ETV1 is a key regulator of enteroendocrine PYY production

This study has investigated the patterns of colocalisation of the conventional K cell marker, glucagon-like insulinotropic peptide (GIP), and the L cell markers, glucagon like peptide-1 (GLP-1) and peptide YY (PYY), in enteroendocrine cells (EEC) of the small intestine and colon of mouse and pig. All combinations of the hormones, 3 in a cell, 2 in a cell and 1 at a time, were encountered. In both species, the three most common EEC types contained (1) both GLP-1 and PYY but not GIP, (2) GLP-1 alone or (3) GIP plus GLP-1 without PYY. Few GIP plus PYY cells and rare cells containing all 3 hormones were encountered. Gradients of cell types occurred along the intestine. For example, in mouse, there were no PYY cells in the duodenum and few in the jejunum, but >50% of labelled EEC in the distal ileum and colon were PYY immunoreactive. By contrast, over 40% of EEC in the pig duodenum contained PYY, and most also contained either GLP-1 or GIP. The gradient in pig was less pronounced. It is concluded that the traditional classification of K and L cells requires revision, and that there are major inter-species differences in the patterns of colocalisation of hormones that have been used to characterise K and L cells.

Accurately identifying γδ T cells in large single-cell RNA sequencing (scRNA-seq) datasets without additional single-cell γδ T cell receptor sequencing (sc-γδTCR-seq) or CITE-seq (cellular indexing of transcriptomes and epitopes sequencing) data remains challenging. In this study, we developed a TCR module scoring strategy for human γδ T cell identification (i.e. based on modular gene expression of constant and variable TRA/TRB and TRD genes). We evaluated our method using 5' scRNA-seq datasets comprising both sc-αβTCR-seq and sc-γδTCR-seq as references and demonstrated that it can identify γδ T cells in scRNA-seq datasets with high sensitivity and accuracy. We observed a stable performance of this strategy across datasets from different tissues and different subtypes of γδ T cells. Thus, we propose this analysis method, based on TCR gene module scores, as a standardized tool for identifying and reanalyzing γδ T cells from 5'-end scRNA-seq datasets.

these proportions had increased to 26% and 22%, respectively, with 55% of women and 66% of men being overweight (BMI > 25 kg/m 2 ; Health Survey for England, 2001), reflecting a worldwide trend which is most marked in, but not restricted to, the developed world. Most of us in affluent countries live in a privileged land of plenty where high calorie foods are easily available and in which we have a limited need for exercise. The rising prevalence of obesity in children is of particular concern (Chinn & Rona, 2001).

Comment on: Effect of laparoscopic Roux-en-Y gastric bypass versus laparoscopic sleeve gastrectomy on fasting gastrointestinal and pancreatic peptide hormones: A prospective nonrandomized trial

