GLP-1-based therapies for type 2 diabetes: from single, dual and triple agonists to endogenous GLP-1 production and L-cell differentiation

Abstract

Glucagon-like peptide-1 (GLP-1) is an incretin peptide hormone mainly secreted by enteroendocrine intestinal L-cells. GLP-1 is also secreted by α-cells of the pancreas and the central nervous system (CNS). GLP-1 secretion is stimulated by nutrient intake and exerts its effects on glucose homeostasis by stimulating insulin secretion, gastric emptying confiding the food intake, and β-cell proliferation. The insulinotropic effects of GLP-1, and the reduction of its effects in type 2 diabetes mellitus (T2DM), have made GLP-1 an attractive option for the treatment of T2DM. Furthermore, GLP-1-based medications such as GLP-1 receptor agonists and dipeptidyl peptidase-4 inhibitors, have been shown to improve diabetes control in preclinical and clinical trials with human subjects. Importantly, increasing the endogenous production of GLP-1 by different mechanisms or by increasing the number of intestinal L-cells that tend to produce this hormone may be another effective therapeutic approach to managing T2DM. Herein, we briefly describe therapeutic agents/compounds that enhance GLP-1 function. Then, we will discuss the approaches that can increase the endogenous production of GLP-1 through various stimuli. Finally, we introduce the potential of L-cell differentiation as an attractive future therapeutic approach to increase GLP-1 production as an attractive therapeutic alternative for T2DM.

Graphical Abstract

Highlights

• 1. Intestinal L cells-derived GLP-1 induce insulin secretion in response to meal intake.

• 2. Insulinotropic potency of GLP-1 decrease in T2DM.

• 3. GLP-1 receptor agonists and DPP4-inhibitor direct the T2DM therapies.

• 4. Increasing the endogenous production of GLP-1 through increasing the L-cells may be an effective therapeutic approach to managing T2DM.

Introduction

Type 2 diabetes mellitus (T2DM) is a chronic metabolic disorder that is characterized by insulin resistance, hyperglycemia, and pancreatic β-cell dysfunction. The statistics are staggering as the incidence of T2DM rises globally. According to the 10th edition of the International Diabetes Federation (IDF) Diabetes Atlas, the global prevalence of diabetes in adults aged 20–79 years is estimated to be 537 million, and this is predicted to rise to 783 million by 2045. T2DM accounts for over ninety percent of all types of diabetes. T2DM is a common and complex but highly treatable disease associated with a multitude of devastating complications such as retinopathy, neuropathy, coronary artery disease, cerebrovascular disease, peripheral vascular disease, and nephropathy [1]. These complications are the important causes of mortality among the affected people which impose a heavy economic burden on the healthcare systems globally.

In response to nutrients, enteroendocrine L-cells and K-cells produce and release the incretin hormones including glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), respectively [2]. GLP-1 and GIP control blood glucose levels by stimulating insulin secretion by pancreatic β-cells while inhibiting glucagon secretion, increasing pancreatic β-cell proliferation, and decreasing their apoptosis [3,4,5,6,7,8]. Apart from pancreatic islets, GIP and GLP-1 receptors have been identified in several other organs, including the intestine, heart, brain, and kidney. This suggests that these hormones have pleiotropic effects beyond lowering blood glucose levels [9,10,11].

Patients with T2DM experience a significant decline in the incretin’s functional properties which causes delayed and reduced insulin release after glucose administration and impairs glucose tolerance [12,13,14]. This decline may be due to the decreased GLP-1 secretion or impairment of L-cell development [15,16,17,18]. However, the functional effects of GLP-1 hormone on healthy and obese subjects in blood glucose control make T2DM a suitable target for treatment. Incretin-based treatments are safe and effective in managing T2DM, and they have beneficial effects on β-cell function in addition to glycemic control.

GLP-1- based treatments for T2DM, such as GLP-1 receptor agonists, dual and triple agonists that activate GLP-1R, GIPR, and glucagon receptors, and dipeptidyl peptidase-4 (DPP-4) inhibitors have been reviewed here. In addition, we discuss the increase in the endogenous secretion of this hormone through various stimuli (e.g., carbohydrates, proteins, gut microbiota, bacteria-derived molecules, bile acids, lipids, fatty acids, polyphenols, etc.) or by the increase of GLP-1-producing cells, i.e., L-cells, which can be an appropriate approach for the management of diabetes.

Differentiation of intestinal stem cells and enteroendocrine cells into L-cells

The incretin hormone GLP-1 is one of the hormones that is produced by post-translational processing of proglucagon by proprotein convertase subtilisin-kexin type 1 (Pcsk1) or Pcsk3 in L-cells of the distal small bowel and colon. L-cells are a type of enteroendocrine cells (EECs) that are found in different parts of the digestive tract and produce hormones that regulate various digestive and metabolic processes in the body. EECs originate from Leucine-rich repeat containing G protein-coupled receptor (Lgr5) positive stem cells in the base of the intestinal crypts. In the crypts, multiple signaling pathways in the niche, including Wnt, BMP, Notch, EGF, and Eph/Ephrin play important roles in the regulation of self-renewal and differentiation of intestinal stem cells (ISCs) [19]. Lgr5⁺ stem cells divide into proliferating transit-amplifying (TA) cells, each of which can differentiate into either a secretory or absorptive progenitor cell. Activation of Notch signaling and the expression of its downstream target gene, hairy and enhancer of split 1 (Hes1) in TA cells repress the expression of transcription factors crucial for entry into the secretory lineage including atonal homolog 1 (Atoh1) and induce absorptive cell fate commitment. Conversely, blocking the Notch signal leads to the expression of Atoh1, which induces secretory progenitors [20]. Subsequently, absorptive progenitors differentiate into enterocyte cells, while secretory progenitors differentiate into Paneth, goblet, tuft cells, and EEC progenitors.

Various transcription factors are responsible for controlling cell differentiation. The expression of the growth factor independence1 (Gfi1) transcription factor in Paneth and Goblet cell precursors prevents their conversion into EEC lineage [21]. Additionally, SRY-box transcription factor 9 (Sox9) and Krüppel-like factor 4 (Klf4) transcription factors, are Paneth and goblet cell-specific differentiation factors [22, 23]. Secretory progenitors differentiate into EEC progenitors through the expression of the neurogenin 3 (Ngn3) transcription factor. Furthermore, several transcription factors, such as Neurod1, Rfx6, Arx, Foxa1, Foxa2, Pax4, and Pax6 regulate the differentiation of EEC subpopulations [24] (Fig. 1).

Fig. 1

A Schematic representation of the crypt-villus unit of the small intestinal epithelium and the major signaling pathways that control the self-renewal and differentiation process of intestinal stem cells (ISCs): Lgr5 + stem cells reside at the very bottom of the crypt and are usually flanked on both sides by Paneth cells. A gradient of BMP signaling with relatively high activity in the villus and less activity within the crypt, and Wnt, and Notch signaling gradients in the highest expression at the crypt base regulates cell renewal and lineage specification. B Schematic representation of the hierarchy of intestinal lineages: Intestinal stem cells differentiate into the mature cell types Paneth cells, Goblet cells, enteroendocrine cells, and absorptive cells. EC enterochromaffin cell, TA-cell transit-amplifying cell, EEC enteroendocrine cell, Sst somatostatin, Tac1 tachykinin

These EECs are classified based on the hormones they produce, such as serotonin-producing enterochromaffin cells (ECs), cholecystokini (CCK)-producing I-cells, GIP-producing K-cells, GLP-1 producing L-cells, somatostatin (SST)-producing D-cells, neurotensin (NTS)-producing N-cells, secretin (SCT)-producing S-cells and ghrelin (GHRL)-producing X cells. However, the majority of EECs express several hormones that overlap between different enteroendocrine cells. The change in the BMP signaling gradient along the crypt-villi axis could induce hormone changes in EECs during the migration of these cells along the crypt-villus axis. For instance, the changes in hormonal expression in EC cells. EC population in the crypt expresses tachykinin (Tac1) and tryptophan hydroxylase-1 (Tph1) but the EC population in the villus expresses Sct and Tph1 [25] (Fig. 1).

Deficiency of L-cells development and GLP-1 secretion in T2DM

GLP-1 has two active forms, GLP-1amide and GLP-1 released in a biphasic manner: the first phase is rapidly released within 15–30 min after nutrient intake, followed by the second phase at 90–120 min. The half-life of this hormone is only approximately two minutes, with cleavage by DPP-4 enzymes occurring at the second residue of incretins, resulting in only 10–15% of secreted GLP-1 reaching the systemic circulation [26]. GLP-1 acts through its receptors, GLP-1R, which is expressed in different tissues, including the pancreas, heart, kidney, adipose, and central nervous system. Therefore, GLP-1 is a multifunction hormone with wide pharmacological potential. Its important action is the stimulation of insulin secretion through binding to GLP-1R on pancreatic β cells. This interaction also increases pancreatic β-cell proliferation while decreasing cellular apoptosis [3,4,5,6,7,8]. Furthermore, this hormone inhibits gastric emptying, gastrointestinal motility, and glucagon secretion [9,10,11]. Patients with T2DM experience a significant decline in the incretin function causing delayed and reduced insulin release after glucose administration, impairing glucose tolerance [12,13,14].

