Gut enterochromaffin cells drive visceral pain and anxiety.

Gastrointestinal (GI) discomfort is a hallmark of most gut disorders and represents an important component of chronic visceral pain1. For the growing population afflicted by irritable bowel syndrome, GI hypersensitivity and pain persist long after tissue injury has resolved2. Irritable bowel syndrome also exhibits a strong sex bias, afflicting women three times more than men1. Here, we focus on enterochromaffin (EC) cells, which are rare excitable, serotonergic neuroendocrine cells in the gut epithelium3-5. EC cells detect and transduce noxious stimuli to nearby mucosal nerve endings3,6 but involvement of this signalling pathway in visceral pain and attendant sex differences has not been assessed. By enhancing or suppressing EC cell function in vivo, we show that these cells are sufficient to elicit hypersensitivity to gut distension and necessary for the sensitizing actions of isovalerate, a bacterial short-chain fatty acid associated with GI inflammation7,8. Remarkably, prolonged EC cell activation produced persistent visceral hypersensitivity, even in the absence of an instigating inflammatory episode. Furthermore, perturbing EC cell activity promoted anxiety-like behaviours which normalized after blockade of serotonergic signalling. Sex differences were noted across a range of paradigms, indicating that the EC cell-mucosal afferent circuit is tonically engaged in females. Our findings validate a critical role for EC cell-mucosal afferent signalling in acute and persistent GI pain, in addition to highlighting genetic models for studying visceral hypersensitivity and the sex bias of gut pain.

A syngeneic inoculation mouse model of endometriosis that develops multiple comorbid visceral and cutaneous pain like behaviours.

ABSTRACT: Endometriosis is a chronic and debilitating condition, commonly characterised by chronic pelvic pain (CPP) and infertility. Chronic pelvic pain can be experienced across multiple pelvic organs, with comorbidities commonly effecting the bowel, bladder, and vagina. Despite research efforts into endometriosis pathophysiology, little is known about how endometriosis induces CPP, and as such, therapeutic interventions are lacking. The aim of this study was to characterise a syngeneic mouse model of endometriosis that mimics naturally occurring retrograde menstruation, thought to precede endometriosis development in patients, and determine whether these mice exhibit signs of CPP and altered behaviour. We characterised the development of endometriosis over 10 weeks following uterine tissue inoculation, measured in vivo and ex vivo hypersensitivity to mechanical stimuli across multiple visceral organs, and assessed alterations in animal spontaneous behaviour. We confirmed that inoculated uterine horn tissue formed into endometriosis lesions throughout the peritoneal cavity, with significant growth by 8 to 10 weeks post inoculation. Additionally, we found that mice with fully developed endometriosis displayed hypersensitivity evoked by (1) vaginal distension, (2) colorectal distension, (3) bladder distension, and (4) cutaneous thermal stimulation, compared to their sham counterparts. Moreover, endometriosis mice displayed alterations in spontaneous behaviour indicative of (5) altered bladder function and (6) anxiety. This model creates a foundation for mechanistical studies into the diffuse CPP associated with endometriosis and the development of targeted therapeutic interventions to improve the quality of life of women with endometriosis.

Olorinab (APD371), a peripherally acting, highly selective, full agonist of the cannabinoid receptor 2, reduces colitis-induced acute and chronic visceral hypersensitivity in rodents.

ABSTRACT: Abdominal pain is a key symptom of inflammatory bowel disease and irritable bowel syndrome, for which there are inadequate therapeutic options. We tested whether olorinab-a highly selective, full agonist of the cannabinoid receptor 2 (CB2)-reduced visceral hypersensitivity in models of colitis and chronic visceral hypersensitivity (CVH). In rodents, colitis was induced by intrarectal administration of nitrobenzene sulfonic acid derivatives. Control or colitis animals were administered vehicle or olorinab (3 or 30 mg/kg) twice daily by oral gavage for 5 days, starting 1 day before colitis induction. Chronic visceral hypersensitivity mice were administered olorinab (1, 3, 10, or 30 mg/kg) twice daily by oral gavage for 5 days, starting 24 days after colitis induction. Visceral mechanosensitivity was assessed in vivo by quantifying visceromotor responses (VMRs) to colorectal distension. Ex vivo afferent recordings determined colonic nociceptor firing evoked by mechanical stimuli. Colitis and CVH animals displayed significantly elevated VMRs to colorectal distension and colonic nociceptor hypersensitivity. Olorinab treatment significantly reduced VMRs to control levels in colitis and CVH animals. In addition, olorinab reduced nociceptor hypersensitivity in colitis and CVH states in a concentration- and CB2-dependent manner. By contrast, olorinab did not alter VMRs nor nociceptor responsiveness in control animals. Cannabinoid receptor 2 mRNA was detected in colonic tissue, particularly within epithelial cells, and dorsal root ganglia, with no significant differences between healthy, colitis, and CVH states. These results demonstrate that olorinab reduces visceral hypersensitivity through CB2 agonism in animal models, suggesting that olorinab may provide a novel therapy for inflammatory bowel disease- and irritable bowel syndrome-associated abdominal pain.

