Enavogliflozin, an SGLT2 Inhibitor, Improves Nonalcoholic Steatohepatitis Induced by High-Fat, High-Cholesterol Diet
Article information
Abstract
Background
Nonalcoholic fatty liver disease, a progressive condition caused by the accumulation of fat in the liver, begins with simple steatosis and can potentially progress to metabolic dysfunction-associated steatohepatitis (MASH) in the presence of inflammation and fibrosis, ultimately leading to cirrhosis or hepatocellular carcinoma. Increasing evidence indicates that sodiumglucose cotransporter 2 (SGLT2) inhibitors effectively alleviate MASH in mouse models. However, there is a lack of research on the effects of enavogliflozin on liver disease. In the present study, we investigated the effects of SGLT2 inhibitors on MASH induced by a high-fat, high-cholesterol (HFHC) diet in mice.
Methods
Male C57BL/6 mice were fed a normal chow diet, HFHC diet, or HFHC diet with enavogliflozin for 12 weeks. LX-2 and HepG2 cells were treated with enavogliflozin in the presence of various pathological stimuli.
Results
The HFHC diet induced excessive hepatic lipid accumulation, inflammation, and severe fibrosis. Administration of enavogliflozin not only ameliorated hepatic steatosis and fibrotic conditions but also suppressed the production of inflammatory cytokines. Positive outcomes were also observed in in vitro experiments, where enavogliflozin demonstrated the ability to impede the activation of hepatic stellate cells and alleviate lipid accumulation in hepatocytes. The potential pathway through which enavogliflozin attenuated liver fibrosis development may be associated with the transforming growth factor β1/Smad signaling pathway.
Conclusion
Our results suggest that enavogliflozin is effective in a mouse model of MASH by attenuating hepatic steatosis, suppressing inflammation, and improving liver fibrosis.
Highlights
• Enavogliflozin reduces steatosis, inflammation, and fibrosis in a mouse MASH model.
• Its effects are linked to inhibiting HSC activation and the TGF-β1/Smad pathway.
• Enavogliflozin may offer a promising treatment for NAFLD and liver fibrosis.
INTRODUCTION
Nonalcoholic fatty liver disease (NAFLD) is a prevalent chronic liver disease affecting a significant portion of the global population. According to a recent study, approximately 46.9 individuals out of every 1,000 are diagnosed with NAFLD [1]. NAFLD typically starts as nonalcoholic fatty liver, which usually does not lead to severe liver complications. However, in some instances, it progresses to a more serious condition known as metabolic dysfunction-associated steatohepatitis (MASH), characterized by inflammation, liver cell damage, and fat accumulation in the liver. If not treated promptly, MASH can progress to liver fibrosis, which results from chronic liver damage and the subsequent formation of scar tissue in the liver [2].
Excessive deposition of hepatic lipids, including triglycerides (TG) and cholesterol esters, is one of the major factors leading to hepatotoxicity, which exacerbates progressive inflammation, oxidative stress, and fibrosis [3,4]. In a physiological context, lipids are stored within dynamic cellular structures referred to as lipid droplets (LDs), predominantly neutral and non-cytotoxic [5,6]. Disruption of liver lipid metabolism, such as elevated fatty acid intake, heightened de novo lipogenesis (DNL), reduced β-oxidation, or impaired TG secretion, leads to excessive hepatic lipid deposition, characteristic of steatosis [7-9]. Disruption of the lipid balance in hepatocytes triggers the generation of detrimental lipids, resulting in dysfunctional organelles that damage liver cells [10]. In addition to stimulating inflammatory cells to attract leukocytes, some apoptotic or injured hepatocytes release reactive oxygen species (ROS) and intermediaries, such as transforming growth factor β1 (TGF-β1). These factors promote fibrosis by activating quiescent hepatic stellate cells (HSCs) to secrete extracellular matrix, ultimately resulting in the formation of scar tissue in the liver, a hallmark of liver fibrosis [11,12].
