Metabolic and hepatic effects of semaglutide and empagliflozin on metabolic dysfunction-associated steatotic liver disease mice

Abstract

BACKGROUND

The molecular mechanisms associated with semaglutide and empagliflozin in metabolic dysfunction-associated steatotic liver disease (MASLD) still require further studies to develop precise therapeutic strategies.

AIM

To investigate the effects and the mechanism of action of semaglutide and empagliflozin on MASLD in obese mice.

METHODS

The experimental subjects consisted of 32 mice, which were arbitrarily allocated into four distinct groups: (1) The control group; (2) The high-fat group; (3) The Sema group; and (4) The Empa group. Mice were assessed for body weight changes, glycolipid metabolic status, inflammatory oxidative stress levels, pathology and metabolomics.

RESULTS

Semaglutide and empagliflozin have been demonstrated to exert a substantial impact on glycolipid reduction, the amelioration of glycolipid metabolism disorders, the attenuation of inflammation and oxidative stress levels, and the restoration of the pathological structure of liver injury to a certain extent in obese mice. No statistically significant differences in the outcomes associated with MASLD were identified between the two cohorts. The results of this study demonstrated that both semaglutide and empagliflozin had the capacity to influence the levels of several lysophosphatidylcholine (LPC).

CONCLUSION

It has been hypothesised that the amelioration of MASLD by semaglutide and empagliflozin may be associated with a decrease in the levels of several LPCs in liver tissue.

Key Words: Metabolic dysfunction-associated steatotic liver disease; Semaglutide; Empagliflozin; Lysophosphatidylcholine; Metabolomics

Core Tip: The present study demonstrated that semaglutide and empagliflozin reduced body weight, ameliorated disorders of glucose and lipid metabolism, lowered levels of inflammation and oxidative stress, and attenuated metabolic dysfunction-associated steatotic liver disease in obese mice. The following section outlines possible mechanisms for reducing the levels of various lysophosphatidylcholines.

INTRODUCTION

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a chronic condition characterised by an abnormal accumulation of fat in the liver (> 5% hepatocellular fat) in the absence of excessive alcohol consumption or other discernible factors of liver damage[1,2]. The pathogenesis of MASLD is complex and involves a multifactorial interaction of metabolic disorders, oxidative stress, inflammatory response and fibrosis[3]. The now widely recognised 'multiple-strike theory' has superseded the earlier 'second-strike theory' and emphasises that multiple factors contribute to the onset and progression of MASLD. The primary driver of MASLD is insulin resistance (IR), particularly in the liver and adipose tissue[4]. The principal underlying factor of MASLD is considered to be IR, particularly the diminished insulin sensitivity of both liver and adipose tissue, in conjunction with lipid metabolism-related disorders. Collectively, these factors result in the accumulation of intracellular lipids within liver cells. The subsequent build-up of these intracellular lipids can trigger a state of oxidative stress and lipotoxicity, which in turn can precipitate the onset of MASLD[5]. Research into the pathological mechanisms of MASLD has provided a solid foundation for the development of precise therapeutic strategies. However, at present there is no specific drug available, and lifestyle interventions remain the mainstay of treatment.

In recent years, there has been an exploration of glucose-lowering drugs, such as glucagon-like peptide-1 receptor agonists (GLP-1RA) (e.g., semaglutide) and sodium-glucose cotransporter-2 inhibitors (SGLT2i) (e.g., empagliflozin), for the treatment of MASLD due to their multiple metabolic modulatory effects[6,7]. Semaglutide has been demonstrated to enhance insulin secretion and inhibit glucagon release through activation of the GLP-1 receptor, thereby improving IR[8]. It has also been shown to reduce caloric intake and significantly decrease body weight through central appetite suppression. In addition, semaglutide has been shown to reduce hepatic steatosis, inflammation, and fibrosis in animal models, possibly through inhibition of hepatic stellate cell activation[9]. The present indication has not been approved for the treatment of MASLD, and further research is required in the form of long-term liver histological endpoint studies. Empagliflozin exerts its pharmacodynamics by inhibiting renal glucose reabsorption, promoting urinary glucose excretion, and reducing blood glucose and body weight[10]. In addition, it has been demonstrated to reduce free fatty acid levels, improve insulin sensitivity, and reduce hepatic glucose xenobiosis. Moreover, evidence from animal studies suggests that empagliflozin may also reduce hepatic inflammatory factors and oxidative stress[11,12]. Phase III trials that utilise liver histology as an endpoint are deficient. Semaglutide and empagliflozin are two glucose-lowering drugs that have gained significant popularity due to their efficacy in reducing blood glucose levels, promoting weight loss, and ameliorating metabolic disorders. However, the precise molecular mechanisms through which these drugs confer protection against MASLD remain to be fully elucidated.

