Introduction

Glucagon-like peptide 1 (GLP-1) is a satiation signal synthesized predominantly in two locations in the body: L cells in the intestine and preproglucagon (PPG) neurons in the caudal brainstem ¹. GLP-1, released in the gut during a meal, acts as an incretin hormone whose dominant physiologic effects are to increase glucose clearance and enhance satiation ². These actions make the GLP-1 system an attractive target to treat people with obesity and type 2 diabetes (T2D). Accordingly, long-acting GLP-1 receptor (GLP-1R) agonists were the fastest growing class of anti-diabetic therapeutics in the past decade and offer patients improvements in glycemic control and weight loss ^3,4. More recently, development of next generation co-agonist strategies targeting multiple hormone systems have been the focus of some academic labs and pharmaceutical companies, with GLP-1R agonists serving as a favorable foundation to pair with a co-agonist ⁵. To that end, glucose-dependent insulinotropic polypeptide (GIP), another GI-derived incretin hormone, has emerged as a natural and promising partner for GLP-1. Indeed, dual GIP/GLP-1 receptor agonism in pre-clinical models and clinical trials shows superior glucose-lowering and greater weight loss when compared to that of GLP-1R agonism ^6–9. Therefore, there is major interest in understanding how GIPR agonism contributes to the enhanced therapeutic profile of dual GIP/GLP-1 receptor agonists.

In pre-clinical models maintained on a diet of exclusively either chow or high-fat diet (HFD), GLP-1R agonism decreases intake of either diet ^10–12. More impressively however, GLP1-R agonist treatment in rats with ad libitum access to both chow and a highly palatable diet causes a shift in food preference to reduce intake of the highly palatable diet in favor of the chow diet ^13–15. Given that over consumption of highly palatable food is linked with the development and perpetuation of obesity and T2D ¹⁶, these aforementioned findings are extremely provocative when thinking about therapeutic strategies to treat these metabolic diseases by limiting the intake of energy dense palatable foods.

The GIP receptor (GIPR) is expressed centrally in nuclei of relevance to energy balance and peripherally administered GIP analogues activate central GIPRs to reduce caloric intake ^17–20. In fact, GIPR activation is required for the synergistic effects of dual GIP/GLP-1 receptor agonism on weight loss and appetite suppression ²⁰. Despite these findings, the role of GIPR agonism alone or in combination with GLP-1 agonism on diet macronutrient preference has not been explored. Indeed, the mechanisms underlying the enhanced effects of GIPR/GLP-1R co-agonism are far from fully elucidated ⁵. The GIP component of this co-agonist pair has recently been found to provide unique benefits including, but not limited to, blockade of GLP-1-induced nausea and vomiting, weight-independent insulin sensitization, augmented insulin secretion, and lowering of triglycerides through a mechanism distinct from selective GLP-1RAs ^21–25. Thus, GIPR agonism may modify or enhance the effects of GLP-1R agonism on food choice and macronutrient intake. As the unimolecular dual GIP and GLP-1 receptor agonist tirzepatide is currently in phase 3 clinical trials and is under regulatory review for indication of T2D in the US, Japan, and EU ^8,26, how this investigational agent interacts with both receptor systems on food choice is critical to understanding its regulation of food intake, metabolism, and energy homeostasis. This work examines how individual and combined GLP-1R and GIPR agonism modulates preference for low and high palatable foods and the contribution of specific macronutrients in these preference choices.

