Tirzepatide alleviated the blood lipid profile and liver damage in the HFD-induced MAFLD model
To evaluate the efficacy of establishing a mouse model of fatty liver, we performed ultrasound imaging on 29 mice after 20 weeks of feeding. Imaging results (Supplementary Fig. 1A) srevealed that in the NCD group, the kidneys were more echogenic than the liver, whereas in the HFD group, the liver echogenicity was notably higher than that in the kidneys. This increased liver echogenicity correlated with lipid accumulation, indicating the development of fatty liver. quantified the grayscale ratio of liver parenchyma to renal cortex echo, which was recorded as the hepatorenal index (HRI). Quantification of the greyscale ratio between the hepatic parenchyma and renal cortical echoes confirmed these observations (Supplementary Fig. 1B).
To assess the effect of tirzepatide on MAFLD, HFD were fed C57BL/6J mice to establish a model. Compared with the NCD group, the HFD group presented a rapid and significant increase in body weight after 1 week (p < 0.05). Body weight significantly decreased in the 1st week of tirzepatide treatment, and by the 12th week, the body weight of HFD + tirzepatide group was approximated the NCD group (Fig. 1A). After 32 weeks of HFD feeding, liver weight increased significantly in the HFD group compared with the NCD group, but decreased in the HFD + tirzepatide group (Fig. 1B). These findings demonstrate the efficacy of tirzepatide in reducing body weight in an HFD-induced MAFLD model.
The TG, TC, HDL-C, and LDL-C levels were significantly higher in the HFD group than in the NCD group. Post-tirzepatide treatment, TG, TC, HDL-C, and LDL-C levels were significantly decreased (Fig. 1C–F). Tirzepatide also mitigated liver damage, as indicated by reduced serum ALT and AST levels (Fig. 1G, H). Thus, tirzepatide appears to alleviate dyslipidaemia and liver injury.
Tirzepatide reduced hepatic steatosis and inflammation in the HFD-induced MAFLD model
Liver histology and quantification results are shown in Fig. 2A–D. HE staining and quantitative analysis revealed a significant increase in the number and volume of fat vacuoles, as well as inflammatory infiltration, in the liver tissues of HFD-fed mice. Treatment with tirzepatide notably ameliorated these histopathological changes, as evidenced by a reduction in both fat vacuoles and inflammatory infiltration (Fig. 2A, C). Furthermore, ORO staining was employed to quantify the hepatic lipid content. The results demonstrated a significant increase in lipid accumulation within the hepatocytes of the HFD group compared with those of the NCD group, with liver fat constituting approximately 50%. This pronounced lipid accumulation indicated severe fatty liver degeneration in the HFD group. However, treatment with tirzepatide significantly reduced hepatic lipid droplet deposition (Fig. 2B, D).
Effects of tirzepatide on liver metabolic profiling and pathways
Using UHPLC-MS/MS in both the ESI (+) and ESI (-) modes to ensure comprehensive metabolite coverage, we identified 2,268 metabolites that were subsequently categorised into 17 superclasses across all samples (Fig. 3A). The detected metabolites predominantly included 613 lipids and lipid-like molecules (27.03%), 521 organic acids and their derivatives (22.97%), and 334 organoheterocyclic compounds (14.73%). The separation of metabolites among the NCD, HFD, and HFD + tirzepatide groups is depicted in the PLS-DA score plots (Fig. 3B), with the corresponding permutation tests confirming the reliability and quality of the PLS-DA models (Fig. 3C). A total of 926 differential metabolites were identified across the three groups via one-way ANOVA. Hierarchical clustering (Supplementary Fig. 2A) and K-means clustering (Fig. 3D) analyses revealed that lipids and lipid-like molecules were significantly elevated in the HFD group compared with the NCD group. This elevation was significantly reversed following tirzepatide treatment. Similarly, organic acids and their derivatives were significantly reduced in the HFD group compared with the NCD group, and this reduction was also significantly reversed by tirzepatide treatment. These results indicate that tirzepatide may exert its therapeutic effects by modulating lipid metabolism and organic acid metabolism.
