Gut hormone stimulation as a therapeutic approach in oral peptide delivery.

In this contribution to the Orations - New Horizons of the Journal of Controlled Release, I discuss the research that we have conducted on gut hormone stimulation as a therapeutic strategy in oral peptide delivery. One of the greatest challenges in oral drug delivery involves the development of new drug delivery systems that enable the absorption of therapeutic peptides into the systemic circulation at therapeutically relevant concentrations. This scenario is especially challenging in the treatment of chronic diseases (such as type 2 diabetes mellitus), wherein daily injections are often needed. However, for certain peptides, there may be an alternative in drug delivery to meet the need for increased peptide bioavailability; this is the case for gut hormone mimetics (including glucagon-like peptide (GLP)-1 or GLP-2). One plausible alternative for improved oral delivery of these peptides is the co-stimulation of the endogenous secretion of the hormone to reach therapeutic levels of the peptide. This oration will be focused on studies conducted on the stimulation of gut hormones secreted from enteroendocrine L cells in the treatment of gastrointestinal disorders, including a critical discussion of the limitations and future perspectives of implementing this approach in the clinical setting.

Circulating profile of the appetite-regulating hormone ghrelin during moult-fast and chick provisioning in southern rockhopper penguins (Eudyptes chrysocome chrysocome).

A multitude of animal species undergo prolonged fasting events at regularly occurring life history stages. During such periods of food deprivation, individuals need to suppress their appetite. The satiety signalling gut hormone ghrelin has received much attention in this context in studies looking at mammalian systems. In wild birds, however, knowledge on the ghrelin system and its role during extended fasts is still scarce. In this study, we collected plasma samples for measurements of circulating ghrelin concentrations from adult southern rockhopper penguins (Eudyptes chrysocome chrysocome) during the three to four week-long moult-fast that they repeat annually to replace their feathers. We further sampled chicks before and after feeding bouts and non-moulting adults. Circulating ghrelin levels did not differ significantly between fed and unfed chicks but chicks had significantly lower plasma ghrelin levels compared to adults. Furthermore, penguins in late moult (i.e. individuals at the end of the prolonged fasting bout) had higher ghrelin levels compared to non-moulting adults. Our results show elevated levels of circulating ghrelin during moult and generally lower levels of ghrelin in chicks than in adults regardless of feeding state. Given the scarcity or absence of knowledge on the function of ghrelin in seabirds and in fasting birds in general, our results add greatly to our understanding of the avian ghrelin system.

Gut hormone multi-agonists for the treatment of type 2 diabetes and obesity: advances and challenges.

The rapidly rising incidence of obesity, coupled with type 2 diabetes mellitus (T2DM), is a growing concern. Glucagon-like peptide 1 (GLP-1), an endogenous peptide secreted by enteroendocrine L-cells, demonstrates exceptional pharmacological potential for the treatment of T2DM and obesity, primarily through its pivotal roles in regulating glucose homeostasis, stimulating glucose-dependent insulin secretion, and promoting satiety. Considering its proven efficacy in glucoregulation and weight loss, GLP-1 receptor agonists (GLP-1RAs) have emerged as a revolutionary breakthrough in the arena of diabetes management and weight control. Additional gastrointestinal hormones, such as glucose-dependent insulinotropic peptide (GIP) and glucagon, exhibit structural similarities to GLP-1 and work synergistically to lower blood glucose levels or aid in weight loss. Today, various classes of gut hormone receptor multiple agonists are steadily progressing through development and clinical trials, including dual GLP-1/glucagon receptor agonists (first discovered in 2009), dual GLP-1/GIP receptor agonists (first described in 2013), and triple GLP-1/GIP/glucagon receptor agonists (initially designed in 2015). The GLP-1/GIP receptor co-agonist, tirzepatide, was approved by the U.S. Food and Drug Administration (FDA) for the treatment of T2DM, outperforming basal insulin or selective GLP-1RAs by providing superior HbA1c reductions. Remarkably, tirzepatide also facilitated unprecedented weight loss of up to 22.5% in non-diabetic individuals living with obesity. This result is comparable to those achieved with certain types of bariatric surgery. Therefore, the advent of gut hormone multi-agonists signifies the dawn of an exciting new era in peptide-based therapy for obesity and T2DM. This review offers a comprehensive summary of the various types of gut hormone multiple agonists, including their discovery, development, action of mechanisms, and clinical effectiveness. We further delve into potential hurdles, limitations, and prospective advancements in the field.

