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. 2023 Nov 7;73(2):197–210. doi: 10.2337/db23-0571

Leptin Reduction as a Required Component for Weight Loss

Shangang Zhao 1,2,3, Na Li 1,4, Wei Xiong 5, Guannan Li 3, Sijia He 3, Zhuzhen Zhang 6, Qingzhang Zhu 1, Nisi Jiang 3, Christian Ikejiofor 3, Yi Zhu 7, May-Yun Wang 1, Xianlin Han 3, Ningyang Zhang 5, Carolina Solis-Herrera 2, Christine Kusminski 1, Zhiqiang An 5, Joel K Elmquist 8, Philipp E Scherer 1,✉
PMCID: PMC10796304  PMID: 37935033

Abstract

Partial leptin reduction can induce significant weight loss, while weight loss contributes to partial leptin reduction. The cause-and-effect relationship between leptin reduction and weight loss remains to be further elucidated. Here, we show that FGF21 and the glucagon-like peptide 1 receptor (GLP-1R) agonist liraglutide rapidly induced a reduction in leptin. This leptin reduction contributed to the beneficial effects of GLP-1R agonism in metabolic health, as transgenically maintaining leptin levels during treatment partially curtailed the beneficial effects seen with these agonists. Moreover, a higher degree of leptin reduction during treatment, induced by including a leptin neutralizing antibody with either FGF21 or liraglutide, synergistically induced greater weight loss and better glucose tolerance in diet-induced obese mice. Furthermore, upon cessation of either liraglutide or FGF21 treatment, the expected immediate weight regain was observed, associated with a rapid increase in circulating leptin levels. Prevention of this leptin surge with leptin neutralizing antibodies slowed down weight gain and preserved better glucose tolerance. Mechanistically, a significant reduction in leptin induced a higher degree of leptin sensitivity in hypothalamic neurons. Our observations support a model that postulates that a reduction of leptin levels is a necessary prerequisite for substantial weight loss, and partial leptin reduction is a viable strategy to treat obesity and its associated insulin resistance.

Article Highlights

  • Weight loss agents in the glucagon-like peptide 1 receptor and FGF21 group induced a rapid suppression of leptin immediately upon agonist exposure.

  • This leptin suppression significantly contributed to the weight loss.

  • Further leptin suppression with a leptin neutralizing antibody enhanced weight loss and further improved insulin sensitivity.

  • Enhanced leptin reduction leads to further reduction in hepatic steatosis and fibrosis.

Graphical Abstract

graphic file with name db230571F0GA.jpg

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Introduction

The increasing rates of obesity and its associated metabolic disorders, including insulin resistance, type 2 diabetes, fatty liver disease, kidney and heart dysfunction, and some types of cancers, constitute major public health issues (1,2). Substantial weight loss is proven to be an effective strategy to reverse diabetes and fatty liver disease (3). Much progress has been made toward effective pharmacological interventions involving single, dual, or triple agonists, taking advantage of glucagon-like peptide 1 receptor (GLP-1R), glucose-dependent insulinotropic polypeptide receptor, and glucagon receptor agonists (4). However, a better understanding is needed about how these new agonists mechanistically achieve their remarkable effects.

The discovery of leptin almost 30 years ago as an adipocyte-derived hormone has attracted attention toward developing antiobesity therapies because of leptin’s potent effects in reducing food intake and enhancing energy expenditure (5–9). However, individuals with obesity display high levels of circulating leptin, reflective of severe leptin resistance. Recombinant leptin therapy remains completely ineffective toward reducing food intake and/or enhancing energy expenditure (10,11). Thus, leptin therapy alone cannot be an option for weight loss, with the exception of patients with severe lipodystrophy or lipoatrophy (12). A combined use of recombinant leptin with other weight loss compounds, including liraglutide, produces some positive effects (13). However, the ability to produce leptin-mediated effects in weight loss requires a significant reduction in body weight to restore a degree of leptin sensitivity prior to leptin administration. This significantly impairs a sensible and widespread use of leptin therapy in treating conventional obesity. Recently, we reported that hyperleptinemia is indeed a driving force for diet-induced obesity, as the isolated intervention involving just an increase in circulating leptin levels, either by a transgenic approach using an adipocyte-specific leptin transgene or by frequent administration of exogenous leptin, are sufficient to promote weight gain in diet-induced obese mice (14–16). In parallel, we demonstrated that reducing circulating leptin levels under obesogenic, leptin-resistant conditions can drive weight loss and provide improvements in insulin sensitivity and overall metabolic health (14). Partial leptin reduction achieved by either partial genetic deletion of the leptin gene in adipose tissue or the administration of a leptin neutralizing antibody slows down body weight gain and improves glucose tolerance. Moreover, the peripherally restricted cannabinoid receptor 1 inverse agonist JD5037 directly targets adipose tissue to reduce circulating leptin levels, mediating beneficial effects on weight loss (17). A recent report indicated that the beneficial effects of the Food and Drug Administration–approved rheumatoid arthritis drug auranofin toward improving obesity and diabetes are largely mediated by leptin lowering effects in white adipose tissue (18). On the basis of all these observations, we propose a new concept that leptin reduction under obesogenic conditions is a powerful mechanism of intervention (2,19). This new concept has great implications for the future development of novel antiobesity and antidiabetes therapies, but existing interventions that lead to weight loss can also critically rely on leptin lowering prior to inducing weight loss in order to understand their full antiobesogenic impact.

