HWL-088, a new and highly effective FFA1/PPARd dual agonist, attenuates nonalcoholic steatohepatitis by regulating lipid metabolism, inflammation and fibrosis
Lijun Hua, Zongtao Zhoua, Liming Denga, Qiang Rena, Zongyu Caia, Bin Wanga, Zheng Lia,b and Guangji Wanga,c
Abstract
Objectives Nonalcoholic fatty liver (NAFLD), a chronic progressive liver disease, is highly correlated with pathoglycemia, dyslipidemia and oxidative stress. The free fatty acid receptor 1 (FFA1) agonists have been reported to improve liver steatosis and fibrosis, and the peroxisome proliferator-activated receptor d (PPARd) plays a synergistic role with FFA1 in energy metabolism and fibrosis. HWL-088, a PPARd/FFA1 dual agonist, exerts better glucose-lowering effects than the representative FFA1 agonist TAK-875. However, the ability of HWL-088 to protect NAFLD was unknown. This study aimed to discover a new strategy for the treatment of NAFLD.
Methods The methionine- and choline-deficient diet (MCD)-induced Nonalcoholic steatohepatitis (NASH) model was constructed to evaluate the effects of HWL-088.
Key findings Administration of HWL-088 exerted multiple benefits on glucose control, lipid metabolism and fatty liver. Further mechanism research indicated that HWL-088 promotes lipid metabolism by decreasing lipogenesis and increasing lipolysis. Moreover, HWL-088 attenuates NASH by regulating the expression levels of genes related to inflammation, fibrosis and oxidative stress.
Conclusions These positive results indicated that PPARd/FFA1 dual agonist HWL-088 might be a potential candidate to improve multiple pathogenesis of NASH. performed in patients with T2DM.[8,9] Moreover, the activation of FFA1 significantly attenuated fibrosis, and thereby
Keywords
fatty liver; FFA1; fibrosis; inflammation; lipid metabolism; PPARd
Introduction
With lifestyle changes, NAFLD becomes one of the most common liver metabolic diseases around the world, including hepatic steatosis, fibrosis, cirrhosis and even hepatocellular carcinoma.[1,2] The mechanisms of NAFLD involved have been attributed to the disruption of glucose metabolism, lipid metabolism and liver damage.[3,4] Although the incidence of NAFLD is rapidly growing, yet there are no drugs were approved by FDA for the treatment of NAFLD.[5] Therefore, there is a considerable need to identify safe and effective therapeutic agents for NAFLD.
Free fatty acid receptor 1 (FFA1) is mainly found in the pancreas, which plays an essential role in insulin secretion.[6,7] Many clinical trials of FFA1 agonists have been FFA1 is also a promising target in fibrosis pathways.[10] Current research indicated that the activation of FFA1 alleviates liver steatosis.[11] Therefore, FFA1 agonists may be beneficial for the treatment of NASH.[12]
The peroxisome proliferator-activated receptor d (PPARd) is a nuclear receptor and belongs to the PPAR family, which includes PPARa, PPARc and PPARd.[13–15] The agonist of PPARa (fenofibrate) or PPARc (rosiglitazone) has been widely used to the treatment of dyslipidemia and T2DM, but the adverse effects limited its application.[16–18] PPARd, another member of the PPAR family, exhibits high expression levels not only in liver but also in skeletal muscle and macrophages, and its activation ameliorates anti-inflammation, insulin sensitization and lipid metabolism.[19–21] Thus, PPARd might be a potential target for diseases related to energy metabolism, such as T2DM, dyslipidemia and NAFLD.[22–24]
In previous studies, we have identified several FFA1/ PPARd dual agonists.[25–28] A comprehensive structureactivity relationship study provided HWL-088 (the structure is shown in Figure 1a), a PPARd/FFA1 dual agonist.[29,30] Herein, the chronic effects of HWL-088 were explored in the methionine- and choline-deficient diet (MCD) diet-fed db/db mice, a typical model of NASH.[31] HWL-088 alleviated fatty liver by maintaining glucose stability, improving fat metabolism, liver fibrosis, inflammation and oxidative stress. Therefore, investigation of this chemical might be a promising candidate for the treatment of NAFLD.
