The control of the proventriculus in the honeybee (Apis mellifera carnica L.) I. A dynamic process influenced by food quality and quantity?

The control of crop emptying in foraging honeybees was investigated in individuals trained to collect defined amounts of sugar solutions. Following feeding, they were dissected after fixed periods of time in order to measure crop content and haemolymph sugar titers. Between feeding and dissection, the metabolic rate of every investigated forager was measured using open-flow respirometry, so as to assess the effects of both food quality (concentration, molarity and viscosity of the fed sugar solution) and food quantity on the transport rate through the proventriculus. The sugar transport rate through the proventriculus was observed to be mainly dependent on the metabolic expenditure of the individual. Bee foragers were able to precisely adjust the sugar transport rate to their metabolic rates, but under certain conditions, an excess of sugars was transported through the proventriculus, more than needed to cover the bee’s energetic demands. This excess depended on the nutritive value and quantity of the fed sugar solution, and on the time after feeding. It did not depend on the metabolic rate of the bee, the molarity, or the viscosity of the fed sugar solution. As long as the bees did not exhaust their crop contents, the haemolymph sugar titers were unaffected by this excess amount transported, by the time after feeding, the concentration and the viscosity of the fed sugar solution. For all feeding conditions assayed, the haemolymph trehalose titer remained constant, while the titers of other haemolymph sugars varied. It is suggested that the trehalose concentration in the haemolymph is regulated in honeybees, and that it represents the controlled variable in the feedback loop responsible for the transport rate through the proventriculus.     

Serotonin depresses feeding behaviour in ants

Feeding behaviour is a complex functional system that relies on external signals and the physiological state of the animal. This is also the case in ants as they vary their feeding behaviour according to food characteristics, environmental conditions and - as they are social insects - to the colony's requirements. The biogenic amine serotonin (5-HT) was shown to be involved in the control and modulation of many actions and processes related to feeding in both vertebrates and invertebrates. In this study, we investigated whether 5-HT affects nectar feeding in ants by analysing its effect on the sucking-pump activity. Furthermore, we studied 5-HT association with tissues and neuronal ganglia involved in feeding regulation. Our results show that 5-HT promotes a dose-dependent depression of sucrose feeding in Camponotus mus ants. Orally administered 5-HT diminished the intake rate by mainly decreasing the volume of solution taken per pump contraction, without modifying the sucrose acceptance threshold. Immunohistochemical studies all along the alimentary canal revealed 5-HT-like immunoreactive processes on the foregut (oesophagus, crop and proventriculus), while the midgut and hindgut lacked 5-HT innervation. Although the frontal and suboesophageal ganglia contained 5-HT immunoreactive cell bodies, serotonergic innervation in the sucking-pump muscles was absent. The results are discussed in the frame of a role of 5-HT in feeding control in ants.     

Urocortin1-induced anorexia is regulated by activation of the serotonin 2C receptor in the brain

This study was conducted to determine the mechanisms by which serotonin (5-hydroxytryptamine, 5-HT) receptors are involved in the suppression of food intake in a rat stress model and to observe the degree of activation in the areas of the brain involved in feeding. In the stress model, male Sprague–Dawley rats (8 weeks old) were given intracerebroventricular injections of urocortin (UCN) 1. To determine the role of the 5-HT2c receptor (5-HT2cR) in the decreased food intake in UCN1-treated rats, specific 5-HT2cR or 5-HT2b receptor (5-HT2bR) antagonists were administered. Food intake was markedly reduced in UCN1-injected rats compared with phosphate buffered saline treated control rats. Intraperitoneal administration of a 5-HT2cR antagonist, but not a 5-HT2bR antagonist, significantly inhibited the decreased food intake. To assess the involvement of neural activation, we tracked the expression of c-fos mRNA as a neuronal activation marker. Expression of the c-fos mRNA in the arcuate nucleus, ventromedial hypothalamic nucleus (VMH) and rostral ventrolateral medulla (RVLM) in UNC1-injected rats showed significantly higher expression than in the PBS-injected rats. Increased c-fos mRNA was also observed in the paraventricular nucleus (PVN), the nucleus of the solitary tract (NTS), and the amygdala (AMG) after injection of UCN1. Increased 5-HT2cR protein expression was also observed in several areas. However, increased coexpression of 5-HT2cR and c-fos was observed in the PVN, VMH, NTS, RVLM and AMG. Whereas, pro-opiomelanocortin mRNA expression was not changed. In an UNC1-induced stress model, 5-HT2cR expression and activation was found in brain areas involved in feeding control.     

