Legume-Derived Bioactive Peptides in Type 2 Diabetes: Opportunities and Challenges

Diabetes mellitus is a complex disorder characterized by insufficient insulin production or insulin resistance, which results in a lifelong dependence on glucose-lowering drugs for almost all patients. During the fight with diabetes, researchers are always thinking about what characteristics the ideal hypoglycemic drugs should have. From the point of view of the drugs, they should maintain effective control of blood sugar, have a very low risk of hypoglycemia, not increase or decrease body weight, improve β-cell function, and delay disease progression. Recently, the advent of oral peptide drugs, such as semaglutide, brings exciting hope to patients with chronic diabetes. Legumes, as an excellent source of protein, peptides, and phytochemicals, have played significant roles in human health throughout human history. Some legume-derived peptides with encouraging anti-diabetic potential have been gradually reported over the last two decades. Their hypoglycemic mechanisms have also been clarified at some classic diabetes treatment targets, such as the insulin receptor signaling pathway or other related pathways involved in the progress of diabetes, and key enzymes including α-amylase, α-glucosidase, and dipeptidyl peptidase-IV (DPP-4). This review summarizes the anti-diabetic activities and mechanisms of peptides from legumes and discusses the prospects of these peptide-based drugs in type 2 diabetes (T2D) management.


Introduction
Diabetes is a metabolic disease characterized by a persistent over-normal level of blood glucose that causes impressive morbidity and mortality worldwide [1]. Persistent hyperglycemia imposes damage on other organs, such as the eye, heart, kidney, and skin, as well as the nervous system, and is strongly correlated with a myriad of diabetes-related complications [2]. Diabetes typically develops when the pancreas does not create enough insulin, or the organs involved in glucose metabolic regulation do not effectively respond to the insulin [3]. Type 1 diabetes (T1D, 5-7%), type 2 diabetes (T2D, 90%), and gestational diabetes (GD, 2-3%) are the three most common forms of diabetes [4]. T1D is a destructive autoimmune disease of pancreatic β-cells, leading to a deficiency of insulin. T2D is the most prevalent kind of diabetes in the general population, and is frequently caused by inadequate insulin production or insulin resistance [5,6]. GD is another type of diabetes that is caused by abnormal glucose tolerance and diabetes during pregnancy [7,8]. The global prevalence of diabetes shows that 537 million adults were troubled by diabetes in 2021, and the diabetic population is estimated to rise to 643 million (11.3%) in 2030 and to 783 million (12.2%) in 2045 [9]. In recent years, the incidence of T2D cases has accelerated rapidly, and it is predicted that the T2D cases may reach 700 million in 2045 [9].
Given the grim reality of T2D, several strategies including appropriate exercise, reasonable diet, and pharmacotherapy have been applied to control postprandial hyperglycemia peptidase-4 (DPP-4), glucagon-like peptide-1 (GLP-1), gastric inhibitory peptide (GIP), gastrointestinal target hormone peptide (PYY), glucose transporters (GLUTs), and sodiumdependent glucose cotransporters (SGLTs) ( Table 1) [5,[19][20][21]. Insulin and its analogs are the most common injectable diabetes drugs for patients with severe pancreatic dysfunction. Oral anti-diabetic drugs mainly include metformin, sulfonylureas, glinides, thiazolidinediones (TZDs), DPP-4 inhibitors, SGLT-2 inhibitors, α-amylase inhibitors, and α-glucosidase inhibitors. The current medications can lessen the harm to the body brought on by hyperglycemia by regulating blood sugar levels and increasing insulin sensitivity [22]. Insulin, the most important regulator of metabolism, reduces blood sugar by preventing hepatic glucose synthesis and promoting intracellular glucose uptake by fat and muscle cells. Insulin and its analogues have a short action period and a risk of minimally invasive infection and other side effects, such as hypoglycemia, resistance, allergy, edema, and lipodystrophy [23]. The market for peptide-based medicines has been firmly established by the creation of human insulin utilizing recombinant technology, as well as other synthetic hormones [4]. Oral insulin is superior to other forms of insulin delivery because it follows the normal physiological pathway, that is, through the portal vein circulation of the liver, first reaching the liver and later the peripheral tissues [24].