Pharmacological Inhibition of the Voltage-Gated Sodium Channel NaV1.7 Alleviates Chronic Visceral Pain in a Rodent Model of Irritable Bowel Syndrome.

The human nociceptor-specific voltage-gated sodium channel 1.7 (hNaV1.7) is critical for sensing various types of somatic pain, but it appears not to play a primary role in acute visceral pain. However, its role in chronic visceral pain remains to be determined. We used assay-guided fractionation to isolate a novel hNaV1.7 inhibitor, Tsp1a, from tarantula venom. Tsp1a is 28-residue peptide that potently inhibits hNaV1.7 (IC50 = 10 nM), with greater than 100-fold selectivity over hNaV1.3-hNaV1.6, 45-fold selectivity over hNaV1.1, and 24-fold selectivity over hNaV1.2. Tsp1a is a gating modifier that inhibits NaV1.7 by inducing a hyperpolarizing shift in the voltage-dependence of channel inactivation and slowing recovery from fast inactivation. NMR studies revealed that Tsp1a adopts a classical knottin fold, and like many knottin peptides, it is exceptionally stable in human serum. Remarkably, intracolonic administration of Tsp1a completely reversed chronic visceral hypersensitivity in a mouse model of irritable bowel syndrome. The ability of Tsp1a to reduce visceral hypersensitivity in a model of irritable bowel syndrome suggests that pharmacological inhibition of hNaV1.7 at peripheral sensory nerve endings might be a viable approach for eliciting analgesia in patients suffering from chronic visceral pain.

Design of a Stable Cyclic Peptide Analgesic Derived from Sunflower Seeds that Targets the κ-Opioid Receptor for the Treatment of Chronic Abdominal Pain.

The rising opioid crisis has become a worldwide societal and public health burden, resulting from the abuse of prescription opioids. Targeting the κ-opioid receptor (KOR) in the periphery has emerged as a powerful approach to develop novel pain medications without central side effects. Inspired by the traditional use of sunflower (Helianthus annuus) preparations for analgesic purposes, we developed novel stabilized KOR ligands (termed as helianorphins) by incorporating different dynorphin A sequence fragments into a cyclic sunflower peptide scaffold. As a result, helianorphin-19 selectively bound to and fully activated the KOR with nanomolar potency. Importantly, helianorphin-19 exhibited strong KOR-specific peripheral analgesic activity in a mouse model of chronic visceral pain, without inducing unwanted central effects on motor coordination/sedation. Our study provides a proof of principle that cyclic peptides from plants may be used as templates to develop potent and stable peptide analgesics applicable via enteric administration by targeting the peripheral KOR for the treatment of chronic abdominal pain.

A Gut-Intrinsic Melanocortin Signaling Complex Augments L-Cell Secretion in Humans.