To assess the effects of tirzepatide on MAFLD metabolomics, a comparative analysis was performed between the HFD and NCD groups, and between the HFD + tirzepatide and HFD groups. The results revealed a total of 636 significant differential metabolites between the HFD and NCD groups (169 upregulated and 467 downregulated), as illustrated in the heat map (Supplementary Fig. 2B). The disrupted metabolic pathways primarily included protein digestion and absorption, glycerophospholipid metabolism, central carbon metabolism in cancer, and metabolic pathway (Fig. 3E).
In the protein digestion and absorption pathway, we observed that in the HFD group, the levels of several amino acids, including L-serine, proline, L-isoleucine, L-histidine,and L-glutamic acid, were significantly downregulated (Fig. 3F). The metabolic disruption of these amino acids may lead to abnormalities in glucose metabolism and protein synthesis. Specifically, the downregulation of L-isoleucine may result in branched-chain amino acid metabolism disorders, subsequently affecting insulin signalling and glucose metabolism[23]. Additionally, the decreased levels of L-glutamic acid and L-serine may impair glutamine synthesis[24] and one-carbon metabolism[25]. Meanwhile, in the analysis of nucleosides, nucleotides, and their analogues, we found that the levels of cytidine, deoxycytidine, adenosine, and adenosine diphosphate (ADP) tended to decrease in the HFD group (Fig. 3G). This downregulation of metabolites may be attributed to decreased amino acid levels affecting one-carbon metabolism, thereby inhibiting nucleotide synthesis pathways, which in turn impacts cellular energy metabolism and DNA/RNA synthesis.
Conversely, compared with the HFD group, after tirzepatide treatment, we identified 526 significant metabolites (377 upregulated, 149 downregulated), as shown in the heat map (Supplementary Fig. 2C). Among these, 56 metabolites were upregulated in the HFD group compared with those in the NCD group and were subsequently reduced after tirzepatide treatment. 241 metabolites were downregulated in the HFD group and then increased in the HFD + tirzepatide group. The altered metabolic pathways post-treatment mainly included protein digestion and absorption, glycerophospholipid metabolism, central carbon metabolism in cancer, as well as taurine and hypotaurine metabolism (Fig. 3H). These pathways are primarily involved in the metabolism, digestion, and absorption of proteins and lipids, highlighting the broad metabolic effects of tirzepatide treatment.
Effects of tirzepatide on liver lipidomic profiling and pathways
The analysis identified 1,378 lipid molecules through UHPLC-MS/MS in both ESI (+) and ESI (-) modes, further classified into 70 distinct lipid species (Fig. 4A). The most prevalent lipid species were 160 TG (11.61%), 136 phosphatidylcholines (PC, 9.87%), and 114 ether-linked lysophosphatidylethanolamines (Ether PE, 8.27%). A total of 772 differential lipids were identified across the three groups (NCD, HFD, and HFD + tirzepatide) via one-way ANOVA. The PLS-DA plots showed distinct clusters among the groups (Fig. 4B). The permutation tests confirmed the stability of these clusters, suggesting that the model was robust and did not overfit (Fig. 4C). Further hierarchical clustering analysis showed significant differences in the lipid profiles among the three groups (Fig. 4D).
In addition, differential analysis was conducted between the HFD and NCD groups, as well as between the HFD + tirzepatide and HFD groups. In total, 490 significantly different lipids were identified between the HFD and NCD groups. Among these differential lipids, TG represented 12.65%, PC 12.24%, and Ether PE 9.59% (Fig. 4E). Between the HFD + tirzepatide and HFD groups, 420 differential lipids were identified, with 280 upregulated and 140 downregulated. The major differential lipids identified were TG (15.71%), Ether PE (10.71%), and PC (6.43%) (Fig. 4F). The relative contents of fatty acids (FA), phosphatidylinositol (PI), and phosphatidylglycerol (PG) were significantly upregulated in the HFD group compared to those in the NCD group. Conversely, PC, phosphatidylethanolamine (PE), and lysophophatidylcholine (LPC) levels were downregulated. In the HFD group, the profiles of Triglycerides (TG, 66.13%) and diacylglycerol (DG, 26.67%) were upregulated (Fig. 4G). In the HFD + tirzepatide group, the relative levels of PC, PE, and LPC were significantly upregulated compared with those in the HFD group, whereas the levels of DG, PI, and FA were downregulated. Furthermore, the TG levels (65.15%) were also decreased (Fig. 4H). These results indicated that tirzepatide treatment induced a notable shift in circulating lipid profiles, suggesting potential therapeutic effects on lipid metabolism in MAFLD.
