Glucagon-like peptide-1 regulation by food proteins and protein hydrolysates

Abstract

Glucagon-like peptide-1 (GLP-1) is an enterohormone with a key role in several processes controlling body homeostasis, including glucose homeostasis and food intake regulation. It is secreted by the intestinal cells in response to nutrients, such as glucose, fat and amino acids. In the present review, we analyse the effect of protein on GLP-1 secretion and clearance. We review the literature on the GLP-1 secretory effects of protein and protein hydrolysates, and the mechanisms through which they exert these effects. We also review the studies on protein from different sources that has inhibitory effects on dipeptidyl peptidase-4 (DPP4), the enzyme responsible for GLP-1 inactivation, with particular emphasis on specific sources and treatments, and the gaps there still are in knowledge. There is evidence that the protein source and the hydrolytic processing applied to them can influence the effects on GLP-1 signalling. The gastrointestinal digestion of proteins, for example, significantly changes their effectiveness at modulating this enterohormone secretion in both in vivo and in vitro studies. Nevertheless, little information is available regarding human studies and more research is required to understand their potential as regulators of glucose homeostasis.

Introduction

The gastrointestinal tract is responsible for the digestion and absorption of nutrients, and acts as a barrier against luminal pathogens. Moreover, the gastrointestinal tract cooperates in controlling the metabolism through hormones secreted from enteroendocrine cells, which are the body’s largest endocrine organ⁽¹⁾. Enteroendocrine cells are capable of responding to luminal content because their apical side has chemosensing machinery such as taste receptors (TASR), G protein-coupled receptors (GPCR), specific transporters and channels. Their secretory products are stored in characterised secretory vesicles, before being secreted through the basolateral membrane by exocytosis^(2,3) . When luminal content moves through the gastrointestinal tract, specific macronutrients stimulate the chemosensing machinery, which leads to the modulation of gut hormone release. Gut hormones exert their effect via vagal nerve or endocrine/paracrine signalling, through the interaction of specific receptors expressed in different tissues of the body. These hormones, which are mainly glucagon-like peptide-1 (GLP-1), cholecystokinin (CCK), peptide YY (PYY), gastric inhibitory polypeptide (GIP) and ghrelin, influence the functioning of the digestive tract, but also modulate insulin secretion from the pancreas, the energy storage of adipose tissue and neuronal signalling in appetite centres in the brain to mediate the regulation of food intake by terminating hunger and inducing satiety.

Since dietary compounds modulate enterohormone secretion, and given the central role of enterohormones in body homeostasis, such an interaction could have beneficial health implications⁽⁴⁾. In this context, protein and protein hydrolysates are currently being studied to determine their effects on GLP-1 modulation, either through secretion or clearance, which may influence the processes regulated by this hormone such as regulation of glycaemia homeostasis and food intake control. The nutrient-sensing machinery of carbohydrates and lipids is better understood than the detection and pathways followed by protein digestion. The main reasons for this gap in knowledge is the redundant signalling in the gut for the different protein digestion products and the complexity of protein digests⁽⁵⁾. Here we review the literature on this subject in order to determine if the evidence supports differential effects of food proteins on GLP-1 profile. We will introduce the relevance of GLP-1 signalling on health. Then we will focus on the effects on GLP-1 secretion of proteins and its hydrolysates, and the suggested mechanisms. Finally, we will briefly review the use of protein hydrolysates as dipeptidyl peptidase-4 (DPP4) inhibitors. We compile a significant number of scientific studies to highlight the importance of the different protein sources, the hydrolysis conditions applied to them, and the resulting digestion products.

Relevance of glucagon-like peptide-1 signalling in health

There is evidence to suggest that specific enterohormones administered at physiological concentrations can influence the appetite of rodents and human subjects (for a review, see Murphy & Bloom⁽⁶⁾). Likewise, the effects of gut hormones on food intake and body weight have been observed in bariatric surgery (such as Roux-en-Y gastric bypass), which induces a huge increase in GLP-1 and peptide YY (PYY) secretion and is used to treat obesity⁽⁷⁾. Therefore, the modulation of enterohormone signalling may be an important target in the prevention of obesity and related/associated pathologies. Moreover, endogenous gut hormones regulate appetite physiologically, unlike the drugs that are currently available, which mainly influence the central neurotransmitter systems. Therefore, gut hormone-based therapies might lead to fewer side effects⁽⁶⁾.

