Direct comparison of the effects of intravenous kisspeptin-10, kisspeptin-54 and GnRH on gonadotrophin secretion in healthy men.

STUDY QUESTION: How potently does the novel hypothalamic stimulator of reproduction, kisspeptin, increase gonadotrophin secretion when compared with GnRH in healthy men? SUMMARY ANSWER: At the doses tested, intravenous administration of either of two major kisspeptin isoforms, kisspeptin-10 and -54, was associated with similar levels of gonadotrophin secretion in healthy men; however, GnRH was more potent when compared with either kisspeptin isoform. WHAT IS KNOWN ALREADY: Kisspeptin-10 and -54 are naturally occurring hormones in the kisspeptin peptide family which potently stimulates endogenous GnRH secretion from the hypothalamus, so have the potential to treat patients with reproductive disorders. Rodent studies suggest that kisspeptin-54 is more potent when compared with kisspepitn-10; however, their effects have not previously been directly compared in humans, or compared with direct pituitary stimulation of gonadotrophin secretion using GnRH. STUDY DESIGN, SIZE AND DURATION: A single-blinded placebo controlled physiological study was performed from January to December 2013. Local ethical approval was granted, and five participants were recruited to each dosing group. PARTICIPANTS/MATERIALS, SETTING, METHODS: Healthy men were administered vehicle, kisspeptin-10, kisspeptin-54 and GnRH intravenously for 3 h on different study days. Each hormone was administered at 0.1, 0.3 and 1.0 nmol/kg/h doses (n = 5 subjects per group). Regular blood sampling was conducted throughout the study to measure LH and FSH. Study visits were conducted at least a week apart. MAIN RESULTS AND THE ROLE OF CHANCE: Serum LH and FSH levels were ∼3-fold higher during GnRH infusion when compared with kisspeptin-10 and ∼2-fold higher when compared with kisspeptin-54 [mean area under the curve serum LH during infusion (in hours times international units per litre, h.IU/l): 10.81 ± 1.73, 1.0 nmol/kg/h kisspeptin-10; 14.43 ± 1.27, 1.0 nmol/kg/h kisspeptin-54; 34.06 ± 5.18, 1.0 nmol/kg/h GnRH, P < 0.001 versus kisspeptin-10, P < 0.01 versus kisspeptin-54]. LIMITATIONS, REASONS FOR CAUTION: This study had a small sample size. WIDER IMPLICATIONS OF THE FINDINGS: Kisspeptin offers a novel means of stimulating the reproductive axis. Our data suggest that kisspeptin stimulates gonadotrophin secretion less potently when compared with GnRH; however, kisspeptin may stimulate gonadotrophins in a more physiological manner when compared with current therapies. Kisspeptin is emerging as a future therapeutic agent, so it is important to establish which kisspeptin hormones could be used to treat patients with infertility. Results of this study suggest that either isoform has similar effects on reproductive hormone secretion in healthy men when administered intravenously. STUDY FUNDING/COMPETING INTERESTS: This work is funded by grants from the MRC and NIHR and is supported by the NIHR Imperial Biomedical Research Centre Funding Scheme. C.N.J. is supported by an NIHR Clinical Lectureship. A.A. is supported by Wellcome Trust Research Training Fellowships. A.N.C. is supported by Wellcome Trust Translational Medicine Training Fellowship. W.S.D. is supported by an NIHR Career Development Fellowship.

Comparison of the utility of Cocaine- and Amphetamine-Regulated Transcript (CART), chromogranin A, and chromogranin B in neuroendocrine tumor diagnosis and assessment of disease progression.