OBJECTIVE: Hypothalamic melanocortin 4 receptors (MC4R) are a key regulator of energy homeostasis. Brain-penetrant MC4R agonists have failed, as concentrations required to suppress food intake also increase blood pressure. However, peripherally located MC4R may also mediate metabolic benefits of MC4R activation. Mc4r transcript is enriched in mouse enteroendocrine L cells and peripheral administration of the endogenous MC4R agonist, α-melanocyte stimulating hormone (α-MSH), triggers the release of the anorectic hormones Glucagon-like peptide-1 (GLP-1) and peptide tyrosine tyrosine (PYY) in mice. This study aimed to determine whether pathways linking MC4R and L-cell secretion exist in humans. DESIGN: GLP-1 and PYY levels were assessed in body mass index-matched individuals with or without loss-of-function MC4R mutations following an oral glucose tolerance test. Immunohistochemistry was performed on human intestinal sections to characterize the mucosal MC4R system. Static incubations with MC4R agonists were carried out on human intestinal epithelia, GLP-1 and PYY contents of secretion supernatants were assayed. RESULTS: Fasting PYY levels and oral glucose-induced GLP-1 secretion were reduced in humans carrying a total loss-of-function MC4R mutation. MC4R was localized to L cells and regulates GLP-1 and PYY secretion from ex vivo human intestine. α-MSH immunoreactivity in the human intestinal epithelia was predominantly localized to L cells. Glucose-sensitive mucosal pro-opiomelanocortin cells provide a local source of α-MSH that is essential for glucose-induced GLP-1 secretion in small intestine. CONCLUSION: Our findings describe a previously unidentified signaling nexus in the human gastrointestinal tract involving α-MSH release and MC4R activation on L cells in an autocrine and paracrine fashion. Outcomes from this study have direct implications for targeting mucosal MC4R to treat human metabolic disorders.

A mouse model of endometriosis that displays vaginal, colon, cutaneous, and bladder sensory comorbidities.

Endometriosis is a painful inflammatory disorder affecting ~10% of women of reproductive age. Although chronic pelvic pain (CPP) remains the main symptom of endometriosis patients, adequate treatments for CPP are lacking. Animal models that recapitulate the features and symptoms experienced by women with endometriosis are essential for investigating the etiology of endometriosis, as well as developing new treatments. In this study, we used an autologous mouse model of endometriosis to examine a combination of disease features and symptoms including: a 10 week time course of endometriotic lesion development; the chronic inflammatory environment and development of neuroangiogenesis within lesions; sensory hypersensitivity and altered pain responses to vaginal, colon, bladder, and skin stimulation in conscious animals; and spontaneous animal behavior. We found significant increases in lesion size from week 6 posttransplant. Lesions displayed endometrial glands, stroma, and underwent neuroangiogenesis. Additionally, peritoneal fluid of mice with endometriosis contained known inflammatory mediators and angiogenic factors. Compared to Sham, mice with endometriosis displayed: enhanced sensitivity to pain evoked by (i) vaginal and (ii) colorectal distension, (iii) altered bladder function and increased sensitivity to cutaneous (iv) thermal and (v) mechanical stimuli. The development of endometriosis had no effect on spontaneous behavior. This study describes a comprehensive characterization of a mouse model of endometriosis, recapitulating the clinical features and symptoms experienced by women with endometriosis. Moreover, it delivers the groundwork to investigate the etiology of endometriosis and provides a platform for the development of therapeutical interventions to manage endometriosis-associated CPP.

A spider-venom peptide with multitarget activity on sodium and calcium channels alleviates chronic visceral pain in a model of irritable bowel syndrome.

ABSTRACT: Chronic pain is a serious debilitating condition that affects ∼20% of the world's population. Currently available drugs fail to produce effective pain relief in many patients and have dose-limiting side effects. Several voltage-gated sodium (NaV) and calcium (CaV) channels are implicated in the etiology of chronic pain, particularly NaV1.1, NaV1.3, NaV1.7-NaV1.9, CaV2.2, and CaV3.2. Numerous NaV and CaV modulators have been described, but with few exceptions, they display poor potency and/or selectivity for pain-related channel subtypes. Here, we report the discovery and characterization of 2 novel tarantula-venom peptides (Tap1a and Tap2a) isolated from Theraphosa apophysis venom that modulate the activity of both NaV and CaV3 channels. Tap1a and Tap2a inhibited on-target NaV and CaV3 channels at nanomolar to micromolar concentrations and displayed moderate off-target selectivity for NaV1.6 and weak affinity for NaV1.4 and NaV1.5. The most potent inhibitor, Tap1a, nearly ablated neuronal mechanosensitivity in afferent fibers innervating the colon and the bladder, with in vivo intracolonic administration reversing colonic mechanical hypersensitivity in a mouse model of irritable bowel syndrome. These findings suggest that targeting a specific combination of NaV and CaV3 subtypes provides a novel route for treatment of chronic visceral pain.