Furthermore, modulation of endogenous incretin hormones (GLP-1 and GIP) could be an interesting strategy for preventing and/or managing type 2 diabetes mellitus (T2DM)⁽⁸⁾. T2DM is the most common endocrine disorder, characterised by insulin resistance and impaired insulin secretion, and it is one of the fastest growing non-communicable diseases in the world⁽⁹⁾. The main goal in the treatment of T2DM is to keep blood glucose levels within the normal physiological range. In this regard, GLP-1 and GIP are therapeutically interesting peptides because they are important mediators of glycaemic homeostasis, as they are responsible for approximately 50–70 % of the total insulin secreted after glucose intake⁽¹⁰⁾. GLP-1, together with GIP, is responsible for the incretin effect, since it binds to GLP-1 receptor in β-cells in the pancreas leading to an increase in intra-cellular Ca and a subsequent insulin secretion in response to glucose⁽¹¹⁾. It has also been shown that GLP-1 enhances markers of proliferation and differentiation, and decreases markers of apoptosis in the pancreas of Zucker diabetic rats^(12,13) . Furthermore, GLP-1 improves the glycaemic profile by inhibiting glucagon secretion and improves glucose disposal in peripheral tissues⁽¹⁰⁾. In that way, for patients with T2DM, a non-pharmacological therapeutic approach could be achieved by targeting these incretins (GLP-1 and GIP) through protein- and protein hydrolysate-based strategies. This approach would be mainly focused on increasing GLP-1 levels rather than stimulating GIP because in these patients the responsiveness of their β-cells to GIP action is decreased⁽¹⁴⁾. Furthermore, only GLP-1 exerts an appetite-suppressing effect, while GIP does not seem to do the same⁽¹⁰⁾. Accordingly, many incretin-based therapies focus on using GLP-1 analogues, promoting endogenous GLP-1 secretion or using DPP4 inhibitors.

DPP4 is a ubiquitous aminodipeptidase that exists essentially as a membrane-anchored cell-surface enzyme⁽¹⁵⁾. It is expressed throughout the body tissues, such as kidneys, the gastrointestinal tract, liver, pancreas, and the endothelial and epithelial cells on the vascular bed. Its soluble form is found in plasma and therefore it is in close proximity with hormones circulating in the blood^(16,17) . The main activity of DPP4 is to remove N-terminal dipeptides from polypeptides⁽¹⁸⁾, which preferably have a proline or alanine in the second position from the N-terminal. Some of the main DPP4 substrates are GLP-1 and the other incretin hormone GIP, which are peptides with N-terminal Tyr–Ala and His–Ala, respectively⁽¹⁹⁾. The intact GLP-1 is rapidly hydrolysed by DPP4 into a shorter, inactive form, once it reaches the plasma. GLP-1 has a half-life of 1–2 min⁽¹⁸⁾. Only 25 % of the active GLP-1 reaches the portal circulation and subsequently the liver, where a further 40–50 % is digested by the DPP4 in hepatocytes. This means that only 15 % of the secreted GLP-1 enters the systemic circulation and may reach other tissues, such as the pancreas or the brain⁽²⁰⁾. Therefore, DPP4 is responsible for inactivating more than 80 % of the secreted GLP-1⁽¹⁸⁾. Studies focus not only in the development of DPP4-inhibitory drugs, but also on peptides derived from food sources with DPP4-inhibitory capacity.

Although pharmacological compounds are being studied⁽²¹⁾, natural compounds might be used to prevent the development of overweight- and obesity-related problems from early preclinical stages through interaction with the enteroendocrine system⁽²²⁾.

Dietary regulation of glucagon-like peptide-1 secretion

Nutrient ingestion is the primary physiological stimulus for inducing GLP-1 secretion by L cells, located in the ileum and colon in the human gastrointestinal tract. GLP-1 secretion occurs in a biphasic pattern, which consists of a rapid release in 15–30 min after a meal, followed by a second minor peak that occurs in 60–120 min. Enteroendocrine cells have been shown to respond to carbohydrates, lipids and proteins.

Glucose and fat have been reported to be strong GLP-1-secretagogues after they have been ingested⁽²³⁾, or directly administered into the intestine^(24,25) or into perfused ileal segments⁽²⁶⁾. In the murine model, glucose-stimulated GLP-1 release is blocked using Na-dependent GLUT-1 (SGLT-1) knockout mice and SGLT-1 inhibitors^(27,28) , which suggests that glucose metabolism uses glucose transport via SGLT-1 to induce GLP-1 secretion. It has also been proposed that sweet taste receptors (T1R2, T1R3) are involved in the glucose-sensing mechanism, but there is still some controversy about whether this is so^(29,30) . On the other hand, it has been reported that G-protein-coupled receptors (GPCR) are activated by dietary fat to stimulate GLP-1 release, including GPR40 and GPR120 by medium-chain fatty acids, long-chain fatty acids and long-chain unsaturated FA; and GPR41 and GPR43 by SCFA (for reviews, see Hirasawa et al. ⁽³¹⁾ and Reimann⁽³²⁾).