Methods and Methods

Results

We first investigated the effect of selective GIPR agonism or dual GIPR/GLP-1R agonism on ad libitum choice intake between chow and high-fat diet (HFD). Mice received daily administration of vehicle, a long-acting GIPR agonist GIP-085, or the dual GIP/GLP-1 receptor agonist tirzepatide for 5 days. To determine whether the treatment effect differed based on the content of fat in the diet, one group of mice had access to chow and 40% HFD and a second group of mice had access to chow and 60% HFD. In mice given a choice of chow or 40% HFD, there was an overall effect of chow intake to differ by treatment group (Fig. 1A). Specifically, tirzepatide increased chow intake on days 3 and 5 compared to GIP-085 (Fig. 1A). Additionally, tirzepatide decreased daily 40% HFD intake compared to GIP-085 and vehicle for the first 3 days and compared to GIP-085 on day 5 (Fig. 1B). For mice given a choice of chow or 60% HFD, tirzepatide increased daily chow and decreased daily 60% HFD intake compared to GIP-085 and vehicle treated mice across all 5 days (Figs. 1C–1D). For both diet choice experiments, tirzepatide increased 5-day cumulative chow intake and decreased 5-day cumulative HFD intake compared to vehicle and GIP-085, and tirzepatide suppressed cumulative intake of 60% HFD more than 40% HFD (Figs. 1E–1F). In both the 40% and 60% HFD choice paradigms, tirzepatide treated mice lost weight over the 5 treatment days and tirzepatide-induced weight loss was more robust in mice on the 60% HFD vs the 40% HFD choice diet (Fig. 1G).

To examine how GIPR and GLP-1R agonism altered total diet composition, we assessed daily average Kcal intake of both chow and HFD as absolute intake and as a % of total intake. Tirzepatide more effectively suppressed average HFD intake in mice with a choice of 60% HFD compared to 40% HFD, both as absolute intake (Fig. 1H) and when expressed as a % of total intake (Fig. 1I). Moreover, while tirzepatide increased average chow intake in both choice diets, the percent of chow intake was significantly higher for mice paired with 60% HFD (79 ± 3.1 %) versus 40% HFD (36.7 ± 8.8 %).

To help interpret these data, we determined the pharmacokinetic profile of tirzepatide in mice. Following a single subcutaneous administration of 625 nmol/kg tirzepatide, the maximum concentration was achieved by 12 hours post dose. The elimination half-life of tirzepatide was approximately 12 hours and the mean clearance of tirzepatide was 8.72 mL/hr/kg (Table 1).

To determine whether the tirzepatide treatment-induced preference shift was an acute response or persisted during chronic treatment, we next treated mice on a choice diet of chow or 60% HFD with vehicle or tirzepatide for 14 days. Tirzepatide treatment elevated daily and 14-day cumulative chow intake, suppressed daily and 14-day cumulative 60% HFD intake, and caused weight loss over the study period (Figs. 2A–2E). The composition of total average daily intake was dramatically shifted in tirzepatide treated mice. Dual GIPR/GLP-1R agonist treatment increased the consumption of chow and decreased the intake of 60% HFD when expressed as absolute Kcal and as a % of total intake (Fig. 2F–2G). In fact, while vehicle treated mice chose to eat only 4.7 ± 1.0 % of Kcal from chow, tirzepatide treated animals increased chow intake to make up 66.1 ± 15.3 % of total Kcal consumed.

Because dual GLP-1R/GIPR agonist therapeutics are being evaluated as anti-obesity agents in phase 3 clinical trials, we next wanted to compare the actions of tirzepatide on diet preference in obese rats. To select an effective dose of tirzepatide, we first performed a dose response study in diet-induced obese (DIO) rats maintained on only 60% HFD diet. Of the doses tested (0.3, 1, 3, 10, 30 nmol/kg), 10 nmol/kg consistently reduced daily and cumulative food intake and induced weight loss (Figs. 3A–3C). Following a single subcutaneous dose of tirzepatide at 31 nmol/kg and 104 nmol/kg, the mean apparent clearance and mean half-life in rats were 8.31 mL/h/kg and 9.3 h and 10.3 mL/h/kg and 10.3 h, respectively (Table 2). Using the gubra DIO model, rats were fed a choice diet of chow and a highly palatable fat- and sugar-rich diet (gubra diet) composed of equal amounts of the chocolate spread Nutella, peanut butter and powdered chow ¹³. Following 19 weeks on this choice diet, rats received daily administration of vehicle, tirzepatide, or the well-known anti-obesity agent and sympathomimetic sibutramine for 21 days.

