Differences Between Therapeutic Mechanisms of Resmetirom and Semaglutide Against MASH in Western diet-fed MC4R Knockout Mice

Differences Between Therapeutic Mechanisms of Resmetirom and Semaglutide Against MASH in Western diet-fed MC4R Knockout Mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Differences Between Therapeutic Mechanisms of Resmetirom and Semaglutide Against MASH in Western diet-fed MC4R Knockout Mice Takumi Sugawara, Kosuke Hitaka, Mitsuharu Matsumoto, Sayuri Nakamura, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6684850/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Nov, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Metabolic dysfunction-associated steatotic liver disease (MASLD) can progress to metabolic dysfunction-associated steatohepatitis (MASH), which is closely linked to obesity and insulin resistance. Resmetirom, the first approved drug for MASH, and semaglutide, a GLP-1 receptor agonist, have shown therapeutic effects in clinical studies. This study aimed to compare their mechanisms in Western diet (WD)-fed melanocortin 4 receptor-knockout (MC4R-KO) mice, a model that mimics human MASH pathology. MC4R-KO mice fed a WD for 6 weeks were treated with resmetirom or semaglutide for 7 weeks. WD-fed MC4R-KO mice showed increased liver weight and plasma aspartate aminotransferase and alanine transaminase levels. Both the resmetirom and semaglutide treatments substantially improved these parameters. Although resmetirom and semaglutide improved liver hydroxyproline deposition and fat mass, semaglutide markedly suppressed lean mass. Moreover, in terms of energy expenditure, resmetirom enhanced oxygen consumption, whereas semaglutide reduced it. In conclusion, the different mechanisms of resmetirom and semaglutide against MASH were revealed. Similar to clinical evidence, semaglutide treatment, unlike resmetirom, may cause muscle mass reduction due to food intake suppression. To our knowledge, this study is the first to simultaneously compare the effects of resmetirom and semaglutide on MASH phenotypes and reveal their mechanism of action using WD-fed MC4R-KO mice. Biological sciences/Drug discovery Health sciences/Diseases Health sciences/Endocrinology Health sciences/Gastroenterology Health sciences/Medical research metabolic dysfunction-associated steatotic liver disease melanocortin 4 receptor knockout resmetirom semaglutide fibrosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) is rapidly increasing worldwide and is now the most common liver disorder in the western world, with an incidence rate of over 25%.[1] [2] [3] [4] MASLD is strongly associated with metabolic abnormalities, such as obesity, insulin resistance, and type 2 diabetes mellitus, and encompasses complicated and extensive liver diseases, including asymptomatic steatosis and more aggressive metabolic dysfunction-associated steatohepatitis (MASH).[5] [6] MASH is characterized by steatosis, cytoskeletal damage (hepatocellular ballooning), lobular inflammation, and fibrosis.[7] MASH is considered to be progressive; hence, the disease progresses to liver cirrhosis, then to hepatocellular carcinoma, and consequently increases mortality rates.[8] [9] Resmetirom, a thyroid hormone receptor (THR)-β agonist, was approved for MASH treatment through its favorable efficacy, safety profiles, and fibrosis improvement.[10] This approval is a critical advancement in MASH therapy and provides a roadmap for additional therapies. The relationship between MASLD and TSH have been researched, hypothyroidism is considerably associated with the presence and severity of MASLD, and titrated levothyroxine dosing decreases hepatic lipid content.[11] [12] However, systemic dosing of thyroid hormone has concerning adverse effects through THR-α expressing in heart.[13] In contrast, THR-β is highly expressed in hepatocytes and regulates systemic lipids levels and metabolic pathways in the liver.[14] In addition, the expression of THR-β in the liver is inversely correlated with nonalcoholic fatty liver disease (NAFLD) activity score (NAS).[15] Resmetirom has higher selectivity for THR-β rather than for THR-α and is preferentially taken up in the liver through the organic anion transporting polypeptides 1B1 receptor.[16] [17] [18] Resmetirom has shown ameliorative effects on liver fibrosis, steatosis, and inflammation in vivo using diet-induced obese (DIO) mice.[14] [19] [20] However, DIO and chemically induced mouse models do not fully reflect human MASH pathology. Although DIO mice are obese, phenotypes of insulin resistance, hyperphagia, steatohepatitis, and fibrosis are not observed or are mild.[21] For example, Lin et al.[22] used a DIO mice model fed a high fat diet (60% of kcal) to evaluate the effect of THR-β agonists on metabolism function; however, its anti-fibrotic effect was not investigated. The DIO mouse model showed mild MASH pathology characterized by NAS but did not exhibit liver inflammation or fibrosis.[22] Nutrient-deficient diets low in or devoid of methionine and/or choline are used to induce severe liver fibrosis. In addition, chemically induced models such as CCl 4 -induced liver damage models have been used to study the mechanisms of hepatic fibrosis progression. However, neither nutrient-deficient nor chemically induced models fully reflect human MASH pathology because these models are not obese but show weight loss.[23] [24] Melanocortin 4 receptor (MC4R) is a seven-transmembrane G-protein-coupled receptor expressed in the hypothalamic nuclei, and MC4R-knockout (KO) mice are implicated in the regulation of appetite, hyperphagia, and body weight.[25] Western diet (WD)-fed MC4R-KO mice and Gubra-Amylin non-alcoholic steatohepatitis (GAN) diet-fed mice are reported that these models are highly resemble human MASH in terms of obesity, dyslipidemia, insulin resistance, liver injury, steatosis, and fibrosis.[21] [26] [27] However, the GAN diet-induced MASH model was established by feeding a GAN diet for approximately 30 weeks. In contrast, MC4R-KO mice show MASH-like pathology after 8 weeks of feeding a WD; therefore, WD-fed MC4R-KO mice have an advantage as a rodent MASH model owing to their ability to be established quickly.[21] Therefore, combining a WD with MC4R-KO mice, which induces substantial obesity and insulin resistance, is an attractive MASH model with considerable fibrosis compared with that of the DIO mouse model. Suitably, in this study, WD-fed MC4R-KO mice were generated and the anti-MASH effects of resmetirom were confirmed. Moreover, the energy expenditure and fatty acid oxidation of resmetirom for anti-MASH effects were evaluated using the Oxymax system after repeated dosing of resmetirom in WD-fed MC4R-KO mice. Furthermore, single and dual agonists of glucagon-like peptide-1 (GLP-1) have ameliorative effects against MASH.[28] [29] GLP-1 analogs were initially developed for type 2 diabetes mellitus, their efficacy against obesity was demonstrated, and they were used as anti-obesity drugs.[30] Moreover, a reduction in body weight of over 7% improve NAS in MASH patients.[31] [32] Therefore, the therapeutic effect of GLP-1 analogs on MASH is attributed to body weight reduction through a decrease in appetite, followed by improvement in dyslipidemia and hepatic inflammation.[33] [34] Accordingly, in this study, the anti-MASH effect of a GLP-1 analog was evaluated using WD-fed MC4R-KO mice, and its efficacy and scores were compared with that of resmetirom by simultaneous administration in the same study. Results MASH Phenotypes of WD-fed MC4R-KO Mice WD-fed MC4R-KO mice were generated after 6 weeks of feeding on a WD from 22 weeks of age. The mice showed pronounced increases in body weight, food intake, and levels of liver injury markers, such as plasma ALT, AST, TIMP-1, TG, cholesterol, and LDL-cholesterol, compared with those of normal mice. In addition, WD-fed MC4R-KO mice showed a substantial increase in plasma insulin levels compared with those in normal mice, indicating insulin resistance similar to that of a patient with MASH (Supplementary Table 1). These changes in parameters resemble those observed in MASH patients. Effects of Resmetirom and Semaglutide on Food intake, Body Weight, Body Composition, and Tissue weight The effects of resmetirom and semaglutide on food intake, body weight, and body composition of WD-fed MC4R-KO mice were confirmed. Semaglutide markedly decreased body weight and food intake compared with that in vehicle-treated mice; however, resmetirom treatment did not change body weight or food intake in WD-fed MC4R-KO mice (Fig. 1 A and B). A detailed analysis of body weight composition was conducted using an Echo-MRI system to evaluate fat mass and lean mass. Resmetirom substantially suppressed fat mass but had no effect on lean mass. In contrast, semaglutide considerably suppressed not only fat mass, but also lean mass in WD-fed MC4R-KO mice (Fig. 1 C). The liver weight of vehicle-treated WD-fed MC4R-KO mice was approximately three times that of the control mice, and both resmetirom and semaglutide markedly reduced the liver weight (Fig. 1 D). After 7 weeks of resmetirom and semaglutide treatment in WD-fed MC4R-KO mice, no effect on the weight of the heart, which expresses THR-α, was observed (Fig. 1 D). In addition to liver enlargement, the livers of WD-fed MC4R-KO mice were pale, and the liver surface had a granular texture (Fig. 1 E). In contrast, the drug-treated group exhibited an appearance similar to that of the control group. Effect of Resmetirom and Semaglutide on Liver Injury Markers and Plasma LDL-C Levels in WD-fed MC4R-KO mice The effects of resmetirom and semaglutide on MASH were confirmed in WD-fed MC4R-KO mice. Both resmetirom and semaglutide treatment considerably reduced plasma ALT, AST, and TIMP-1 levels in WD-fed MC4R-KO mice, thus indicating a reduction in liver injury and fibrosis (Fig. 2 A, B). Resmetirom showed a notable decrease of plasma LDL-C levels compared with those in vehicle-treated MC4R-KO mice, thereby indicating this is on-target effect through THR-β (Fig. 2 C). In contrast, plasma total T3 was not influenced by resmetirom, thus indicating that resmetirom did not disrupt thyroid hormone homeostasis (Fig. 1 D). In terms of other lipid parameters, total plasma cholesterol also increased in WD-fed MC4R-KO mice and decreased in the drug-treated groups (Fig. 2 E). There were no notable changes in TG levels in any group (Fig. 2 E). Evaluation of Liver Hydroxyproline, TG, and TC contents To confirm the qualitative changes in the liver, cholesterol, TG, and hydroxyproline levels were measured. Similar to the plasma cholesterol level, cholesterol in the liver was increased in WD-fed MC4R-KO mice and was reduced by resmetirom and semaglutide treatment (Fig. 3 A). Interestingly, the change in TG levels in the liver was similar to that of cholesterol, even though plasma TG levels were constant