Abstract
Background/Aim: Non-alcoholic fatty liver disease is a major cause of liver-related morbidity and mortality. Metformin is a widely used medication and may have additional benefits beyond glycemic control. Liraglutide, a novel treatment for diabetes and obesity, also has beneficial effects on non-alcoholic steatohepatitis (NASH). Metformin and liraglutide have both benefited NASH treatment. However, no study has reported the effects of combination therapy with liraglutide and metformin on NASH. Materials and Methods: We investigated the in vivo effects of metformin and liraglutide on NASH in a methionine/choline-deficient (MCD) diet-fed C57BL/6JNarl mouse model. Serum triglyceride, alanine aminotransferase and alanine aminotransferase levels were documented. Histological analysis was performed according to the NASH activity grade. Results: After treatment with liraglutide and metformin, body weight loss improved, and the liver/body weight ratio decreased. The metabolic effects and liver injury improved. Liraglutide and metformin alleviated MCD-induced hepatic steatosis and injury. Histological analysis revealed that NASH activity was reduced. Conclusion: Our results provide evidence for the anti-NASH activity of liraglutide in combination with metformin. Liraglutide with metformin may offer the potential for a disease-modifying intervention for NASH.
Non-alcoholic fatty liver disease (NAFLD) is emerging as a major cause of liver-related morbidity and mortality, with a global prevalence of 25.24% (1). It is linked to obesity, insulin resistance and metabolic syndrome, and its appearance ranges from fat accumulation to advanced fibrosis and cirrhosis (2). It is now widely recognized that NAFLD represents a continuum of liver damage, ranging from simple steatosis to non-alcoholic steatohepatitis (NASH), advanced fibrosis and cirrhosis (3, 4). The risk of developing NASH and fibrosis is significantly higher in patients with metabolic comorbidities, such as obesity, diabetes and dyslipidemia (5). While current treatment options focus on lifestyle modifications and management of associated comorbidities, ongoing research is exploring the potential of pharmacological therapies. Recent clinical trials have explored the efficacy of various agents for NASH treatment, including insulin sensitizers, lipid-lowering drugs, and agents targeting inflammation and fibrosis (6-8). However, there is still a lack of consensus regarding the most effective therapeutic approach, and further research is needed to establish the safety and long-term efficacy of these agents (7).
Liraglutide is used to treat type 2 diabetes mellitus (T2DM) and obesity (9, 10). It is a glucagon-like peptide-1 (GLP1) receptor agonist that increases insulin secretion, reduces glucagon secretion and slows gastric emptying (11). This medication has been extensively studied in recent years, and several new findings have emerged regarding its safety and efficacy [reviewed in (12)]. It is recommended as a first-line treatment option by expert organizations such as the American Diabetes Association and European Association for the Study of Diabetes (12, 13). Recent studies have shown that liraglutide may also have a beneficial effect on NASH (14-16). A randomized controlled trial published in The Lancet in 2019 reported that liraglutide was associated with a significant reduction in liver fat content and liver stiffness compared with placebo in patients with NAFLD (16). These studies also found that liraglutide improved insulin sensitivity and lipid metabolism (17-20).
Metformin has widely been used for the management of T2DM for decades (21). Recent studies suggest that metformin may have additional benefits beyond glycemic control (21, 22) and may also be effective in treating NAFLD (23-25). A systematic review and meta-analysis reported that metformin was associated with a significant reduction in liver fat content in patients with NASH (26, 27). Moreover, studies reported that metformin is associated with improvements in liver function and insulin sensitivity in patients with NASH (28-32). These findings suggest that metformin may be a promising treatment option for patients with NASH, particularly for those with T2DM.
Metformin and liraglutide have shed light on NASH treatment. However, to the best of our knowledge, no study has reported the effects of combination therapy with liraglutide and metformin on NASH. In this study, we used a methionine/choline-deficient (MCD) diet-fed C57BL/6JNarl mouse model to study the combined effects of liraglutide and metformin on NASH (33, 34).
Materials and Methods
Chemicals and materials. Liraglutide was purchased from Novo Nordisk, Denmark. Metformin was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). The basic diet (Prolab®,
RMH2500, 5P14) was purchased from LabDiet, Inc. (St. Louis, MO, USA) and an MCD diet (A02082002B) was purchased from Research Diet Inc. (New Brunswick, NJ, USA). The nutritional composition of the basic and MCD diets are presented in Table I. Table II shows the amino acid composition of the MCD diet.
Nutritional composition of standard diet and methionine and choline-deficient diet (MCD).