Sodium-glucose cotransporter 2 (SGLT2) inhibitors are glucose-lowering medications that lower blood glucose levels by inhibiting its reabsorption in the proximal tubules, thereby promoting urinary glucose excretion [13]. Numerous studies have demonstrated that SGLT2 inhibitors not only offer beneficial effects in the treatment of type 2 diabetes mellitus (T2DM) but also have a favorable impact on cardiovascular and obesity-related risks [14-16]. Additionally, some SGLT2 inhibitors have been shown to improve liver function in patients with NAFLD as well as those with T2DM [17,18]. Enavogliflozin is the first investigational SGLT2 inhibitor in Korea. As it is a new drug, there has been little research on the effects of enavogliflozin on liver diseases. By investigating the effects of enavogliflozin in a diet-induced mouse model of NAFLD, the study aims to elucidate its impact on hepatic steatosis, inflammation, and liver fibrosis. If enavogliflozin demonstrates efficacy in attenuating these pathological features of MASH, it could offer a promising pharmacological intervention for patients with NAFLD/MASH, addressing a significant unmet clinical need in the management of this increasingly prevalent liver condition.
METHODS
All experiments were approved by the Animal Care and Use Committee of Kangwon National Univeristy (KW-220218-2).
Materials
The reagents utilized in this study were purchased from the following suppliers: SGLT2 inhibitor enavogliflozin (#DWP-16001) from Daewoong Pharmaceutical Co. Ltd. (Seoul, Korea); recombinant human TGF-β1 (#240-B) from Bio-Techne R&D systems (Minneapolis, MN, USA); palmitic acid (PA, #P0500-10G), Oil Red O (#O0625-25G) and lipopolysaccharide from Sigma-Aldrich Inc. (St. Louis, MO, USA); high glucose Dulbecco’s Modified Eagle’s Medium (DMEM; #LM001-05) and Minimum Essential Medium (MEM; #LM007-07) from WELGENE Inc. (Gyeongsan, Korea); Quanti-MAXTM WST-8 Cell Viability Assay Kit (#QM2500) and OxiTecTM Glutathione Assay Kit (#BO-GLU-200) from Biomax Inc. (Guri, Korea); glutamic pyruvic transaminase (alanine aminotransferase [ALT]) Assay kit (#AM102-K) and GOT (aspartate aminotransferase [AST]) assay kit (#AM103-K) from Asan Pharmaceutical Co. Ltd. (Seoul, Korea); TG assay kit (#Ab65336) and 2’,7’-dichlorofluorescin diacetate (DCFDA/H2DCFDA)-Cellular ROS Assay Kit (#ab113851) from Abcam plc (Cambridge, United Kingdom).
The primary antibodies used were: anti-SGLT2 from Thermo Fisher Scientific (#PA5-101893, Waltham, MA, USA); anti-collagen type I alpha 1 (COL1A1) from Sigma-Aldrich Inc. (#SAB1402151); anti-α-smooth muscle actin (α-SMA) from Abcam plc (#ab5694); anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from GenTex (#GT239, Redlands, CA, USA). The antibodies: anti-phospho-Smad2 (pSmad2, #3108), anti-SMAD2 (Smad2, #5339), anti-phospho-Smad3 (pSmad3, #9520), anti-SMAD3 (Smad3, #9513), anti-TGF-β1 (#3711), anti-interleukin 6 (IL-6, #12912), anti-IL-1β (#12242), and antifatty acid-binding protein 4 (FABP4, #2120) were sourced from Cell Signaling Technology Inc. (Danvers, MA, USA).
Cell culture and treatment
The LX-2 human HSCs and HepG2 human hepatoma cells were maintained in high glucose DMEM and MEM upplemented with 10% fetal bovine serum, penicillin, and streptomycin at 37°C in a humidified incubator with 5% CO2. LX-2 cells were seeded at the density of 1×105 cells/mL overnight and then treated with 4 ng/mL of TGF-β1, with or without enavogliflozin at concentrations of 50 or 100 nM for 24 hours. HepG2 cells were plated at a density of 7×105 cells/mL overnight and then exposed to 300 μM PA alone or in combination with either 50 or 100 nM enavogliflozin for 3 days.
