Advances in Using GLP-1 Receptor Agonists to Treat Type 2 Diabetes

Transcription factors regulate gene expression by binding to specific enhancer sequences. During the development and maturation of pancreatic β cells, key transcription factors include Pancreatic/duodenal homeobox 1 (Pdx1), neurogenin 3 (NEUROG3), and V-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MafA) (Zhu et al., 2017). Pdx1, in particular, plays a critical role in the development of the pancreas and the function of β cells by binding to regulatory elements and promoting the transcription of the insulin gene (Yang et al., 2012). However, prolonged exposure of β cells to high-glucose environments can lead to decreased expression levels of Pdx1 and MafA, potentially inhibiting insulin biosynthesis and secretion (Kaneto and Matsuoka, 2015). Consequently, patients with T2DM often exhibit reduced Pdx1 expression levels (Ofori et al., 2022). Cordycepin has been found to promote insulin synthesis by upregulating the mRNA levels of insulin, Pdx1, and glucose transporter 1 (GLUT1), as well as the protein expression of Pdx1, GLUT1, Akt, and phosphorylated Akt (Sun et al., 2022).

GLP-1-induced insulin synthesis is initiated by Pdx1. On one hand, GLP-1 activates PKA, increasing the expression of Pdx1 and causing Pdx1 to translocate into the nucleus. Within the nucleus, Pdx1 binds to the insulin promoter, initiating insulin expression and synthesis (Wang et al., 2001; Müller et al., 2019). On the other hand, B-cell translocation gene 2 (BTG2) can upregulate Pdx1-induced insulin gene expression. Studies have shown that GLP-1 treatment significantly increases the expression levels of BTG2, Pdx1, and the insulin gene in pancreatic β cells, indicating that GLP-1 positively regulates insulin gene expression through BTG2, thereby promoting insulin synthesis (Hwang et al., 2013).

GLP-1 stimulates insulin secretion in a glucose-dependent manner through specific receptors expressed on pancreatic βcells.GLP-1R is a family of secretins (class B) that couples GLP-1 to downstream βcell responses, and conformational changes in the receptor upon ligand binding facilitate the interaction between its intracellular region and signaling proteins (Jones et al., 2018). GLP-1R is highly expressed in pancreatic βcells, providing a clear pathway to directly promote insulin secretion. After the knockout of the GLP-1R gene, in transgenic mice expressing human GLP-1R in pancreatic islets and pancreatic ductal cells restored cAMP and serine threonine kinase (Akt) phosphorylation levels in isolated islets, increased GLP-1-stimulated insulin secretion and GLP-1R-dependent β-cell proliferation, and promoted transgenic mice for glucose regulation in response to feeding (Lamont et al., 2012). β-cell-selective knockout of the GLP-1R in mice with impaired glucose tolerance after intraperitoneal injection of glucose and exogenous supplementation of GLP-1 did not induce insulin secretion, but glucose tolerance returned to normal after oral glucose, suggesting that the extra-islet GLP-1R plays a regulatory role in oral glucose tolerance (Smith et al., 2014). GLP-1 is able to stimulate cAMP formation in RIN 1046-38 cells, which in turn causes an increase in insulin mRNA levels and insulin release, and insulin accumulation in the culture fluid was observed by adding GLP-1 (7-37) to RIN 1046-38 cells for 1 h incubation (Drucker et al., 1987). In addition, GLP-1 (7-37) concentrations as low as 50 p.m. stimulated insulin release in perfused rat pancreas (Mojsov et al., 1987).