Materials and Methods
Materials and animals
HWL-088 was synthesized with reference to reported papers, purity >98%.[30] Eight-week-old male C57BL/6 mice were purchased from Guangdong Medical Laboratory Animal Center (Guangdong, China), and eight-week-old male db/db mice were purchased from Model Animal
Figure 1 Effects of HWL-088 on body weight, liver-to-brain ratio and on glucose tolerance of MCD-fed db/db mice. (a) The structure of HWL088. (b) Evolution of body weight. (c) Evolution of body weight changes. (d) Accumulated food intake. (e) Liver weight. (f) Liver to brain weight radio. (g) Liver morphology of the normal, model, and HWL-088. (h) Fasting blood glucose. (i) Effects of HWL-088 on OGTT after treatment for 42 days. Data are presented as mean SD values. n = 6 mice per group. Statistical comparison between different groups was made using oneway analysis of variance. #P < 0.05 (#Significant differences Model vs Normal).
Research Center of Nanjing University (Jiangsu, China). All animals were acclimatized for one week before the experiments. Unless otherwise stated, Mice were allowed to obtain standard pellets and water. For all animal studies, the vehicle was 0.5% CMC-Na. All experimental protocols were approved by the ethical committee at Guangdong Pharmaceutical University and conducted according to the
Laboratory Animal Management Regulations in China and adhered to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication NO. 85-23, revised 2011).
Chronic administration in MCD diet-fed db/ db mice
The male C57BL/6 mice fed normal diet were set as normal control (n = 6). The male db/db mice were randomly divided into two groups (n = 6): a group of mice fed the MCD for 45 days without treatment to induce NASH as model control, the other group mice were fed the MCD in parallel with HWL-088 (60 mg/kg) by gavage once daily. The body weight is evaluated every 5 days and the measurement of food consumption at regular intervals daily. After 42 days of MCD mice were fasted overnight, oral glucose tolerance test was performed with glucose (3 g/kg). The plasma glucose was measured at 30, 0, 15, 30, 60 and 90 min. At the end of treatment period, mice were sacrificed under deep anaesthesia. Liver and brain weights were used to calculate liver to brain weight ratio through the following formula: liver to brain weight ratio = (liver weight/brain weight) 9 100%. The levels of ALT, AST, TG, CHOL, HDLC and LDLC were evaluated by the automatic biochemical analyser (Beckman Coulter, Tokyo, Japan). Liver hepatic triglyceride, total cholesterol and nonesterified fatty acid (NEFA) content were measured using the commercial kits, and the methods were according to the manufacturer’s instructions (Nanjing Jiancheng Co., Ltd., Nanjing, China). Liver tissue was washed with ice-cold saline before fixed in 10% (v/v) formalin, embedded in paraffin, sectioned, and stained with haematoxylin and eosin (H&E) and Masson’s trichrome staining according to a standard procedure. The deparaffinized sections of liver from each mouse were exposed to anti-a-SMA antibody (Servicebio, Cat#GB13044) overnight at 4°C. The sections were developed using corresponding genus IgG (HRP, Servicebio, Cat#GB23303) and counterstained with haematoxylin. Digital images were obtained with a Model Eclipse Ci-L (Nikon, Tokyo, Japan). Histological scoring of the liver lesions was assessed histologically by NASH activity score (NAS), as described.[32] NAS included assessments of three histological features: steatosis (0–3), hepatocellular ballooning (0–2) and lobular inflammation (0–3). Samples with scores >5 were designated as ‘NASH’, and samples with scores <3 were designated ‘not NASH’. Using Image-Pro Plus version 6.0.0.260 software to analyse the images of Masson and a-SMA.
