Lead-in calorie restriction enhances the weight-lowering efficacy of incretin hormone-based pharmacotherapies in mice

Abstract

Objectives

The potential benefits of combining lifestyle changes with weight loss pharmacotherapies for obesity treatment are underexplored. Building on recent clinical observations, this study aimed to determine whether “lead-in” calorie restriction before administering clinically approved weight loss medications enhances the maximum achievable weight loss in preclinical models.

Methods

Diet-induced obese mice (DIO) were exposed to 7 or 14 days of calorie restriction before initiating treatment with semaglutide (a glucagon-like peptide-1 receptor (GLP-1R) agonist), tirzepatide (a GLP-1R/glucose insulinotropic peptide receptor (GIPR) co-agonist), or setmelanotide (a melanocortin-4 receptor (MC4R) agonist). Follow-up assessments using indirect calorimetry determined the contributions of energy intake and expenditure linked to consecutive exposure to dieting followed by pharmacotherapy.

Results

Calorie restriction prior to treatment with semaglutide or tirzepatide enhanced the weight loss magnitude of both incretin-based therapies in DIO mice, reflected by a reduction in fat mass and linked to reduced energy intake and a less pronounced adaptive drop in energy expenditure. These benefits were not observed with the MC4R agonist, setmelanotide.

Conclusions

Our findings provide compelling evidence that calorie restriction prior to incretin-based therapy enhances the achievable extent of weight loss, as reflected in a weight loss plateau at a lower level compared to that of treatment without prior calorie reduction. This work suggests that more intensive lifestyle interventions should be considered prior to pharmacological treatment, encouraging further exploration and discussion of the current standard of care.

Highlights

• •Combining lifestyle interventions with drug treatments presents untapped potential for obesity management.
• •Lead–in calorie restriction enhances the maximal achievable weight loss with semaglutide and tirzepatide in mice.
• •The amplified weight loss is reflected in loss of fat mass and preserved energy expenditure.
• •Calorie restriction prior to treatment with the MC4R agonist setmelanotide has no additional weight loss benefits.

1. Introduction

The prevalence of obesity continues to rise, with recent estimates indicating that over 1.9 billion adults are currently living with overweight or obesity [1]. Excessive body fat is a major risk factor for numerous co-morbidities, including type 2 diabetes, metabolic dysfunction-associated steatohepatitis (MASH), and cardiovascular diseases [[2], [3], [4], [5]]. Accordingly, reducing body weight and fat mass represents a key goal of obesity treatment, with the magnitude of weight loss correlating with a reduction in obesity-related complications [[6], [7], [8]].

Lifestyle modifications, including diet and exercise regimens, are still the first-line interventions for patients with obesity. A calorie-reduced diet combined with increased physical activity and behavioral counseling can lead to a reduction in body weight of 5–8% [9]. This level of weight loss is clinically meaningful and has health benefits for obesity-linked co-morbidities [[6], [7], [8]]. Sustaining weight loss, however, presents a significant challenge, as many patients experience rapid weight regain despite their intentions and efforts to maintain a lower body weight [10,11]. The regain of lost body weight is governed by homeostatic feedback control mechanisms, as evidenced by persistent hormonal changes that promote a compensatory increase in hunger and energy intake as well as an adaptive decrease in energy expenditure [12,13].

Over the last decades, key technological advancements in transforming short-acting gut peptide hormones into innovative pharmaceutical solutions have yielded anti-obesity pharmacotherapies that safely deliver 15–20% weight loss in people with obesity [14,15]. Notably, these incretin-based pharmacotherapies appear to alter the strength of the feedback control circuits governing weight regain [16] and demonstrate a stable weight plateau for up to 4 years with continued treatment [17]. The clinical weight loss trials are based on a background regimen of lifestyle interventions, including dietary guidance, increased physical activity, and behavioral counseling. Recently, there has been an intensified interest in understanding the potential benefits of a coordinated combination of specific lifestyle changes with weight loss drugs, particularly for weight loss maintenance and protection against the loss of lean body mass [18,19]. Whereas the combination of an extensive behavioral intervention and semaglutide did not elicit greater weight loss than semaglutide alone [19,20], an intensive lifestyle intervention prior to the initiation of tirzepatide treatment was found to significantly enhance the maximal achievable weight loss in patients with overweight or obesity [21].