Animals model
Male C57BL/6 mice, aged 4 weeks and weighing approximately 20 to 21 g, were obtained from Doo Yeol Biotech (Seoul, Korea). All mice were handled in accordance with the guidelines outlined in the 13th Article of the Korean Animal Protection Law. These mice were housed at the Kangwon National University animal care facility under controlled conditions, including an ambient temperature of 22°C±1°C and a 12/12-hour light/dark cycle. Throughout the experiment, the mice had unrestricted access to food and water. After 1 week of acclimation, the mice were randomly divided into three groups: one group received a chow diet (n=8), another received a high-fat, high-cholesterol (HFHC) diet (n=10), and the third group received an HFHC diet along with enavogliflozin at a dose of 1.28 mg/kg/day (n=10). The composition of each diet is detailed in Table 1. Food was replaced every 2 days, and food intake was documented. Body weight was recorded weekly. After 12 weeks, each mouse was anesthetized with N2O in an induction chamber and euthanized. Blood samples were collected in tubes and centrifuged to obtain serum for subsequent analysis. A portion of the liver from each mouse sample was submerged in a 10% formaldehyde solution, while the remainder was immediately placed into a nitrogen-filled container and stored in a –80°C refrigerator.
Food composition for different groups
Cell viability assay
The cell viability of LX-2 cells and HepG2 cells was assessed using Quanti-MAXTM WST-8 Cell Viability Assay Kit. Cells were seeded in 24-well plates at a density of 1×105 cells/well for LX-2 cells and 7×105 cells/well for HepG2 cells and incubated overnight. The culture medium was then replaced with the designated treatment medium. Following the treatment period, 50 μL of cell counting kit-8 reagent and 450 μL of medium were added to each well, and the cells were further incubated for an additional 2 hours. The absorbance values were then analyzed using a microplate reader at a wavelength of 450 nm.
Wound healing migration assay
LX-2 cells were seeded in a 6-well culture plate at a density of 5×105 cells/well and cultured for 24 hours. Once the confluence reached above 90%, the cells were treated with 1 μg/mL mitomycin C, diluted in serum-free DMEM. After 1 hour, a yellow pipette tip was used to create a wound in the cell monolayer, followed by gentle washing of the cells twice with DMEM to remove loose cells. Subsequently, the cells were exposed to treatment media containing 4 ng/mL TGF-β1 with or without enavogliflozin for 6 hours. The injured lines were observed and imaged using a microscope at 0- and 6-hour time points. The covered area, representing cell migration ability, was quantified using ImageJ version 1.8.0 112 64bit (National Institutes of Health, Bethesda, MD, USA).
Enzyme-linked immunosorbent assay
After cells were exposed to TGF-β1 treatment, either with or without enavogliflozin, the culture medium was collected to evaluate inflammatory cytokine expression. Utilizing a KOMA enzyme-linked immunosorbent assay (ELISA) kit (Komabiotech, Seoul, Korea), the levels of IL-6 and IL-1β were quantified in the supernatant of various samples following the manufacturer’s guidelines. Briefly, the conditioned medium was applied to coat the plate, and the detection antibody was incubated for 2 hours. Subsequently, horseradish peroxidase conjugated with streptavidin was added and incubated for 30 minutes. The 3,3ʹ,5,5ʹ-tetramethylbenzidine solution was then utilized for an appropriate duration to facilitate color development before the addition of the stop solution. The optical density was measured at 450 nm using a microplate reader.