While daily chow and gubra diet intake varied among treatment groups over the 3-week period, all drug treatments suppressed intake of the gubra diet for the first 9-days compared to vehicle (Figs. 3D–3E). Dual GIPR/GLP-1R agonism most consistently decreased gubra diet intake, as tirzepatide treated rats ate less gubra diet than vehicle treated rats on 16 of the 21 study days (Fig. 3E). Tirzepatide treatment increased 21-day cumulative chow intake compared to sibutramine treatment, but neither group was different from vehicle (Fig. 3F). Both drug treatments suppressed 21-day cumulative gubra diet intake and body weight change relative to vehicle treated rats (Figs. 3G–3H). Average daily gubra diet intake was decreased by sibutramine treatment compared to vehicle and further suppressed by tirzepatide (Fig. 3I). However, when looking at each diet choice as a percentage of total average daily intake, only tirzepatide treated rats showed a shift in diet preference to eat more chow and less gubra diet than the vehicle group (Fig. 3J). Although not significant, sibutratmine treatment decreased chow intake (3.6 ± 1.2 % of total Kcal versus 9.7 ± 1.3 % of total Kcal for vehicle) while the tirzepatide treatment did not (23.0 ± 4.5 % of total Kcal). Thus, while sibutramine suppressed intake of both food choices, tirzepatide selectively suppressed gubra diet intake. Whole body composition measured at baseline and during week 3 of treatment determined that while both drug treatment groups lost fat mass and gained lean mass as a percentage of body weight relative to their baseline levels, only tirzepatide treated rats had lower fat mass and elevated lean mass relative to vehicle treated rats at week 3 (Figs. 3K–3L).

We’ve established that dual GIP/GLP-1 agonism selectively suppresses intake of the high-fat high-sugar palatable diet in a choice paradigm. However, as HFD and the gubra diet both have elevated sucrose and lipid content we wanted to determine whether this suppression was selective towards a certain palatable macronutrient. To test the individual and combined roles of GIPR and GLP-1R activation on altered preference for lipid and carbohydrate intake, rats were placed on an ad libitum diet with access to chow, Crisco, a 10% sucrose solution, and water for 8 days. Rats were treated daily with vehicle, the long-acting GIPR agonist GIP-085, a long-acting GLP-1R agonist (GLP-140) alone, or a combination of GIP-085 and GLP-140. In this case, having two separate molecules instead of a single dual GIP/GLP-1 analog allowed us to evaluate the effects of each individual component separately. Daily and 8-day cumulative chow intake was not different between treatment groups (Figs. 4A–4B). Starting on day 2 of treatment, daily intake of Crisco as well as 8-day cumulative Crisco intake was substantially reduced in GLP-140 and combo treated rats relative to vehicle and GIP-085 treated rats (Figs. 4C–4D). Interestingly, sucrose intake was not significantly affected by any drug treatment (Figs. 4E–4F). GLP-140 treatment increased 8-day cumulative water intake compared to vehicle and GIP-085 treatment (Figs. 4G–4F). GLP-140 and the combo treatment suppressed cumulative weight gain relative to vehicle and GIP-085 treatment (Fig. 4I).

Comparing absolute average daily Kcal intake of chow, Crisco, and sucrose, GLP-140 and combo treatment decreased the consumption of Crisco compared against vehicle or GIP-085, but there was no difference between treatment groups in the Kcal intake of chow or sucrose (Fig. 4J). However, when comparing the Kcal intake from chow, Crisco, and sucrose as a % of total Kcal intake, GLP-140 and combo treatment decreased intake of Crisco and increased intake of chow relative to vehicle and GIP-085 treatment (Fig. 4K). The percentage of total Kcal intake made up by sucrose was similar between all treatments. While vehicle and GIP-085 treated rats consumed roughly 25% less Kcals from sucrose than Crisco (Sucrose:Crisco ratio 0.75 ± 0.12 vehicle, 0.77 ± 0.16 GIP-085), GLP-140 and combo treated rats consumed over 800% more Kcals from sucrose than Crisco (Sucrose:Crisco ratio 13.93 ± 2.81 GLP-140, 8.59 ± 1.85 combo). Thus, rats treated with GLP-140 and the combination of GLP-140/GIP-085 robustly favored the intake of carbohydrates over lipids.