According to studies, the reduced incretin effect in patients with T2DM may be due to a decrease in the response of β-cell to GIP and/or GLP-1 or a decrease in GLP secretion [15, 18, 27, 28]. However, the results regarding postprandial GLP-1 secretion and fasting GLP-1 between individuals with versus without T2DM are inconsistent. These discrepancies may arise from variations in sample sizes and the methods used to measure GLP-1 hormones, as well as poor medication reporting during data collection. Consequently, further investigation is warranted [29]. However, in vitro, evidence suggests that hyperglycemia, a primary metabolic feature of T2DM, can lead to impaired GLP-1 secretion and L-cell differentiation. For example, under high-glucose conditions, the overexpressed transforming growth factor β1 (TGFβ1) creates a highly inflammatory environment in NCI–H716 cells and decreases GLP-1 secretion. TGFβ1, via its downstream Ras homolog family member A (RhoA)/rho-associated coiled coil-containing protein kinase (ROCK) signaling pathway, stimulates the abnormal distribution of F-actin and G-actin and stress fiber accumulation, creating a barrier for GLP-1 secretion. Additionally, yes-associated protein (YAP) protein expression, a major determinant of cell fate and downstream of actin dynamics and the RhoA/ROCK, is inhibited under high-glucose conditions [30].

Chronic exposure to high glucose in intestinal organoids impairs the expression of transcription factors related to the differentiation of early and late endocrine progenitors and L-cell development including Ngn3 and Neurod1. Moreover, hyperglycemia impairs the expression of Sox9, a key factor of Paneth cell differentiation involved in stem cell maintenance and the expression of stem cell markers Lgr5 and Musashi RNA-binding protein 1 (Msi1) and transit-amplifying cells marker prominin 1 (Prom1). Therefore, the decrease in the differentiation of L-cells may be due to the reduction in stem cell proliferative capacity induced by chronic hyperglycemia [31].

In the era of personalized medicine, single-cell sequencing at genetic and epigenetic levels may contribute to a better understating of the pathogenicity of T2DM and its treatment. For example, the RNA sequencing analysis on isolated enteroendocrine cells from obese individuals with or without T2DM revealed decreased expression of the PCSK1 gene in the jejunal epithelium, contributing to the biosynthesis of GLP-1 [32]. Additionally, in high-fat-diet-induced obese mice, miR-194 overexpression through transcription factor 7-like 2(Tcf7l2) and forkhead box a1(Foxa1) inhibits proglucagon (Gcg) and Pcsk1 gene transcription, ultimately suppressing GLP-1 synthesis in L-cells [33].

Considering these changes in T2DM and the critical role of GLP-1 in glucose homeostasis, strategies for managing T2DM based on this hormone include addressing the differentiation of L-cells, increasing the endogenous production and secretion of GLP-1 through stimulants, extending the half-life of GLP-1 by inhibiting its degrading enzyme and targeting GLP-1R using its agonists.

Targeting GLP-1/GLP-1 receptors

While altered GLP-1 secretion in T2DM remains contested, native GLP-1 has demonstrated efficacy in improving blood glucose levels in patients. However, native GLP-1 has low stability in vivo and is inactivated by the enzyme DPP-4 [34].Two strategies exist for overcoming this problem: first, the development of GLP-1 receptor agonists (GLP-1RAs) that activate GLP-1 receptors similarly to native GLP-1. Moreover, they are resistant to DPP-4 activity and have a longer half-life than native GLP-1. Second, the use of DPP-4 inhibitors to prevent the proteolytic degradation of GLP-1 by the DPP-4 enzyme, thereby increasing native GLP-1 levels in the plasma.

GLP-1 receptor agonists

GLP-1 receptor monoagonists

According to their pharmacodynamics and pharmacokinetic properties, the GLP-1RAs are categorized as either short-acting or long-acting agents. Short-acting GLP-1RAs, such as exenatide and lixisenatide, have a half-life of 2–4 h, requiring daily administration once or twice a day. Long-acting GLP-1RAs, such as liraglutide with a half-life > 12 h and albiglutide, dulaglutide, exenatide, and semaglutide, have a half-life of up to 14 days, allowing for once-weekly administration of longer-acting GLP-1RAs. Short-acting GLP-1Ras have postprandial anti-hyperglycemic effects by inhibiting gastric emptying. By contrast, long-acting GLP-1RAs lower fasting blood glucose and hemoglobin A1c (HbA1c) by stimulating insulin secretion [35].

Several methods are introduced to increase the half-life of GLP-1 including: (i) modifying peptides to make them resistant to cleavage by DPP-4, such as exenatide twice daily and lixisenatide, (ii) attaching free fatty acid side chains to liraglutide and semaglutide, which enhances their binding to plasma albumin thereby preventing renal filtration of GLP-1 and prolonging their action in vivo [36, 37], (iii) conjugation of albumin or the Fc fragment of IgG to GLP-1 molecule is used in albiglutide and dulaglutide [37, 38], (iv) development of modified nanoparticles for the controlled release of exenatide-LAR (long-acting release) that provide prolonged release of the peptide [39], and finally, (v) chemical permeation enhancers such as sodium salcaprozate (SNAC) could be utilized to overcome the low permeability and high enzymatic degradation of the gastrointestinal tract of GLP-1 analog that is administrated orally such as semaglutide [40].

GLP-1 receptor agonist exerts its action through the GLP-1R. GLP-1 receptor agonist by activating the GLP-1 receptor in β-cell of the pancreas leads to activation of adenylyl cyclase and increased cAMP. cAMP activates PKA and Epac-2 signal transduction pathways, PKA and Epac-2 lead to the closure of ATP-sensitive potassium channels, which in turn causes depolarization of the cell membrane and opening of voltage-dependent calcium channels, resulting in uptake of calcium and stimulation of the exocytosis of insulin granules and blood glucose control of T2DM patients. GLP-1RAs stimulate insulin secretion in a glucose-dependent manner, therefore, reduce the risk of hypoglycemia. In addition, GLP-1RAs, reduce pancreatic islet β-cell apoptosis, stimulate the proliferation and differentiation of β -cells, and decrease body weight by suppressing food intake in patients with T2DM [8, 41,42,43].

The results of a meta-analysis indicate that GLP-1RAs can reduce major adverse cardiovascular events (MACE) such as cardiovascular death, stroke, or myocardial infarction, as well as hospital admissions for heart failure and all-cause mortality, in patients with T2D [44].

In a large trial with a median follow-up of 3.8 years, liraglutide (1.78 mg) significantly reduced cardiovascular risk factors such as weight, systolic, and diastolic blood pressure. However, the heart rate was 3.0 beats per minute higher in the liraglutide group. Additionally, liraglutide decreased the occurrence of nonfatal myocardial infarction and nonfatal stroke. It reduced cardiovascular mortality compared to placebo in patients with T2DM who were at high risk for cardiovascular events while on standard therapy [45]. Moreover, semaglutide and dulaglutide have also demonstrated cardiovascular benefits in clinical trials [46, 47].

Among GLP-1RAs, oral semaglutide has a distinct absorption profile compared to once-weekly subcutaneous semaglutide. However, In patients with T2DM, oral semaglutide showed better glycemic control than placebo over 26 weeks [48]. Additionally, patients with T2DM and comorbid cardiovascular or chronic kidney disease who received once-daily oral semaglutide (14 mg) exhibited a cardiovascular safety profile similar to that of the subcutaneous form [49]. Ongoing studies, such as the SOUL trial, are exploring the potential cardiovascular benefits of oral semaglutide in T2DM patients with established atherosclerotic cardiovascular disease and/or chronic kidney disease [50].

The protective cardiovascular effects of GLP-1RAs can be through improving glycemic control, as reducing HbA1c can improve blood pressure, body weight, hyperlipoproteinemia, and inflammation. Also, these effects can be attributed to direct action on GLP-1R in cardiac and vascular tissues [50].

GLP-1RAs have shown promising results in treating non-alcoholic fatty liver disease (NAFLD) in patients with T2DM [51]. These agonists directly activate the GLP-1R in hepatocytes and help alleviate hepatic steatosis through signaling pathways and genes involved in lipid metabolism. Therefore, these drugs are considered potential options for treating and slowing the progression of NAFLD [52, 53].

Most studies have reported that GLP-1RAs have renoprotective effects. GLP-1RAs can reduce the development of macroalbuminuria and reduce the estimated glomerular filtration rate (eGFR) decline in participants with T2D [54, 55].This reduction is attributed to various mechanisms, including the reduction of oxidative stress and inflammation and inhibition of the immune system’s response. How this renal protection occurs is still unknown. GLP-1RAs functions effects may involve the reduction of glucose and blood pressure, as well as weight loss. Additionally, GLP-1R, expressed in human kidneys’ proximal tubular cells, the renal vasculature, the preglomerular vascular smooth muscle cells, and juxtaglomerular cells could directly contribute to these functions. Furthermore, GLP-1RAs can increase diuretic and natriuretic actions through changes in renal hemodynamics and inhibition of the sodium-hydrogen exchanger 3 in the renal proximal tubule [56].