Metabolomics provides a unique perspective for understanding life processes, disease mechanisms and environmental interactions by comprehensively analysing the metabolic networks of organisms. The potential for clinical application of this technology is set to be realised at an accelerated pace, driven by advancements in technology and improvements in database management. The scientific study of small molecule metabolites (having a molecular weight of less than 1500 Da) in organisms, known as metabolomics, has proven effective in the identification of metabolic abnormalities associated with the development of MASLD[13]. Furthermore, metabolomics has facilitated the discernment of systematic imbalances within the metabolic networks of lipids, amino acids, bile acids, and related compounds. This innovative approach has not only enhanced our understanding of the disease's heterogeneity but has also opened new avenues for the development of targeted therapeutic interventions. Moreover, metabolomics offers novel insights into disease classification, enabling more precise diagnosis and the identification of therapeutic targets.

Notably, MASLD represents the hepatic manifestation of metabolic syndrome, and its disease progression is insidious yet deleterious. Lifestyle interventions are the current mainstay of treatment, with the potential benefits of semaglutide and empagliflozin yet to be determined. Further elucidation of the molecular mechanisms involved and the development of precise diagnostic strategies are required.

MATERIALS AND METHODS

Animals

A total of 32 male C57 mice (aged six weeks) were procured from Hebei Ivivo Biotechnology Co. Ltd (Shijiazhuang, Hebei Province, China). The experimental diets used were obtained from Beijing Huafukang Bio-technology Co. Specifically, the diets used were a low-fat control diet and high-fat diets designed for laboratory mice (Mus musculus). The Co60 breeding chow displayed a total calorie content of 3628 kcal/kg, exhibiting an amino acid composition comprising 23.2% protein, 12.9% fat and 63.9% carbohydrates. The high-fat feed (H10060) exhibited a total calorie content of 5.24 kcal/kg, comprising 20% protein, 20% carbohydrate, and 60% fat. The animals were accommodated in housing structures that maintained optimal temperature and humidity levels. The animals were provided with sufficient food and water, and their bedding and feed were changed regularly. Body weight of each group of mice was recorded on a weekly basis.

Experimental design

Following a week of acclimatisation, the mice were randomly divided into two groups: (1) A control group (NCD group, n = 8); and (2) A high-fat group (HFD group, n = 24). The NCD group was administered a diet consisting of 60% carbohydrates, 20% protein and 20% fat, while the HFD group was given a diet with a high fat content (60% fat, 20% protein, 20% carbohydrates). The modelling results were evaluated at the conclusion of the 12-week period according to the criterion that the body weights of mice in the high-fat group were 20% higher than that of the NCD group. Eligible obese mice were randomly assigned to three groups (n = 8). The HFD group was provided with a high-fat diet and administered 120 μg/kg/day of saline intraperitoneally and 10 mg/kg/day of saline by gavage. The semaglutide intervention group, or Sema group, underwent an intensive dietary intervention involving a high-fat diet, in conjunction with intraperitoneal administration of semaglutide (Novo Nordisk, Copenhagen, Denmark) at a dosage of 120 μg/kg/day. The Empa group was administered a high-fat diet and 10 mg/kg/day of empagliflozin (Boehringer Ingelheim Pharmaceuticals) by gavage. As per the protocol established in a preceding study, both semaglutide and empagliflozin were diluted using saline[14,15]. The intervention was administered across a 12-week period, with participants allocated to one of the following groups and subjected to different treatments.