CONTEXT: Prognosis in patients with neuroendocrine tumors (NETs) is often poor, frequently reflecting delayed diagnosis. Hence, accurate and practical NET markers are needed. Cocaine- and amphetamine-regulated transcript (CART) peptide is a potential novel NET marker. DESIGN AND PARTICIPANTS: Circulating levels of CART peptide and the established NET markers chromogranin A (CgA) and chromogranin B (CgB) were measured using RIA in 353 patients with NET (normal renal function) and in controls. Clinical data were collected retrospectively. MAIN OUTCOME MEASURE(S): The comparative and combined utility of CART, CgA, and CgB for diagnosis and assessment of disease progression was measured in different NET subtypes. RESULTS: CgA and CgB in combination improved diagnostic accuracy in patients with gut NETs, nongastroenteropancreatic NETs, and NETs with an unknown primary origin compared with each biomarker alone. Measuring CART did not further improve diagnosis in these NET subtypes. For pancreatic NETs, CgB was superior to CgA and CART in detecting stable disease (P < .007), whereas CgA and CART in combination were most effective in identifying progressive disease. In phaeochromocytomas/paragangliomas (PCC/PGL), CART was the most useful biomarker for identifying stable (P < .001) and progressive (P = .001) disease. Consistent with this, plasma CART decreased following PCC/PGL tumor resection, remaining low in all patients in remission, but increasing in those with progressive disease. CONCLUSIONS: CART is a useful marker for identifying progressive pancreatic NETs. CART is superior to CgA and CgB in detecting stable and progressive PCC/PGLs, and may have a role as a surveillance marker for PCC/PGL patients.

The short chain fatty acid propionate stimulates GLP-1 and PYY secretion via free fatty acid receptor 2 in rodents.

BACKGROUND AND OBJECTIVES: The gut hormones peptide YY (PYY) and glucagon-like peptide 1 (GLP-1) acutely suppress appetite. The short chain fatty acid (SCFA) receptor, free fatty acid receptor 2 (FFA2) is present on colonic enteroendocrine L cells, and a role has been suggested for SCFAs in appetite regulation. Here, we characterise the in vitro and in vivo effects of colonic propionate on PYY and GLP-1 release in rodents, and investigate the role of FFA2 in mediating these effects using FFA2 knockout mice. METHODS: We used Wistar rats, C57BL6 mice and free fatty acid receptor 2 knockout (FFA(-/-)) mice on a C57BL6 background to explore the impact of the SCFA propionate on PYY and GLP-1 release. Isolated colonic crypt cultures were used to assess the effects of propionate on gut hormone release in vitro. We subsequently developed an in vivo technique to assess gut hormone release into the portal vein following colonic infusion of propionate. RESULTS: Propionate stimulated the secretion of both PYY and GLP-1 from wild-type primary murine colonic crypt cultures. This effect was significantly attenuated in cultures from FFA2(-/-) mice. Intra-colonic infusion of propionate elevated PYY and GLP-1 levels in jugular vein plasma in rats and in portal vein plasma in both rats and mice. However, propionate did not significantly stimulate gut hormone release in FFA2(-/-) mice. CONCLUSIONS: Intra-colonic administration of propionate stimulates the concurrent release of both GLP-1 and PYY in rats and mice. These data demonstrate that FFA2 deficiency impairs SCFA-induced gut hormone secretion both in vitro and in vivo.

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.

Reduced levels of plasma kisspeptin during the antenatal booking visit are associated with increased risk of miscarriage.

CONTEXT: Kisspeptin is a recently identified hormone encoded by the KISS1 gene, playing a critical role in human reproduction. Plasma kisspeptin levels rise dramatically during normal pregnancy due to placental synthesis, which implicates it as a potential tool for assessing risks of pregnancy complications. No previous prospective study has investigated the association between plasma kisspeptin and risk of miscarriage. OBJECTIVE: The objective of the study was to determine whether a single plasma kisspeptin or serum human chorionic gonadotropin (hCG) measurement in asymptomatic women attending their booking antenatal visit is associated with miscarriage. DESIGN: This was a prospective cohort study. SETTING: The study was conducted at a tertiary obstetric center. PARTICIPANTS: A total of 993 asymptomatic pregnant women with a gestation of 6 weeks or longer attending routine antenatal booking visit were recruited between January 2010 and December 2012. MAIN OUTCOME MEASURES: Plasma kisspeptin and serum hCG were measured during the antenatal booking visit. Pregnancy outcome was recorded prospectively. RESULTS: Plasma kisspeptin correlated with gestation (r(2) = 0.57; P < .0001). Gestational age-corrected (multiples of median) plasma kisspeptin was 60.4% lower (P < .001), and multiples of median-hCG was 36.1% lower (P < .001) in women later diagnosed with miscarriage compared with women without miscarriage. Increased plasma kisspeptin was associated with reduced miscarriage risk, even after adjusting for age, body mass index, gestational age, smoking, and blood pressure [odds ratio 0.13 (95% confidence interval 0.08-0.22), P = .0001]. Kisspeptin had a higher diagnostic performance for miscarriage than hCG (receiver-operator characteristic-area under the curve 0.899 ± 0.025 plasma kisspeptin; 0.775 ± 0.040, serum hCG, P < .01 vs plasma kisspeptin). CONCLUSION: Our data suggest for the first time that a single plasma kisspeptin measurement taken during the antenatal booking visit provides a potential novel marker for identifying asymptomatic pregnant women at a gestation of 6 weeks or greater at increased risk of miscarriage.

