Abstract
Objective
Combination therapies with gut hormone analogs represent promising treatment strategies for obesity. This pilot study investigates the therapeutic potential of modulators of the glucagon-like peptide 1 (GLP-1) and neuropeptide Y (NPY) system using GLP-1 receptor agonists (semaglutide) and antagonists (exendin 9-39), as well as non-selective and NPY-Y2-receptor selective peptide tyrosine tyrosine (PYY) analogs (PYY3-36/NNC0165-0020 and NNC0165-1273) and an NPY-Y2 receptor antagonist (JNJ31020028).
Methods
High-fat diet (HFD)-induced obese rats were randomized into following treatment groups: group 1, nonselective PYY analog + semaglutide (n = 4); group 2, non-selective and NPY-Y2 receptor selective PYY analog + semaglutide (n = 2); group 3, GLP-1 receptor antagonist + NPY-Y2 receptor antagonist (n = 3); group 4, semaglutide (n = 5); and group 5, control (n = 5). Animals had free access to HFD and low-fat diet. Food intake, HFD preference and body weight were measured daily.
Results
A combinatory treatment with a non-selective PYY analog and semaglutide led to a maximum body weight loss of 14.0 ± 4.9% vs 9.9 ± 1.5% with semaglutide alone. Group 2 showed a maximum weight loss of 20.5 ± 2.4%. While HFD preference was decreased in group 2, a strong increase in HFD preference was detected in group 3.
Conclusions
PYY analogs (especially NPY-Y2 selective receptor agonists) could represent a promising therapeutic approach for obesity in combination with GLP-1 receptor agonists. Additionally, combined GLP-1 and PYY3-36 receptor agonists might have beneficial effects on food preference.
Introduction
The prevalence of obesity has almost tripled since 1975, with almost two-thirds of the U.S. population having excess body weight (1, 2). The obesity pandemic is particularly devastating in the western hemisphere, with high and continuously rising health-care costs (3). Being overweight correlates with an increased risk of various serious comorbidities associated with a significantly shortened life expectancy (4, 5, 6, 7). Non-invasive treatment options with adequate success are limited. In contrast, bariatric surgery, especially Roux-en-Y gastric bypass (RYGB), is associated with a pronounced reduction in body weight, due to a reduction of energy intake of up to 50% (8, 9, 10, 11, 12, 13, 14). Most likely, the success of RYGB lies in changes of gut hormone levels relevant for food intake behavior. Elevated levels of glucagon-like peptide 1 (GLP-1), peptide tyrosine tyrosine (PYY), glucose-dependent insulinotropic polypeptide (GIP) and oxyntomodulin were found following bariatric surgery (15, 16, 17, 18).
With the rising prevalence of obesity, the need for effective non-invasive treatment options is of highest priority. Gut hormones and peptides and their hypothalamic actions in appetite regulation, as well as additional peripheral mechanisms like a delay in gastric emptying, hold promise for effective therapeutic approaches. GLP-1 is mainly involved in central appetite regulation, pancreatic function, energy balance and gastric motility (19, 20). Long-acting GLP-1 receptor (GLP-1-R) agonists, e.g. semaglutide, are already in clinical use for the treatment of obesity. In adults with overweight or obesity and without diabetes mellitus, semaglutide leads to a mean weight loss of about 10% compared to placebo (21).
PYY, structurally related to neuropeptide Y, is co-secreted with proglucagon-derived peptides like GLP1 from L cells in the lower gut. Serum levels of PYY increase after food intake (22). Following secretion, PYY is rapidly cleaved by dipeptidyl peptidase 4 into the major circulating and active form PYY3-36 (23). PYY3-36 mediates satiety and influences energy homeostasis. It has a high affinity to Y2 receptors (Y2-R), lower affinity to Y1-R and Y4-R and a 10-fold lower selectivity for Y5-R (24, 25). Injection of PYY3-36 into the arcuate nucleus of the hypothalamus leads to a reduction of food intake in rodents (26, 27). Additionally, PYY knockout mice developed larger subcutaneous and visceral fat depots (28). In humans some studies indicate an association of obesity and lower fasting PYY levels, and an attenuated meal stimulated PYY response (28, 29, 30). One study also showed that obese subjects have to consume more calories to reach PYY levels comparable to those seen in nonobese subjects (30). Exogenous PYY3-36 infusions reduce food intake and increase energy expenditure in lean and obese humans (31, 32). In a previous study with diet-induced obese rats, a combinatory treatment with PYY3-36 and the GLP-1-R agonist liraglutide led to a similar and plateaued weight loss as RYGB and was more effective than monotherapy with PYY3-36 or liraglutide. Additionally, the combinatory treatment led to a reduced overall food intake and a decreased high-fat diet (HFD) preference, again mimicking the effects of RYGB (33).
