The novel chimeric multi-agonist peptide (GEP44) reduces energy intake and body weight in male and female diet-induced obese mice in a glucagon-like peptide-1 receptor-dependent manner

Obesity is a major worldwide health concern as it increases the risk of cardiovascular disease, obstructive sleep apnea, cancer, osteoarthritis, depression, COVID-19 related hospitalizations and type 2 diabetes. According to the NCD Risk Factor Collaboration, more than one billion people are obese worldwide (1). Approximately 1 in 2 US adults are predicted to have obesity by 2030 (2) and the costs to treat obesity in the US are estimated to be approximately 3 trillion/year by 2030 (3). More recently developed monotherapies to treat obesity such as the long-acting glucagon-like peptide-1 receptor (GLP-1R) agonists, liraglutide and semaglutide (4), produce more pronounced effects on weight loss relative to previous analogues. Weight loss in response to once-weekly treatment with semaglutide has ranged from ≈6.7% over 40 weeks (5) to ≈14.9% weight loss over 68 weeks (4). However, improvements still need to be made in terms of improving overall weight loss effectiveness over more prolonged periods [≈10.2% weight loss over 208 weeks (6)]. Furthermore, there is rapid recovery of weight when discontinued (7). This is likely due to the activation of counter-regulatory orexigenic mechanisms that increase energy intake and/or reduce energy expenditure to promote weight regain (8).

Recent studies suggest that combination therapy aimed at suppressing energy intake and/or increasing energy expenditure at low-dose or subthreshold dose combinations may be more effective for producing sustained weight loss than monotherapy (9) and may minimize the potential for unwanted side effects. The overall ineffectiveness of monotherapies to evoke prolonged weight loss in humans with obesity is assumed to occur, in part, by recruitment of robust orexigenic mechanisms that drive energy intake and decrease energy expenditure, resulting in body weight (BW) gain and thus preventing further weight loss. In addition to combination therapy, a recent innovative approach involves the targeting of two or more signaling pathways using a single compound such as monomeric multi-agonists (dual- or triple-agonists) based on glucose-dependent insulinotropic polypeptide (GIP) and GLP-1R agonists, with and without glucagon receptor (GCGR) agonism. One such drug, tirzepatide (Zepbound™), which targets both GLP-1R and and the GIP receptor (GIPR), was found to elicit a robust 20.9% and 25.3% weight loss in humans with obesity over 72- (10) and 88-week periods (11), and was recently approved by the FDA for weight management. Recent data indicate that the triple-agonist, retatrutide, which targets GLP-1R, GIPR and GCGR, was able to elicit 24.2% weight loss over 48-week period (12) (2 to 12 mg). While such therapies show considerable promise for effective and sustained reduction of BW during drug treatment, there are still mild to moderate adverse gastrointestinal side effects including nausea, diarrhea, abdominal pain and vomiting (10). Given the rise of the obesity epidemic in the US and worldwide, there remains an urgent need to develop newer and more effective anti-obesity treatment strategies in order to reduce obesity rates across a broader spectrum of the population. We recently designed the novel chimeric peptide, GEP44, which binds to GLP-1R, Y1R and Y2R (13). We found that systemic administration of GEP44 reduced energy intake and BW in both lean (14) and diet-induced obese rats (13, 14). Importantly, GEP44 also reduced energy intake at doses that were not associated with significant pica behavior (kaolin intake) in rats (14) or emesis in musk shrews (14). A critical remaining question is whether GEP44 reduces energy intake, BW, impacts thermogenesis and improves glucose homeostasis in a GLP-1R dependent manner, either alone or in-part. Given the role of GLP-1R in the control of energy intake and BW, we hypothesized that chimeric peptide GEP44 reduces energy intake and BW through a GLP-1R dependent mechanism. To test this hypothesis, we determined the extent to which GEP44 reduces energy intake and BW, impacts core temperature (core temperature as surrogate marker of energy expenditure) and gross motor activity and improves fasting blood glucose in male and female DIO mice that lack GLP-1R (GLP-1R^-/-) relative to age-matched cohorts of male and female GLP-1R^+/+ mice. Male and female mice were also weight-matched within a given cohort of GLP-1R^-/- and GLP-1R^+/+ mice prior to treatment. The selective GLP-1R agonist, exendin-4, was included as a control to assess GLP-1R mediated effects on energy intake (15).

