GLP-1R Signaling and Functional Molecules in Incretin Therapy

Diabetes is considered the fastest-growing global health problem, affecting about 10% of adults around the world [ 1 , 2 ]. The diabetic population will reach 783 million globally in 2045 according to an estimate from the International Diabetes Federation [ 2 ]. All types of diabetes share the common clinical manifestation of hyperglycemia and several characteristic symptoms, including thirst, polyuria, constant hunger, fatigue, weight loss, and blurred vision [ 2 ]. As the disease progresses, complications such as retinopathy, nephropathy, and neuropathy may occur, and the risk of cardiovascular diseases, obesity, and nonalcoholic fatty liver disease will increase, which significantly affects the quality of life. T2DM accounts for more than 90% of diabetes cases [ 1 , 2 ], although this percentage might be higher since approximately one-third of people living with T2DM are undiagnosed [ 2 ]. Adopting a healthy lifestyle and taking metformin are the cornerstone for T2DM management. A combination of sulfonylureas, alpha-glucosidase inhibitors, thiazolidinediones, and insulin injections can be added when the single antidiabetic medication is insufficient. However, these drugs can cause side effects such as hypoglycemia, weight gain, and cardiovascular risk [ 3 ]. Pancreatic β-cell dysfunction and resultant insulin deficiency are the key features of T2DM [ 4 ], however, most medications do not target β-cell and become less effective as diabetes progress [ 5 ]. Fortunately, a couple of gut-derived natural peptides termed incretins that can stimulate insulin secretion [ 6 ] have inspired novel T2DM treatments. Incretins were first discovered upon observing that oral glucose administration leads to greater insulinotropic effects than intravenous administration [ 7 ]. Since then, researchers began to investigate gut-derived insulin secretagogues; and incretin-based therapies currently have become the preferred first injection therapy for T2DM treatment, due to their strong glycemic control effect and remarkable safety profile [ 8 ]. Incretins include glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). Although both GLP-1 and GIP could promote insulin secretion in a healthy state, the therapeutic potential of GIP alone is controversial [ 9 , 10 ]. In contrast, the GLP-1 receptor (GLP-1R) is thought to be one of the most important potential drug targets for glucose-dependent T2DM treatment, lowering hypoglycemia risk compared to insulin and sulfonylureas [ 11 ]. GLP-1, as the endogenous agonist of GLP-1R [ 12 , 13 , 14 ], is a promising natural antidiabetic product due to its anorexigenic, insulinotropic, and weight-reducing effects [ 15 ]. However, in vivo GLP-1 can be cleaved by dipeptidyl peptidase 4 (DPP-4) immediately after secretion at the second amino acid (alanine) from its N-terminal [ 16 ]. This will lead to an instant degradation of GLP-1 and a short circulation time in the human body. Although GLP-1 has many potential advantages, its short circulation time of about 2 min [ 17 ] limits its application in treatment since such frequent administration is incompatible with patient compliance and thereby reducing drug effectiveness. DPP-4 inhibitors and GLP-1 analogs with prolonged circulation time have already been applied in incretin therapy and have performed well. However, GLP-1R agonists are favored because they have superior body weight control [ 18 ] and cardiovascular outcomes [ 19 ]. In this review, we discuss the current GLP-1R signaling and ligand development strategies, trends in incretin therapy, and perspectives on T2DM treatment.