This suggests that implementing calorie restriction before administering a weight loss drug may enhance the extent of weight loss achievable with anti-obesity treatments. To investigate this hypothesis, we conducted experiments using diet-induced obese (DIO) mice exposed to varying durations of calorie restriction before initiating pharmacological treatment with semaglutide (GLP-1R agonist), tirzepatide (dual GIPR/GLP-1R agonist), or setmelanotide (MC4R agonist). Our findings provide compelling evidence supporting the concept that calorie restriction prior to an incretin-based treatment enhances weight loss efficacy and leads to a weight loss plateau at a lower body weight compared to treatment without a lead-in hypocaloric diet. Notably, these benefits were not observed with the MC4R agonist.

2. Methods

2.1. Animals and housing conditions

All experiments were performed under approval of the Danish Ethical Committee for Animal Research and the Danish Animal Experimentation Inspectorate. Male C57BL/6J mice were maintained on ad libitum high-fat, high-sucrose diet (HFD, 58 kcal% fat, #D12331i, Research Diets, New Brunswick, NJ, USA) from 8 weeks of age and for a minimum of 16 weeks. Mice had an average body weight of >45 g before initiating pharmacological studies. The mice were either single- or double-housed in a humidity- (45–65%) and temperature-controlled environment (21–23 °C) with a 12-hour light/dark cycle (6 am - 6 pm).

2.2. In vivo pharmacology

For pharmacological studies, mice were blocked in experimental groups according to body weight. Semaglutide (NNC0113-0217) was obtained through Novo Nordisk Compound Sharing, while tirzepatide (MedChemExpress) and Setmelanotide (MedChemExpress) were purchased from commercial sources. Semaglutide and tirzepatide were formulated in an aqueous vehicle consisting of 240 mM propylene glycol in 8 mM phosphate buffer at pH 8.0, while setmelanotide was formulated in a vehicle consisting of 0.1% bovine serum albumin in phosphate-buffered saline at pH 7.4. Compounds and vehicle were administered as once-daily subcutaneous injections at an injection volume of 5 μL per gram body weight (between 3 pm and 5 pm). Calorie restriction was performed to match the weight loss trajectory of the groups treated with pharmacotherapies. Mice subjected to calorie restriction received vehicle injections during the restriction period. Calorie restriction was done by breaking food pellets into small pieces, weighing out the exact amount of food, and providing each cage with one pellet per mouse at the indicated meal sizes. For double-housed mice, the pellets were placed in opposite sides of the cage. Body weight and food intake were measured every day at the time of injection. Upon switching the calorie restriction groups to pharmacotherapy (day 7 or day 14), mice were provided ad libitum access to the HFD at the time of injection. Mice were euthanized by decapitation for the collection of trunk blood and tissues. Tissues were frozen immediately after collection, on dry ice. Blood was collected in EDTA-coated microvette tubes and immediately chilled on wet ice until centrifugation at 3,000×g and 4 °C for 10 min. Plasma was aliquoted and stored together with tissues at −70 °C until analysis.

2.3. Body composition analysis

Body composition was determined using nuclear magnetic resonance (Bruker LF90II Body Composition Analyzer).

2.4. Plasma parameters

Plasma was sampled under non-fasted conditions. Plasma triglycerides (Stanbio Laboratory, ref. no. 2100-430) and plasma total cholesterol (Stanbio Laboratory, ref. no. 1010-430) were determined according to manufacturer's protocols. Plasma aspartate transaminase (AST) (Stanbio Laboratory, ref. no. 2920-430) and plasma alanine aminotransferase (ALT) (Stanbio Laboratory, ref. no. 2930-430) were quantified according to manufacturer's protocols. Plasma insulin levels (Crystal Chem Ultra-Sensitive Mouse Insulin Elisa Kit, cat. no. #90080, Denmark) was quantified by sandwich ELISA according to the manufacturer's protocol.