All results are expressed as mean ± SE. Planned comparisons within respective genotypes and sex between vehicle and drug (plasma measures, tail vein glucose, and gene expression data) involving between subjects design were made using one-way ANOVA. Planned comparisons within respective genotypes and sex to examine treatment means of vehicle and drug (BW change, energy intake, core temperature, activity and T_IBAT) involving within-subjects designs were made using a one-way repeated-measures ANOVA. A two-way ANOVA was used to examine sex*drug interactive effects on BW change and energy intake. In addition, planned comparisons were used to examine sex differences on BW change and energy intake using a one-way ANOVA. Analyses were performed using the statistical program SYSTAT (Systat Software, Point Richmond, CA). Differences were considered significant at P<0.05, 2-tailed.

The overall goal of these studies was to use the GLP-1R^-/- mouse as a strategy to determine the extent to which chimeric peptide, GEP44, reduces BW and energy intake, impacts core temperature (as surrogate for energy expenditure) and activity, and improves glucose homeostasis through the GLP-1R in both male and female DIO mice. In addition, we incorporated use of the selective GLP-1R agonist, exendin-4, to assess GLP-1R mediated effects on BW, energy intake, core temperature, activity, and glucose levels in male and female DIO mice. DIO GLP-1R^+/+ and GLP-1R^-/- mice underwent a 3-day vehicle period, 3-day dose escalation drug treatment (5, 10, 20 and 50 nmol/kg; GEP44 vs the selective GLP-1R agonist, exendin-4) and a 3-day washout in sequential order with the same mice receiving the escalating dose. Energy intake, core temperature and activity were averaged throughout each sequential 3-day vehicle and 3-day drug treatment period. Note that energy intake, core temperature and activity were averaged across two-day vehicle treatments (prior to 20 and 50 nmol/kg) for one of the four cohorts used in the dose escalation studies.

Baseline BW-matching in Male and Female DIO GLP-1R^+/+ and GLP-1R^-/- Mice at Study Onset (post-dietary intervention/pre-drug treatment). By design, there were no differences in baseline BW between designated drug treatment groups (GEP44 vs exendin-4) of male GLP-1R^+/+ mice [F(1,19) = 0.009, P=NS] or male GLP-1R^-/- mice [F(1,18) = 0.682, P=NS] prior to drug treatment (Table 1). Similarly, there were also no differences in baseline BW between designated treatment groups (GEP44 vs exendin-4) of female GLP-1R^+/+ mice [F(1,18) = 0.071, P=NS] or GLP-1R^-/- mice [F(1,16) = 0.037, P=NS] prior to drug treatment (Table 1).

GEP44 treatment significantly reduced energy intake only in male (Figures 3A, B) and female DIO GLP-1R^+/+ mice (Figures 3C, D) but not in male (with exception of the lowest and highest dose) and female DIO GLP-1R ^-/- mice. GEP44 treatment decreased energy intake (10, 20 and 50 nmol/kg); P<0.005) in both male and female mice. Similar results on FI were obtained when normalizing to BW (Supplementary Figures 1A–D).

Exendin-4 treatment also significantly reduced energy intake in male (Figures 4A, B) and female DIO GLP-1R^+/+ mice (Figures 4C, D) but not in male (with exception of the lowest dose) and female DIO GLP-1R ^-/- mice. Specifically, exendin-4 treatment decreased energy intake (10, 20 and 50 nmol/kg; P<0.05) in male GLP-1R^+/+ mice and also tended to decrease energy intake at the low dose (5 nmol/kg) in male GLP-1R ^-/- mice (P=0.055). However, exendin-4 reduced energy intake in female GLP-1R^+/+ mice across all doses (5, 10, 20 and 50 nmol/kg; P<0.05).

Similar results on energy intake were obtained when normalizing to BW (). 2

Here we report effects of the novel chimeric peptide (GEP44), which targets GLP-1R, Y1R and Y2R, on energy intake, BW, thermoregulation (core temperature) and gross motor activity in DIO mice and characterize the extent to which these effects are mediated through GLP-1R. We tested the hypothesis that GEP44 reduces energy intake and BW through a GLP-1R dependent mechanism. We found that GEP44 reduced BW in both male and female DIO male GLP-1R^+/+ mice whereas these effects were absent in male and female DIO GLP-1R^-/- mice. These findings suggest that GLP-1R signaling contributes to GEP44-elicited reduction of BW in both male and female mice. Additionally, GEP44 decreased energy intake in both male and female DIO GLP-1R^+/+ mice, but GEP44 produced more robust effects across multiple doses in males. These findings suggest that 1) GEP44 reduces BW, in part, through reductions in energy intake in DIO mice and 2) male mice might have enhanced sensitivity to the anorexigenic effects of GEP44. In GLP-1R^-/- mice, the effects on energy intake were observed only at low and high doses in males suggesting that GEP44 may reduce energy intake in males, in part, through a GLP-1R independent mechanism. GEP44 reduced both core temperature and activity in both male and female GLP-1R^+/+ mice suggesting that reductions in energy expenditure and/or spontaneous activity-induced thermogenesis may also contribute to the weight lowering effects of GEP44 in mice. Lastly, we show that GEP44 reduced fasting blood glucose in DIO male and female mice through GLP-1R signaling. Together, these findings support the hypothesis that the chimeric peptide, GEP44, reduces energy intake, BW, core temperature, and glucose levels, in part, through a GLP-1R dependent mechanism.