The Effects of a Cinchona Supplementation on Satiety, Weight Loss and Body Composition in a Population of Overweight/Obese Adults: A Controlled Randomized Study.

Obesity is a risk factor for several diseases present worldwide. Currently, dietary changes and physical activity are considered the most effective treatment to reduce obesity and its associated comorbidities. To promote weight loss, hypocaloric diets can be supported by nutraceuticals. The aim of this study was to evaluate the effects of a hypocaloric diet associated with Cinchona succirubra supplementation on satiety, body weight and body composition in obese subjects. Fifty-nine overweight/obese adults, were recruited, randomized into two groups and treated for 2 months. The first group (32 adults) was treated with a hypocaloric diet plus cinchona supplementation (the T-group); the second one (27 adults) was treated with a hypocaloric diet plus a placebo supplementation (the P-group). Anthropometric-measurements as well as bioimpedance analysis, a Zung test and biochemical parameters were evaluated at baseline and after 60 days. T-group adults showed significant improvement in nutritional status and body composition compared to those at the baseline and in the P-group. Moreover, T-group adults did not show a reduction in Cholecystokinin serum levels compared to those of P-group adults. In conclusion, our data demonstrate that a hypocaloric diet associated with cinchona supplementation is effective in inducing more significant weight loss and the re-establishment of metabolic parameters than those obtained with a hypocaloric diet.

Effects of secretin gene knockout on the diversity, composition, and function of gut microbiota in adult male mice.

The gut microbiota plays a vital role in maintaining gastrointestinal homeostasis, however, whether it is influenced by gut hormones remains unknown. Secretin is a well-known gastrointestinal hormone produced by enteroendocrine S cells. This study utilized 16S rRNA amplicon sequencing to characterize the effect of SCT deficiency on the gut microbiota. Our results show that systemic SCT knockout alters the composition and abundance of the mouse gut microbiota but does not affect fecal short-chain fatty acids and lipids concentrations. At the genus level, the abundance of Turicibacter, Bacteroides, Ruminococcu, Romboutsia, Asaccharobacter, and Parasutterella increased in SCT-/- mice, whereas the abundance of Akkermansia and Escherichia decreased. Functional prediction results showed that lack of SCT reduced the abundance of carbohydrate metabolism-related pathways but increased the abundance of linoleic acid metabolism and branched-chain amino acid degradation. Overall, systemic SCT knockout had only minor effects on gut microbiota composition and function in adult male mice fed a standard chow diet.

High-calorie diets uncouple hypothalamic oxytocin neurons from a gut-to-brain satiation pathway via κ-opioid signaling.

Oxytocin-expressing paraventricular hypothalamic neurons (PVNOT neurons) integrate afferent signals from the gut, including cholecystokinin (CCK), to adjust whole-body energy homeostasis. However, the molecular underpinnings by which PVNOT neurons orchestrate gut-to-brain feeding control remain unclear. Here, we show that mice undergoing selective ablation of PVNOT neurons fail to reduce food intake in response to CCK and develop hyperphagic obesity on a chow diet. Notably, exposing wild-type mice to a high-fat/high-sugar (HFHS) diet recapitulates this insensitivity toward CCK, which is linked to diet-induced transcriptional and electrophysiological aberrations specifically in PVNOT neurons. Restoring OT pathways in diet-induced obese (DIO) mice via chemogenetics or polypharmacology sufficiently re-establishes CCK's anorexigenic effects. Last, by single-cell profiling, we identify a specialized PVNOT neuronal subpopulation with increased κ-opioid signaling under an HFHS diet, which restrains their CCK-evoked activation. In sum, we document a (patho)mechanism by which PVNOT signaling uncouples a gut-brain satiation pathway under obesogenic conditions.

Applications of Enteroendocrine Cells (EECs) Hormone: Applicability on Feed Intake and Nutrient Absorption in Chickens.

This review focuses on the role of hormones derived from enteroendocrine cells (EECs) on appetite and nutrient absorption in chickens. In response to nutrient intake, EECs release hormones that act on many organs and body systems, including the brain, gallbladder, and pancreas. Gut hormones released from EECs play a critical role in the regulation of feed intake and the absorption of nutrients such as glucose, protein, and fat following feed ingestion. We could hypothesize that EECs are essential for the regulation of appetite and nutrient absorption because the malfunction of EECs causes severe diarrhea and digestion problems. The importance of EEC hormones has been recognized, and many studies have been carried out to elucidate their mechanisms for many years in other species. However, there is a lack of research on the regulation of appetite and nutrient absorption by EEC hormones in chickens. This review suggests the potential significance of EEC hormones on growth and health in chickens under stress conditions induced by diseases and high temperature, etc., by providing in-depth knowledge of EEC hormones and mechanisms on how these hormones regulate appetite and nutrient absorption in other species.

