L-cysteine suppresses ghrelin and reduces appetite in rodents and humans.

BACKGROUND: High-protein diets promote weight loss and subsequent weight maintenance, but are difficult to adhere to. The mechanisms by which protein exerts these effects remain unclear. However, the amino acids produced by protein digestion may have a role in driving protein-induced satiety. METHODS: We tested the effects of a range of amino acids on food intake in rodents and identified l-cysteine as the most anorexigenic. Using rodents we further studied the effect of l-cysteine on food intake, behaviour and energy expenditure. We proceeded to investigate its effect on neuronal activation in the hypothalamus and brainstem before investigating its effect on gastric emptying and gut hormone release. The effect of l-cysteine on appetite scores and gut hormone release was then investigated in humans. RESULTS: l-Cysteine dose-dependently decreased food intake in both rats and mice following oral gavage and intraperitoneal administration. This effect did not appear to be secondary to behavioural or aversive side effects. l-Cysteine increased neuronal activation in the area postrema and delayed gastric emptying. It suppressed plasma acyl ghrelin levels and did not reduce food intake in transgenic ghrelin-overexpressing mice. Repeated l-cysteine administration decreased food intake in rats and obese mice. l-Cysteine reduced hunger and plasma acyl ghrelin levels in humans. CONCLUSIONS: Further work is required to determine the chronic effect of l-cysteine in rodents and humans on appetite and body weight, and whether l-cysteine contributes towards protein-induced satiety.

Cerebellin1 is a novel orexigenic peptide.

AIM: Cerebellin1 (Cbln1) is highly expressed in the hypothalamus, a region of the brain involved in appetite regulation. However, the effects of Cbn1 on food intake are not known. The present study aimed to investigate the effect of Cbln1 on appetite regulation in rats. METHODS: We determined the effect of (i) intracerebroventricular (ICV) injection of Cbln1 on food intake, behaviour and plasma pituitary hormone levels in male Wistar rats; (ii) Cbln1 on the release of hypothalamic neuropeptides known to modulate food intake from hypothalamic explants and (iii) fasting on hypothalamic Cbln1 mRNA expression. RESULTS: (i) ICV administration of Cbln1 significantly increased food intake in rats and caused no adverse behaviours. ICV administration of Cbln1 significantly reduced plasma thyroid stimulating hormone (TSH) levels 10 min postinjection in rats. (ii) Cbln1 significantly increased the release of neuropeptide Y (NPY) from hypothalamic explants. (iii) Cbln1 mRNA expression levels were increased in the ventromedial nucleus of the hypothalamus in fasted rats. CONCLUSIONS: These data suggest that Cbln1 is a novel orexigenic peptide, which may mediate its effects via hypothalamic NPY.

Alarin stimulates food intake and gonadotrophin release in male rats.

BACKGROUND AND PURPOSE: Alarin is a recently discovered member of the galanin peptide family encoded by a splice variant of galanin-like peptide (GALP) mRNA. Galanin and GALP regulate energy homeostasis and reproduction. We therefore investigated the effects of alarin on food intake and gonadotrophin release. EXPERIMENTAL APPROACH: Alarin was administered into the third cerebral ventricle (i.c.v.) of rats, and food intake or circulating hormone levels were measured. The effect of alarin on the hypothalamo-pituitary-gonadal axis was investigated in vitro using hypothalamic and anterior pituitary explants, and immortalized cell lines. Receptor binding assays were used to determine whether alarin binds to galanin receptors. KEY RESULTS: The i.c.v. administration of alarin (30 nmol) to ad libitum fed male rats significantly increased acute food intake to 500%, and plasma luteinizing hormone (LH) levels to 170% of responses to saline. In vitro, 100 nM alarin stimulated neuropeptide Y (NPY) and gonadotrophin-releasing hormone (GnRH) release from hypothalamic explants from male rats, and 1000 nM alarin increased GnRH release from GT1-7 cells. In vivo, pretreatment with the GnRH receptor antagonist cetrorelix prevented the increase in plasma LH levels observed following i.c.v. alarin administration. Receptor binding studies confirmed alarin did not bind to any known galanin receptor, or compete with radiolabelled galanin for hypothalamic binding sites. CONCLUSIONS AND IMPLICATIONS: These results suggest alarin is a novel orexigenic peptide, and that it increases circulating LH levels via hypothalamic GnRH. Further work is required to identify the receptor(s) mediating the biological effects of alarin.