One of the largest challenges in medicine today is to break the curve of the increasing prevalence of obesity and its deleterious effects on health including type 2 diabetes, cardiovascular disease and cancer in most societies across the world. Although feeding behaviour and exercise habits to a large extent are considered to be deliberate and voluntary choices of human beings, research conducted over the last couple of decades has unravelled a significant and non-voluntary contribution of peripheral appetite regulating hormones like leptin, ghrelin, serotonin, glucagon like peptide-1 (GLP-1) and peptide YY on feeding behaviour, and thereby on risk of development of obesity. Genetic or non-genetic permanent changes in secretion or action, or in the ‘set point control’, of these hormones may provide the clue to understand why behaviour changes are most often unsuccessful to prevent, or in particular to revert, obesity. While appetite may be controlled in different areas of the brain, the hypothalamic arcuate, ventromedial and paraventricular nuclei as well as the lateral hypothalamic area seems to take centre stage in the hormonal and metabolic control of feeding behaviour. Interestingly, the metabolic actions of most appetite controlling hormones goes far beyond hypothalamic regulation of feeding behaviour per se, and may influence other direct and indirect obesity and diabetes related functions including energy expenditure (leptin, peptide YY), sleep (serotonin), insulin secretion (GLP-1, leptin) and insulin action (leptin, peptide YY). To this end, metabolic substrates including glucose and fatty acids exhibit more direct effects on central neuronal activities and functions than previously thought, all together unmaking a complex scenario of metabolic and hormonal cross-talk(s) between peripheral tissues, including adipose tissue and gut, on one side, and the central nervous system on the other side, with potential utmost importance for understanding the molecular mechanisms causing obesity and diabetes. In this issue of The Journal of Physiology, we focus on novel aspects of hormonal and metabolic effects on neuronal and CNS functions relevant to appetite, feeding behaviour, and ultimately to the understanding of the mechanisms causing – as well as the potentials to treat – obesity and type 2 diabetes. The discovery of the appetite regulating gut hormones glucagon like peptide-1 (GLP1), peptide YY and ghrelin has created increased awareness of the gut being perhaps the largest endocrine organ, with metabolic and behavioural effects going far beyond food digestion and dissemination. GLP1 and peptide YY are both produced and secreted form specialized enteroendocrine L-cells within the distal gastrointestinal tract in response to food intake, and they share the effect on a hypothalamic level to reduce appetite and food intake. Hypothalamic Y2-receptors seems to mediate the anorectic effect of peptide PYY, and as reviewed by Karra et al. (2009), plasma levels of PYY have been reported to associate negatively with obesity in humans, and knock-out experiments have documented a hyperphagic and obese phenotype in mice lacking PYY. Most interestingly, the surprising almost complete normalization of glucose metabolism in obese patients with type 2 diabetes occurring the first day(s) after gastric bypass surgery may be caused by significantly elevated PYY levels due to a more rapid passage of food through the gut after operation. Altogether, pharmacological targeting of the PYY system represents a promising strategy to treat obesity in the future. As for GLP1, analogues mimicking its documented metabolic and appetite reducing effects have in fact already been approved and launched as drugs to treat obese patients with type 2 diabetes. Native GLP1 is cleared rapidly after secretion by the enzyme dipeptidyl-peptidase, and native GLP1 therefore cannot be used clinically as a drug without some kind of prolongation of action. The GLP1 analogues have primarily been approved due to their incretin effects to enhance insulin secretion, and they are not (yet) approved to treat obesity per se. However, despite increased insulin secretion, treatment with GLP1 analogues does result in less weight gain as compared with antidiabetic drugs with the same potency, and it may be a question of time before GLP1 analogues may be used to treat obesity per se. In the review by Tolhurst et al. (2009), they focus on cellular and physiological mechanisms, as well as on nutritional effects, of GLP1 production and release from the intestinal L-cells. Obviously, the development of tools to increase endogenous GLP1 secretion and plasma levels represent yet another way to reduce appetite in obesity, and to increase insulin secretion in type 2 diabetes. Hypocretin (Hcrt) peptides are produced in neurones in the lateral hypothalamus playing a key role in the control of sleep and in the diurnal variation of a number of metabolic functions including appetite, basal metabolic rate, insulin action as well as insulin secretion. The role of Hcrt neurons in metabolism has become a topic of increased interest with the awareness of sleep deprivation as a significant cause of increased appetite, possibly due to reduced leptin and increased ghrelin levels, as well as insulin resistance and decreased insulin secretion. Recent evidence suggest that Hcrt neurones my act as ‘primary’ sensors responding to various different changing neuroendocrine and metabolic signals including leptin, ghrelin and glucose levels. In the review by Adamantidis & de Lecca (2009), they discuss the intriguing potential role of Hrct neurones in coordinating metabolic and physiological inputs into appropriate goal-orientated behaviour. Gonzalez et al. (2009) review the physiological significance of hypothalamic glucose sensing neurones on appetite and sleep regulating circuits. Hypothalamic neuronal firing rates may either decrease or increase in response to extracellular glucose influencing wakefulness-promoting hypocretin/orexin related signalling, as well as appetite-regulating neuropeptide Y (NPY) and pro-opiomelanocortin (POMC) biological functions. Interestingly, dissociation between sensing and metabolism of glucose represents a homeostatic mechanism allowing the brain rapidly to respond to changes in extracellular glucose without adverse effects on the vital and fine tuned flow of intracellular fuel in the brain. It has long been known that increased availability of serotonin (5-hydroxy-tryptamine, 5-HT) in the CNS attenuate food intake. However, induction of tolerance and side-effects of serotonin raising compounds including fenfluramine and dexfenfluramine has limited their clinical usefulness. In the review by Garfield & Heisler (2009), they discuss the molecular mechanism of action of serotonin to reduce appetite by modulating firing of pro-opiomelanocortin (POMC)/cocaine and amphetamine regulated transcript (CART) as well as agouti related protein (AgRP)/neuropeptide Y (NPY) neurones in the arcuate nucleus of the hypothalamus. Serotonin activates a number of different receptors in different tissues, some of them being responsible for the undesirable side-effects, and therefore the potential selectively to target the most central appetite regulating serotonin receptors including the 5HT1b and 5HT2c receptors may represent a promising way forward to reduce appetite in obesity. After reading the reviews on recent progress in knowledge concerning neuroendocrine and metabolic hypothalamic – as well as gut – control of feeding behaviour in this issue of The Journal of Physiology, it is difficult not to become somewhat less defaitistic regarding the possibility to develop more effective and safe tools and drugs to combat obesity in the future.

Glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) are anti-diabetes/obesity hormones secreted from the gut after meal ingestion. We have shown that dietary-resistant starch (RS) increased GLP-1 and PYY secretion, but the mechanism remains unknown. RS is a fermentable fiber that lowers the glycemic index of the diet and liberates short-chain fatty acids (SCFAs) through fermentation in the gut. This study investigates the two possible mechanisms by which RS stimulates GLP-1 and PYY secretion: the effect of a meal or glycemic index, and the effect of fermentation. Because GLP-1 and PYY secretions are stimulated by nutrient availability in the gut, the timing of blood sample collections could influence the outcome when two diets with different glycemic indexes are compared. Thus we examined GLP-1 and PYY plasma levels at various time points over a 24-h period in RS-fed rats. In addition, we tested proglucagon (a precursor to GLP-1) and PYY gene expression patterns in specific areas of the gut of RS-fed rats and in an enteroendocrine cell line following exposure to SCFAs in vitro. Our findings are as follows. 1) RS stimulates GLP-1 and PYY secretion in a substantial day-long manner, independent of meal effect or changes in dietary glycemia. 2) Fermentation and the liberation of SCFAs in the lower gut are associated with increased proglucagon and PYY gene expression. 3) Glucose tolerance, an indicator of increased active forms of GLP-1 and PYY, was improved in RS-fed diabetic mice. We conclude that fermentation of RS is most likely the primary mechanism for increased endogenous secretions of total GLP-1 and PYY in rodents. Thus any factor that affects fermentation should be considered when dietary fermentable fiber is used to stimulate GLP-1 and PYY secretion.

Effects on GLP-1, PYY, and leptin by direct stimulation of terminal ileum and cecum in humans: implications for ileal transposition

The 2 gut hormones glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) are well known to be coexpressed, costored, and released together to coact in the control of key metabolic target organs. However, recently, it became clear that several other gut hormones can be coexpressed in the intestinal-specific lineage of enteroendocrine cells. Here, we focus on the anatomical and functional consequences of the coexpression of neurotensin with GLP-1 and PYY in the distal small intestine. Fluorescence-activated cell sorting analysis, laser capture, and triple staining demonstrated that GLP-1 cells in the crypts become increasingly multihormonal, ie, coexpressing PYY and neurotensin as they move up the villus. Proglucagon promoter and pertussis toxin receptor-driven cell ablation and reappearance studies indicated that although all the cells die, the GLP-1 cells reappear more quickly than PYY- and neurotensin-positive cells. High-resolution confocal fluorescence microscopy demonstrated that neurotensin is stored in secretory granules distinct from GLP-1 and PYY storing granules. Nevertheless, the 3 peptides were cosecreted from both perfused small intestines and colonic crypt cultures in response to a series of metabolite, neuropeptide, and hormonal stimuli. Importantly, neurotensin acts synergistically, ie, more than additively together with GLP-1 and PYY to decrease palatable food intake and inhibit gastric emptying, but affects glucose homeostasis in a more complex manner. Thus, neurotensin is a major gut hormone deeply integrated with GLP-1 and PYY, which should be taken into account when exploiting the enteroendocrine regulation of metabolism pharmacologically.