Intraperitoneal glucose tolerance test

The blood glucose levels of the subjects in question were monitored by means of a Roche blood glucose meter of German manufacture. The Mouse Insulin Enzyme-Linked Immunosorbent Assay kit was supplied by Wuhan Elite Biotechnology Co. Prior to the commencement of the experiment, the mice were subjected to a fasting period of 6-8 hours and provided with unrestricted access to water. Following this, the tail tips of the mice were clipped and blood was extracted for the purpose of measuring fasting blood glucose. This was followed by the intraperitoneal injection of glucose (2 g/kg) according to the body weight of the mice. Tail tip blood was collected at 15 minutes, 30 minutes, 60 minutes, 90 minutes and 120 minutes for the purpose of glucose measurement.

Serological indicators

The collection of blood was followed by a period of standing, during which the process of centrifugation was initiated. This resulted in the extraction of serum. The following parameters were measured: (1) Serum alanine aminotransferase (ALT); (2) aspartate aminotransferase (AST); (3) Triglycerides (TG); (4) Total cholesterol (TC); (5) Low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C); (6) Malondialdehyde (MDA); (7) Superoxide dismutase (SOD); (8) Tumour necrosis factor-α (TNF-α); (9) Interleukin (IL)-6 and IL-1β; and (10) Uric acid (UA) (Nanjing Jiancheng, Nanjing, Jiangsu Province, China).

Hematoxylin-eosin staining

Liver tissues were subjected to a series of processing steps, including fixation, dehydration, transparency, paraffin-embedding and sectioning. Subsequent to this, the dewaxed and dehydrated sections were immersed sequentially in hematoxylin staining for a duration of five minutes, hydrochloric acid ethanol differentiation for five seconds, running water re-blue for ten minutes, eosin staining and re-staining for one to two minutes. The final step involved a gradient ethanol dehydration process, followed by xylene transparency, and sealing with neutral gum. The images were captured using a microscope (ECLIPSE Ci-L, Nikon, Tokyo, Japan).

Oil red O staining

Frozen sections of liver tissue were subjected to fixation in 4% paraformaldehyde for a period of 10 minutes. Thereafter, the samples were rinsed in distilled water and washed in 60% isopropanol for a duration of one minute. The staining process involved an initial incubation in an oil red O working solution for 15 minutes, followed by differentiation in 60% isopropanol until the background was deemed clear. Subsequently, the samples were stained with hematoxylin for one minute, washed in water, and then sealed with glycerol gelatin. Subsequent microscopic (Nikon ECLIPSE Ci-L, Tokyo, Japan) observation revealed pathological changes.

Masson staining

The procedure involved the dewaxing and hydration of paraffin-sectioned tissue from a liver sample. This was followed by sequential immersion in solutions designed for staining: (1) Ferric hematoxylin; (2) Lichun red acidic magenta mixture; (3) Phosphomolybdic acid solution; and (4) Aniline blue. Finally, the tissue was dehydrated using a gradient of ethanol, then xylene transparency was applied, followed by neutral gum sealing. Subsequent microscopic observation revealed pathological changes.

Transmission electron microscopy

Mouse liver tissue was fixed in a special liquid called 2.5% glutaraldehyde solution at 4 °C. After washing, the tissue samples were put into a 2% osmium tetroxide solution to fix them. After washing, the tissue samples were put into a 2% osmium tetroxide solution to fix them, then graded using ethanol and propylene oxide replacement, and finally embedded in epoxy resin. The liver tissue was cut into very thin slices and stained with special chemicals. After drying, the samples were examined and the images were taken using a special microscope called a transmission electron microscope model HT7700 from Hitachi, Japan.