Oil Red O staining
Lipid accumulation was detected and measured by Oil Red O (ORO) staining. After 3 days of treatment with PA and enavogliflozin, HepG2 cells were washed and fixed with formaldehyde solution for 30 minutes, followed by staining with 60% Oil Red O taining solution for 15 minutes. Subsequently, cells were washed with 60% isopropanol and immersed in distilled water. The red-stained LDs were visualized and captured using an Olympus IX73 (Olympus, Tokyo, Japan) inverted microscope at different magnifications.
Statistical analysis
The results are presented as the mean±standard error of the mean. Statistical analyses were performed using GraphPad Prism version 6.02 (GraphPad Software Inc., San Diego, CA, USA). Statistical significance was set at P<0.05.
RESULTS
Expression of SGLT2 in LX-2 cells, HepG2 cells, and mouse liver
Stimulation with TGF-β1 enhanced SGLT2 expression in LX-2 cells and HepG2 cells, whereas treatment with enavogliflozin significantly reduced SGLT2 expression at concentrations of 50 and 100 nM (Fig. 1A and B). Also, palmitate treatment increased the expression of SGLT2 in HepG2 cells, whereas enavogliflozin reduced the increased expression of SGLT2 (Fig. 1B). To ensure enavogliflozin safety, its potential cytotoxic effects on cell viability were examined. Supplementary Fig. 1 indicate that concentrations of 50 and 100 nM of enavogliflozin are not cytotoxic. Therefore, these concentrations were chosen for subsequent experiments. Furthermore, western blot analysis conducted on mouse liver tissue demonstrated that the HFHC condition led to elevated expression of SGLT2. However, the co-administration of enavogliflozin resulted in a reduction in SGLT2 expression (Fig. 1C and D).
Expression of sodium-glucose cotransporter 2 (SGLT2) in LX-2 cells, HepG2 cells, and mouse liver. (A) LX-2 and (B) HepG2 cells were treated with transforming growth factor β1 (TGF-β1) or palmitate with or without enavogliflozin (Envlo) to evaluate SGLT2 expression using Western blot analysis. (C) Western blot analysis shows the expression of SGLT2 in mouse liver tissue. (D) Liver sections of mice after being fed within 12 weeks were stained with SGLT2 (scale bars: 100 and 50 μm). All data are presented as mean±standard error of the mean. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CD, chow diet; HFHC, high-fat, high-cholesterol. aP<0.05, bP<0.005, c,dMeans without a common letter differ, P<0.05.
Enavogliflozin prevented HSC activation and inflammation
Enavogliflozin treatment significantly reduced the expression levels of collagen I and α-SMA, the markers of HSC activation induced by TGF-β1 (Fig. 2A). TGF-β1 also increased IL-6 and IL-1β levels, and enavogliflozin supplementation effectively reversed the elevation of these cytokines (Fig. 2B and C). Wound healing in LX-2 cells was notably enhanced by TGF-β1 stimulation compared to the control condition (Fig. 2D). Although treatment with 50 nM enavogliflozin did not yield statistically significant results for HSC migration, a noticeable decrease was observed after treatment with 100 nM enavogliflozin.
Enavogliflozin (Envlo) prevented hepatic stellate cells activation and inflammation. (A) Western blot analysis illustrates the expression of collagen I and α-smooth muscle actin (α-SMA) in LX-2 cells. The release of inflammatory factors, (B) interleukin 6 (IL-6), and (C) IL-1β, in LX-2 cell supernatant was evaluated using enzyme-linked immunosorbent assay. (D) Representative images of wound healing migration (scale bars: 50 μm) when LX-2 cells were treated with 4 ng/mL of transforming growth factor β1 (TGF-β1) in the presence of either 50 or 100 nM Envlo for 0 or 6 hours. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. aP<0.05, bP<0.01, cP<0.001, means without a common letter differ, P<0.05.