Metformin, an oral medication, is used to reduce blood sugar levels in people with non-insulin-dependent diabetic mellitus. It promotes the uptake and utilization of glucose in adipocytes and muscles and improves insulin sensitivity by reducing glucose synthesis in the liver [12]. Metformin has good security, but 5-15% of the administrated patients are still troubled by side effects, such as diarrhea, nausea, indigestion, and other gastrointestinal symptoms [20]. TZDs also ameliorate insulin resistance in adipocytes, liver, and muscle [25]. Rosiglitazone and pioglitazone, peroxisome proliferator-activated receptor c (PPARc) agonists of TZDs, act as insulin sensitizers that bring benefits by maintaining long-term glycemic balance [25]. Presently, TZDs are rarely considered for the treatment of T2D due to side effects, such as cardiac issues, edema, and weight gain.
Sulfonylureas are currently used as second-line drugs for T2D or in combination with other medications. They promote insulin secretion by blocking K+ channels in pancreatic tissue, limiting gluconeogenesis, reducing the insulin clearance rate in the liver, and reducing fatty acid decomposition [26,27]. First-generation and second-generation sulfonylureas are the two categories. The tolbutamide and chlorpropamide sulfonylureas are first-generation sulfonylureas. Glyburide (also known as glibenclamide), glipizide, glimepiride, and gliclazide are second-generation sulfonylureas. These days, second-generation sulfonate drugs are used in clinical settings more frequently. All sulfonyl medications have a common side effect called hypoglycemia, along with less serious side effects like headache, nausea, dizziness, allergic reactions, and weight gain [6]. Meglitinides, non-sulfonylureas insulin secretagogues, also target pancreatic β-cell tissue, stimulating the pancreatic β-cells to secrete insulin, without hypoglycemic risk [26].
SGLT-2 inhibitors are oral hypoglycemic medications that lower blood sugar by blocking renal glucose reabsorption. They prevent about 90% of the filtered glucose reabsorption by directly inactivating the SGLT-2, a glucose transporter located in the proximal tubules' first section [20,28]. SGLT-2 inhibitors include dapagliflozin, canagliflozin, empagliflozin, ertugliflozin, and sotagliflozin. They also have side effects, such as urinary and genital infection risk (6.4%) and ketoacidosis risk [6]. Glucose reabsorption is blocked in the kidney when SGLT-2 is inhibited, resulting in insulin-independent glucose lowering as well as a reduction in weight and blood pressure [20].
GLP-1 receptor agonists, GLP-1 analogues, and incretin mimics are common hypoglycemic drugs. Injectable GLP-1 receptor agonists include exenatide, liraglutide, lixisenatide, albiglutide, and dulaglutide. They control the blood glucose balance via the enhancement of insulin release and suppression of excessive glucagon secretion by activating the GLP-1 receptor [7]. Exedin-4 is one of the most promising GLP-1 receptor agonists currently utilized to treat diabetes, and is injected twice daily [29]. Liraglutide is a once-daily GLP-1 analogue, whose homology of amino acids sequence with human GLP-1 is 97% [30]. The modification of a fatty acid at position 26, and the replacement of lysine with arginine at position 34, result in an extension of the plasma half-life of liraglutide (13 h), an increase of selfassociation, and a resistance to digestion of DPP-4 [31,32]. In 2019, semaglutide (Rybelsus ® ), the first oral hypoglycemic peptide drug, was approved by the U.S. Food and Drug Administration (FDA) to improve glycemic control in adults with T2D [33]. The combination of semaglutide and a pharmaceutical sodium 8-[(2-hydroxybenzoyl)amino]octanoate (SNAC), an absorption enhancer, leads to a better solubility of semaglutide and an increased resistance to degradation at a low pH value [31]. Despite being combined with SNAC, the oral bioavailability of Rybelsus ® is only 0.4-1% [30].
DPP-4 inhibitors are oral medications that extend the half-lives of endogenous incretins by preventing the DPP-4 enzyme from digesting incretins [28,34]. These medications include sitagliptin, vildagliptin, saxagliptin, linagliptin, and alogliptin. The hypoglycemic effect of DPP-4 inhibitors is characterized by glucose concentration dependence. Therefore, they generally may not cause hypoglycemia.