Chemical modifications of GLP-1RAs have increased the medicinal value of these agonists for the treatment of diabetes. However, not all patients respond effectively to GLP-1R monoagonists, and higher doses aimed at increasing efficacy result in adverse gastrointestinal effects. As discussed below, the development of unimolecular multi-agonists that stimulate GLP-1 receptors and other receptors such as GIP receptors and glucagon receptors (GCGR) can have beneficial effects on metabolism and increase metabolic rate at tolerable doses [57].

GLP-1 receptor dual agonists

GIP and GCG which are involved in nutrient and energy metabolism, can be one of the components of these multi-functional single molecules. Studies on obese diabetic (ob/ob) mice showed that GIP (Lys16PAL) and GIP (Lys37PAL) long-acting analogs for GIPR increased the insulin response to glucose and improved glucose tolerance [58]. Therefore, this hormone could be a promising candidate for combination therapy with GLP-1 for better control of T2D. Studies show that single-molecule dual GIPR/GLP-1R agonists have better insulinotropic and antihyperglycemic effects than GLP-1 agonists in rodents, primates, and humans, without causing gastrointestinal disturbances [58].

NNC0090-2746 (RG7697) is a compound that has been tested in multiple preclinical and clinical studies as a dual GIPR/GLP-1R agonist [58,59,60]. In Phase 1 and 2a clinical trials, once-daily subcutaneous injections of RG7697 (0.25–2.5 mg) for 14 days in 56 patients with T2DM and 1.8 mg of RG7697 for 12 weeks in patients with T2DM who were poorly responsive to metformin reduced body weight and improved glycemic control. Although the most commonly reported adverse events were gastrointestinal side effects such as nausea, vomiting, and diarrhoea, the treatment was well tolerated in the study population [59, 60].

Tirzepatide is a new dual GIPR/GLP-1R agonist recently approved by both the European Medicines Agency and the FDA. It is used as an adjunctive therapy, along with diet and exercise, to help manage blood sugar levels and promote weight reduction in adults with T2DM [61]. Tirzepatide's amino acid sequence is derived from GLP-1, GIP, semaglutide, and a few unique residues. Conjugation of a fatty acid chain via hydrophilic linkers to lysine residue at the C20 position prolongs its half-life and enables once‐weekly subcutaneous administration [62]. Based on Phase 2 clinical trials, patients with T2DM who were inadequately managed with diet and exercise alone or were on a stable metformin regimen for at least 3 months before screening received once-weekly subcutaneous injections of tirzepatide at doses of 1 mg, 5 mg, 10 mg, or 15 mg for 26 weeks. The trials indicated that all doses of tirzepatide resulted in greater weight loss and enhanced glycemic control in comparison to placebo. Furthermore, the 5 mg, 10 mg, and 15 mg doses exhibited superior weight loss and glycemic control relative to dulaglutide (1.5 mg), while maintaining an acceptable safety and tolerability profile. The most commonly observed side effects in the trial were nausea, diarrhea, and vomiting. However, the severity of gastrointestinal adverse events was mild to moderate [63]. Another study indicates that starting treatment with a lower dose and gradually increasing it helped reduce the incidence of gastrointestinal adverse events [64]. Moving forward, the results of the phase 3 SURPASS clinical trial program demonstrated that tirzepatide was as effective and safe as other anti-diabetic agents, such as semaglutide and dulaglutide (SURPASS-2) (SURPASS J-mono), long-acting insulin analogs (degludec and glargine) (SURPASS-3,4), and placebo (SURPASS-1) (SURPASS-5), in managing T2DM diabetes [65,66,67,68,69].

The SURPASS-1 trial demonstrated significant improvements in glycemic control and substantial reductions in body weight with all three doses of tirzepatide (5 mg, 10 mg, or 15 mg) compared to placebo over a 40-week period. These results were observed in patients with T2DM who were inadequately controlled by diet and exercise alone and who had no prior experience with injectable diabetes therapy [70]. In the second trial, SURPASS-2, tirzepatide administered at doses of 5 mg, 10 mg, or 15 mg showed superior efficacy compared to semaglutide (1 mg) in reducing body weight and HbA1c levels over 40 weeks in patients with T2DM inadequately controlled with metformin [66].

Following this, the SURPASS-3 trial evaluated tirzepatide in patients with T2DM inadequately controlled by metformin, with or without SGLT2 inhibitors. In this study, once-weekly subcutaneous injections of tirzepatide (5 mg, 10 mg, or 15 mg) were compared to once-daily subcutaneous injections of titrated insulin degludec. Tirzepatide showed greater reductions in HbA1c and body weight at week 52, along with a lower risk of hypoglycemia [67].

In the SURPASS-4 trial, adults with T2DM at heightened cardiovascular risk, who were inadequately managed with oral glucose-lowering agents, once-weekly tirzepatide (5 mg, 10 mg, or 15 mg) were compared to insulin glargine (100 U/mL). The findings were compelling, tirzepatide not only achieved greater reductions in HbA1c but also delivered significant body weight loss, all while showing a lower incidence of hypoglycemia at the 52-week mark. More importantly, the hazard ratio for major adverse cardiovascular events (including cardiovascular death, myocardial infarction, stroke, or hospitalization for unstable angina) for the pooled tirzepatide groups versus glargine was 0.74 (95% CI 0.51–1.08), suggesting no increased cardiovascular risk with tirzepatide compared to insulin glargine. These findings highlight tirzepatide's potential to revolutionize treatment for high-risk T2DM patients, offering not only improved metabolic control but also a favorable cardiovascular safety profile [71].

The addition of tirzepatide to insulin glargine was evaluated in the SURPASS-5 study, focusing on patients with T2DM who had inadequate glycemic control. After 40 weeks, tirzepatide significantly outperformed placebo in both glycemic control and weight loss. Mean HbA1c reductions were − 2.40% for the 10-mg dose, − 2.34% for the 15-mg dose, and − 2.11% for the 5-mg dose of tirzepatide, compared to just − 0.86% for placebo (all P < 0.001). Furthermore, body weight reductions were notable: − 5.4 kg with 5 mg, − 7.5 kg with 10 mg, and − 8.8 kg with 15 mg, compared to a 1.6 kg increase with placebo (all P < 0.001). These results highlight the significant benefits of adding tirzepatide to insulin therapy, offering improved glycemic control and meaningful weight loss in patients with T2DM who are inadequately controlled on insulin glargine alone [72].

Individuals diagnosed with T2DM and at high risk for cardiovascular issues were studied to determine the effects of tirzepatide on their heart health. The results showed that tirzepatide did not exacerbate their existing heart problems and improved their heart health markers [68, 73, 74]. Currently, the SURPASS-CVOT trial is evaluating the impact of dulaglutide and tirzepatide on cardiovascular events in patients with T2D (NCT04255433). The trial aims to ensure complete cardiovascular safety. Additionally, in a post-hoc analysis of the SURPASS-4 trials, tirzepatide showed a slower rate of eGFR decline and lower urinary albumin creatinine values as compared to patients who were treated with glargine insulin [75, 76].

GCGR activation plays a multifaceted role in energy and glucose metabolism. In the liver, GCGR signaling stimulates hepatic glucose production through glycogenolysis and gluconeogenesis, promotes lipolysis, and inhibits fat accumulation. Importantly, GCGR agonist is also associated with increased energy expenditure, a key factor in weight management and metabolic health. However, the hyperglycemic effects of glucagon pose a challenge for its therapeutic application [77].

Combining GLP-1R and GCGR agonists has emerged as a promising strategy to overcome these challenges. Co-infusion of GLP-1 and GCG has been shown to reduce glucagon-induced hyperglycemia, lower blood glucose levels, suppress food intake, and enhance energy expenditure in both diabetic obese mice and healthy human volunteers [78]. Similarly, oxyntomodulin, a natural peptide hormone secreted by intestinal L-cells following a meal, functions as a dual GLP-1R/GCGR agonist. By activating both receptors, oxyntomodulin increases energy expenditure, reduces appetite, promotes weight loss, and improves glucose tolerance, showcasing the therapeutic potential of this dual mechanism [79]. These insights have driven the development of synthetic GLP-1R/GCGR co-agonists aimed at achieving long-term metabolic benefits. Preclinical and early clinical trials reveal that these dual-acting peptides can deliver multiple pharmacological effects, including enhanced glucose regulation and significant weight reduction. Their unique ability to target multiple metabolic pathways makes them highly promising for treating T2D and obesity. Currently, several long-acting dual GLP-1R/GCGR agonists are in various stages of clinical development. These advanced therapeutics hold the potential to revolutionize the management of metabolic disorders by providing sustained benefits for glucose control, weight loss, and overall metabolic health [80,81,82] (Table 1).