Relaxin-3 stimulates the neuro-endocrine stress axis via corticotrophin-releasing hormone.

Relaxin-3 is a member of the insulin superfamily. It is expressed in the nucleus incertus of the brainstem, which has projections to the hypothalamus. Relaxin-3 binds with high affinity to RXFP1 and RXFP3. RXFP3 is expressed within the hypothalamic paraventricular nucleus (PVN), an area central to the stress response. The physiological function of relaxin-3 is unknown but previous work suggests a role in appetite control, stimulation of the hypothalamic-pituitary-gonadal axis and stress. Central administration of relaxin-3 induces c-fos expression in the PVN and increases plasma ACTH levels in rats. The aim of this study was to investigate the effect of central administration of human relaxin-3 (H3) on the hypothalamic-pituitary-adrenal (HPA) axis in male rodents in vivo and in vitro. Intracerebroventricular (i.c.v) administration of H3 (5 nmol) significantly increased plasma corticosterone at 30 min following injection compared with vehicle. Intra-PVN administration of H3 (1.8-1620 pmol) significantly increased plasma ACTH at 1620 pmol H3 and corticosterone at 180-1620 pmol H3 at 30 min following injection compared with vehicle. The stress hormone prolactin was also significantly raised at 15 min post-injection compared with vehicle. Treatment of hypothalamic explants with H3 (10-1000 nM) stimulated the release of corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP), but H3 had no effect on the release of ACTH from in vitro pituitary fragments. These results suggest that relaxin-3 may regulate the HPA axis, via hypothalamic CRH and AVP neurons. Relaxin-3 may act as a central signal linking nutritional status, reproductive function and stress.

Neuromedin B stimulates the hypothalamic-pituitary-gonadal axis in male rats.

Neuromedin B (NMB) is a highly conserved bombesin-related peptide found in mammals. NMB mRNA is detected in the central nervous system (CNS) and is highly expressed in the rat hypothalamus, in particular the medial preoptic area and the arcuate nucleus. The mammalian bombesin family of receptors consists of three closely related G protein coupled receptors, BB1, BB2 and BB3. The BB1 receptor subtype has the highest affinity for NMB. NMB has well documented roles in the regulation of the thyroid axis and the stress axis in rats. However, there is little available data regarding the role of NMB in the regulation of the hypothalamic-pituitary-gonadal (HPG) axis. It is known that the NMB receptor is expressed in immortalised gonadotrophin releasing hormone (GnRH) releasing GT1-7 cells and murine forebrain GnRH neurons, and that anterior pituitary NMB-immunoreactivity is altered by changes in the sex steroid environment. The objective of these studies was thus to further investigate the effects of NMB on the HPG axis. Intracerebroventricular (ICV) administration of NMB (10 nmol) to adult male rats significantly increased plasma luteinising hormone (LH) levels 30 min after injection (plasma LH ng/ml; saline 0.69±0.07, 10 nmol NMB 1.33±0.17, P<0.01). In vitro, NMB stimulated GnRH release from hypothalamic explants from male rats and from hypothalamic GT1-7 cells. NMB had no significant effect on LH release from anterior pituitary explants from male rats, or from pituitary LßT2 cells in vitro. These results suggest a previously unreported role for NMB in the stimulation of the HPG axis via hypothalamic GnRH. Further work is now required to determine the receptor mediating the effects of NMB on the reproductive axis and the physiological role of NMB in reproduction.