Mounjaro (tirzepatide) is a "first-in-class" injectable drug that activates both GIP and GLP-1 receptors, controlling hyperglycemia through a dual mechanism of action [35,36]. Imeglimin, another "first-in-class" oral tablet, can improve insulin secretion disorders with a novel mechanism of action targeting mitochondrial bioenergetics [37]. It rebalances respiratory chain activity, resulting in less reactive oxygen species formation and the prevention of mitochondrial permeability transition pore opening [37]. Chiglitazar, a PPAR complete agonist, is a new oral drug approved by China's national medical products administration for T2D treatment in 2021; it can activate three subunits of PPAR (α, γ, and δ) [38,39].
α-glucosidase inhibitors (acarbose, miglitol, voglibose) and α-amylase inhibitors are the first-line drugs for T2D. They lower blood glucose levels by slowing down the digestion rate and inhibiting intestinal glucose absorption [40]. Their main side effect is gastrointestinal reaction.
Over the past few decades, there has been a significant increase in therapy alternatives, but none of them has prevented the decline of β-cell function. The current drugs available to treat T2D listed in Table 1 have side effects, and most of them are administered by injection. As a result, many scientists have made efforts to create alternative delivery methods, such as transdermal, oral, nasal, and ocular delivery. Due to the higher safety and compliance, the oral route remains the most appealing option for T2D patients. Currently, various oral peptide drugs have reached clinical trials, including oral insulin, calcitonin, parathyroid hormone, and vasopressin. The approval of an oral formulation of the GLP-1 receptor agonist semaglutide for the treatment of T2D represents a significant landmark in the development of oral peptide drugs [33].

Legume-Derived Peptides and Their Anti-Diabetic Activity
The legume is the fruit or seed of plants of the legume family, which are used for food, including lentils (Lens culinaris), common beans (Phaseolus vulgaris), broad beans (Vicia faba), dry peas (Pisum sativum), chickpeas (Cicer arietinum), cowpeas (Vigna sinensis), and mung beans (Vigna radiata) [41]. Legumes are an excellent source of good quality protein (20-45% protein), providing high amounts of the essential amino acids such as lysine and leucine, and lower amounts of methionine and tryptophan, complex carbohydrates, dietary fiber, unsaturated fats, vitamins, and essential minerals for the human diet [42,43].
The intake of legumes brings many benefits and positive effects for physical health, especially on metabolic syndromes, such as cardiovascular protection and the regulation of glucose and lipid metabolism [11,44]. Legume proteins can be converted into a variety of peptides that control vital biological activities, especially those involved in the endocrinology of living organisms [45,46]. Several observations of clinical statistics indicate that intake of legumes (lentils, chickpeas, and soybeans) or their protein significantly lowers the risk of T2D among Caucasians and Xanthoderms in different countries [47][48][49][50]. The relationship between the risk of T2D and the intake of legumes is listed in Table 2.
Given that legumes and their proteins benefit T2D patients, various legume-derived peptides are identified as anti-hyperglycemia agents due to their metabolic regulating bioactivity in vivo or in vitro. Plant-derived peptides have been shown to have hypoglycemic effects as well, which overcome security hurdles and yield an efficient treatment for T2D [4,51]. These agents can be divided into native proteins, peptides, and protein hydrolysates (hydrolytic peptides) [4,52]. The native proteins and peptides can be directly extracted and purified from legume storage or germinating seeds. Hydrolytic peptides, also called oligopeptides, are a mixture derived from the legume protein by digestion of various enzymes, fermentation of microorganisms, or mechanical cutting. Several studies have demonstrated the potential anti-diabetic activity of native preparations and hydrolysates in vitro, in vivo, or in clinical trials [53][54][55]. Animal models are the most common approaches to investigate the anti-diabetic activity of drugs in vivo, including streptozotocin (STZ) and high-fat diet (HFD)-induced diabetic mice [14,56,57], KKA y diabetic mice [58], T1D Sprague Dawley (SD) rats [14,59], STZ-induced Wistar rats [60,61], HFD-C57BL/6J mice [15], and alloxan-induced diabetic mice [62]. The potential anti-diabetic benefits of legume hydrolysates and fractions or synthesized pure peptides have been investigated by several cell line models. Glucose consumption activities were evaluated by human HepG2 [15,57,59,63,64], Caco-2 [64], 3T3-L1 [64,65], and INS-1E cells [14,66]. Furthermore, Rat L6 skeletal muscle cells [67], Min6 cell NCI-H716 [65], and human embryo kidney 293 cell models [65] were used to evaluate the activation of the insulin signaling pathway. The legume-derived peptides related to anti-diabetic activities, experimental models, and anti-diabetic mechanisms are summarized in Table 3. Table 3. Hypoglycemic effect and mechanism of legume peptides and hydrolysates in vivo and in vitro.