Table 1 Clinical trials of GLP-1-baseddual/triple agonist in patients with T2DM

Cotadutide, a dual GLP-1R/GCGR agonist, has shown significant potential in managing T2D and obesity. In early phase 2a trials, it effectively improved glycemic control, increased post-meal insulin levels, and facilitated weight loss in participants with obesity and patients with T2DM [83]. An MRI-based exploratory analysis also indicated that cotadutide significantly reduced hepatic fat compared to placebo [84].

In phase 2b trials, a 54-week regimen of daily subcutaneous cotadutide injections (100, 200, or 300 µg) significantly lowered HbA1c levels compared to placebo. However, there were no significant differences in HbA1c reductions between any of the cotadutide dose groups and the liraglutide (1.8 mg) group. In terms of weight loss, cotadutide 200 µg produced effects similar to liraglutide, while the 300-µg dose resulted in greater weight reduction. Further analyses revealed that cotadutide 300 µg improved lipid profiles, liver enzyme levels (AST and ALT), and markers of liver fibrosis, including propeptide of type III collagen, and fibrosis-4 index, compared to placebo. However, these improvements were not observed with liraglutide. These hepatic improvements, which may be associated with cotadutide's glucagon activity, indicate its potential as a therapeutic option for nonalcoholic steatohepatitis (NASH). Collectively, these findings highlight cotadutide’s dual benefits for glycemic control and liver health, supporting its further development for T2D, obesity, and NASH treatment [85].

Survodutide (BI 456906) is another promising dual GLP-1R/GCGR agonist under development for the treatment of T2D, obesity, and NASH. By incorporating a C18 fatty acid into its acylated peptide structure, survodutide’s half-life is extended, enabling weekly administration. Preclinical studies in animal models have demonstrated that survodutide effectively activates both GLP-1R and GCGR, resulting in reduced body weight, slower gastric emptying, decreased energy intake, increased energy expenditure, and improved glucose tolerance [86]. In Phase 1 studies, survodutide was generally well tolerated, with no significant safety or tolerability issues observed in either healthy individuals with overweight or obesity [87, 88]. In Phase 2 trials, treatment with survodutide led to greater reductions in both HbA1c levels and body weight compared to placebo after 16 weeks. Furthermore, survodutide at doses of 1.8 mg or higher resulted in more substantial weight loss than semaglutide 1.0 mg after the same treatment period. Additionally, the mean HbA1c reduction with survodutide at a dose of 0.9 mg was similar to that observed with semaglutide.

Gastrointestinal disorders, primarily nausea, were the most common treatment-emergent adverse events, which could be mitigated with slower dose escalation. These findings highlight the importance of optimizing escalation protocols in future studies. This study also demonstrated modest reductions in several key NASH-related scores, including the Fibrosis-4 index, aspartate aminotransferase-to-platelet ratio, and NAFLD fibrosis score. These findings suggest that survodutide has the potential to reduce liver fibrosis and improve liver function in patients with NASH, making it a promising therapeutic option for this complex liver disease [89].

In addition to the hormones GIP and glucagon, other hormones such as estrogen, peptide tyrosine tyrosine (PYY), gastrin, xenin, and fibroblast growth factor 21 can be good candidates to pair with GLP-1 and create dual agonists that have anti-diabetic and anti-obesity effects. However, these new emergent dual agonists need further investigation to understand the mechanisms of action, how to interact with the respective receptors, increase efficacy, and good safety profile [90,91,92,93,94]. For example, a recent study indicated that unlike in mouse islets where tirzepatide stimulates insulin secretion predominantly through the GLP-1R, the therapeutic effects of tirzepatide on human beta and alpha cells require engagement of both the GLP-1R and the GIPR, and in addition, tirzepatide increased glucagon secretion in isolated islets  [207]. Of note, ex vivo isolated islet experiments do not simulate the full range of regulation present in vivo under T2DM conditions [207], which requires further investigation to clarify.

GLP-1 receptor triple agonists

Building upon the success of monoagonists and dual agonists that target multiple hormone receptors (like GLP-1, GCG, and GIP), researchers have developed triple agonists such as SAR441255 and LY3437943. These innovative peptides simultaneously activate these receptors, demonstrating significant potential in preclinical and early clinical trials [104,105,106].

Retatrutide, a single peptide targeting GIP, GLP-1, and glucagon receptors, has shown remarkable results in early-phase studies. In subsequent Phase 2 trials, once-weekly subcutaneous injections of retatrutide at doses of 4 mg, 8 mg, and 12 mg resulted in marked improvements in glycemic control compared to placebo, along with substantial weight loss when contrasted with placebo and dulaglutide (1.5 mg). The safety profile of retatrutide in individuals with T2DM was also favorable [103].

SAR441255, derived from the exendin-4 sequence, this synthetic peptide exhibits strong agonist activity at GLP-1, GCG, and GIP receptors. SAR441255 demonstrated superior weight loss compared to dual GLP-1/GCG agonists in animal models and did not exhibit major cardiovascular adverse effects in lean monkeys.

SAR441255 exhibits significantly greater potency at the GCG receptor compared to dual GLP-1R/GCGR agonists. This enhanced GCG receptor activity, combined with the effects of GIP receptor engagement, is believed to contribute to the superior weight loss observed with this triple agonist. Importantly, this increased GCG receptor activity did not adversely impact glycemic control.

Early human trials with single subcutaneous doses of SAR441255 in healthy subjects demonstrated improved glycemic control during a mixed-meal tolerance test, while also being well-tolerated. These findings suggest that simultaneous engagement of GIP, GCG, and GLP-1 receptors in humans may offer a significant therapeutic advantage in the management of T2DM [104].

While retatrutide and SAR441255 demonstrate significant potential, several important aspects must be addressed to fully optimize the clinical application of triple agonists. Long-term studies are crucial to assess their safety and effectiveness across diverse patient populations. Exploring the mechanisms of receptor synergy and its influence on weight loss and glycemic control could help in designing more targeted and potent agonists. Additionally, evaluating how these therapies interact with current antidiabetic treatments and determining their suitability for combination therapies may further enhance their clinical value.

In addition to GLP-1/GIP/GCG receptors tri-agonists, there are also triagonists targeting Xenopus GLP-1/GCG/Cholecystokinin-2 (CCK2) receptors [107], GLP-1/GCG/ neuropeptide Y₂ receptors [108], and GLP-1R, neuropeptide Y1, and neuropeptide Y2 receptors [109]. Preclinical studies have validated the therapeutic potential of these compounds for the treatment of obesity and diabetes.

Dipeptidyl peptidase-4 inhibitors

DPP-4 is a part of the serine protease family that is found in two membrane-bound and soluble forms in various tissues such as the heart, lung, kidney and liver. DPP4 can cleave various peptides, leading to inactivation and/or generation of new bioactive peptides. For instance, it is capable of inactivating GIP and GLP-1 peptides. Therefore, DPP-4 inhibitors extend the half-life of endogenous GLP-1 lead to a 2–threefold elevation of endogenous GLP-1 concentration, and provide beneficial effects in T2DM [110, 111]. To date, 5 types of DPP-4 inhibitors are approved for clinical use: sitagliptin, saxagliptin, vildagliptin, linagliptin and alogliptin. The other three DPP-4 inhibitors, teneligliptin, anagliptin and trelagliptin, are only approved in the Japanese and Korean markets. The peptide mimetic DPP-4 inhibitors including vildagliptin, saxagliptin and teneligliptin, were identified by peptide-based substrate substitution experiments, while the non-peptide mimetic group such as sitagliptin, alogliptin and linagliptin, were derived from the inhibitor and initially found in random screening. The diverse chemical structure also explains the unique binding pattern of DPP-4 inhibitors [112].

The six inhibitors are classified into three types based on their different binding to the active site of the DPP-4 enzyme. Type 1 contains vildagliptin and saxagliptin, which bind only to the S1 and S2 subsites and form a covalent bond with the nitrile group of their cyanopyrrolidine moiety and Ser630 of DPP-4. Alogliptin and linagliptin in Group 2, interact with the S1' subsite. Linagliptin also interacts with the S2' subsite. The uracil rings of both inhibitors induce a conformational change at Tyr547 of subsite S1'. In the third Group, teneligliptin and sitagliptin interact with the extended S2 subsite of the DPP-4 active site and exhibit the highest inhibitory function against DPP-4 [113].

As mentioned, DPP-4 inhibitors increase the concentration of endogenous GLP-1, leading to increased insulin secretion, decreased glucagon secretion, and decreased HbA1c in patients with T2DM. DPP-4 inhibitors carry a minimal risk of hypoglycemia and gastrointestinal symptoms. Additionally, they enhance β-cell proliferation and neogenesis while inhibiting apoptosis [114, 115]. Unlike GLP-1 agonists, DPP-4 inhibitors do not affect satiety, gastric emptying, or induce weight loss. GLP-1 receptor agonists have been shown to provide superior glycemic control and weight loss relative to the DPP-4 inhibitors. However, the combination of GLP-1 receptor agonists with DPP-4 inhibitors have not demonstrated clinically significant additional benefits [116]. Furthermore, in addition to incretin hormones, DPP-4 cleaves many other non-incretin peptides and regulates various biological processes. Therefore, DPP-4 inhibitors may have pleiotropic effects.