Identification of key protein targets and potential mechanisms of tirzepatide
A total of 6838 quantifiable proteins were identified via LC-MS/MS. Visualisation of protein separation between the HFD and NCD groups, as well as the HFD + tirzepatide and HFD groups, was accomplished using PCA score plots (Fig. 5A, B). The plot showed that the protein profiles were distinctly segregated, indicating considerable variability, primarily through two principal components.
The study identified 753 differentially regulated proteins (356 upregulated and 397 downregulated) in the HFD group compared with the NCD group (Fig. 5C), and 794 differentially regulated proteins (511 upregulated and 283 downregulated) in the HFD group compared with the HFD group (Fig. 5D). Among these, 162 upregulated proteins in the HFD group were modulated by tirzepatide treatment and 201 downregulated proteins in the HFD group were restored to normal levels by tirzepatide treatment (Fig. 5E).
To further understand the biological functions and key pathways involved in tirzepatide treatment for MAFLD, we conducted functional enrichment analysis via Gene Ontology (GO) and KEGG. Compared with the NCD group, the HFD group exhibited significant changes in biological processes (BP) such as cellular lipid metabolism and fatty acid metabolic. In the cellular component (CC) category, enrichment analysis revealed significant alterations in peroxisome and autophagosome membrane. Molecular function (MF) analysis indicated significant changes in oxidoreductase activity and catalytic activity (Fig. F). KEGG pathway enrichment analysis further revealed that the differential proteins were primarily involved in key pathways such as retinol metabolism, steroid hormone biosynthesis, PPAR signalling pathway, lysosome, and fatty acid degradation, all of which are closely related to lipid metabolism and protein degradation (Fig. 5H).
In the PPAR signalling pathway, the expression of FA transport proteins (Cd36), fatty acid-binding proteins (Fabp2, Fabp4), and perilipins (Plin2, Plin4, and Plin5) associated with lipid accumulation were significantly increased. This was consistent with the lipidomic results showing increased FA and TG in the HFD group. Furthermore, the upregulation of FAO related proteins such as Cpt1a, Acaa1b, Ehhadh, and Cyp4a10 suggests that the liver is attempting to counteract lipid accumulation by increasing FAO.
Additional differential enrichment analysis found that proteins related to the mitophagy pathway, such as Sqstm1, Bnip3, Map1lc3b, and Optn, were significantly upregulated, suggesting an increase in mitochondrial damage. However, in the lysosomal pathway, most acid hydrolases and Atp6v0d1 were downregulated. The downregulation of Atp6v0d1 indicates a potential reduction in the conversion of ATP to ADP, which may affect the pH of lysosomes. Metabolomics data also showed decreased ADP levels, further supporting the hypothesis of altered lysosomal pH.
Compared to the HFD group, tirzepatide treatment exhibited significant changes in various BP, particularly in cellular lipid catabolic and peroxisomal transport. In the CC category, enrichment analysis revealed significant changes in the microbody membrane and peroxisomal membrane, suggesting the critical role of these membrane structures in metabolic regulation. MF analysis indicated that tirzepatide treatment significantly regulated steroid hydroxylase activity and acyl-CoA hydrolase activity. These changes in enzyme activity may directly impact lipid metabolic pathways (Fig. 5I).
KEGG pathway enrichment analysis indicated that the differential proteins following tirzepatide treatment were primarily enriched in key metabolic pathways such as PPAR signalling pathway, lysosome, cholesterol metabolism, retinol metabolism, and fatty acid degradation (Fig. 5J). This suggests that tirzepatide may reduce lipid accumulation and protect hepatocytes through multiple mechanisms.