NNC0165-0020 (NNC-20) is a physiologically acting non-selective PYY analog (25). NNC0165-1273 (NNC-1273) is a modified form of PYY with high selectivity for the Y2-R (>5000-fold over Y1-R, 1250-fold over Y4-R, and 650-fold over Y5-R). The application of NNC-1273 resulted in a reduction in nighttime feeding at a dose, at which PYY3-36 loses efficacy and decreased body weight in obese mice dose dependently (25, 34).
In the present pilot study with male Wistar rats, a treatment with non-selective PYY analogs (PYY3-36 or NNC-20) in combination with semaglutide was compared to semaglutide alone. In an additional treatment group, a combination of a selective Y2-R agonist (NNC-1273) and, to avoid excessive and possibly life-threatening body weight loss, a non-selective PYY3-36 analog (NNC-20) was used. Unlike on NNC-20, there is no prior data on the Y2-R selective agonist NNC-1273 regarding effects on body weight in long-term use. To further investigate the GLP-1 and NPY-system, rats of an additional group were treated with the Y2-R antagonists JNJ-31020028 (JNJ) and the GLP-1-R antagonist exendin(939) amide (exendin 9-39) (35, 36). JNJ is a brain penetrant antagonist that binds to Y2 receptors in humans and rats with high affinity (36). Exendin 9-39 is a selective competitive antagonist of the GLP1R (35, 37). The combination of both increased HFD preference additively in RYGB-operated rats (38). However, their long-term effects on eating behavior and body weight have not been reported so far. Levels of GLP-1 and PYY were measured with enzyme-linked immunosorbent assays (ELISA) to show treatment efficiency. Levels of ALT, AST, cholesterol, triglycerides and glucose were measured to show possible effects on liver health, lipid- and glucose metabolism.
The approach to use agonists and antagonists may help to further scrutinize the relevance of GLP-1-R and Y2-R related pathways for the regulation of food intake behavior and body weight.
Materials and methods
Peptides
PYY3-36 (Cayman Chemical) and NNC-0020 (NNC0165-0020, Novo Nordisk) are physiologically acting PYY3-36 analogs (Fig. 1A and Table 1).
Two-dimensional molecular structure of (A) the sequence of native PYY3-36 (NNC0165-0020 (Novo Nordisk), PYY (Cayman Chemical)) and (B) the sequence of NNC0165-1273 (Novo Nordisk) (structure from Novo Nordisk’s data sheet) and (C) JNJ-31020028 (36). Image created with ChemDraw®.
Citation: Endocrine Connections 13, 3; 10.1530/EC-23-0398
Calculated properties for NNC0165-1273 and PYY3-36 (NNC0165-0020) (data from Novo Nordisk’s data sheet and (25)).
| Compound | Y1 | Y2 | Y4 | Y5 |
|---|---|---|---|---|
| Receptor binding data (Ki (nM)) | ||||
| PYY3-36 (NNC0165-0020) | 40 | 0.40 | 13 | 3.2 |
| NNC0165-1273 | 1000 | 2.0 | 2500 | 1300 |
| Potency data (EC50 (nM)) | ||||
| PYY3-36 | 16 | 1.0 | >30 | 7.9 |
| NNC0165-1273 | >1000 | 5.0 | >1000 | 790 |
NNC-1273 (NNC0165-1273, Novo Nordisk) is a modified PYY peptide with increased selectivity for Y2R compared to the other Y receptors. It is modified in C-terminal position 35 (30 tryptophan and 35 beta-homo-arginine as sequence substitution), which results in a strongly improved selectivity for the Y2-R and stabilization against C-terminal degradation (Fig. 1B). In vitro metabolism studies showed a half-life of 570 min compared to 200 min for PYY3-36 (substance 29 in (25)). Table 1 shows the inhibitory constant (Ki) of NNC-1273 and physiologically active PYY3-36 for the different Y receptors. NNC-1273 has a Ki for of 2 nM for the Y2 receptor and >10000, 2500 and 1300 nM for Y1, Y4 and Y5 receptors in binding assays. This leads to a more than 250-, 192- and 400-fold selectivity for the Y1, Y4 and Y5 receptors.