Serotonin 5-HT2C receptor-independent expression of hypothalamic NOR1, a novel modulator of food intake and energy balance, in mice

NOR1, Nur77 and Nurr1 are orphan nuclear receptors and members of the NR4A subfamily. Here, we report that the expression of hypothalamic NOR1 was remarkably decreased in mildly obese β-endorphin-deficient mice and obese db/db mice with the leptin receptor mutation, compared with age-matched wild-type mice, whereas there were no genotypic differences in the expression of hypothalamic Nur77 or Nurr1 in these animals. The injection of NOR1 siRNA oligonucleotide into the third cerebral ventricle significantly suppressed food intake and body weight in mice. On the other hand, the decreases in hypothalamic NOR1 expression were not found in non-obese 5-HT2C receptor-deficient mice. Moreover, systemic administration of m-chlorophenylpiperazine (mCPP), a 5-HT2C/1B receptor agonist, had no effect on hypothalamic NOR1 expression, while suppressing food intake in β-endorphin-deficient mice. These findings suggest that 5-HT2C receptor-independent proopiomelanocortin-derived peptides regulate the expression of hypothalamic NOR1, which is a novel modulator of feeding behavior and energy balance.     

The control of the proventriculus in the honeybee (Apis mellifera carnica L.) II. Feedback mechanisms

The mechanisms underlying the control of solution transport rates through the proventriculus in foraging honeybees were investigated in individuals trained to collect defined amounts of sugar solutions. Following feeding, bees were injected either with metabolisable (glucose, fructose, trehalose), or non-metabolisable (sorbose) sugars, in order to distinguish between haemolymph osmolarity and haemolymph sugar levels as factors controlling the solution transport rates through the proventriculus. After a fixed period, workers were dissected in order to measure crop content and haemolymph sugar titers. Between feeding and dissection, the metabolic rate of every investigated forager was measured using open-flow respirometry. Bees injected with metabolisable sugars 15 min after feeding were observed to reduce their solution transport rates through the proventriculus, but injection of non-metabolisable sugars had no influence on them. This suggests that the solution transport rate through the proventriculus is controlled by the concentration of metabolisable compounds in the haemolymph, and not by the haemolymph osmolarity. A period of 10 min after injection of metabolisable sugars was enough to observe reduced solution transport rates. However, if bees were injected only 5 min after feeding, no reduced solution transport rates were observed 10 min after injection.     

Multiple Serotonin Receptors: Opportunities for New Treatments for Obesity?

Recent progress in the molecular pharmacology of 5-HT receptors and the development of selective ligands for various 5-HT receptor subtypes has advanced our understanding of the role of 5-HT mechanisms in the control of food intake and body weight The most intensively investigated 5-HT receptor subtypes have been the 5-HT_1A receptor, the 5-HT_1B receptor andthe5-HT_2C receptor. The overall pattern of results to date suggests that selective 5-HT_2C agonists may be novel anorectic drugs and prove useful in the treatment of obesity. However, a number of issues remain unresolved, particularly regarding potential side-effects, as the 5-HT_2C receptor agonist mCPP has been reported to induce anxiety and nausea in humans, actions that would clearly limit its therapeutic utility. In addition, the possible role of recently cloned 5-HT receptor subtypes such as 5-ht₅,5-ht₆ and 5-ht₇ remains unexplored and the development of selective ligands for these sites has the potential to lead to new treatments for obesity .     