in all groups (Fig. 3 B). In terms of hydroxyproline, which is a component of collagen and is used for assessment of tissue fibrosis, its levels were higher in WD-fed MC4R-KO mice than in control mice. This suggests liver fibrosis development in this MASH mouse model (Fig. 3 C). Both resmetirom and semaglutide markedly lowered the hepatic hydroxyproline content. Comparison of Oxygen Consumption, Energy Expenditure, and Respiratory Exchange Ratio (RER) Between Resmetirom and Semaglutide Activation of THR-β in the liver affects fatty acid metabolism and improves hepatic fat content. Therefore, in this study, energy expenditure, oxygen consumption, and RER in WD-fed MC4R-KO mice treated with resmetirom and semaglutide was analyzed using the Oxymax system. Resmetirom substantially increased oxygen consumption but had no effect on energy expenditure, whereas semaglutide considerably reduced the energy expenditure and RER (Fig. 4 A–F). These results may be attributed to a reduction in food intake. Effect of Gene Expression After 7 Weeks of Resmetirom and Semaglutide Administration Considering that the pathology of MASH is related to inflammation, fibrosis and lipid metabolism, the expression of related genes was examined using qRT-PCR. Among the inflammation-related genes, Il1b and Ccl2 expression markedly increased, and Il6 expression tended to increase in the livers of WD-fed MC4R-KO mice. Resmetirom markedly suppressed Ccl2 expression and tended to suppress Il1b and Il6 expression. In contrast, semaglutide resulted in a lower reduction in the expression of these genes (Table 1 ). Expression of fibrosis-related genes, such as Col1a1 , Col3a1 and Spp1 , was upregulated in WD-fed MC4R-KO mice, and both resmetirom and semaglutide tended to decrease the expression of these genes. In lipid metabolism-related genes, Scd1 , Fabp4 and Mogat2 was significantly upregulated in WD-fed MC4R-KO mice. Resmetirom and semaglutide significantly suppressed of Mogat2 expression and tended to suppress Scd1 and Fabp4 expression. Table 1 Effect of gene expression after 7 weeks of resmetirom and semaglutide administration Mice Control MC4R KO Treatment Vehicle Vehicle Resmetirom Semaglutide (n = 4) (n = 8) (n = 8) (n = 4) Inflammation-related gene expression Tnf 1.00 ± 0.14 7.45 ± 3.89** 5.96 ± 3.91 5.91 ± 3.99 Il1b 1.00 ± 0.24 4.43 ± 1.75** 2.43 ± 0.88 3.66 ± 3.16 Il6 1.00 ± 0.40 3.78 ± 2.74 2.00 ± 1.51 2.45 ± 1.19 Ccl2 1.00 ± 0.18 16.54 ± 6.39** 7.54 ± 3.81 $$ 11.12 ± 5.34 Fibrosis-related gene expression Acta2 1.00 ± 0.13 2.66 ± 0.74** 2.33 ± 0.66 2.73 ± 1.31 Col1a1 1.00 ± 0.48 22.06 ± 12.86** 11.44 ± 10.15 11.14 ± 8.14 Col3a1 1.00 ± 0.31 9.58 ± 5.26** 5.38 ± 4.92 4.99 ± 2.84 Spp1 1.00 ± 0.26 10.84 ± 5.16** 5.45 ± 4.71 5.03 ± 3.35 Lipid metabolism-related gene expression Scd1 1.00 ± 0.41 2.13 ± 0.43** 1.73 ± 0.31 1.99 ± 0.74 Fabp4 1.00 ± 0.16 3.74 ± 1.16** 2.03 ± 0.79 4.43 ± 4.93 Mogat2 1.00 ± 0.23 2.65 ± 0.57** 0.85 ± 0.32 $$ 1.35 ± 0.73 $$ Fold-changes in the mRNA expression of inflammation-, fibrosis-, and lipid metabolism-related genes in the liver were normalized to those in the control group. Glyceraldehyde-3-phosphate dehydrogenase ( Gapdh ) was used as the endogenous control. Data are presented as the mean ± standard deviation (SD). MC4R, melanocortin 4 receptor; KO, knockout; Tnf , tumor necrosis factor; Il1b , interleukin 1 beta; Il6 , interleukin 6; Ccl2 , C-C motif chemokine ligand 2; Col1a1 , collagen type 1 alpha 1; Col3a1 , collagen type 1 alpha 1; Spp1 , osteopontin; Scd1 , stearoyl-Coenzyme A desaturase 1; Fabp4 , fatty acid binding protein 4; Mogat2 , monoacylglycerol O-acyltransferase 2. ** p < 0.01, vs Control (Student’s t-test) $$ p < 0.01, vs Vehicle (Dunnett test) Histopathological Evaluation for NAS and fibrosis Liver steatosis and lobular inflammation were evaluated using HE-staining specimens (Fig. 5 A, Table 2 ). Vehicle-treated, WD-fed MC4R-KO mice showed an increase in these scores, with a prominent increase in liver steatosis. In terms of ballooning degeneration of NAS, these mice did not show the histopathological change clearly. These results indicated that WD-fed MC4R-KO mice exhibited severe steatosis with mild inflammation in the liver. Treatment with resmetirom and semaglutide considerably improved the liver steatosis score, suggesting resmetirom and semaglutide decreased the NAS compared to the vehicle treatment. In terms of the lobular inflammation score, no remarkable effects were observed with either resmetirom or semaglutide. Table 2 Evaluation of NAFLD activity score and quantification of Sirius red-positive areas Mice Control MC4R KO Treatment Vehicle Vehicle Resmetirom Semaglutide (n = 4) (n = 8) (n = 8) (n = 4) Histopathology Steatosis 0.0 ± 0.0 3.0 ± 0.0 1.8 ± 0.7 2.3 ± 1.0 Lobular inflammation 0.0 ± 0.0 1.0 ± 0.0 1.0 ± 0.0 0.8 ± 0.5 NAFLD activity score 0.0 ± 0.0 4.0 ± 0.0 2.8 ± 0.7 3.0 ± 1.4 Fibrosis analysis Sirius red-positive area (%) 0.24 ± 0.04 2.07 ± 0.91 2.29 ± 1.37 1.20 ± 0.90 Individual scores for steatosis (0–3) and inflammation (0–2) were provided and were added to determine the NAFLD activity score as a semi-quantitative measure of disease severity. The Sirius red-positive areas were quantified using Halo AI. Data are presented as the mean ± standard deviation (SD). MC4R, melanocortin 4 receptor; KO, knockout; NAFLD, nonalcoholic fatty liver disease. Histological evaluation of liver fibrosis was also performed using Sirius Red-staining specimens (Fig. 5 B, Table 2 ). In accordance with the elevation of plasma TIMP-1 levels, the Sirius red-positive area in the liver of WD-fed MC4R-KO mice was broader than that in the control group. Despite the reduction in plasma TIMP-1, hepatic hydroxyproline, and expression of fibrosis-related genes by resmetirom and semaglutide, there was no change in the Sirius red-positive area with either resmetirom or semaglutide. Discussion Resmetirom was approved by the FDA as the first and thus far only MASH therapeutic drug, and the GLP-1 analog semaglutide showed positive effects on MASH patients in a phase III clinical trial.[37] This study simultaneously evaluated resmetirom and semaglutide in WD-fed MC4R-KO mice. Animal models of MASH induced by diet or chemicals are often used for drug development. However, these models show only mild liver fibrosis and do not fully reflect human MASH pathology.[23] [24] Contrary to this, the WD-fed MC4R-KO mouse model develops liver and metabolic dysfunctions, such as obesity, dyslipidemia, insulin resistance, liver damage, and histological activity (development of steatosis and lobular inflammation), similar to human MASH.[21] In this study, WD-fed MC4R-KO mice showed characteristics similar to those of human MASH patients, such as increased ALT, AST, TIMP-1, TG, TC, and insulin levels (Supplementary Table 1). THR is highly expressed in the liver and the severity of MASLD is associated with reduced THR-β expression in the liver.[15] Moreover, THR-β in the liver regulates plasma LDL-C concentrations by tuning of LDL receptor (LDLR) expression in the liver; LDLR incorporates LDL into cells.[38] [39] Many MASLD patients also have obesity, diabetes, and a risk of cardiovascular events; therefore, an LDL-lowering effect is preferable for MASH patients. [40] Furthermore, THR-β controls fat synthesis, regulates fatty acid oxidation and cholesterol metabolism, improves mitochondrial function, and reduces inflammation and fibrosis.[41] [42] Accordingly, resmetirom, an approved drug for MASH treatment, decreases LDL-C levels and improves liver fibrosis.[43] A decrease in LDL-C levels is attributed to LDLR upregulation in the liver.[38] [39] In this study, the effect of resmetirom on THR-β was confirmed by a decrease in plasma LDL-C levels of WD-fed MC4R-KO mice. This result indicates that resmetirom changed LDL-C as a pharmacodynamic marker by activating THR-β through an on-target effect. In contrast, plasma total T3 was not influenced by resmetirom, thus indicating that resmetirom did not disrupt the homeostasis of thyroid hormones. MASLD is strongly associated with metabolic abnormalities, such as obesity, and anti-obesity is thought to be a potential therapeutic mechanism. GLP-1 analogues are actively developed as candidates for MASH therapeutic drugs because of their role in reducing body weight.[44] Like resmetirom, GLP-1 analogs show indirect anti-inflammatory and anti-fibrotic effects by improving metabolic abnormalities. The therapeutic effect of GLP-1 on MASH is thought to be due to a reduction in body weight caused by a decrease in appetite, and insulin resistance leads to an improvement in dyslipidemia and hepatic inflammation.[33] [34] Suitably, this study also showed that semaglutide reduced food intake and body weight. As described above, although resmetirom and semaglutide have different mechanism, they both improved liver injury reflected by decline in ALT and AST and the liver hydroxyproline content; however, some parameters differed in this study. According to body mass analysis using an Echo-MRI system, semaglutide reduced both lean and fat mass. Muscle and bone weights are included in lean mass, loss of lean mass results in reduction in energy expenditure and bone density, and skeletal muscle reduction is considered as the main reason for body weight regain after discontinuation of semaglutide.[45] [46] In this study, a notable reduction in energy expenditure was confirmed in semaglutide-treated WD-fed MC4R-KO mice for the first time. These effects could be derived from a reduction in lean mass, including muscle and bone mass. Therefore, semaglutide treatment may cause muscle atrophy and osteoporosis. In contrast, resmetirom reduced fat mass but not lean mass. Furthermore, resmetirom-treated mice showed increased oxygen consumption, which may lead to increased muscle mass. Therefore, combination therapy with resmetirom and semaglutide would be an ideal treatment with a synergistic effect on MASH and improve the risk of muscle mass reduction. Similar to plasma parameters reflecting MASH pathology, lipid accumulation in the liver, enhanced transcription of inflammation-related genes, and liver histopathology, such as steatosis and lobular inflammation, was also exacerbated in WD-fed MC4R-KO mice. However, lobular inflammation was weak, and ballooning degeneration was not clearly observed in a previous study.[35] Although the NAS scoring method for humans includes ballooning degeneration, the evaluation of ballooning in MASH model mice is subject to observer variation, and disagreement exists even among experts.