In rats and mice, we’ve observed that treatment with a GIPR agonist had no impact on food preference (Figs. 1 and 4). Furthermore, combined GIP/GLP-1 receptor agonism did not differentially suppress intake of Crisco beyond that seen with GLP-1R agonism. To determine whether the effects of dual GIPR and GLP-1 receptor agonism treatment on choice intake are mediated by engagement of the GLP-1R, we treated WT and GLP-1R knockout mice (GLP-1RKO) with ad libitum access to chow and 60% HFD with vehicle or tirzepatide for 5 days. Tirzepatide treated WT mice ate more chow than tirzepatide treated GLP-1RKO mice on treatment days 2–5, and more than vehicle treated WT mice on days 3 and 4 (Fig. 5A). Daily intake of 60% HFD was decreased by tirzepatide treatment in WT mice relative to tirzepatide treated GLP-1RKO mice and vehicle treated WT mice on all 5 days (Fig. 5B). On no day was chow or 60% HFD intake for tirzepatide treated GLP-1RKO mice different from vehicle treated mice. Tirzepatide treatment in WT mice increased 5-day cumulative chow intake, decreased cumulative intake of 60% HFD, and promoted cumulative body weight loss compared to all other treatment groups (Figs. 5C–5E). In WT mice, tirzepatide treatment increased the contribution of chow and respectively decreased the contribution of 60% HFD when expressed as absolute Kcal and as a % of total intake (Figs. 5F–5G).

Discussion

The food intake suppressive effects of dual GIP and GLP-1 receptor agonism have been investigated only in rodents maintained on one type of diet ^6,9,19. However, this does not model the complexity of food choices in human eating behavior. Understanding how GIPR/GLP-1R co-agonism influences the preference for low and high palatability foods and the role of different macronutrients in these choices will help inform the effects of this treatment on energy homeostasis in clinical applications. Herein, we show for the first time that in mice and rats, treatment with the dual GIPR and GLP-1R agonist tirzepatide simultaneously reduced the intake of a palatable high-fat/high-sugar diet and increased the consumption a low-fat chow diet (Figs. 1–3). However, utilizing two different experimental approaches we found that GIPR agonism does not contribute the effect of tirzepatide on food choice in mice or rats. Specifically, we found that dosing a GIPR agonist alone had no impact on food or macronutrient choice (Figs. 1 and 4), and the effect of tirzepatide on food preference was absent in GLP-1RKO mice (Fig. 5). Together, these data suggests that despite GIPRAs established synergistic effect with GLP-1RAs to decrease overall caloric intake ^7,28, in this choice paradigm GIPR agonism does not modify the action of GLP-1R agonism to strongly favor consumption of a low-palatability chow diet despite access to a highly palatable HFD.

GLP-1R agonists have been shown to promote intake of chow over high-fat/high-sugar diets or even candy ^13–15. One study using the gubra DIO model reported that the GLP-1R agonist liraglutide decreased intake of the gubra diet and simultaneously increased intake of chow ¹³. In our study following the gubra DIO choice paradigm, we found that tirzepatide also increased chow intake and suppressed gubra diet intake, supporting that both GLP-1R agonism and dual GIPR/GLP-1R agonism selectively suppresses intake of palatable food under choice conditions. In fact, the magnitude of preference shift induced by liraglutide and tirzepatide in the gubra choice DIO model are very similar. Compared to controls, both liraglutide and tirzepatide decreased cumulative gubra diet intake by approximately 60% and approximately doubled cumulative chow intake ¹³. This supports the hypothesis that the effect of tirzepatide on food preference may be driven by GLP-1R agonism. Interestingly, we observed that tirzepatide more robustly shifted food preference away from HFD in mice when given a choice of chow and 60% HFD, compared to mice given a choice of chow and 40% HFD (Fig. 1). In agreement with these findings, treatment with the GLP-1R agonists in rats maintained on low-fat (17%), medium-fat (40%) or high-fat (81%) diets elicits a stronger and longer-lasting anorectic response and body weight loss as the percentage of fat in the diet increases ¹². Collectively, these data suggest that GLP-1R activation may strongly regulate intake of lipids.

