Glucagon-like peptide-1 (GLP-1) and glucagon-like peptide-2 (GLP-2) are related intestinal L-cell derived secretory products. GLP-1 has been extensively studied in terms of its influence on metabolism, but less attention has been devoted to GLP-2 in this regard. The current study compares the effects of these proglucagon-derived peptides on pancreatic beta-cell function, as well as on glucose tolerance and appetite. The insulin secretory effects of GLP-1 and GLP-2 (10−12–10−6 M) were investigated in BRIN-BD11 beta-cells as well as isolated mouse islets, with the impact of test peptides (10 nM) on real-time cytosolic cAMP levels further evaluated in mouse islets. The impact of both peptides (10−8–10−6 M) on beta-cell growth and survival was also studied in BRIN BD11 cells. Acute in vivo (peptides administered at 25 nmol/kg) glucose homeostatic and appetite suppressive actions were then examined in healthy mice. GLP-1, but not GLP-2, concentration dependently augmented insulin secretion from BRIN-BD11 cells, with similar observations made in isolated murine islets. In addition, GLP-1 substantially increased [cAMP]cyt in islet cells and was significantly more prominent than GLP-2 in this regard. Both GLP-1 and GLP-2 promoted beta-cell proliferation and protected against cytokine-induced apoptosis. In overnight fasted healthy mice, as well as mice trained to eat for 3 h per day, the administration of GLP-1 or GLP-2 suppressed appetite. When injected conjointly with glucose, both peptides improved glucose disposal, which was associated with enhanced glucose-stimulated insulin secretion by GLP-1, but not GLP-2. To conclude, the impact of GLP-1 and GLP-2 on insulin secretion is divergent, but the effects of beta-cell signaling and overall health are similar. Moreover, the peripheral administration of either hormone in rodents results in comparable positive effects on blood glucose levels and appetite.
Glucagon-like peptide-1 (GLP-1) and glucagon-like peptide-2 (GLP-2) are peptide hormones derived from the same precursor gene, known as proglucagon, and co-secreted in equimolar concentrations by enteroendocrine L-cells in response to nutrient ingestion. The proglucagon gene product undergoes tissue-specific post-translational processing to yield different bioactive peptides, including GLP-1 and GLP-2. Thus, in the intestine and brain, proglucagon is processed by prohormone convertase 1/3 (PC1/3) at Arg-Arg sites to yield glicentin, oxyntomodulin (OXM), intervening peptide-2 (IP-2), GLP-1 and GLP-2. Conversely, in alpha-cells of the endocrine pancreas, proglucagon is cleaved by PC2 to generate glicentin-related pancreatic polypeptide (GRPP) intervening peptide-1 (IP-1), the major proglucagon fragment (MPGF) and glucagon. Whilst there is evidence of islet synthesis and the secretion of GLP-1 and GLP-2 under conditions of islet stress, these hormones are still largely considered as intestinal-derived peptides.
To date, there is a plethora of literature relating to the metabolic benefits of GLP-1 receptor activation, highlighted by the clinical approval of GLP-1 mimetics for both type 2 diabetes and obesity. Thus, the positive modulation of GLP-1 receptor signaling leads to an enhancement of glucose-stimulated insulin secretion (GSIS) from pancreatic beta-cells, together with the inhibition of glucagon secretion from alpha-cells, the slowing of gastric emptying and the promotion of satiety. On the other hand, GLP-2 receptor activation has been shown to exert benefits on intestinal growth and repair through the promotion of epithelial proliferation, leading to the clinical application of GLP-2 drugs for short bowel syndrome. Despite the structural similarities of GLP-1 and GLP-2, alongside the fact that both hormones are secreted in response to nutrient ingestion, there is a lack of information about the possible metabolic benefits of GLP-2. However, GLP-2 may have the potential to protect against the dysregulation of glucose metabolism, as well as positively modulate energy homeostasis.
Therefore, the present study directly compares the impact of GLP-1 and GLP-2 on pancreatic BRIN BD11 beta-cell proliferation, survival and overall secretory function. The effects of both peptides on insulin secretion were then verified in isolated murine islets, along with their influence on islet cell cytosolic cAMP concentrations. In addition, we investigated the effects of the intraperitoneal injection of GLP-1 and GLP-2 on glucose tolerance and GSIS in mice. Finally, the influence of both peptides on appetite regulation in mice fasted overnight, as well as mice trained to eat for 3 h per day, was examined. Overall, the data further emphasize the prominent anti-diabetic and -obesity effects of GLP-1 receptor signaling. Furthermore, we also reveal, for the first time, the positive impact of GLP-2 receptor signaling on islet cell cAMP levels, as well as beta-cell turnover, meriting further investigation in terms of therapeutic strategies for diabetes.
Peptides (95% purity) were obtained from a commercial source (Synpeptide, Shanghai, China) and fully characterized in our laboratory, as previously described.