Native Legume Peptides
The primary elements of legume seeds are proteins and peptides generated by the connection of amino acids via peptide bonds. Native legume peptides (aglycin, vglycin and soymorphin-5) have been reported as anti-T2D agents due to their bioactivities in decreasing blood sugar levels, improving insulin sensitivity and glucose tolerance, and promoting the proliferation of β-cells (Table 3).
Aglycin, a biopeptide containing 37 amino acid residues, was identified as matching to the residues 27-63 of plant albumin 1 B precursor (PA1B) from pea seeds [56,77]. Aglycin has a high affinity with a 43 kDa basic 7S globulin protein [78]. This protein binding mode is similar to the interaction of insulin and insulin receptor; therefore, aglycin is also called leginsulin [79]. Structurally, it is made up of six cysteines embedded in three disulfide bonds (C3-C20, C7-C22, and C15-C32), which are highly conserved and can resist the degradation of pepsin, trypsin, and Glu-C protease [56,57]. Aglycin was initially thought to be a leginsulin that participates in plant signal transduction to control growth and differentiation [80]. Subsequently, Chen's group found that the oral administration of aglycin in diabetic mice effectively controlled hyperglycemia by improving the insulinsignaling pathway and enhancing glucose uptake in peripheral tissues [77]. Furthermore, a long-term administration of aglycin benefited the impaired pancreatic restoration and insulin secretion [56].
Vglycin, another leginsulin, is highly similar to aglycin in amino acid sequence and structural domain [57]. It may function with a similar mechanism to aglycin, which could be useful in T2D treatment for restoring impaired insulin signaling, glucose tolerance, and pancreatic function [14]. Other leginsulin variants identified in different cultivars, as well as their bacteria-expressed recombinants, also exert insulin-like activities in myotube-like differentiated L6 and C2C12 cells [13]. Thus, leginsulins, such as aglycin and vglycin, could be considered as potential orally administrated drugs to be developed.

Legume Proteins
Legume seed proteins are the main dietary source of plant protein, in which antinutritional substances include lectins, hydrolase inhibitors, ribosome-inactivating proteins (RIPs), and allergens [81]. Recently, soy and lupin proteins have been reported to have hypoglycemic effects.
Most of the protein in lupin seeds (around 44% w/w of the total conglutins) belongs to the conglutin family, whose molecular mass is between 143 and 260 kDa [82]. β1-, β3-, and β6-conglutin proteins can bind to insulin in vitro, suggesting that β-conglutin proteins have potential benefits for the prevention and treatment of diabetes [83]. The second most prevalent globulin, α-conglutin, shares 33% w/w of all conglutins. The third conglutin is δ-conglutin, which accounts for 12% w/w of all conglutins and 4-5% w/w of all globulins [82]. γ-conglutin (~50 kDa) is a glycoprotein composed of two subunits (17 kDa and 29 kDa). γ-conglutin has excellent hypoglycemic effects in vitro and in vivo (Table 3). In mouse C2C12 cells, γ-conglutin activated the kinases of the synthetic protein pathway and increased glucose consumption by up-regulating muscle-specific gene MHC transcription and GLUT4 gene translocation [73]. In the STZ rat model, Ins1 gene expression and pancreatic insulin content were elevated by γ-conglutin [72]. Bertoglio's group reported that lupin γ-conglutin had a glucose-lowering effect in a clinical trial [54].
When different dosages of γ-conglutin (630, 315, and 157.5 mg) were eaten 30 min before the carbohydrate supply for 7 weeks, a relevant hypoglycemic effect could be observed in human subjects [54]. Lupin seed protein could be transported by Caco-2 cell monolayers and everted intestinal sacs, suggesting it has the potential to be developed as an oral agent to manage T2D [84].