To distinguish the effects of individual or combined GIPR/GLP-1R agonism on preference of the high-fat versus high-sugar component of these diets, we tested rats with choice access to lipid (Crisco), carbohydrate (10% sucrose solution), and chow (Fig. 4). GLP-1R agonism alone or in combination with GIPR agonism selectively and effectively inhibited intake of Crisco while having no effect on sucrose intake and increased the contribution of chow to total Kcals consumed. Again, GIPR agonism alone did not alter food preference and had no effect to modify the actions of GLP-1R agonism. Vehicle and GIPR agonist treated rats consumed on average 30% more daily Kcals from Crisco than the sucrose solution. In contrast, GLP-1R agonist and combo treated rats vastly preferred sugar, consuming a daily average of 8–14 times more Kcals from sucrose solution than Crisco.

One study testing the role of liraglutide on macronutrient preference in rats presented with a cafeteria diet that included a low-fat low-sugar, low-fat high-sugar, high-fat low-sugar, and high-fat high-sugar option found that while GLP-1R agonism decreased total Kcals consumed, the intake of any one food type or macronutrient was not significantly affected ²⁹. Differences between the results of Hyde et al. and our findings that GLP-1R agonism and GIPR/GLP-1R co-agonism do selectively suppress fat intake could be explained by several experimental differences in food choice, exposure paradigm, route of administration, drug, and dose. In addition to macronutrient composition, the taste, smell, and consistency of a diet likely influence the animal’s preference for consumption of that food. However, evidence in humans also support that GLP-1R agonists most strongly decreases intake of fat in a diet. Indeed, treatment with GLP-1R agonists in people with obesity, T2D, or type 1 diabetes not only show decreased overall food intake and body weight, but show specific reductions in the % of Kcals consumed from fat and a lower preference for high-fat foods ^30–32. Fat is more energy dense (9 kcal/g) than carbohydrates or protein (4 kcal/g), and reducing the energy density of meals has been shown to induce weight loss even when people are not told to restrict calories ³³. Thus, diminished caloric intake through specific reduction of fats in a diet may contribute to GLP-1R agonist-induced weight loss.

Food choices following GLP-1R agonist treatment could be a consequence of reduced preference for lipids or an increased preference for carbohydrates. Similarly, these effects could be due in part to an altered perceived taste and reward encoded valuation of these macronutrients that may influence these altered food choices. Liraglutide treatment in T2D patients not only decreased the pleasure response for fatty foods but increased their sensitivity for sweets ³⁴. Interestingly, in addition to the brainstem preproglucagon neurons and L cells in the intestine, GLP-1 is also synthesized locally in taste bud cells of the tongue and modulates taste perception through GLP-1Rs expressed on neighboring taste nerve fibers ³⁵. GLP-1 increases taste sensitivity to solutions of sucrose but not lipids, and surprisingly the addition of lipids to a sucrose solution further enhances the GLP-1R dependent attraction for sucrose ³⁶. When rats are presented with only a sucrose solution or water, GLP-1R agonists do decrease intake of sucrose ^15,37. Thus, while GLP-1R agonists may increase the sensitivity for sweet tastes, they don’t overtly increase the consumption of sugar. One explanation could be that given a choice between high-fat or high-sugar foods, GLP-1R agonists induce a preference for carbohydrates over lipids. However, the contribution of lingual GLP-1R signaling in food choice induced by systemic delivery of a GLP-1R agonist has not been directly investigated.