As circulating leptin levels are highly proportional to total fat mass, a significant reduction in body fat mass is always associated with a high level of circulating leptin (20). Thus, the commonly widely used antiobesity compounds, including liraglutide, orlistat, phentermine-topiramate, semaglutide, and setmelanotide, induce a substantial reduction in body fat mass, leading to a great reduction in circulating leptin levels (21). As for mechanism of action, the resulting leptin reduction is generally regarded as a consequence of weight loss or even an antagonistic driver causing weight rebound (22). This would, however, not be consistent with our current model regarding the beneficial effects of leptin reduction. Here, we aimed to systematically delineate the effects of leptin reduction in weight loss and weight rebound. We hypothesized that leptin reduction is a prerequisite for these compounds to fully unravel their weight loss potential. By focusing on two specific representatives of two distinct classes of weight reducing agents, liraglutide and FGF21, in combination with our monoclonal leptin neutralizing antibodies, we demonstrate that leptin reduction acts as a common mechanism for weight loss and improvement in metabolic disorders, including diabetes and liver fibrosis. In addition, prevention of leptin surge during weight gain can delay and partially prevent weight rebound after drug removal.

Research Design and Methods

Animals

All animal experimental protocols have been approved by the institutional animal care and use committee of The University of Texas Southwestern Medical Center at Dallas. Mice were housed under standard laboratory conditions (12 h light/dark, with lights on at 6:00 a.m.) in a temperature-controlled environment with food and water available ad libitum. Male mice were obtained from The Jackson Laboratory at ∼8 weeks of age. We selected male mice instead of female mice because male mice are prone to develop diet-induced obesity and its associated metabolic disorders, including adipose tissue inflammation, fatty liver, and liver fibrosis.

Antibody Preparation

The parental monoclonal antileptin antibody (LepAB) was isolated from a phage-displayed human single-chain fragment variable antibody library. To avoid immunogenicity against the human antibody during long-term and multiple dosing treatment in mice, we mouserized the parental LepAB. Mouserization was accomplished by using a combined Kabat/IMGT complementarity determining region (CDR)–grafting method. The VH and VL DNA sequences of the parental antibody were blasted against the mouse germline gene sequence database with IgBlast tool or IMGT/V-Quest software. The most similar mouse germline VH and VL sequences were selected as templates. The CDRs defined by Kabat/IMGT were grafted onto the framework regions of corresponding templates. The CDR-grafted VH and VL were cloned into mouse IgG1 and light chain backbone to express the full-length antibody. Antibody expression and purification were based on protocols described previously (14). LepAB and isotype control (IgG) were transiently expressed in ExpiHEK293 cells in shake flask cultures according to the manufacturer’s protocol (Thermo Fisher Scientific, Invitrogen). Briefly, variable heavy and light sequences (patent no. 17/124,481; US2021-0188970-A1) were constructed into two separate expression vectors for cotransfection and expression in HEK293 cells using polyethylenimine (Sigma-Aldrich) to mediate cell transfection. Cell culture supernatants were harvested by centrifugation at 4,000g for 10 min after 7 days of culturing in a shaker incubator with 8% CO2 and 80% humidity. Monoclonal antibodies secreted in cultures were purified using protein A affinity resin (Repligen), as described previously (14).

Mouse Treatment

After 1 week’s acclimation in the mouse room, mice were placed either on a high-fat diet (HFD) diet (60% fat; Bio-Serv) for at least 8 weeks to induce diet-induced obesity (∼50 g). Then, the mice were single housed for at least 1 week to adapt to new cage conditions. Before injection of liraglutide, FGF21, or LepAB, all the mice received PBS injections for 2 weeks to allow for reduced stress and anxiety. In these studies, we tried two different doses of liraglutide (0.025 vs. 0.1 mg/kg body weight). FGF21 was injected at a dose of 0.1 mg/kg body weight daily. LepAB was injected at a dose of 4 mg/kg body weight every other day.

Food Intake and Body Weight

To measure food intake and body weight gain, all the male mice were single housed. Before each experiment, the mice were acclimated in the single cage for at least 1 week to reduce stress. Food intake and body weight were measured before each injection.