Dorothy Hodgkin lecture 2023: The enteroendocrine system-Sensors in your guts.

Glucagon-like peptide-1 (GLP-1)-based medication is now widely employed in the treatment of type 2 diabetes and obesity. Like other gut hormones, GLP-1 is released from eneteroendocrine cells after a meal and in this review, based on the Dorothy Hodgkin lecture delivered during the annual meeting of Diabetes UK in 2023, I argue that there is sufficient spare capacity of GLP-1 and other gut hormone expressing cells that could be recruited therapeutically. Years of research has revealed several receptors expressed in enteroendocrine cells that could be targeted to stimulate hormone release: although from this research it seems unlikely to find agents that selectively boost GLP-1, release of a mixture of hormones might be the more desirable outcome anyway, given the recent promising results of new peptides combining GLP1-receptor with other gut hormone receptor activation. Alternatively, the fact that GLP-1 and peptideYY (PYY) expressing cells are found in greater density in the ileum might be exploited by increasing the delivery of chyme to the distal small intestine.

Endogenous glucagon-like peptide (GLP)-1 as alternative for GLP-1 receptor agonists: Could this work and how?

In recent years, we have witnessed the many beneficial effects of glucagon-like peptide (GLP)-1 receptor agonists, including the reduction in cardiovascular risk in patients with type 2 diabetes, and the reduction of body weight in those with obesity. Increasing evidence suggests that these agents differ considerably from endogenous GLP-1 when it comes to their routes of action, although their clinical effects appear to be the same. Given the limitations of the GLP-1 receptor agonists, could it be useful to develop agents which stimulate GLP-1 release? Here we will discuss the differences and similarities between GLP-1 receptor agonists and endogenous GLP-1, and will detail how endogenous GLP-1-when stimulated appropriately-could have clinically relevant effects.

Higher circulating levels of proneurotensin are associated with increased risk of incident NAFLD.

BACKGROUND: Neurotensin (NT), an intestinal peptide able to promote fat absorption, is implicated in the pathogenesis of obesity. Increased levels of proneurotensin (pro-NT), a stable NT precursor fragment, have been found in subjects with nonalcoholic fatty liver disease (NAFLD); however, whether higher pro-NT levels are associated with an increased NAFLD risk independently of other metabolic risk factors is unsettled. METHODS: Ultrasound-defined presence of NAFLD was assessed on 303 subjects stratified into tertiles according to fasting pro-NT levels. The longitudinal association between pro-NT levels and NAFLD was explored on the study participants without NAFLD at baseline reexamined after 5 years of follow-up (n = 124). RESULTS: Individuals with higher pro-NT levels exhibited increased adiposity, a worse lipid profile, and insulin sensitivity as compared to the lowest tertile of pro-NT. Prevalence of NAFLD was progressively increased in the intermediate and highest pro-NT tertile as compared to the lowest tertile. In a logistic regression analysis adjusted for several confounders, individuals with higher pro-NT levels displayed a raised risk of having NAFLD (OR = 3.43, 95%CI = 1.48-7.97, p = 0.004) than those in the lowest pro-NT tertile. Within the study cohort without NAFLD at baseline, subjects with newly diagnosed NAFLD at follow-up exhibited higher baseline pro-NT levels than those without incident NAFLD. In a cox hazard regression analysis model adjusted for anthropometric and metabolic parameters collected at baseline and follow-up visit, higher baseline pro-NT levels were associated with an increased risk of incident NAFLD (HR = 1.52, 95%CI = 1.017-2.282, p = 0.04). CONCLUSION: Higher pro-NT levels are a predictor of NAFLD independent of other metabolic risk factors.

Weight loss by calorie restriction does not alter appetite-regulating gut hormone responses from perfused rat small intestine.