Central GIPRs and both peripheral and central GLP-1Rs mediate the anorectic actions of these hormones ^10,20. Two feeding relevant nuclei that express both receptors and have circumventricular access to circulating ligands are the dorsal vagal complex in the brainstem and the hypothalamus ^21,38,39. Interestingly, GIPR agonism attenuates GLP-1 induced neuronal activation in the hindbrain dorsal vagal complex, while subthreshold doses of GIP and GLP-1 synergistically increase hypothalamic neuronal activation ^19,21. While these nuclei are involved in the hypophagic effects of these hormones, the hindbrain and hypothalamus primarily regulate homeostatic feeding behavior and are unlikely to directly regulate palatable food preferences. Third ventricle exendin-4 administration, which bathes both the hypothalamus and hindbrain, similarly suppress intake of diets with a low or high fat content ¹². Higher-order feeding relevant nuclei like the ventral tegmental area (VTA) and nucleus accumbens (NAc) both express GLP-1Rs, receive PPG neuron projections from the nucleus of the solitary tract in the hindbrain, and strongly regulate palatable food intake ^15,40. In fact, direct injections of a GLP-1R agonist into the VTA or NAc decreases intake of HFD and increase intake of chow in rats on a choice diet ¹⁵. Furthermore, GLP-1R antagonist treatment in the VTA partially blocks peripheral exendin-4 induced hypophagia in rats on a HFD, suggesting that direct VTA GLP-1R signaling is potentially clinically relevant for food intake control ⁴¹. Systemically administered exendin-4 was recently demonstrated to penetrate the brain to the mesolimbic reward system and bind VTA GLP-1Rs ^41,42. The mechanism by which peripherally administered GLP-1R/GIPR co-agonists regulate palatable food intake, and specifically suppress lipid intake, may very well involve direct and/or polysynaptic routes to modulate the mesolimbic reward system ^15,42,43. Future work utilizing GLP-1 and GIP receptor knockout/knockdown models in combination with targeted antagonism of GLP-1Rs will be necessary to elucidate the site of actions underlying incretin regulation of macronutrient preference.

This work is the first to investigate the role of GIPR agonism and dual GIPR/GLP-1R agonism on changes in food preference. Although we report that GIPR agonism does not affect food choice and does not contribute to the tirzepatide mediated preference shift in food intake, possible GIPR-specific effects may be evident under more chronic experimental conditions. As this work focused on the regulation of palatable macronutrients, we specifically investigated the effects of GIPR/GLP-1R agonism on lipid and sugar intake but did not investigate any possible regulation of protein intake. Still, these findings demonstrate that GIP/GLP-1 co-agonism promotes the consumption of healthier food choices over more palatable high-fat high-sugar foods. Given some of the emerging metabolic and antiemetic benefits of GIPR/GLP-1R co-agonism ^21,22, understanding their regulation of food preference is pertinent to improving the control of food intake, body weight, and metabolic health of people with obesity and T2D.

Introduction

Metabolic dysfunction-associated steatotic liver disease (MASLD) encompasses a range of disorders characterized by excessive accumulation of triglyceride (TG) in hepatocytes, replacing the previous term non-alcoholic fatty liver disease (NAFLD), which relies on exclusionary confounder terms¹. MASLD progresses from TG accumulation (metabolic dysfunction-associated steatotic liver, MASL) to inflammation and hepatocellular injury (metabolic dysfunction-associated steatohepatitis, MASH), hepatic fibrosis, and, ultimately, cirrhosis and/or hepatocellular carcinoma (HCC)². MASLD is a rapidly growing health concern³ that has made an increasing contribution to end-stage liver disease in recent years, thus imposing a substantial socioeconomic burden^4–6. Owing to the lack of suitable MASLD animal models, limited studies have focused on the exploration of the underlying mechanisms and treatment strategies⁷. Therefore, establishment of an animal model that adequately reflects the entire spectrum of MASLD remains a crucial unmet need.