2.5. Metabolic phenotyping and indirect calorimetry

DIO male C57BL/6J mice were single-housed in metabolic cages (16-channel Promethion, Sable Systems International, NV, USA) and allowed to habituate for 3 days before initiation of the study. Oxygen consumption (VO₂), carbon dioxide production (VCO₂), respiratory exchange ratio, energy expenditure (kcal h⁻¹), and locomotor activity (beam breaks) were recorded and collated in 15-minute intervals for analysis. Water was available ad libitum throughout the study period. HFD was available ad libitum for mice treated with vehicle or 10 nmol kg⁻¹ semaglutide. Calorie restriction was performed by weighing out food pellets and providing each cage with a pellet at the indicated meal size, before injecting mice with vehicle. For ad libitum fed mice, new food was provided every day, and old food removed from the cages. Compounds were injected at the indicated doses at a volume of 5 μL per gram of body weight (between 3 pm and 5 pm). Mice assigned to the different experimental groups were randomly distributed across two systems to correct for potential space-related bias. On the final day of the experiment, mice were euthanized by decapitation for the collection of trunk blood and tissues. Raw data for each individual mouse was plotted in GraphPad Prism and carefully inspected for aberrant datapoints. For respiratory exchange ratio datasets, datapoints related to the opening of cages were excluded from the analysis. Finally, data was analyzed and visualized using the online tool CalR (CalR, version 1.3) and GraphPad Prism, respectively.

2.6. Statistical analyses

Statistical analyses were performed in GraphPad Prism version 10; statistical tests used are described in respective figure legends. All data are presented as mean ± SEM and all P values ≤ 0.05 were considered statistically significant. ∗P ≤ 0.05, ∗∗P ≤ 0.01, ∗∗∗P ≤ 0.001, ∗∗∗∗P ≤ 0.0001.

3. Results

3.1. Lead-in calorie restriction amplifies semaglutide-induced weight loss

To assess if pre-intervention calorie restriction amplifies the maximal weight loss that can be obtained with semaglutide, we conducted a 28-day study in which mice were calorie-restricted for 7 or 14 days to match the weight loss of mice receiving repeated daily dosing with 10 nmol kg⁻¹ semaglutide before initiating treatment with 10 nmol kg⁻¹ semaglutide. These groups were compared to a group of mice that received daily injections with 10 nmol kg⁻¹ semaglutide for all 28 days (Figure 1A). We observed that 10 nmol kg⁻¹ semaglutide induced a weight loss of 31.7% over 28 days, while 7-day or 14-day calorie restriction before treatment with 10 nmol kg⁻¹ semaglutide induced a significantly greater weight loss of 37.5% or 39.3%, respectively (Figure 1B,C). Both calorie restriction groups had a greater reduction in total energy intake over the 28-day intervention period. This difference in energy intake was established during the lead-in body weight matching period as semaglutide had similar food intake-lowering effect in all groups irrespective of the lead-in paradigm (Supplementary Figs. 1A and B). Accordingly, mice exposed to 14 days of pre-intervention calorie restriction had a lower total energy intake than those exposed to 7 days of pre-intervention calorie restriction (Figure 1D,E). However, there were no significant difference in weight loss between the two groups (Figure 1B,C). This mismatch between energy intake and body weight loss might reflect compensatory dynamics in energy expenditure [22]. Previous work has demonstrated that semaglutide treatment can offset weight loss-related compensatory metabolic adaptations in rodents [23]. Finally, examination of body composition revealed that the additional weight loss was exclusively reflected by an amplified loss of fat mass relative to semaglutide-treated animals (Figure 1F,G).

Figure 1.