In addition, we employed the STRING database to perform a protein-protein interaction (PPI) network analyses on 188 proteins enriched in significant KEGG pathways (p < 0.05) between the HFD group and the HFD + tirzepatide group. Subsequently, we visualised the network using Cytoscape software (Fig. 5H). This analysis showed that several critical proteins, including Cyp7a1, Scarb1, Pck1, Abcg5, Abcg8, and Nr1h4 were significantly upregulated after post-treatment, indicating enhanced liver function and bile acid metabolism. In contrast, proteins such as CD36, Acox3, Acaa1b, Ehhadh, Cyp4a10, Hsd17b4, Adipoq, and Lipe were significantly downregulated, suggesting reduced lipid peroxidation and inflammation. These proteins are known for their substantial roles in liver metabolism, lipid transport, and the inflammatory response.
Integration analysis of the mechanistic effects of tirzepatide in MAFLD
To further investigate the molecular mechanisms through which tirzepatide alleviates MAFLD, we conducted a comprehensive analysis of liver tissues via metabolomics, lipidomics, and proteomics. Our findings indicate that tirzepatide treatment reverses the key disrupted metabolic and mitophagic pathways identified in MAFLD mice, as shown in Fig. 6. After treatment with tirzepatide, the expression of free fatty acids (FFA) and FA transport proteins (Fabp2, Fabp5, and Cd36) was significantly downregulated, potentially reducing hepatic lipid uptake. In addition, the levels of DG, TG, and lipid droplet proteins (Plin2, Plin4, and Plin5) were significantly reduced, which was consistent with the observed decrease in hepatic lipid accumulation. Further analysis revealed that key proteins involved in gluconeogenesis, pentose phosphate pathway, and glycogen synthesis were significantly upregulated after administration, indicating the restoration of glucose metabolism. FA oxidation-associated proteins (Ehhadh, Acaa1a, Acaa1b, and Cyp4a10) were downregulated after tirzepatide treatment, suggesting a reduction in compensatory lipid oxidation. This may have a positive effect on alleviating oxidative stress and protecting the mitochondria from damage. Furthermore, the lysosome-related compounds D-mannose-6-phosphate (M6P) and Tcirg1 protein were upregulated (p < 0.05), ensuring proper transport and activity of lysosomal acid hydrolases. Concurrently, the metabolic disturbances in phospholipids (PC, PI, PG, and PS), amino acids, cholesterol, and bile acids were corrected (p < 0.05) following tirzepatide treatment. In summary, tirzepatide alleviates MAFLD pathology through multiple pathways, including the improvement of cholesterol, glucose, and lipid metabolism, as well as the enhancement of mitophagy.
Validation of multiple mechanisms of tirzepatide treatment in MAFLD mice
To further elucidate the molecular mechanisms by which tirzepatide treats MAFLD in mice, we conducted PRM experiments on liver tissues to verify the reliability of protein expression levels.
In the HFD group, the expression levels of Cd36, Fabp2, Fabp4, Plin2, Plin4, and Plin5 were significantly upregulated. After tirzepatide treatment, the expression of these proteins was significantly reduced (Fig. 7A). FAO-related proteins Cpt1a, Acaa1b, and Cyp4a10 were significantly upregulated in the HFD group (Fig. 7B), along with antioxidant stress proteins Gstm2, Hmox1, and Hspb1. In the tirzepatide treatment group, the expression of these proteins was significantly reduced, suggesting that tirzepatide can alleviate oxidative stress (Fig. 7C). Mitochondrial autophagy-related proteins (Bnip3 and Optn) were significantly elevated in the HFD group, while lysosome-related protein Atp6v0d1 was significantly downregulated. After tirzepatide treatment, mitochondrial autophagy-related protein Optn was downregulated, and lysosome-related proteins (Lamp1 and Lamp2) were upregulated (Fig. 7D). Additionally, we found that proteins involved in cholesterol metabolism, such as Abcg5, Abcg8, Cyp7a1, and HNF4A, were significantly upregulated after tirzepatide treatment (Fig. 7E), which increased the direct transport of cholesterol to the gallbladder and its excretion via faeces, thereby reducing the amount of cholesterol in systemic circulation.