Oral Glucose Tolerance Test

Oral glucose tolerance test (OGTT) was performed as previously described (23). For the OGTTs, mice were fasted for 4–6 h in the morning and then received oral gavage of glucose 2 g/kg body weight (dissolved in PBS) (cat. no. 806552; Sigma-Aldrich). Blood glucose was measured using a Contour glucometer.

Blood Parameters

Blood was taken from fed animals in the morning, allowed to clot, and centrifuged for 5 min at 8,000g to isolate serum for multiple analyses. Leptin and adiponectin were measured using appropriate ELISA kits (#90080 [Crystal Chem] and EZMADP-60K [Thermo Fisher Scientific]).

Quantitative RT-PCR

RNA was extracted from fresh or frozen tissues by homogenization in TRIzol reagent (Thermo Fisher Scientific) as previously described (24). cDNA was prepared using the iScript Reverse Transcription Kit (Bio-Rad), and analyses were performed using PowerUp SYBR Green Master Mix (Thermo Fisher Scientific) on a QuantStudio 6 system (Thermo Fisher Scientific). Most of the quantitative RT-PCR primers were from Harvard PrimerBank (https://pga.mgh.harvard.edu/primerbank/). The relative expression levels were calculated using the comparative threshold cycle method and normalized to the housekeeping gene Rps16.

Histology

Histology was performed as previously described (25). In brief, adipose tissue and liver tissues were collected and fixed overnight in 10% PBS-buffered formalin and stored thereafter in 50% ethanol. Tissues were further processed by The University of Texas Southwestern Molecular Pathology Core.

Statistical Analysis

All values are expressed as the mean ± SEM. The significance between the mean values for each study was evaluated using Student t tests for comparisons of two groups. One-way or two-way ANOVA was used for comparisons of more than two groups. P ≤ 0.05 was regarded as statistically significant.

Data and Resource Availability

All primary data are available upon request from the corresponding author.

Results

Liraglutide and FGF21 Induce a Rapid Reduction in Circulating Leptin Levels

To confirm observations that weight loss can reduce leptin levels, we used the GLP-1R agonist liraglutide as well as recombinant FGF21, both of which are established weight loss agents (26). Previous studies have indicated that the weight loss effects of liraglutide and FGF21 are largely mediated by central actions of these agents through the brain (27,28). Chronic treatment with liraglutide or FGF21 reduces fat mass and concomitantly reduces circulating leptin levels (29). However, whether acute exposure to either liraglutide or FGF21 can exert a similar impact on leptin remains unexplored. Diet-induced obese mice were singled housed for 2 weeks and then received one injection of liraglutide or FGF21, as shown in our research strategy (Fig. 1A). One day after injection, liraglutide, but not FGF21, significantly reduced food intake (Fig. 1B) and induced significant weight loss (Fig. 1C). Despite a great differential in food intake and acute weight loss, both liraglutide and FGF21 exerted potent effects in reducing circulating leptin levels (Fig. 1D). These results clearly indicate that acute treatment with both liraglutide and FGF21 leads to a significant reduction in leptin levels within 24 h. We also measured circulating adiponectin levels after treatment and found that FGF21, but not liraglutide, significantly increased circulating adiponectin levels (Fig. 1E), while liraglutide had no significant effects. The increased adiponectin levels were regarded as a crucial mediator for FGF21’s beneficial effects. Furthermore, we assessed Lep and AdipoQ gene expression in gonadal fat depots. We found that FGF21 could potently inhibit Lep gene expression (Fig. 1F) but had no impact on AdipoQ expression (Fig. 1G), while liraglutide did not show any significant effects in regulating Lep and AdipoQ mRNA expression within the 24-h treatment period. These results clearly indicate that liraglutide and FGF21 exert different effects within this acute setting of 24 h of treatment.

Figure 1.

Figure 1

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Acute effects of liraglutide (Lira) and FGF21 on body weight and adipokine regulation. A: Diet-induced obese mice (n = 5 per group) were single housed for 2 weeks. Then, the mice received one injection of Lira and FGF21. After 24 h, the mice were euthanized for analysis. B: Food intake during the treatment period. C: Body weight during the treatment period. D: Circulating leptin levels before and after treatment. E: Circulating adiponectin levels before and after treatment. F: Leptin expression in gonadal fat after treatment. G: AdipoQ expression in gonadal fat after treatment. H: Leptin expression in fully differentiated mature adipocytes after treatment. I: PPARλ expression in fully differentiated mature adipocytes after treatment. J: AdipoQ expression in fully differentiated mature adipocytes after treatment. K: ATGL expression in fully differentiated mature adipocytes after treatment. L: HSL expression in fully differentiated mature adipocytes after treatment. **P < .01, ***P < .001. Ctrl, control.