The activation of the classical cAMP pathway is achieved by releasing irritating G protein subunit Gαs through the exchange of guanine nucleotide and the activation/dissociation of heterogeneous trimer G protein, and in pancreatic βcells, GLP-1 binds to the specific guanine nucleotide-binding protein-coupled receptor GLP-1R, leading to adenylyl cyclase activation and elevated cAMP levels (Mayo et al., 2003; Dalvi et al., 2012). cAMP is an important second messenger driving the acute insulin effect of GLP-1. Overexpression of phosphodiesterase 3B promotes cAMP hydrolysis and reduces GLP-1-induced insulin secretion, an effect evident in both isolated islets and cultured insulinoma cells (Härndahl et al., 2002). cAMP elevation induced by GLP-1 can lead to its downstream protein kinase A (PKA) activation and enhanced signaling via cAMP-activated guanine nucleotide exchange factor (Epac) (Holz, 2004; Doyle and Egan, 2007). cAMP activates PKA, which will phosphorylate the β2 subunit of the L-type VDC channel and the Kir6.2 and SUR1 subunits of the K_ATP channel, increasing K_ATP channel sensitivity to ATP and leading to K_ATP channel closure, cell membrane depolarization and VDC channel opening, followed by Ca²⁺ influx through VDC channels into βcells to promote the exocytosis of insulin granules and the acute secretion of insulin and into circulation (Bünemann et al., 1999; Müller et al., 2019) (Figure 2). GLP-1-activated PKA inhibits voltage-gated potassium channels, prevents membrane repolarization, and prolongs VDC channel opening to promote Ca²⁺ inward currents, contributing to GLP-1 stimulate insulin secretion, and the antagonistic effect of GLP-1R activation on Kv currents in βcells requires transactivation of epidermal growth factor receptors, accompanied by signal conduction of cAMP/PKA and phosphatidylinositol-3-kinase (PI3K)/PKCζ signaling (MacDonald et al., 2003). Both Epac1 and Epac2 are expressed in the pancreas (Leech et al., 2000), and under high glucose conditions, Ca²⁺ flows into βcells through VDC channels, and Epac2 opens RYR calcium channels in the endoplasmic reticulum, further increasing intracellular Ca²⁺ concentrations and enhancing exocytosis of insulin-secreting vesicles (Holz, 2004; Doyle and Egan, 2007). In isolated perfused rat pancreas, GLP-1 did not stimulate insulin release at glucose concentrations below 2.8 mM and could only promote insulin release when glucose concentrations were above 6.6 mM (Weir et al., 1989). As a result, compared with other hypoglycemic drugs, GLP-1RAs has a lower risk of hypoglycemia. In human islets, GLP-1 promotes increased connectivity between individual βcells to improve calcium kinetics and insulin secretion throughout the islets, and promotes insulin secretion from islet cells when βcells detect both elevated blood glucose levels and hormonal signals from the intestine.

The proliferation potential of pancreatic β cells is very limited. Under normal conditions, new β cells generated through mitosis in adult rats constitute about 3% per day, a rate that rapidly declines with age (Swenne, 1983). However, in vitro, certain nutrients and growth factors can enhance their replicative activity. The size of β cell clusters is a major determinant of the total insulin secretion by the pancreas. Expanding β cell clusters through recruitment of β cell proliferation could help maintain normal blood glucose levels, potentially compensating for β cell loss or dysfunction in diabetes (Sjöholm, 1996).

GLP-1 and GLP-1RAs promote the expansion of β-cell mass by stimulating β-cell proliferation, inducing islet neogenesis, and inhibiting β-cell apoptosis (Wajchenberg, 2007). In isolated rat islets, GLP-1 acts as a growth factor in a dose-dependent manner to increase the incorporation of tritiated thymidine and promotes cell proliferation through the upregulation of PI3K-mediated pathways (Buteau et al., 1999). cAMP is a potent mitogenic factor for β cells, and GLP-1 increases intracellular cAMP levels, thereby promoting β-cell proliferation (Sjöholm, 1996; Susini et al., 1998) (Figure 3). GLP-1 promotes islet neogenesis, lowers blood glucose levels, upregulates Pdx1 expression, and increases β-cell mass in db/db mice (Stoffers et al., 2000). Furthermore, endogenous GLP-1 receptor signaling is crucial for the adaptive proliferative response of islets to metabolic stress and pancreatic injury (Drucker, 2003). In a partially pancreatectomized model of type 2 diabetic rats, continuous treatment with GLP-1RAs for 10 days mitigated the progression of diabetes (Xu et al., 1999). Therefore, GLP-1RAs hold promise as a novel therapeutic approach for diabetic patients with reduced β-cell mass, stimulating β-cell proliferation and differentiation (Iqbal et al., 2021).

Apoptosis plays an important role in the pathophysiology of diabetes. The reduction in β-cell mass in patients with T2DM can be attributed to β-cell apoptosis triggered by multiple factors, including islet amyloid deposition, lipotoxicity, glucotoxicity, and inflammation (Costes et al., 2021). Stress pathways induced by these factors, including endoplasmic reticulum stress, oxidative stress, mitochondrial dysfunction, and autophagy, are all involved in the induction of β-cell apoptosis, and these pathways may occur simultaneously at the same level or may interact to further exacerbate β-cell death in the form of superimposed harmful stress. In addition, some non-coding RNAs, such as Bcl-2, Ago, MafA, Pdx1, MTPN, and caspase-3, etc., also play an important role in β-cell apoptosis in T2DM patients by regulating the expression of key β-cell factors (You et al., 2022). β-cell apoptosis is mediated by a series of cascade mechanisms of the cysteine protease family, and current studies on hyperglycemia-induced β-cell apoptosis have focused on the balance between the pro-apoptotic Bcl-2 proteins (Bad, Bid, Bik, and Bax) and the anti-apoptotic Bcl family (Bcl-2 and Bcl-xl) (Tomita, 2016), and apoptosis occurs only when the concentration of the pro-apoptotic protein Bcl-2 on the mitochondrial membrane of the intrinsic pathway exceeds the concentration of the anti-apoptotic protein. In patients with T2DM, where islet neogenesis and β-cell replication are normal, increased apoptosis is the main cause of the decrease in their β-cell numbers, so preventing β-cell apoptosis may become an effective means of treating T2DM (Butler et al., 2003).