Figure 1C shows the molecular structure of JNJ (JNJ-31020028, Janssen, Titusville, NJ, USA), a selective brain penetrant small molecule antagonist of the NPY Y2 receptor. Receptor binding assays demonstrated that JNJ competed with high affinity at PYY receptor binding sites (Hill slope = 1). It showed no significant affinity in a panel of 50 different receptor, ion channel and transporter assays and did not inhibit any kinases in a panel comprised of 65 kinases at concentrations up to 10 μM. In a calcium mobilization assay the antagonist did not stimulate a calcium response itself and inhibited PYY-induced calcium response (pKB = 7.8 ± 0.1) (36).
Exendin 9-39 (exendin (9-39) amide, Abcam) is a potent, selective GLP-1 receptor antagonist (Kd = 1.7 nM), that inhibits the generation of intracellular cAMP induced by GLP-1 (37). It reduces dose-dependently the insulinotropic actions of GLP-1 and shows no agonistic properties. At 300 pmol kg−1 min−1 it blocks the insulinotropic effects of physiological doses of GLP-1 (35).
Animals
The study design is illustrated in Fig. 2. Male Wistar rats (Charles River Laboratories, n = 19) aged about 6 weeks, weighing 264 ± 27 g were initially group-housed at an ambient room temperature of 22°C and a 12-h light/12-h darkness cycle. To induce obesity, the animals had free access to a HFD (D12451 HF diet from Ssniff, Germany, 4615 kcal/kg; 45 kJ% fat, 20 kJ% protein, 35 kJ% carbohydrates) for 8 weeks. Thereafter, animals (mean body weight 542 ± 13 g) were randomized into the following treatment groups. Group 1 (n = 4) received non-selective PYY3-36 analogs (0.015 mol/kg/day, PYY: Cayman Chemical or NNC-20: Novo Nordisk, depending on availability) via osmotic mini-pump in combination with semaglutide s.c. (120 μg/kg/day, Novo Nordisk). Animals of group 2 (n = 2) received the non-selective PYY3-36 analog NNC-20 (0.04 µmol/kg/d) and the Y2-R selective PYY analog NNC-1273 (0.04 µmol/kg/d) via osmotic minipump and semaglutide s.c. (120 μg/kg/day). Animals of group 3 (n = 3) received a combination of JNJ-31020028 (2.5 mg/kg/day) and exendin 9-39 (30 µg/kg/day,), both via osmotic minipumps. Animals of group 4 (n = 5) received a monotherapy with semaglutide s.c. (120 μg/kg/day). The control group (n = 5) underwent sham surgery for minipump implantation and received a saline treatment. Subcutaneous injections were administered at about 09:00 h. To minimize adverse effects, semaglutide was dosed up over 4 days at the beginning of the treatment period.
Overview of the experiment and the different treatment groups. (A) Over a period of 8 weeks, all animals received a high-fat diet to induce obesity. Thereafter, the rats were randomized into different treatment groups. During the treatment phase, the animals had free access to high- and low-fat diet. Data on food preference and body weight was collected in the first 6 weeks of the treatment phase (’observation period’). After 8 weeks of treatment, blood was collected for further analysis. (B) Animals were randomized into five groups. Group 1 (n = 4) received a treatment with a non-selective PYY analog (PYY3-36 or NNC0165-0020) via subcutaneous osmotic minipump and semaglutide s.c.. Group 2 (n = 2) received semaglutide and a non-selective PYY analog (NNC0165-0020), as well as the selective Y2-R agonist NNC0165-1273 via subcutaneous osmotic minipump. Group 3 (n = 3) received the Y2-R antagonist JNJ-31020028 and the GLP-1-R antagonist exendin 9-39 via intraperitoneal osmotic minipumps. Group 4 (n = 5) received semaglutide s.c. only. Group 5 (n = 5, control group) received a saline treatment via subcutaneous osmotic minipump. Image created with BioRender.com.