In addition to the in vivo studies, we isolated the stromal vascular fraction from mouse inguinal fat and fully differentiated it into mature adipocytes. We then treated the cells with liraglutide and FGF21. We found that FGF21 could potently inhibit Lep gene expression (Fig. 1H) without affecting expression of other genes, including PPARγ, AdipoQ, ATGL, and HSL (Fig. 1I–L). Regarding liraglutide, our results indicate that it did not have any significant effect on gene expression of the targets examined (Fig. 1H–L). These results further indicate that FGF21 and liraglutide may exert their differential impact on leptin reduction via different mechanisms.

Leptin Reduction as a Requirement for Liraglutide-Induced Weight Loss

As liraglutide acutely triggers a leptin reduction, we wondered whether this leptin reduction contributed to liraglutide-induced weight loss. To test this, we used doxycycline-inducible leptin transgenic mice that ectopically produced leptin from adipocytes to maintain circulating leptin levels during the period of liraglutide injection. A cohort of leptin transgenic mice (ALep) and littermate control mice were single housed and exposed to a regular HFD for several weeks to reach a body weight of ∼45 g. Control and ALep mice were subsequently switched to HFD plus doxycycline 600 mg/kg body weight to induce transgene Lep expression. After 1 week, all the mice were treated with liraglutide for 2 weeks (Fig. 2A). As expected, after 1 week’s doxycycline induction, circulating leptin levels were physiologically increased compared with control mice (Fig. 2B). Furthermore, after 2 weeks’ liraglutide treatment, circulating leptin levels were significantly reduced in control mice, while ALep mice maintained circulating leptin at the pretreatment levels (Fig. 2B). In addition, there was no significant difference in circulating adiponectin levels (Fig. 2C). During the period of liraglutide injection, daily food intake was slightly increased in the ALep mice (Fig. 2D), which was more evident when cumulative food consumption was calculated (Fig. 2E). With increasing food intake, liraglutide-induced weight loss was partially curtailed in ALep mice (Fig. 2F and G). These results indicate that leptin reduction is an integral contributor to liraglutide-induced weight loss.

Figure 2.

Figure 2

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Prevention of leptin reduction curtails the beneficial effects of liraglutide (Lira). A: ALep and control (Ctrl) mice were single housed for 1 week. Then, doxycycline 600 mg/kg body weight (DOX600) was supplemented into the HFD to induce Lep transgene expression. One week later, Lira 0.1 mg/kg body weight was injected into each mouse for 2 weeks. Body weight and food intake were measured daily. B: Circulating leptin levels before and after Lira injection. C: Circulating adiponectin levels before and after Lira injection. D: Daily food intake during Lira injection. E: Food accumulation during injection period. F: Total body weight. G: Calculated body weight loss during injection. H: Inflammatory gene expression in liver after Lira injection. I: Fibrosis markers in liver after Lira injection. Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by Student t test or one-way ANOVA compared with the Ctrl group.

Previous reports indicated that liraglutide and leptin reduction both alleviate adipose tissue inflammation and liver fibrosis (14). We wondered whether prevention of leptin reduction after liraglutide treatment would affect the inflammatory state of adipose tissue and the degree of liver fibrosis. We analyzed markers of adipose tissue inflammation and liver fibrosis in the ALep and control mice after liraglutide treatment. Our results indicated that the beneficial effects of liraglutide treatment toward improving inflammation in adipose tissue (Fig. 2H) and fibrosis in the liver (Fig. 2I) were largely impacted and deteriorated in the ALep mice. On the basis of these observations, we conclude that the liraglutide-induced leptin reduction contributes to its beneficial effects on metabolic health in addition to its impact on weight loss.

Combination of Liraglutide and LepAB Further Lowers Leptin Levels and Increases Weight Loss

If the reduction of leptin levels is indeed a major driver for liraglutide-induced weight loss, we wondered whether a higher degree of leptin reduction would lead to more weight loss in diet-induced obese mice. Previously, we described that LepAB can effectively bind leptin and reduce bioactive, circulating leptin levels. Therefore, we combined LepAB and liraglutide to achieve a higher degree of leptin reduction and examined their impact on weight loss. The treatment plan is outlined in Fig. 3A. Liraglutide by itself triggered a significant reduction in food intake. The combination of liraglutide and LepAB further reduced daily food intake and food accumulation (Fig. 3B and C). In addition, while liraglutide treatment leads to a significant reduction in total body weight as judged by weight loss in absolute terms and as a percentage of body weight, the combination of liraglutide and LepAB further potentiated these effects on weight loss (Fig. 3D–F). Besides its effects on weight loss, we have explored the impact of the combination therapy using an OGTT. Prior to treatment, all four groups showed comparable glucose tolerance. After 2 weeks of treatment, the liraglutide-treated group had significantly improved glucose tolerance, while a further improvement in glucose tolerance was observed in the liraglutide plus LepAB combination group. After EchoMRI analysis, we found that the reduced body weight was accompanied by a further reduction in fat mass, with no impact on lean mass (Fig. 3I and J). These results support the notion that a higher degree of leptin reduction in diet-induced obese mice is beneficial and synergizes with GLP-1R agonism to yield a more dramatic weight loss.