Twice-daily subcutaneous injection of kisspeptin-54 does not abolish menstrual cyclicity in healthy female volunteers.

BACKGROUND: Kisspeptin is a critical hypothalamic regulator of reproductive function. Chronic kisspeptin administration causes profound tachyphylaxis in male monkeys and in women with functional hypothalamic amenorrhea. The pharmacological effects of chronic kisspeptin exposure in healthy women with normal menstrual cycles have not been studied previously. AIM: Our aim was to determine the effects of follicular-phase kisspeptin-54 treatment on menstrual cyclicity in healthy women. METHODS: We performed a prospective, single-blinded, 1-way crossover study. Healthy women received twice-daily sc injections of kisspeptin (6.4 nmol/kg) or 0.9% saline during menstrual days 7-14 (n = 5 per treatment arm). Serial assessments of basal reproductive hormones, ultrasound parameters, LH pulsatility, and acute sensitivity to GnRH and kisspeptin-54 injection were performed. RESULTS: Menstrual cyclicity persisted in all women after follicular-phase kisspeptin-54 treatment. Chronic exposure to kisspeptin-54 did not abolish acute stimulation of LH after injection of kisspeptin-54 or GnRH. In addition, kisspeptin-54 treatment was associated with a shorter mean length of the menstrual cycle (mean length of menstrual cycle was 28.6 ± 1.4 days with saline vs 26.8 ± 3.1 days with kisspeptin, P < .01), earlier onset of highest recorded serum LH (mean menstrual day of highest LH was 15.2 ± 1.3 with saline vs 13.0 ± 1.9 with kisspeptin, P < .05), and earlier onset of the luteal phase (mean menstrual day of progesterone increase was 18.0 ± 2.1 with saline vs 15.8 ± 0.9 with kisspeptin, P < .05). CONCLUSION: Our data suggest that 1 week of exogenous kisspeptin-54 does not abolish menstrual cyclicity in healthy women. Further work is needed to determine whether kisspeptin could be used to treat certain anovulatory disorders.

A single injection of kisspeptin-54 temporarily increases luteinizing hormone pulsatility in healthy women.

BACKGROUND: Kisspeptin is a novel hypothalamic peptide which stimulates endogenous gonadotrophin releasing hormone (GnRH) secretion. A single subcutaneous bolus injection of kisspeptin-54 increases circulating luteinizing hormone (LH) levels in women, but its acute effects on LH pulsatility are not known. AIMS: To investigate the effects of a single subcutaneous (sc) injection of kisspeptin-54 administration on LH pulsatility in healthy female volunteers. METHODS: Six healthy female adult volunteers underwent 10-minute blood sampling for serum LH measurement for 8 h during the follicular phase of menstrual cycle. Sc bolus injection of saline or kisspeptin-54 (0·15, 0·30 or 0·60 nmol/kg) was administered 4 h after commencing the study. A previously described, blinded deconvolution method was used to detect LH pulses. RESULTS: Mean number of LH pulses was increased significantly following 0·30 and 0·60 nmol/kg kisspeptin-54 when compared with saline (mean increase in number of LH pulses per 4 h, following injection: -0·17 ± 0·54, saline; +2·33 ± 0·56, 0·30 nmol/kg kisspeptin-54, P < 0·05 vs saline; +2·33 ± 0·80, 0·60 nmol/kg kisspeptin-54, P < 0·05 vs saline). LH pulse secretory mass increased following injection of 0·60 nmol/kg in five of six subjects, but the mean change in all subjects was non-significant when compared with saline (mean increase in pulse secretory mass in IU/l following injection: +0·35 ± 0·40, saline; +2·61 ± 1·17, 0·60 nmol/kg kisspeptin-54, P = 0·10 vs saline). CONCLUSIONS: A single injection of kisspeptin-54 temporarily stimulates the number of LH pulses in healthy women. Further studies are required to investigate the therapeutic potential of kisspeptin-54 injection to restore LH pulsatility in patients with reproductive disorders caused by impaired GnRH secretion.