Aims/hypothesisTargeting the secretion of gut peptides such as glucagon-like peptide 1 (GLP-1) and peptide YY (PYY) is a strategy under development for the treatment of diabetes and obesity, aiming to mimic the beneficial alterations in intestinal physiology that follow gastric bypass surgery. In vitro systems are now well established for studying the mouse enteroendocrine system, but whether these accurately model the human gut remains unclear. The aim of this study was to establish and characterise human primary intestinal cultures as a model for assessing GLP-1 and PYY secretion in vitro.MethodsFresh surgical biopsies of human colon were digested with collagenase to generate primary cultures from which GLP-1 and PYY secretion were assayed in response to test stimuli. GLP-1 and PYY co-localisation were assessed by flow cytometry and immunofluorescence microscopy.ResultsGLP-1 and PYY were found localised in the same cells and the same secretory vesicles in human colonic tissue samples. GLP-1 release was increased to 2.6-fold the control value by forskolin + isobutylmethylxanthine (10 μmol/l each), 2.8-fold by phorbol myristate acetate (1 μmol/l) and 1.4-fold by linoleic acid (100 μmol/l). PYY release was increased to 2.0-, 1.8- and 1.3-fold by the same stimuli, respectively. Agonists of G-protein-coupled receptor (GPR)40/120 and G-protein-coupled bile acid receptor 1 (GPBAR1) each increased GLP-1 release to 1.5-fold, but a GPR119 agonist did not significantly stimulate secretion.Conclusions/interpretationPrimary human colonic cultures provide an in vitro model for interrogating the human enteroendocrine system, and co-secrete GLP-1 and PYY. We found no evidence of PYY-specific cells not producing GLP-1. GLP-1 secretion was enhanced by small molecule agonists of GPR40/120 and GPBAR1.

OBJECTIVEBariatric surgery causes durable weight loss. Gut hormones are implicated in obesity pathogenesis, dietary failure, and mediating gastrointestinal bypass (GIBP) surgery weight loss. In mice, we determined the effects of diet-induced obesity (DIO), subsequent dieting, and GIBP surgery on ghrelin, peptide YY (PYY), and glucagon-like peptide-1 (GLP-1). To evaluate PYY’s role in mediating weight loss post-GIBP, we undertook GIBP surgery in PyyKO mice.RESEARCH DESIGN AND METHODSMale C57BL/6 mice randomized to a high-fat diet or control diet were killed at 4-week intervals. DIO mice underwent switch to ad libitum low-fat diet (DIO-switch) or caloric restriction (CR) for 4 weeks before being killed. PyyKO mice and their DIO wild-type (WT) littermates underwent GIBP or sham surgery and were culled 10 days postoperatively. Fasting acyl-ghrelin, total PYY, active GLP-1 concentrations, stomach ghrelin expression, and colonic Pyy and glucagon expression were determined. Fasting and postprandial PYY and GLP-1 concentrations were assessed 30 days postsurgery in GIBP and sham pair-fed (sham.PF) groups.RESULTSDIO progressively reduced circulating fasting acyl-ghrelin, PYY, and GLP-1 levels. CR and DIO-switch caused weight loss but failed to restore circulating PYY to weight-appropriate levels. After GIBP, WT mice lost weight and exhibited increased circulating fasting PYY and colonic Pyy and glucagon expression. In contrast, the acute effects of GIBP on body weight were lost in PyyKO mice. Fasting PYY and postprandial PYY and GLP-1 levels were increased in GIBP mice compared with sham.PF mice.CONCLUSIONSPYY plays a key role in mediating the early weight loss observed post-GIBP, whereas relative PYY deficiency during dieting may compromise weight-loss attempts.

The relationship between postprandial peptides at circulating physiological levels and short-term appetite control is not well understood. The purpose of this study was first to compare the postprandial profiles of ghrelin, glucagon-like peptide 1 (GLP-1), and peptide YY (PYY) after isoenergetic meals differing in fat and carbohydrate content and second to examine the relationships between ghrelin, GLP-1, and PYY with hunger, fullness, and energy intake. Plasma was collected before and periodically after the meals for 180 minutes, after which time ad libitum food was provided. Simultaneous ratings of hunger and fullness were tracked for 180 minutes through phases identified as early (0-60 minutes) and late (60-180 minutes) satiety. This study was conducted at the Psychobiology and Energy Balance Research Unit, University of Leeds. The participants were 16 healthy overweight/obese adults. Changes in hunger and fullness and metabolic markers were indicators of the impact of the meals on satiety. Ghrelin was influenced similarly by the 2 meals [F(1, 12) = 0.658, P = .433] and was significantly associated with changes in hunger (P < .05), which in turn correlated with food intake (P < .05). GLP-1 and PYY increased more by the high-fat meal [F(1, 15) = 5.099 and F(1, 14) = 5.226, P < .05]. GLP-1 was negatively associated with hunger in the late satiety phase and with energy intake (P < .05), but the PYY profile was not associated with hunger or fullness, nor was PYY associated with food intake. The results demonstrate that under these conditions, these peptides respond differently to ingested nutrients. Ghrelin and GLP-1, but not PYY, were associated with short-term control of appetite over the measurement period.