Metabolomics

Firstly, liver tissue samples were subjected to a series of pre-processing steps, encompassing sample collection, protein precipitation, metabolite extraction and centrifugation purification, with the objective of acquiring high-purity metabolite extracts. Subsequently, the metabolites were separated and detected by high-throughput using liquid chromatography-mass spectrometry/global chloroform-mass spectrometry, and then amalgamated with a database of matched standards to facilitate qualitative and quantitative metabolite analyses. Following the acquisition of the data, the metabolites exhibiting significant disparities between the groups were subjected to screening through the utilisation of principal component analysis and partial least squares discriminant analysis. The metabolic pathway enrichment tool was employed to conduct a comprehensive analysis of the pivotal biochemical pathways implicated in the metabolic pathways. This approach ultimately led to the revelation of the metabolic phenotypic alterations and the potential biological mechanisms underlying the samples.

Statistical analysis

The data presented in the manuscript was all presented as the mean ± SD. The analysis of said data was conducted using GraphPad Prism 9 and the subsequent visualization of the data was undertaken. The statistical significance of differences between various groups was determined by conducting one-way analysis of variance followed by least significant difference tests. The statistical significance of the findings was established as P < 0.05.

RESULTS

Changes in body weight and glucose metabolism

The results of the study demonstrated that, following a fortnight of further high-fat dietary intervention, there was a significant increase in body weight of high-fat mice in comparison to non-high-fat dietary mice (P < 0.01; Figure 1A). This increase in body weight was observed to gradually increase over time. Following a fortnight-long intervention period during which both semaglutide and empagliflozin were administered, a significant decrease in body weight was observed in the mice. At the conclusion of the experimental phase, which spanned a total of 17 weeks, a marked difference in body weight was evident between the semaglutide and empagliflozin groups (P < 0.01; Figure 1A). This difference in body weight persisted until the culmination of the trial. In relation to glucose metabolism, semaglutide and empagliflozin both demonstrated improvements in IR and glucose tolerance, with no significant difference observed between the two (Figure 1B and C). Furthermore, the investigation revealed that both drugs exhibited no substantial impact on fasting glucose levels (Figure 1D).

Figure 1 Changes in body weight and glucose metabolism. A: Folding line graph of body weight change over time for each group of mice (n = 8). The black arrow indicates the point in time when semaglutide and empagliflozin intervened. The ^aP < 0.01 on the first straight line indicates high-fat group (HFD group) vs control group (NCD group); ^bP < 0.01 indicates HFD vs Sema; ^cP < 0.01 indicates HFD vs Empa; B: Comparison of insulin levels in four groups of mice (n = 8). ^aP < 0.01 indicates NCD vs Sema, ^bP < 0.01 indicates HFD vs Sema, ^cP < 0.01 indicates HFD vs Empa, ^dP < 0.01 indicates NCD vs Empa; C: Intraperitoneal glucose tolerance results. ^aP < 0.01 indicates HFD vs NCD, Sema and Empa; D: Comparison of fasting blood glucose levels in the four groups of mice (n = 8). ^aP < 0.01 indicates NCD vs HFD, ^bP < 0.01 indicates NCD vs Sema, ^cP < 0.01 indicates NCD vs Empa. FBG: Fasting blood glucose; HFD: A high-fat group; NCD: A control group.

Changes in serological indicators

In terms of the convenience of lipid metabolism, both semaglutide and empagliflozin reduced TC and LDL-C levels and increased HDL-C levels, with no significant difference between the two (Figure 2A-C). However, for TG, both drugs reduced the levels to a limited extent, while semaglutide appeared to reduce it to a greater degree than empagliflozin (Figure 2D). The analysis of the findings revealed that inflammation and oxidation stress exhibited a downward trend with both semaglutide and empagliflozin, as indicated by a decrease in MDA, IL-6, IL-1β, and TNF-α, and an increase in SOD levels. However, the study found no significant differences between the two substances (Figure 2E-I). Furthermore, both semaglutide and empagliflozin demonstrated a reduction in serum ALT and AST levels (Figures 2J and K). It is noteworthy that semaglutide led to a substantial reduction in UA levels, a phenomenon that was not observed with empagliflozin (Figure 2L).