Composition of amino acids in the methionine/choline deficient diet (MCD).
Animal studies. Six-week-old male C57BL/6JNarl mice were purchased from the National Laboratory Animal Center (Taipei City, Taiwan, ROC). They were housed in wire-mesh cages (39×26×21 cm) at 60% humidity with a 12 h-12 h light/dark cycle (light phase was 8:00 a.m. to 8:00 p.m.). The temperature was maintained at 23±1°C. The Institutional Animal Care and Use Committee of the China Medical University approved the experimental protocol (No. CMUIACUC-2020-013). For the 9-week study period, mice were randomly divided into five experimental groups.
Normal control group fed a standard diet for all 9 weeks (n=3).
NASH control group fed an MCD diet for all 9 weeks (n=3).
Liraglutide-treated group fed an MCD diet for 4 weeks then MCD combined with treatment with 0.5 mg/kg liraglutide (subcutaneous, daily, 5 times a week) for 5 more weeks (n=3).
Metformin-treated group fed an MCD diet for 4 weeks then MCD combined with treatment with 100 mg/kg metformin (oral administration, daily, 5 times a week) for 5 more weeks (n=3).
Combination-treated group fed an MCD diet for 4 weeks then MCD combined with treatment with liraglutide and metformin as above for 5 more weeks (n=3).
Animals were monitored for the study period, with measurement of body weight at intervals; a weight loss of over 25% fulfilled the criterion for euthanasia. At the end of the 9 weeks, animals were sacrificed and serum triglycerides (TG), alanine aminotransferase (ALT) and alanine aminotransferase (AST) levels were documented. Livers were excised and histological analysis was performed.
Analysis of serum TG, ALT and AST levels. Serum ALT, AST and TG levels were determined using a Fujifilm Dri-Chem NX-500 automated clinical chemistry analyzer. All consumables used for testing (Fuji Dri-Chem Slide GOT/AST-P III, #3150; GPT/ALT-P III, #3250; TG-P III, #1650) were manufactured by Fujifilm (Tokyo, Japan), and each batch of consumables was calibrated using the original quality control card before use.
Histological analysis. Liver tissues from the C57BL/6JNarl mice were fixed in 10% neutral formalin, dehydrated, paraffin-embedded and cut into 5 mm-thick sections for hematoxylin-eosin staining. The NASH activity grade was evaluated according to the World Gastroenterology Organisation (35).
Statistical analysis. The results are expressed as the mean±standard error of the mean (n=3) and significance among multiple experimental groups was estimated using one-way analysis of variance (ANOVA), followed by Dunnett’s test or Tukey’s post hoc test using SPSS 16.0 software (SPSS, Inc., IBM, Armonk, NY, USA). A value of p<0.05 indicated statistical significance.
Results
Liraglutide and metformin ameliorated body weight loss and reduced the liver/body weight ratio. After 4 weeks of standard or MCD diet, the mice were treated with metformin, liraglutide, or liraglutide plus metformin for another 5 weeks. Figure 1 shows that MCD control mice weighed 33.3% less (17.10±0.97 g) than age- and sex-matched normal control mice (27.67±0.67 g). Initially, the MCD control mice lost more weight than those of the normal control group. After 2 weeks on the MCD diet, these mice had lost 25% of their body mass/week, 3% in week 3 and 2% thereafter. There was no significant increase in body weight with metformin, compared to MCD control mice; however, there was a significant increase in body weight in mice treated with liraglutide or metformin plus liraglutide, compared to MCD control mice. Figure 2 shows liraglutide led to a significant reduction in the liver/body weight ratio in the liraglutide-treated and liraglutide plus metformin-treated groups; however, the liver/body weight ratio under treatment with metformin did not significantly differ from that under MCD alone.
Body weight of C57BL/6JNarl mice in the five treatment groups. After 4 weeks of standard (control) or methionine/choline-deficient (MCD) diet, the mice were treated with 100 mg/kg metformin, 0.5 mg/kg liraglutide, or liraglutide plus metformin for another 5 weeks. The results are expressed as the mean±standard error of the mean (n=3). Means with different letters at 35 days are significantly different at p<0.05.
Liver/body weight ratio of C57BL/6JNarl mice in five treatment groups. After 4 weeks of standard (control) or methionine/choline-deficient (MCD) diet, the mice were treated with 100 mg/kg metformin, 0.5 mg/kg liraglutide, or liraglutide plus metformin for another 5 weeks. The results are expressed as the mean±standard error of the mean (n=3). Means with different letters are significantly different at p<0.05.