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.

Exercise suppresses appetite in an intensity‐dependent manner partly due to changes in peripheral appetite‐regulating hormones. While the underlying mechanisms are unclear, lactate release from active musculature may be important as its binding to gastric cell lines inhibits the release of the orexigenic hormone ghrelin. The contraction‐induced myokine interleukin‐6 (IL‐6) may also be involved, as it stimulates the production and release of anorexigenic factors such as glucagon‐like peptide‐1 (GLP‐1) and peptide YY (PYY) from intestinal cells. If these mechanisms contribute to the intensity‐dependent suppression of appetite following exercise, post‐exercise increases in lactate and IL‐6 should coincide with changes in ghrelin, GLP‐1, and PYY. To test this hypothesis, the current study examined changes in blood lactate, IL‐6, and appetite‐regulating hormones in response to running at various intensities. Eight males performed four experimental sessions: 1) Moderate‐intensity continuous training (MICT, 30 min at 65% VO2max); 2) High‐intensity continuous training (HICT, 30 min at 85% VO2max); 3) Sprint interval training (SIT, 4 × 30 sec maximal efforts, 4 min rest); 4) Control (CTRL, no exercise). Lactate, IL‐6, ghrelin, GLP‐1, and PYY concentrations were measured pre‐, post‐, 30 min post‐, and 90 min post‐exercise. Appetite perceptions were assessed at the same time‐points using a visual analog scale. Exercise suppressed ghrelin and appetite in an intensity‐dependent manner (HICT and SIT vs. CTRL, p&lt;0.001), with SIT resulting in a greater (SIT vs. MICT at 30 min post‐exercise, p&lt;0.020) and more prolonged (SIT vs. all other sessions at 90 min post‐exercise, p&lt;0.005) response. GLP‐1 increased immediately after MICT (p&lt;0.001 vs. all other sessions) and at 30 min post‐exercise after HICT (p&lt;0.002 vs. all sessions) and SIT (p=0.001 vs. CTRL). PYY increased immediately post‐exercise (p&lt;0.001 vs. CTRL) though more so after HICT versus MICT (p=0.027). Exercise‐induced changes in appetite‐regulating hormones correlated significantly (p&lt;0.009) with appetite perceptions (ghrelin: r=0.40; GLP‐1: r=−0.31; PYY: r=−0.36). Post‐exercise increases in blood lactate correlated negatively (p&lt;0.036) with ghrelin (MICT: r=−0.43; HICT: r=−0.50; SIT: r=−0.45) and appetite (MICT: r=−0.43; HICT: r=−0.47; SIT: r=−0.45). Changes in IL‐6 correlated with GLP‐1 following SIT only (r=0.48, p=0.018), though no relationship between IL‐6 and PYY was observed. These findings support an intensity‐dependent paradigm for appetite regulation following exercise, which appears to be closely associated with reductions in ghrelin though increases in GLP‐1 and PYY also contribute. The results also highlight the potential involvement of lactate and IL‐6 in mediating changes in appetite‐regulating hormones, particularly after intense exercise.Support or Funding InformationNatural Sciences and Engineering Research Council of Canada

Ghrelin suppresses cholecystokinin (CCK), peptide YY (PYY) and glucagon-like peptide-1 (GLP-1) in the intestine, and attenuates the anorectic effects of CCK, PYY and GLP-1 in goldfish (Carassius auratus)

Enhanced transcription of pancreatic peptide YY by 1α-hydroxyvitamin D3 administration in streptozotocin-induced diabetic mice