Researchers have been pursuing functional studies of GLP-1R for many years to illuminate the mechanism of GLP-1R signaling. GLP-1R downstream signaling pathways network can be activated through coupling with the intracellular transducers [ 29 ]. The diverse protein-binding forms will lead to complex downstream pathways. Figure 1 Signaling pathways of GLP-1R in pancreatic β-cell [ 15 , 30 ]. Three downstream signaling pathways initiated from Gαs (blue), Gαq (pink), and β-arrestin (green) are shown in the left half of the β-cell. The GLP-1R internalization process and insulin secretion after GLP-1R activation are also shown. Colored circles show mono-agonists targeting GLP-1R (red), GIPR (green), and GCGR (blue), and multi-agonists targeting GLP-1R/GIPR (red/green), GLP-1R/GCGR (red/blue), and GLP-1R/GIPR/GCGR (red/green/blue). The top left inset shows the structure of GLP-1R with a seven-transmembrane helix bundle and a large ECD. Abbreviations: VDCC, voltage-dependent calcium channel; TRPM, the transient receptor potential melastatin; GCGR, glucagon receptor; GIPR, gastric inhibitory polypeptide receptor. Currently, it is believed that GLP-1R predominantly signals through the Gα s /cAMP pathway; however, there is evidence that GLP-1R couples with Gα q and other G proteins. After activation, GLP-1R undergoes phosphorylation at the C-terminal, which further recruits β-arrestin, leading to internalization and desensitization of the receptor ( Figure 1 ). The Gα s /cAMP pathway directly leads to the glucose-induced secretion of insulin granules [ 31 , 32 ]. After activation by full agonists such as GLP-1, GLP-1R couples with Gα s , activates adenylate cyclase (AC), and causes the accumulation of cAMP [ 33 ]. With increasing cAMP levels, protein kinase A (PKA) [ 31 ] and the exchange protein directly activated by cAMP-2 (Epac-2) [ 34 ] are also activated. PKA and Epac-2 trigger the closure of K ATP and K V channels, which depolarizes the cell membrane, opens voltage-dependent calcium channels (VDCC), and causes Ca 2+ influx [ 35 , 36 ]. In addition to the classical function of cAMP, the cAMP/CREB pathway could also induce the expression of insulin receptor substrate 2 (IRS2) and promote β-cell survival, demonstrating the protective effects of GLP-1 analogs on β-cells [ 37 ]. In addition to the Gα s /cAMP pathway, GLP-1R is also able to couple with other G protein subtypes including Gα i , Gα q , Gα o , and Gα 11 [ 38 , 39 ]. Recent research has demonstrated the importance of the Gα q pathway in the pancreatic β-cell. First, the Gα q pathway initiates from the coupling of phospholipase-C (PLC), transforming phosphatidylinositol 4,5-bisphosphate (PIP2) to inositol triphosphate 3 (IP3) and diacylglycerol (DAG), and accompanies the activation of protein kinase C (PKC) and intracellular Ca 2+ influx production via IP3 receptor [ 39 ]. PKC also triggers the closure of the K ATP channel and activation of TRPM4/5 [ 40 ]. Gα s and Gα q regulate the cAMP and Ca 2+ levels, respectively; however, variational mechanisms exist in the complex network of G protein signaling pathways. A switch of Gα s and Gα q pathways has been found under certain conditions, including persistent depolarization of cell membrane [ 30 ], and reduction in GLP-1 concentration to picomolar [ 41 ]. The switch of G protein pathways clarifies a possible mechanism for basal insulin secretion under drug treatment and provides important direction for incretin therapy in T2DM. Two types of β-arrestin, β-arrestin-1 and β-arrestin-2, mediate the GLP-1R downstream signaling in β-cells, and also elicit the receptor internalization process. β-arrestin-1 mediates the phosphorylation of CREB and ERK1/2 [ 42 ], further phosphorylating Bad (Bcl-xL/Bcl-2-associated death promoter homolog) and inhibiting cell apoptosis [ 43 ]. β-arrestin-2 functions as an indispensable insulin regulator. Knocking out β-arrestin-2 in the mice model and leading to impaired insulin secretion [ 44 ]. With the treatment of sulfonylureas, direct interaction between β-arrestin-1 and Epac-2 can upregulate the Ca 2+ concentration [ 45 ]. GLP-1R is a fast-internalized and recycling receptor [ 46 ]. After activation, GLP-1R-ligand complexes enter the endosome [ 32 ]. A portion of that is eventually transported to lysosomes for degradation, while the other portion returns to the cell membrane [ 46 ]. Different agonists may show different effects on GLP-1R internalization and recycling. For example, GLP-1 is apt to receptor recycling, while exendin-4 may favor slower recycling and lysosome targeting [ 47 ]. The latest study suggested that internalized endosomal GPCR-Gα s complexes are the origin of induced ERK activity [ 48 ], but the detailed mechanism remains implicit. Due to advantageous physiological effects and rapid response in GLP-1R signaling pathways, functional molecules targeting GLP-1R are attracting increasing attention in drug discovery. Optimization of the current molecules and disc