Evidence suggests that DPP-4 inhibitors reduce urinary albumin excretion, oxidative stress, and renal dysfunction in patients with T2D independent of their glucose-regulatory activity [117, 118]. Additionally, DPP-4 inhibitors have been shown to improve abnormal liver transaminases levels such as aspartate aminotransferase and alanine transaminase, in patients with T2DM with liver dysfunction. This improvement is attributed to increased GLP-1 activity and direct inhibition of DPP-4 expressed in the liver [119]. Moreover, GLP-1RA and DPP-4 inhibitors are approved for the treatment of T2DM and exhibit potential pleiotropic effects on the heart and kidneys, which may be independent of or dependent on the hypoglycemic effects.

Increasing the endogenous secretion of GLP-1

The apical surface of L-cells in the intestinal lining plays a critical role in sensing luminal compounds such as carbohydrates, proteins, gut microbiota, bacteria-derived molecules, bile acids, lipids, fatty acids, and polyphenols. These compounds interact with receptors on L-cells, triggering signaling pathways that stimulate the secretion of GLP-1. Leveraging this natural mechanism to enhance the endogenous secretion of GLP-1 presents a promising avenue for managing T2D. By increasing GLP-1 production or stimulating its release, this approach can complement existing therapies, such as GLP-1RAs and DPP-4 inhibitors, offering additional benefits while potentially reducing side effects.

Endogenous GLP-1 and GLP-1RAs differ significantly in their pharmacokinetics, mechanisms of action, and physiological effects. As noted, endogenous GLP-1 has a very short half-life of 1–2 min because it is rapidly degraded by the enzyme DPP-4, which results in low systemic levels. Only about 10% of the secreted GLP-1 is delivered to the pancreas in its active form, while the rest is rapidly converted to inactive metabolites. However, even small increases in GLP-1 levels achieved by enhanced secretion or slowed degradation can have significant effects on glucose homeostasis, appetite regulation, and gastrointestinal motility. Importantly, localized increases in GLP-1, such as that occur in the gut and portal vein, often have a more profound effect than systemic elevations, emphasizing the therapeutic potential of targeting regional activity rather than relying solely on systemic concentrations.

In contrast, GLP-1RAs are designed to resist degradation by DPP-4, enabling sustained high plasma concentrations and prolonged systemic effects. Unlike the transient and localized actions of endogenous GLP-1, GLP-1RAs maintain consistent levels across arterial, portal, and venous compartments. The molecular stability of lipid-tailed GLP-1RAs, such as liraglutide and semaglutide, allows them to achieve active concentrations of 180–350 pmol/L, while non-acylated agents like exenatide yield slightly lower levels (~ 50 pmol/L). Interestingly, these pharmacological concentrations align closely with peak levels of endogenous GLP-1 observed during intense physiological stimulation [120, 121].

The signaling pathways activated by endogenous GLP-1 and GLP-1RAs also differ based on their concentrations. Endogenous GLP-1, at physiological picomolar levels, primarily engages the PLC/PKC pathway, which is critical for natural regulation. In contrast, the pharmacological nanomolar concentrations of GLP-1RAs predominantly activate the cAMP/PKA pathway, potentially explaining their broader therapeutic effects [122].

In the CNS, endogenous GLP-1 acts primarily through vagal afferent signaling, transmitting information from the gastrointestinal tract to the brainstem [123, 124]. This neural pathway plays a key role in regulation of appetite and satiety. Conversely, GLP-1RAs may exert direct effects on the brain, depending on their ability to cross the blood–brain barrier (BBB). Non-acylated GLP-1RAs, such as exenatide, significantly penetrate the BBB and affect brain regions like the hypothalamus, which controls appetite. Acylated GLP-1RAs, like liraglutide and semaglutide, heve limited direct BBB penetration but still reach critical areas such as the arcuate nucleus (ARH) through indirect pathways [125].

Moreover, both endogenous GLP-1 and GLP-1RAs interact with circumventricular organs (CVOs), such as the area postrema and subfornical organ, which lack a fully intact BBB. These specialized regions detect circulating GLP-1 and GLP-1RAs and relay signals to the hypothalamus, further influencing appetite and body weight regulation. While the brain uptake of GLP-1RAs has been extensively studied in animals, the translation of these findings to human clinical outcomes remains uncertain [126].

GLP-1 also regulates gastric motility by slowing gastric emptying in a dose-dependent manner, helping to control postprandial glucose levels. However, prolonged exposure to GLP-1RAs can lead to tachyphylaxis, reducing their effect on gastric emptying over time. Short-acting GLP-1RAs, such as lixisenatide, maintain robust effects on gastric emptying, whereas long-acting agents, like liraglutide and semaglutide, exhibit reduced efficacy due to continuous receptor activation. These differences may also explain differences in gastrointestinal adverse reactions, such as nausea and vomiting, which are common with GLP-1RAs but typically diminish over time [127, 128].

In conclusion, endogenous GLP-1 functions as a short-lived, localized signal that primarily acts through neural pathways, offering precise and physiological regulation of glucose and appetite. In contrast, GLP-1RAs provide prolonged systemic effects, making them well-suited for managing chronic conditions like T2D and obesity. However, enhancing the endogenous secretion of GLP-1 through dietary, microbial, or pharmacological stimuli represents a promising complementary strategy. By increasing the activity or number of L-cells producing GLP-1, this approach can potentially improve therapeutic outcomes, reduce reliance on synthetic agonists, and minimize associated side effects. Identifying the compounds and pathways that stimulate endogenous GLP-1 secretion remains a critical step in advancing this therapeutic avenue.

Proteins

Casein protein [129], whey proteins [130], peptones [131], and different amino acids such as l-glutamine [132], l-phenylalanine [133], L-tryptophan [134], and l-arginine induce GLP-1 secretion through activation of the calcium-sensing receptor (CaSR), the taste receptor TAS1R1/TAS1R3, G-protein-coupled receptor 6A (GPRC6A) [135], GPR142, peptide transporter-1 (PEPT1) [136], and amino acid (AA) transporter [137] (Fig. 2). These compounds play an important role in insulin secretion and improving glucose clearance by inducing GLP-1 secretion [138]. Therefore, nutritional or pharmaceutical treatments based on amino acids and proteins can be useful in diabetic individuals by improving GLP-1 secretion.

Fig. 2

Signaling pathways are activated by peptides and amino acids involved in glucagon-like peptide 1 (GLP-1) secretion and synthesis in L-cells. Peptides from protein gastrointestinal digestion and amino acids released in the lumen stimulate GLP-1 secretion via CaSR or GPRC6A activation. GPRC6A and CaSR triggered GLP-1 exocytosis by activating PLC- and IP3-dependent signaling pathways and intracellular Ca²⁺ increase. In addition, Na⁺-coupled amino acid (AA) transport and PEPT1 by membrane depolarization and subsequent opening of voltage-dependent calcium channels involved in GLP-1 secretion. Some amino acids were sensed by Gq-coupled receptor GPR142 and caused an increase in intracellular Ca²⁺ concentration and GLP-1 secretion. Protein hydrolysates stimulate proglucagon gene transcription by cAMP increase and CREB transcription factor phosphorylation [139]. ER endoplasmic reticulum, IP3 inositol 1,4,5-triphosphate, PLC phospholipase C, ∆Ψ membrane depolarization, VDCC voltage-dependent calcium channels

Lipids

Lipids and fatty acids can affect glucose homeostasis by stimulating GLP secretion via free fatty acid receptors (FFARs) known as G protein-coupled receptors (GPCRs). The effects of fatty acids usage depend on the type and duration of their use [140]. Long-term treatment with palmitate, a type of long-chain saturated fatty acid, can disrupt the rhythmic secretion of GLP-1 by suppressing the expression of the Bmal1 clock gene [141]. Also, it increases the death of GLP-1-producing cells through increased ceramide production, caspase-3 activity, and DNA fragmentation [140]. Long-chain fatty acids (LCFAs) such as α-linolenic acid (αLA) promote GLP-1 secretion through the G-protein-coupled receptor, GPR120 [142]. However, the carbonic anhydrase 8 (CAR8), which is highly expressed in L-cells, has been found to reduce GLP-1 secretion in response to LCFAs. This effect has been observed both in vitro and in vivo. Therefore, inhibiting CAR8 could be a promising strategy for improving GLP-1 secretion, a new approach to treating diabetes [143].

Protein kinase Cζ and fatty acid transport protein 4 (FATP4) [144] play an important role in the increase of GLP-1 secretion and improvement in glycemic tolerance caused by oleic acid (OA), both in vivo and in vitro [145]. Additionally, oleic acid reduces ceramide content and ROS production, which increase due to prolonged exposure to high levels of palmitate [140] (Fig. 5A).