Other food components could also modulate GLP-1 secretion. Flavonoid structures, present in several vegetables, also stimulate GLP-1 secretion⁽³³⁾. In both ex vivo ⁽³⁴⁾ and rat models⁽³⁵⁾, these compounds have been shown to improve the metabolic status altered by a cafeteria diet treatment⁽³⁶⁾.

Effects of proteins on glucagon-like peptide-1 secretion

Dietary proteins undergo digestion by gastric (pepsin) and pancreatic (chymotrypsin and trypsin) proteases and membrane digestion by peptidases associated with the brush-border membrane of enterocytes. The different digestive proteases cleave the peptide bonds at preferential positions. The primary endproducts are dipeptides and tripeptides, which will enter the cell through peptide transporters. Free amino acids are also released after luminal protein digestion and after peptide hydrolysis within the intestinal cells, and then exit across the basolateral membrane via specific amino acid transporters.

GLP-1 release is activated by luminal intestinal chemosensors, which could be reached by peptides of different sizes, mixed with free amino acids.

Studies in human, animal and enteroendocrine cells have shown increased GLP-1 secretion by free amino acids such as l-phenylalanine, l-alanine and l-glutamine^(37,38) and l-asparagine⁽³⁹⁾. The effect of glutamine has been confirmed in healthy, obese and diabetic human subjects^(40,41) . Tolhurst et al. ⁽⁴²⁾ demonstrated this effect in isolated mouse L cells and reported that the mechanisms were associated with an increase in cyclic AMP (cAMP) and cytosolic Ca²⁺ levels. They also found evidence to suggest that electrogenic Na-coupled amino acid uptake is responsible for initiating membrane depolarisation and voltage gated Ca²⁺, while a second pathway increases intracellular cAMP levels. Young et al. ⁽⁴³⁾ also reported similar results with l-proline, l-serine, l-alanine, l-glycine, l-histidine, l-cysteine and l-methionine in the STC-1 cell line.

When analysing the effects of protein on GLP-1 release, many studies focus on the effects of protein hydrolysates, produced by the hydrolysis of food protein with commercial enzymes (summarised in Tables 1–3). Sometimes, especially in in vitro studies, these are digestive enzymes that simulate intestinal digestion. However, many different hydrolysates are obtained through treatment with enzymes other than pepsin, chymotrypsin or trypsin. Protein hydrolysis can have two main benefits: (1) protein will be more quickly digested after intake; and (2) bioactive peptides^(44–52) might be released. Thus, the degree of protein digestion may impact the capability of protein to stimulate GLP-1 release, as discussed below.

Table 1. Stimulation of glucagon-like peptide-1 (GLP-1) secretion by protein and protein hydrolysates in humans

↑, GLP-1 secretion is incremented v. the control, specified in each row; N.D., hydrolysis conditions not described; T2DM, type 2 diabetes mellitus; CGMP, casein glycomacropeptide.

* Number of subjects per group.

† Time not known.

Table 2. Stimulation of glucagon-like peptide-1 (GLP-1) secretion by protein and protein hydrolysates in vitro*

↑ GLP-1 secretion is incremented v. the control, specified in each row; –, GLP-1 secretion is not altered v. the control, specified in each row; ↓ GLP-1 secretion is reduced v. the control, specified in each row; BSA, bovine serum albumin; Corolase PP, a porcine pancreatic enzyme preparation; DH32, 32 % degree of hydrolysis; DH45, 45 % degree of hydrolysis; DPS, Dutch Protein Services; H, hydrolysis; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; IVD, in vitro digestion with pepsin and pancreatin, always indicates the same hydrolysis conditions as the protein that is compared with; KRB, Krebs–Ringer modified buffer; N.D., hydrolysis conditions not described; PSE, proline-specific endoprotease; UCVP, undigested cuttlefish viscera protein.

* The salivary fluid does not contain enzymes.

† Pea protein origin: DPS, from Dutch Protein Services; Pisane, from Cosucra; SM, from Nutralys; HP90, from Triballat.

‡ Temperature or time not known.

§ This pea hydrolysate did not stimulate GLP-1 secretion; nor did the 10 kDa permeate. Nevertheless, the supernatant fraction obtained after centrifugation increased GLP-1 secretion compared with the control.

‖ Hydrolysis with cuttlefish hepatopancreas digestive proteases.

¶ Hydrolysis with cuttlefish smooth hound intestine digestive proteases.