[47] Therefore, ballooning was not included in NAS scoring in this study, but both resmetirom and semaglutide improved NAS, mainly due to a reduction in the steatosis score. The reduction of the steatosis score is reflected in the decrease in liver TG and TC contents and the lipid metabolism-related gene expression. These mice showed increased plasma TIMP-1 and liver hydroxyproline content, and an enhanced Sirius red-positive area, indicating fibrosis progression. However, histological evaluation of liver fibrosis did not show differences between vehicle and resmetirom or semaglutide, although these drugs improved the plasma and liver biochemical markers. Similar results were observed for the other MASH models. Nielsen et al. [48] reported that interventional treatment with resmetirom and semaglutide did not result in a considerable reduction in the Sirius red-positive area in choline-deficient L-amino acid-defined high-fat diet (CDAA-HFD)-fed mice, although CDAA-HFD-fed mice showed body weight reduction. In addition, GAN diet-induced MASH model mice also showed liver fibrosis, and resmetirom reduced steatosis and lobular inflammation but did not improve the liver fibrosis score.[49] Kannt et al.[14] reported that mice fed a Amylin liver non-alcoholic steatohepatitis (AMLN) diet for 34 weeks and administered resmetirom for 8 weeks did not show a decrease in the picrosirius staining fractional area and postulated that a longer treatment duration is needed. In contrast, Wang et al.[19] reported that resmetirom treatment for 8 weeks after 25 weeks of feeding mice an AMLN diet improved liver fibrosis; therefore, the effect of resmetirom against liver fibrosis varies depending on the fibrosis level attributed to the duration of diet feeding and the type of MASH model. A longer resmetirom treatment duration may be necessary to improve liver fibrosis in WD-fed MC4R-KO mice. Given that the phase 3 clinical trial of resmetirom also had a longer duration of 52 weeks, it is possible that more repeated dosing times are needed to improve fibrosis in WD fed-MC4R-KO mice. Another possibility is that the antifibrotic effect of resmetirom is not strong. Similar to this study using WD- fed MC4R-KO mice, plasma biochemical parameters were considerably improved in the resmetirom-treated group in a phase 3 clinical trial.[43] However, the percentage of patients with improved fibrosis without worsening of NAFLD was approximately 25% after 52 weeks of treatment. Taken together, the anti-fibrotic effect of resmetirom is not very strong and resmetirom indirectly improves fibrosis through improvement of hepatic steatosis and suppression of inflammation. Moreover, the mechanism of action of semaglutide is not a direct effect on fibrosis; however, it reduces appetite. Therefore, further development of MASH treatment drugs with more potent and direct antifibrotic effects is necessary. In conclusion, these results revealed the different mechanisms of the anti-MASH effects of resmetirom and semaglutide (Fig. 6 ). The main effects of semaglutide were appetite suppression and body weight reduction, thereby improving fat accumulation in the liver and improving MASH pathology. In contrast, resmetirom did not reduce body weight or fat mass. Resmetirom increases energy expenditure and improves MASH pathology. This study is the first to simultaneously evaluate and compare the effects of resmetirom and semaglutide on MASH phenotypes and reveal their mechanisms of action using WD-fed MC4R-KO mice. In this study, the different mechanisms of action of resmetirom and semaglutide were confirmed in MASH models by simultaneous treatment. Although resmetirom is a novel MASH therapeutic drug, approximately 70% of patients who received resmetirom did not achieve the primary endpoint in clinical trials.[43] The study results suggest that combination therapy with resmetirom and semaglutide instead of monotherapy may be beneficial for MASH treatment and may improve MASH treatment outcomes. Methods Compounds Resmetirom and semaglutide were purchased from MedChemExpress (HY-12216, HY-114118; MedChemExpress, USA). The methyl cellulose solution was purchased from Fujifilm Wako Pure Chemical Industries. Animals Generation of MC4R-KO mice has been previously described.[35] A targeting vector for homologous recombination was constructed by insertion of an mCherry unit and a neomycin resistant unit between the transcription start site and the initiation codon of the MC4R gene with BAC clone RP23-112M22 using the Red/ET recombination kit (Gene Bridges GmbH, Land Baden-Württemberg, Germany). Male MC4R-KO mice were fed with WD (D12079B; Research Diets, New Brunswick, Canada) for 13 weeks starting at 22 weeks of age. Normal chow-fed wild-type male C57BL/6J mice of the same age were used as lean controls. Both groups were allowed ad libitum access to food and water. The mice were individually housed under controlled temperature and humidity and a 12-hour light-dark cycle (lights on 7:00–19:00). Repeated dosing study of Resmetirom and Semaglutide Efficacy in WD-fed MC4R-KO Mice WD-fed MC4R mice were randomly divided into the following three groups: vehicle (0.5% methyl cellulose [MC], orally [p.o.]) (n = 8), resmetirom (5 mg/kg, p.o.) (n = 8), semaglutide (0.1 mg/kg subcutaneously, s.c.) (n = 4). These groups were based on the following plasma parameters: alanine transaminase (ALT), aspartate aminotransferase (AST), and low-density lipoprotein cholesterol (LDL-C) levels and body weight. The age-matched normal CE-2-fed wild-type mice were also used, and wild-type mice were administered the vehicle (0.5% MC, p.o.) (n = 4). 0.5% MC and resmetirom were orally administered, and semaglutide was subcutaneously injected once daily for 7 weeks. The body weight and food intake were measured weekly. After 2 and 4 weeks of treatment, plasma parameters were measured. After 7 weeks of treatment, all mice were anesthetized with isoflurane (3–5%), blood was collected from the abdominal vena cava, and the liver were harvested for histopathological and gene expression analyses in the same manner. All animal experiments were conducted with the approval of the Institutional Animal Care and Use Committee and Shonan Health Innovation Park. Analysis of Plasma and Liver Biochemistry Blood was collected from the tail vein before treatments and at 2, 4 weeks after treatment, or from the posterior vena cava at 7 weeks after treatment. Plasma ALT, AST, LDL-C, triglyceride (TG), and total cholesterol (TC) levels were measured enzymatically using the Clinical Analyzer 7180 (Hitachi High-Technologies, Tokyo, Japan). Plasma tissue inhibitor of metalloproteinase-1 (TIMP-1) concentrations were measured using the mouse TIMP-1 Quantikine enzyme-linked immunosorbent assay (ELISA) Kit (R&D Systems, Minneapolis, USA). Plasma insulin concentrations were measured using an ultrasensitive mouse insulin ELISA kit (Morinaga Institute of Biological Science, Kanagawa, Japan). Total plasma T3 concentration was measured using a Total Triiodothyronine ELISA KIT (Alpha Diagnostic International, USA). To measure hepatic TGs and cholesterol, aliquots of the liver were homogenized at a concentration of 100 mg of tissue per 1 mL of saline, and the homogenate was then mixed thoroughly with a mixture of hexane and 2-propanol (3:2). After centrifugation, lipid-containing upper organic layers were collected. Hexane and 2-propanol solutions were added and the upper layer was collected again. The collected upper layers were dried and the residue was dissolved in 2-propanol. TG and cholesterol concentrations were measured using TG and cholesterol E tests, respectively (Fujifilm Wako Pure Chemical Industries, Osaka, Japan). Hepatic hydroxyproline content was measured using a commercially available Total Collagen Kit (Quickzyme Biosciences, Leiden, Netherlands), according to the manufacturer’s instructions. Measurement of Body Composition Fat and lean mass compositions were measured at 7 weeks after treatment using a quantitative magnetic resonance method without anesthesia, according to the manufacturer’s instructions (EchoMRI-900; Hitachi Aloka Medical Ltd., Tokyo, Japan). Analysis of Energy Expenditure via Oxymax WD-fed MC4R KO mice treated with the compounds for 7 weeks were housed individually in the metabolic chamber of the Oxymax system (Columbus Instructions, Columbus, OH, USA) according to the manufacturer’s instructions. At 5:00 pm, the mice were administered vehicle (0.5% MC solution), resmetirom, or semaglutide, and their metabolic rate and respiratory quotient were measured from 6 pm to 1 pm (19:00–7:00, dark phase; 7:00–19:00, light phase). Gene Expression Analysis via Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Total RNA was isolated from 50–100 mg of liver tissue using the RNeasy Mini kit (Qiagen, Tokyo, Japan), followed by reverse transcription using a high-capacity RNA-to-cDNA kit (Thermo Fisher Scientific, Tokyo, Japan) according to the manufacturer’s instructions. The cDNA was amplified using TaqMan Universal Master Mix II (Invitrogen, Tokyo, Japan) and ABI7900 (Life Technologies, Tokyo, Japan) according to the manufacturer’s instructions. Commercially available primer-probe sets were used (Applied Biosystems, Waltham, MA, USA). The sets of qRT-PCR probes were as follows: tumor necrosis factor ( Tnf ; Mm00443260), interleukin 1 beta ( Il1b ; Mm00434228), interleukin 6 ( Il6 ; Mm00446190), C-C motif chemokine ligand 2 ( Ccl2 ;Mm00441242), collagen type 1 alpha 1 ( Col1a1 ; Mm00801666), collagen type 1 alpha 1 ( Col3a1 ; Mm00802300), osteopontin ( Spp1 ; Mm00436767), stearoyl-Coenzyme A desaturase 1 ( Scd1 ; Mm00772290), monoacylglycerol O-acyltransferase 2 ( Mogat2 ; Mm00624192) and fatty acid binding protein 4 ( Fabp4 ; Mm00445878). Glyceraldehyde-3-phosphate dehydrogenase ( Gapdh ; Mm99999915) was used as an endogenous control gene, and relative mRNA expression was calculated via the ΔΔCt method. Histopathological Analysis The dissected liver tissues were fixed in 10% neutral formalin and embedded in paraffin. Paraffin sections (3 µm) were stained with hematoxylin and eosin (HE) and NAS was determined by pathologists.