Serotonin,Eating Behavior,and Fat Intake

There is an intimate relationship between nutritional intake (eating) and serotonin activity. Experimental manipulations (mainly neuropharmacological) of serotonin influence the pattern of eating behavior, subjective feelings of appetite motivation, and the response to nutritional challenges. Similarly, nutritional manipulations (food restriction, dieting, or altered nutrient supply) change the sensitivity of the serotonin network. Traditionally, serotonin has been linked to the macronutrient carbohydrate via the intermediary step of plasma amino acid ratios. However, it has also been demonstrated that 5-HT drugs will reduce energy intake and reverse body weight gain in rats exposed to weight increasing high fat diets. 5-HT drugs can also reduce food intake and block weight gain of rats on a high fat cafeteria diet. Some diet selection studies in rats indicate that the most prominent reduction of macronutrient intake is for fat. These data indicate that 5-HT activity can bring about a reduction in fat consumption. In turn, different types of dietary fat can alter brain 5-HT activity. In human studies the methodology of food choice experiments has often precluded the detection of an effect of 5-HT manipulation on fat intake. However, there is evidence that in obese and lean subjects some 5-HT drugs can readily reduce the intake of high fat foods. Data also suggest that 5-HT activation can lead to a selective avoidance of fat in the diet. These effects of 5-HT on the intake of dietary fat may involve a pre-absorptive mechanism and there is evidence that 5-HT is linked to cholecystokinin and enterostatin. These proposals have theoretical and practical implications and suggest possible strategies to intensify or advance fat-induced satiety signals.     

RNA interference suggests sulfakinins as satiety effectors in the cricket Gryllus bimaculatus

In the Mediterranean field cricket, Gryllus bimaculatus, the action of sulfakinin (SK) gene expression on food intake, food transport in the gut and carbohydrate digestion (alpha-amylase activity) was investigated by using the RNA interference (RNAi) method. Injection of SK double-stranded (ds) RNA into the abdomen of female adults and last instar larvae led to a systemic silencing of the SK gene, as was shown by RT-PCR studies. In adults, suppression of SK gene expression was effective from the first day after injection up to at least the third day. Treatment of the adult crickets by injection or feeding of dsRNA led to a stimulation of the food intake. Assuming that the gene silencing is followed by a depletion of the SK in tissues and/or haemolymph implies an inhibitiory role of the native SK peptides on food intake. The alpha-amylase activity in vitro in the midgut tissue and in the secretions of adult females was not affected by silencing the SK gene.     

The contribution of serotonin 5-HT2C and melanocortin-4 receptors to the satiety signaling of glucagon-like peptide 1 and liragultide, a glucagon-like peptide 1 receptor agonist, in mice

Glucagon-like peptide 1 (GLP-1), an insulinotropic gastrointestinal peptide produced mainly from intestinal endocrine L-cells, and liraglutide, a GLP-1 receptor (GLP-1R) agonist, induce satiety. The serotonin 5-HT2C receptor (5-HT2CR) and melanoroctin-4 receptor (MC4R) are involved in the regulation of food intake. Here we show that systemic administration of GLP-1 (50 and 200 μg/kg)-induced anorexia was blunted in mice with a 5HT2CR null mutation, and was attenuated in mice with a heterozygous MC4R mutation. On the other hand, systemic administration of liraglutide (50 and 100 μg/kg) suppressed food intake in mice lacking 5-HT2CR, mice with a heterozygous mutation of MC4R and wild-type mice matched for age. Moreover, once-daily consecutive intraperitoneal administration of liraglutide (100 μg/kg) over 3 days significantly suppressed daily food intake and body weight in mice with a heterozygous mutation of MC4R as well as wild-type mice. These findings suggest that GLP-1 and liraglutide induce anorexia via different central pathways.     

Antifeeding properties of myosuppressin in a generalist phytophagous leafworm, Spodoptera littoralis (Boisduval)

Insect myosuppressins are a family of peptides with a characteristic HV/SFLRFamide carboxy terminus. They are expressed in brain, neurohemal organs, stomatogastric nervous system, and in midgut endocrine cells. From a functional point of view, myosuppressins inhibit contractions of different visceral muscles, stimulate certain skeletal muscles and activate enzyme secretion from the gut. Moreover, in the omnivorous cockroach Blattella germanica, myosuppressin inhibits food intake. Based on these results, we studied the antifeeding activity of myosuppressin in the phytophagous leafworm Spodoptera littoralis. Firstly, we isolated the cDNA corresponding to the S. littoralis myosuppressin precursor encoding the typical myosuppressin peptide of lepidopterans: pQDVVHSFLRFamide. Then, we determined the expression patterns (in terms of mRNA and peptide) of myosuppressin in brain and midgut, and peptide levels in the haemolymph. Myosuppressin patterns in the brain and haemolymph were similar, and symmetrical to that of food consumption, thus suggesting that myosuppressin might inhibit feeding in S. littoralis. Moreover, synthetic myosuppressin effectively inhibited food intake in non-choice antifeeding tests. Taken together, the obtained results point to the hypothesis that myosuppressin represses feeding in S. littoralis.     