AIM: Postprandial secretion of the appetite-inhibiting hormones, glucagon-like peptide-1 (GLP-1), and peptide YY are reduced with obesity. It is unclear if the reduced secretion persists following weight loss (WL), if other appetite-inhibiting hormones are also reduced, and if so whether reduced secretion results from intrinsic changes in the gut. METHODS: To address whether WL may restore secretion of GLP-1 and other appetite-inhibiting hormones, we performed a gut perfusion study of the small intestine in diet-induced obese (DIO) rats after WL. A 20% weight loss (means ± SEM (g): 916 ± 53 vs. 703 ± 35, p < 0.01, n = 7) was induced by calorie restriction, and maintained stable for ≥7 days prior to gut perfusion to allow for complete renewal of enteroendocrine cells. Age-matched DIO rats were used as comparator. Several gut hormones were analyzed from the venous effluent, and gene expression was performed on gut tissue along the entire length of the intestine. RESULTS: Secretion of cholecystokinin, gastrin, glucose-dependent insulinotropic peptide, GLP-1, neurotensin, and somatostatin was not affected by WL during basal conditions (p ≥ 0.25) or in response to macronutrients and bile acids (p ≥ 0.14). Glucose absorption was indistinguishable following WL. The expression of genes encoding the studied peptides, macronutrient transporters (glucose, fructose, and di-/tripeptides) and bile acid receptors did also not differ between DIO and WL groups. CONCLUSIONS: These data suggest that the attenuated postprandial responses of GLP-1, as well as reduced responses of other appetite-inhibiting gut hormones, in people living with obesity may persist after weight loss and may contribute to their susceptibility for weight regain.

The gut odorant receptor and taste receptor make sense of dietary components: A focus on gut hormone secretion.

Odorant receptors (ORs) and taste receptors (TRs) are expressed primarily in the nose and tongue in which they transduce electrical signals to the brain. Advances in deciphering the dietary component-sensing mechanisms in the nose and tongue prompted research on the role of gut chemosensory cells. Acting as the pivotal interface between the body and dietary cues, gut cells "smell" and "taste" dietary components and metabolites by taking advantage of chemoreceptors-ORs and TRs, to maintain physiological homeostasis. Here, we reviewed this novel field, highlighting the latest discoveries pertinent to gut ORs and TRs responding to dietary components, their impacts on gut hormone secretion, and the mechanisms involved. Recent studies indicate that gut cells sense dietary components including fatty acid, carbohydrate, and phytochemical by activating relevant ORs, thereby modulating GLP-1, PYY, CCK, and 5-HT secretion. Similarly, gut sweet, umami, and bitter receptors can regulate the gut hormone secretion and maintain homeostasis in response to dietary components. A deeper understanding of the favorable influence of dietary components on gut hormone secretion via gut ORs and TRs, coupled with the facts that gut hormones are involved in diverse physiological or pathophysiological phenomena, may ultimately lead to a promising treatment for various human diseases.

Effects of esterification, saturation and amount of fatty acids infused into the rumen or abomasum in lactating dairy cows.

Our objective was to determine the effects of chemical structure, amount, and site of infusion of long-chain fatty acids (LCFA) in lactating dairy cows. Six multiparous Holstein cows were used in a 6 × 6 Latin square design with 21-d periods. During d 1 to 14, 250 g/d of LCFA and during d 15 to 21, 500 g/d of LCFA were infused continuously into either the rumen or abomasum. Treatments were 1) Control (CONT); 200 g/d of meat solubles plus 12 g/d of Tween 80 in 10 L of water, administered half in the rumen and half in abomasum; 2) control plus mostly saturated LCFA into the abomasum (SFAA); 3) control plus mostly saturated LCFA into the rumen (SFAR); 4) control plus soy (mostly unsaturated LCFA) free fatty acids (FFA) into the abomasum (UFAA); 5) control plus soy triglycerides (TG) into the abomasum (TGA); and 6) control plus soy TG into the rumen (TGR). The first 10 d of each period were for adaptation and washout from the previous treatment. The diet consisted of 30% (dry matter basis) corn silage, 20% alfalfa silage and 50% concentrate. Cows infused with UFAA had lower dry matter intake and milk yield than those infused with SFAA or TGA and reductions were greater at the higher infusion amount. Milk fat yield was decreased by UFAA relative to other treatments. Unsaturated LCFA decreased milk fat yield more than saturated LCFA. All LCFA treatments decreased short- and medium-chain FA in milk relative to CONT, with greatest decreases for UFAA. Apparent total tract digestibilities of nutrient fractions were decreased by UFAA compared with TGA and SFAA and tended to be lower at the higher infusion amount. Apparent digestibility of total fatty acids (FA) was greater for SFAR than for SFAA. Plasma glucagon-like peptide-1 was greater for cows infused with UFAA than SFAA or TGA and increased at the higher amount. Plasma cholecystokinin was greater for cows infused with LCFA compared with CONT. Postruminal unsaturated FFA reduced intake and digestibility of nutrients and FA compared with postruminal TG infusion; saturated FA did not decrease dry matter intake or disrupt nutrient digestion. Glucagon-like peptide-1 may be involved in regulation of feed intake by long-chain fatty acids.