Protein isolation and Western blot
The protein extracts (twenty microgram) were separated in 10% SDS-PAGE, followed by transferring onto PVDF membranes and blocked with 5% dried milk in ddH2O. Membranes were incubated overnight with primary antibodies at four degrees Celsius, including SREBP-1c (ab28481), ATGL (ab109251), LPL (ab21356), LCAD (ab196655) and SCD1(ab19862), After the membranes had been washed with TBS-T, the blots were incubated with a 1/2000 dilution of PBS secondary antibodies at indoor temperature. The specific bands were visualized using F-6 (Clinx Science Instruments, Co., Ltd, Shanghai, China). The relative signal intensity was quantified using ImageJ software (NIH, Bethesda, MD, USA).
Quantitative real-time PCR
Total liver RNA was extracted by using the RNA-Quick Purification Kit (ESciencie, Guangzhou Squirrel Biotechnology Co., Ltd., Guangzhou, China), with the ReverTra Ace qPCR RT (Toyobo, Toyobo life science, Shanghai, China) to synthesize cDNA. Real-time RT-PCR was performed with the SYBR Green qPCR Master Mix (ESciencie) on Bio-Rad IQ5 instrument following the manufacturer’s instructions. The gene expression levels were calculated using the DDCT method and normalized against GAPDH mRNA. Primer sequences were provided in Table 1.
Statistical analysis
Data are expressed as mean SD values. Experimental comparison between groups was analysed by using one-way analysis of variance. A value of P < 0.05 was considered to be significant.
Results
Long-term effects of HWL-088
To investigate whether HWL-088 has protective effects on NASH, MCD-fed db/db mice were treated with HWL-088 for 45 days. Compared with model group, the body weight was lower in HWL-088 group, while no significant difference on the food intake between the groups (Figure 1b–d). Moreover, HWL-088 slightly reduces the liver weight and the ratio of liver to brain weight (Figure 1e–g). To evaluate the glucose-lowering effect of HWL-088, the fasting glucose and oral glucose tolerance test were performed. As shown in Figure 1h–i, HWL-088 slightly decreased fasting blood glucose levels throughout this study and improved glucose tolerance.
Effects of HWL-088 on fatty liver
The histopathology analysis of H&E staining indicated that db/db mice induced by MCD developed NASH score >5 with severe steatosis, inflammation, and ballooning in the liver. As expected, long-term treatment of HWL-088 significantly decreased these hepatic pathological. To assess the effects of HWL-088 on liver fibrosis, the liver sections were stained with Masson staining and a-SMA. As shown in Figure 2a–d, Masson staining and a-SMA indicated that HWL-088 effectively reduces liver fibrosis in MCD-induced db/db mice. Moreover, HWL-088 markedly decreased the levels of ALT and AST, the markers for liver injury (Figure 3a and 3b). MCD-induced db/db mice had higher serum levels of triglyceride, cholesterol, and LDLC compared to normal mice. HWL-088 slightly decreased serum triglyceride and cholesterol, significantly reduced LDLC and increased HDLC compared to model mice (Figure 3c– f). The triglyceride and total cholesterol in liver were significantly elevated in model control, which was obviously decreased by the treatment of HWL-088(Figure 3g–h). The liver nonesterified fatty acid (NEFA) was reduced compared to the model control in the drug administration group (Figure 3i). These positive results indicated that HWL-088 attenuates MCD-induced NASH.
Effects of HWL-088 in the protein expression related to lipid metabolism
To investigate the mechanism of HWL-088 on the improvement of liver steatosis and plasma lipid levels, the protein expression levels associated with lipogenesis and lipolysis were determined. The lipogenesis-related protein levels of sterol regulatory element-binding protein 1c (SREBP-1c) and its downstream genes stearoyl-CoA desaturase 1 (SCD1) were increased in MCD induced db/db mice. Notably, HWL-088 significantly decreased the expressions of SREBP-1c and SCD1. Long-chain acyl-CoA dehydrogenase (LCAD), lipoprotein lipase (LPL), and adipose triglyceride lipase (ATGL) are critical enzymes for lipoprotein lipolysis. As expected, HWL-088 significantly increased the expressions of LCAD, LPL and ATGL (Figure 4). These results indicated that HWL-088 promotes lipid metabolism by decreasing lipogenesis and increasing lipolysis.