Figure 2 Changes in serological indicators. A: Total cholesterol. ^aP < 0.01 indicates high-fat group (HFD group) vs control group (NCD group), ^bP < 0.01 indicates HFD vs Sema, ^cP < 0.01 indicates NCD vs Sema, ^dP < 0.01 indicates HFD vs Empa, ^eP < 0.01 indicates NCD vs Empa; B: Low-density lipoprotein cholesterol. ^aP < 0.01 indicates HFD vs NCD, ^bP < 0.01 indicates HFD vs Sema, ^cP < 0.01 indicates HFD vs Empa; C: High-density lipoprotein cholesterol. ^aP < 0.01 indicates HFD vs NCD, ^bP < 0.01 indicates HFD vs Sema, ^cP < 0.01 indicates HFD vs Empa; D: Triglycerides. ^aP < 0.01 indicates HFD vs NCD, ^bP < 0.01 indicates Sema vs Empa, ^cP < 0.01 indicates NCD vs Empa; E: Malondialdehyde. ^aP < 0.01 indicates HFD vs NCD, ^bP < 0.01 indicates HFD vs Sema, ^cP < 0.01 indicates HFD vs Empa; F: Superoxide dismutase. ^aP < 0.01 indicates HFD vs NCD, ^bP < 0.01 indicates HFD vs Sema, ^cP < 0.01 indicates NCD vs Sema, ^dP < 0.01 indicates HFD vs Empa, ^eP < 0.01 indicates NCD vs Empa; G: Tumour necrosis factor-α. ^aP < 0.01 indicates HFD vs NCD, ^bP < 0.01 indicates HFD vs Sema, ^cP < 0.01 indicates NCD vs Sema, ^dP < 0.01 HFD vs Empa, ^eP < 0.01 indicates NCD vs Empa; H: Interleukin (IL)-6; ^aP < 0.01 indicates HFD vs NCD, ^bP < 0.01 indicates HFD vs Sema, ^cP < 0.01 indicates HFD vs Empa; I: IL-1β. ^aP < 0.01 indicates HFD vs NCD, ^bP < 0.01 indicates HFD vs Sema, ^cP < 0.01 indicates HFD vs Empa, ^dP < 0.01 indicates NCD vs Empa; J: Alanine aminotransferase. ^aP < 0.01 indicates HFD vs NCD, ^bP < 0.01 indicates HFD vs Sema, ^cP < 0.01 HFD vs Empa; K: Aspartate aminotransferase. ^aP < 0.01 indicates HFD vs NCD, ^bP < 0.01 indicates HFD vs Sema, ^cP < 0.01 indicates HFD vs Empa; L: Uric acid. ^aP < 0.01 indicates HFD vs NCD, ^bP < 0.01 indicates HFD vs Sema, ^cP < 0.01 indicates NCD vs Empa; n = 4/group. ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; HDL-C: High-density lipoprotein cholesterol; HFD: A high-fat group; IL: Interleukin; LDL-C: Low-density lipoprotein cholesterol; MDA: Malondialdehyde; NCD: A control group; SOD: Superoxide dismutase; TC: Total cholesterol; TG: Triglycerides; TNF-α: Tumour necrosis factor-α; UA: Uric acid.

Changes in pathological structures

A comprehensive evaluation of the study cohort revealed that the HFD group exhibited substantial diffuse steatosis of hepatocytes, accompanied by significant hydropic degeneration of hepatocytes, the presence of spherical vacuoles, and cellular swelling. Masson's staining revealed comparable outcomes, with the HFD group exhibiting localised lymphocytic infiltration but no discernible liver necrosis. While both semaglutide and empagliflozin demonstrated efficacy in enhancing these indices, semaglutide was found to be more efficacious than empagliflozin, with the exception of lymphocytic infiltration (Figure 3A and B). Furthermore, the administration of both semaglutide and empagliflozin resulted in a reduction of the number of lipid droplets (Figure 3C). The results of the transmission electron microscopy investigation revealed that the mitochondria in the HFD group exhibited slight swelling. Additionally, the ridges were found to be contracted, and the intramembrane matrix was observed to be partially disintegrated. The intervention of semaglutide and empagliflozin resulted in enhanced mitochondrial microautophagy, reduced mitochondrial structural swelling, and relatively more ordered mitochondrial cristae in comparison to the HFD group (Figure 3D).