Rather than single agonists targeting only GLP-1R, dual or triple agonists targeting additional receptors (GIPR, GCGR, and GLP-2R) involved in the incretin axis and/or other pathways ( Table 2 ) are expected to have stronger glucoregulatory, weight-reducing, and even cardiovascular protective effects [ 4 ]. Although the mechanism by which multi-agonists have superior effects is unknown, many clinical results have shown their therapeutic potential. Thus far, GIPR/GLP-1R is the most attractive target combination. Instead of playing a redundant role, GIP may protect β-cells from dysfunction and destruction independently of GLP-1 [ 83 ]. Although the therapeutic potential of GIPR agonists is debatable, clinical trials of dual GIPR/GLP-1R agonists have produced promising results. The combination of these two GPCR pathways is assumed to increase glucose-dependent insulin secretion, decrease energy consumption, improve white adipose tissue function, and increase insulin sensitivity [ 84 ]. Tirzepatide, the first dual agonist for the treatment of T2DM and obesity targeting GIPR and GLP-1R, was approved by the U.S. FDA in 2022. Compared with existing drugs such as semaglutide, once-weekly tirzepatide showed better results in glycated hemoglobin A1c (HbA1c) [ 85 ] and body weight reduction [ 86 ] with a similar safety profile. GCGR/GLP-1R dual agonists have also received much attention. OXM can naturally activate both GCGR and GLP-1R with lower potency than their primary ligands [ 87 ]. Although glucagon and GLP-1 have distinct effects on glucose levels, their effects on food intake may be additive [ 88 ], leading to more significant body-weight reduction. Several OXM derivatives are under clinical trials, for example, cotadutide, a once-daily GCGR/GLP-1R dual agonist, has shown promising impacts on glycemic control, body weight, and liver fat reduction [ 89 ]. molecules-28-00751-t002_Table 2 Table 2 Dual/triple-agonists targeting GLP-1R and other GPCRs. Name Trade Name Administration Indications Status 1 Ref. GLP-1R/GIPR Tirzepatide Mounjaro / SC, qw T2DM Obesity 2022 2022 FTD 2 [ 90 , 91 ] [ 86 ] CT-868 / SC, qd T2DM Phase 1 / NNC0090-2746 (RG7697) / SC, qd T2DM Phase 2 [ 92 ] GLP-1R/GLP-2R Dapiglutide (ZP7570) / SC, qw SBS Phase 1 / GLP-1R/GCGR SAR425899 / SC, qd T2DM Phase 2 (discontinued) [ 93 ] Pemvidutide (ALT-801) / SC, qw Obesity NASH Phase 1 Phase 1 / / Pegapamodutide (LY2944876) / SC, qw T2DM Phase 2 (discontinued) / Cotadutide (MEDI0382) / SC, qd T2DM NASH CKD Obesity Phase 2 Phase 2 Phase 2 Phase 1 [ 94 ] [ 95 ] [ 96 ] [ 97 ] Efinopegdutide (MK-6024) / SC, qw NASH Phase 2 / Mazdutide (IBI-362) / SC, qw T2DM Obesity Phase 2 Phase 1 / [ 98 ] BI456906 / SC, qw T2DM Obesity Phase 2 Phase 2 / / MK-8521 / SC, qd T2DM Phase 2 / PB-718 / SC, qw NASH Phase 1 / NN9277 (NNC 9204 1177) / SC, qw Obesity Phase 1 (discontinued) / MOD6031 / SC, qw Obesity Phase 1 / GLP-1R/GCGR/GIPR HM-15211 / SC, qw NAFLD Phase 1 / LY-3437943 / SC, qw T2DM Phase 1 [ 99 ] 1 Year of approval, the final phase reached. 2 The U.S. FDA granted fast track designation (FTD) to Tirzepatide in October 2022. Among these drug candidates, only tirzepatide has been approved. Abbreviations: SC, subcutaneous injection; bid, twice daily; qw, once weekly; qd, once daily; T2DM, type 2 diabetes mellitus; SBS, short bowel syndrome; CKD, chronic kidney disease; NASH, non-alcoholic steatohepatitis; NAFLD, non-alcoholic fatty liver disease. Data were manually collected from clinicaltrials.gov and Drugs@FDA database on 15 December 2022. Clinical trial failures may occur in some cases of dual or triple agonists, caused by their side effects. In order to develop safer therapeutic drugs, it is essential to study the mechanism of these dual and triple agonists [ 88 ] from structural, functional, and pharmacologic aspects.