Hypothalamic mapping of orexigenic action and Fos-like immunoreactivity following relaxin-3 administration in male Wistar rats.

The insulin superfamily, characterized by common disulphide bonds, includes not only insulin but also insulin-like peptides such as relaxin-1 and relaxin-3. The actions of relaxin-3 are largely unknown, but recent work suggests a role in regulation of food intake. Relaxin-3 mRNA is highly expressed in the nucleus incertus, which has extensive projections to the hypothalamus, and relaxin immunoreactivity is present in several hypothalamic nuclei. In the rat, relaxin-3 binds and activates both relaxin family peptide receptor 1, which also binds relaxin-1, and a previously orphaned G protein-coupled receptor, RXFP3. These receptors are extensively expressed in the hypothalamus. The aims of these studies were twofold: 1) map the hypothalamic site(s) of the orexigenic action of relaxin-3 and 2) examine the site(s) of neuronal activation following central relaxin-3 administration. After microinjection into hypothalamic sites, human relaxin-3 (H3; 180 pmol) significantly stimulated 0- to 1-h food intake in the supraoptic nucleus (SON), arcuate nucleus (ARC), and the anterior preoptic area (APOA) [SON 0.4+/-0.2 (vehicle) vs. 2.9+/-0.5 g (H3), P<0.001; ARC 0.7+/-0.3 (vehicle) vs. 2.7+/-0.2 g (H3), P<0.05; and APOA 0.8+/-0.1 (vehicle) vs. 2.2+/-0.2 g (H3), P<0.05]. Cumulative food intake was significantly increased

Implantation of fibre encapsulated RIN 1056a cells transfected with NPY cDNA into the lateral ventricle of rats alters body weight.

Neuropeptide Y (NPY) is a hypothalamic neuropeptide thought to play an important role in the regulation of food intake and energy expenditure. Our aim was to over-express bioactive NPY in the lateral ventricle by implanting cells transfected with NPY cDNA. Cells from the RIN 1056a clonal rat islet cell line were transfected with NPY cDNA. Radioimmunoassay, chromatography and receptor binding assays were used to ensure the secreted NPY was bioactive, before and after implantation. NPY cDNA transfected and untransfected control cells were encapsulated in PVDF hollow fibres to prevent tumour formation and implanted into the lateral ventricle of male Wistar rats. The effects on body weight and food intake were measured for 15 days. Animals implanted with NPY cDNA transfected RIN 1056a cells showed a greater rise in body weight than controls. This difference was statistically significant five days after implantation, and remained so until the end of the experiment. Cumulative food intake was also increased in rats implanted with NPY cDNA transfected RIN 1056a cells, but this difference failed to reach statistical significance. We have demonstrated that implantation of NPY over-expressing cells into the lateral hypothalamus of rats increases body weight gain.

Central relaxin-3 administration causes hyperphagia in male Wistar rats.

Relaxin-3 (INSL-7) is a recently discovered member of the insulin superfamily. Relaxin-3 mRNA is expressed in the nucleus incertus of the brainstem, which has projections to the hypothalamus. Relaxin-3 binds with high affinity to the LGR7 receptor and to the previously orphan G protein-coupled receptor GPCR135. GPCR135 mRNA is expressed predominantly in the central nervous system, particularly in the paraventricular nucleus (PVN). The presence of relaxin-3 and these receptors in the PVN led us to investigate the effect of central administration of relaxin-3 on food intake in male Wistar rats. The receptor involved in mediating these effects was also investigated. Intracerebroventricular injections of human relaxin-3 (H3) to satiated rats significantly increased food intake 1 h post administration in the early light phase [0.96 +/- 0.16 g (vehicle) vs. 1.81 +/- 0.21 g (180 pmol H3), P < 0.05] and the early dark phase [2.95 +/- 0.45 g (vehicle) vs. 4.39 +/- 0.39 g (180 pmol H3), P < 0.05]. Intra-PVN H3 administration significantly increased 1-h food intake in satiated rats in the early light phase [0.34 +/- 0.16 g (vehicle) vs. 1.23 +/- 0.30 g (18 pmol H3), P < 0.05] and the early dark phase [4.43 +/- 0.32 g (vehicle) vs. 6.57 +/- 0.42 g (18 pmol H3), P < 0.05]. Feeding behavior increased after intra-PVN H3. Equimolar doses of human relaxin-2, which binds the LGR7 receptor but not GPCR135, did not increase feeding. Hypothalamic neuropeptide Y, proopiomelanocortin, or agouti-related peptide mRNA expression did not change after acute intracerebroventricular H3. These results suggest a novel role for relaxin-3 in appetite regulation.

