Peptides as central regulators of feeding

During the past decade there has been an increased awareness of the role peptides play as neuromodulators. In this article we review the available data on peptides as central regulators of food ingestion. We stress the possible problems of non-specific effects. We stress that whereas many peptides decrease feeding after central injection, only two families of peptides have been shown to increase feeding after central injection. These are the opioid family and the pancreatic polypeptide-neuropeptide Y family. The putative role of corticotropin releasing factor as the mediator of norepinephrine and serotonin effects on feeding is discussed.     

Brain dynorphin and enkephalin systems in Fischer and Lewis rats: effects of morphine tolerance and withdrawal

Lewis rats are more likely to self-administer various drugs of abuse than Fischer rats. Here these two strains of rats were compared with regard to basal brain opioid peptide levels and the response to chronic morphine treatment and to naloxone-precipitated withdrawal. Lewis rats had lower basal dynorphin peptides in the substantia nogra, striatum (not Leu-enkephalinArg⁶) and VTA (not dynorphin B) and the pituitary gland. Leu-enkephalinArg⁶ levels were also lower in these structures (with the exception of striatum which had higher levels) and in the nucleus accumbens. There were also strain differences in the response to chronic morphine treatment; in the nucleus accumbens, morphine treatment increased dynorphin A levels in Fischer rats only, in the ventral tegmental area effects were opposite with increased dynorphin levels in Fischer and decreased levels in Lewis rats, in the hippocampus dynorphin levels were markedly reduced in Lewis rats only. In Fischer rats, chronic morphine strongly affected peptide levels in the substantia nigra and striatum, whereas Lewis rats responded less in these areas. Leu-enkephalin, which derives from both prodynorphin and proenkephalin, and Met-enkephalin, which derives from proenkephalin, were effected by chronic morphine mainly in Fischer rats, increasing levels in most of the brain areas examined. The results in this study show (1) strain differences in basal levels of prodynorphin-derived opioid peptides, (2) the prodynorphin system to be differently influenced by morphine in Lewis rats than in Fischer rats and 3) the proenkephalin system to be influenced by chronic morphine in brain areas related to reward processes only in Fischer rats.     

Effects of 2% sodium chloride imbibition on various opiate related hyperphagic conditions

Dynorphin is one of the most potent appetite stimulants among the endogenous opioids. In this study, we describe the anorexic effects of 5 days of forced 2% NaCl drinking in rats, a regimen which depletes vasopressin as well as dynorphin in the neurohypophysis. Feeding induced by direct activation of kappa-opioid receptors with ketocyclazocine was unaffected by the NaCl regimen. However, 2% NaCl imbibition reduced 2-deoxy-D-glucose (2-DG) induced feeding by 65% and spontaneous nocturnal feeding by 38%. Feeding subsequent to 24 hour food deprivation was not decreased. Naloxone-resistant hyperphagia induced by insulin and spontaneous daytime feeding were also not reduced. The combination of naloxone (3.0 mg/kg) and the NaCl regimen produced an additional 50% reduction in 2-DG induced feeding and an extra 40% decrease in nocturnal feeding. Naloxone, given with 2% NaCl to food deprived animals, retained its appetite suppressing activity, indicating that the NaCl regimen did not deplete the endogenous opioid which mediates food deprivation hyperphagia. These results demonstrate that 2% NaCl imbibition suppresses certain opioid mediated hyperphagias. However, the failure of 2% NaCl to affect all of the naloxone-sensitive types of feeding and the independence of naloxone-sensitive and NaCl-sensitive components suggests that NaCl drinking does not deplete dynorphin in the brain areas which mediate opiate-sensitive hyperphagias.     

Effects of chronic food restriction on prodynorphin-derived peptides in rat brain regions

Chronic food restriction produces a variety of physiological and behavioral adaptations including a potentiation of the reinforcing effect of food, drugs and lateral hypothalamic electrical stimulation. Previous work in this laboratory has revealed that the lowering of self-stimulation threshold by food restriction is reduced by μ- and κ-selective opioid antagonists. In the present study, the effect of chronic food restriction on levels of three prodynorphin-derived peptides, namely dynorphin A_1–17 (A_1–17), dynorphin A_1–8 (A_1–8) and dynorphin B_1–13 (B_1–13) were measured in eleven brain regions known to be involved in appetite, taste and reward. Food restriction increased levels of A_1–17 in dorsal medial (+ 19.6%), ventral medial (+ 24.2%) and medial preoptic (+ 82.9%) hypothalamic areas. Levels of A_1–17 decreased in the central nucleus of the amygdala (− 35.1%). Food restriction increased levels of A_1–8 in nucleus accumbens (+ 34.4%), bed nucleus of the stria terminalis (+ 24.5%) and lateral hypothalamus (+ 41.9%). Food restriction had no effect on levels of B_1–13. A_1–17 is highly κ-preferring and the brain regions in which levels increased all have a high ratio of κ:μ and δ receptors. A_1–8 is less discriminating among opioid receptor types and the brain regions in which levels increased have a low ratio of κ:μ and δ receptors. The present results suggest that food restriction alters posttranslational processing within the dynorphin A domain of the prodynorphin precursor, possibly leading to a change in the balance between κ and non-κ opioid receptor stimulation in specific brain regions.     