Liraglutide and metformin alleviated metabolic effects and liver injury in mice on an MCD diet. TGs are affected by metformin (36). Figure 3 shows serum TG levels in MCD control mice were significantly increased compared to normal controls. Metformin treatment alone did not significantly affect the TG level. Treatment with liraglutide, alone and combined with metformin, did significantly reduce the serum TG level compared to MCD control mice.
Serum triglyceride levels of C57BL/6JNarl mice in five treatment groups. After 4 weeks of standard (control) or methionine/choline-deficient (MCD) diet, the mice were treated with 100 mg/kg metformin, 0.5 mg/kg liraglutide, or liraglutide plus metformin for another 5 weeks. The results are expressed as the mean±standard error of the mean (SEM) (n=3). Means with different letters are significantly different at p<0.05.
The enzyme AST is found in the liver, heart, skeletal muscle and other tissues in the body. An elevated AST level may indicate liver damage or other underlying medical conditions (19, 37). Liraglutide significantly reduces the level of ALT, an enzyme produced in liver cells that leaks into the bloodstream when cells are damaged (19, 37). Liver function in non-diabetic NAFLD can be improved by metformin therapy (26, 27). The ALT and AST levels were, therefore, examined in the study groups. MCD control mice had a significantly higher serum AST level, more than double that of normal control mice. Similarly, the serum ALT level in MCD control mice was significantly higher, by over 5-fold, than in normal control mice. Our results showed that a reduction in AST and ALT levels was observed in the liraglutide-only and metformin plus liraglutide groups compared to MCD control mice (Figure 4). Our results suggest that the combination of metformin and liraglutide may have positive effects on liver enzymes and liver function in mice with MCD-induced hepatic steatosis.
Serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) of C57BL/6JNarl mice in five treatment groups. After 4 weeks of standard (control) or methionine/choline-deficient (MCD) diet, the mice were treated with 100 mg/kg metformin, 0.5 mg/kg liraglutide, or liraglutide plus metformin for another 5 weeks. The results are expressed as the mean±standard error of the mean (n=3). Means with different letters are significantly different at p<0.05.
Liraglutide and metformin alleviate MCD-induced hepatic steatosis and injury. Several animal studies have demonstrated the effectiveness of liraglutide or metformin in preventing hepatic steatosis and injury in animals of NAFLD [reviewed in (38)]. We further investigated the effect of this combination on MCD-induced hepatic steatosis. The hematoxylin and eosin staining results in Figure 5 and NASH activity grade in Table III demonstrate that liraglutide and metformin plus liraglutide attenuated MCD-induced hepatocyte steatosis, inflammation and ballooning. Our results suggest that the combination of metformin and liraglutide may have positive effects on hepatocyte steatosis and injury.
Hematoxylin-eosin staining in liver tissue of C57BL/6JNarl mice in five treatment groups. After 4 weeks of standard (control) or methionine/choline-deficient (MCD) diet, the mice were treated with 100 mg/kg metformin, 0.5 mg/kg liraglutide, or liraglutide plus metformin for another 5 weeks. The results showed that liraglutide alone and in combination with metformin attenuated MCD-induced hepatocyte steatosis, inflammation and ballooning.
The grade of non-alcoholic steatohepatitis (NASH) (35) of liver from C57BL/6JNarl mice in five treatment groups. After 4 weeks of standard (control) or methionine/choline-deficient (MCD) diet, the mice were treated with 100 mg/kg metformin, 0.5 mg/kg liraglutide, or liraglutide plus metformin for another 5 weeks. Means with different letters are significantly different at p<0.05.
Discussion
NASH, an NAFLD, is a metabolic steatohepatitis syndrome with progressive pathological changes (39, 40). However, then are currently no U.S. Food and Drug Administration-approved therapy agents for NASH or related liver diseases (39). Because T2DM is associated with an increased risk of NASH (41, 42), many research groups have focused on liraglutide and metformin (T2DM therapy agents) for anti-NASH activity (43-47). Early studies showed that liraglutide reduced hepatic steatosis and insulin resistance in in vitro (48-51) and in vivo models (48, 49, 52, 53). The results of a phase II multicenter clinical trial (ClinicalTrials.gov-NCT01237119) showed that 39% of patients who received liraglutide had resolution of definite NASH compared with 9% of such patients in the placebo group (relative risk=4.3, 95% confidence interval=1.0-17.7; p=0.019) and suggested that liraglutide is a safe agent and leads to histological resolution of NASH (16). In this study, we focused on the anti-NASH activity of liraglutide and metformin in C57BL/6J mice fed an MCD diet. The MCD diet-fed C57BL/6J mouse model is a typical and representative nutritional NASH model (54, 55). The MCD diet fed to C57BL/6J mice for 4 weeks caused body weight loss, an increase in liver to body weight ratio, and increased serum TG, ALT and AST levels (56-58). Our results demonstrate that liraglutide significantly improved body weight loss (Figure 1), reduced the liver to body weight ratio (Figure 2), and reduced serum TG (Figure 3) and ALT and AST levels (Figure 4), as well as ameliorating hepatic steatosis and liver injury (Figure 5 and Table III). To the best of our knowledge, this is the first study to report that metformin plus liraglutide alleviates MCD-induced hepatic steatosis in this model.