Citation: Endocrine Connections 13, 3; 10.1530/EC-23-0398
For the administration of PYY3-36, NNC-1273 and NNC-20, osmotic minipumps (pump model 2006, purchased from ALZET, Cupertino, CA, USA) with a delivery rate of at least 6 weeks were implanted subcutaneously into the interscapular region of the animals under isoflurane anesthesia (3–5% at 1.8 L/min) with buprenorphine (0.05 mg/kg) and carprofen (5 mg/kg) analgesia. JNJ and exendin 9-39 were administered via osmotic minipumps implanted intraperitoneally under the same regimen. Drugs were diluted following their solubility properties: PYY3-36, NNC-1273 and NNC-20 were diluted with 0.9% saline, whereas JNJ and exendin 9-39 were diluted with 50% DMSO.
Under therapy, animals were single housed for another 8 weeks and had free access to HFD and low-fat diet (LFD; D12450J LF, Ssniff, 3630 kcal/kg; 10 kJ% fat, 20 kJ% protein, 70 kJ% carbohydrates). The body weights were monitored for 6 weeks on a daily basis in all groups. Food intake and preference were monitored in groups 2, 3 and 5 over 6 weeks on a daily basis. Based on the daily consumed grams of each diet, the average calories per gram of the diets were used to calculate the daily kilocalories (kcal) consumed. HFD preference was calculated by dividing the daily calories consumed from the HFD by the total daily calories consumed (HFD and LFD).
The animals were screened daily for severe adverse effects with a special focus on breathing, signs of pain, seizures, micturition and defecation.
All animal procedures were approved by the local regulatory authority (Regierung von Unterfranken, Würzburg, Germany, AZ-RUF-55.2.2-2532-2-1412). All experiments were performed following German and European laws and regulations (TierSchG, TierSchVersV, Directive 2010/63/EU).
Enzyme-linked immunosorbent assay
Blood samples (pretreated with DPP-IV inhibitor (Merck)) were taken directly from the abdominal aorta at the end of the experimental period. Serum levels of GLP-1 (EK-028-11, Phoenix Pharmaceuticals, Burlingame, CA, USA) and PYY (EK-059-02, Phoenix Pharmaceuticals) were measured using ELISA. Glucose was measured with handheld measuring devices. Measurements of aspartate transaminase (AST) alanine transaminase (ALT), triglyceride (TG) and cholesterol (CHOL) were performed in an external laboratory (Laboklin, Bad Kissingen, Germany) with a cobas® 8000 modular analyzer (Roche Deutschland Holding GmbH).
Statistical analysis
Statistical analysis was performed using GraphPad Prism (Version 10.0.1) software. Kolmogorov–Smirnov test was used to test for normality. Two-way ANOVA with Tukey’s post hoc comparison test was used for statistical analysis of changes in body weight over time, food intake and food preference. For the statistical analysis of the blood parameter results and the comparison of starting weights of the different treatment groups, an ordinary one-way ANOVA with Tukey’s multiple comparison test was used. Data are presented as mean with standard error of the mean. P values of ≤ 0.05 were considered statistically significant. For group 2, only descriptive statistics were performed due to the small sample size.
Results
The combination of a non-selective or selective Y2 receptor agonist and semaglutide leads to a sustained body weight loss
Figure 3 shows the weight course in weekly intervals within the delivery time of the osmotic minipumps (42 days) for all treatment groups. At the time of treatment initiation, all treatment groups (starting at different time points) had comparable body weights (567 ± 48 g for group 1, 576 ± 6 g for group 2, 474 ± 10 g for group 3, 559 ± 69 g for group 4 and 533 ± 42 g for the control group, P = 0.14). Animals of the control group gained weight continuously and ended up with a 14.4 ± 1.0% higher body weight compared to their initial weight. Semaglutide treated animals (group 4) lost a maximum of 9.9 ± 1.5% of their initial body weight in week 2 but regained weight thereafter. At the end of the observation period, semaglutide s.c. treated animals were 16.0 ± 2.0% lighter than the control group (P ≤ 0.001). Animals treated with non-selective PYY3-36 analogs and semaglutide (group 1) lost 14.0 ± 4.9% of their initial body weight until week 2 and ended up 16.7 ± 3.4% lighter than the control group (P ≤ 0.05). Animals of group 1 ended up 0.8 ± 3.7% lighter than the semaglutide only group (P = 0.1). Animals, that were co-treated with a non-selective and a Y2-R selective PYY analog and semaglutide (group 2) showed the most pronounced reduction of their initial body weight (20.5 ± 2.4%) in week 2, regained weight afterward and plateaued at week 5. They ended up 25.3 ± 4.0% lighter than the control group and 9.3 ± 4.3% lighter than animals of group 4 at the end of the observation period. The mean body weight loss from baseline at the end of the observation period was 10.8 ± 3.9% in group 2 and 2.2 ± 3.3% in group 1 (mean difference: 8.6 ± 5.1%, P = 0.56).