Calorie restriction before semaglutide treatment leads to additional lowering of body weight in DIO mice. (A) Schematic of experimental design in which DIO mice were treated with once-daily subcutaneous injections of vehicle (n = 5 mice), 10 nmol kg⁻¹ semaglutide (n = 7 mice), 7 days of calorie restriction (together with daily vehicle injections) from day 0 to day 7 followed by 10 nmol kg⁻¹ semaglutide (n = 6 mice), or 14 days of calorie restriction (together with daily vehicle injections) from day 0 to day 14 followed by 10 nmol kg⁻¹ semaglutide (n = 8 mice) for 28 days. (B) Percentage change in body weight. (C) Total percentage change in body weight over 28 days. (D) Daily energy intake in kcal. (E) Total energy intake in kcal over 28 days. (F) Percentage change in fat mass from day −3 to day 28. (G) Percentage change in lean mass from day −3 to day 28. Data was analyzed by one-way ANOVA, multiple comparison, Bonferroni post hoc test (C, E, F and G). Data are presented as mean ± SEM; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

3.2. Metabolic phenotyping of sequential calorie restriction and semaglutide treatment

To further elucidate the physiological mechanisms underlying the amplified weight loss linked to pre-intervention calorie restriction followed by semaglutide treatment, we conducted a 21-day metabolic phenotyping study using metabolic cages (indirect calorimetry). In our initial study, we observed that 14 days of calorie restriction before starting semaglutide treatment did not enhance the weight loss compared to a 7-day calorie restriction period. Consequently, we designed the indirect calorimetry study to compare the effects of 10 nmol kg⁻¹ semaglutide relative to 7-day calorie restriction before treatment initiation with 10 nmol kg⁻¹ semaglutide (Figure 2A). During the 21-day study, 10 nmol kg⁻¹ semaglutide reduced body weight by 27.4% while the group receiving 7-day calorie restriction before 10 nmol kg⁻¹ semaglutide lost 35.2% body weight (Figure 2B,C). These data confirm our initial findings (Figure 1A–C). The weight loss for both semaglutide-based treatment groups coincided with a reduction in caloric intake, which was more pronounced for the group undergoing calorie restriction before being switched to semaglutide treatment (Figure 2D,E). Consistent with the findings in the first study, the difference in total energy intake is established in the weight matching period as the anorexigenic effect of semaglutide was similar for both groups (Supplementary Fig. 2A, B).

Figure 2.

Metabolic phenotyping of mice subjected to calorie restriction before semaglutide treatment. (A) Schematic of experimental design in which DIO mice were treated with once-daily subcutaneous injections of vehicle (n = 9 mice), 10 nmol kg⁻¹ semaglutide (n = 10 mice) or 7 days of calorie restriction (together with daily vehicle injections) before 10 nmol kg⁻¹ semaglutide (n = 10 mice) in an indirect calorimetry system for 21 days. (B) Percentage change in body weight. (C) Total percentage change in body weight over 21 days. (D) Daily energy intake in kcal. (E) Total energy intake over 21 days in kcal. (F) Energy expenditure during the 21 days of treatment. (G) Total energy expenditure over the 21 days of treatment. (H) Average energy expenditure between days 0–7, 8–14 and 15–21 of the study. (I) Respiratory exchange ratio during the 21 days of treatment. (J) Average respiratory exchange ratio between days 0–7, 8–14 and 15–21 of the study. (K) Locomotor activity during the 21 days of treatment. (L) Daily locomotor activity during the 21 days of treatment. (M) Plasma insulin levels. (N) Plasma total cholesterol levels. (O) Plasma triglyceride levels. (P) Plasma aspartate aminotransferase (AST) levels. (Q) Plasma alanine aminotransferase (ALT) levels. Data was analyzed by one-way ANOVA, multiple comparison, Bonferroni post hoc test (C, E, G, H, J and L-Q) and two-way ANOVA, multiple comparison, Bonferroni post hoc test (B). Data are presented as mean ± SEM; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗∗P < 0.0001.