Glucagon and GLP-1 inhibit food intake and increase c-fos expression in similar appetite regulating centres in the brainstem and amygdala.

BACKGROUND: Glucagon and glucagon-like peptide-1 (GLP-1) are evolutionarily related anorectic hormones. Glucagon also increases energy expenditure. The combination of glucagon and GLP-1 could cause weight loss through a simultaneous reduction in food intake and increased energy expenditure. However, the effect of combined administration of glucagon and GLP-1 on food intake and neuronal activation has not previously been studied. Furthermore, the effect of glucagon on neuronal activation in appetite regulating centres has not been assessed. Characterisation of the effects of glucagon when administered singly and in combination with GLP-1 on neuronal activation will be important for determining the mechanism of action of related potential antiobesity therapies. OBJECTIVES: To investigate the effects of peripherally administered GLP-1 and glucagon on food intake, neuronal activation and blood glucose in mice when administered individually and in combination. METHODOLOGY: Food intake, blood glucose and c-fos expression in the hypothalamus, amygdala and brainstem were measured in response to GLP-1 and glucagon, alone and in combination. RESULTS: Peripherally administered GLP-1 and glucagon decreased food intake and increased c-fos expression in the brainstem and amygdala. Doses of GLP-1 and glucagon that individually did not significantly affect feeding, in combination were anorectic and stimulated neuronal activation in the area postrema (AP) and central nucleus of the amygdala. Combined administration of GLP-1 and glucagon prevented the acute hyperglycemic effect of glucagon alone. CONCLUSION: Anorectic doses of glucagon and GLP-1 induced similar patterns of c-fos expression. Combined administration of low dose GLP-1 and glucagon inhibited food intake and induced c-fos expression in the AP and amygdala. The combination of both hormones may offer the opportunity to utilise the beneficial effects of reduced food intake and increased energy expenditure, and may therefore be a potential treatment for obesity.

Peripheral administration of prokineticin 2 potently reduces food intake and body weight in mice via the brainstem.

BACKGROUND AND PURPOSE: Prokineticin 2 (PK2) has recently been shown to acutely reduce food intake in rodents. We aimed to determine the CNS sites and receptors that mediate the anorectic effects of peripherally administered PK2 and its chronic effects on glucose and energy homeostasis. EXPERIMENTAL APPROACH: We investigated neuronal activation following i.p. administration of PK2 using c-Fos-like immunoreactivity (CFL-IR). The anorectic effect of PK2 was examined in mice with targeted deletion of either prokineticin receptor 1 (PKR1) or prokineticin receptor 2 (PKR2), and in wild-type mice following administration of the PKR1 antagonist, PC1. The effect of IP PK2 administration on glucose homeostasis was investigated. Finally, the effect of long-term administration of PK2 on glucose and energy homeostasis in diet-induced obese (DIO) mice was determined. KEY RESULTS: I.p. PK2 administration significantly increased CFL-IR in the dorsal motor vagal nucleus of the brainstem. The anorectic effect of PK2 was maintained in mice lacking the PKR2 but abolished in mice lacking PKR1 and in wild-type mice pre-treated with PC1. DIO mice treated chronically with PK2 had no changes in glucose levels but significantly reduced food intake and body weight compared to controls. CONCLUSIONS AND IMPLICATIONS: Together, our data suggest that the anorectic effects of peripherally administered PK2 are mediated via the brainstem and this effect requires PKR1 but not PKR2 signalling. Chronic administration of PK2 reduces food intake and body weight in a mouse model of human obesity, suggesting that PKR1-selective agonists have potential to be novel therapeutics for the treatment of obesity.

Exogenous peptide YY3-36 and Exendin-4 further decrease food intake, whereas octreotide increases food intake in rats after Roux-en-Y gastric bypass.