[36] For fibrosis evaluation, paraffin sections (3 µm) were stained with 0.1% Sirius Red and 0.1% Fast Green FCF solution. Whole slide digital images were acquired using NanoZoomer S60 (Hamamatsu Photonics, Shizuoka, Japan). Detection of Sirius Red-positive area was performed using Halo ® Area Quantification analysis software version v2.3.1 combined with DenseNet classifier of Halo AI v4.0 (plugin) (Indica Labs, Albuquerque, NM, USA). First, the region of interest (ROI) was set by detecting hepatic parenchyma excluding connective tissues of capsules and around the large blood vessels using DenseNet classifier. Next, the fibrosis area was detected using Area Quantification software based on the staining of Sirius Red, and the percentage of Sirius Red-positive areas in the total ROIs was evaluated. Statistical Analysis All data are represented as mean + standard deviation. To confirm the establishment of the disease state, statistical differences between normal CE-2-fed wild-type mice and vehicle-treated WD-fed MC4R-KO mice were analyzed using the Student’s t-test. To evaluate the effects of the drugs, statistical differences between the vehicle and drug treatment groups were analyzed using Dunnett’s test. The Bonferroni correction was employed to compare multiple time points. Statistical significance was set at P < 0.05. Abbreviations MASLD, metabolic dysfunction-associated steatotic liver disease; MASH, metabolic dysfunction-associated steatohepatitis; WD, western diet; MC4R, melanocortin 4 receptor; KO, knockout; THR, thyroid hormone receptor; NAFLD, nonalcoholic fatty liver disease; NAS, nonalcoholic fatty liver disease activity score; DIO, diet-induced obese; GAN, Gubra-Amylin non-alcoholic steatohepatitis; GLP-1, glucagon-like peptide-1; MC, methyl cellulose; p.o., per os; ALT, alanine transaminase; AST, aspartate aminotransferase; LDL-C, low-density lipoprotein cholesterol; TG, triglyceride; TC, total cholesterol; TIMP-1, tissue inhibitor of metalloproteinase-1; ELISA, enzyme-linked immunosorbent assay; qRT-PCR, quantitative real-time polymerase chain reaction; Tnf, tumor necrosis factor; Il1b, interleukin 1 beta; Il6, interleukin 6; Ccl2, C-C motif chemokine ligand 2; Col1a1, collagen type 1 alpha 1; Col3a1, collagen type 1 alpha 1; Spp1, osteopontin; Scd1, stearoyl-Coenzyme A desaturase 1; Fabp4, fatty acid binding protein 4; Mogat2, monoacylglycerol O-acyltransferase 2; Gapdh, glyceraldehyde-3-phosphate dehydrogenase; HE, hematoxylin and eosin; ROI, regions of interest; RER, respiratory exchange ratio; LDLR, LDL receptor; CDAA-HFD, choline-deficient L-amino acid defined high fat diet; AMLN, Amylin liver non-alcoholic steatohepatitis. Declarations Acknowledgments The authors thank the lab members for their cooperation in this study. The authors also thank Masayuki Goto and Manami Kaneko for advice. Authorship contribution statement Takumi Sugawara : Writing – original draft, Visualization, Investigation, Methodology, Formal analysis, Data curation, Conceptualization. Kosuke Hitaka :Investigation, Methodology. Mitsuharu Matsumoto, Sayuri Nakamura , Ryosuke Kobayashi , Hitoshi Kandori : Investigation, Methodology. Yasunori Nio : Writing – review & editing, Investigation, Methodology, Supervision, Conceptualization. Data availability The data supporting the findings of this study are included in this published article. Raw data generated and/or analysed during the current study are available from the corresponding author, upon reasonable request. Funding sources This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. 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Supplementary Files SupplementaryInformation.docx Cite Share Download PDF Status: Published Journal Publication published 20 Nov, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 11 Aug, 2025 Reviews received at journal 08 Aug, 2025 Reviews received at journal 10 Jul, 2025 Reviewers agreed at journal 01 Jul, 2025 Reviewers agreed at journal 30 Jun, 2025 Reviewers invited by journal 30 Jun, 2025 Editor assigned by journal 03 Jun, 2025 Editor invited by journal 03 Jun, 2025 Submission checks completed at journal 31 May, 2025 First submitted to journal 17 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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(B) Cumulative food intake for 7 weeks. (C) Fat mass and lean mass determined via EchoMRI at week 7. (D) Liver and heart weight. (E) Representative pictures of liver after treatment. Data are presented as the mean + standard deviation (SD).\u003c/p\u003e\n\u003cp\u003e#p \u0026lt; 0.05, ##p \u0026lt; 0.01, vs Vehicle\u003c/p\u003e\n\u003cp\u003e(Dunnett’s test followed by the Bonferroni correction).\u003c/p\u003e\n\u003cp\u003e*p \u0026lt; 0.05, **p \u0026lt; 0.01, vs Control (Student’s t-test)\u003c/p\u003e\n\u003cp\u003e$p \u0026lt; 0.05, $$p \u0026lt; 0.01, vs Vehicle (Dunnett’s test)\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6684850/v1/49840fe05947a8f2b1df4ee6.png"},{"id":83428211,"identity":"a0154242-c7e7-4693-ae30-8cae9396334b","added_by":"auto","created_at":"2025-05-26 06:00:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":524750,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of resmetirom and semaglutide on liver injury markers and plasma LDL-C levels in WD-fed MC4R-KO mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Time course of plasma ALT and AST levels for 7 weeks. (B) Plasma TIMP-1 concentration. (C) Time course of rate of plasma LDL-C concentration from the values before treatment. (D) Plasma total T3 concentration. (E) Plasma total cholesterol and triglyceride concentration. Data are represented as the mean + SD. LDL-C, low-density lipoprotein cholesterol; WD, western diet; MC4R, melanocortin 4 receptor; KO, knockout; ALT, alanine transaminase; AST, aspartate; TIMP-1, tissue inhibitor of metalloproteinase-1; SD, standard deviation.\u003c/p\u003e\n\u003cp\u003e# p \u0026lt; 0.05, ## p \u0026lt; 0.01, vs Vehicle\u003c/p\u003e\n\u003cp\u003e(Dunnett’s test followed by the Bonferroni correction).\u003c/p\u003e\n\u003cp\u003e* p \u0026lt; 0.05, ** p \u0026lt; 0.01, vs Control (Student’s t-test)\u003c/p\u003e\n\u003cp\u003e$ p \u0026lt; 0.05, $$ p \u0026lt; 0.01, vs Vehicle (Dunnett’s test)\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6684850/v1/753ad30cbccf88c0501287d2.png"},{"id":83428207,"identity":"b6030a08-52ba-43c5-bb66-12da0c78838a","added_by":"auto","created_at":"2025-05-26 06:00:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":298611,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvaluation of liver TG, TC and hydroxyproline contents\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Total cholesterol, (B) triglyceride, and (C) hydroxyproline content in the liver. Data are presented as the mean + standard deviation (SD).\u003c/p\u003e\n\u003cp\u003e** p \u0026lt; 0.01, vs Control (Student’s t-test)\u003c/p\u003e\n\u003cp\u003e$ p \u0026lt; 0.05, $$ p \u0026lt; 0.01, vs Vehicle (Dunnett’s test)\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6684850/v1/429663105a578ed56118b543.png"},{"id":83428215,"identity":"1311a2b5-03f8-484f-b7d4-e83e3a8483f7","added_by":"auto","created_at":"2025-05-26 06:00:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":697749,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of oxygen consumption, energy expenditure, and RER between resmetirom and semaglutide\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe time course of metabolic rates was monitored continuously for 19 h using the Oxymax system. (A) Oxygen consumption, (C) energy expenditure, and (E) RER. The shaded regions represent the dark phase of a 12-hour light-dark cycle. (B) Average oxygen consumption, (D) energy expenditure, and (F) RER during light and dark periods. Data are represented as the mean +SD. RER, respiratory exchange ratio; SD, standard deviation.\u003c/p\u003e\n\u003cp\u003e$ p \u0026lt; 0.05, $$ p \u0026lt; 0.01, vs Vehicle (Dunnett’s test)\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6684850/v1/e53460dd7b3293d445a55447.png"},{"id":83428220,"identity":"bc3e6b38-a15b-4e07-a3ba-4024edc6a2d1","added_by":"auto","created_at":"2025-05-26 06:00:32","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":8093685,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistopathology for NAS and fibrosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative images of liver sections stained with hematoxylin \u0026amp; eosin. Large-sized lipid droplets were increased in the Vehicle group compared to the Control group, whereas amelioration was observed in both the resmetirom and semaglutide treated groups. (B) Representative images of liver sections stained with Sirius Red. HE, hematoxylin \u0026amp; eosin\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6684850/v1/68f9c3ad7055373a38e517d6.png"},{"id":83428545,"identity":"cda6692d-917d-45c2-bb4c-842ac052e284","added_by":"auto","created_at":"2025-05-26 06:08:32","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":985712,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchema of amelioration effects of resmetirom and semaglutide.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLDL-C, low-density lipoprotein cholesterol; VO\u003csub\u003e2\u003c/sub\u003e, oxygen consumption; ALT, alanine transaminase; AST, aspartate aminotransferase; TC, total cholesterol; HYP, hydroxyproline; BW, body weight; MASH, metabolic dysfunction-associated steatohepatitis.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6684850/v1/ca7cd82e0f7f45041b1cd7fb.png"},{"id":96650384,"identity":"624a2ffc-fa4d-44d9-929c-97f4e5f6263f","added_by":"auto","created_at":"2025-11-24 16:11:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12684961,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6684850/v1/9cd1f2e1-c519-41cf-8c11-4ac98550b900.pdf"},{"id":83428541,"identity":"52e8fadb-8553-442a-a540-b5ce9ac2548b","added_by":"auto","created_at":"2025-05-26 06:08:32","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":20704,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-6684850/v1/e4d46485ef85019c520e5679.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Differences Between Therapeutic Mechanisms of Resmetirom and Semaglutide Against MASH in Western diet-fed MC4R Knockout Mice","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) is rapidly increasing worldwide and is now the most common liver disorder in the western world, with an incidence rate of over 25%.[1] [2] [3] [4] MASLD is strongly associated with metabolic abnormalities, such as obesity, insulin resistance, and type 2 diabetes mellitus, and encompasses complicated and extensive liver diseases, including asymptomatic steatosis and more aggressive metabolic dysfunction-associated steatohepatitis (MASH).[5] [6] MASH is characterized by steatosis, cytoskeletal damage (hepatocellular ballooning), lobular inflammation, and fibrosis.[7] MASH is considered to be progressive; hence, the disease progresses to liver cirrhosis, then to hepatocellular carcinoma, and consequently increases mortality rates.[8] [9] Resmetirom, a thyroid hormone receptor (THR)-β agonist, was approved for MASH treatment through its favorable efficacy, safety profiles, and fibrosis improvement.[10] This approval is a critical advancement in MASH therapy and provides a roadmap for additional therapies. The relationship between MASLD and TSH have been researched, hypothyroidism is considerably associated with the presence and severity of MASLD, and titrated levothyroxine dosing decreases hepatic lipid content.[11] [12] However, systemic dosing of thyroid hormone has concerning adverse effects through THR-α expressing in heart.[13] In contrast, THR-β is highly expressed in hepatocytes and regulates systemic lipids levels and metabolic pathways in the liver.[14] In addition, the expression of THR-β in the liver is inversely correlated with nonalcoholic fatty liver disease (NAFLD) activity score (NAS).