Oleoylethanolamide (OEA), an ethanolamide fatty acid synthesized in the body, can regulate glucose homeostasis and weight loss by stimulating GLP-1 secretion from intestinal L-cells and insulin secretion from β-cells in a GPR119-dependent manner and by affecting the abundance of the bacterium Akkermansia muciniphila [146, 147]. Medium-chain triglycerides (MCTs) consisting of decanoate (C10:0) can improve glucose tolerance and inhibit HFD-induced obesity in mice by stimulating GLP-1 secretion through GPR84 [148]. Isoprenoid derivatives of lysophosphatidylcholines (LPCs), such as cytronellic acid- lysophosphatidylcholine (1-CA-LPC) stimulated GLP-1 secretion from GLUTag cells through GPR55, GPR119, and GPR120 [149] (Fig. 4).

The expression and distribution of FFARs such as GPR40, GPR119, and GPR120 in L and β cells and glucose, along with their dual effects on insulin and GLP-1 secretion, have made them attractive therapeutic targets, leading to the development of agonists for these receptors for the treatment of T2DM [150,151,152,153,154]. Fatty acids (FAs) have therapeutic potential by stimulating GLP-1 secretion. However, delivering FAs orally is challenging because they are absorbed before reaching the target site. Therefore, a delivery system is needed to enable FAs to reach the distal part of the gastrointestinal (GI) tract, which is rich in L-cells. For instance, oral administration of αLA loaded into thermally hydrocarbonized porous silicon (THCPSi) particles stimulated GLP‐1 release via GPR120 in a sustained manner and inhibited the food intake in mice [155] (Fig. 3).

Fig. 3

Sustained GLP-1 secretion in the distal intestine by α-glucosidase inhibitors, SGLT-1 inhibitors, and αLA loaded into THCPSi particles. Inhibition of α-glucosidase and SGLT-1 causes more un-digested and unabsorbed carbohydrates to transport to the distal intestine and L-cell-rich part of the small intestine and in distal are utilized by the gut microbiome to produce SCFAs, which increased sustained release of GLP-1 through activating their receptor (GPR41/43). Also, delivery of αLA through thermally hydrocarbonized porous silicon particles (THCPSi) leads to the transfer of this fatty acid to the distal intestine and releases GLP-1 through GPR120. SGLT-1 sodium-glucose transporter 1, SCFA short-chain fatty acids, GPR41/43 G protein-coupled receptor, αLA α-linolenic acid

Carbohydrates and gut microbiota

Evidence shows that gut microbiota is crucial for maintaining human health. It is involved in GLP-1 secretion pathways and other intestinal hormones such as leptin, ghrelin, peptide YY, and CCK. Dysbiosis, or an imbalance in gut microbiota, is associated with metabolic diseases, especially T2DM, and can cause changes in the signaling pathways of intestinal hormone secretion, leading to various health problems [156].

Studies have shown that certain bacterial genera such as Bacteroides, Akkermansia, Faecalibacterium, Roseburia, and Bifidobacterium are negatively associated with T2DM, while others like Blautia, Fusobacterium, and Ruminococcus are positively associated with T2DM [156]. Further analysis revealed a decrease in the abundance of Ruminococcaceae, Akkermansiaceae, and Lactobacillaceae in db/db mice compared to control mice and a significant increase in the abundance of members belonging to Enterococcaceae and Bacteroidaceae [157]. These findings suggest that it is possible to increase GLP-1 secretion and manage T2DM and its complications by restoring and improving the gut microbiota [158]. Gut microbiota is involved in GLP-1 secretory pathways through various mechanisms. Short-chain fatty acids (SCFAs) produced by gut bacteria through activation of the G protein-coupled free fatty acid receptors FFAR2 (GPR43) and FFAR3 (GPR41) induce GLP-1 Secretion [159]. Bacterial components, such as the P9 protein secreted by A. muciniphila, induce GLP-1 secretion and improve glucose homeostasis in C57BL/6J mice fed a high-fat diet (HFD) through interaction with intercellular adhesion molecule 2 (Icam-2), which activates GPCR-like downstream signals. Additionally, P9 induces interleukin-6 (IL-6) secretion from the intestine, which subsequently stimulates GLP-1 secretion by intestinal L-cells and pancreatic α-cells, and also affects Icam2 mRNA expression, which suggests the normal expression of Icam-2 and the effects of P9 in glucose homeostasis may require IL-6 [160] (Fig. 4).

Fig. 4

Pathways involved in GLP-1 secretion through fatty acids, carbohydrates, gut microbiota, and polyphenols. A Fatty acids by G protein-coupled receptors and fatty acid transport protein (FATP) increase intracellular Ca²⁺ concentration and intracellular cAMP concentration, respectively, leading to GLP-1 secretion. B, C Carbohydrates stimulate GLP-1 secretion via SGLT-1 and monosaccharide transporter. Glucose entry into L-cells via SGLT-1 and GLUT-2 causes an increase in ATP, which leads to K ATP channel closure. Together with Na entry via SGLT-1, this results in a change in membrane potential, which opens voltage-gated calcium channels and leads to GLP-1 release. In addition, carbohydrate-induced GLP-1 secretion is mediated by STRs and increases in intracellular calcium. Gut microbiota via fermentation of glucose to SCFAs is involved in GLP-1 secretion. Bacterial components such as the P9 protein interact with intercellular adhesion molecule 2 (ICAM-2) and induce GLP-1 secretion. D Polyphenols stimulate GLP-1 secretion by stimulating GPR40/120 and the Ca²⁺-calmodulin-stimulated protein kinase II (CaMKII) pathway activation and reducing the damage of L-cells caused by pro-inflammatory mediators (IL-6, TNF-α, and CRP) and oxidative stress. GLUT-2 glucose transporter 2, SGLT-1 sodium-glucose transporter 1, K ATP channel ATP-sensitive potassium channels, ∆Ψ membrane depolarization, VDCC voltage-dependent calcium channels, STR sweet taste receptors, SCFA short-chain fatty acids, GRP 41/43, G-protein coupled receptor41/43, ICAM-2 intercellular adhesion molecule 2, GRP120 G-protein coupled receptor 120, FATP4 fatty acid transport protein 4, PLC phospholipase C, PKCζ protein kinase Cζ, TAS2R50 taste 2 receptor member 50, CaMKII Ca²⁺-calmodulin-stimulated protein kinase II, IL-6 interleukin 6, TNF-α tumor necrosis factor-alpha, CRP c-reactive protein

Glucose [161] and other carbohydrates such as d-allulose [162], Astragalus polysaccharide (APS) [163], polysaccharides from Dendrobium officinale [164], indigestible polysaccharides resistant maltodextrin (RMD) [165], β-glucan or arabinoxylan [166], and enzymatically-synthesized glycogen [167]stimulate GLP-1 secretion by utilizing glucose sensors, including sweet taste receptors (STRs), sodium-glucose transporter 1 (SGLT-1), and monosaccharide transporter [168, 169] (Fig. 4B). Acacia tortilis polysaccharide and also α-glucosidase inhibitors such as miglitol, by inhibiting α-glucosidase enzyme, increase the level of GLP-1 [170,171,172] (Fig. 3). Additionally, carbohydrates induce GLP-1 secretion by changing the gut microbiota and subsequently improving the production of bacteria-derived molecules such as SCFAs, and bile acids [173].

Despite the direct effects of SGLT-1 in glucose-induced GLP-1 hormone secretion, inhibition of this transporter can indirectly increase GLP-1 secretion. Inhibition of SGLT-1 in the small intestine causes more glucose transport to the distal intestine and colon, resulting in greater fermentation of glucose to SCFAs by luminal bacteria and increased sustained release of GLP-1 in the distal intestine [174] (Fig. 4C).

Bile acid

Bile acids (BAs) play an important role in controlling the body's glucose response after a meal by stimulating GLP-1 secretion. These amphipathic molecules are made from cholesterol and are synthesized and conjugated in the liver. Afterward, they are stored in the gallbladder, released into the intestinal lumen after a meal, and further metabolized by the gut microbiota into secondary bile acids. Bile acids are known to be ligands of the nuclear farnesoid X receptor (FXR) and Takeda G protein-coupled receptor 5 (TGR5), also known as G-protein-coupled bile acid receptor 1 (GPBAR1). TGR5 and FXR are expressed in enteroendocrine L-cells in the intestine. Bile acids can promote GLP-1 secretion via TGR5 in enteroendocrine cell lines. However, FXR activation decreases both proglucagon mRNA levels and GLP-1 secretion in response to glucose [175, 176]. For instance, the rectal administration of taurocholic acid increased GLP-1 secretion in both healthy subjects [177] and obese type 2 diabetic volunteers [178]. Acute administration of CDCA (chenodeoxycholic acid) also increased GLP-1 secretion in patients with T2DM and healthy control subjects [179]. These findings led to the production of BA analog or TGR5 agonist to induce GLP-1 production in vivo. For example, INT-777 and MN6, TGR5 selective agonists, stimulated GLP-1 secretion and improved insulin sensitivity in diet-induced obesity (DIO) mice [180, 181]. The therapeutic effects of TGR5 agonists could be improved in combinatorial therapy. Co-treatment of 4d (A2-thio-imidazole derivative), a novel TGR5 agonist, and Sitagliptin (DPP-4 inhibitor) exhibited a GLP-1 secretion and glucose-lowering effect in animal models, while this compound alone exhibits only a slight 1.3-fold increase in GLP-1 secretion [182].