Figure 3 Changes in pathological structures. A: Hematoxylin-eosin staining (400 ×); B: Masson staining (400 ×); C: Oil red staining (400 ×); D: Transmission electron microscopy images showed the mitochondria, mitochondrial microautophagy structure and ribosomes. HE: Hematoxylin-eosin; HFD: A high-fat group; NCD: A control group; TEM: Transmission electron microscopy.

Changes in liver metabolomics

In order to explore the mechanisms by which the improvement of MASLD is achieved in obese mice by semaglutide and empagliflozin, we performed metabolomic analyses. The findings of the differential metabolite analysis demonstrated that lysophosphatidylcholine (LPC) exhibited the most significant differential expression among the four groups (Figure 4). Moreover, correlation analysis indicated that LPC plays a pivotal role in the progression of MASLD (Figure 5). The findings of the differential metabolite pathway enrichment analysis between the four groups indicated that these metabolites were predominantly enriched in Histidine metabolism, Arachidonic acid metabolism, and adenosine triphosphate-binding cassette transporters (Figure 6). The present study was conducted with the objective of providing further clarification on the effects of semaglutide and empagliflozin on LPC. To this end, the study analysed the differences in the concentrated LPC of the four groups, noting significant comparative differences between them. The results demonstrated that a high-fat diet resulted in increased levels of hepatic LPC (16:0), LPC [18:1 (11Z)], LPC [20:4 (5Z, 8Z, 11Z, 14Z)], LPC [22:4 (7Z, 10Z, 13Z, 16Z)], LPC [20:4 (8Z, 11Z, 14Z, 17Z)], and LPC (0:0/16:0), LPC (17:0), and LPC [22:5 (7Z, 10Z, 13Z, 16Z, 19Z)] levels. It was observed that both semaglutide and empagliflozin reduced the levels of the aforementioned metabolites to varying degrees (Figure 7).

Figure 4 Heatmap of the top 50 difference metabolites.

Figure 5 Heatmap pearson correlation analysis for assessing the correlation between different metabolites. Some metabolites are negatively correlated (blue) and some are positively correlated (red). The darker the colour the stronger the correlation.

Figure 6 Kyoto Encyclopedia of Genes and Genomes enrichment analysis pointed out the top 20 hepatic pathways by bubble mapping.

Figure 7 Line plots of the variation of several lysophosphatidylcholine between the four groups. A: LysoPC [20:1 (11Z)]; B: LysoPC (16:0); C: LysoPC [18:1 (11Z)]; D: LysoPC [20:4 (5Z, 8Z, 11Z, 14Z)]; E: LysoPC [20:3 (5Z, 8Z, 11Z)]; F: LysoPC [20:5 (5Z, 8Z, 11Z, 14Z, 17Z)]; G: LysoPC (15:0); H: LysoPC [22:4 (7Z, 10Z, 13Z, 16Z)]; I: LysoPC [20:4 (8Z, 11Z, 14Z, 17Z)]; J: LysoPC (0:0/16:0); K: LysoPC (17:0); L: LysoPC [22:5 (7Z, 10Z, 13Z, 16Z, 19Z)]. ^aP < 0.01 indicates high-fat group (HFD group) vs control group, ^bP < 0.05 Sema vs HFD, ^cP < 0.05 indicates Empa vs HFD. HFD: A high-fat group; NCD: A control group.