[15] Resmetirom has higher selectivity for THR-β rather than for THR-α and is preferentially taken up in the liver through the organic anion transporting polypeptides 1B1 receptor.[16] [17] [18] Resmetirom has shown ameliorative effects on liver fibrosis, steatosis, and inflammation \u003cem\u003ein vivo\u003c/em\u003e using diet-induced obese (DIO) mice.[14] [19] [20] However, DIO and chemically induced mouse models do not fully reflect human MASH pathology. Although DIO mice are obese, phenotypes of insulin resistance, hyperphagia, steatohepatitis, and fibrosis are not observed or are mild.[21] For example, Lin et al.[22] used a DIO mice model fed a high fat diet (60% of kcal) to evaluate the effect of THR-β agonists on metabolism function; however, its anti-fibrotic effect was not investigated. The DIO mouse model showed mild MASH pathology characterized by NAS but did not exhibit liver inflammation or fibrosis.[22] Nutrient-deficient diets low in or devoid of methionine and/or choline are used to induce severe liver fibrosis. In addition, chemically induced models such as CCl\u003csub\u003e4\u003c/sub\u003e-induced liver damage models have been used to study the mechanisms of hepatic fibrosis progression. However, neither nutrient-deficient nor chemically induced models fully reflect human MASH pathology because these models are not obese but show weight loss.[23] [24] Melanocortin 4 receptor (MC4R) is a seven-transmembrane G-protein-coupled receptor expressed in the hypothalamic nuclei, and MC4R-knockout (KO) mice are implicated in the regulation of appetite, hyperphagia, and body weight.[25] Western diet (WD)-fed MC4R-KO mice and Gubra-Amylin non-alcoholic steatohepatitis (GAN) diet-fed mice are reported that these models are highly resemble human MASH in terms of obesity, dyslipidemia, insulin resistance, liver injury, steatosis, and fibrosis.[21] [26] [27] However, the GAN diet-induced MASH model was established by feeding a GAN diet for approximately 30 weeks. In contrast, MC4R-KO mice show MASH-like pathology after 8 weeks of feeding a WD; therefore, WD-fed MC4R-KO mice have an advantage as a rodent MASH model owing to their ability to be established quickly.[21]\u003c/p\u003e \u003cp\u003eTherefore, combining a WD with MC4R-KO mice, which induces substantial obesity and insulin resistance, is an attractive MASH model with considerable fibrosis compared with that of the DIO mouse model. Suitably, in this study, WD-fed MC4R-KO mice were generated and the anti-MASH effects of resmetirom were confirmed. Moreover, the energy expenditure and fatty acid oxidation of resmetirom for anti-MASH effects were evaluated using the Oxymax system after repeated dosing of resmetirom in WD-fed MC4R-KO mice.\u003c/p\u003e \u003cp\u003eFurthermore, single and dual agonists of glucagon-like peptide-1 (GLP-1) have ameliorative effects against MASH.[28] [29] GLP-1 analogs were initially developed for type 2 diabetes mellitus, their efficacy against obesity was demonstrated, and they were used as anti-obesity drugs.[30] Moreover, a reduction in body weight of over 7% improve NAS in MASH patients.[31] [32] Therefore, the therapeutic effect of GLP-1 analogs on MASH is attributed to body weight reduction through a decrease in appetite, followed by improvement in dyslipidemia and hepatic inflammation.[33] [34] Accordingly, in this study, the anti-MASH effect of a GLP-1 analog was evaluated using WD-fed MC4R-KO mice, and its efficacy and scores were compared with that of resmetirom by simultaneous administration in the same study.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMASH Phenotypes of WD-fed MC4R-KO Mice\u003c/h2\u003e \u003cp\u003eWD-fed MC4R-KO mice were generated after 6 weeks of feeding on a WD from 22 weeks of age. The mice showed pronounced increases in body weight, food intake, and levels of liver injury markers, such as plasma ALT, AST, TIMP-1, TG, cholesterol, and LDL-cholesterol, compared with those of normal mice. In addition, WD-fed MC4R-KO mice showed a substantial increase in plasma insulin levels compared with those in normal mice, indicating insulin resistance similar to that of a patient with MASH (Supplementary Table\u0026nbsp;1). These changes in parameters resemble those observed in MASH patients.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEffects of Resmetirom and Semaglutide on Food intake, Body Weight, Body Composition, and Tissue weight\u003c/h3\u003e\n\u003cp\u003eThe effects of resmetirom and semaglutide on food intake, body weight, and body composition of WD-fed MC4R-KO mice were confirmed. Semaglutide markedly decreased body weight and food intake compared with that in vehicle-treated mice; however, resmetirom treatment did not change body weight or food intake in WD-fed MC4R-KO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and B). A detailed analysis of body weight composition was conducted using an Echo-MRI system to evaluate fat mass and lean mass. Resmetirom substantially suppressed fat mass but had no effect on lean mass. In contrast, semaglutide considerably suppressed not only fat mass, but also lean mass in WD-fed MC4R-KO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The liver weight of vehicle-treated WD-fed MC4R-KO mice was approximately three times that of the control mice, and both resmetirom and semaglutide markedly reduced the liver weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). After 7 weeks of resmetirom and semaglutide treatment in WD-fed MC4R-KO mice, no effect on the weight of the heart, which expresses THR-α, was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). In addition to liver enlargement, the livers of WD-fed MC4R-KO mice were pale, and the liver surface had a granular texture (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). In contrast, the drug-treated group exhibited an appearance similar to that of the control group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of Resmetirom and Semaglutide on Liver Injury Markers and Plasma LDL-C Levels in WD-fed MC4R-KO mice\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe effects of resmetirom and semaglutide on MASH were confirmed in WD-fed MC4R-KO mice. Both resmetirom and semaglutide treatment considerably reduced plasma ALT, AST, and TIMP-1 levels in WD-fed MC4R-KO mice, thus indicating a reduction in liver injury and fibrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). Resmetirom showed a notable decrease of plasma LDL-C levels compared with those in vehicle-treated MC4R-KO mice, thereby indicating this is on-target effect through THR-β (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). In contrast, plasma total T3 was not influenced by resmetirom, thus indicating that resmetirom did not disrupt thyroid hormone homeostasis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). In terms of other lipid parameters, total plasma cholesterol also increased in WD-fed MC4R-KO mice and decreased in the drug-treated groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). There were no notable changes in TG levels in any group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eEvaluation of Liver Hydroxyproline, TG, and TC contents\u003c/h3\u003e\n\u003cp\u003eTo confirm the qualitative changes in the liver, cholesterol, TG, and hydroxyproline levels were measured. Similar to the plasma cholesterol level, cholesterol in the liver was increased in WD-fed MC4R-KO mice and was reduced by resmetirom and semaglutide treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Interestingly, the change in TG levels in the liver was similar to that of cholesterol, even though plasma TG levels were constant in all groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). In terms of hydroxyproline, which is a component of collagen and is used for assessment of tissue fibrosis, its levels were higher in WD-fed MC4R-KO mice than in control mice. This suggests liver fibrosis development in this MASH mouse model (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Both resmetirom and semaglutide markedly lowered the hepatic hydroxyproline content.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eComparison of Oxygen Consumption, Energy Expenditure, and Respiratory Exchange Ratio (RER) Between Resmetirom and Semaglutide\u003c/h3\u003e\n\u003cp\u003eActivation of THR-β in the liver affects fatty acid metabolism and improves hepatic fat content. Therefore, in this study, energy expenditure, oxygen consumption, and RER in WD-fed MC4R-KO mice treated with resmetirom and semaglutide was analyzed using the Oxymax system. Resmetirom substantially increased oxygen consumption but had no effect on energy expenditure, whereas semaglutide considerably reduced the energy expenditure and RER (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u0026ndash;F). These results may be attributed to a reduction in food intake.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eEffect of Gene Expression After 7 Weeks of Resmetirom and Semaglutide Administration\u003c/h3\u003e\n\u003cp\u003eConsidering that the pathology of MASH is related to inflammation, fibrosis and lipid metabolism, the expression of related genes was examined using qRT-PCR. Among the inflammation-related genes, \u003cem\u003eIl1b\u003c/em\u003e and \u003cem\u003eCcl2\u003c/em\u003e expression markedly increased, and \u003cem\u003eIl6\u003c/em\u003e expression tended to increase in the livers of WD-fed MC4R-KO mice. Resmetirom markedly suppressed \u003cem\u003eCcl2\u003c/em\u003e expression and tended to suppress \u003cem\u003eIl1b\u003c/em\u003e and \u003cem\u003eIl6\u003c/em\u003e expression. In contrast, semaglutide resulted in a lower reduction in the expression of these genes (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Expression of fibrosis-related genes, such as \u003cem\u003eCol1a1\u003c/em\u003e, \u003cem\u003eCol3a1\u003c/em\u003e and \u003cem\u003eSpp1\u003c/em\u003e, was upregulated in WD-fed MC4R-KO mice, and both resmetirom and semaglutide tended to decrease the expression of these genes. In lipid metabolism-related genes, \u003cem\u003eScd1\u003c/em\u003e, \u003cem\u003eFabp4\u003c/em\u003e and \u003cem\u003eMogat2\u003c/em\u003e was significantly upregulated in WD-fed MC4R-KO mice. Resmetirom and semaglutide significantly suppressed of \u003cem\u003eMogat2\u003c/em\u003e expression and tended to suppress \u003cem\u003eScd1\u003c/em\u003e and \u003cem\u003eFabp4\u003c/em\u003e expression.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of gene expression after 7 weeks of resmetirom and semaglutide administration\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMice\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eMC4R KO\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVehicle\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVehicle\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eResmetirom\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSemaglutide\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;4)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eInflammation-related gene expression\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTnf\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.45\u0026thinsp;\u0026plusmn;\u0026thinsp;3.89**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.96\u0026thinsp;\u0026plusmn;\u0026thinsp;3.