Histamine induces peripheral and central hypersensitivity to bladder distension via the histamine H1 receptor and TRPV1.

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a common chronic pelvic disorder with sensory symptoms of urinary urgency, frequency, and pain, indicating a key role for hypersensitivity of bladder-innervating sensory neurons. The inflammatory mast cell mediator histamine has long been implicated in IC/BPS, yet the direct interactions between histamine and bladder afferents remain unclear. In the present study, we show, using a mouse ex vivo bladder afferent preparation, that intravesical histamine enhanced the mechanosensitivity of subpopulations of afferents to bladder distension. Histamine also recruited "silent afferents" that were previously unresponsive to bladder distension. Furthermore, in vivo intravesical histamine enhanced activation of dorsal horn neurons within the lumbosacral spinal cord, indicating increased afferent signaling in the central nervous system. Quantitative RT-PCR revealed significant expression of histamine receptor subtypes (Hrh1-Hrh3) in mouse lumbosacral dorsal root ganglia (DRG), bladder detrusor smooth muscle, mucosa, and isolated urothelial cells. In DRG, Hrh1 was the most abundantly expressed. Acute histamine exposure evoked Ca2+ influx in select populations of DRG neurons but did not elicit calcium transients in isolated primary urothelial cells. Histamine-induced mechanical hypersensitivity ex vivo was abolished in the presence of the histamine H1 receptor antagonist pyrilamine and was not present in preparations from mice lacking transient receptor potential vanilloid 1 (TRPV1). Together, these results indicate that histamine enhances the sensitivity of bladder afferents to distension via interactions with histamine H1 receptor and TRPV1. This hypersensitivity translates to increased sensory input and activation in the spinal cord, which may underlie the symptoms of bladder hypersensitivity and pain experienced in IC/BPS.

Activation of pruritogenic TGR5, MrgprA3, and MrgprC11 on colon-innervating afferents induces visceral hypersensitivity.

Itch induces scratching that removes irritants from the skin, whereas pain initiates withdrawal or avoidance of tissue damage. While pain arises from both the skin and viscera, we investigated whether pruritogenic irritant mechanisms also function within visceral pathways. We show that subsets of colon-innervating sensory neurons in mice express, either individually or in combination, the pruritogenic receptors Tgr5 and the Mas-gene-related GPCRs Mrgpra3 and Mrgprc11. Agonists of these receptors activated subsets of colonic sensory neurons and evoked colonic afferent mechanical hypersensitivity via a TRPA1-dependent mechanism. In vivo intracolonic administration of individual TGR5, MrgprA3, or MrgprC11 agonists induced pronounced visceral hypersensitivity to colorectal distension. Coadministration of these agonists as an "itch cocktail" augmented hypersensitivity to colorectal distension and changed mouse behavior. These irritant mechanisms were maintained and enhanced in a model of chronic visceral hypersensitivity relevant to irritable bowel syndrome. Neurons from human dorsal root ganglia also expressed TGR5, as well as the human ortholog MrgprX1, and showed increased responsiveness to pruritogenic agonists in pathological states. These data support the existence of an irritant-sensing system in the colon that is a visceral representation of the itch pathways found in skin, thereby contributing to sensory disturbances accompanying common intestinal disorders.

Sugar Responses of Human Enterochromaffin Cells Depend on Gut Region, Sex, and Body Mass.

Gut-derived serotonin (5-HT) is released from enterochromaffin (EC) cells in response to nutrient cues, and acts to slow gastric emptying and modulate gastric motility. Rodent studies also evidence a role for gut-derived 5-HT in the control of hepatic glucose production, lipolysis and thermogenesis, and in mediating diet-induced obesity. EC cell number and 5-HT content is increased in the small intestine of obese rodents and human, however, it is unknown whether EC cells respond directly to glucose in humans, and whether their capacity to release 5-HT is perturbed in obesity. We therefore investigated 5-HT release from human duodenal and colonic EC cells in response to glucose, sucrose, fructose and α-glucoside (αMG) in relation to body mass index (BMI). EC cells released 5-HT only in response to 100 and 300 mM glucose (duodenum) and 300 mM glucose (colon), independently of osmolarity. Duodenal, but not colonic, EC cells also released 5-HT in response to sucrose and αMG, but did not respond to fructose. 5-HT content was similar in all EC cells in males, and colonic EC cells in females, but 3 to 4-fold higher in duodenal EC cells from overweight females (p < 0.05 compared to lean, obese). Glucose-evoked 5-HT release was 3-fold higher in the duodenum of overweight females (p < 0.05, compared to obese), but absent here in overweight males. Our data demonstrate that primary human EC cells respond directly to dietary glucose cues, with regional differences in selectivity for other sugars. Augmented glucose-evoked 5-HT release from duodenal EC is a feature of overweight females, and may be an early determinant of obesity.