Bile acid sequestrants (BASs) are anion exchange resins that prevent ileal uptake of bile acids by trapping BAs in the intestinal lumen, resulting in induced de novo synthesis of bile acids from cholesterol in the liver, which decreases plasma cholesterol. This lipid-lowering drug, in addition to lipid control, is used as an adjunct therapy for reducing plasma glucose levels in T2D [183]. However, the effects of the BASs on GLP-1 secretion are controversial. Colesevelam in DIO mice, TGR5-mediated induces GLP-1 secretion and improved glucose tolerance [184, 185]. However, in another study, colesevelam alone or co-administered with CDCA does not have an acute effect on GLP-1 secretion in humans with or without diabetes [179]. In addition, BAS sevelamer suppresses the acute bile acid-induced GLP-1 secretion in patients with T2DM. Nonetheless, further investigation is needed to elucidate the biological effect of this type of agent [186].

Polyphenols

Polyphenols are secondary metabolites of plants with antidiabetic effects, whose benefits are related to the stimulation of insulin and GLP-1 secretion, as well as increasing GLP-1 half-life by inhibiting DPP-4 activity [187]. Polyphenolic compounds are divided into phenolic acids, phenolic alcohols, flavonoids, stilbenoids, and lignans [188]. These compounds can enhance GLP-1 secretion via various mechanisms. Delphinidin 3-rutinoside (D3R) through GPR40/120 and the Ca²⁺-calmodulin-stimulated protein kinase II (CaMKII) pathway activation stimulates GLP-1 secretion [189]. Additionally, anthocyanins (ACs) (cyanidin and delphinidin), protocatechuic acid (PCA), and (–)-epicatechin (EC) induce the GLP-1 secretion through activation of PKA and cAMP [190]. Moreover, Genistein, by reduction of pro-inflammatory mediators (IL-6, tumor necrosis factor-α (TNF-α) and C-reactive protein (CRP)) in serum and intestine in alloxan-induced diabetic rats, reduces the damage of L-cells and leads to an increase in the expression and secretion of GLP-1[191]. Studies have shown that bitter-tasting pharmaceuticals and substances, including polyphenols such as isosinensetin, bind to bitter taste receptors, and unlike other TAS2R agonists, stimulate GLP-1 secretion in NCI-H716 cells [192] (Fig. 4D).

Berberine, an alkaloid extracted from traditional Chinese medicine, binds to the bitter taste receptor TAS2R38 and activates the PLC signaling pathway to stimulate GLP-1 secretion in STC-1 cells [193]. Additionally, Cucurbitacin B (CuB) stimulates GLP-1 secretion via the bitter taste receptor TAS2R10 and, similar to Metformin, induces hypoglycemic effects in diabetic mice [194]. Furthermore, the administration of denatonium benzoate, in conjunction with gavage of glucose, causes the secretion of GLP-1 and subsequently elevates plasma insulin levels and reduces plasma glucose in db/db mice [195]. Studies on T2R gene expression in the human pancreas confirmed the presence of multiple TAS2R receptors. However, their precise role within the pancreas remains largely unexplored. Interestingly, certain bitter compounds, such as denatonium benzoate (DB), have been found to stimulate the secretion of insulin, glucagon, somatostatin, and GLP-1 from pancreatic islets independently of glucose levels. DB enhances insulin release through mechanisms involving KATP channels, bitter taste receptor signaling, and intraislet GLP-1 secretion. Nonetheless, exposure to high doses of DB (5 mM) has been associated with cellular apoptosis in pancreatic islets. These findings suggest that while bitter substances promise to improve glucose homeostasis, their potential adverse effects beyond the gastrointestinal system warrant careful consideration [196].

The available data on the effects of bitter compounds in humans are limited and inconsistent. For example, 600 mg of quinine stimulates the secretion of GLP-1 and plasma C-peptide. Furthermore, quinine has been found to reduce plasma glucose levels and slow down gastric emptying, both on its own and after the consumption of a mixed-nutrient drink in healthy men. However, the contribution of GLP-1 to insulin stimulation was likely modest, and further evaluation using a GLP-1 receptor antagonist would be necessary. Additionally, studies in patients with T2DM are now needed to explore potential clinical benefits [197].

Increasing the number of intestinal endocrine L-cells

According to the impairment of L-cells development in T2DM, a promising strategy for treating T2DM is to increase the number of L-cells in the intestine, thereby increasing the production of GLP-1. This approach can also result in increased production of other hormones produced by L-cells such as oxyntomodulin and peptide YY (PYY), which can enhance the metabolic effects of GLP-1.

Several strategies have been discovered for the potential augmentation of L-cells. One notable approach involves the use of dibenzazepine (DBZ), a γ-secretase/Notch pathway inhibitor. Continuous exposure to DBZ at concentrations of 1 nM or higher for 96 h has been shown to increase the number of L-cells in both mouse and human ileum organoids (Fig. 5). Additionally, a single-pulse regimen of 5 μM DBZ applied for 3 h resulted in an eightfold increase in L-cell numbers after 96 h, while preserving the overall structure of the organoid. Furthermore, administering DBZ at a dose of 10 mg/kg over two consecutive days has been found to enhance L-cell numbers and GLP-1 secretion in rats fed a high-fat diet. Notably, a stronger effect was observed in the jejunum compared to the ileum. DBZ increases the expression of transcription factors Ngn3 and Neurod1. Expression of Ngn3 directs differentiating secretory progenitors toward endocrine fate, while expression of Neurod1 directs differentiating EEC progenitors toward EECs such as L-cells. For this reason, the effect of Notch inhibition is not selective for L-cells, and the number of K, goblet, and Paneth cells also increases. However, increasing the K cell by DBZ can increase GIP release, which may act synergistically with GLP-1 signaling. Besides GLP-1 secretion, mice treated with DBZ show increased insulin secretion and improved glucose tolerance. The proliferative effect of DBZ can be amplified by SCFAs in small intestinal organoids, highlighting the benefits of combination therapies [198]. In addition, combined inhibition of Wnt, Notch, and epidermal growth factor receptor (EGFR) pathways, as well as combined inhibition of Wnt, Notch, and mitogen-associated protein kinase (MAPK), increase differentiation of Lgr5 + stem cells towards EECs and abolished differentiation of goblet cells and Paneth cells in intestinal Organoids [199] (Fig. 1).

In mouse and human organoids, treatment with SCFA led to an increase in the number of L-cells and glucose-induced GLP-1 secretion. This effect was observed without any impact on the marker expression of Lgr5 + stem cells, goblet cells, and Paneth cells. In mouse organoids, the effect of SCFAs was limited to L-cells alone because markers of other endocrine cell types (Sct, Tph, and Gip) remained unchanged. However, in human organoids, SCFA treatment showed an extra impact on enterochromaffin EC cells and S cells. Also, SCFA treatment increased the expression of transcription factors Neurod1 and Foxa1/2, which are associated with late enteroendocrine precursors of L-cells. This shows that the effects of SCFAs on the development of L-cells are through the effect on endocrine progenitors [200].

The RhoA/ROCK pathway plays a significant role in the differentiation of L-cells and other secretory cells by regulating cytoskeleton dynamics and YAP signaling. When YAP is activated in organoids, it increases the expression of downstream target genes, including connective tissue growth factor (Cgtf) and cysteine-rich angiogenic inducer 61 (Cyr61). Consequently, the expression of secretory lineage markers is dramatically reduced. When ROCK is inhibited with Y-27632, it causes a redistribution of F-actin, leading to decreased nuclear localization of YAP and reduced expression of Cgtf and Cyr61. This, in turn, increases the numbers of L-cells, other intestinal secretory lineages, and GLP-1 secretion in both intestinal organoids and mice fed with normal chow and high-fat diet. Moreover, this treatment also improves glucose tolerance and insulin levels in mice on both types of diets. However, the impact of inhibiting ROCK was not as significant in vivo as in vitro [201] (Fig. 5).