BACKGROUND: Patients show an elevated postprandial satiety gut hormone release after Roux-en-Y Gastric bypass (gastric bypass). The altered gut hormone response appears to have a prominent role in the reduction of appetite and body weight (BW) after gastric bypass. Patients with insufficient BW loss after gastric bypass have an attenuated postprandial gut hormone response in comparison with patients who lost an adequate amount of BW. The effects of additional gut hormone administration after gastric bypass are unknown. METHODS: The effects of peripheral administration of peptide YY3-36 (PYY3-36; 300 nmol kg(-1)), glucagon-like peptide-1 (GLP-1) analogue Exendin-4 (20 nmol kg(-1)) and somatostatin analogue octreotide (10 µg kg(-1)) on feeding and BW were evaluated in rats after gastric bypass. RESULTS: Gastric bypass rats weighed (P<0.01) and ate less on postoperative day 5 (P<0.001) and thereafter, whereas postprandial plasma PYY and GLP-1 levels were higher compared with sham-operated controls (P<0.001). Administration of both PYY3-36 and Exendin-4 led to a further decrease in food intake in bypass rats compared with saline treatment (P=0.02 and P<0.0001, respectively). Similar reduction in food intake was observed in sham rats (P=0.02 and P<0.001, respectively). Exendin-4 treatment resulted in a significant BW loss in bypass (P=0.03) and sham rats (P=0.04). Subsequent treatment with octreotide led to an increase in food intake in bypass (P=0.007), but not in sham rats (P=0.87). CONCLUSION: Peripheral administration of PYY3-36 and Exendin-4 reduces short-term food intake, whereas octreotide increases short-term food intake in rats after gastric bypass. The endogenous gut hormone response after gastric bypass can thus potentially be further enhanced by additional exogenous therapy with pharmacological doses of gut hormones in patients with insufficient weight loss or weight regain after surgery.

Alterations of sucrose preference after Roux-en-Y gastric bypass.

Roux-en-Y gastric bypass (gastric bypass) patients reportedly have changes in perception and consumption of sweet-tasting foods. This study aimed to further investigate alterations in sweet food intake in rats and sucrose detection in humans after gastric bypass. Wistar rats were randomized to gastric bypass or sham-operations and preference for sucrose (sweet), sodium chloride (salty), citric acid (sour) and quinine hydrochloride (bitter) was assessed with standard two-bottle intake tests (vs. water). Intestinal T1R2 and T1R3 expression and plasma levels of glucagon-like-peptide 1 (GLP-1) and peptide YY (PYY) were measured. Furthermore, obese patients and normal weight controls were tested for sucrose taste detection thresholds pre- and postoperatively. Visual analogue scales measuring hedonic perception were used to determine the sucrose concentration considered by patients and controls as "just about right" pre- and postoperatively. Gastric bypass reduced the sucrose intake relative to water in rats (p<0.001). Preoperative sucrose exposure reduced this effect. Preference or aversion for compounds representative of other taste qualities in naïve rats remained unaffected. Intestinal T1R2 and T1R3 expression was significantly decreased in the alimentary limb while plasma levels of GLP-1 and PYY were elevated after bypass in rats (p=0.01). Bypass patients showed increased taste sensitivity to low sucrose concentrations compared with controls (p<0.05), but both groups considered the same sucrose concentration as "just about right" postoperatively. In conclusion, gastric bypass reduces sucrose intake relative to water in sucrose-naïve rats, but preoperative sucrose experience attenuates this effect. Changes in sucrose taste detection do not predict hedonic taste ratings of sucrose in bypass patients which remain unchanged. Thus, factors other than the unconditional affective value of the taste may also play a role in determining food preferences after gastric bypass.

Ghrelin does not orchestrate the metabolic changes seen in fasting but has significant effects on lipid mobilisation and substrate utilisation.