gamma-MSH increases intracellular cAMP accumulation and GnRH release in vitro and LH release in vivo.

The roles of the melanocortin 3 receptor (MC3-R) and its agonist, gamma(2)-melanocyte-stimulating hormone (gamma(2)-MSH) in the regulation of the hypothalamo-pituitary-gonadal (HPG) axis are poorly understood. Here we show gamma(2)-MSH stimulated intracellular cAMP accumulation and gonadotrophin-releasing hormone (GnRH) secretion in the immortalised GnRH cell line GT(1)-7. The MC3/4-R antagonist Agrp blocked these actions. Reverse transcriptase polymerase chain reaction demonstrated GT(1)-7 cells express MC3-R mRNA. gamma(2)-MSH also stimulated GnRH release from hypothalamic explants. In vivo, gamma(2)-MSH administration into the medial preoptic area significantly increased plasma luteinising hormone. MC3-R and gamma(2)-MSH may modulate the HPG axis.

Orexins: effects on behavior and localisation of orexin receptor 2 messenger ribonucleic acid in the rat brainstem.

The orexins are neuropeptides originally reported to be involved in the stimulation of food intake. However, analysis of orexin immunoreactive fibres have revealed the densest innervation in brain sites involved in arousal and sleep-wake control, notably the noradrenergic locus coeruleus, an area that also expresses orexin receptor 1 (OX1R) messenger RNA (mRNA). We report here that, in the rat, a single intracerebroventricular injection of orexin A (1 and 3 nmol) or orexin B (3 nmol), during the early light phase, did not increase food intake over the first 4 h postinjection. However, the frequency of active behaviors such as grooming, rearing, burrowing and locomotion increased. Feeding behavior and food intake subsequently decreased over the following 20 h (4-24 h postinjection period) in the orexin A 3 nmol injected group whilst the frequency of inactive behavior (still or asleep) in this group increased. Using riboprobes, we performed in situ hybridization histochemistry to map the distribution of orexin receptor 2 (OX2R) mRNA within the rat brainstem. We report here, for the first time, the presence of OX2R mRNA in the nucleus of the solitary tract and the lateral reticular field (LRt). The LRt is a brainstem site that, amongst other functions, is implicated in attention and wakefulness. This distribution of OX2R and the effects on behavior support recent reports that the orexins might modulate central nervous system arousal and sleep-wake mechanisms rather than exclusively being involved in the control of food intake.

A comparison of the effects of lamotrigine on neuroma-induced action potential firing and normal behaviour in rat: implications for establishing a pre-clinical 'therapeutic index'.

The effects of lamotrigine on rat neuroma and behavioural paradigms were evaluated to determine a pre-clinical therapeutic index. Lamotrigine blocked neuroma-induced burst pattern firing at a free plasma concentration of 13.7+/-1.7 microM (n=5). Oral dosing of lamotrigine (50-200 mg/kg) had no significant effects on behaviour but measurements of plasma concentrations of free drug showed non-linear oral absorption and lower than predicted drug levels (5-27 microM). Given intravenously (10-100 mg/kg), lamotrigine did affect behaviour at a free plasma concentration of 42.0 microM (n=2). By comparing free plasma concentrations, a therapeutic index of 3 was calculated, which is lower than published data based on comparing oral doses. We propose that a therapeutic index should only be derived with reference to plasma drug concentrations to prevent non-linear or incomplete drug absorption from confounding accurate estimation.