Herbal compounds, based on their components can stimulate GLP-1 secretion and provide glycemic control effects in T2DM in different ways. For example, ginseng-derived ginsenoside stimulated GLP-1 secretion in L-cells and decreased hyperglycemia in diabetic models through a sweet taste receptor-mediated signal transduction pathway [202]. In addition, 2.0 μM Compound K (CK), a metabolite of ginsenoside produced by intestinal flora, increases the expression of Gcg and GLP-1 secretion and promotes L-cell differentiation under high-glucose conditions (50 mM) in NCI-H716 cells. CK, through the reduction of TGFβ1 and inhibiting its downstream pathway, RhoA/ROCK, ameliorates the G/F-actin ratio, which under glucotoxicity creates a barrier for GLP-1 secretion. Finally, amelioration of the G/F-actin ratio restores the disruption of the YAP signal induced by high glucose and upregulates the transcription factors Neurod1, Foxa1, and Foxa2, which are responsible for controlling L-cell differentiation [30] (Fig. 5). In db/db mice, CK treatment increases secondary BAs, such as lithocholic acid (LCA) and deoxycholic acid (DCA) through changes in the gut microbiota, especially the Ruminococcaceae family, and up-regulation of cytochrome P450 family 7 subfamily B member 1 (Cyp7b1) and cytochrome P450 family 27 subfamily A member 1 (Cyp27a1) and down-regulation of cytochrome P450 family 8 subfamily B member 1 (Cyp8b1), which changes BA biosynthesis from the classical pathway to the alternative pathway. As a result, the increased secondary BAs can activate TGR5, which increases L-cells and GLP-1 secretion by inhibiting the RhoA/ROCK pathway and inducing YAP activation [157, 203].

Considering the potential of secondary bile acids to increase L-cells and GLP-1 secretion by TGR5, TGR5 agonists are promising drug candidates for increasing L-cells and GLP-1 secretion [180, 181]. One such agonist is L3740. Incubation with 1 μM L3740 for 48 h has increased GLP-1 secretion and the number of L-cells in human and mouse intestinal organoids through GLP-1R signaling and serotonin signaling. When GLP-1 is secreted from L-cells through the TGR5 agonist, it stimulates GLP-1R in enterochromaffin cells and induces serotonin or 5-hydroxytryptamine (5-HT) secretion from these cells. Serotonin signaling in progenitor cells causes an increase in the expression of transcription factors guiding the differentiation of L-cells, thus increasing the number of L-cells [204] (Fig. 5).

Dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs) are a type of serine/threonine kinases that are involved in many different cellular processes, such as regulation of incretin-expressing cell number and regulation of GLP-1 expression. DYRK inhibitors, such as AZ Dyrk1B 33 and ID-8, increase the GLP-1 protein levels in GLUTag cells and the number of L and K cells in zebrafish and diabetic rats. Furthermore, AZ Dyrk1B 33 improves glucose control in larval and juvenile zebrafish and diabetic mice. Dyrk1b knock-down with small interfering RNA suggests that DYRK1B is the major mediator of this signaling pathway in enteroendocrine cells. Apart from DYRK inhibitors, other compounds like allarline, gonadotropin-releasing hormone receptor agonist, and AZD7687, diacylglycerol acyltransferase 1 inhibitor, increase the number of L and K cells in zebrafish [205].

Finally, sirtuin 1 (SIRT1) has the potential to become a treatment strategy for T2DM. SIRT1 is a nicotinamide adenine dinucleotide (NAD⁺)-dependent protein deacetylase that is involved in the cell-cycle regulation of ISCs and EEC progenitors. Sirt1 deficiency, by regulating Wnt/β-catenin activity increases the proliferation of EEC progenitors and subsequently increases the number of EECs, including L-cells in HFD-fed mice and the organoid culture system. This leads to an increase in plasma GLP-1 level and improves the metabolic phenotype in HFD-fed mice [206] (Fig. 5).

Fig. 5

The strategies for enhancement of L-cell: γ-secretase/Notch pathway inhibitor, ROCK inhibitor, Compound K, TGR5 agonist, and Sirtuin 1. 5-HTR 5-hydroxytryptamine receptor, 5-HT 5-hydroxytryptamine, DBZ dibenzazepine, LCA lithocholic acid. DCA deoxycholic acid, TGR5 Takeda G protein-coupled receptor 5

Although the enrichment of L-cells by pharmacological agents can be a novel approach for improving insulin secretion, glucose tolerance and the treatment of T2DM, more research for the specific differentiation of L-cells and the translation of in vitro and animal data to humans, are needed for the success of this approach.

Conclusion

The GLP-1 hormone is recognized as an effective target for the treatment of T2DM due to its multifaceted role in glucose regulation. It contributes to blood glucose control by stimulating glucose-dependent insulin secretion, inhibiting glucagon release, delaying gastric emptying, and reducing appetite. These characteristics make GLP-1 a promising therapeutic target, with several strategies developed to enhance its effects.

The first strategy involves GLP-1RAs, which mimic the action of natural GLP-1 and activate its receptors. These agents are engineered for increased stability and longer half-life compared to native GLP-1. Examples include exenatide, liraglutide, and semaglutide. The second strategy focuses on DPP-4 inhibitors, which prevent the rapid degradation of endogenous GLP-1 by inhibiting the DPP-4 enzyme, thereby extending the hormone's activity. These strategies compared to currently available drug medications such as thiazolidinediones, sulphonylureas, and insulin, have the potential to treat diabetes without causing side effects of hypoglycemia and weight gain. Additionally, have shown remarkable advantages, including weight loss, cardiovascular benefits, improved blood pressure, a better lipid profile, and potential beta-cell regeneration. However, substantial research is needed to expand their utility, optimize delivery methods, and improve patient outcomes.

Currently, most GLP-1 RAs require subcutaneous injection, which can limit patient adherence. The development of oral formulations, such as oral semaglutide, has been a notable advancement in improving treatment acceptance. Existing extended-release options, like weekly formulations of semaglutide and dulaglutide, have reduced injection frequency, and future innovations may include monthly formulations or implantable devices that deliver continuous therapy. These improvements could significantly enhance adherence and quality of life for patients managing chronic conditions like diabetes. GLP-1 RAs have also demonstrated cardioprotective effects by reducing the risk of major cardiovascular events, such as heart attacks and strokes, in individuals with T2DM. Understanding the precise mechanisms of these benefits and whether they extend to non-diabetic populations remains a critical area of research. Emerging data also suggest potential renoprotective effects, offering hope for managing or preventing CKD in patients with diabetes or hypertension. Future trials are essential to confirm these renal benefits and their applicability across diverse patient populations.

The combination of GLP-1 RAs with other therapeutic agents, such as SGLT-2 inhibitors and insulin, is another promising approach. Fixed-dose combinations could optimize outcomes, particularly for patients with advanced disease or those unresponsive to monotherapy. Moreover, increasing focus on pediatric and elderly populations is vital, as T2DM prevalence rises in these groups. Research into safety, efficacy, and specific needs in these demographics will ensure comprehensive care.

The cost of GLP-1 RAs remains a barrier, particularly in low- and middle-income countries. Future efforts should address affordability and accessibility through health economic analyses and policy-driven approaches.

Simultaneously, dual and triple agonists are gaining attention as a promising therapeutic approach. Despite the development of GLP-1 monoagonists, new peptides that combine the pharmacology of GLP-1 with other gut peptides increase the efficacy of these agonists while reducing their dose-related gastrointestinal side effects. However, despite the anti-diabetic and anti-obesity functions, of these dual and triple agonists, the long-term safety, cardiovascular, and renal benefits of these multi-target therapies need further validation.

The third approach aims to enhance endogenous GLP-1 production from intestinal L-cells. This can be achieved through dietary stimulants, pharmacological agents, or interventions targeting the gut microbiota. The final strategy emphasizes L-cell differentiation, with the goal of increasing the number of GLP-1-producing L-cells in the intestine. These strategies offer several potential advantages over GLP-1 analogs, including a more natural stimulation of GLP-1 pathways, reduced risk of side effects, and the ability to address metabolic dysregulation at its source However, clinical applicability for pharmacological targets to increase sufficient GLP-1 production and promote L-cell differentiation has not been identified. Nevertheless, recent advances in understanding the molecular mechanisms, regulatory factors of GLP-1 secretion, signaling pathways, and transcription factors that are effective in endocrine cell differentiation may pave the way for the development of effective applicable medications that can increase GLP-1 secretion and improve L-cell differentiation. Furthermore, long-term studies focusing on safety, efficacy, and patient-specific factors will be essential to translate these innovations into viable therapeutic options. Bridging the gap between findings in animal models and human applications is a critical step toward achieving clinical success. Combining these advancements with other treatment modalities may further optimize their potential, creating a more comprehensive framework for managing T2DM and related metabolic disorders. Additionally, advancements in gene-editing technologies, such as CRISPR, could enable precise genetic modifications to enhance L-cell functionality and proliferation. By directly targeting the genes and molecular pathways involved in L-cell differentiation, gene therapy holds the potential for long-term or even permanent solutions. Research into efficient viral and non-viral delivery systems will be essential to advancing these therapies.

One of the most exciting future directions for GLP-1-mediated therapeutic interventions is personalized diabetes care. Understanding an individual's genetic, dietary, and microbiome profiles could improve the effectiveness of these interventions and minimize the potential adverse effects of these treatments. Together, these advancements could revolutionize diabetes treatment, offering holistic and patient-centered solutions for managing T2DM and metabolic disorders.