From analyzing energy expenditure data, we observed that 7 days of severe calorie restriction-induced a progressive decrease in energy expenditure. In contrast, semaglutide-treated mice, which achieved similar weight loss to the calorie-restricted mice, did not decrease their energy expenditure (Figure 2F–H). The prevention of a compensatory downregulation of energy expenditure by semaglutide is consistent with previous work [23]. Interestingly, switching the calorie restricted group to semaglutide treatment gradually restored energy expenditure (Figure 2F–H, Supplementary Fig. 2C). The combinatorial treatment displayed a prolonged amplification of whole-body fat oxidation compared to semaglutide alone, indicated by the reduced respiratory exchange ratio (RER) (Figure 2I,J). The RER normalized in agreement with increased food intake and body weight stabilizing at a new plateau for both intervention groups (Figure 2I). The changes in energy balance coincided with a subtle lowering of total locomotor activity over the entire period for the mice subjected to the combinatorial treatment regimen, and possibly also for the mice in the semaglutide group although this was not significant (Figure 2K,L). Next, we assessed whether the additive weight loss following the combinatorial treatment was associated with additive improvements in plasma parameters of metabolic health. We found that the combinatorial treatment was associated with a significant improvement in plasma insulin levels, which was not observed for the semaglutide-treated group (Figure 2M). In addition, we observed a decrease in plasma cholesterol levels, but not triglycerides for both semaglutide-treated groups (Figure 2N,O). Finally, a reduction in the plasma levels of the liver enzymes AST and ALT was observed for both semaglutide-based treatment groups, albeit only statistically significant for ALT (Figure 2P,Q).

3.3. Lead-in calorie restriction potentiates tirzepatide-induced weight loss but not setmelanotide-induced weight loss

To explore if the amplified weight loss benefits of calorie restriction prior to pharmacological treatment extends beyond semaglutide, we conducted additional experiments in which the 7-day calorie restriction regimen was introduced before 14-day exposure to other clinically approved obesity therapies, i.e. tirzepatide (a GIPR/GLP-1R co-agonist) and setmelanotide (a MC4R agonist).

In the first study, tirzepatide was dosed at 10 nmol kg⁻¹, a dose that has been shown to potently lower body weight in mice [[24], [25], [26]] (Figure 3A). Treatment of DIO mice with once-daily subcutaneous injections of 10 nmol kg⁻¹ tirzepatide resulted in an average weight loss of 23.2% after 7 days, which was slightly more than the 19.5% weight loss for the group that was calorie restricted during the same period (Figure 3B). Nonetheless, following the switch from calorie restriction to treatment with tirzepatide and ad libitum HFD access these mice continued to lower their body weight and ended up realizing a significantly larger weight loss of 41.4% compared to 34.4% for mice treated only with tirzepatide (Figure 3C). The additional reduction in body weight was reflected in a significantly lower energy consumption (Figure 3D,E), a difference that was established during the first 7 days (Supplementary Fig. 3A, B).

Figure 3.