Effects of HWL-088 in inflammation, fibrosis and oxidative stress
To further illuminate the mechanism of HWL-088 in the improvement of fatty liver, the gene expressions related to inflammation, fibrosis, and oxidative stress were evaluated. As shown in Figure 5a, the inflammatory marker genes including tumour necrosis factor-alpha (TNF-a), transforming growth factor beta (TGF-b) and interleukin-6 (IL6) were downregulated by the administration of HWL-088 relative to the Model group. As shown in Figure 5a, the macrophage marker F4/80 was slightly reduced by treatment of HWL-088. Furthermore, fibrosis marker genes including collagen type 1 alpha (col1a1) and tissue inhibitor of matrix metalloproteinase (TIMP) were evaluated. HWL-088 markedly suppressed the expressions of these factors (Figure 5b). Oxidative stress in liver promotes inflammation and fibrosis. We quantified the expression of antioxidant genes catalase, and glutathione peroxidase (GPx1), SOD1 and SOD2. The expression of GPx1, which quenches H2O2, was significantly enhanced in HWL-088 group (Figure 5c). Furthermore, HWL-088 significantly increased the expressions of catalase and SOD. These results suggested that HWL-088 decreases oxidative stress by upregulating the expression of antioxidant-related genes.
Discussion and Conclusions
NAFLD is regarded as the manifestations of hepatic disorders, which is closely related to diabetes and obesity.[1] With extensive research on the pathogenesis of NAFLD, multi-target drug which reduces liver steatosis, lipid peroxidation, inflammation and fibrosis could achieve good clinical results.[5,33,34] Many FFA1 agonists are widely studied in type 2 diabetes, which is highly correlated with In the present study, HWL-088 treatment-induced weight loss without affecting appetite compared with the model group, suggesting that HWL-088 might improve the pathogenesis of NAFLD by partially regulating metabolic tissue. Moreover, HWL-088 reduced liver damage and maintained glucose homeostasis in a NASH model induced by MCD. There are two main methods to maintain lipid homeostasis. One is to regulate lipogenesis by inhibiting the expression of lipid synthetic proteins, and the other is to promote lipid metabolism by regulating lipoprotein lipolysis and fatty acid b-oxidation. SREBP-1c is a member of the family of sterol regulatory elements, which can directly participate in the regulation of fatty acid, triglyceride and glucose metabolism-related enzyme genes, thereby affecting the accumulation of fat particles in the liver.[38] LPL and ATGL play an important role in lipid metabolism as rate-limiting enzyme hydrolyzing plasma TG-rich lipoproteins and very-low-density lipoprotein.[39–41] LCAD is a crucial enzyme participating in fatty acid oxidation.[42] HWL-088 reduced the liver protein levels of SREBP-1c and its downstream genes including SCD1. Besides, HWL-088 promoted the expression of key enzymes related lipid metabolism, such as LPL, ATGL and LCAD. Additionally, treatment with HWL-088 can decrease the mRNA levels of TNF-a, TGF-b, IL-6, col1a1 and TIMP to prevent inflammation and fibrosis.
In general, hepatic oxidative stress is closely connected Fasiglifam with the development of liver inflammation and fibrosis.[43,44] Antioxidant enzymes including superoxide dismutase (SOD) and catalase.[45] Glutathione peroxidase (Gpx1) is the downstream factor of SOD and also plays an important role in antioxidant activity. As we expected, the antioxidative enzyme SOD was significantly upregulated in the liver and consequently upregulates downstream Gpx1 in HWL-088 treated group. These antioxidant mechanisms might be contributed to the improvement of NASH.