Figure 3.

Figure 3

Figure 3

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Synergistic effects of LepAB and liraglutide for food intake, body weight, and metabolic parameters. A: A cohort of diet-induced obese mice (n = 8 per group) were single housed for 1 week, then received PBS injections for 2 weeks. Then, the mice were categorized into four groups (vehicle [CtrlAB] vs. LepAB vs. liraglutide vs. LepAB + liraglutide) for 2-week treatment. B: Daily food intake during treatment. C: Food accumulation during treatment. D: Body weight change during treatment. E: Calculated weight loss by grams. F: Calculated weight loss by percentage. G: OGTT before treatment. H: OGTT after treatment. I: Fat mass measured by EchoMRI. J: Lean mass measured by EchoMRI. K: Trichrome staining of liver (scale bars = 100 μm). L: Fibrotic gene expression in liver. M: Hematoxylin-eosin (H&E) staining of subcutaneous (SubQ) fat. N: H&E staining of gonadal fat. Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.00, CtrlAB vs. LepAB, liraglutide + CtrlAB, or liraglutide +LepAB; #P < 0.05, ###P < 0.001 by Student t test or one-way or two-way ANOVA, LepAB vs. liraglutide + LepAB or liraglutide vs. liraglutide + LepAB.

In general terms, weight loss leads to improved adipose tissue health, which in turn is positively associated with improvements in liver histology and function. At the end of the treatment period, we therefore explored the impact of these treatments on liver histology and gene expression. Our results indicate that liraglutide-induced weight loss significantly reduced steatosis in the liver, and the combination of liraglutide and LepAB led to a further reduction in lipid accumulation. Importantly, despite a minimal impact on body weight and hepatic steatosis, LepAB treatment alone had a profound impact on liver fibrosis, as evidenced by a trichrome stain and hepatic expression of fibrosis markers. The liraglutide plus LepAB combination further potentiated the improvement in liver fibrosis induced by the monotherapy of LepAB and liraglutide (Fig. 3K and L). In addition, we examined the overall morphology of subcutaneous and gonadal fat after the treatment. In line with a reduction in body weight, the size of adipocytes in subcutaneous fat depots was reduced in the LepAB, liraglutide, and combination groups compared with the control group (Fig. 3M). Moreover, massive macrophage accumulation was observed in gonadal fat of the control group, which was greatly alleviated in the LepAB, liraglutide, and combination groups (Fig. 3N). These observations imply that leptin reduction not only may be an effective therapy for weight loss but also may have other applications in reversing liver fibrosis.

As LepAB could potentiate the effect of a high dose of liraglutide in food intake and weight loss, we wondered whether LepAB could act similarly in the presence of a low dose of liraglutide. We reduced the dose of liraglutide from 0.1 to 0.025 mg/kg body weight and repeated the same experiments. As expected, the lower dose of liraglutide led to reduced food intake, but the reduction level was much smaller compared with the higher dose. In addition, a combination of LepAB with low-dose liraglutide significantly reduced food intake. In line with this observation, the combination of LepAB with low-dose liraglutide significantly potentiated the effects of low-dose liraglutide on total body weight and weight loss calculated based on percentage and net loss (in grams). These observations firmly support that leptin reduction is a universal mechanism to induce weight loss and that LepAB could be combined with any weight loss strategy (Supplementary Fig. 1A–E).

FGF21 Effects Are Potentiated by Further Leptin Reduction

To further confirm the association of leptin reduction and weight loss, we looked at a second well-established weight loss mediator. We replaced liraglutide with FGF21 and assessed the impact of further leptin reduction. Previous studies have indicated that increasing FGF21 levels using a transgenic approach or exogenous administration of recombinant FGF21 leads to significant weight loss (26). Similar to liraglutide treatment, we treated diet-induced obese mice using a similar strategy, as outlined in Fig. 4A.

Figure 4.

Figure 4

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Synergistic effects of LepAB and FGF21 on food intake, body weight, and metabolic parameters. A: A cohort of diet-induced obese mice (n = 8 per group) were single housed for 1 week, then received PBS injection for 2 weeks. Then, the mice were categorized into four groups (vehicle [control or CtrlAB] vs. LepAB vs. FGF21 vs. LepAB plus FGF21) for 2-week treatment. B: Daily food intake during treatment. C: Food accumulation during treatment. D: Body weight change during treatment. E: Calculated weight loss by grams. F: Calculated weight loss by percentage. G: OGTT after treatment. H: Trichrome staining of liver. I: Fibrotic gene expression in liver. Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by Student t test or one-way or two-way ANOVA compared with either the CtrlAB, LepAB, or FGF21 group.