The catcher in the gut: Tirzepatide, a dual incretin analog for the treatment of type 2 diabetes mellitus and obesity.

The ever-increasing burden of obesity, type 2 diabetes mellitus (T2DM) and related comorbidities is demanding a better pathophysiological understanding as well as new treatment options. Incretin based therapies are already available while the recent Food and Drug Administration (FDA) approval of the dual glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) receptor agonist tirzepatide appears to revolutionize the treatment of T2DM and obesity. GLP-1 and GIP exert pleiotropic physiological actions, including enhancement of insulin secretion, glycemic and appetite control, cardioprotection, and adipose tissue improved function among others. Evidence from recent clinical trials has shown that tirzepatide is at least or more effective compared to classic metabolic therapeutic agents, including insulin, when it comes to glycemic control in T2DM. Of importance, it also exerts remarkable weight-lowering actions, emerging as an alternative to bariatric surgery for obesity treatment. Overall, current data show that tirzepatide is a highly effective therapeutic option for T2DM. Numerous ongoing randomized controlled clinical trials are further examining its potential as a treatment for obesity.

Glucose-Dependent Insulinotropic Polypeptide Plasma Level Influences the Effect of n-3 PUFA Supplementation.

Elevated glucose-dependent insulinotropic peptide (GIP) levels in obesity may predict the metabolic benefits of n-3 PUFA supplementation. This placebo-controlled trial aimed to analyze fasting and postprandial GIP response to 3-month n-3 PUFA supplementation (1.8 g/d; DHA:EPA, 5:1) along with caloric restriction (1200-1500 kcal/d) in obese subjects. Compliance was confirmed by the incorporation of DHA and EPA into red blood cells (RBCs). Blood analyses of glucose, insulin, non-esterified fatty acids (NEFAs), GIP and triglycerides were performed at fasting, and during an oral glucose tolerance test and a high fat mixed-meal tolerance test. Fatty acid composition of RBC was assessed by gas chromatography and total plasma fatty acid content and composition was measured by gas-liquid chromatography. The DHA and EPA content in RBCs significantly increased due to n-3 PUFA supplementation vs. placebo (77% vs. -3%, respectively). N-3 PUFA supplementation improved glucose tolerance and decreased circulating NEFA levels (0.750 vs. 0.615 mmol/L), as well as decreasing plasma saturated (1390 vs. 1001 µg/mL) and monounsaturated (1135 vs. 790 µg/mL) fatty acids in patients with relatively high GIP levels. The effects of n-3 PUFAs were associated with the normalization of fasting (47 vs. 36 pg/mL) and postprandial GIP levels. Obese patients with elevated endogenous GIP could be a target group for n-3 PUFA supplementation in order to achieve effects that obese patients without GIP disturbances can achieve with only caloric restriction.

Butylated starch alleviates polycystic ovary syndrome by stimulating the secretion of peptide tyrosine-tyrosine and regulating faecal microbiota.

Polycystic ovary syndrome (PCOS) is an endocrine disorder that affects women of reproductive age. Resistant starch can be fermented by gut microbes then produce short-chain fatty acids (SCFAs). It was reported SCFAs might play a key role in gut microbiota-dependent therapy of PCOS. However, the effects of resistant starch on PCOS symptoms have not yet been investigated. Here, maize starch was acylated with specific SCFAs. Our results indicated that acylated starch can release acetate, propionate, and butyrate into the caecum and colon. Treatment with butylated starch (BS) alleviated abnormal ovarian morphology, metabolic disorders, and sex hormone imbalance in letrozole-treated rats, whereas treatment with acetylated starch and propylated starch did not exhibit such effects. Furthermore, BS stimulated the secretion of peptide tyrosine-tyrosine into the serum by activating a G protein-coupled receptor, GPR41, which further affected disease phenotypes. In addition, compared with caecal microbiota, faecal microbiota was more affected by BS. Butyrate-producing microbes were enriched in faeces after BS treatment and may have helped further to relieve PCOS symptoms.