4.1. GLP-1R Agonists Based T2DM Therapy in Clinical Trials In the early stage of diabetes, metformin monotherapy is widely applied as the first-line drug and is generally sufficient for glycemic control in T2DM [ 118 ]. With the progression of the disease, metformin alone cannot maintain blood glucose at the desired level, and combination therapies become necessary. The selection of combined medication, including sulfonylureas, alpha-glucosidase inhibitors, thiazolidinediones, dipeptidyl peptidase 4 inhibitors (DPP4i), GLP-1 agonists, and sodium-dependent glucose transporters 2 inhibitors (SGLT2i) [ 2 , 119 ], is based on clinical characteristics and patients’ preferences. Among these drugs, thiazolidinediones and sulfonylureas are commonly used and cost-effective. However, they can cause congestive heart failure and hypoglycemia, respectively, thus limiting their further application. GLP-1R agonists and SGLT2i both have fewer side effects and are recommended for patients with comorbidities. Meta-analysis showed that the combination of a GLP-1R agonist and metformin could effectively reduce blood glucose levels [ 120 ]. Insulin injection is inevitable for glycemic control after the dysfunction of non-insulin treatment. Among the non-insulin medication, incretin therapy is an advanced treatment for T2DM patients. The therapeutic potential of incretin in T2DM has been explored for more than one hundred years. In 1906, Moore et al. used extracts from the duodenal mucous membrane to treat T2DM [ 121 ]. Half a century later, Nauck et al. discovered an impaired incretin response in T2DM patients, leading to a decrease in incretin-stimulated insulin release [ 122 ]. A further study identified that the reduction in GLP-1 accounted for the diabetic state [ 6 ]. A 6-week pilot study investigated the long-term effect of GLP-1 in T2DM by continuous administration of this peptide hormone. Patients had lower fasting glucose (4.3 mmol/L) and HbA1c (−1.3%) under the administration of GLP-1 [ 123 ]. However, due to the short circulation time of native GLP-1, it is infeasible to continuously administrate native GLP-1 for long-term blood glucose management [ 124 ]. Some GLP-1 analogs were developed to overcome this problem. Exenatide, the first approved GLP-1 analog, has significant effects on HbA1c level reduction [ 125 ], weight loss, and glycemic control [ 125 ], but no cardioprotective effect [ 67 ]. As the plasma drug concentration could be maintained for 12 h, the method of administration was by subcutaneous injection twice a day. Liraglutide was another novel GLP-1 analog that was approved by the FDA in 2010. A series of phase 3 trials (Liraglutide Effect and Action in Diabetes, LEAD) was launched to compare the efficacy and safety between liraglutide and other oral antidiabetic drugs [ 69 , 126 , 127 ]. In the LEAD-3 study, a significant reduction in HbA1c level was found with a dosage of 1.8 mg liraglutide compared with glimepiride monotherapy [ 126 ]. Moreover, compared with exenatide, liraglutide carries a lower risk of cardiovascular death [ 120 ]. Throughout the LEAD trials, patients showed high tolerance to the drug, with the most frequently reported side effects being GI events such as nausea and vomiting. Liraglutide was found to maintain a maximum plasma drug concentration for about 12–14 h, so the period of administration became once daily. Most recently, the GLP-1 analogs have continuously improved, representing great advances in treating T2DM, obesity, and cardiovascular diseases. Semaglutide Unabated Sustainability in Treatment of Type 2 Diabetes (SUSTAIN) trials showed the positive effects of subcutaneous semaglutide on glycemic control [ 128 ]. Weekly 2.4 mg semaglutide, compared with daily 3 mg liraglutide, shows a superior effect on weight loss [ 129 ]. Oral semaglutide administered once per week showed favorable efficacy and safety in the treatment of T2DM patients and the large, randomized control clinical trial is ongoing [ 130 ]. In addition to GLP-1R agonists, the discovery of multi-agonists is also ongoing in recent years. The first approved dual agonist, tirzepatide, is a potent agonist for both GLP-1R and GIPR. At dosages of 5, 10, and 15 mg, tirzepatide showed non-inferior treatment in glycemic control and superior behavior in weight loss than 1 mg semaglutide [ 85 ], further demonstrating the therapeutic efficacy of multi-agonists. Many other multi-agonists have emerged and are in the process of early-stage clinical trials ( Table 2 ).