Liraglutide is a GLP1 receptor agonist (59). In the pancreas, brain and adipose tissue, the mechanism of action of liraglutide, similar to physiological GLP1, for anti-diabetic effect is via controlling food intake, energy absorption and glucose-dependent insulin secretion (38). Liraglutide improves the histological features of NASH, including hepatocyte steatosis, inflammation and ballooning, but has no effect on fibrosis (38). In vitro and in vivo studies have demonstrated that liraglutide decreases de novo lipogenesis, increases fatty acid oxidation and reduces steatosis in human hepatocytes (60-62). Our results suggest that liraglutide improves anti-NASH activity by improving hepatic and adipose insulin sensitivity and reducing hepatic de novo lipogenesis. Metformin, a biguanide glucose-lowering agent, is a first-line therapy for T2DM (22). The mechanism of action of metformin for anti-diabetic effects are as follows: reducing hepatic gluconeogenesis and then reducing glucose production from the liver; reducing intestinal glucose absorption; reducing total cholesterol, low-density lipoprotein, or TG levels; activating AMP-activated protein kinase and regulating energy homeostasis such as lipid and glucose metabolism; reducing cholesterol biosynthesis; inhibiting mitochondrial complex I, preventing hepatic glucose production and increasing glucose utilization; increasing glucose uptake in the periphery; increasing muscle gluconeogenesis; promoting fatty acid oxidation; and improving insulin resistance (22, 63, 64). NAFLD-related studies demonstrated that metformin reduces fat accumulation in the liver and alleviates lipid peroxidation (27, 65-67). Li et al. demonstrated that metformin alleviated hepatocyte inflammation through signal transducer and activator of transcription 3-mediated autophagy (68). Autophagy is a double-edged sword for cell survival and death (69), providing nutrients to maintain cellular energy during fasting and removing damaged organelles, lipids and misfolded proteins in T2DM (69). Dan Zhang et al. demonstrated that metformin improved hepatic steatosis and insulin resistance, promoting transcription factor EB (TFEB)-dependent autophagy processes in a high-fat diet-induced model of NAFLD (70); it has been demonstrated that liraglutide improves hepatic steatosis, with similar effects in the same model (50). Our results suggest that liraglutide significantly improved hepatic steatosis and liver injury. Literature revealed that metformin enhanced anti-NASH activity possibly through promoting TFEB-dependent autophagy processes. In the future, we will test this hypothesis using next-generation sequencing and TFEB activity analysis (71).
In conclusion, our results provide evidence for the anti-NASH activity of liraglutide in combination with metformin. Liraglutide with metformin may offer a potential disease-modifying intervention for NASH.
Acknowledgements
The Authors wish to acknowledge the work of Mr Yung-Shuan Chang (Bio-Cando Ltd. Taiwan) for his excellent technical and equipment support in this study.
This study was supported by a project (grant no. TCRD109-63) from the Hualien Tzu Chi Hospital, Taiwan. This work was also supported in part by projects DMR-112-135 from China Medical University Hospital, Taiwan; V111B-040 from Taipei Veterans General Hospital, Taiwan; and MOST 111-2314-B-075 -083 -MY2 from the Ministry of Science and Technology, Taiwan.
Footnotes
Authors’ Contributions
HY Chiu, JS Yang, FJ Tsai and YJ Chiu conceived and designed the experiments. SC Tsai, YH Lo and SR Jhan performed the experiments. CC Cheng, TY Liu and JS Yang analyzed the data. HY Chiu, SC Tsai, CC Cheng and JS Yang wrote and modified the article. All Authors read and approved the final article.
Conflicts of Interest
The Authors declared no potential conflicts of interest with respect to the research, authorship and publication of this article.
- Received March 6, 2023.
- Revision received March 27, 2023.
- Accepted March 29, 2023.
- Copyright © 2023 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