Overall, our results indicated that HWL-088 prevents NASH by regulating the protein and gene expressions related to hepatic lipid metabolism, inflammation, fibrosis and oxidative stress (Figure 6). These results indicated that HWL-088 might be a promising NASH candidate worthy of further evaluation and development.
References
1. Younossi ZM et al. Global epidemiology of nonalcoholic fatty liver diseaseMeta-analytic assessment of prevalence, incidence, and outcomes. Hepatology 2016; 64: 73–84.
2. Younossi ZM et al. Association of nonalcoholic fatty liver disease (NAFLD) with hepatocellular carcinoma (HCC) in the United States from 2004 to 2009. Hepatology 2015; 62: 1723–30.
3. Huh JH et al. Obesity is more closely related with hepatic steatosis and fibrosis measured by transient elastography than metabolic health status. Metabolism 2017; 66: 23–31.GPR84. Am J Pathol 2018; 188: 1132–48.
4. Noureddin M, Sanyal AJ. Pathogenesisof NASH: the impact of multiple pathways. Curr Hepatol Rep. 2018; 17: 350–60.
5. Younossi ZM et al. Current and future therapeutic regimens for nonalcoholic fatty liver disease and nonalcoholic steatohepatitis. Hepatology 2018; 68: 361–71.
6. Itoh Y et al. Free fatty acids regulate insulin secretion from pancreatic b cells through GPR40. Nature 2003; 422: 173–6.
7. Briscoe CP et al. The orphan G protein-coupled receptor GPR40 is activated by medium and long chain fatty acids. J Biol Chem 2003; 278: 11303–11.
8. Proschak E et al. Opportunities and challenges for fatty acid mimetics in drug discovery. J Med Chem 2017; 60: 5235–66.
9. Milligan G et al. Complex pharmacology of free fatty acid receptors. Chem Rev 2017; 117: 67–110.
10. Gagnon L et al. A newly discovered antifibrotic pathway regulated by two fatty acid receptors GPR40 and funding acquisition, writing – review and editing.
11. Ou HY et al. Activation of free fatty acid receptor 1 improves hepatic steatosis through a p38-dependent pathway. J Mol Endocrinol 2014; 53: 165–74.
12. Musso G et al. Cholesterol metabolism and the pathogenesis of non-alcoholic steatohepatitis. Prog Lipid Res 2013; 52: 175–91.
13. Hong F et al. PPARs as nuclear receptors for nutrient and energy metabolism. Molecules 2019; 24: 2545.
14. Gross B et al. PPARs in obesity-induced T2DM, dyslipidaemia and NAFLD. Nat Rev Endocrinol 2017; 13: 36–49.
15. Zhao Z-H et al. Promoter methylation status and expression of PPAR-c gene are associated with prognosis of acute-on-chronic hepatitis B liver failure. Clin Epigenet 2015; 7: 115.
16. Monsalve FA et al. Peroxisome proliferator-activated receptor targets for the treatment of metabolic diseases. Mediat Inflamm 2013; 2013: 1–18.
17. Ahmadian M et al. PPAR gamma signaling and metabolism: the good, the bad and the future. Nat Med 2013;19: 557–66.
18. Verges B. Clinical interest of PPARsligands – particular benefit in type 2 diabetes and metabolic syndrome. Diabetes Metab 2004; 30: 7–12.
19. Tanaka T et al. Activation of peroxisome proliferator-activated receptor delta induces fatty acid beta-oxidation in skeletal muscle and attenuates metabolic syndrome. Proc Natl Acad Sci U S A 2003; 100: 15924–9.
20. Adhikary T et al. The transcriptional PPARbeta/delta network in human macrophages defines a unique agonist-induced activation state. Nucleic Acids Res 2015; 43: 5033–51. 21. Sprecher DL et al. Triglyceride: highdensity lipoprotein cholesterol effects in healthy subjects administered a peroxisome proliferator activated receptor delta agonist. Arterioscl Throm Vas 2007; 27: 359–65.