Soybean storage proteins, such as β-conglycinin and glycinin, also promote antidiabetic activity (Table 3). Nordentoft reported that soy protein improved insulin sensitivity and up-regulated the expression of key insulin-regulatory genes [69]. A clinical study compared the effects of the intake of 6% (w/v) whey protein isolate (WPI), whey protein hydrolysate (WPH), soy protein isolate (SPI), and 2.66% WPI, or a control (no protein added) on insulin and glucose responses in 25 healthy men. The results suggested that SPI with 6% protein enhanced insulin response and decreased the plasma glucose level compared with a control beverage [55]. Another study demonstrated that the SPI increased the insulin response compared to WPH [85].

Hydrolytic Peptides
Bioactive hydrolytic peptides are organic substances formed from amino acids and released mainly through enzymatic proteolysis or food processing (cooking, fermentation, ripening) [86,87]. Currently, the commonly used peptide and protein extraction methods mainly include aqueous, organic solvent, enzymatic, ultrasonic, two-phase aqueous, and reverse micelle extraction [86]. The two most common methods for making bioactive peptides are enzyme hydrolysis and fermentation [88]. Pepsin, trypsin, chymotrypsin, alcalase, and flavourzyme are frequently utilized for hydrolysis. Compared with microbial fermentation, these bioactive oligopeptides, occurring in different sizes and produced by enzymes, have higher scalability [4]. The peptides, after enzymatic digestion, can be recovered by desalination, membrane ultrafiltration, column chromatography, and freeze-drying [87].
Crucially, some oligopeptides with short sequences of 2-20 amino acids can be absorbed by the intestine into the blood circulation and exert systemic or local physiological anti-diabetic activities, such as decreasing blood glucose levels, improving glucose and insulin sensitivity in target tissues, and inhibiting key enzymes like α-amylase, α-glucosidase, and DPP-4 related to T2D [4,89,90].
Legume-derived hydrolysates and fractions have also been investigated in cell and animal models (Table 3). Administered with semipurified γ-conglutin, hydrolysates (5 mg/mL) from Andean lupin legumes induced a 6.5-fold glucose uptake and decreased gluconeogenesis in male SD rats by 50% [64]. In Caco-2 cells, treatment with 10 mg/mL black bean hydrolyzed protein isolate (HPI) decreased glucose absorption by 21.5%, and intake of 100-200 mg of HPI/kg BW/day lowered postprandial glucose levels by 22.7-47.7% in a hyperglycemic rat model [60]. The intake of common bean peptide fractions reduced glucose levels in male Wistar rats at the same rate as glibenclamide, keeping a stable basal level without a surge in postprandial hyperglycemia [75]. Jiang found that soybean protein hydrolysate (<5 kDa) made by ultrafiltration significantly reduced the level of fasting blood glucose (FBG) in mice [62]. Wei reported that pea oligopeptides significantly reduced blood glucose levels, lipid profiles, and liver fat deposition in diabetic mice [74].
Some pure sequence-known peptides have also shown hypoglycemic effects in animal and cell models (Table 3). LPYP, IAVPGEVA, and IAVPTGVA from soy glycinin improved glucose metabolism by increasing glucose uptake via up-regulating the expression of GLUT1 and GLUT4 in cultured hepatic cells [63]. Lammi's group developed a fast, sensitive, and cost-effective ex vivo DPP-4 assay for human serum by collecting venous blood from a healthy female volunteer and analyzing how peptides inactivated the enzyme. They discovered that hydrolytic peptides like LTFPGSAED from lupin β-conglutin and IAVPTGVA from soybean glycinin inhibited the activity of DPP-4 [91].
Collectively, legume peptides and hydrolysates show remarkable hypoglycemic effects through various approaches. Interest has been directed toward the acceptable oral administration of bioactive peptides, one of the main attractions for T2D patients. Based on their inherent amino acid composition and sequence, bioactive peptides exhibit different activities in regulating glucose and lipid metabolism, which exert beneficial effects on the control of diabetic progress.

Potential Hypoglycemic Mechanism of Bioactive Legume Peptides
The hypoglycemic mechanism of legume diabetic peptides mainly includes reducing glucose absorption, promoting pancreatic β-cells proliferation, enhancing insulin secretion and sensitivity through signaling pathways associated with diabetes, and inhibiting carbohydrate-digesting enzymes (α-amylase and α-glucosidase) and DPP-4 in target organs (Figure 1).