Table 3. Stimulation of glucagon-like peptide-1 (GLP-1) secretion by protein and protein hydrolysates in animals

N.D., hydrolysis conditions not described; ↑, GLP-1 secretion is incremented v. the control, specified in each row; –, GLP-1 secretion is not altered v. the control, specified in each row; SPF, specific pathogen-free; BW, body weight; ZDF, Zucker diabetic fatty.

* Number of animals per group.

† Sprague–Dawley streptozotocin-induced diabetic rats.

‡ Changes in plasma GLP-1 after oral administration of the protein under the oral glucose tolerance test.

§ Changes in plasma GLP-1 after oral administration of the protein under the intraperitoneal glucose tolerance test.

In vitro studies on the STC-1 cell line showed a clear stimulation by whole dairy proteins (whey, casein, α-lactalbumin, β-lactoglobulin)^(53–55). Moreover, the stimulation of GLP-1 by whey protein β-lactoglobulin in STC-1 cells was partially lost when treated with trypsin (β-lactoglobulin 7·3-fold increase and hydrolysates 2–5·8-fold increase, all v. vehicle control), and totally lost when digested with chymotrypsin for 60 min or more⁽⁵⁴⁾. In the same cell line, the stimulatory effects of whey protein on GLP-1 were lost after extensive hydrolysis with microbial (not described) enzymes, or after a simulated gastrointestinal digestion that included a 90-min treatment with pepsin and a 150-min treatment with Corolase PP⁽⁵⁶⁾. Another study showed that treating whey or casein with trypsin or DPP4 for 30 min did not lead to any loss of GLP-1-stimulatory properties⁽⁵³⁾.

In humans, dairy protein is one of the most studied protein sources involving GLP-1 secretion. Intraduodenal infusion of whey protein hydrolysate has stimulated plasma GLP-1 in lean and obese subjects⁽⁵⁷⁾, reduced glucose concentration and suppressed energy intake⁽⁵⁸⁾ compared with saline. In these studies, hydrolysed, rather than intact, whey protein was selected because it more closely resembles partially digested protein. Also in patients with T2DM, a whey preload increased GLP-1 secretion, lowered plasma glucose levels and increased the insulin response^(59,60) compared with water and sucralose, respectively. It has been shown that whey, casein and casein hydrolysates increase GLP-1 secretion^(61-63). However, there is no agreement about whether there are any differences between their effect on GLP-1 secretion. Hall et al. ⁽⁶²⁾ showed that 120 min after being ingested, whey protein induced a 2-fold increase in postprandial GLP-1 levels compared with casein protein. On the other hand, when comparing whey, casein and their hydrolysates, Calbet & Holst⁽⁶³⁾ showed that the release of GLP-1 was not influenced by the source or hydrolysis process. Also, a commercially available whey protein hydrolysate showed a higher GLP-1 release 30 min after an oral glucose tolerance test (OGTT) than did casein glycomacropeptide (CGMP), but not compared with whey isolate or α-lactalbumin-enriched whey⁽⁶⁴⁾ (incremental AUC_30min median; 593 (hydrolysate), 270 (CGMP); P=0·045). Thus, the studies performed with whey and whey hydrolysates do not show any differences in the effects of the two sources in terms of GLP-1 secretion. Calbet & Holst⁽⁶³⁾ suggested that this is because the dairy protein hydrolyses rapidly in the intestine and there is a subsequent rise in peripheral amino acids independent of the fractionation.

Other protein sources have also been shown to stimulate GLP-1 release in vivo. A similar rise in rat plasma GLP-1 levels, comparable with that caused by dairy protein, has been observed after pea-protein meals⁽⁶⁵⁾. Furthermore, also in rats, pea protein and pea-protein hydrolysate have been shown to similarly stimulate GLP-1 release, although the hydrolysate showed stronger eating-inhibitory properties⁽⁶⁶⁾ (total energy intake: 63 (SEM 6) kJ, 46 (SEM 3) kJ, 67 (SEM 5) kJ after pea protein, the hydrolysate and the control, respectively). In vitro studies with STC-1 cells showed that intact pea protein increases GLP-1 release. On the other hand, various pea-protein hydrolysates obtained by enzymic hydrolysis with subtilisin were tested, and only one of them maintained its GLP-1-secretory capacity⁽⁵³⁾.