变色树蜥消化道5-羟色胺细胞的免疫组织化学定位

用免疫组织化学ABC法,应用5-羟色胺(5-HT)特异性抗血清,对变色树蜥(Calotes versicolor)消化道内含有5-HT的内分泌细胞进行了免疫组织化学的定位研究和形态学观察。结果显示,5-HT细胞在变色树蜥消化道的各个部位均有分布,分布密度近似呈“M”形,其中以空肠分布密度最高,胃体次之,食管最低。5-HT细胞的形态多样,呈圆形、椭圆形、锥体形、长锥体形等,其中胃、幽门和直肠以圆形和椭圆形为主,小肠则以长锥体形为主,其细长突起指向肠腔或固有膜;细胞分布于上皮基部、上皮细胞之间、腺泡上皮细胞之间或固有膜内;多数细胞以内分泌功能为主,少数细胞具有外分泌功能。比较分析表明,5-HT细胞分布型可能与动物的食性有关。     

Age-dependent reduction of ghrelin- and motilin-induced contractile activity in the chicken gastrointestinal tract

Ghrelin is an endogenous ligand for growth hormone secretagogue-receptor 1a (GHS-R1a) and stimulates gastrointestinal (GI) motility in the chicken. Since ghrelin stimulates GH release, which regulates growth, it might be interesting to compare ghrelin-induced responses in GI tract of different-aged chickens. Motilin is a ghrelin-related gut peptide that induces strong contraction in the small intestine. Aim of this study was to clarify age-dependent changes in ghrelin- and motilin-induced contractions of the chicken GI tract and expression of their receptor mRNAs. Chicken ghrelin caused contraction of the crop and proventriculus. Ghrelin-induced contraction in the proventriculus decreased gradually up to 100 days after hatching, but the responses to ghrelin in the crop were the same during the growth period. GHS-R1a mRNA expression in the crop tended to increase, but that in the proventriculus decreased depending on the age. Chicken motilin caused contraction of the chicken GI tract. Atropine decreased the responses to motilin in the proventriculus but not in the ileum. Motilin-induced contraction in the proventriculus but not that in the ileum decreased depending on post-hatching days. On the other hand, motilin receptor mRNA expression in every region of the GI tract decreased with age, but the decrease was more marked in the proventriculus than in the ileum. In conclusion, ghrelin- and motilin-induced GI contractions selectively decreased in the chicken proventriculus depending on post-hatching days, probably due to the age-related decrease in respective receptors expression. The results suggest an age-related contribution of ghrelin and motilin to the regulation of chicken GI motility.     

Serotonin modulates a photic response in circadian locomotor rhythmicity of adults of the blow fly, Calliphora vicina

Abstract. The present experiments were undertaken to explore a role for serotonin (5-hydroxytryptamine, 5-HT) in modulating photic signal transduction in photoreceptors of the blow fly, Calliphora vicina. Injection of p-chlorophenylalanine (pCPA) into the haemolymph appeared to reduce sensitivity to the photic effects of constant ‘bright’ light (LL hyperactivity and circadian arrhythmicity). After drug injection in bright LL, flies continued with a free-running rhythm as in constant darkness (DD) or with a lengthened period τ as in ‘dim’ LL. When 5-HT was injected into flies kept in dim LL, they became hyperactive and arrhythmic as in bright LL. This finding suggests a potential role for serotonin as mediator of circadian changes in the insect visual system including extraretinal photoreceptors.     