We extend previous findings from our laboratory (13, 14) and demonstrate that the effects of GEP44 to elicit weight loss in both male and female mice are primarily driven by GLP-1R. These effects are mediated, at least in part, by reductions of energy intake. Similar to GLP-1R driven effects of GEP44 on BW, based on our findings in both male and female GLP-1R^-/- mice, the ability of GEP44 to reduce energy intake also appears to be largely mediated by GLP-1R. However, the finding that the low and high dose of GEP44 also reduced energy intake in male GLP-1R^-/- mice suggests other mechanisms are involved in contributing to these effects. Given the role of Y2R in the control of energy intake and BW (21), and results showing synergism/additive effects between co-administered GLP-1R and Y2-R agonists (22, 23), it is likely that the Y2R is also playing a role in the beneficial effects of GEP44 in terms of BW.

We incorporated the use of exendin-4 as a positive control for GLP-1R mediated effects on both food intake and BW. Similar to what others have reported (15, 24), we also report that exendin-4 reduced BW and energy intake in both male and female DIO mice through GLP-1R signaling. Exendin-4 treatment appeared to be more effective at reducing BW in males at the lowest dose (5 nmol/kg). These findings are consistent with previous findings from Baggio and colleagues who reported that a single dose of exendin-4 (1.5 μg or 0.356 nmol/mouse) failed to reduce chow diet intake in male GLP-1R^-/- mice (15). Another study also found that chronic subcutaneous administration of a single dose (0.126 mg/kg/day or 30 nmol/kg/day) also failed to reduce high fat diet intake and BW (vehicle corrected) in male GLP-1R^-/- mice (24). In contrast to our studies, only a single dose of exendin-4 was examined in male mice in both studies (15, 24) and neither study examined the effects of exendin-4 in male and female DIO mice.

We also extend previous findings from our laboratory (13, 14) to demonstrate that GEP44 reduces core temperature (surrogate measure of energy expenditure) in both male and female mice, similar to what we described earlier herein following exendin-4 treatment. These data are also consistent with a previous report by Hayes and colleagues showing that the GLP-1R agonist, exendin-4, produced a long-lasting reduction of core temperature (hypothermia) that lasted for 4 hours following systemic [intraperitoneal (IP)] injections in rats (25). The hypothermic effects may be due, in part, to the reduced thermic effect of food (diet-induced thermogenesis) in exendin-4 and GEP44 treated mice. Furthermore, Baggio and colleagues (15) found that central and peripheral (IP; 1.5 μg or 0.356 nmol/mouse) administration of exendin-4 reduced VO2 and resting energy expenditure in adult male wild-type mice. These effects occurred over 2 and 21 hours following peripheral and central administration, respectively. Similarly, Krieger and colleagues found that peripheral exendin-4 reduced energy expenditure over 4-h post-injection in rats (26). In addition, van Eyk and colleagues found that liraglutide treatment reduced resting energy expenditure after 4 weeks of treatment in humans with type 2 diabetes (27). Gabery initially found that semaglutide reduced energy expenditure during the dark phase in DIO mice through treatment day 6 but these differences were no longer significant after adjusting for lean mass (28). Similarly, Blundell and colleagues initially found that semaglutide reduced resting energy expenditure after 12 weeks of treatment in humans but these effects were no longer significant after adjusting for lean mass (29). Despite previous reports indicating that acute central (ICV) administration of GLP-1 increases core temperature over 4-h post-injection (30), IBAT temperature over 6-h post-injection (31) and activates SNS fibers that innervate BAT (31), to our knowledge, the majority of studies that administered GLP-1R agonists peripherally have either found a reduction or no change in energy expenditure. Given the dearth of studies that have measured both core temperature and energy expenditure in the same group of animals, it will be important to measure both endpoints simultaneously in the same animal model. Ongoing studies are currently in the process of addressing the extent to which systemic administration of GEP44 and exendin-4 alters core temperature and more directly impacts energy expenditure (measured by indirect calorimetry) in male and female DIO rats.