Targeting the Pancreas
One of the main factors contributing to the development of T2D is assumed to be a decrease in glucose-stimulated insulin secretion from pancreatic β-cells [105]. An appealing idea for treating diabetes and its complications is to restore the function of poor β-cells. The IRS (insulin receptor substrate)/AKT (protein kinase B) pathway is the primary mechanism of the hypoglycemic effect of the body's response to insulin stimulation [106]. Vglycin peptide exhibited beneficial effects on hyperglycemia by facilitating glucose metabolism and enhancing insulin sensitivity, but it could not increase insulin secretion [57]. Another study supports an apparent impact of vglycin on regulating β-cell preservation; a selfrenew program was initiated in which vglycin directly promoted the proliferation of β-cells via activating the IR/Akt/Erk pathway [14]. These results reveal the protective effects of vglycin on improving β-cell function in long-term glucolipotoxicity.
Some hydrolysates and fractions also exhibited a protective effect on the dysfunctional pancreas. Hydrolysates (<3 kDa) from hard-to-cook beans and common beans were reported to increase glucose-stimulated insulin secretion up to 57% and 45% from the basal state in INS-1E pancreatic β-cells, respectively [66,76]. In another study, fermented meju water extracts (W-60), containing 15 kDa peptides, increased glucose-stimulated insulin secretion capacity and β-cell viability in Min6 insulinoma cells [65]. Previous studies have identified that amino acid metabolism, especially glutamine and alanine, is essential for the functional maintenance of pancreatic cells and insulin secretion [107]. Therefore, it can be inferred that the amino acids or oligopeptides in the hydrolysates of legumes may be beneficial for the exertion of hypoglycemic function of the pancreas directly or indirectly.

Targeting the Liver
Soymorphin-5 (YPFVV), derived from the soy conglycinin subunit, plays a role in glucose and lipid metabolism [58]. Although no differences were observed in the weights of mesenteric and epididymal fat and brown adipose tissues, plasma TG levels and liver weights in KKA y mice were significantly reduced by soymorphin-5 treatment, suggesting that soymorphin-5 enhanced the liver lipid metabolism. In terms of mechanism, soymorphin-5 improved fatty acid β-oxidation and energy expenditure by increasing the hepatic expression of PPAR and its target genes, including Adipor2, AOX1, CPT1, and UCP2 [58]. In another study, vglycin was probably responsible for increased fatty acid β-oxidation via the AMPK pathway and inhibited fatty acid synthesis (FAS) by downregulating expression of FAS in HFD-fed C57BL/6J mice [15]. Soy peptides IAVPGEVA, IAVPTGVA, and LPYP enhanced glucose uptake through GLUT1 and GLUT4 activation, and modulated glucose metabolism by activating Akt and AMPK pathways in HepG2 cells [63]. In addition, γ-conglutin hydrolysates decreased gluconeogenesis by 50% in a dual-layered enterocyte/hepatocyte system and down-regulated the expression of phosphoenolpyruvate carboxykinase (PEPCK) in HepG2 cells [64].

Targeting Muscle and Adipose Tissue
Increasing glucose absorption and transformation in muscle and adipose tissue is an effective method to control hyperglycemia. As shown in a STZ/HFD-induced diabetic mice model, oral administration of aglycin could potentially attenuate or prevent hyperglycemia via enhancing the IR/IRS1 signaling pathway in skeletal muscle [56]. Additionally, in C2C12 cells, aglycin increased glucose uptake by recruiting glucose transporter GLUT4 to the surface of cells [56]. Cowpea peptides induced the Akt phosphorylation in skeletal cells and mimicked the actions of insulin by activating the same signaling pathway [67]. Prior findings strongly implied that the translocation of GLUT4 was regulated by activation of the Akt signaling pathway in the skeletal muscle cells, which facilitated glucose uptake [108,109].
Studies of fermented soy products have suggested that the small bioactive peptides produced during fermentation may contribute to preventing or slowing the progression of T2D [65,110]. Water extracts (<3 kDa) from chungkookjang increased PPAR-r activity by about 59%, indicating that there exist certain small peptides that work as mild PPAR-r agonists in glucose and lipid metabolism [65]. γ-conglutin hydrolysates also significantly increased the insulin-induced glucose uptake in mature 3T3-L1 adipocytes through an enhanced expression of GLUT4, similar to metformin treatment [64]. Similarly, in C2C12 cells, γ-conglutin increased flottilin-2, caveolin-3 concentrations, and CBL phosphorylation, resulting in GLUT4 translocated on the cell membrane, which suggested that γ-conglutin plays an essential role in muscle energy metabolism through insulin-mimetic action [73].