In contrast to liraglutide’s potent effect in reducing food intake, the effects of FGF21 on food intake were less pronounced (Fig. 4B and C), similar to our previous observations. However, the combination of FGF21 and LepAB significantly reduced daily food intake and food accumulation (Fig. 4B and C). In addition, as expected, FGF21 induced significant weight loss. The combination of FGF21 and LepAB further potentiated the effects of FGF21 on weight loss (Fig. 4D–F). Furthermore, FGF21 treatment significantly increased glucose tolerance. The combination of FGF21 and LepAB led to a further improvement in glucose tolerance (Fig. 4G). These observations further support a model in which an additional reduction in leptin levels leads to synergistic effects on weight loss.

We further examined liver function after the treatments. Similar to what we observed with liraglutide and LepAB, FGF21 greatly reduced lipid accumulation and liver fibrosis, as seen in a trichrome stain (Fig. 4H). Importantly, the combination of FGF21 and LepAB further improved liver function by reducing lipid droplet accumulation and liver fibrosis (Fig. 4H and I). In addition to the fibrosis stain, we assessed inflammatory and fibrotic gene expression in the liver. We found that the LepAB, FGF21, and combination groups had greatly reduced inflammation and fibrosis in livers (Fig. 4I). These observations further highlight the potent synergistic impact of leptin reduction on conventional interventions leading to weight loss.

Liraglutide Washout-Induced Weight Rebound Is Partially Mediated by an Early Rise in Leptin

Maintaining reduced body weight over the long term is a big challenge for weight loss therapies. In both clinical and preclinical models, upon removal of GLP-1R agonists, body weight rebounds rapidly to its initial level or an even higher level. Upon withdrawal the GLP-1R agonists, a rapid increase in circulating leptin is observed. This occurs even before any weight gain is observed (Supplementary Fig. 2). The contributions of the rapid leptin increase toward this weight rebound are not clear. As we previously reported, hyperleptinemia contributes to leptin resistance and weight gain (14), and we believe that the increase in circulating leptin levels contributes to the weight rebound upon drug removal. To directly test this, a cohort of obese mice were treated with liraglutide for 2 weeks. Upon removal of liraglutide, we applied the leptin neutralizing antibody to prevent the leptin surge upon liraglutide withdrawal (Fig. 5A). Liraglutide strongly inhibited daily food intake and food accumulation for the 14 days of treatment, with an initial strong reduction that rebounded to a higher level during the rest of the liraglutide exposure (Fig. 5B and C). After liraglutide withdrawal (indicated by the dotted lines in Fig. 5E and F), we added either LepAB or control antibody to the mice. In both cases, a dramatic increase in daily food intake was observed. The prevention of the leptin surge by leptin neutralizing antibody had a small, but significant effect on food accumulation (Fig. 5B and D). In line with this observation, the weight rebound was significantly reduced by prevention of the leptin surge (Fig. 5E and F). In addition, administration of LepAB significantly improved glucose tolerance after liraglutide removal (Fig. 5G). These observations indicate that the leptin surge after liraglutide removal significantly contributes to the weight surge and the associated deteriorated glucose tolerance. This is also reflected in expression levels of a number of different genes at the level of visceral adipose tissue (Fig. 5H).

Figure 5.

Figure 5

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Effects of leptin reduction in liraglutide (Lira) removal–induced weight gain. A: A cohort of diet-induced obese mice (n = 8 per group) were single housed for 1 week, then received PBS injection for 2 weeks. Then, all the mice received Lira injection for 2 weeks. After that, Lira was switched to LepAB for another 2 weeks. B: Daily food intake during treatment. C: Food accumulation during Lira injection. D: Food accumulation during LepAB injection. E: Body weight change during treatment. F: Calculated weight loss by grams during treatment. G: OGTT after LepAB treatment. H: Gene expression in visceral fat after LepAB treatment. Data are mean ± SEM. *P < 0.05, **P < 0.01 by Student t test or one-way ANOVA compared with the vehicle(CtrlAB) group.

Leptin Reduction Slows Down Weight Rebound After FGF21 Withdrawal

To further confirm the findings made with liraglutide, a similar experiment with FGF21 was performed (Fig. 6A). Compared with liraglutide, FGF21 did not have a significant effect on daily food intake and food accumulation, as previously reported (26). However, upon FGF21 withdrawal, a significant increase in daily food intake was observed (Fig. 6B and C). Reduction of leptin with LepAB treatment decreased the surge in daily food intake and food accumulation (Fig. 6B and D). In line with the reduction in food intake, LepAB treatment slowed down the weight rebound (Fig. 6E–G). In addition, the mice that received LepAB treatment had improved OGTT results (Fig. 6H). All these observations further confirm that prevention of leptin surge slows down weight rebound upon cessation of the intervention and highlight the beneficial effects of leptin reduction during drug withdrawal.