Enavogliflozin improved HFHC-induced liver injuries
After 12 weeks of HFHC diet administration, there were noticeable color changes in the liver morphology in the enavogliflozin-treated group compared to the HFHC diet group (Fig. 3A), although the difference in liver-to-body weight ratio was not statistically significant (Fig. 3B and C). Specifically, enavogliflozin co-treatment significantly mitigated ALT and AST levels compared to the HFHC diet group (Fig. 3D and 3E). Additionally, Western blot experiments were conducted to assess the expression of IL-1β and IL-6 in mouse liver samples. The data revealed that HFHC-induced severe inflammation in liver tissue, whereas treatment with enavogliflozin significantly improved this inflammatory condition (Fig. 3F). These findings suggest that enavogliflozin may have a beneficial effect in attenuating inflammation associated with steatosis and fibrosis in the liver.
Enavogliflozin (Envlo) improved high-fat, high-cholesterol (HFHC)-induced liver injuries. (A) Images of mouse livers after 12 weeks of feeding with different diets. (B) Liver weight. (C) The ratio between liver weight and body weight. (D) Serum alanine aminotransferase (ALT) and (E) serum aspartate aminotransferase (AST) levels were investigated. (F) Western blot analysis evaluated the protein expression levels of interleukin 1β (IL-1β) and IL-6 in liver tissue. CD, chow diet; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. aP<0.05, bP<0.005, cP<0.001.
Enavogliflozin reduced lipid accumulation in HepG2 cells and HFHC-induced hepatic steatosis.
Exposure to 0.3 mM PA for 3 days significantly increased intracellular lipid accumulation in HepG2 cells (Fig. 4A). However, co-treatment with 100 nM enavogliflozin notably attenuated PA-induced lipid accumulation. To ensure that this reduction in lipid content was not attributable to a decrease in cell number, a WST-8 assay was conducted to evaluate the cytotoxic effects of enavogliflozin. The results indicated that after 3 days of co-treatment with PA, enavogliflozin exhibited no cytotoxic effects on the viability of HepG2 cells at concentrations of 50 and 100 nM (Fig. 4B). In addition, PA significantly increased the expression of FABP4. However, enavogliflozin exhibited a reversing effect on this upregulation (Fig. 4C).
Enavogliflozin (Envlo) reduced lipid accumulation in HepG2 cells and high-fat, high-cholesterol (HFHC)-induced hepatic steatosis. (A) HepG2 cells were stained with Oil Red O (scale bars: 200 and 50 μm). (B) Cell viability of HepG2 cells after 3 days of treatment with palmitic acid (PA) and Envlo was assessed through WST-8 assays. (C) Western blot analysis shows the expression of fatty acid-binding protein 4 (FABP4) in HepG2 cells. (D) Liver triglyceride levels were evaluated in groups using the adipogenesis detection assay kit. (E) Liver samples underwent staining using the H&E method (scale bars: 100 and 50 μm). (F) Western blot analysis evaluated the protein expression levels of acetyl-CoA carboxylase (ACC), phospho-acetyl-CoA carboxylase (pACC), FASN fatty acid synthase (FASN), peroxisome proliferator-activated receptor γ (PPARγ), sterol regulatory element-binding protein 1 (SREBP1c), peroxisome proliferator-activated receptor α (PPARα), carnitine palmitoyltransferase I (CPT1A), and FABP4. SGLT2, sodium-glucose cotransporter 2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TG, triglyceride; CD, chow diet. aP<0.05, bP<0.01, cP<0.005, dP<0.001.
Enavogliflozin co-treatment significantly ameliorated the HFHC-induced increase in liver TG (Fig. 4D). Histological examination using H&E staining revealed that the administration of enavogliflozin noticeably alleviated hepatic steatosis by reducing fatty degeneration of hepatocytes and intracellular LD formation (Fig. 4E). Western blot data shows that HFHC regimen did not result in an upregulation of DNL genes, including acetyl-CoA carboxylase (ACC), phospho-acetyl-CoA carboxylase (pACC), FASN, peroxisome proliferator-activated receptor γ (PPARγ), and sterol regulatory element-binding protein 1 (SREBP1c). Moreover, it did not lead to a reduction in fatty acid beta oxidation genes, namely PPARα and carnitine palmitoyltransferase I (CPT1A). Nevertheless, there was a notable increase in the expression of FABP4, and treatment with enavogliflozin resulted in a significant reduction in the expression of this protein (Fig. 4F).