5-hydroxytryptamine regulates the (Na++K+)ATPase activity in malpighian tubules of Rhodnius prolixus: Evidence for involvement of G-protein and cAMP-dependent protein kinase

5-Hydroxytryptamine (5-HT, serotonin) acts as a diuretic hormone in Rhodnius prolixus, where it increases to 0.1 μM in the haemolymph during feeding and stimulates the fluid secretion in isolated Malpighian tubules. The ouabain-sensitive (Na⁺+K⁺)ATPase activity present in homogenates of Malpighian tubules from unfed Rhodnius prolixus is inhibited 60% by 0.01 μM 5-HT. This inhibition is reversed by ketanserin, a 5-HT₂ receptor antagonist in mammals, and also by GDPβS, a competitive inhibitor of G-protein GTPase activity. GTPγS, a nonhydrolysable analog of GTP, and cholera toxin, a G_s-protein activator, also inhibit the ouabain-sensitive (Na⁺+K⁺)ATPase activity, while pertussis toxin, a G_i-protein inhibitor, has no effect. The (Na⁺+K⁺)ATPase activity is inhibited 55% by 0.4–100 μM dibutyryl-cAMP in the presence of IBMX, a phosphodiesterase inhibitor, which also potentiates the effect of a low concentration of 5-HT. The cAMP-dependent protein kinase inhibitor peptide abolishes the 5-HT effect. These data suggest that the (Na⁺+K⁺)ATPase activity in Malpighian tubules is inhibited by 5-HT through activation of G_s-protein and a cAMP-dependent protein kinase. Inhibition of the Na⁺+K⁺ pump would contribute to the diuretic effect of 5-HT. Arch. Insect Biochem. Physiol. 36:203–214, 1997. © 1997 Wiley- Liss, Inc.     

Serotonin systems upregulate the expression of hypothalamic NUCB2 via 5-HT2C receptors and induce anorexia via a leptin-independent pathway in mice

NEFA/nucleobindin2 (NUCB2), a novel satiety molecule, is associated with leptin-independent melanocortin signaling in the central nervous system. Here, we show that systemic administration of m-chlorophenylpiperazine (mCPP), a serotonin 5-HT1B/2C receptor agonist, significantly increased the expression of hypothalamic NUCB2 in wild-type mice. The increases in hypothalamic NUCB2 expression induced by mCPP were attenuated in 5-HT2C receptor mutant mice. Systemic administration of mCPP suppressed food intake in db/db mice with leptin receptor mutation as well as lean control mice. On the other hand, the expression of hypothalamic NUCB2 and proopiomelanocortin (POMC) was significantly decreased in hyperphagic and non-obese 5-HT2C receptor mutants compared with age-matched wild-type mice. Interestingly, despite increased expression of hypothalamic POMC, hypothalamic NUCB2 expression was decreased in 5-HT2C receptor mutant mice with heterozygous mutation of β-endorphin gene. These findings suggest that 5-HT systems upregulate the expression of hypothalamic NUCB2 via 5-HT2C receptors, and induce anorexia via a leptin-independent pathway in mice.     

Contractile effects of ghrelin-related peptides on the chicken gastrointestinal tract in vitro

Ghrelin is an endogenous ligand for growth hormone secretagogue receptor (GHS-R), and it stimulates growth hormone (GH) release, food intake and gastrointestinal motility in mammals. Ghrelin has also been identified in the chicken, but this peptide inhibits food intake in the chicken. We examined the effects of ghrelin and related peptides on contractility of the isolated chicken gastrointestinal tract in vitro. Among ghrelin-related peptides examined (1 microM of rat ghrelin, human ghrelin, chicken ghrelin and growth hormone releasing peptide-6 (GHRP-6)), only chicken ghrelin was effective on contraction of the chicken gastrointestinal tract. Des-acyl chicken ghrelin was ineffective, suggesting that octanoylation at Ser3 residue of chicken ghrelin was essential for inducing the contraction. Amplitude of chicken ghrelin-induced contraction was region-specific: highest in the crop and colon, moderate in the esophagus and proventriculus, and weak in the small intestine. The contractile response to chicken ghrelin in the crop was not affected by tetrodotoxin (TTX), but that in the proventriculus was decreased by TTX and atropine to the same extents. D-Lys3-GHRP-6 (a GHS-R antagonist) caused a transient contraction and inhibited the effect of chicken ghrelin without affecting the high-K+-induced contraction. Chicken ghrelin potentiated electrical field stimulation-induced cholinergic contraction without affecting the responsiveness to bath-applied carbachol in the proventriculus. The location of GHS-R differs in the crop (smooth muscle) and proventriculus (smooth muscle and enteric neurons). These results indicate that ghrelin has contractile activity on gastrointestinal tract in the chicken in vitro, and the effect was region-specific. The action would be mediated through the GHS-R, which is highly sensitive to chicken ghrelin.     