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.91\u0026thinsp;\u0026plusmn;\u0026thinsp;3.99\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eIl1b\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.43\u0026thinsp;\u0026plusmn;\u0026thinsp;1.75**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.66\u0026thinsp;\u0026plusmn;\u0026thinsp;3.16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eIl6\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.78\u0026thinsp;\u0026plusmn;\u0026thinsp;2.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.45\u0026thinsp;\u0026plusmn;\u0026thinsp;1.19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCcl2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16.54\u0026thinsp;\u0026plusmn;\u0026thinsp;6.39**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.54\u0026thinsp;\u0026plusmn;\u0026thinsp;3.81\u003csup\u003e$$\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11.12\u0026thinsp;\u0026plusmn;\u0026thinsp;5.34\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eFibrosis-related gene expression\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eActa2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.74**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.73\u0026thinsp;\u0026plusmn;\u0026thinsp;1.31\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCol1a1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22.06\u0026thinsp;\u0026plusmn;\u0026thinsp;12.86**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.44\u0026thinsp;\u0026plusmn;\u0026thinsp;10.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11.14\u0026thinsp;\u0026plusmn;\u0026thinsp;8.14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCol3a1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.58\u0026thinsp;\u0026plusmn;\u0026thinsp;5.26**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.38\u0026thinsp;\u0026plusmn;\u0026thinsp;4.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.99\u0026thinsp;\u0026plusmn;\u0026thinsp;2.84\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSpp1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.84\u0026thinsp;\u0026plusmn;\u0026thinsp;5.16**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.45\u0026thinsp;\u0026plusmn;\u0026thinsp;4.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.03\u0026thinsp;\u0026plusmn;\u0026thinsp;3.35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eLipid metabolism-related gene expression\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eScd1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.74\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFabp4\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.74\u0026thinsp;\u0026plusmn;\u0026thinsp;1.16**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.43\u0026thinsp;\u0026plusmn;\u0026thinsp;4.93\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMogat2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003csup\u003e$$\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73\u003csup\u003e$$\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eFold-changes in the mRNA expression of inflammation-, fibrosis-, and lipid metabolism-related genes in the liver were normalized to those in the control group. Glyceraldehyde-3-phosphate dehydrogenase (\u003cem\u003eGapdh\u003c/em\u003e) was used as the endogenous control. Data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). MC4R, melanocortin 4 receptor; KO, knockout; \u003cem\u003eTnf\u003c/em\u003e, tumor necrosis factor; \u003cem\u003eIl1b\u003c/em\u003e, interleukin 1 beta; \u003cem\u003eIl6\u003c/em\u003e, interleukin 6; \u003cem\u003eCcl2\u003c/em\u003e, C-C motif chemokine ligand 2; \u003cem\u003eCol1a1\u003c/em\u003e, collagen type 1 alpha 1; \u003cem\u003eCol3a1\u003c/em\u003e, collagen type 1 alpha 1; \u003cem\u003eSpp1\u003c/em\u003e, osteopontin; \u003cem\u003eScd1\u003c/em\u003e, stearoyl-Coenzyme A desaturase 1; \u003cem\u003eFabp4\u003c/em\u003e, fatty acid binding protein 4; \u003cem\u003eMogat2\u003c/em\u003e, monoacylglycerol O-acyltransferase 2.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e** p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, vs Control (Student\u0026rsquo;s t-test)\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e$$ p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, vs Vehicle (Dunnett test)\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eHistopathological Evaluation for NAS and fibrosis\u003c/h2\u003e \u003cp\u003eLiver steatosis and lobular inflammation were evaluated using HE-staining specimens (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Vehicle-treated, WD-fed MC4R-KO mice showed an increase in these scores, with a prominent increase in liver steatosis. In terms of ballooning degeneration of NAS, these mice did not show the histopathological change clearly. These results indicated that WD-fed MC4R-KO mice exhibited severe steatosis with mild inflammation in the liver. Treatment with resmetirom and semaglutide considerably improved the liver steatosis score, suggesting resmetirom and semaglutide decreased the NAS compared to the vehicle treatment. In terms of the lobular inflammation score, no remarkable effects were observed with either resmetirom or semaglutide.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEvaluation of NAFLD activity score and quantification of Sirius red-positive areas\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMice\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eMC4R KO\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVehicle\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVehicle\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eResmetirom\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSemaglutide\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;4)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eHistopathology\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSteatosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLobular inflammation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNAFLD activity score\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eFibrosis analysis\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSirius red-positive area (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.29\u0026thinsp;\u0026plusmn;\u0026thinsp;1.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.90\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eIndividual scores for steatosis (0\u0026ndash;3) and inflammation (0\u0026ndash;2) were provided and were added to determine the NAFLD activity score as a semi-quantitative measure of disease severity. The Sirius red-positive areas were quantified using Halo AI. Data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). MC4R, melanocortin 4 receptor; KO, knockout; NAFLD, nonalcoholic fatty liver disease.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eHistological evaluation of liver fibrosis was also performed using Sirius Red-staining specimens (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In accordance with the elevation of plasma TIMP-1 levels, the Sirius red-positive area in the liver of WD-fed MC4R-KO mice was broader than that in the control group. Despite the reduction in plasma TIMP-1, hepatic hydroxyproline, and expression of fibrosis-related genes by resmetirom and semaglutide, there was no change in the Sirius red-positive area with either resmetirom or semaglutide.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eResmetirom was approved by the FDA as the first and thus far only MASH therapeutic drug, and the GLP-1 analog semaglutide showed positive effects on MASH patients in a phase III clinical trial.[37] This study simultaneously evaluated resmetirom and semaglutide in WD-fed MC4R-KO mice. Animal models of MASH induced by diet or chemicals are often used for drug development. However, these models show only mild liver fibrosis and do not fully reflect human MASH pathology.[23] [24] Contrary to this, the WD-fed MC4R-KO mouse model develops liver and metabolic dysfunctions, such as obesity, dyslipidemia, insulin resistance, liver damage, and histological activity (development of steatosis and lobular inflammation), similar to human MASH.[21] In this study, WD-fed MC4R-KO mice showed characteristics similar to those of human MASH patients, such as increased ALT, AST, TIMP-1, TG, TC, and insulin levels (Supplementary Table\u0026nbsp;1). THR is highly expressed in the liver and the severity of MASLD is associated with reduced THR-β expression in the liver.[15] Moreover, THR-β in the liver regulates plasma LDL-C concentrations by tuning of LDL receptor (LDLR) expression in the liver; LDLR incorporates LDL into cells.[38] [39] Many MASLD patients also have obesity, diabetes, and a risk of cardiovascular events; therefore, an LDL-lowering effect is preferable for MASH patients. [40] Furthermore, THR-β controls fat synthesis, regulates fatty acid oxidation and cholesterol metabolism, improves mitochondrial function, and reduces inflammation and fibrosis.[41] [42] Accordingly, resmetirom, an approved drug for MASH treatment, decreases LDL-C levels and improves liver fibrosis.[43] A decrease in LDL-C levels is attributed to LDLR upregulation in the liver.[38] [39] In this study, the effect of resmetirom on THR-β was confirmed by a decrease in plasma LDL-C levels of WD-fed MC4R-KO mice. This result indicates that resmetirom changed LDL-C as a pharmacodynamic marker by activating THR-β through an on-target effect. In contrast, plasma total T3 was not influenced by resmetirom, thus indicating that resmetirom did not disrupt the homeostasis of thyroid hormones.\u003c/p\u003e \u003cp\u003eMASLD is strongly associated with metabolic abnormalities, such as obesity, and anti-obesity is thought to be a potential therapeutic mechanism. GLP-1 analogues are actively developed as candidates for MASH therapeutic drugs because of their role in reducing body weight.[44] Like resmetirom, GLP-1 analogs show indirect anti-inflammatory and anti-fibrotic effects by improving metabolic abnormalities. The therapeutic effect of GLP-1 on MASH is thought to be due to a reduction in body weight caused by a decrease in appetite, and insulin resistance leads to an improvement in dyslipidemia and hepatic inflammation.[33] [34] Suitably, this study also showed that semaglutide reduced food intake and body weight.