In contrast to the effects of GEP44 and exendin-4 to reduce core temperature, we found that both GEP44 and exendin-4 stimulated thermogenic markers (biochemical readout of BAT thermogenesis) within IBAT and T_IBAT (functional readout of BAT thermogenesis). It is not clear why GEP44 and exendin-4 elicited opposing effects on core temperature and T_IBAT given that BAT thermogenesis helps maintain core temperature in other contexts such as fever or stress (32, 33). Future studies that examine temporal profile will help identify if the elevated BAT thermogenesis may be in response to the reduction of core temperature. Our findings, however, that exendin-4 stimulated thermogenic markers within IBAT and IBAT temperature are consistent with what others have found following systemic (intraperitoneal) exendin-4 (34) or liraglutide (35) administration in mice. On the other hand, Krieger (26) found that systemic (IP) exendin-4 reduced BAT thermogenesis, skin temperature above the interscapular area over 2-h post-injection and BAT Adrb3 in a rat model. Note that measurements of skin temperature above the interscapular area might not necessarily reflect T_IBAT as the interscapular subcutaneous area may also be influenced by heating and cooling of the skin temperature. We found that the majority of thermogenic genes in IBAT were elevated in response to both GEP44 and exendin-4. Furthermore, our findings suggest that GEP44 may also elicit BAT thermogenesis through similar mechanisms (PPargc1a) in both male and female GLP-1R^+/+ mice while exendin-4 may increase BAT thermogenesis via multiple thermogenic genes (UCP-1, Gpr120, and Ppargc1a) in female mice. In contrast to the effects of exendin-4 in GLP-1R^+/+ mice, the chimeric peptide, GEP44, appears to promote BAT thermogenesis in both male and female GLP-1R^+/+ mice through different genes. In contrast, GEP44 stimulated GPR120 only in female GLP-1R^-/- mice raising the possibility that other receptor subtypes may contribute to these effects at the high dose in female mice. Overall, our findings suggest that systemic administration of both GEP44 and exendin-4 may promote BAT thermogenesis through different mechanisms in a mouse model. It will be important in future studies to examine if SNS outflow to BAT is a predominant mediator of GEP44 or exendin-4-elicited weight loss in male and female DIO mice.

Our findings also demonstrate that GEP44 reduced gross motor activity through what appears to be both GLP-1R and Y2R signaling in male mice; while this effect appears to be entirely mediated by GLP-1R in female mice. In contrast, we found that exendin-4 treatment reduces activity strictly through GLP-1R in both male and female mice, as anticipated. Similarly, others have also found that exendin-4 at lower [0.5-5 μg/kg (0.2-2 μg/rat, IP)] (36) or higher doses [30 μg/kg (9 μg/rat, IP)] (37) reduces locomotor activity in lean male Sprague Dawley rats (36, 37). While Hayes and colleagues (25) did not find any change in activity in response to exendin-4 at [3 μg/kg (0.9 μg/rat, IP)] in lean male Sprague-Dawley rats, it might be possible that differences in paradigm and/or timing of injections may account for discrepancies between studies. Given that we found reductions in both core temperature and gross motor activity, our data raise the possibility that reductions in spontaneous physical activity-induced thermogenesis (38) and/or shivering and non-shivering thermogenesis in skeletal muscle (39) may have contributed to the hypothermic effects of both GEP44 and exendin-4. Moreover, our findings indicate that GEP44 may produce GLP-1R independent effects on 1) activity in male mice and 2) core temperature in both male and female mice while exendin-4 appears to impact both activity and core temperature through GLP-1R dependent mechanism in both male and female mice.

We have now extended our previous findings on glucoregulation (14) by showing that the effects of GEP44 to reduce fasting blood glucose in DIO male and female mice were mediated, in part, by GLP-1R. In addition, the effects of peripheral GEP44 and exendin-4 to reduce cholesterol in female GLP-1R^+/+ and GLP-1R^-/- mice are also consistent with the potential GLP-1R and/or Y2-receptor mediated cholesterol lowering effects observed following the GLP-1R agonists, liraglutide (40) and exendin-4 (41, 42), Y2 receptor agonist, lipidated PYY_3-36 analog, [Lys₇(C16- γGlu)PYY3−36] (similar potency on Y2R as PYY_3-36) (40), and dual GLP-1R/Y2 receptor agonists, 6q and peptide 19 (40), in DIO rodents.