Cereal protein has also been shown to stimulate GLP-1. Maize protein zein (a major maize protein) hydrolysate attenuated glycaemia in rats under the intraperitoneal glucose tolerance test, associated with enhanced secretions of GLP-1 and GIP⁽⁶⁷⁾ compared with water. In vitro (GLUTag cells), zein hydrolysate was shown to stimulate GLP-1 release more than egg albumin, country bean and meat hydrolysates⁽⁶⁸⁾. However, the type of hydrolysis was different in the various sources, so the effect of the protein source per se cannot be concluded from this paper. The stimulation of GLP-1 secretion by maize zein hydrolysate in GLUTag cells is not affected by treatment with pepsin/pancreatin for 60 min, although it is reduced after pronase treatment⁽⁶⁷⁾ compared with the positive control, KCl 70 mm. The authors suggested that the hydrolysate is not further cleaved by pepsin treatment (the degree of hydrolysis was only 8·6 %).

Oral administration of rice protein hydrolysates also increased total GLP-1 in plasma, and improved glycaemic response in rats⁽⁶⁹⁾ (the control used was 2 g/kg of glucose solution). In the same study, rice protein hydrolysates (degree of hydrolysis 5–10 %) stimulated GLP-1 in GLUTag cells, with the potency depending on the enzyme and the time of digestion⁽⁶⁹⁾ compared with the blank treatment. The effect of the whole rice protein was not assessed. The authors found that GLP-1 secretion was weaker after 60 min digests with pepsin in rice endosperm protein hydrolysates than after 30 min digests, which suggests that oligo- or larger peptides, rather than small peptides or free amino acids, might be responsible for this stimulation. The results for wheat protein were just the opposite. In GLUTag cells, a low-molecular fraction of wheat protein hydrolysate enhanced GLP-1 secretion while a high-molecular fraction did not⁽⁷⁰⁾. The low-molecular fraction of wheat protein hydrolysate had a glucose-lowering effect mediated by GLP-1 in rats⁽⁷⁰⁾ after an oral administration compared with 0·9 % NaCl. Also, in another study in a distal enteroendocrine cell model (GLUTag cells), the effect of wheat hydrolysate on the stimulation of GLP-1 secretion was largely enhanced by pepsin/pancreatin digestion relative to the blank⁽⁷¹⁾.

For other protein sources, in vitro studies also showed that GLP-1-secreting activity of digested protein was greater than that of the original source. In a study performed with cuttlefish (Sepia officinalis) viscera, a hydrolysate (obtained from digestion with cuttlefish hepato-pancreatic enzymes) was found to exert GLP-1-secreting action while the undigested protein did not⁽⁷²⁾. These results were found with the samples solubilised in saliva, but they were subjected to further in vitro simulated gastrointestinal digestion (including treatment with pepsin and pancreatin). Results showed that gastrointestinal digestion increased the GLP-1-secretory effects of both the hydrolysate and the initially undigested protein, leading to no differences between the hydrolysate and the non-hydrolysate gastrointestinally digested samples. Also, intestinal digested bovine Hb protein had a greater effect on GLP-1 release than partially digested protein (saliva and gastric digest) in STC-1 cells⁽⁷³⁾.

Taken together, all these studies prove that several protein sources increase GLP-1 secretion, which is associated to benefits such as food intake or glucose homeostasis regulation. In vivo studies do not fully clarify whether previous hydrolysis of the protein sources with commercial enzymes leads to stronger GLP-1-secreting effects. In vitro data show that many protein sources, including purified proteins, activate GLP-1 release. However, digestion as it might physiologically happen upon protein intake might stimulate or reduce the effect of the undigested protein, depending on the original source. This suggests that some high-molecular-weight peptides might reach enteroendocrine cells and activate GLP-1 secretion, while in other cases the lower-molecular-weight peptides or the amino acids released after digestion are responsible for the secretion.

Mechanisms involved in the effects of protein as glucagon-like peptide-1 secretagogue

The mechanisms through which the proteins and peptides released after protein hydrolysis (either ‘synthetic’ or simulated digestion) act as secretagogues are still not fully understood, but several pathways have been shown to be involved. Studies on the mechanisms through which protein and protein hydrolysates stimulate GLP-1 secretion are carried out using in vitro (i.e. enteroendocrine cell lines such as STC-1 and GLUTag) and ex vivo (i.e. perfused intestine and intestinal explants) models, and also primary cultures.

Many of the studies that focus on the mechanisms that stimulate GLP-1 secretion use commercial meat peptones, that is meat hydrolysates produced by the digestion of meat with proteolytic enzymes which lead to a complex mixture of partially metabolised proteins.