Enhanced weight loss from lead-in calorie restriction extends beyond semaglutide treatment. (A–E) Treatment of DIO mice with once-daily s.c. injections of 10 nmol kg⁻¹ tirzepatide (n = 7 mice), 7 days of calorie restriction (together with daily vehicle injections) to match the weight loss trajectory of 10 nmol kg⁻¹ tirzepatide followed by 14 days of 10 nmol kg⁻¹ tirzepatide (n = 7 mice) or vehicle (n = 7 mice) for 21 days. (A) Schematic. (B) Percentage change in body weight. (C) Total percentage change in body weight over 21 days. (D) Daily energy intake in kcal. (E) Total energy intake over 21 days in kcal. (F–J) Treatment of DIO mice with once-daily s.c. injections of 3.6 μmol kg⁻¹ setmelanotide (n = 7 mice), 7 days of calorie restriction (together with daily vehicle injections) to match the weight loss trajectory of 3.6 μmol kg⁻¹ setmelanotide followed by 14 days of 3.6 μmol kg⁻¹ setmelanotide (n = 8 mice) or vehicle (n = 8 mice) for 21 days. (F) Schematic. (G) Percentage change in body weight. (H) Total percentage change in body weight over 21 days. (I) Daily energy intake in kcal. (J) Total energy intake over 21 days in kcal. Data was analyzed by one-way ANOVA, multiple comparison, Bonferroni post hoc test (C, E, H and J) and two-way ANOVA, multiple comparison, Bonferroni post hoc test (B and G). Data are presented as mean ± SEM; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Next, we wanted to explore if the sequential calorie restriction followed by drug treatment was equally effective for a non-incretin-based anti-obesity drug. For this, we used the MC4R agonist, setmelanotide, a cyclized octapeptide that is clinically approved for the treatment of patients with rare genetic forms of obesity linked to pro-opiomelanocortin (POMC), proprotein convertase subtilisin/kexin type 1 (PCSK1) or leptin receptor deficiency [27]. In rodent models of diet-induced obesity, setmelanotide is typically administered as once-daily subcutaneous injections at a dose of 3.6 μmol kg⁻¹ [28,29]. Hence, mice were treated for 7 days with daily subcutaneous injections of 3.6 μmol kg⁻¹ setmelanotide or exposed to calorie restriction to match the weight loss of the setmelanotide-only treatment mice, before being switched to the same setmelanotide treatment regimen for 14 days (Figure 3F). In contrast to what was observed for semaglutide and tirzepatide, calorie restriction preceding treatment with setmelanotide failed to amplify the weight loss efficacy relative to mice treated with setmelanotide for the full duration of the study (Figure 3G,H). Consistent with this, no difference in total energy intake was observed between the setmelanotide-treated groups (Figure 3I,J), suggesting that the calorie restriction dampens the sensitivity to MC4R agonism (Supplementary Fig 3C, D). Additional work is needed to determine if the enhanced weight loss observed with calorie restriction before pharmacology is confined to incretin-based drugs.

4. Discussion

For decades, obesity management has focused on lifestyle interventions, particularly diet and exercise, as cornerstones of weight management [30]. However, the limited long-term success of these lifestyle changes in sustaining weight loss and the recent emergence of effective incretin hormone-based weight loss pharmacotherapies have shifted the focus of obesity treatment towards a more drug-centric approach. Accordingly, various clinical guidelines now recommend anti-obesity medications for individuals with obesity or overweight and associated health problems [31,32]. Incretin hormone-based weight loss therapies, such as semaglutide and tirzepatide, reduce body weight by 15–20% in clinical trials involving individuals with obesity [20,33]. Furthermore, next-generation combinatorial pharmacotherapies are anticipated to enhance weight loss outcomes even further [[34], [35], [36]]. Importantly, recent clinical data suggest that we should expand our perspective on combinatorial treatment beyond merely pharmacotherapies [18,21].

In 2023, a phase 3 clinical trial was published in which tirzepatide treatment was prescribed following an initial 12-week intensive lifestyle intervention resulting in an initial 6.9% weight loss [21]. This, in combination with subsequent pharmacological intervention at the maximum tolerated dose of tirzepatide (10 or 15 mg), resulted in a cumulative 24.3% weight loss from baseline body weight, thus exceeding the 20.9% seen in a previous clinical trial with a weekly dose of 15 mg tirzepatide given for 72 weeks without prior lifestyle treatment [33]. This observation is supported by earlier findings with the GLP-1R agonist liraglutide demonstrating that an 8-week lead-in period with low-calorie dieting improved markers of cardiometabolic health beyond liraglutide treatment alone in adults with obesity [18,37]. However, it is not clear from these trials if the initial period of calorie restriction directly modulated the weight loss magnitude of the subsequent treatment.