DISCUSSION

MASLD is a condition devoid of secondary elements, in which fatty deterioration of the liver is identified through imaging or histological inspection. It is the most widespread chronic liver illness in Western populations, with roughly 50%-60% of overweight adults exhibiting fatty liver[16]. Corpulence considerably heightens the likelihood of developing serious hepatic steatosis, with hepatocellular degeneration and predominantly lobular inflammation, as well as cirrhosis[17]. In addition, the risk of MASLD was found to have been doubled in all analyses of patients with obesity, and a higher risk of developing hepatocellular carcinoma was also found to have been presented by patients with MASLD[18-20]. Also, there is a higher chance of getting heart disease, which is one of the main causes of death[21].

The effects of GLP-1RA on the liver are characterised by a series of indirect pathways, which include alterations in portal and peripheral plasma insulin and glucagon concentrations. Furthermore, improvements in hepatocyte mitochondrial function and hepatic insulin sensitivity have been observed, in addition to adipose tissue lipotoxicity induced by reduced body weight and body weight-independent mechanisms[22]. The findings of this study demonstrated that semaglutide led to a substantial reduction in body weight, along with the amelioration of glucose-lipid metabolism disorders, and a reduction in inflammation and oxidative stress levels. In addition to these observations, a decrease in lipid deposition in hepatocytes was noted. This is consistent with the previously identified mechanism of GLP-1RA improvement in MASLD. As demonstrated in previous clinical studies, semaglutide has been shown to have a beneficial effect on patients suffering from MASLD. In patients diagnosed with both type 2 diabetes mellitus (T2DM) and MASLD, treatment involving the administration of semaglutide in combination with metformin resulted in a substantial decrease in ALT and AST levels, as well as a notable reduction in liver fibrosis markers[23,24]. However, these effects were not directly observed on hepatocytes, as recent evidence suggests that hepatocytes lack GLP-1 receptor. Consequently, the precise mechanism by which these effects occur requires further exploration.

SGLT2i is a medication that has become increasingly popular due to its proven efficacy in the treatment of patients diagnosed with T2DM. It is evident that, in addition to the hypoglycaemic properties of the substance in question, there is an array of cardiovascular and renal protection, along with minor reductions in both blood pressure and body weight[25]. As well as exerting direct effects on hepatic inflammation, reactive oxygen species (ROS) production, and mitochondrial function[26], the administration of SGLT2i treatments may also have beneficial effects in cases of MASLD. It is evident that the remaining mechanisms encompass a reduction in macrophage polarisation, autophagy, endoplasmic reticulum stress, and attenuation of steatosis and fibrosis, amongst others[27-29]. In the present study, the hepatoprotective agent empagliflozin was found to demonstrate clinical evidence indicative of its efficacy in improving MASLD, as indicated by a reduction in body weight, enhancement in glycolipid metabolism, and reduction in inflammatory and oxidative stress markers. Furthermore, the treatment resulted in improvements to hepatic pathological structures. In preclinical studies, the administration of empagliflozin resulted in increased energy expenditure, adipose tissue browning, and decreased adipose tissue inflammation in obese mice. A number of randomised controlled trials have demonstrated that a period of 6-12 months of treatment with empagliflozin results in a significant reduction in intrahepatic fat content, accompanied by improvements in IR and liver enzyme levels[30-32]. In summary, the potential benefits of empagliflozin in MASLD are manifold, including the improvement of metabolic disorders, the reduction of hepatic fat accumulation, and the exertion of anti-inflammatory and anti-fibrotic effects. The extant evidence supports the utilisation of this intervention as part of a comprehensive management strategy. Nevertheless, further research is required in order to ascertain its efficacy in patients with non-diabetic MASLD, and to explore more deeply the specific mechanisms involved.

Semaglutide and empagliflozin have been demonstrated to intervene in the process of mitochondrial microautophagy, thereby reducing the structural expansion effect that is associated with this process. This effect may be dependent on the regulation of lysozyme. Lysosomal lysozyme has been identified as a pivotal factor in the terminal degradation phase of mitochondrial microautophagy. The potential for a synergistic effect between lysosomal lysozyme and other factors, such as microautophagy initiation signals, has been postulated. However, the absence of direct evidence hinders the confirmation of this hypothesis. Nonetheless, it is hypothesised that lysosomal lysozyme function plays a central role in this process. Despite the absence of direct evidence, it is hypothesised that lysozyme function constitutes the primary support, a hypothesis that can be tested in future studies through lysozyme knockdown/overexpression and other experimental approaches. The potential involvement of non-lysosomal pathways remains a possibility.