With this protein source, it seems that one key player in the oligopeptide stimulation of GLP-1 release is peptide transporter 1 (PepT1) (Fig. 1). Meat peptone was shown to stimulate GLP-1 secretion in mouse colonic primary culture through PepT1-dependent uptake, followed by an increase in intracellular Ca, and activation of Ca-sensing receptor (CaSR)⁽⁷⁴⁾. Very recently Modvig et al. ⁽⁷⁵⁾ used isolated perfused rat small intestine to study GLP-1 secretion stimulated by meat peptone. The sensory mechanisms underlying the response depended on di-/tripeptide uptake through PepT1 and subsequent basolateral activation of the amino acid-sensing receptor (CaSR) (Fig. 2). CaSR might also be activated by free amino acids taken up from the intestinal lumen by different amino acid transporters⁽⁷⁵⁾.

Fig. 1. The intestinal transporter form PEPT1 (SLC15A1) is located in apical membranes with a functional coupling to the apical Na⁺/H⁺ antiporter (NHE3) for pH recovery from the peptide-transport-induced intracellular acid load. Adapted from Daniel et al. ⁽¹⁰³⁾.

Fig. 2. Illustration of the endocrine L cell and the proposed mechanisms by which peptone stimulates glucagon-like peptide-1 (GLP-1) release. Di-/tripeptides are taken up by PepT1 and are degraded by cytosolic peptidases to their respective amino acids (AA). Intracellular amino acids are then transported to the interstitial side through basolateral amino acid transporters, wherefrom they stimulate the L cells by activating amino acid sensors, like calcium-sensing receptor (CaSR), situated on the basolateral membrane. IP₃, inositol trisphosphate; PLC, phospholipase C. Adapted from Modvig et al. ⁽⁷⁵⁾.

It has been pointed out that it is difficult to determine the PepT1-dependent oligopeptide-sensing pathway in GLUTag and STC-1 cell lines, because the expression of endogenous PepT1 is lower than in native L cells⁽⁷⁴⁾. Therefore, the effects of peptones observed in both cell lines may be due to the free amino acids that some of these peptones contain, as has been suggested in an in vitro study on the effects of salmon hydrolysate⁽⁷⁶⁾ carried out in GLUTag cells. However, other studies on these cell lines do not share this view. As mentioned above, GLP-1 secretion is activated by dairy proteins^(53–55), low-molecular-weight wheat (with less than 1 % free amino acids)⁽⁷⁰⁾, intact pea-protein⁽⁵³⁾ or peptin-resistant zein hydrolysate⁽⁶⁷⁾. Furthermore, three synthetic peptide sequences (ANVST, TKAVEH and KAAT) were reported to be able to enhance GLP-1 secretion in STC-1 cells⁽⁷⁷⁾. The authors concluded that the incretin effect of proteins is associated with the amino acid profile, but the specific amino acid motif that triggers GLP-1 secretion stimulation was not determined. Thus, receptor or peptide transporters other than PepT1 expressed in STC-1 and GLUTag cells might be involved in the peptide stimulation of GLP-1. For instance, one of the mediators suggested was the G protein-coupled receptor family C group 6 subtype A (GPRC6A)⁽⁷⁰⁾ (Fig. 3).

Fig. 3. Signalling through G protein-coupled receptor family C group 6 subtype A (GPRC6A) in β- or gut cells. GPRC6A can be directly activated by amino acids and use calcium as an allosteric regulator. IP₃, inositol triphosphate; PLCβ, phospholipase Cβ; GLP-1, glucagon-like peptide-1; VDCC, voltage-dependent calcium channel. ‡ Described in enterocyte L cells of the small intestine. Adapted from Wauson et al. ⁽¹⁰⁴⁾.

Protein hydrolysates are also detected by the umami receptor (T1R1–T1R3 heterodimer)⁽⁷⁸⁾ (Fig. 4) and G protein-coupled receptor 92/93 (GPR92/93)⁽⁷⁹⁾, which leads to the release of the gut-derived satiety factor cholecystokinin. There is no direct evidence of umami stimulation and GLP-1 secretion, but the T1R1 receptors were co-expressed with GLP-1-expressing STC-1 cells⁽⁸⁰⁾, which suggests that umami receptors play a role in GLP-1 signalling.

Fig. 4. The T1R1/T1R3 heterodimer is coupled to a heteromeric G protein, where the Gbc subunit appears to mediate the predominant leg of the signalling pathway. Ligand-binding activates Gbg, which results in activation of phospholipase Cβ2 (PLCβ2), which produces inositol trisphosphate (IP₃) and diacylglycerol (DAG). IP₃ activates IP₃ receptor type 3 (IP₃R3) which results in the release of Ca²⁺ from intracellular stores. AC, adenylyl cyclase; cAMP, cyclic AMP; PDE, phosphodiesterase; PIP2, phosphatidylinositol 4,5-bisphosphate. Adapted from Kinnamon⁽¹⁰⁵⁾.