Augmented capacity for peripheral serotonin release in human obesity.

BACKGROUND/OBJECTIVES: Evidence from animal studies highlights an important role for serotonin (5-HT), derived from gut enterochromaffin (EC) cells, in regulating hepatic glucose production, lipolysis and thermogenesis, and promoting obesity and dysglycemia. Evidence in humans is limited, although elevated plasma 5-HT concentrations are linked to obesity. SUBJECTS/METHODS: We assessed (i) plasma 5-HT concentrations before and during intraduodenal glucose infusion (4 kcal/min for 30 min) in non-diabetic obese (BMI 44 ± 4 kg/m2, N = 14) and control (BMI 24 ± 1 kg/m2, N = 10) subjects, (ii) functional activation of duodenal EC cells (immunodetection of phospho-extracellular related-kinase, pERK) in response to glucose, and in separate subjects, (iii) expression of tryptophan hydroxylase-1 (TPH1) in duodenum and colon (N = 39), and (iv) 5-HT content in primary EC cells from these regions (N = 85). RESULTS: Plasma 5-HT was twofold higher in obese than control responders prior to (P = 0.025), and during (iAUC, P = 0.009), intraduodenal glucose infusion, and related positively to BMI (R2 = 0.334, P = 0.003) and HbA1c (R2 = 0.508, P = 0.009). The density of EC cells in the duodenum was twofold higher at baseline in obese subjects than controls (P = 0.023), with twofold more EC cells activated by glucose infusion in the obese (EC cells co-expressing 5-HT and pERK, P = 0.001), while the 5-HT content of EC cells in duodenum and colon was similar; TPH1 expression was 1.4-fold higher in the duodenum of obese subjects (P = 0.044), and related positively to BMI (R2 = 0.310, P = 0.031). CONCLUSIONS: Human obesity is characterized by an increased capacity to produce and release 5-HT from the proximal small intestine, which is strongly linked to higher body mass, and glycemic control. Gut-derived 5-HT is likely to be an important driver of pathogenesis in human obesity and dysglycemia.

Intragastric administration of leucine or isoleucine lowers the blood glucose response to a mixed-nutrient drink by different mechanisms in healthy, lean volunteers.

BACKGROUND: The branched-chain amino acids leucine and isoleucine lower blood glucose after oral glucose ingestion, and the intraduodenal infusion of leucine decreases energy intake in healthy, lean men. OBJECTIVE: We investigated the effects of the intragastric administration of leucine and isoleucine on the gastric emptying of, and blood glucose responses to, a physiologic mixed-macronutrient drink and subsequent energy intake. DESIGN: In 2 separate studies, 12 healthy, lean subjects received on 3 separate occasions an intragastric infusion of 5 g leucine (leucine-5g) or an intragastric infusion of 10 g leucine (leucine-10g), an intragastric infusion of 5 g isoleucine (isoleucine-5g) or an intragastric infusion of 10 g isoleucine (isoleucine-10g), or a control. Fifteen minutes later, subjects consumed a mixed-nutrient drink (400 kcal, 56 g carbohydrates, 15 g protein, and 12 g fat), and gastric emptying (13C-acetate breath test) and blood glucose, plasma insulin, C-peptide, glucagon, glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), and cholecystokinin (leucine study only) were measured for 60 min. Immediately afterward, energy intake from a cold, buffet-style meal was assessed. RESULTS: Compared with the control, leucine-10g decreased the blood glucose area under the curve (AUC) (P < 0.05) and tended to reduce peak blood glucose (P = 0.07), whereas effects of leucine-5g were NS. Leucine-10g, but not leucine-5g, increased plasma insulin and C-peptide AUCs (P < 0.01 for both), but neither dose affected glucagon, GLP-1, GIP, cholecystokinin, gastric emptying, or energy intake. Compared with the control, isoleucine-10g reduced the blood glucose AUC and peak blood glucose (P < 0.01), whereas effects of isoleucine-5g were NS. Neither load affected insulin, C-peptide, glucagon, GLP-1, or GIP. Isoleucine-10g, but not isoleucine-5g, slowed gastric emptying (P < 0.05), but gastric emptying was not correlated with the blood glucose AUC. Isoleucine did not affect energy intake. CONCLUSIONS: In healthy subjects, both leucine and isoleucine reduced blood glucose in response to a mixed-nutrient drink but did not affect subsequent energy intake. The mechanisms underlying glucose lowering appear to differ; leucine stimulated insulin, whereas isoleucine acted insulin independently. These trials were registered at www.anzctr.org.au as 12613000899741 and 12614000837628.