Immunohistological localization of serotonin in the CNS and feeding system of the stable fly Stomoxys calcitrans L. (Diptera: Muscidae)

Serotonin, or 5-hydroxytryptamine (5-HT), plays critical roles as a neurotransmitter and neuromodulator that control or modulate many behaviors in insects, such as feeding. Neurons immunoreactive (IR) to 5-HT were detected in the central nervous system (CNS) of the larval and adult stages of the stable fly, Stomoxys calcitrans, using an immunohistological technique. The location and pattern of the 5-HT IR neurons are described and compared for these two different developmental stages. Anatomical features of the fly feeding system were analyzed in third instar larvae and adult flies using a combination of histological and immunohistological techniques. In third instar larvae, the cibarial dilator muscles were observed within the cibarial pump skeleton and innervated by 5-HT IR neurons in nerves arising from the brain. There were four pairs of nerves arising from the frontal surface of the larval brain that innervate the cibarial pump muscles, pharynx, and muscles controlling the mouth hooks. A strong serotoninergic innervation of the anterior stomatogastric system was observed, which suggests 5-HT may play a role in the coordination of different phases of food ingestion by larvae. Similarly, many 5-HT IR neurons were found in both the brain and the thoracico-abdominal ganglia in the adult, some of which innervate the cibarial pump dilator muscles and the stomatogastric muscles. This is tnhe first report describing neuromuscular structures of the stable fly feeding system. The results reported here suggest 5-HT may play a critical role in feeding behaviors of stable fly larvae and adults.     

FMRFamide-like immunoreactivity in the stomatogastric nervous system innervating the gut of the fly,Sarcophaga bullata

1. Distribution of FMRFamide-like peptide activity was examined in the stomatogastric nervous system of the adult fly, Sarcophaga bullata by the indirect immunofluorescent method.2. The neurons of the hypocerebral ganglion exhibit intense immunoreactivity and extend a thick axon bundle ventrally towards the proventriculus and crop.3. Near the mouth of the stomodeal valve a dense network of radial and circular immunoreactive processes branch off and innervate the proventriculus.4. Beyond the proventriculus, the crop duct and anterior midgut wall are also innervated by the FMRFamide-like immunoreactive processes of the nerve from the hypocerebral ganglion.5. From the pattern of innervation of the gut by FMRFamide-like immunoreactive processes it is suggested that this neuropeptide may regulate feeding activities in the adult fly.     

Synthesis and SAR of potent and selective tetrahydropyrazinoisoquinolinone 5-HT2C receptor agonists

The 5-HT_2C receptor has been implicated as a critical regulator of appetite. Small molecule activation of the 5-HT_2C receptor has been shown to affect food intake and regulate body weight gain in rodent models and more recently in human clinical trials. Therefore, 5-HT_2C is a well validated target for anti-obesity therapy. The synthesis and structure–activity relationships of a series of novel tetrahydropyrazinoisoquinolinone 5-HT_2C receptor agonists are presented. Several members of this series were identified as potent 5-HT_2C receptor agonists with high functional selectivity against the 5-HT_2A and 5-HT_2B receptors and reduced food intake in an acute rat feeding model upon oral dosing.     

Alpha-ethyltryptamines as dual dopamine–serotonin releasers

The dopamine (DA), serotonin (5-HT), and norepinephrine (NE) transporter releasing activity and serotonin-2A (5-HT_2A) receptor agonist activity of a series of substituted tryptamines are reported. Three compounds, 7b, (+)-7d and 7f, were found to be potent dual DA/5-HT releasers and were >10-fold less potent as NE releasers. Additionally, these compounds had different activity profiles at the 5-HT_2A receptor. The unique combination of dual DA/5-HT releasing activity and 5-HT_2A receptor activity suggests that these compounds could represent a new class of neurotransmitter releasers with therapeutic potential.