Enavogliflozin improved HFHC-induce liver fibrosis by inhibiting TGF-β1/Smad2 signaling pathway
We observed a significant reduction in mouse body weight in the HFHC diet group, and no discernible difference in body weight was observed between the HFHC-only group and the group co-treated with enavogliflozin (Fig. 5A). Additionally, we observed lower food intake in the HFHC group, whereas there was no significant difference in food intake between the control group and the enavogliflozin-treated group (Fig. 5B).
Enavogliflozin (Envlo) improved high-fat, high-cholesterol (HFHC)-induced liver fibrosis through inhibiting transforming growth factor β1 (TGF-β1)/Smad2 signaling pathway. (A) Body weight and (B) food intake of mice after 12-week feeding. (C) Liver sections of mice after being fed within 12 weeks were stained with Masson’s trichrome to assess the extracellular matrix (scale bars: 100 and 50 μm). (D) Western blot analysis showed the protein expression levels of α-smooth muscle actin (α-SMA), TGF-β1, pSmad2, and Smad2 in mouse liver tissue. (E) pSmad2, Smad2, pSmad3, and Smad3 in LX-2 were evaluated after treatment with TGF-β1 and Envlo in 24 hours using Western blot. CD, chow diet; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NS, not significant. aP<0.05, bP<0.01, cP<0.005, dP<0.001.
Masson’s trichrome staining revealed that after 12 weeks of feeding with the HFHC diet, the livers of these mice developed marked fibrosis, and treatment with enavogliflozin contributed to the reversal of this condition (Fig. 5C). Western blotting results demonstrated a significant increase in α-SMA in HFHC diet-fed mice, whereas enavogliflozin treatment significantly reduced the expression of this protein (Fig. 5D). Treatment of LX-2 cells with TGF-β1 led to an increase in the phosphorylation of Smad2 and Smad3, a canonical downstream component of the TGF-β1 pathway. However, enavogliflozin treatment significantly blocked the TGF-β1-induced phosphorylation of Smad2 in LX-2 cells (Fig. 5E). In vivo, the HFHC diet efficiently increased the expression of TGF-β1 and pSmad2, and enavogliflzin alleviated this response (Fig. 5D).
DISCUSSION
NAFLD has become a significant global public health challenge without a standard treatment due to its complex pathogenesis [2,19]. Pharmacotherapies target both pathogenic factors and metabolic disturbances, with emerging evidence suggesting that SGLT2 inhibitors may confer benefits in metabolic and hepatic disorders [20-25]. This study demonstrates enavogliflozin’s beneficial effects in reducing hepatic steatosis, inflammation, and fibrosis in NAFLD.
Previous research shows SGLT2 expression increases in various tissues, including liver cells, upon TGF-β1 treatment [26-28]. Our study found TGF-β1 elevated SGLT2 expression in LX-2 and HepG2 cells, and modest overexpression in mouse liver tissues fed an HFHC diet. Our findings showed that enavogliflozin reduced PA-induced lipid accumulation in HepG2 cells, linked to decreased FABP4 expression. Similarly, in HFHC diet-fed mice, enavogliflozin reduced liver lipid accumulation and FABP4 expression.
Intrahepatic lipid accumulation and lipotoxic intermediates contribute to insulin resistance and endoplasmic reticulum (ER) stress, leading to hepatic dysfunction [29-31]. Oleic acid/PA elicits heightened free fatty acid uptake, characterized by elevated expression of CD36, a free fatty acid transport protein, in National Cell Type Culture (NCTC) 1469 cells [32]. Our findings showed that enavogliflozin reduced PA-induced lipid accumulation in HepG2 cells, linked to decreased FABP4 expression. High-fat diets (HFD) generally reduce the expression of certain lipogenesis-related genes [33], but our HFHC diet study observed decreased DNL-related protein expression. Different diets can complexly affect lipid metabolism genes [34].