The BRIN-BD11 beta-cell line was used to examine the insulin secretory actions of the test peptides (n = 8; 20 min incubation; 10−12–10−6 M) at 5.6 and 16.7 mM of glucose, as described previously. Furthermore, the impact of the peptides on insulin secretion (n = 4; 60 min incubation; 10−8 and 10−6 M) was also examined in an islet isolated from 12-week-old C57BL/6 mice by collagenase digestion. The subsequent acid–ethanol extraction of test islets allowed for the presentation of islet secretion data as a percentage of the islet insulin content. Samples were kept at −20 °C prior to insulin determination using an in-house radioimmunoassay.
C57BL/6 male mouse (12 weeks old) islets were isolated as above. To quantify the cytosolic cAMP levels, a recombinant fluorescent sensor (Upward Green cADDis, Montana Molecular, Bozeman, MT, USA) was used. The sensor was delivered to the islets via adenoviral transduction, allowing 24–48 h for gene expression. Time-lapse imaging of [cAMP]cyt was performed using the Green Upward cADDis sensor, as described previously, with image acquisition managed using μManager 2.0 software, capturing the cAMP levels in the islets every 60 s (16 mHz). For imaging, a bath perfusion solution (140 mM of NaCl, 4.6 mM of KCl, 2.6 mM of CaCl 2, 1.2 mM of MgCl 2, 1 mM of NaH 2 PO 4, 5 mM of NaHCO 3, 10 mM of glucose, 10 mM of HEPES, pH 7.4) was used, along with GLP-1, GLP-2 (both at 10 nM) or IBMX (100 µM) as a positive control. Image sequences were analyzed with the use of the open-source FIJI software version 2.9.0 (National Institutes of Health (NIH), Bethesda, MD, USA).
The impact of GLP-1 and GLP-2 on BRIN-BD11 beta-cell proliferation (40,000 cells per well) was assessed using the Ki67 primary antibody (Ab15580, AbCam, Cambridge, UK) and the Alexa Fluor® 594 secondary antibody, as described previously in our laboratory. For apoptosis studies, cellular stress was induced by the incubation of BRIN BD11 beta-cells with a cytokine cocktail (IL-1β 100 U/mL, IFN-γ 20 U/mL, TNF-α 200 U/mL), and the rate of apoptosis was monitored through TUNEL staining (Fluorescein, Roche Diagnostics, Burgess Hill, UK). The effects were visualized using a fluorescence microscope (Olympus system microscope, model BX51; Southend-on-Sea, UK) and a DP70 camera adapter system using DAPI (350 nm), TRITC (594 nm) and FITC (488 nm) filters, alongside an Olympus XM10 camera. For quantification, the cell-counter function within ImageJ Software Version 1.54 (National Institutes of Health (NIH), Bethesda, MD, USA) was employed to establish the number of positively stained Ki-67 or TUNEL cells, as appropriate; the data were then presented as a percentage of the total cells investigated.
Animal experiments were performed in male C57BL/6 (12–14 weeks of age) or NIH Swiss male mice (30 weeks of age), as appropriate; both were purchased from Harlan Ltd., Huntingdon, UK. Animals were housed individually in the Biomedical and Behavioural Research Unit (BBRU) at Ulster University for pre-clinical studies, with a standard temperature and light cycle, namely 22 ± 2 °C and a 12 h light/dark cycle, respectively. All procedures were performed in compliance with the UK Animal Scientific Procedures Act 1986.
The effects of GLP-1 or GLP-2 (25 nmol/kg bw, an intraperitoneal (i.p.) administration) on food intake, glucose homeostasis and insulin secretion were studied in overnight fasted C57BL/6 mice, as previously documented in our laboratory. The dosing regimen employed for the test peptides was based on previous positive observations with GLP-1 and GLP-2, as well as related gut-derived peptide hormones, within the same experimental systems. In a separate series, NIH male mice habituated to a daily feeding regime of 3 h/day were also used to evaluate the impact of GLP-1 and GLP-2 (25 nmol/kg bw) on the cumulative food intake, using the same protocol as described above. These mice were subject to a progressive reduction in the daily feeding period over 3 weeks at 12 weeks of age, as detailed previously. Mice were maintained on this 3 h/day feeding regimen until 30 weeks of age, and experiments were then conducted.
Blood glucose levels were quantified using a blood glucose meter (Ascencia Contour; Bayer Healthcare, Berkshire, UK). For plasma insulin analysis, blood samples were collected into chilled fluoride/heparin glucose micro-centrifuge tubes (Sarstedt, Numbrecht, Germany), immediately centrifuged at 13,000× g for 1 min, and retained at −20 °C before insulin quantification by radioimmunoassay.
Statistical analyses were conducted using GraphPad PRISM software (Version 8.0, Irvine, CA, USA). Data are presented as mean ± S.E.M. Comparative analyses between groups were performed using one-way ANOVA with a Bonferroni post hoc test or Student’s unpaired t-test, as appropriate. Statistical significance was defined as p< 0.05.
At 5.6 and 16.7 mM of glucose, GLP-1 (10⁻10–10⁻6 M) significantly (p< 0.05–0.001) augmented insulin secretion when compared to the controls. Conversely, GLP-2 did not impact insulin release from BRIN BD11 cells at any of the concentrations tested at either 5.6 or 16.7 mM glucose.