Orexin A immunoreactivity and preproorexin mRNA in the brain of Zucker and WKY rats.

The primary role of the orexins was originally believed to be appetite regulation, but is now believed to be the regulation of sleep, arousal and locomotor activity. Orexin A immunoreactivity (orexin A-IR) and prepro-orexin mRNA were measured in the CNS of obese and lean Zucker rats. There were no differences in orexin A-IR or prepro-orexin mRNA levels between obese and lean Zucker rats. The orexins are therefore unlikely to be important in this model of obesity. Levels of orexin A-IR and prepro-orexin mRNA were measured in the CNS of Wistar-Kyoto (WKY) rats, which are hypoactive and have abnormal sleep architecture. Compared to Wistar rats, WKY rats had significantly lower orexin A-IR (with differences of up to 100% in some brain regions) and prepro-orexin mRNA levels. These observations suggest that the sleep and activity phenotype of the WKY strain may be related to orexin deficiency and that this strain may be a useful model of partial orexin deficiency.

Quantification and synthesis of cocaine- and amphetamine-regulated transcript peptide (79-102)-like immunoreactivity and mRNA in rat tissues.

The distribution of cocaine- and amphetamine-regulated transcript peptide (79-102)-like immunoreactivity (CART-LI) was quantified in brain and peripheral tissues of male and female Wistar rats, and male obese (fa/fa) and heterozygous (Fa/+) Zucker rats using a specific RIA. CART-LI tissue levels have not been quantified previously. The assay, using cocaine- and amphetamine-regulated transcript (CART) (79-102) as a standard and radioactive tracer and an antibody to CART (79-102) fragment, detected CART-LI in all brain regions examined, the anterior and posterior pituitary, the spinal cord and throughout the gastrointestinal tract of both male and female Wistar rats. The highest concentrations were found in the hypothalamus, duodenum, anterior pituitary and posterior pituitary (50.6+/-4.4, 26.1+/-4.2, 50.0+/-1.3 and 373.0+/-55.2 pmol/g wet tissue respectively, means+/- s.e.m., n=6-10 male animals). There was no significant variation between the sexes. The concentrations of CART-LI in hypothalami and anterior and posterior pituitaries from fa/fa rats were significantly (P<0002) lower than those of Fa/+ controls (35.9+/-2.1 vs 53.9+/-4.9,<0.6 vs 1.8+/-0.4 and 114+/-9.1 vs 255.5+/- 20.9 pmol/g wet tissue respectively, means+/- s.e.m., n=7). Gel permeation chromatography of regions of rat brain and gastrointestinal tract showed possible differential processing between regions. CART-LI was released from hypothalamic tissue slices in a calcium-dependent fashion by potassium-induced depolarisation. Northern blot analysis detected CART mRNA in the hypothalamus, anterior pituitary, brain stem, cerebellum and spinal cord. The pattern o! f distribution of CART mRNA and CART-LI in various neural and other tissues is in accord with a role for CART as a neurotransmitter.

Acupressure and the prevention of nausea and vomiting after laparoscopy.

The efficacy of currently available antiemetics remains poor. Concern with their side effects and the high cost of the newer drugs has led to renewed interest in non-pharmacological methods of treatment. We have studied the efficacy of acupressure at the P6 point in the prevention of nausea and vomiting after laparoscopy, in a double-blind, randomized, controlled study of acupressure vs placebo. We studied 104 patients undergoing laparoscopy and dye investigation. The anaesthetic technique and postoperative analgesia were standardized. Failure of treatment was defined as the occurrence of nausea and/or vomiting within the first 24 h after anaesthesia. The use of acupressure reduced the incidence of nausea or vomiting from 42% to 19% compared with placebo, with an adjusted risk ratio of 0.24 (95% CI 0.08-0.62; P = 0.005). Other variables were similar between groups.

Experiences of remembering, knowing, and guessing.