Other SGLT2 inhibitors like dapagliflozin also reduce hepatic lipid accumulation and inflammation [21,35]. In our investigation, enavogliflozin exhibited anti-inflammatory properties, reducing the expression of inflammatory cytokines such as IL-6 and IL-1β induced by an HFHC diet. Additionally, a study showed that ipragliflozin treatment lowered serum ALT, a marker of liver damage, and liver TG levels in HFD mice compared to HFD control mice [36]. Enavogliflozin further showed efficacy in lowering serum ALT, AST, and liver TG levels, indicating its potential in mitigating liver damage and metabolic dysfunction. Other SGLT2 inhibitors have been reported to ameliorate liver fibrosis and inhibit LX-2 stellate cell activation via different pathways [37,38]. In our experiment, we showed that enavogliflozin significantly blocked the TGF-β1-induced phosphorylation of Smad2 in LX-2 cells. In vivo, enavogliflozin alleviated HFHC diet-induced overexpression of TGF-β1 and pSmad2.
There are limited studies regarding the role of SGLT2 inhibitors in HSC activation or liver fibrosis. However, other studies provide some insights. For instance, empagliflozin activates Hippo signaling and decreased Yes-associated protein (YAP) activity in mice model of liver fibrosis [37] and downregulates miRNA-34a-5p, targeting gremlin 2, DAN family BMP antagonist (GREM2) to inactivate hepatic stellate cells (HSCs) and ameliorate NAFLD-associated fibrosis [38]. Additionally, empagliflozin has been shown to suppress the CCl4-induced hedgehog pathway and alleviate ER stress [39]. In other study, empagliflozin reduced the body weight largely attributable to decreases in both visceral and subcutaneous fat depots under pair-fed HFD conditions and taken together, empagliflozin increased energy expenditure, heat production, and the expression of uncoupling protein 1 in brown fat and in inguinal and epididymal white adipose tissue [40]. It remains to be clarified whether the observed improvement in liver fibrosis is due to the amelioration of insulin resistance resulting from weight loss or is a direct effect of enavogliflozin through its regulation of SGLT2 expression in HSCs or hepatocytes, independent of weight loss and glucose metabolism. Additional experiments are needed to determine whether enavogliflozin also decreases fat composition and increase energy expenditure through a similar mechanism like empagliflozin and to clarify the mechanism by which enavogliflozin may show beneficial effects on liver fibrosis.
In summary, we demonstrated the effectiveness of enavogliflozin in a MASH mouse model by reducing hepatic steatosis, suppressing inflammation, and improving liver fibrosis. Enavogliflozin also inhibited HSC activation and migration and prevented lipid accumulation in vitro. Additionally, our findings suggest that enavogliflozin prevents HSC activation and liver fibrosis through the TGF-β1/Smad2 signaling pathway.
SUPPLEMENTARY MATERIALS
Supplementary materials related to this article can be found online at https://doi.org/10.4093/dmj.2024.0259.
Enavogliflozin (Envlo) did not affect cell viability. The impact of Envlo at 50 and 100 nM on the viability of (A) LX-2 cells and (B) HepG2 cells was evaluated through WST-8 assays. TGF-β1, transforming growth factor β1. aP<0.05.
Notes
CONFLICTS OF INTEREST
No potential conflict of interest relevant to this article was reported.
AUTHOR CONTRIBUTIONS
Conception or design: S.M.K., E.H.C.
Acquisition, analysis, or interpretation of data: P.T.M.P., S.M.K., E.H.C.
Drafting the work or revising: all authors.
Final approval of the manuscript: all authors.
FUNDING
This study was carried out with the support of a research grant from Daewoong Pharmaceuticals, 2022.
ACKNOWLEDGMENTS
None