\u003c/p\u003e \u003cp\u003eAs described above, although resmetirom and semaglutide have different mechanism, they both improved liver injury reflected by decline in ALT and AST and the liver hydroxyproline content; however, some parameters differed in this study. According to body mass analysis using an Echo-MRI system, semaglutide reduced both lean and fat mass. Muscle and bone weights are included in lean mass, loss of lean mass results in reduction in energy expenditure and bone density, and skeletal muscle reduction is considered as the main reason for body weight regain after discontinuation of semaglutide.[45] [46]\u003c/p\u003e \u003cp\u003eIn this study, a notable reduction in energy expenditure was confirmed in semaglutide-treated WD-fed MC4R-KO mice for the first time. These effects could be derived from a reduction in lean mass, including muscle and bone mass. Therefore, semaglutide treatment may cause muscle atrophy and osteoporosis. In contrast, resmetirom reduced fat mass but not lean mass. Furthermore, resmetirom-treated mice showed increased oxygen consumption, which may lead to increased muscle mass. Therefore, combination therapy with resmetirom and semaglutide would be an ideal treatment with a synergistic effect on MASH and improve the risk of muscle mass reduction.\u003c/p\u003e \u003cp\u003eSimilar to plasma parameters reflecting MASH pathology, lipid accumulation in the liver, enhanced transcription of inflammation-related genes, and liver histopathology, such as steatosis and lobular inflammation, was also exacerbated in WD-fed MC4R-KO mice. However, lobular inflammation was weak, and ballooning degeneration was not clearly observed in a previous study.[35] Although the NAS scoring method for humans includes ballooning degeneration, the evaluation of ballooning in MASH model mice is subject to observer variation, and disagreement exists even among experts.[47] Therefore, ballooning was not included in NAS scoring in this study, but both resmetirom and semaglutide improved NAS, mainly due to a reduction in the steatosis score. The reduction of the steatosis score is reflected in the decrease in liver TG and TC contents and the lipid metabolism-related gene expression.\u003c/p\u003e \u003cp\u003eThese mice showed increased plasma TIMP-1 and liver hydroxyproline content, and an enhanced Sirius red-positive area, indicating fibrosis progression. However, histological evaluation of liver fibrosis did not show differences between vehicle and resmetirom or semaglutide, although these drugs improved the plasma and liver biochemical markers. Similar results were observed for the other MASH models. Nielsen et al. [48] reported that interventional treatment with resmetirom and semaglutide did not result in a considerable reduction in the Sirius red-positive area in choline-deficient L-amino acid-defined high-fat diet (CDAA-HFD)-fed mice, although CDAA-HFD-fed mice showed body weight reduction. In addition, GAN diet-induced MASH model mice also showed liver fibrosis, and resmetirom reduced steatosis and lobular inflammation but did not improve the liver fibrosis score.[49] Kannt et al.[14] reported that mice fed a Amylin liver non-alcoholic steatohepatitis (AMLN) diet for 34 weeks and administered resmetirom for 8 weeks did not show a decrease in the picrosirius staining fractional area and postulated that a longer treatment duration is needed. In contrast, Wang et al.[19] reported that resmetirom treatment for 8 weeks after 25 weeks of feeding mice an AMLN diet improved liver fibrosis; therefore, the effect of resmetirom against liver fibrosis varies depending on the fibrosis level attributed to the duration of diet feeding and the type of MASH model. A longer resmetirom treatment duration may be necessary to improve liver fibrosis in WD-fed MC4R-KO mice. Given that the phase 3 clinical trial of resmetirom also had a longer duration of 52 weeks, it is possible that more repeated dosing times are needed to improve fibrosis in WD fed-MC4R-KO mice. Another possibility is that the antifibrotic effect of resmetirom is not strong. Similar to this study using WD- fed MC4R-KO mice, plasma biochemical parameters were considerably improved in the resmetirom-treated group in a phase 3 clinical trial.[43] However, the percentage of patients with improved fibrosis without worsening of NAFLD was approximately 25% after 52 weeks of treatment. Taken together, the anti-fibrotic effect of resmetirom is not very strong and resmetirom indirectly improves fibrosis through improvement of hepatic steatosis and suppression of inflammation. Moreover, the mechanism of action of semaglutide is not a direct effect on fibrosis; however, it reduces appetite. Therefore, further development of MASH treatment drugs with more potent and direct antifibrotic effects is necessary.\u003c/p\u003e \u003cp\u003eIn conclusion, these results revealed the different mechanisms of the anti-MASH effects of resmetirom and semaglutide (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The main effects of semaglutide were appetite suppression and body weight reduction, thereby improving fat accumulation in the liver and improving MASH pathology. In contrast, resmetirom did not reduce body weight or fat mass. Resmetirom increases energy expenditure and improves MASH pathology. This study is the first to simultaneously evaluate and compare the effects of resmetirom and semaglutide on MASH phenotypes and reveal their mechanisms of action using WD-fed MC4R-KO mice. In this study, the different mechanisms of action of resmetirom and semaglutide were confirmed in MASH models by simultaneous treatment. Although resmetirom is a novel MASH therapeutic drug, approximately 70% of patients who received resmetirom did not achieve the primary endpoint in clinical trials.[43] The study results suggest that combination therapy with resmetirom and semaglutide instead of monotherapy may be beneficial for MASH treatment and may improve MASH treatment outcomes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv\u003e\n \u003cp\u003eCompounds\u003c/p\u003e\n \u003cp\u003eResmetirom and semaglutide were purchased from MedChemExpress (HY-12216, HY-114118; MedChemExpress, USA). The methyl cellulose solution was purchased from Fujifilm Wako Pure Chemical Industries.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv\u003e\n \u003cp\u003eAnimals\u003c/p\u003e\n \u003cp\u003eGeneration of MC4R-KO mice has been previously described.[35] A targeting vector for homologous recombination was constructed by insertion of an mCherry unit and a neomycin resistant unit between the transcription start site and the initiation codon of the MC4R gene with BAC clone RP23-112M22 using the Red/ET recombination kit (Gene Bridges GmbH, Land Baden-W\u0026uuml;rttemberg, Germany). Male MC4R-KO mice were fed with WD (D12079B; Research Diets, New Brunswick, Canada) for 13 weeks starting at 22 weeks of age. Normal chow-fed wild-type male C57BL/6J mice of the same age were used as lean controls. Both groups were allowed \u003cem\u003ead libitum\u003c/em\u003e access to food and water. The mice were individually housed under controlled temperature and humidity and a 12-hour light-dark cycle (lights on 7:00\u0026ndash;19:00).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv\u003e\n \u003cp\u003eRepeated dosing study of Resmetirom and Semaglutide Efficacy in WD-fed MC4R-KO Mice\u003c/p\u003e\n \u003cp\u003eWD-fed MC4R mice were randomly divided into the following three groups: vehicle (0.5% methyl cellulose [MC], orally [p.o.]) (n\u0026thinsp;=\u0026thinsp;8), resmetirom (5 mg/kg, p.o.) (n\u0026thinsp;=\u0026thinsp;8), semaglutide (0.1 mg/kg subcutaneously, s.c.) (n\u0026thinsp;=\u0026thinsp;4). These groups were based on the following plasma parameters: alanine transaminase (ALT), aspartate aminotransferase (AST), and low-density lipoprotein cholesterol (LDL-C) levels and body weight. The age-matched normal CE-2-fed wild-type mice were also used, and wild-type mice were administered the vehicle (0.5% MC, p.o.) (n\u0026thinsp;=\u0026thinsp;4). 0.5% MC and resmetirom were orally administered, and semaglutide was subcutaneously injected once daily for 7 weeks. The body weight and food intake were measured weekly. After 2 and 4 weeks of treatment, plasma parameters were measured. After 7 weeks of treatment, all mice were anesthetized with isoflurane (3\u0026ndash;5%), blood was collected from the abdominal vena cava, and the liver were harvested for histopathological and gene expression analyses in the same manner. All animal experiments were conducted with the approval of the Institutional Animal Care and Use Committee and Shonan Health Innovation Park.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv\u003e\n \u003cp\u003eAnalysis of Plasma and Liver Biochemistry\u003c/p\u003e\n \u003cp\u003eBlood was collected from the tail vein before treatments and at 2, 4 weeks after treatment, or from the posterior vena cava at 7 weeks after treatment. Plasma ALT, AST, LDL-C, triglyceride (TG), and total cholesterol (TC) levels were measured enzymatically using the Clinical Analyzer 7180 (Hitachi High-Technologies, Tokyo, Japan). Plasma tissue inhibitor of metalloproteinase-1 (TIMP-1) concentrations were measured using the mouse TIMP-1 Quantikine enzyme-linked immunosorbent assay (ELISA) Kit (R\u0026amp;D Systems, Minneapolis, USA). Plasma insulin concentrations were measured using an ultrasensitive mouse insulin ELISA kit (Morinaga Institute of Biological Science, Kanagawa, Japan). Total plasma T3 concentration was measured using a Total Triiodothyronine ELISA KIT (Alpha Diagnostic International, USA). To measure hepatic TGs and cholesterol, aliquots of the liver were homogenized at a concentration of 100 mg of tissue per 1 mL of saline, and the homogenate was then mixed thoroughly with a mixture of hexane and 2-propanol (3:2). After centrifugation, lipid-containing upper organic layers were collected. Hexane and 2-propanol solutions were added and the upper layer was collected again. The collected upper layers were dried and the residue was dissolved in 2-propanol. TG and cholesterol concentrations were measured using TG and cholesterol E tests, respectively (Fujifilm Wako Pure Chemical Industries, Osaka, Japan). Hepatic hydroxyproline content was measured using a commercially available Total Collagen Kit (Quickzyme Biosciences, Leiden, Netherlands), according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv\u003e\n \u003cp\u003eMeasurement of Body Composition\u003c/p\u003e\n \u003cp\u003eFat and lean mass compositions were measured at 7 weeks after treatment using a quantitative magnetic resonance method without anesthesia, according to the manufacturer\u0026rsquo;s instructions (EchoMRI-900; Hitachi Aloka Medical Ltd., Tokyo, Japan).