This article presents and discusses transcripts of some 270 explanations subjects provided subsequently for recognition memory decisions that had been associated with remember, know, or guess responses at the time the recognition decisions were made. Only transcripts for remember responses included reports of recollective experiences, which seemed mostly to reflect either effortful elaborative encoding or involuntary reminding at study, especially in relation to the self. Transcripts for know responses included claims of just knowing, and of feelings of familiarity. These transcripts indicated that subjects were often quite confident of the accuracy of their decisions, compared with those for guess responses. Transcripts for decisions associated with guess responses also expressed feelings of familiarity but additionally revealed various strategies and inferences that did not directly reflect memory for studied items. The article concludes with a historical and theoretical overview of some interpretations of the states of awareness measured by these responses.

The effect of different concentrations of sugars in two foods (yoghurts and baked beans) on plaque pH.

The effect of different concentrations of sugars in two foods (strawberry yoghurt and baked beans) on plaque acidogenicity was investigated using plaque sampling with a 'Beetrode' electrode. Six varieties of yoghurt, four varieties of baked beans and positive and negative control solutions of 10 per cent sucrose and sorbitol, respectively, were tested on six volunteers, selected according to the guidelines agreed at the San Antonio Conference of 1985. Plaque was harvested immediately before a one minute challenge with the test condition and at 5, 10, 15, 20 and 30 minutes thereafter. Parameters investigated were minimum plaque pH reached after a one minute consumption/rinse of the test food or control solution, maximum drop in plaque pH and 'area of the Stephan curve below pH 6.5'. An Acidogenic Potential Index was calculated for each test food and the results did not suggest a linear relationship between sucrose concentration in a food and acidogenic potential.

Repetition of previously novel melodies sometimes increases both remember and know responses in recognition memory.

Recognition memory for previously novel melodies was tested in three experiments in which subjects usedremember andknow responses to report experiences of recollection, or of familiarity in the absence of recollection, for each melody they recognized. Some of the melodies were taken from Polish folk songs and presented vocally, but without the words. Others were taken from obscure pieces of classical music, presented as single-line melodies. Prior to the test, the melodies were repeated for varying numbers of study trials. Repetition of the Polish melodies increased both remember and know responses, while repetition of classical melodies increased remember but not know responses. When subjects were instructed to report guesses, guess responses were inversely related to remember and know responses and there were more guesses to lures than to targets. These findings establish that remembering and knowing are fully independent functionally and, by the same token, they provide further evidence against the idea that response exclusivity causes increases in remembering to force decreases in knowing. The findings also suggest that simultaneous increases in remembering and knowing occurred because the Polish melodies came from a genre for which the subjects had relatively little previous experience.

Distribution, molecular characterization of pituitary adenylate cyclase-activating polypeptide and its precursor encoding messenger RNA in human and rat tissues.

The distribution of a novel neuropeptide, pituitary adenylate cyclase-activating polypeptide (PACAP), was studied in the brain of the rat and man and a variety of other rat tissues using Northern blot hybridization and two radioimmunoassays for PACAP 1-38 and PACAP 1-27. The assay, using PACAP 1-38 as standard and an antibody to PACAP 21-38 and radiolabelled tracer, revealed immunoreactive PACAP in all brain regions examined, with the highest concentrations in the rat being in the hypothalamus, nucleus accumbens and substantia nigra (380 +/- 34, 310 +/- 37 and 346 +/- 30 pmol/g wet tissue, means +/- S.E.M., n = 5 respectively), whilst in man the highest concentrations were found in the pituitary gland (15.8 +/- 4.7 pmol/g). Immunoreactive PACAP 1-38 was also detected in the rat gastrointestinal tract, adrenal gland and testis. The assay using PACAP 1-27 as standard and label and an antibody to PACAP 1-27 detected immunoreactive PACAP only in the rat hypothalamus (12.6 +/- 1.8 pmol/g wet tissue, n = 5). PACAP mRNA of approximately 2.7 kb in size was detectable in all brain regions of both rat and man, and its distribution paralleled that of the immunoreactive peptide. Gel permeation chromatography of different regions of human and rat hypothalamus, and also rat spinal cord and small intestine, showed a broad immunoreactive peak corresponding to PACAP 1-38. Fast protein liquid chromatography (FPLC) resolved this peak into two immunoreactive peaks, the majority eluting in the position of synthetic PACAP 1-38.(ABSTRACT TRUNCATED AT 250 WORDS)

A comparative study of transcutaneous electrical nerve stimulation (TENS), entonox, pethidine + promazine and lumbar epidural for pain relief in labor.