Weekly body weight change from baseline (in %) of the different treatment groups. The data of the two-way ANOVA with Tukey’s post hoc test are presented as mean ± standard error of the mean. Animals of group 2 are shown individually. Mean body weight of group 1 (n = 4) differed significantly from the control group (n = 5) in week 1, 5 and 6 (*P ≤ 0.05). Mean body weight of group 4 (n = 5) differed significantly from the control group at week 1 (P ≤ 0.05), week two and three (P ≤ 0.01), and week 5–6 (P ≤ 0.001) (not indicated in the graph). Group 3 (n = 3) did not differ significantly from the control group. Group 2 was excluded from statistical tests due to its small sample size. Error bars that are not visible were too short to be mapped.
Citation: Endocrine Connections 13, 3; 10.1530/EC-23-0398
The body weight course of animals co-treated with the GLP-1-R and Y2R antagonists (group 3) did not differ significantly from the control group (P = 0.53), both groups gaining weight continuously.
The combination of an NPY-Y2-R selective PYY analog and semaglutide leads to reduced food intake and HFD preference, while their antagonists lead to an increased HFD preference
Co-treatment with semaglutide, a non-selective PYY3-36 analog and a selective Y2R agonist (group 2) led to a reduced overall food intake (in kcal) in the first 16 days of treatment (Fig. 4A/B in 4-day intervals). While the overall food intake increased over the first 12 days in all groups, it remained stable thereafter. Figure 4C and D shows the HFD preference in percent in 4-day intervals. Animals of group 2 had a lower (reduced by 43.8 ± 12.7%) HFD preference at the end of the observation period compared to the control group (P = 0.10 at day 4144).
Effects on overall food intake (A, B) and HFD preference (C, D) in different treatment groups calculated with a two-way ANOVA and Tukey’s post hoc test. (A) Food intake in kcal in 4-day intervals. (B) Food intake of the individual animals of group 2 and 3 in kcal in 4-day intervals. (C) Preference for HFD in percent in 4-day intervals. (D) Food preference of the individual animals of group 2 and 3 in percent in 4-day intervals. Data in (A) and (C) are presented as mean ± s.e.m. Group 2 was excluded from statistical tests due to its small sample size. Error bars that are not visible were too short to be mapped.
Citation: Endocrine Connections 13, 3; 10.1530/EC-23-0398
While there was no significant difference in overall calorie intake between the control group and the antagonistic treated animals (group 3), animals of group 3 consumed HFD almost exclusively over the whole treatment period (mean preference at all-time points: 99.2 ± 0.3%, P ≤ 0.05 compared to controls).
No severe adverse events were observed. Apart from one animal that showed temporary diarrhea at the beginning of treatment with semaglutide and the non-selective PYY3-36 analog, other general side effects such as pain, constipation or behavioral changes in daily interaction were not observed in the monitoring procedure.
Groups receiving semaglutide had significantly higher serum levels of GLP-1 after week 8
At 8 weeks, serum levels of PYY and GLP-1 were measured. Groups receiving semaglutide subcutaneously had higher GLP-1 levels compared to controls (P < 0.01, semaglutide: 2.02 ± 0.58 ng/mL, post hoc: P < 0.01; selective PYY analog + semaglutide: 2.30 ± 0.26 ng/mL; non-selective PYY + semaglutide 1.91 ± 0.20 ng/mL, post hoc: P = 0.01; control group: 0.91 ± 0.21 ng/mL) (see Fig. 5). Animals receiving NNC-20 or PYY via an osmotic minipump with a guaranteed delivery of 42 days showed no significant differences in PYY levels beyond this period at week 8 (P = 0.12, non-selective PYY analog + semaglutide: 0.66 ± 0.11 ng/mL, post hoc: P = 0.973; selective PYY analogue + semaglutide: 1.15 ± 0.71 ng/mL; control group: 0.58 ± 0.48 ng/mL).