GLP-1 has a beneficial effect on β-cell function by promoting β-cell proliferation and neogenesis and reducing apoptosis (Zheng et al., 2022). GLP-1 has anti-apoptotic effects in vivo, and its promotion of cells expressing the GLP-1 receptor directly resists β-cell apoptosis (Drucker, 2003). In addition, GLP-1 increases the activity of protein kinase C (PKC) in islet β cells in a dose-dependent manner, promotes pancreatic β-cell survival, and inhibits mitochondria-dependent apoptosis by regulating Bcl-2/Bax expression levels (Zhang et al., 2015). When GLP-1 binds to GLP-1R on the surface of β-cells, cAMP-dependent PKA is activated and subsequently activates the downstream signal PI3K, which inhibits apoptosis in islet β-cells by blocking the expression of the pro-apoptotic protein Bax (Li et al., 2021) (Figure 4). IKKε phosphorylates targets in the NF-κB signaling pathway, thereby activating the NF-κB pathway. NF-κB is an important nuclear transcription factor, which can promote the regulation of inflammation and apoptosis. The expression levels of IL-6, IKK-ε and NF-κB were elevated in pancreatic cells from T2DM rats, accompanied by increased Bax expression and decreased Bcl-2 expression, as well as decreased pancreatic β-cell function; The GLP-1 receptor agonist liraglutide was able to reduce Bax expression in pancreatic cells from T2DM rats, and Bcl-2 expression was increased, islet tissue structure was improved, insulin-positive expression was increased, and β-cell apoptosis was reduced (Liu et al., 2021). Liraglutide attenuates the inflammatory response by inhibiting IKK-ε/NF-κB, thereby reducing islet β-cell apoptosis.

Considered an anti-insulin hormone, 2022 marks the 100th anniversary of the discovery of glucagon, which is produced in islet α cells through the processing of prohormone-converting enzyme 2 (Pcsk2) and acts through glucagon receptor (GCGR) (Hayashi, 2011). Under normal physiological conditions, glucagon secreted by pancreatic islet α cells plays an important role in maintaining blood glucose homeostasis by regulating hepatic glucose production. Fasting and postprandial hyperglucagonemia in T2DM patients stimulates glucose production in the liver, leading to hyperglycemia. Inhibition of glucagon secretion or antagonism of glucagon receptors has become a potentially effective treatment strategy for T2DM patients (Lund et al., 2014). Both glucagon and GLP-1 receptors belong to the GPCR superfamily. Glucagon acts in the liver via cAMP (Abbas et al., 2020), and glucagon is secreted by α cells as an immediate response to glucose reduction. Compared with the saline group, the physiological dose of GLP-1 injected during the hyperglycemic clamp can significantly reduce the glucagon level in post-transplantation diabetic patients, suggesting that GLP-1 has a clear glucagon inhibitory effect and can restore the altered insulin and glucagon secretion in post-transplantation diabetic patients in a glucose-dependent manner (Halden et al., 2016). Giving exogenous GLP-1 analogues or increasing endogenous GLP-1 levels can enhance the signaling of GLP-1R, and the activation of GLP-1R can effectively inhibit glucagon secretion in human body. The inhibitory effect of GLP-1 on glucagon secretion in vivo was observed only when the blood glucose level was equal to or higher than the fasting level, at higher blood glucose levels, the inhibition of glucagon gradually increased (Holst, 2007), and when the blood glucose concentration was less than 3.7 m mol/L, GLP-1 lost its effect on inhibiting glucagon secretion (Nauck et al., 2002). GLP-1R expression on α cells is low. In pancreas, the main expression sites of GLP-1R are located in pancreatic β cells and δ cells (Richards et al., 2014), and the expression of GLP-1R in α cells was about 0.2% of that in β cells (De Marinis et al., 2010), or even absent (Tornehave et al., 2008). Therefore, indirect regulation, via β- or δ-cell products has been thought to be the primary mechanism by which GLP-1 inhibits glucagon secretion (Gromada et al., 2007; Christensen et al., 2011; Müller et al., 2019; Davis and Sandoval, 2020). However, GLP-1 can activate GCGR in α cells, and GCGR is able to reduce the number of docking granules by coupling and activating inhibitory GTP binding protein (Gi/o), thus inhibiting the secretion of glucagon, which is independent of GLP-1R (Gandasi et al., 2024). The mechanisms of GLP-1 inhibiting glucagon secretion are shown in Figure 5.