Despite the numerous anti-hyperglycemic agents that are already available on the market for T2DM treatment, there are still a surprising number of drug candidates in clinical trials, of which no less than 40% are with novel therapeutic molecules or targets [ 144 , 145 ]. With this rapid development of clinical therapeutic solutions for treating diabetes, a burst of potential first-in-class drugs is expected that would further reduce adverse effects while maintaining or even enhancing the efficacy of current drugs. Such advances would provide more choices for diabetes management and would be beneficial to personalize medicine with better patient compliance. Many strategies have been conducted to further optimize GLP-1R agonists. For example, in the quest for longer-lasting peptides, a series of GLP-1R G-protein biased agonists have been developed via backbone modifications with normal or enhanced G-protein signaling, but significantly reduced β-arrestin response, leading to the improved duration of action of the peptides [ 133 ]. Compared with “nonbiased” agonists, these biased GLP-1 agonists would lead to reduced receptor desensitization, prolonged availability of GLP-1R at the cell surface, enhanced insulinotropic and glucose-lowering properties, and greater therapeutic value. Among all these agents, replacing the first eight amino acids of exendin-4 (HGEGTFTS) with novel sequence ELVDNAVGG, the modified agent named P5, was a potent and selective biased GLP-1 agonist that was investigated, which was found to have a higher acute anti-hyperglycemia efficacy than exendin-4 [ 135 ]. A recent study reported that PX17, an updated agent based on P5, shows greater blood glucose modulation and body weight reduction compared with semaglutide [ 146 ]. Exendin-phe1, with minimal substitutions within the N-terminus of exendin-4, was found to promote glycemic benefits without decreasing tolerability [ 147 ]. Structure and functional studies, aided by molecular dynamics simulation, illustrate that the peptide agonist conformation plasticity, especially the dynamic interaction of the receptor with the N-terminal activation domain of the peptide, may be an essential determinant for agonist efficacy [ 148 ]. Thus, biased GLP-1R agonists generated via backbone modification provide a powerful strategy for improving therapeutic efficacy and have led to novel treatments for T2DM. Recent studies have reported strategies, which altered the brain penetration property of GLP-1R agonists, to mitigate nausea or other unwanted gastrointestinal responses, the most prominent adverse effects of GLP-1R agonist treatment [ 134 , 149 ]. The adverse and glucose-lowing effects probably possess at least partially different signaling response profiles. Thus, researchers could rationally design for separating these two signaling pathways to tune down the unwanted adverse effects via directed modifications. Nausea, or other unwanted gastrointestinal responses, may be triggered by the effects of GLP1R agonists targeting the receptors expressed in the central nervous system (CNS). Blood glucose regulation effects mainly depend on the targeting of the pancreas or other peripheral systems. One possible strategy would be to downregulate the effects of GLP1R agonists on the CNS. Since all the agents gain access to the brain by penetrating the blood–brain barrier, this protection mechanism could be utilized as a key step to filter out unwanted agents. Studies utilizing the corrination method, conjugating GLP1R agonists with vitamin B12 or related compounds containing corrin ring, affect pharmacokinetics and modify the solubility of GLP1R agonists and prevent them from penetrating into the CNS. Specifically, covalent conjugation of extendin-4 to vitamin B12, which possesses a corrin ring structure, forms B12-exendin-4 with reduced brain penetrance, leading to glucose lowering while significantly abolishing the unwanted emetic events [ 134 ]. Conjugation of extendin-4 with Dicyanocoinamide, the B12 precursor, could also exhibit similar glucoregulatory capability and nausea reduction effect [ 149 ]. These approaches have effectively reduced emetic effects, while retaining efficient glycemic control.