LPC has been defined as a hydrolysate of phosphatidylcholine, which is catalytically generated by phospholipase A2 (PLA2) and constitutes an important class of bioactive lipids[33]. In the context of lipid metabolism, LPC has been implicated in cell membrane repair and signalling processes. However, elevated levels of LPC have been observed to be associated with various pathological conditions. Recent studies have demonstrated that LPC plays a pivotal role in the development of MASLD, and its aberrant metabolism is closely related to the pathological mechanisms of MASLD[34]. LPC is implicated in the pathological progression of MASLD through several mechanisms: The substance under scrutiny has been demonstrated to promote the uptake of fatty acids in hepatocytes, while concomitantly inhibiting mitochondrial β-oxidation and exacerbating the accumulation of intrahepatic fat. In addition, it has been shown to activate pro-inflammatory pathways, such as toll like receptor/nuclear factor-kappa B (TLR/NF-κB), and to induce immune cell infiltration. It has been demonstrated that the activation of pro-inflammatory pathways, such as TLR/NF-κB, serves to induce immune cell infiltration and to amplify the hepatic inflammatory response. It has been demonstrated that the promotion of activation of hepatic stellate cells, in conjunction with the inhibition of extracellular matrix degradation, is a key factor in the progression of hepatic fibrosis. Furthermore, the induction of ROS generation has been shown to exacerbate oxidative stress and hepatocellular injury[35-38]. Furthermore, LPC and its isoforms have the potential to serve as biomarkers for diagnosing, staging, and prognostising MASLD. Targeting the LPC metabolic pathway, for instance by regulating phospholipase A2 activity or interfering with its receptor signalling, offers novel avenues for treating MASLD.

Semaglutide and empagliflozin have been observed to exert a regulatory effect on LPC levels, albeit indirectly, through the improvement of metabolic disorders. Semaglutide has been observed to reduce serum and intrahepatic LPC concentrations by decreasing PLA2 activity or activating LPC-producing receptors through a reduction in body weight, an improvement in IR, and an inhibition of inflammation. This may be more efficacious in MASLD patients with obesity or T2DM[39]. Conversely, empagliflozin has been shown to impede LPC-mediated fatty acid uptake and endoplasmic reticulum stress through the promotion of renal glucose excretion, enhanced energy metabolism, and diminished intrahepatic fat accumulation, thereby concomitantly attenuating LPC concentrations[40]. These effects may be concomitant with enhanced mitochondrial function and reduced oxidative stress. In the present study, metabolomics was employed to demonstrate that semaglutide and empagliflozin could reduce multiple LPC levels in the livers of obese mice. Furthermore, pathway analysis revealed a close relationship with lipid metabolism, suggesting that these two drugs may share a common mechanism of action in the improvement of MASLD. However, it is important to acknowledge that the majority of extant studies have been predicated on indirect indicators of metabolic enhancement. Consequently, there is a necessity for further research that will elucidate the disparities in the regulation of particular subtypes of LPC by the two pharmaceutical agents, as well as the dose-dependent consequences within the context of clinical application. Moreover, the present study does not establish a causal relationship, despite demonstrating a reduction in LPC levels and improvement in MASLD after semaglutide and empagliflozin intervention. The present study is limited by a paucity of mechanistic experiments that would directly demonstrate the causal role of LPC in this process. Further research is required to validate the results of this study through exogenous LPC supplementation or interventions that inhibit LPC degradation.

CONCLUSION

Semaglutide and empagliflozin have been demonstrated to reduce body weight, ameliorate disorders of glucose and lipid metabolism, reduce levels of inflammation and oxidative stress, and attenuate MASLD in obese mice. The underlying mechanism for this effect may involve a common pathway leading to the reduction of multiple LPC levels.