The effect of U-50,488H tolerance-dependence and abstinence on the levels of dynorphin (1–13) in brain regions, spinal cord, pituitary gland and peripheral tissues of the rat

Male Sprague-Dawley rats were rendered tolerant to and physically dependent on U-50,488H, a κ-opiate agonist, by injecting 25 mg/kg of the drug intraperitoneally twice a day for 4 days. Two sets of rats were used. Rats labeled as tolerant-dependent were injected with U-50,488H (25 mg/kg) 1 h before sacrificing on day 5, whereas the abstinent rats were sacrificed on day 5 without the injection of U-50,488H. Of all the tissues examined, the pituitary gland had the highest level of dynorphin (1–13), whereas the heart had the lowest level. The levels of dynorphin (1–13) increased in the hypothalamus, hippocampus and pons/medulla of U-50,488H tolerant-dependent rats, whereas in abstinent rats the levels of dynorphin (1–13) were elevated only in the midbrain. The levels of dynorphin (1–13) in the pituitary gland of U-50,488H tolerant-dependent or abstinent rats were unchanged. In peripheral tissues, the levels of dynorphin (1–13) in the heart of U-50,488H tolerant-dependent rats were increased. In the abstinent rats they were elevated in the adrenals, spleen, and the heart but were decreased in the kidneys. Compared to morphine tolerant-dependent and abstinent rats, significant differences in the levels of dynorphin (1–13) in tissues of 50,488H tolerant-dependent and abstinent rats were observed and may explain many pharmacological differences in the μ- and κ-opiate induced tolerance-dependence and abstinence processes.     

Effect of Chronic Ethanol on Enkephalin in the Hypothalamus and Extra‐Hypothalamic Areas

Background: Ethanol may be consumed for reasons such as reward, anxiety reduction, or caloric content, and the opioid enkephalin (ENK) appears to be involved in many of these functions. Previous studies in Sprague–Dawley rats have demonstrated that ENK in the hypothalamic paraventricular nucleus (PVN) is stimulated by voluntary consumption of ethanol. This suggests that this opioid peptide may be involved in promoting the drinking of ethanol, consistent with our recent findings that PVN injections of ENK analogs stimulate ethanol intake. To broaden our understanding of how this peptide functions throughout the brain to promote ethanol intake, we measured, in rats trained to drink 9% ethanol, the expression of the ENK gene in additional brain areas outside the hypothalamus, namely, the ventral tegmental area (VTA), nucleus accumbens shell (NAcSh) and core (NAcC), medial prefrontal cortex (mPFC), and central nucleus of the amygdala (CeA). Methods: In the first experiment, the brains of rats chronically drinking 1 g/kg/d ethanol, 3 g/kg/d ethanol, or water were examined using real‐time quantitative polymerase chain reaction (qRT‐PCR). In the second experiment, a more detailed, anatomic analysis of changes in gene expression, in rats chronically drinking 3 g/kg/d ethanol compared to water, was performed using radiolabeled in situ hybridization (ISH). The third experiment employed digoxigenin‐labeled ISH (DIG) to examine changes in the density of cells expressing ENK and, for comparison, dynorphin (DYN) in rats chronically drinking 3 g/kg/d ethanol versus water. Results: With qRT‐PCR, the rats chronically drinking ethanol plus water compared to water alone showed significantly higher levels of ENK mRNA, not only in the PVN but also in the VTA, NAcSh, NAcC, and mPFC, although not in the CeA. Using radiolabeled ISH, levels of ENK mRNA in rats drinking ethanol were found to be elevated in all areas examined, including the CeA. The experiment using DIG confirmed this effect of ethanol, showing an increase in density of ENK‐expressing cells in all areas studied. It additionally revealed a similar change in DYN mRNA in the PVN, mPFC, and CeA, although not in the NAcSh or NAcC. Conclusions: While distinguishing the NAc as a site where ENK and DYN respond differentially, these findings lead us to propose that these opioids, in response to voluntary ethanol consumption, are generally elevated in extra‐hypothalamic as well as hypothalamic areas, possibly to carry out specific area‐related functions that, in turn, drive animals to further consume ethanol. These functions include calorie ingestion in the PVN, reward and motivation in the VTA and NAcSh, response‐reinforcement learning in the NAcC, stress reduction in the CeA, and behavioral control in the mPFC.     