Serum levels of (A) GLP-1 and (B) PYY after 8 weeks of treatment in ng/ml of group 1 (non-selective PYY + semaglutide (n = 3)), group 2 (Y2-receptor selective PYY analog + semaglutide (n = 2)), group 4 (semaglutide (n = 5)) and control (n = 5) calculated with one-way ANOVA. Data are presented as mean ± s.e.m . *P ≤ 0.05, **P ≤ 0.01. Group 2 was excluded from statistical tests due to its small sample size. Each dot represents an animal of the respective groups.
Citation: Endocrine Connections 13, 3; 10.1530/EC-23-0398
Treatment groups showed no significant differences in transaminase levels (ALT and AST), cholesterol and triglycerides
ANOVA of serum level ALT-levels (39.08 ± 3.89 U/L (group 1), 35.10 ± 1.20 U/L (group 2), 43.90 ± 5.74 U/L (group 3)), 58.68 ± 20.26 U/L (group 4) and 37.20 ± 1.87 U/L (control group)) and AST levels (91.88 ± 10.73 U/L (group 1), 120.30 ± 3.60 U/L (group 2), 117.40 ± 40.29 U/L (group 3), 202.8 ± 113.7 U/L (group 4) and 102.70 ± 3.65 U/L (control group)) revealed no significant difference between the treatment groups (ALT: P = 0.65, AST: P = 0.75). Furthermore, no significant difference was found regarding cholesterol (2.10 ± 0.12 mmol/L (group 1), 1.95 ± 0.15 mmol/L (group 2), 2.10 ± 0.30 (group 3), 2.46 ± 0.11 mmol/L (group 4) and 2.52 ± 0.15 mmol/L (control group), P = 0.13) and triglycerides (1.07 ± 0.50 mmol/L (group 1), 2.67 ± 2.28 mmol/L (group 2), 0.49 ± 0.23 mmol/L (group 3), 0.39 ± 0.12 mmol/L (group 4) and 0.66 ± 0.16 mmol/L (control group), P = 0.14) (Fig. 6).
Serum levels of (A) alanine aminotransferase (ALT), (B) aspartate aminotransferase (AST), (C) cholesterol and (D) triglycerides after 8 weeks of treatment in ng/mL of group 1 (non-selective PYY + semaglutide (n = 4)), group 2 (Y2-receptor selective PYY analog + semaglutide (n = 2)), group 3 (JNJ + exendin 9-39 (n = 3)), group 4 (semaglutide (n = 5)) and control group (n = 5) calculated with one-way ANOVA. Data are presented as mean ± s.e.m. Each dot represents an animal of the respective groups.
Citation: Endocrine Connections 13, 3; 10.1530/EC-23-0398
Treatment groups showed no significant differences in blood glucose levels
One-way ANOVA of serum glucose levels revealed no significant difference between the treatment groups (P = 0.18; semaglutide: 193.5 ± 26.38 mg/dL, selective PYY analog + semaglutide: 149.0 ± 1.0 mg/dL, nonselective PYY + semaglutide 152.6 ± 07.7 mg/dL, control group: 130.6 ± 19.5 ng/mL).
Discussion
Obesity and its comorbidities represent a global health crisis. The identification of effective non-invasive treatment options is urgently needed. It was shown before that PYY3-36 in a combination with the GLP1 receptor agonist liraglutide can lead to similar body weight loss as RYGB in a rat model of HFD-induced obesity (33). Since the combination of liraglutide and PYY3-36 has proven to be more effective than each single agent alone, an additive anorexigenic effect of these substances was suggested. In this pilot study, we assessed the combination of semaglutide, the currently most potent GLP-1 receptor agonist already in clinical use in the treatment of obesity, with unselective and Y2-R selective PYY analogs.