Targeting the Intestine and Colon
In STZ-induced Wistar rats and the Caco-2 cell models, black bean peptides AKSPLF, ATNPL, FFEELN, and LSVSVL may have reduced glucose absorption via blocking glucose transporters GLUT2 and SGLT1 (Figure 1) [60]. In Table 4, we summarized 19 α-amylase inhibitors, 12 α-glucosidase inhibitors, and 16 DPP-4 inhibitors from legume peptides, all of which worked as potential anti-diabetic agents targeting the intestine and colon (Figure 1). The mechanism of lowering postprandial hyperglycemia by inhibition of α-amylase and αglucosidase is to inhibit the rate of glucose release and absorption in the intestine [12]. The hypoglycemic mechanism of inhibiting the DPP-4 enzyme is to retard the decomposition of GLP-1 and increase the endogenous physiological level of GLP-1 in the human body [34]. Recently, the structure of oligopeptides and their binding modes to these enzymes have been systematically investigated, which may provide a clear anti-diabetic mechanism for these peptides [40].
α-amylase contains three distinct structural domains (A, B, and C). The active site region in domain A is associated with an extended substrate binding cleft, allowing it to recognize polymeric starch molecules [111]. Asp197, Glu233, and Asp300 are three essential catalytic carboxylic acid residues located within this active site cleft between the carboxyl ends of domains A and B. Domain B is responsible for substrate specificity and enzyme stability, and domain C helps to maintain the stability of the catalytic site. Therefore, blocking the catalytic sites was an effective way to inhibit the activity of α-amylase [40]. Peptides amply containing aromatic residues such as Leu, Pro, Gly, and Phe were effective inhibitors of α-amylase because the substrate-binding pockets of the α-amylase enzyme have many aromatic residues [111]. Hence, apart from interactions involving hydrogen bonds and electrostatic and van der Waals interactions, aromatic-aromatic interactions have also played a crucial role in α-amylase inhibitory activity [4].
The bioactive peptides LSSLEMGSLGALFVCM, PLPLHMLP, PPMHLP, PPHMLP, PPHMGGP, and PLPWGAGF, extracted from pinto beans, interacted or bound to the indicated domain of α-amylase, ultimately restricting protein conformation and affecting the degree of enclosure of the substrate. Mechanically, when the (α-amylase)-starch complex was formed, the uncompetitive peptide inhibitors bound to the complex, resulting in the starch no longer being able to hold on at its original position due to the altered conformation of α-amylase. Consequently, the starch was detached from the (α-amylase)-starch complex [92]. The competitive inhibitors GSR and EAK formed hydrogen bonds with the catalytic residues [62].
Structural analysis has clearly shown the intimate interaction between bean-derived peptides and α-amylase catalytic residues. The interaction prevented residue Asp300 from adopting its functional position and destroyed the water channel leading from the "flexible loop" to the heart of the active-site depression [112]. Another inhibitory mechanism was identified in the interaction of porcine pancreatic α-amylase and the bean P. vulgarisderived peptide, in which residue His305 participated in substrate binding and structural changes [113].
Similar to peptides that inhibit α-amylase, certain peptides have the potential to inhibit α-glucosidase, reducing glucose absorption and delaying carbohydrate digestion, thereby reducing postprandial blood glucose levels. Pure peptides such as KTYGL were able to inhibit the enzyme mainly through polar interactions (Asn32, Asp34, Asp38, and Asp89), hydrophobic interactions (Trp36 and Trp81), and hydrogen bonds (Ser31) with the residues from the binding pocket of the α-glucosidase [98]. AKSPLF showed only hydrogen bonding and polar interactions with α-glucosidase, and Ala and Lys were the only two amino acids interacting with ASP34, THR83, ASP89 and ASN32 [95]. Besides, α-glucosidase inhibitors also played crucial roles in the secretion of GLP-1 in T2D [114].