Adenosine modulation of dynorphin B release by hippocampal synaptosomes

A rat hippocampal preparation enriched in mossy fiber synaptosomes was employed in an attempt to expose any relationship between endogenous adenosine and the release of dynorphin B-like immunoreactivity (DynB-LI). Presumptive blockade of purinergic receptors increased the spontaneous release of DynB-LI, and reducing synaptic adenosine by exogenous adenosine deaminase increased the K(+)-evoked release. Evoked release of DynB-LI was reduced by inhibitors of adenosine uptake and 5'-nucleotidase. Taken together, these data suggest that adenosine endogenous to hippocampal mossy fiber synaptosomes serves to inhibit the release of one of the peptide neuromodulators of this preparation, and provide support for the concept of autoregulation of release.     

The cloned μ, δ and κ receptors and their endogenous ligands: Evidence for two opioid peptide recognition cores

The opioid peptides are derived from three prohormone precursors referred to as proopiomelanocortin (POMC), proenkephalin (ProEnk) and prodynorphin (ProDyn). Following specific cleavage, several biologically active peptides are generated that can bind the μ, δ and κ receptors. The present study examines the receptor binding affinities of the POMC, ProEnk and ProDyn peptides to the cloned μ, δ and κ receptors expressed transiently in transfected COS-1 cells. Consistent with previous findings using brain homogenates, competition studies demonstrate that no opioid peptide family can be exclusively associated with a specific opioid receptor type. Short ProEnk peptides, such as Leu- and Met-enkephalin are selective for δ, but C-terminally extended peptides such as Met-Enk-Arg-Gly-Leu and Met-Enk-Arg-Phe have a high affinity to μ, δ and κ. Similarly, Peptide E, the BAM peptides, and metorphamide have a high affinity for all three opioid receptors types. While dynorphin A peptides and - and β-neoendorphin have a preference for κ, they also bind the cloned δ and μ receptors. Our findings do not easily fit a simple ‘message-address’ model where the Try-Gly-Gly-Phe core is extended and this gradually alters selectivity. Rather, the pattern appears more discontinuous, and would fit better with the idea of two similar but distinct cores; a Tyr-Gly-Gly-Phe Met- or Leu core that is necessary and sufficient for μ and δ but not κ and a Tyr-Gly-Gly-Phe-Met or Leu core with an Arg-X extension that is equally necessary and sufficient for κ.     

Opiates induce long-term increases in prodynorphin-derived peptide levels in the guinea-pig myenteric plexus

Summary The subcutaneous administration of a single dose of an opiate agonist (levorphanol) or antagonist (naloxone) to guinea pigs results in an at least 3-fold elevation of dynorphin and alpha-neoendorphin-immunoreactivity in the longitudinal muscle myenteric plexus preparation. The effects are time- and dose-dependent, significant elevations first being observed 6 h after treatment and lasting for up to 24 h. Pretreatment levels of opioid peptides were observed after 8 days. Combined injection of the narcotic agonist and antagonist, at sufficiently high doses, resulted in an additive effect of the individual drugs. The respective stereoisomers dextrorphan and (+)-naloxone did not affect prodynorphin-derived peptide concentrations. An increase of endogenous opioids was also observed after administration of the nonopiate clonidine, a compound which, like opiates, alters the activity of the myenteric plexus. It is suggested that feedback mechanisms in the myenteric plexus are responsible for the elevation of endogenous opioid peptides following exposure to exogenous opiates.Using a monoclonal antibody (3-E7), which recognizes virtually all endogenous opioid peptides, it was found that levels of higher molecular material were also increased upon opiate challenge. This suggests that a single dose of an exogenous opiate results in an increase in peptide synthesis.     

Functional κ-opioid receptors on oxytocin and vasopressin nerve terminals isolated from the rat neurohypophysis

Opioids intrinsic to the rat neurohypophysial system act to inhibit secretion from the terminals of magnocellular neurones. Opioid receptors in the neurohypophysis are predominantly of the kappa-subtype and selective kappa-agonists suppress electrically evoked release of oxytocin (OXT) and vasopressin (AVP). We have looked for the presence of functional kappa-receptors on neurohypophysial nerve terminals by examining effects of kappa-agonists on secretion from suspensions of isolated neurohypophysial nerve terminals (neurosecretosomes) retained on filters in a perifusion system. Release of both OXT and AVP evoked by K+-depolarisation was inhibited by the kappa-agonists U-50,488H (34% and 45% respectively) and dynorphin A1-13 (68% and 51% respectively). Inhibition by dynorphin A was only observed in the presence of peptidase inhibitors. The actions of both kappa-agonists were prevented by the opioid receptor antagonist naloxone. The experiments indicate the presence of kappa-receptors on terminals of OXT and AVP neurones. This receptor population is in addition to those previously described on pituicytes and those influencing release of neurohypophysial noradrenaline.