An increase in intracellular Ca has been reported to be a pathway activated by protein hydrolysates to mediate GLP-1 secretion. Pais et al. ⁽³⁷⁾ reported that meat peptone-stimulated GLP-1 secretion from primary L cells was also associated with Ca influx through voltage gate Ca channels (Fig. 3). In NCI-H716 human enteroendocrine cells, tetrapeptides, but not single amino acids or any of the dipeptides, tripeptides and pentapeptides tested, were found to induce a robust and selective [Ca²⁺]_i response associated with increased secretion of GLP-1⁽⁸¹⁾. Moreover, these effects were not observed in either STC-1 or in GLUTag rodent cells. Interestingly, in the same paper, the authors showed that casein protein hydrolysate elicited an increase in GLP-1 without modulating intracellular Ca.

It has been suggested that GLP-1 secretion is mediated by other intracellular pathways such as extracellular signal-regulated kinase 1/2 (ERK1/2), mitogen-activated protein kinase (MAPK) and p38 MAPK, activated by peptones and mixtures of essential amino acids in NCI-H716 cells⁽⁸²⁾.

Altogether, the studies show that which signalling pathways are involved in GLP-1 secretion by different peptide mixtures will depend on the peptide length, the sequences and/or the amino acid composition, and whether there are free amino acids in the mixture. Furthermore, the model studied has to be carefully considered since there are differences in the expression of key genes (such as pepT-1) and some effects might depend on the vectoriality of the system (the capacity to differentiate basolateral and apical processes).

Protein bioactivity on glucagon-like peptide-1 clearance

Like the studies on the effects of protein on GLP-1 secretion, most of the studies on the effects of protein on DPP4 inhibition are performed with protein hydrolysates. Over the past few years, bioactive peptides have shown their potential as DPP4 inhibitors, a research area that is currently expanding. In vitro simulated gastrointestinal digestion has been reported to produce DPP4-inhibitory protein hydrolysates^(83,84) . Also, hydrolysis with a range of enzymes is used to release DPP4-inhibitory peptides^(69,85–90) . Thus, a wide range of protein sources has been used to obtain hydrolysates, for which DPP4-inhibitory activity has been screened mainly in vitro.

Research has shown that the amino acid sequence plays a much greater role in DPP4-inhibitory activity than other physico-chemical parameters such as length, isoelectric point, hydrophobicity and net charge^(91,92) . DPP4 preferentially cleaves substrates that bear proline or alanine at their P₁ position (Xaa-Pro and Xaa-Ala; where Xaa represents any amino acid) and also acts on substrates that bear other residues, such as glycine, serine, valine and leucine⁽⁹³⁾. Hydrophobic and basic residues at the P₂ position enhance the affinity for cleavage compared with acidic residues⁽⁹⁴⁾. The presence of tryptophan residue at the N-terminal position increases the susceptibility to cleavage. Although the residues at the N-terminal position may have a major impact by inhibiting DPP4, the authors pointed out that the C-terminal amino acid also affects the potency of DPP4 because it is involved in the interaction with the enzyme⁽⁹⁵⁾.

To date, some studies have been carried out on the in vivo DPP4-inhibitory effects of the hydrolysates and peptides from dietary proteins. Peptides derived from milk and bean proteins, which have been shown to inhibit the activity of DPP4 in vitro, were also found to have glycaemic effects in mice^(96,97) as plasma glucose levels decreased after an OGTT. A β-casein-derived peptide LPQNIPPL found in Gouda-type cheese with in vitro DPP4-inhibitory effects has also been tested with animal models. Oral administration of this octapeptide resulted in 1·8-fold lower postprandial glucose AUC; however, insulin plasma levels did not differ⁽⁹⁸⁾. In these studies, the authors did not measure plasma DPP4 activity, so it is not known whether the lower blood glucose was caused by inhibition of DPP4 activity. Chicken feet hydrolysates with DPP4-inhibitory activity in vitro improved hyperglycaemia in diet and aged models of glucose homeostasis impairment⁽⁹⁹⁾.