GLP-1RAs has been shown to delay gastric emptying (Jelsing et al., 2012), thereby extending the retention of food in the stomach and small intestine and reducing postprandial glucose levels (Flint et al., 2011). GLP-1 is an energy sensor that reduces intestinal motility, prolonging the retention of food in the gut, contributing to more efficient absorption of nutrients and better bioavailability (Winkler et al., 2019). GLP-1 infusion slows gastric emptying of solid and liquid components of food and alters the distribution of food in the stomach so that a greater proportion of food is retained in the distal stomach (Marathe et al., 2011). Even intravenous administration of GLP-1 at a physiological postprandial dose of 0.3 pmol/kg/min significantly slowed the rate of gastric emptying in healthy subjects, bringing them into a state of "gastroparesis" (Little et al., 2006). Exogenous GLP-1 improves gastric compliance in a dose-dependent manner (Schirra et al., 2002), reduces the frequency and amplitude of gastric antrum and duodenum contraction, inhibits the contractile force of gastric antrum and duodenum, increases pyloric tension, and stimulates pyloric tetanic and phase motion (Schirra et al., 2000). Endogenous GLP-1 affects postprandial blood glucose by slowing gastric emptying and glucose absorption in healthy subjects (Deane et al., 2010) by inhibiting antral and duodenal motility and stimulating pyloric motility (Schirra et al., 2006). Other studies (Delgado-Aros et al., 2002) have shown that GLP-1 can increase fasting and postprandial stomach capacity and delay gastric emptying by inhibiting the cholinergic function of vagus nerve, without inducing postprandial symptoms. GLP-1 also helps with ileal braking, a circuit that regulates the rate of gastric emptying so that the rate of nutrients entering the duodenum is balanced with that of nutrients in the upper small intestine. If the flow of nutrients from the stomach is too fast, duodenum and jejunum absorption is incomplete, and the increased nutrient load in the ileum triggers increased GLP-1 and PYY secretion, which in turn feeds back regulation and further slows gastric emptying, thus restoring balance to the system. To sum up, the potential mechanisms of delayed gastric emptying by GLP-1 are mainly related to: (1) decreasing the contractile force of the gastric antrum and duodenum and increasing the pyloric tension (Schirra et al., 2000; Schirra et al., 2006); (2) inhibiting the cholinergic function of the vagus nerve (Delgado-Aros et al., 2002); (3) ileal braking (Layer et al., 1995; Holst, 2022).

Appetite and weight regulation are controlled by the central nervous system in a fairly complex manner, with the human brain playing a central role in integrating internal and external inputs and regulating energy homeostasis (Farr et al., 2016a). Farr et al. first demonstrated that GLP-1 receptors exist in the parietal cortex, hypothalamus and medulla of the human brain and are involved in regulating food intake (Farr et al., 2016b). GLP1-RAs is clinically used to treat T2DM and promote weight loss in obese individuals. Liraglutide can enter specific brain regions associated with appetite regulation, electrophysiological measurements of mouse brain sections showed that GLP-1 directly stimulated promelanocortin (POMC)/cocaine and amphetamine regulated transcript (CART) neurons and indirectly inhibited neurotransmission of neuropeptide Y (NPY) and agouti-related peptide (AgRP) in arcuate nucleus neurons through GABA-dependent signal transduction. GLP-1R on arcuate nucleus neurons expressing POMC/CART can mediate Liraglutide-induced weight loss (Secher et al., 2014). GLP-1RAs directly stimulate POMC/CART neurons and indirectly inhibit NPY and AgRP to increase measures of satiety and decrease hunger, resulting in reduced energy intake, thereby facilitating weight loss (Ard et al., 2021) (Figure 6). Consumption of high-calorie, high-sugar and high-fat foods is conducive to the development and maintenance of obesity (Berthoud and Zheng, 2012). Obese subjects were given liraglutide 3 mg once a day for 16 weeks. Compared with placebo group, liraglutide increased subjects' satiety, reduced intake of sweet, salty and high-fat foods, and increased plasma PYY level after meals. As well as reduced fat storage throughout the body, trunk, and upper and lower body, these results suggest that liraglutide has a central role in altering appetite cravings (Kadouh et al., 2020). GLP-1RAs affects the central nervous system, including reducing appetite, improving diet control, increasing satiety, reducing calorie intake, etc. In general, GLP-1RAs provides a highly effective and well-tolerated treatment regimen to help obese patients lose weight while improving weight-related complications (Ard et al., 2021).