OBJECTIVE: Short-term fasting is associated with increased GH pulsatility and mobilisation of fats, but underlying mechanisms are unclear. We studied ghrelin's role during fasting and the effects of exogenous ghrelin on lipid mobilisation. DESIGN: Randomised placebo-controlled study. METHODS: In this study, ten controls (body mass index (BMI) 23.3±3.2), ten morbidly obese subjects (BMI 50.1±10.6) and six post-gastrectomy subjects (BMI 25.2±1.0) were fasted for 36  h undergoing regular blood sampling. On a separate occasion, subjects were infused with either i.v. ghrelin (5  pmol/kg per min) or saline over 270  min. RESULTS: Obese and post-gastrectomy subjects had lower ghrelin compared with controls (ANOVA, P=0.02) during the fast. Controls and gastrectomy subjects showed a similar increase in GH pulsatility, circulating non-esterified fatty acids (NEFA) and 3ß-hydroxybutyrate (3 HB). Obese subjects had an impaired GH response (P<0.001), reduced excursions of 3 HB (P=0.01) but no change in NEFA excursions (P=0.09) compared with controls. Ghrelin infusion increased GH, NEFA and ketone bodies (ANOVA, P<0.0001) in all the three groups, but GH response was impaired in the obese subjects (P=0.001). Ghrelin also induced a significant (ANOVA, P=0.004) biphasic NEFA response to meals in all the subjects. CONCLUSIONS: Despite low circulating ghrelin, gastrectomy subjects maintain a normal metabolic response to fasting, implying that ghrelin plays a minimal role. In contrast, infused ghrelin has significant effects on lipid mobilisation and induces a marked biphasic NEFA response to meals. Hence, ghrelin may play a significant role in meal-related substrate utilisation and metabolic flexibility.

Effects of oral ingestion of sucralose on gut hormone response and appetite in healthy normal-weight subjects.

BACKGROUND/OBJECTIVE: The sweet-taste receptor (T1r2+T1r3) is expressed by enteroendocrine L-cells throughout the gastrointestinal tract. Application of sucralose (a non-calorific, non-metabolisable sweetener) to L-cells in vitro stimulates glucagon-like peptide (GLP)-1 secretion, an effect that is inhibited with co-administration of a T1r2+T1r3 inhibitor. We conducted a randomised, single-blinded, crossover study in eight healthy subjects to investigate whether oral ingestion of sucralose could stimulate L-cell-derived GLP-1 and peptide YY (PYY) release in vivo. METHODS: Fasted subjects were studied on 4 study days in random order. Subjects consumed 50 ml of either water, sucralose (0.083% w/v), a non-sweet, glucose-polymer matched for sweetness with sucralose addition (50% w/v maltodextrin+0.083% sucralose) or a modified sham-feeding protocol (MSF=oral stimulation) of sucralose (0.083% w/v). Appetite ratings and plasma GLP-1, PYY, insulin and glucose were measured at regular time points for 120 min. At 120 min, energy intake at a buffet meal was measured. RESULTS: Sucralose ingestion did not increase plasma GLP-1 or PYY. MSF of sucralose did not elicit a cephalic phase response for insulin or GLP-1. Maltodextrin ingestion significantly increased insulin and glucose compared with water (P<0.001). Appetite ratings and energy intake were similar for all groups. CONCLUSIONS: At this dose, oral ingestion of sucralose does not increase plasma GLP-1 or PYY concentrations and hence, does not reduce appetite in healthy subjects. Oral stimulation with sucralose had no effect on GLP-1, insulin or appetite.

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.

Central and peripheral administration of human relaxin-2 to adult male rats inhibits food intake.

