphine (4 mg/kg, i. v.) by 73% (P less than 0.001), and reduced the analgesic effect of electroacupuncture by 67% (P less than 0.001). These effects were dose-dependent within the range from 1.5 ng to 6.0 ng. The effect of CCK-8 was reversed by CCK receptor blocker proglumide (4 microliters, intra-PAG injection). Unsulfated CCK-8 (CCK-us) had no effect in this regard. These results indicate that in the PAG of the rabbit, exogenously administered CCK-8 was capable of antagonizing opioid analgesia by the activation of CCK receptors. 2. Two groups of rabbits were given with morphine (2 mg/kg, i. v.) and simultaneous injection of CCK-8 antiserum (CCK-AS, 1 microliter) or normal rabbit serum (NRS) into the PAG.(ABSTRACT TRUNCATED AT 250 WORDS)

Cholecystokinin octapeptide (CCK-8): antagonism to electroacupuncture analgesia and a possible role in electroacupuncture tolerance.

The analgesic effect produced by electroacupuncture (EA) stimulation in the rat was dose-dependently antagonized by cholecystokinin octapeptide (CCK-8) administered intracerebroventricularly (i.c.v.) or intrathecally (i.th) at a dose range of 0.25-4 ng. This effect had an immediate onset and lasted for at least 4 h. CCK-8 per se, however, did not affect baseline tail flick latency. Rats subjected to prolonged EA stimulation developed EA tolerance as well as cross-tolerance to morphine. These tolerances could be postponed or reversed by i.c.v. or i.th injection of antiserum against CCK-8. While CCK-8 antagonized opioid analgesia, it did not affect analgesia induced by 5-hydroxytryptamine (5-HT) or norepinephrine (NE). Moreover, CCK-8 antiserum did not alter the basic level of nociception, nor did it potentiate EA analgesia in naive rats. It is concluded that prolonged EA stimulation results in a profound release of opioids which may trigger the release of CCK-8 in the central nervous system to counteract the opioid component of EA analgesia. This mechanism may account, at least in part, for the development of EA tolerance.