Collectively, legume peptides exhibit great inhibitory potential due to their affinity for and specificity of action on key enzymes such as α-amylase, α-glucosidase, and DPP-4, which are promising candidates for the development of anti-diabetic drugs. However, the structure-activity relationships between peptides and these enzymes remain to be uncovered.

Challenges of Legume Peptides' Development in T2D Treatment
Despite their tremendous potential value in anti-T2D therapies, legume-derived peptides have intrinsic disadvantages, including poor chemical and physical stability, poor bioavailability, short effective half-life, and inefficiency of oral delivery, which must be considered for their application as nutraceuticals or medicines [12,116]. Effectively delivering therapeutic peptides via the oral route is still a big challenge, even though some drawbacks can be solved by rational design with the increase of biological activity, selectivity, or bioavailability. Numerous studies have concentrated on creating novel technologies through physical and chemical methods, including enteric coating, enzyme inhibitors, permeability enhancers, nanoparticles, and intestinal microdevices, to improve the oral bioavailability and stability of the peptides, which overcome the absorptive barriers in the gastrointestinal tract [4,22,117].
Low selectivity, resistance to protein hydrolysis, and the demands of large-scale production are also some of the main challenges [22]. Approaches such as proteomic analysis, bioinformatics tools, pretreatments, hydrolysis mechanisms and isolation, and effective cell and animal screening models all contribute to obtaining hydrolysates or peptides with hypoglycemic activity [118]. Regarding the delivery and administration of peptides in the human body, the effectivity and stability of the peptides in the gastrointestinal tract are worth considering. Since oligopeptides are predicted to be resistant to digestive enzymes, they can be utilized as an ingredient or supplement in nutraceutical foods. Those specific peptides with long amino acid sequences or hydrolysates are still susceptible to digestive enzymes.
In addition, some legume-derived anti-diabetic peptides are mainly studied by integrated computational methods or in vitro assays, due to a lack of sufficient animal and human clinical trials. These potential anti-diabetic peptides need a deep investigation geared towards the development of nutraceuticals and drugs. On the other hand, hydrolytic peptides are generated from dietary proteins during gastrointestinal digestion or are produced by processing proteins. Their bioavailability after oral administration depends on their absorption and distribution to reach target organs [119]. The peptides in the legume hydrolysates (Table S1) should be identified, and their anti-diabetic activities and precise mechanisms need further investigation. Undoubtedly, legume-derived peptides research can benefit from pharmaceutical approaches for the discovery, design, and development of nutraceutical or oral medicinal peptides for T2D.

Conclusions and Perspectives
Peptides are becoming one of the most attractive candidates for the development of oral anti-diabetic drugs. The FDA has approved Rybelsus ® (semaglutide) oral tablets to improve control of blood sugar in adult patients with T2D, along with diet, exercise, and other anti-diabetic drugs. The efficacy and safety of the treatment of Rybelsus ® , assisted with Salcaprozate sodium, an absorption enhancer, have been considered acceptable in several clinical trials [31,120,121]. It provides a viable design for oral legume-derived drug development for T2D. The biopeptides and protein hydrolysates from legumes summarized in this review are confirmed to have anti-diabetic activities, and their anti-diabetic mechanisms are illustrated in vivo and in vitro.
The biological activity of a peptide mainly depends on its chemical structure, which determines its molecular function in target organs [44,94]. Structural modifications can enhance the enzymatic tolerance of polypeptide drugs, thereby increasing the half-life of drugs. As a native peptide, aglycin consists of 37 amino acids and contains the special cystine knot structure in the molecule, which can resist the hydrolysis of proteases in the digestive tract. This self-protective structural feature makes aglycin viable as an oral agent in the management of T2D and other metabolic syndromes.
Since its discovery, insulin has been one of the major therapeutic molecules used to control diabetes. The most common route of insulin administration is the subcutaneous route. However, oral delivery is the most convenient and patient-centered route. The human physiological system acts as a formidable barrier to insulin, limiting its bioavailability. Currently, the delivery methods enhancing the bioavailability of oral insulin and GLP-1 receptor agonists include the use of ionic liquids, hydrogels, nanoparticles, microparticles, and nano-in-microparticles [122]. These strategies provide new insights into the development of the effective delivery of orally administered protein and peptide drugs.

Conflicts of Interest:
The authors declare no conflict of interest.