AIM: Relaxin is a polypeptide hormone involved in pregnancy and lactation. It is mainly secreted by the corpus luteum and placenta, but is expressed in a number of other tissues, including heart and brain. Within the brain, relaxin is expressed in the olfactory and limbic systems, the cortex and the hypothalamic arcuate nucleus (ARC). Its cognate receptor, relaxin family peptide receptor 1 (RXFP1), is also widely expressed in the brain, including the hypothalamic ARC and paraventricular nucleus (PVN), areas important in appetite regulation. The aim of this study was to investigate whether relaxin influences food intake through central hypothalamic circuits. METHODS: The human form of relaxin, human relaxin-2 (H2) was administered centrally and peripherally to male Wistar rats and food intake measured. Behaviour was also assessed. RESULTS: Intracerebroventricular (ICV) administration of H2 significantly decreased 1-h food intake in the early dark phase [2.95 ± 0.45 g (saline) vs. 0.95 ± 0.18 g (180 pmol H2), p < 0.001]. ICV administration of H2 decreased feeding behaviour and increased grooming and headdown behaviour. Intraparaventricular injections of H2 significantly decreased 1-h food intake in the early dark phase [3.13 ± 0.35 g (saline) vs. 1.35 ± 0.33 g (18 pmol H2), p < 0.01, 1.61 ± 0.31 g (180 pmol H2), p < 0.05 and 1.23 ± 0.32 g (540 pmol H2), p < 0.001]. Intraperitoneal (IP) administration of H2 significantly decreased 1-h food intake in the early dark phase [4.63 ± 0.46 g (vehicle) vs. 3.08 ± 0.15 g (66 nmol H2), p < 0.01, 3.00 ± 0.17 g (200 nmol H2), p < 0.01 and 2.26 ± 0.36 g (660 nmol H2), p < 0.001]. CONCLUSIONS: Central and peripheral administration of H2 reduces the food intake in rats. This effect may be mediated via the PVN and/or other brain regions.

Twice-weekly administration of kisspeptin-54 for 8 weeks stimulates release of reproductive hormones in women with hypothalamic amenorrhea.

Kisspeptin is a novel therapeutic target for infertility. A single kisspeptin-54 (KP-54) injection acutely stimulates the release of reproductive hormones in women with hypothalamic amenorrhea (HA), a commonly occurring condition characterized by absence of menstruation; however, twice-daily administration of KP-54 results in tachyphylaxis. We determined the time course of desensitization to twice-daily KP-54 injections, compared the effects of twice-daily and twice-weekly administration regimens of KP-54, and studied the effects of long-term twice-weekly administration of KP-54 on the release of reproductive hormones in women with HA. When KP-54 was administered twice daily, responsiveness to luteinizing hormone (LH) diminished gradually, whereas responsiveness to follicle-stimulating hormone (FSH) was nearly abolished by day 2. Twice-weekly KP-54 administration resulted in only partial desensitization, in contrast to the complete tolerance achieved with twice-daily administration. Women with HA who were treated with twice-weekly KP-54 injections had significantly elevated levels of reproductive hormones after 8 weeks as compared with treatment with saline. No adverse effects were observed. This study provides novel pharmacological data on the effects of KP-54 on the release of reproductive hormones in women with HA.

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.

Subcutaneous oxyntomodulin analogue administration reduces body weight in lean and obese rodents.

OBJECTIVE: To determine the efficacy of a long-acting oxyntomodulin (OXM) analogue, OXM6421, in inhibiting food intake and decreasing body weight in lean and diet-induced obese (DIO) rodents. RESEARCH DESIGN AND METHODS: The glucagon-like peptide-1 (GLP-1) receptor binding affinity and efficacy, sensitivity to enzymatic degradation in vitro and persistence in the circulation after peripheral administration were investigated for OXM6421 and compared with native OXM. The chronic effect of OXM6421 on food intake, body weight and energy expenditure was examined in lean rats, and its anti-obesity potential was evaluated in DIO mice. RESULTS: OXM6421 showed enhanced GLP-1 receptor binding affinity and cyclic adenosine monophosphate (cAMP) stimulation, and higher resistance to enzymatic degradation by dipeptidyl peptidase IV (DPP-IV) and neutral endopeptidase (NEP) compared with native OXM. OXM6421 persisted longer in the circulation than OXM after peripheral administration. Acute administration of OXM6421 potently inhibited food intake in lean rodents, with cumulative effects lasting up to 24 h. In lean rats, daily subcutaneous (s.c.) administration of OXM6421 caused greater weight loss than the pair-fed animals, and a higher rate of oxygen consumption than both the pair-fed and the saline controls. In DIO mice, continuous s.c. infusion of OXM6421 resulted in a significant weight loss, accompanied by an improvement in glucose homeostasis and an increase in circulating adiponectin levels. Once-daily s.c. administration of OXM6421 for 21 days caused sustained weight loss in DIO mice. CONCLUSION: OXM6421 induces negative energy balance in both lean and obese rodents, suggesting that long-acting OXM analogues may represent a potential therapy for obesity.