One limitation to our studies is the lack of weight restricted or pair-fed controls for studies that assessed the effects of GEP44 and exendin-4 on gene expression as well as plasma measurements (total cholesterol, leptin, and glucose) in adult DIO male and female mice. As a result, it is possible that the effects of GEP44 and exendin-4 on thermogenic gene expression or glucose, total cholesterol or leptin lowering in DIO mice may also be due, in part, to the weight loss in response to GEP44 or exendin-4 treatment. In addition, we only collected T_IBAT between 120 and 360 min-post-injection. It is possible that, due to absence of being able to measure T_IBAT at earlier time points, we might have missed any potential hypothermic effects that might have preceded the elevations of T_IBAT that occurred at the later time points. In addition, we were unable to assess the roles of Y1R or Y2R signaling more fully in contributing to the effects of GEP44. Future studies incorporating the use of Y2R deficient mice will be helpful in more fully establishing the role of Y2R signaling in the anorectic response to GEP44.

In summary, the results presented in this manuscript highlight the beneficial effects of the dual-agonist, GEP44, on BW, energy intake, and glucoregulation in adult male and female DIO mice at doses that have not been found to elicit visceral illness in rats or emesis in shrews (13, 14). Our findings further demonstrate that effects on changes of BW, energy intake, activity, and glucose levels are largely mediated through GLP-1R signaling. However, given that GEP44 may also elicit GLP-1R independent effects on 1) activity in male mice and 2) core temperature in both male and female mice, it is possible that other receptor subtypes, including the Y1R (43) and Y2R, may contribute to these effects. The findings to date indicate that GEP44 is a promising drug targeting limitations associated with current GLP-1R agonist medications (5, 44) for the treatment of obesity and/or T2DM.

All relevant data is contained within the article: The original contributions presented in the study are included in the article/, further inquiries can be directed to the corresponding author/s. 1

The animal study was approved by VA Puget Sound Health Care System IACUC and the US Army Medical Research and Development Command (USAMRDC) Animal Care and Use Review Office (ACURO). The study was conducted in accordance with the local legislation and institutional requirements.

JB: Writing – review & editing, Writing – original draft, Supervision, Resources, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. MH: Writing – review & editing, Writing – original draft, Resources, Project administration, Methodology, Investigation, Data curation. JS: Writing – review & editing, Writing – original draft, Resources, Project administration, Methodology, Investigation, Data curation. MG: Writing – review & editing, Writing – original draft, Resources, Project administration, Methodology, Investigation, Data curation. JR: Writing – review & editing, Writing – original draft, Resources, Project administration, Methodology, Investigation, Data curation. ET: Writing – review & editing, Writing – original draft, Resources, Project administration, Methodology, Investigation, Data curation. AD: Writing – review & editing, Writing – original draft, Resources, Project administration, Methodology, Investigation, Data curation. KS: Writing – review & editing, Writing – original draft, Project administration, Investigation. TS: Writing – review & editing, Writing – original draft, Methodology, Investigation, Conceptualization. CE: Writing – review & editing, Writing – original draft, Methodology, Investigation, Funding acquisition, Conceptualization. KC: Writing – review & editing, Writing – original draft, Resources, Methodology, Investigation. EA: Writing – review & editing, Writing – original draft, Resources, Methodology, Investigation. SZ: Writing – review & editing, Writing – original draft, Resources, Project administration, Methodology, Investigation. RD: Writing – review & editing, Writing – original draft, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Conceptualization. CR: Writing – review & editing, Writing – original draft, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Conceptualization.

Plasma sample analysis was performed by the UC Davis MMPC Live M&MH Core (NIH U2CDK135074). The authors thank the technical support of Dr. Tami Wolden-Hanson with body composition measurements and James Graham and Dr. Peter Havel for plasma analyses.

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This material was based upon work supported by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs (VA) and the VA Puget Sound Health Care System Rodent Metabolic Phenotyping Core and the Metabolic and Cellular Phenotyping Core of the Diabetes Research Center at the University of Washington and supported by National Institutes of Health (NIH) grant P30DK017047. This work was supported by Department of Defense Grant 6W81XWH201029901 to CR and RD (JB, subaward PI). This work was also supported by the VA Merit Review Award 5I01BX004102, from the United States (U.S.) Department of Veterans Affairs Biomedical Laboratory Research and Development Service. Research reported in this publication is a project of the Seattle Institute for Biomedical and Clinical Research, supported by the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health under Award Number R01DK115976 (JB) and R01DK135125 (CR, RD, SZ). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The contents do not represent the views of the U.S. Department of Veterans Affairs or the United States Government.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fendo.2024.1432928/full#supplementary-material↗