Contributions of upper gut hormones and motility to the energy intake-suppressant effects of intraduodenal nutrients in healthy, lean men - a pooled-data analysis.

We have previously identified pyloric pressures and plasma cholecystokinin (CCK) concentrations as independent determinants of energy intake following administration of intraduodenal lipid and intravenous CCK. We evaluated in healthy men whether these parameters also determine energy intake in response to intraduodenal protein, and whether, across the nutrients, any predominant gastrointestinal (GI) factors exist, or many factors make small contributions. Data from nine published studies, in which antropyloroduodenal pressures, GI hormones, and GI /appetite perceptions were measured during intraduodenal lipid or protein infusions, were pooled. In all studies energy intake was quantified immediately after the infusions. Specific variables for inclusion in a mixed-effects multivariable model for determination of independent predictors of energy intake were chosen following assessment for collinearity, and within-subject correlations between energy intake and these variables were determined using bivariate analyses adjusted for repeated measures. In models based on all studies, or lipid studies, there were significant effects for amplitude of antral pressure waves, premeal glucagon-like peptide-1 (GLP-1) and time-to-peak GLP-1 concentrations, GLP-1 AUC and bloating scores (P < 0.05), and trends for basal pyloric pressure (BPP), amplitude of duodenal pressure waves, peak CCK concentrations, and hunger and nausea scores (0.05 < P ≤ 0.094), to be independent determinants of subsequent energy intake. In the model including the protein studies, only BPP was identified as an independent determinant of energy intake (P < 0.05). No single parameter was identified across all models, and effects of the variables identified were relatively small. Taken together, while GI mechanisms contribute to the regulation of acute energy intake by lipid and protein, their contribution to the latter is much less. Moreover, the effects are likely to reflect small, cumulative contributions from a range of interrelated factors.

Diacylglycerol acyltransferase-1 inhibition enhances intestinal fatty acid oxidation and reduces energy intake in rats.

Acyl CoA:diacylglycerol acyltransferase-1 (DGAT-1) catalyzes the final step in triacylglycerol (TAG) synthesis and is highly expressed in the small intestine. Because DGAT-1 knockout mice are resistant to diet-induced obesity, we investigated the acute effects of intragastric (IG) infusion of a small molecule diacylglycerol acyltransferase-1 inhibitor (DGAT-1i) on eating, circulating fat metabolites, indirect calorimetry, and hepatic and intestinal expression of key fat catabolism enzymes in male rats adapted to an 8 h feeding-16 h deprivation schedule. Also, the DGAT-1i effect on fatty acid oxidation (FAO) was investigated in enterocyte cell culture models. IG DGAT-1i infusions reduced energy intake compared with vehicle in high-fat diet (HFD)-fed rats, but scarcely in chow-fed rats. IG DGAT-1i also blunted the postprandial increase in serum TAG and increased ß-hydroxybutyrate levels only in HFD-fed rats, in which it lowered the respiratory quotient and increased intestinal, but not hepatic, protein levels of Complex III of the mitochondrial respiratory chain and of mitochondrial hydroxymethylglutaryl-CoA synthase. Finally, the DGAT-1i enhanced FAO in CaCo2 (EC50 = 0.3494) and HuTu80 (EC50 = 0.00762) cells. Thus, pharmacological DGAT-1 inhibition leads to an increase in intestinal FAO and ketogenesis when dietary fat is available. This may contribute to the observed eating-inhibitory effect.