The combination of semaglutide and either PYY analog led to a significant weight loss compared to the control group and a more pronounced weight loss compared to a monotherapy with semaglutide. Although numbers were small, it was shown that the addition of a selective Y2-R agonist holds the potential to be even more effective, as this therapy group showed the strongest weight reducing effects. As the experimental group with the selective Y2 receptor agonist only included a small sample size, no statistical conclusions can be drawn yet, but when looking at the individual animals a similar body weight course in both treated animals, more pronounced than in the other groups, could be shown. It was apparent in all agonist treatment groups that the greatest weight loss occurred in the first 2 weeks. Subsequently, there was an increase in body weight in all treatment groups. However, all agonistic groups remained below baseline until the end of treatment, in contrast to the control and the antagonistic group. The effect that body weight is not maintained at the rodents' nadir but increases again over time, was observed before with PYY3-36 monotherapy (39, 40, 41), treatment with GLP-1 receptor agonists (33, 42), a combination of both (33) or even other obesity treatments (43). However, compared to the groups 1 and 4, both animals receiving an Y2-R selective agonist showed either a plateau or a weight loss at week 6. To evaluate tachyphylaxis, longer treatment periods are necessary. Previous studies showed that the direct injection of PYY3-36 into the hypothalamic arcuate nucleus, where Y2 receptors are highly expressed, inhibits food intake, while no effect could be detected when a Y2 receptor antagonist was given together with PYY3-36 (27, 44). However, intracerebroventricular injection of PYY3-36 in mice resulted in increased food intake by activating Y1 and Y5 receptors instead of Y2 (45, 46). Elevated postprandial levels of PYY3-36 and GLP-1 might also be a relevant factor for reduced food intake and healthier food choices following RYGB (38, 47). Similarly, exogenous administration of PYY3-36 and GLP-1 receptor agonists in earlier studies resulted in reduced food intake in both rodents and humans (30, 39, 40, 48, 49). In this study, the Y2-R selective agonist group presented with reduced food intake in the first 16 days only. Other studies also showed an only temporary effect on food intake under monotherapy with PYY3-36 analogs, where the effect only lasted for 3–7 days after the start of treatment (40, 41, 50, 51). The transient effect on feed intake could be due to receptor downregulation and tolerance (52) as well as redundancy and plasticity in the systems involved in the regulation of energy homeostasis (53). However, there is no increase in body weight after 2 weeks. This corresponds to an earlier study that showed a dose-dependent body weight reduction independent of food intake (39). One possible reason for this could be a reduction in the respiratory exchange rate, suggesting that increased lipid oxidation contributes to the weight-reducing effects of PYY3-36 (41, 50). Another reason could be healthier food choices. The combination of semaglutide and an Y2-R selective PYY analog led to a reduced HFD preference. This reduction was particularly prominent from day 12 on, at the time animal’s food intake increased again. The reduced high fat preference was evident in this group until the end of therapy and the healthier food choice could be another reason why PYY3-36 or the Y2 receptor exert an anorectic effect. Additionally, the treatment with JNJ and exendin 9-39, specifically blocking the action of Y2-R and GLP1 receptors in the brain, led to a permanent elevated high fat preference in all animals, clearly underlining that these two signaling pathways have an important influence on food preference. These results are in accordance with a previous study, which showed that PYY3-36 reduced the motivation to seek high-fat food after exposure to pellet priming or pellet cues, and that this effect could be wiped out by co-administration of a Y2-R antagonist (54).
With increasing knowledge of the gut–brain axis and the involved peptide hormones, the development of polyagonists therapies is only at its beginning and a co-administration of PYY3-36 and leptin (55) or PYY3-36 and amylin (56) may be further promising approaches, as these regimes already showed their weight-reducing potentials in rodent studies.
All groups receiving semaglutide (groups 1, 2 and 4) showed increased GLP-1 concentrations compared to the control group. PYY was only tendentially elevated in respective treatment groups. Since the guaranteed pump delivery time was exceeded at the time of blood sampling, this finding is not surprising.
GLP-1 receptor agonists are already known to have positive effects on blood pressure, glucose and lipids in addition to weight loss when used in humans (57, 58). PYY is also thought to play a role in lipid metabolism (29). Therefore, in this study, additional parameters were determined at the end of the experiment to analyze general effects of semaglutide or long-term effects of therapy with a non-selective or Y2-R selective PYY analog or a combination of both. Studies suggest that semaglutide improves cardiovascular risk by influencing lipid balance (e.g. reduction in cholesterol (59) or triglycerides (60)), among other things. GLP-1 receptor agonists also appear to have a positive effect on non-alcoholic fatty liver disease/non-alcoholic steatohepatitis (61, 62), but the reasons for this are not yet fully understood. Precise data for PYY on these issues are lacking. In our study we could not detect significant differences in serum levels of cholesterol, triglycerides, and transaminases, which is in accordance with a previous study also not showing significant differences in ALT and AST in rats treated with GLP-1 receptor agonists and PYY3-36 (63).