Patients with metabolic disorders, such as type 2 diabetes mellitus (T2DM) and obesity, have a higher prevalence of MASLD and exacerbated risk of developing MASH, advanced fibrosis (defined as fibrosis stage ≥3), and HCC^8–10. In a recent study on T2DM patients who had undergone liver biopsies, MASH and advanced fibrosis were detected in 58% and 38% cases, respectively¹¹. Another study reported a higher incidence of MASLD and advanced fibrosis in T2DM patients with obesity compared to those without¹². However, the mechanism underlying the rapid progression of MASLD in individuals with T2DM and obesity is not well understood.

From the therapeutic aspect, pharmacological interventions based on lifestyle may be effective in blocking pathological progression in T2DM patients with obesity, since MASLD develops rapidly in this population¹³. Tirzepatide, a dual glucose-dependent insulinotropic polypeptide/glucagon-like peptide-1 receptor agonist (GIP/GLP-1 RA), is a commonly used therapeutic agent for both T2DM and obesity^14–16, with clinically proven efficacy against MASL¹⁷. However, the issue of whether continuous use of tirzepatide can prevent the development of advanced fibrosis or HCC remains to be resolved. Furthermore, there is a paucity of evidence regarding the optimal time to administer tirzepatide across the broad spectrum of MASLD.

In this study, we developed a mouse model of MASLD encompassing the whole spectrum of the disease, including MASL, MASH, advanced fibrosis, and HCC. The model was generated by treatment with low-dose streptozotocin (STZ) in conjunction with a high-fat diet (HFD) and utilized to demonstrate the therapeutic efficacy of tirzepatide against MASH, fibrosis, and HCC.

Results

Discussion

In this study, we established a murine MASLD model with histopathological characteristics encompassing the full spectrum of MASLD using low-dose STZ and HFD. Notably, from 32 weeks of age, STZ + HFD mice developed MASH and significant fibrosis. At 38 weeks, some of STZ + HFD mice developed HCC which was observed in all mice up to 68 weeks. The transcriptomic features of our model closely reflected those of MASH and MASLD-related HCC in patients. Furthermore, we demonstrated that interventions at various stages of MASLD using dietary changes or tirzepatide treatment improved MASH, hepatic fibrosis, and hepatic tumorigenesis.

The coexistence of metabolic dysfunction with the whole spectrum of MASLD, observed in the clinical setting in patients³¹, is a distinguishing feature of our animal model. STZ + HFD mice showed body weight gain and metabolic dysfunction including increased fat mass, insulin resistance, dyslipidemia, and visceral adipocyte hypertrophy and inflammation. Previous MASLD models based on STZ and HFD had limitations in that they did not show the whole spectrum of MASLD or metabolic dysfunction^32,33. In one study, mice were fed HFD for 15 weeks and then administered STZ at a dose of 65 mg/kg for two consecutive days³². The study found that hepatic fibrosis was increased in the group that received both HFD and STZ compared to those administered HFD or STZ only, but advanced fibrosis or MASLD-related HCC was not induced. In another study, 200 μg STZ was administered to mice two days after birth, and HFD was provided from 4 weeks of age (STAM mice)³³. Although STAM mice showed rapid MASH progression and HCC, they lacked the characteristics of metabolic dysfunction, such as obesity and insulin resistance. STAM mice developed microvesicular steatosis and mild zone 3 perivenular/pericellular fibrosis but no ballooning, indicating that the model does not mimic MASH in patients³⁴. Furthermore, the extent to which STZ and HFD contributed to the rapid progression of MASH and development of MASLD-related HCC in STAM mice is unclear, since this study did not include a control group that received STZ injections during the neonatal period but was not administered HFD.

The current STZ + HFD mouse model was characterized by metabolic dysfunction similar to severe T2DM^35–37. In terms of MASH histopathology, the STZ + HFD model presented hepatocyte ballooning as well as hepatic fibrosis dominant in zone 3, which is typically observed in MASH patients³⁴. In our experiments, neither MASH progression nor HCC development was observed in STZ + SCD mice. These findings imply that rapid MASH progression in our model does not occur solely as a result of STZ-induced direct toxic effects^38,39 or hyperglycemia. Instead, prolonged metabolic stress caused by HFD is essential for the progression of MASH and development of HCC.