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv\u003e\n \u003cp\u003eAnalysis of Energy Expenditure via Oxymax\u003c/p\u003e\n \u003cp\u003eWD-fed MC4R KO mice treated with the compounds for 7 weeks were housed individually in the metabolic chamber of the Oxymax system (Columbus Instructions, Columbus, OH, USA) according to the manufacturer\u0026rsquo;s instructions. At 5:00 pm, the mice were administered vehicle (0.5% MC solution), resmetirom, or semaglutide, and their metabolic rate and respiratory quotient were measured from 6 pm to 1 pm (19:00\u0026ndash;7:00, dark phase; 7:00\u0026ndash;19:00, light phase).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv\u003e\n \u003cp\u003eGene Expression Analysis via Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)\u003c/p\u003e\n \u003cp\u003eTotal RNA was isolated from 50\u0026ndash;100 mg of liver tissue using the RNeasy Mini kit (Qiagen, Tokyo, Japan), followed by reverse transcription using a high-capacity RNA-to-cDNA kit (Thermo Fisher Scientific, Tokyo, Japan) according to the manufacturer\u0026rsquo;s instructions. The cDNA was amplified using TaqMan Universal Master Mix II (Invitrogen, Tokyo, Japan) and ABI7900 (Life Technologies, Tokyo, Japan) according to the manufacturer\u0026rsquo;s instructions. Commercially available primer-probe sets were used (Applied Biosystems, Waltham, MA, USA). The sets of qRT-PCR probes were as follows: tumor necrosis factor (\u003cem\u003eTnf\u003c/em\u003e; Mm00443260), interleukin 1 beta (\u003cem\u003eIl1b\u003c/em\u003e; Mm00434228), interleukin 6 (\u003cem\u003eIl6\u003c/em\u003e; Mm00446190), C-C motif chemokine ligand 2 (\u003cem\u003eCcl2\u003c/em\u003e ;Mm00441242), collagen type 1 alpha 1 (\u003cem\u003eCol1a1\u003c/em\u003e; Mm00801666), collagen type 1 alpha 1 (\u003cem\u003eCol3a1\u003c/em\u003e; Mm00802300), osteopontin (\u003cem\u003eSpp1\u003c/em\u003e; Mm00436767), stearoyl-Coenzyme A desaturase 1 (\u003cem\u003eScd1\u003c/em\u003e; Mm00772290), monoacylglycerol O-acyltransferase 2 (\u003cem\u003eMogat2\u003c/em\u003e; Mm00624192) and fatty acid binding protein 4 (\u003cem\u003eFabp4\u003c/em\u003e; Mm00445878). Glyceraldehyde-3-phosphate dehydrogenase (\u003cem\u003eGapdh\u003c/em\u003e; Mm99999915) was used as an endogenous control gene, and relative mRNA expression was calculated via the \u0026Delta;\u0026Delta;Ct method.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv\u003e\n \u003cp\u003eHistopathological Analysis\u003c/p\u003e\n \u003cp\u003eThe dissected liver tissues were fixed in 10% neutral formalin and embedded in paraffin. Paraffin sections (3 \u0026micro;m) were stained with hematoxylin and eosin (HE) and NAS was determined by pathologists.[36] For fibrosis evaluation, paraffin sections (3 \u0026micro;m) were stained with 0.1% Sirius Red and 0.1% Fast Green FCF solution. Whole slide digital images were acquired using NanoZoomer S60 (Hamamatsu Photonics, Shizuoka, Japan). Detection of Sirius Red-positive area was performed using Halo\u003csup\u003e\u0026reg;\u003c/sup\u003e Area Quantification analysis software version v2.3.1 combined with DenseNet classifier of Halo AI v4.0 (plugin) (Indica Labs, Albuquerque, NM, USA). First, the region of interest (ROI) was set by detecting hepatic parenchyma excluding connective tissues of capsules and around the large blood vessels using DenseNet classifier. Next, the fibrosis area was detected using Area Quantification software based on the staining of Sirius Red, and the percentage of Sirius Red-positive areas in the total ROIs was evaluated.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv\u003e\n \u003cp\u003eStatistical Analysis\u003c/p\u003e\n \u003cp\u003eAll data are represented as mean\u0026thinsp;+\u0026thinsp;standard deviation. To confirm the establishment of the disease state, statistical differences between normal CE-2-fed wild-type mice and vehicle-treated WD-fed MC4R-KO mice were analyzed using the Student\u0026rsquo;s t-test. To evaluate the effects of the drugs, statistical differences between the vehicle and drug treatment groups were analyzed using Dunnett\u0026rsquo;s test. The Bonferroni correction was employed to compare multiple time points. Statistical significance was set at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eMASLD, metabolic dysfunction-associated steatotic liver disease; MASH, metabolic dysfunction-associated steatohepatitis; WD, western diet; MC4R, melanocortin 4 receptor; KO, knockout; THR, thyroid hormone receptor; NAFLD, nonalcoholic fatty liver disease; NAS, nonalcoholic fatty liver disease activity score; DIO, diet-induced obese; GAN, Gubra-Amylin non-alcoholic steatohepatitis; GLP-1, glucagon-like peptide-1; MC, methyl cellulose; p.o., per os; ALT, alanine transaminase; AST, aspartate aminotransferase; LDL-C, low-density lipoprotein cholesterol; TG, triglyceride; TC, total cholesterol; TIMP-1, tissue inhibitor of metalloproteinase-1; ELISA, enzyme-linked immunosorbent assay; qRT-PCR, quantitative real-time polymerase chain reaction; Tnf, tumor necrosis factor; Il1b, interleukin 1 beta; Il6, interleukin 6; Ccl2, C-C motif chemokine ligand 2; Col1a1, collagen type 1 alpha 1; Col3a1, collagen type 1 alpha 1; Spp1, osteopontin; Scd1, stearoyl-Coenzyme A desaturase 1; Fabp4, fatty acid binding protein 4; Mogat2, monoacylglycerol O-acyltransferase 2; Gapdh, glyceraldehyde-3-phosphate dehydrogenase; HE, hematoxylin and eosin; ROI, regions of interest; RER, respiratory exchange ratio; LDLR, LDL receptor; CDAA-HFD, choline-deficient L-amino acid defined high fat diet; AMLN, Amylin liver non-alcoholic steatohepatitis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank the lab members for their cooperation in this study. The authors also thank Masayuki Goto and Manami Kaneko for advice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTakumi Sugawara\u003c/strong\u003e:\u0026nbsp;Writing – original draft,\u0026nbsp;Visualization, Investigation, Methodology, Formal analysis, Data curation, Conceptualization. \u003cstrong\u003eKosuke Hitaka\u003c/strong\u003e:Investigation, Methodology. \u003cstrong\u003eMitsuharu Matsumoto,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eSayuri Nakamura\u003c/strong\u003e\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eRyosuke Kobayashi\u003c/strong\u003e\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eHitoshi Kandori\u003c/strong\u003e: Investigation, Methodology. \u003cstrong\u003eYasunori Nio\u003c/strong\u003e: Writing – review \u0026amp; editing, Investigation, Methodology, Supervision, Conceptualization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data supporting the findings of this study are included in this published article. Raw data generated and/or analysed during the current study are available from the corresponding author, upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding sources\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBellentani S. The epidemiology of non-alcoholic fatty liver disease. Liver Int 2017;37:81-84.\u003c/li\u003e\n \u003cli\u003eEstes C, Razavi H, Loomba R, Younossi Z, Sanyal AJ. 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Acta Physiol (Oxf) 2024;240(10):e14217.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"metabolic dysfunction-associated steatotic liver disease, melanocortin 4 receptor knockout, resmetirom, semaglutide, fibrosis","lastPublishedDoi":"10.21203/rs.3.rs-6684850/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6684850/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMetabolic dysfunction-associated steatotic liver disease (MASLD) can progress to metabolic dysfunction-associated steatohepatitis (MASH), which is closely linked to obesity and insulin resistance. Resmetirom, the first approved drug for MASH, and semaglutide, a GLP-1 receptor agonist, have shown therapeutic effects in clinical studies. This study aimed to compare their mechanisms in Western diet (WD)-fed melanocortin 4 receptor-knockout (MC4R-KO) mice, a model that mimics human MASH pathology. MC4R-KO mice fed a WD for 6 weeks were treated with resmetirom or semaglutide for 7 weeks. WD-fed MC4R-KO mice showed increased liver weight and plasma aspartate aminotransferase and alanine transaminase levels. Both the resmetirom and semaglutide treatments substantially improved these parameters. Although resmetirom and semaglutide improved liver hydroxyproline deposition and fat mass, semaglutide markedly suppressed lean mass. Moreover, in terms of energy expenditure, resmetirom enhanced oxygen consumption, whereas semaglutide reduced it. In conclusion, the different mechanisms of resmetirom and semaglutide against MASH were revealed. Similar to clinical evidence, semaglutide treatment, unlike resmetirom, may cause muscle mass reduction due to food intake suppression. To our knowledge, this study is the first to simultaneously compare the effects of resmetirom and semaglutide on MASH phenotypes and reveal their mechanism of action using WD-fed MC4R-KO mice.\u003c/p\u003e","manuscriptTitle":"Differences Between Therapeutic Mechanisms of Resmetirom and Semaglutide Against MASH in Western diet-fed MC4R Knockout Mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-26 05:59:49","doi":"10.21203/rs.3.rs-6684850/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-11T06:48:53+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-08T08:18:32+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-10T22:25:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"60046289412288932802406465399536191279","date":"2025-07-01T05:09:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"244893351863601270279925932572420053240","date":"2025-06-30T16:21:20+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-30T16:08:27+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-03T07:31:20+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-06-03T04:38:32+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-31T11:09:26+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-05-17T06:08:13+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a357f744-ecb9-4c91-bdc1-29124f586d42","owner":[],"postedDate":"May 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":48663933,"name":"Biological sciences/Drug discovery"},{"id":48663934,"name":"Health sciences/Diseases"},{"id":48663935,"name":"Health sciences/Endocrinology"},{"id":48663936,"name":"Health sciences/Gastroenterology"},{"id":48663937,"name":"Health sciences/Medical research"}],"tags":[],"updatedAt":"2025-11-24T16:07:29+00:00","versionOfRecord":{"articleIdentity":"rs-6684850","link":"https://doi.org/10.1038/s41598-025-24927-3","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-11-20 15:58:47","publishedOnDateReadable":"November 20th, 2025"},"versionCreatedAt":"2025-05-26 05:59:49","video":"","vorDoi":"10.1038/s41598-025-24927-3","vorDoiUrl":"https://doi.org/10.1038/s41598-025-24927-3","workflowStages":[]},"version":"v1","identity":"rs-6684850","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6684850","identity":"rs-6684850","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}