While GLP-1 receptor agonists have already been established in diabetes treatment (64), the effect of PYY on glucose balance has not yet been clarified in detail. This study revealed no significant differences between the treatment groups regarding blood glucose levels. Initial studies also showed no effect of intravenously administered PYY on plasma levels of glucose (65). Another study revealed that a single intraperitoneal injection of PYY3-36 administered to fasted ob/ob and db/db mice did not affect plasma glucose concentrations and furthermore, a 4-week infusion of PYY3-36 via osmotic pump to ob/ob mice and fa/fa rats did not influence fasting plasma glucose levels (39). Therefore, PYY3-36 might not play an important role in glucose homeostasis, especially regarding long-term effects after completion of treatment as seen in our study. Semaglutide, on the other hand, is already established in diabetes therapy. However, neither the monotherapy group nor groups in which semaglutide was used in combination showed a significant reduction of glucose levels. One possible reason could be that the rats were only obese and not suffering from diabetes and the effect on glucose metabolism was therefore not significant.
The strength of the present study is a controlled study design, demonstrating the promising potential of the combination of two gut hormone analogs for the treatment of obesity. However, this pilot study has various limitations. First, longer treatment periods are necessary to identify tachyphylaxis as well as potential adverse effects. Second, better powered and equal group sizes are necessary to overcome a potential statistical bias. Therefore, a higher and more concise animal number will be included in future experiments. Third, we did not include a study group with PYY analog monotherapy, as we saw no effect of a PYY3-36 monotherapy in an earlier study (according to the 3R principles) (33). Fourth, in mice, the effect of a selective Y2-R agonist on food intake was dose dependent; therefore, in order to observe more pronounced effects, it might be necessary to increase the dose (34). Fifth, a specific behavioral testing was not part of this study design, and it must be mentioned, that the NPY system might play a role in psychiatric diseases like anxiety and depression (66, 67, 68, 69, 70, 71).
Conclusion
In summary, we demonstrated that the combination of a PYY3-36 analog and semaglutide led to a pronounced body weight loss. Comparison of a selective Y2-R agonist and a non-selective PYY3-36 analog (both combined with semaglutide) showed that the former resulted in a higher weight loss pointing toward the Y2-R as an effective therapeutic target for obesity. We also demonstrated that the GLP-1 and Y2-R pathways are involved in food choice, as their blockade leads to an almost complete HFD preference. Future studies with bigger group sizes are necessary to strengthen our findings.
Declaration of interest
UD was supported by Novo Nordisk and Janssen via material transfer agreements (see Acknowledgements). The companies had no influence on the study design, the collection, analyses, and interpretation of data; on the writing of the manuscript; or on the decision to publish the results.
Funding
This study was supported by the IZKF Würzburg (Z-3BC/01, to UD). CGZ was supported by the Deutsche Forschungsgemeinschaft (DFG; grant TRR-CRC 205). CM is supported by the DFG (Ma 2528/8-1, SFB 1525/project #453989101).
Institutional review board statement
The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the local regulatory authority (Regierung von Unterfranken, Würzburg, Germany, AZ: 55.2.2-2532-2-1412). All experiments were performed in accordance with German and European laws and regulations (TierSchG, TierSchVersV, Directive 2010/63/EU). All efforts were made to minimize suffering.
Author contribution statement
Conceptualization, UD; data curation, MO, CGZ and UD; formal analysis, MO; funding acquisition, UD; investigation, UD, MO, CGZ; methodology, UD, MO and CGZ; project administration, UD; resources, UD and MF.; supervision, UD; validation, UD; visualization, MO and SK; writing – original draft, MO; writing – review and editing, SK, UD, CGZ, MF, AN, CM and VS. All authors have read and agreed to the published version of the manuscript.
Acknowledgements
We thank Helen Göhler for her support in the animal experiments and Niklas Haerting for his laboratory work support. We thank Novo Nordisk for substance NNC0165-1273, NNC0165-0020 and semaglutide. We thank Janssen for substance JNJ31020028.
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