Figure 6.

Figure 6

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Effects of leptin reduction in FGF21 removal–induced weight gain. A: A cohort of diet-induced obese mice (n = 8 per group) were single housed for 1 week, then received PBS injection for 2 weeks. Then, all the mice received FGF21 injections for 2 weeks. After that, FGF21 was switched to LepAB for another 2 weeks. B: Daily food intake during treatment. C: Food accumulation during FGF21 injection. D: Food accumulation during LepAB injection. E: Body weight change during treatment. F: Calculated weight gain by grams during treatment. G: Calculated weight gain by percentage during treatment. H: OGTT after LepAB treatment. Data were mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by Student t test or one-way ANOVA compared with the vehicle(CtrlAB) group.

Enhanced Leptin Signaling in the Hypothalamus Contributes to Weight Loss Upon Leptin Reduction

Our previous reports have indicated that enhanced leptin signaling in the hypothalamus accounts for the beneficial effects of leptin reduction in weight loss (14). Here, we aimed to delineate the underlying mechanisms associated with leptin reduction and weight loss. We performed phosphorylated STAT3 (p-STAT3) staining in the whole brain. Our results clearly indicate a significant increase in acute leptin-induced p-STAT3 staining in the hypothalamus in mice that received leptin neutralizing antibody with either liraglutide or FGF21 compared with that of liraglutide or FGF21 monotherapy (Fig. 7A and C). We also checked the p-STAT3 expression in the area postrema region and found a minimal signal and observed no differences, indicating that this part of the brain is unlikely to be involved in the differential response (Fig. 7B and D). On the basis of these observations, we believe that hypothalamic leptin action is a key component of centrally mediated effects and complements the direct peripheral consequences of reduced leptin action. Further studies are needed to address the broader impact on a wider array of brain regions not examined here.

Figure 7.

Figure 7

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Effects of combination therapy in leptin signaling in the mediobasal hypothalamus and area postrema (AP) region. A: Staining of p-STAT3 in hypothalamic region after treatment with either liraglutide or liraglutide + LepAB. B: Staining of p-STAT3 in the AP region after treatment with either liraglutide or liraglutide + LepAB. C: Staining of p-STAT3 hypothalamic region after treatment with either FGF21 or FGF21 + LepAB. D: Staining of p-STAT3 in the AP region after treatment with either FGF21 or FGF21 + LepAB. Magnification x1.5.

Discussion

By taking advantaging of multiple transgenic mouse models (adipocyte-specific leptin overexpressing mice; adipocyte-specific leptin deletion mice), as well as pharmacological interventions (the GLP-1R agonists liraglutide and FGF21), we demonstrate that a reduction in circulating leptin levels contributes to FGF21- and liraglutide-induced weight loss and improvements in metabolic health. Emerging evidence suggests that GLP-1 treatment in mice may also require an intact leptin pathway, since ob/ob or db/db mice are mostly refractory to weight loss. Moreover, the leptin surge after treatment cessation is an underestimated driver for the weight rebound, underscoring that a reduction of leptin per se (i.e., even prior to any weight loss) is a direct driver of reduced fat mass. As such, the ability to reduced leptin expression is an integral part of the mechanism by which these compounds achieve weight loss. On the other hand, cessation of treatment with liraglutide and FGF21 leads to an acute increase in leptin expression, which is a driver of subsequent weight gain. Together, these findings substantiate our previously made statements that leptin is not a mere passenger to obesity but, rather, a direct driver for weight gain (14).

Leptin therapy has been successfully applied to treat patients with lipodystrophy and has been proven to be very useful for the small group of individuals with obesity with low circulating leptin levels (30,31). In the general population, a subset of individuals with obesity maintains a low level of leptin, which helps to sustain a higher degree of leptin sensitivity (32). These individuals could indeed benefit from leptin therapy. In contrast, individuals with high circulating leptin levels uniformly display severe leptin resistance and lack any response to leptin treatment. To counteract the severe leptin resistance, interventions that lead to a leptin reduction constitute an alternative strategy to restore leptin sensitivity and achieve weight control.

In this study, we investigated two commonly used compounds (liraglutide and FGF21) with entirely different underlying mechanisms to induce weight loss. However, both had an impact on the leptin axis in common. On the basis of these observations, we can conclude that leptin neutralizing antibody is well tolerated and can be combined with any weight loss therapies to induce higher degrees of weight reduction.