As well as hydrolysates from milk and bean protein, in in vivo models hydrolysate from the egg protein lysosyme has also shown a 25 % reduction in blood serum DPP4 activity and a trend towards higher serum GLP-1 levels after 90 min in diabetic rats undergoing chronic treatment⁽¹⁰⁰⁾. Streptozotocin-induced diabetic rats were used to evaluate the effects of porcine skin gelatin hydrolysates⁽⁴⁸⁾, Atlantic salmon skin gelatin⁽⁴⁷⁾, and halibut and tilapia skin gelatin⁽⁴⁹⁾. In all these studies, diabetic animals showed reduced blood glucose levels during OGTT, increased plasma insulin and active GLP-1 levels, and reduced plasma DPP4 activity after a chronic treatment with these proteins compared with water. Diabetic rats treated for 42 d with a daily dose of 300 mg/kg of porcine skin gelatin showed their plasma glucose AUC reduced from 30 000 to 28 000 mg × min/dl (1665 to 1554 mmol × min/l), insulin levels increased 2-fold, active GLP-1 levels reduced from 15 to 13·5 pm and DPP4 activity reduced by half⁽⁴⁸⁾. In another study in which the animals were treated for 35 d with a daily dose of 300 mg/kg of Atlantic salmon skin gelatin hydrolysate, blood glucose levels were reduced to less than 200 mg/dl (11·1 mmol/l) during OGTT, insulin levels increased 3-fold, active GLP-1 levels increased 1·6-fold and DPP4 activity was reduced from 115·5 to 82·6 % (lower than in normal rats)⁽⁴⁷⁾. When these animals received a 30 d treatment involving a daily dose of 750 mg/kg of halibut (HSGH) or tilapia skin gelatin hydrolysate (TSGH) the plasma glucose was lower than 200 mg/dl (11·1 mmol/l) in the TSGH-treated group. When TSGH was administered, insulin levels were 1·56 g/l, higher than that of HSGH (1·14 g/l) and the diabetic control group (0·43 g/l). The active GLP-1 plasma levels of the diabetic control rats (5·14 pm) were lower than those for TSGH-treated group (13·32 pm) and for HSGH-treated group (7·37 pm) and the DPP4 activity reduced from 115·5 in the diabetic group to 86·6 and 71·6 % in the HSGH- and TSGH-treated groups, respectively⁽⁴⁹⁾. Moreover, rodents receiving halibut and tilapia skin gelatin hydrolysates also showed increased total GLP-1 levels. Therefore, the findings of this study suggest that these hydrolysates exert their anti-hyperglycaemic effect via dual actions of DPP4 inhibition and GLP-1 secretion enhancement. Similarly, the ileal administration of zein protein hydrolysate to rats was found to potentiate the incretin effect when administered before an intraperitoneal glucose tolerance test, resulting in decreased glucose concentration, increased insulin levels, decreased plasma DPP4 activity, and increased total and active GLP-1 secretion compared with water⁽¹⁰¹⁾. Rice-derived peptides were likewise found to act via dual action. Oral administration increased plasma GLP-1 levels compared with water during an intraperitoneal glucose tolerance test, and ileal administration reduced plasma DPP4 activity and increased the ratio of active GLP-1 to total GLP-1⁽⁶⁹⁾ in rats. In vitro studies also showed dual mechanisms for protein hydrolysates; both enhanced GLP-1 secretion and inhibited DPP4, as has been shown for the cuttlefish (Sepia officinalis) viscera protein hydrolysate and bovine Hb hydrolysate^(72,77) , whey proteins⁽⁵⁶⁾ and chicken feet hydrolysate⁽⁹⁹⁾. Therefore, these two mechanisms might also take part in vivo for some protein sources, leading to an increase in active GLP-1 and improve glycaemia.

Human studies, although limited, offer some evidence that food-derived peptides, mostly from dairy protein, act as DPP4 inhibitors⁽¹⁰²⁾. It was shown that a whey preload, consumed before the breakfast meal, reduced glucose levels by 28 % and increased insulin and total GLP-1 levels by 105 and 141 %, respectively, compared with water. Nevertheless, no significant differences in plasma DPP4 activity were found. This could be interpreted as whey protein acting as an endogenous inhibitor of DPP4 in the proximal small intestine, but not in the plasma (intestinal DPP4 activity was not assessed)⁽⁶⁰⁾. Further studies are needed to examine the potential of casein- and whey-derived peptides, as well as peptides derived from other sources, to act with DPP4 inhibitors in human subjects.

Conclusions

Food proteins target the enteroendocrine system. They directly enhance GLP-1 release from enteroendocrine cells. Current studies suggest that the source of the protein might lead to differences in GLP-1 secretion, although there is not enough literature to enable the different proteins to be compared. The effect of gastrointestinal digestion can also enhance or decrease GLP-1-secreting capacity depending on the protein type. Thus, it is important to consider this digestion when discussing the effects of protein on GLP-1 secretion in vitro. In addition, peptides with DPP4-inhibitory effects can be released during the digestion process, which could modulate the life span of target enterohormones. However, whether this hydrolysis remains important after intestinal digestion in vivo remains to be clarified. Thus, the use of protein/protein hydrolysates to ameliorate situations of glucose derangements is promising, but more research, specifically human studies, is required to define the most effective sources/treatments.