In the present study, we confirm and extend these clinical observations. Using DIO mice, we demonstrate that calorie restriction prior to pharmacological treatment with semaglutide or tirzepatide enhances the maximum achievable weight loss of both incretin-based therapies. This amplified loss of body weight is driven by loss of fat mass and is likely to be clinically relevant and mediate additional health benefits [[6], [7], [8]]. Thus, instead of focusing solely on developing novel combinatorial pharmacotherapies to maximize weight loss efficacy, our results encourage further research into understanding the benefits of combining lifestyle changes with incretin-based drugs. It is possible that specific sub-groups of patients may respond better to this type of intervention or that changes in diet and exercise can be further refined and optimized in terms of type, duration, intensity, and sequence [38].

We have begun to explore the physiological mediators of the amplified weight loss observed when calorie restriction precedes pharmacotherapy. As demonstrated in the literature, extensive calorie restriction lowers energy expenditure in a compensatory manner to counteract weight loss [23]. Once the restricted animals are switched to pharmacotherapy and given full access to their diet, we observed that this adaptive slowing of metabolism is reversed, despite the animals continuing to experience a further negative energy balance. This indicates that the initial homeostatic attempt to regain the lost body weight may be temporally alleviated once the drugs are introduced. Furthermore, the anorexigenic efficacy of the incretin hormones are not influenced by the diet-restricted weight loss.

While the benefits of calorie restriction prior to drug-induced weight loss are clear for semaglutide and tirzepatide, no additional weight loss was found when calorie restriction preceded treatment with setmelanotide. Setmelanotide is reported to increase energy expenditure [29], [39], leading to the notion that MC4R agonism should be effective in reversing the decline in energy expenditure following calorie restriction. This raises important questions: Are the benefits observed with calorie restriction preceding anti-obesity medication treatment exclusive to the incretin-based class of anti-obesity drugs? Or is the absence of beneficial interaction unique to drugs targeting the leptin-melanocortin pathway? The latter seems less likely, as the lack of lifestyle–drug interaction has also been observed with the lipase inhibitor orlistat [40]. Further research is needed to clarify these issues.

In summary, our findings in mice reinforce clinical data suggesting that a body weight-lowering lifestyle intervention before starting incretin-based anti-obesity medication can enhance the maximal achievable weight loss with subsequent pharmacotherapy. However, the details of the optimal combination of lifestyle changes and pharmacotherapy are still unclear. Additional preclinical and clinical studies are required to establish the optimal type, duration, intensity, and sequence of both treatment approaches. Finally, consistent with the SURMOUNT-3 trial [21], our findings suggest that more intensive lifestyle interventions could be considered as part of the eligibility criteria before initiating pharmacological treatment, prompting further exploration and discussion on the current standard of care.

Funding

C.C. is supported by a research grant from the Novo Nordisk Foundation, Denmark (grant number NNF22OC0073778). S.H. is supported by a research grant from the Danish Diabetes Academy, Denmark, which is funded by the Novo Nordisk Foundation (grant number NNF17SA0031406). J.L. is supported by the BRIDGE, Denmark – Translational Excellence Programme (www.bridge.ku.dk) funded by the Novo Nordisk Foundation (grant NNF20SA0064340). The Novo Nordisk Foundation Center for Basic Metabolic Research is an independent Research Center, based at the University of Copenhagen, Denmark, and partially funded by an unconditional donation from the Novo Nordisk Foundation (www.cbmr.ku.dk) (grant numbers NNF18CC0034900 and NNF23SA0084103).

CRediT authorship contribution statement

Jonas Petersen: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Methodology, Investigation, Formal analysis, Conceptualization. Christoffer Merrild: Writing – review & editing, Investigation. Jens Lund: Writing – review & editing, Investigation, Conceptualization. Stephanie Holm: Writing – review & editing, Investigation, Conceptualization. Christoffer Clemmensen: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Project administration, Funding acquisition, Conceptualization.

Declaration of competing interest

J.P. and C.C. are co-founders of Ousia Pharma ApS, a biotech company developing therapeutics for obesity treatment. The remaining authors declare no competing interests.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Data availability

Data will be made available on request.