Beyond histopathologic and metabolic characteristics, hepatic transcriptome changes in our STZ + HFD model also precisely recapitulated the entire MASLD spectrum in patients. Similarities were identified between STZ + HFD mice at 14 to 20 weeks of age and early-stage MASH patients (NAS 2-6 and fibrosis stage 0-1), between STZ + HFD mice at 32 weeks of age and late-stage MASH patients (NAS 4-7 and fibrosis stage 4), and between HCC occurring in STZ + HFD mice and MASLD-related HCC patients. In addition, we observed a strong transcriptomic correlation between STZ + HFD mice and MASH patients with both obesity and diabetes compared to those with obesity but without diabetes. Our results demonstrated that each stage shared transcriptomic characteristics with the hepatic transcriptome of MASLD in patients. These findings support the utility of the STZ + HFD mouse model as a valuable tool for investigating the underlying pathophysiological mechanisms and development of therapeutic strategies for MASLD.

MASLD patients with obesity and T2DM show rapid MASH progression frequently accompanied by severe fibrosis^11,12, making them a target population for lifestyle intervention-based pharmacological treatments¹³. While tirzepatide is an effective treatment for T2DM and obesity^14–16 with significant promise as a therapeutic option for MASLD¹⁷, its potential long-term benefits in preventing the progression to advanced fibrosis or HCC are unknown. Since MASLD is associated with metabolic dysfunction in our model, it provides a suitable platform to examine the above issues. In this study, we validated the therapeutic efficacy of tirzepatide against MASLD. Tirzepatide attenuated MASH and fibrosis when administered in the early stage and improved fibrosis and prevented hepatic tumorigenesis when administered in the middle stage of MASH. However, administration of tirzepatide after advanced fibrosis did not improve MASH or fibrosis, only reducing hepatic tumorigenesis. These results suggest that the most appropriate time to initiate pharmacological treatment for MASLD is prior to the onset of advanced fibrosis. Intriguingly, when administered after the onset of advanced fibrosis, tirzepatide only improved HCC without effect on MASH or fibrosis. Tirzepatide downregulated the expression of genes related to the Wnt signaling pathway which is associated with HCC development in various liver diseases⁴⁰. Therefore, improving metabolic dysfunction with tirzepatide may prevent hepatic tumorigenesis regardless of changes in MASH or hepatic fibrosis by limiting metabolic reprogramming and inhibiting Wnt signaling pathway during tumor development⁴¹. Overall, our findings support the potential of tirzepatide as an effective therapeutic approach for the management of the whole spectrum of MASLD.

MASLD progression^42,43 and MASLD-related HCC development^44,45 have been linked to susceptibility to oxidative cellular damage. To examine the underlying cause of rapid MASH progression in our MASLD model, we compared the histopathologic, transcriptomic, and epigenetic phenotypes of four groups of mice (SCD-only, STZ + SCD, HFD-only, and STZ + HFD) at 20 weeks of age. Compared to the HFD-only group, STZ + HFD mice showed greater NAS, downregulation of genes linked to the tissue repair response, and epigenetic repression of genes related to the DNA damage response. These findings imply that a decrease in compensatory responses to metabolic stress may contribute to the rapid progression of MASH in STZ + HFD mice. Furthermore, oxidative cellular damage is known to play a significant role in the development of MASLD-related HCC⁴⁵ which could explain why HCC is frequently observed in our model. However, since additional factors, such as genomic instability, contribute to the occurrence of HCC²⁸, further research on the underlying cause of HCC development in STZ + HFD mice is warranted.

In conclusion, using low-dose STZ and HFD, we successfully established a murine MASLD model exhibiting the full spectrum of histopathologic, transcriptomic, and metabolic characteristics. With the aid of this model, we demonstrated the potential of tirzepatide as an effective therapeutic agent for MASLD. The collective findings indicate that the STZ + HFD murine model established in this study shows promise as a useful tool for advancing our knowledge of the pathophysiology of MASLD and developing innovative therapeutic options.

Methods