Targeting the Enteroendocrine System for Treatment of Obesity.

Mimetics of the anorexigenic gut hormone glucagon-like peptide 1 (GLP-1) were originally developed as insulinotropic anti-diabetic drugs but also evoke significant weight loss, leading to their recent approval as obesity therapeutics. Co-activation of receptors for GLP-1 and other gut hormones which reduce food intake - peptide YY (PYY3-36), cholecystokinin (CCK) and glucose-dependent insulinotropic peptide (GIP) - is now being explored clinically to enhance efficacy. An alternative approach involves pharmacologically stimulating endogenous secretion of these hormones from enteroendocrine cells (EECs) to recapitulate the metabolic consequences of bariatric surgery, where highly elevated postprandial levels of GLP-1 and PYY3-36 are thought to contribute to improved glycaemia and weight loss.

The Enteroendocrine System in Obesity.

The enteroendocrine system coordinates the physiological response to food intake by regulating rates of digestion, nutrient absorption, insulin secretion, satiation and satiety. Gut hormones with important anorexigenic and/or insulinotropic roles include glucagon-like peptide 1 (GLP-1), peptide YY (PYY3-36), cholecystokinin (CCK) and glucose-dependent insulinotropic peptide (GIP). High BMI or obesogenic diets do not markedly disrupt this enteroendocrine system, which represents a critical target for inducing weight loss and treating co-morbidities in individuals with obesity.

Dietary Medium-Chain Triglyceride Decanoate Affects Glucose Homeostasis Through GPR84-Mediated GLP-1 Secretion in Mice.

Background: Dietary triglycerides are an important energy source; however, their excess intake causes metabolic diseases such as obesity and type 2 diabetes. Medium-chain triglycerides (MCTs) as triglyceride forms of medium-chain fatty acids (MCFAs) are applied to meet the energy demands of athletes, the elderly, and people with stunted growth, because MCFAs are efficiently converted into energy for immediate utilization by the organs and do not accumulate as fat. Although the intake of each MCT type (octanoate; C8:0, decanoate; C10:0, and dodecanoate; C12:0) exhibits beneficial metabolic effects, individual functional differences remain unclear. Methods: MCTs or MCFAs were administrated to male GPR84-deficient mice with a C57BL/6J background and mouse enteroendocrine cell line STC-1, and the effects on glucose homeostasis and gut hormone GLP-1 secretion were evaluated. Results: C10:0 intake improves glucose metabolism through the MCFA receptor GPR84-mediated GLP-1 secretion. Each MCT intake showed resistance to obesity and improved metabolic parameters compared with lard intake. Moreover, oral administration of MCTs enhanced glucose tolerance, especially C10:0 administration, which sufficiently increased plasma GLP-1 levels. Additionally, C10:0 stimulation promoted GLP-1 secretion via GPR84 in STC-1, enhanced glucose tolerance through GPR84-mediated GLP-1 secretion, and showed resistance to high-fat diet (HFD)-induced obesity in mice. Conclusions: Dietary MCT (C10:0) intake efficiently may protect against obesity and improve insulin resistance via GLP-1 secretion.

Blood Sampling From Rat Ileal Mesenteric Vein Revealed a Major Role of Dietary Protein in Meal-Induced GLP-1 Response.

The present study was conducted to examine region-dependent glucagon-like peptide-1 (GLP-1) responses to "meal ingestion" under physiological (conscious and unrestrained) conditions using rats with a catheter inserted into either the portal vein (PV) or the ileal mesenteric vein (ILMV). After recovery from the cannulation surgery, blood samples were collected from either PV or ILMV catheter before and after the voluntary ingestion of test diets. After an AIN-93G standard diet ingestion, GLP-1 concentration was higher in ILMV than in PV, and postprandial responses of peptide-YY (PYY) had similar trend, while that of glucose dependent-insulinotropic polypeptide showed an opposite trend to GLP-1/PYY responses. In a separated experiment, a protein-enriched diet containing casein at 25% wt/wt transiently increased GLP-1 concentration only in ILMV; however, a protein-free diet did not increase GLP-1 concentrations in PV or ILMV. These results indicate that postprandial GLP-1 is immediately released from the distal intestine under physiological conditions, and that dietary protein has a critical role in the enhancement of postprandial GLP-1 response.