Analgesic effect, labor outcome, safety and consumer satisfaction were compared in 170 primigravid women; 50 using TENS initially for pain relief, 20 using entonox, 50 pethidine + promazine and 50 lumbar epidural. 88% choosing epidural related it fully effective. 90% using entonox, 96% using TENS and 54% given pethidine + promazine found partial relief. 82% of patients given TENS and 80% given pethidine + promazine required additional analgesia. This was also needed by one of the 20 patients choosing entonox. Women using entonox alone had the shortest labors and women using lumbar epidural, the longest. Operative delivery was significantly more common in women receiving lumbar epidural. No significant inter-group differences were noted in cord pH or Apgar scores. Parturients and midwives both gave high consumer satisfaction ratings to all methods--except for pethidine + promazine, whose use must therefore be questioned. The analgesic efficacy of lumbar epidural outweighs any possible side effects. Entonox appears suited to those able to cope with the earlier part of labor, drug-free. Realization of the potential of TENS requires the design of machines specifically to cope with the quality of the pain of labor.

The distribution in the rabbit of choline administered by injection or infusion.

1. The concentration of choline in plasma, erythrocytes, skeletal muscle, heart, lung, liver, small intestine and kidneys and the changes that follow the injection or infusion of choline have been measured in rabbits anaesthetized with pentobarbitone.2. The concentration of choline in the plasma of arterial blood was 11.8 +/- 0.6 n-mole/ml. and in the erythrocytes 28.4 +/- 1.3 n-mole/ml. blood.3. All tissues contained a higher concentration of free choline than did plasma. The values range from 19.1 +/- 2.2 n-mole/g in skeletal muscle to 500 +/- 25 n-mole/g in the kidney.4. In order of their choline concentrations the tissues were intestine (duodenal end) > kidney > intestine (caecal end) > liver > lung > brain > heart > erythrocytes > (blood) > skeletal muscle > plasma, while in order of the contribution to the total body choline they were liver > intestine > skeletal muscle > (blood) > kidney > erythrocytes > lung > brain > plasma > heart. The total free choline determined by these analyses was between 30-40 mumole/kg body weight, about one third being present in the liver.5. The choline content of the small intestine varied along its length. The lowest amount being present in the portion adjoining the caecum.6. Within 1 min of the injection of choline 100 or 300 mumole/kg, 70-90% had left the circulation. The proportionate loss was higher after 100 mumole/kg than after 300 mumole/kg.7. The loss following 300 mumole/kg was increased if that dose were preceded by a dose of 100 mumole/kg 40 min earlier; this suggests some additional disposal mechanism(s) had been activated by the first dose.8. Three minutes after the injection of choline 300 mumole/kg, about 60% was present as free choline in the tissues studied. The order of the concentration increases was kidneys > liver > muscle > lung > small intestine (caecal end) > heart > intestine > small intestine (duodenal end) > brain.9. Forty minutes after the injection of choline 300 mumole/kg, only 11% could be accounted for as free choline. Only the levels in the kidney, liver, muscle and lung were significantly above normal at this time.10. Infusion of choline 0.8 mumole/kg. min or greater produced rises in plasma choline that corresponded to a clearance of 32 ml. plasma/kg. min.11. After the infusion of 300 mumole/kg over a period of 1 hr, raised levels of choline were detected in all tissues assayed, but the amount found accounted for only 14% of the choline administered. The concentrations in the kidney, liver and lung were similar to those found 40 min after the injection of 300 mumole/kg.12. There was no change in the concentration of choline in the erythrocytes after the injection of choline 100 or 300 mumole/kg, nor during the infusion of choline at the rate of 5 mumole/kg. min for 1 hr.13. The plasma volume appeared to be affected by the injection of the large doses of choline; after choline 300 mumole/kg the plasma volume was reduced. No effect on the plasma volume was observed during the infusion of the same dose.