Another exciting finding is that leptin reduction may be an alternative strategy toward reversing liver fibrosis, independent of weight loss. Prolonged HFD exposure leads to severe fatty liver and a degree of liver fibrosis in obese mice (33,34). Our observations clearly indicate that even after a short-term treatment (as little as 2 weeks) with LepAB, liver fibrosis is significantly improved. Whether the beneficial effects of leptin reduction on liver fibrosis is mediated through its actions on the central nervous system or a direct peripheral effect is currently unknown. Previous studies have suggested that leptin is a profibrotic factor (35,36). Increasing leptin levels strongly promotes the development of liver fibrosis (37). Several mechanisms have been proposed to explain these effects of leptin on liver fibrosis. Whether leptin reduction works via these pathways needs to be examined further (38). Meanwhile, our previous studies have shown that mice deficient in adiponectin develop severe liver fibrosis in the absence of lipid accumulation in the liver (39). This highlights the important role that adipose tissue and adipokines play in this process, with a configuration of high leptin and low adiponectin predisposing the liver to extreme fibrosis.

A big question regarding interventions that lead to a reduction in leptin is what degree of leptin reduction should be aimed for. Would near-complete leptin reduction in obese mice mimic a complete leptin-deficient state, leading to weight gain rather than weight loss, as seen in ob/ob mice (40)? Alternative studies have indicated that complete inhibition of leptin signaling (e.g., by using leptin receptor antagonists) leads to weight gain in obese mice, substantiating that leptin signaling remains active even in obese mice (41). However, targeting circulating leptin is entirely different from the effects of leptin receptor inactivation. The continuous production of leptin from adipose tissue fuels the system and creates a unique situation with a limited but constant supply that an antibody does not completely scavenge. The lower concentrations of leptin lead to an actual leptin sensitization in the central nervous system, while peripheral leptin signaling is reduced in parallel. Circulating leptin levels in obese mice are up to 10 times higher compared with what is seen in lean mice. As such, leptin reduction by 90% still falls into the normal physiological range, so the opportunity to create a complete leptin-deficient mouse with a neutralizing antibody is unlikely and has not been achieved in our hands. On the basis of this wide therapeutic window, we believe that leptin reduction therapy represents a safe approach to treat obesity and its associated metabolic disorders, including insulin resistance and fatty liver disease. The absence of any apparent side effects renders this approach a powerful tool for combination therapies for existing interventions, as demonstrated here.

This article contains supplementary material online at https://doi.org/10.2337/figshare.24498310.

Article Information

Funding. This work was supported by National Institutes of Health (NIH) grants R01-DK55758, R01-DK127274, R01-DK131537, R01-DK099110, P01-AG051459, and RC2-DK118620 to P.E.S. and R01-DK118725 and R01-DK088423 to J.K.E. S.Z. was supported by NIH grant R00-AG068239 and S.Z. holds a Voelcker Fund Young Investigator Pilot Award from the Max and Minnie Tomerlin Voelcker Fund. Y.Z. was supported by NIH grants R01-DK136619 and R01-DK136532. Q.Z. was supported by an American Heart Association career development award. W.X., N.Z., and Z.A. were supported by the grants from the Cancer Prevention and Research Institute of Texas (RP220032, RP150551, and 632 RP190561) and the Welch Foundation (AU-0042-20030616).

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. S.Z. wrote the original draft of the manuscript. S.Z., N.L., G.L., S.H., Z.Z., Q.Z., N.J., C.I., Y.Z., and M.-Y.W. contributed to the investigation. S.Z., N.L., and Q.Z. designed the methodology. S.Z. and P.E.S. conceptualized the study. W.X., N.Z., and Z.A. generated the antibodies. X.H., C.S.-H., C.K., J.K.E., and P.E.S. contributed to writing, reviewing, and editing the manuscript. P.E.S. provided supervision. P.E.S. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Prior Presentation. Parts of this study were presented in oral form at the 83rd Scientific Sessions of the American Diabetes Association, San Diego, CA, 23–26 June 2023.

Funding Statement

This work was supported by National Institutes of Health (NIH) grants R01-DK55758, R01-DK127274, R01-DK131537, R01-DK099110, P01-AG051459, and RC2-DK118620 to P.E.S. and R01-DK118725 and R01-DK088423 to J.K.E. S.Z. was supported by NIH grant R00-AG068239 and S.Z. holds a Voelcker Fund Young Investigator Pilot Award from the Max and Minnie Tomerlin Voelcker Fund. Y.Z. was supported by NIH grants R01-DK136619 and R01-DK136532. Q.Z. was supported by an American Heart Association career development award. W.X., N.Z., and Z.A. were supported by the grants from the Cancer Prevention and Research Institute of Texas (RP220032, RP150551, and 632 RP190561) and the Welch Foundation (AU-0042-20030616).

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