22. Lee C et al. Peroxisome proliferatoractivated receptor delta promotes very low-density lipoprotein-derived fatty acid catabolism in the macrophage. Proc Natl Acad Sci U S A 2006; 103:2434–9.
23. Luquet S et al. Roles of PPAR delta in lipid absorption and metabolism: a new target for the treatment of type 2 diabetes. Biochim Biophys Acta 2005; 1740: 313–7.
24. Chen J et al. Insights into the role of PPAR beta/delta in NAFLD. Int J Mol Sci 2018; 19: 1893–23.
25. Li Z et al. Design, synthesis, and biological evaluation of novel pan agonists of FFA1, PPARgamma and PPARdelta. Eur J Med Chem 2018;159: 267–76.
26. Li Z et al. Discovery of first-in-class thiazole-based dual FFA1/PPARdelta agonists as potential anti-diabetic agents. Eur J Med Chem 2019; 164:352–65.
27. Li Z et al. Design, synthesis, and biological evaluation of novel dual FFA1 (GPR40)/PPARdelta agonists as potential anti-diabetic agents. Bioorg Chem 2019; 92: 103254.
28. Li Z et al. A novel FFA1 agonist, CPU025, improves glucose-lipid metabolism and alleviates fatty liver in obese-diabetic (ob/ob) mice. Pharmacol Res 2020; 153: 104679.
29. Chen Y et al. HWL-088, a new potent free fatty acid receptor 1 (FFAR1) agonist, improves glucolipid metabolism and acts additively with metformin in ob/ob diabetic mice. Br J Pharmacol 2020; 177: 2286–16.
30. Li Z et al. Discovery of HWL-088: a highly potent FFA1/GPR40 agonist bearing a phenoxyacetic acid scaffold. Bioorg Chem 2019; 92: 103209.
31. Rinella ME et al. Mechanisms of hepatic steatosis in mice fed a lipogenic methionine choline-deficient diet. J Lipid Res 2008; 49: 1068–76.
32. Huang K et al. Polydatin promotes Nrf2-ARE anti-oxidative pathway through activating Sirt1 to resist AGEs-induced upregulation of fibronetin and transforming growth factor-b1 in rat glomerular messangial cells. Mol Cell Endocrinol 2015; 399: 178–89.
33. Byrne CD, Targher G. EASL–EASD– EASO Clinical Practice Guidelines for the management of non-alcoholic fatty liver disease: is universal screening appropriate? Diabetologia 2016; 59: 1141–4.
34. Rotman Y, Sanyal AJ. Current andupcoming pharmacotherapy for nonalcoholic fatty liver disease. Gut 2017; 66: 180–90.
35. Li Z et al. Free fatty acid receptor agonists for the treatment of type 2 diabetes: drugs in preclinical to phase II clinical development. Expert Opin Inv Drug 2016; 25: 871–90.
36. Li Z et al. Free fatty acid receptor 1 (FFAR1) as an emerging therapeutic target for type 2 diabetes mellitus: recent progress and prevailing challenges. Med Res Rev 2018; 38: 381–
37. Li Zheng et al. Current status of GPR40/FFAR1 modulators in medicinal chemistry (2016–2019): a patent review. Expert Opin Ther Pat 2020; 30: 27–38.
38. Watanabe M et al. Bile acids lower triglyceride levels via a pathway involving FXR, SHP, and SREBP-1c. J Clin Investig 2004; 113: 1408–18.
39. Zhang R. Lipasin, a novel nutritionally-regulated liver-enriched factor that regulates serum triglyceride levels. Biophys Res Commun 2012; 424: 786–
40. Li J et al. Triglyceride metabolism and angiopoietin-like proteins in lipoprotein lipase regulation. Clin Chim Acta 2020; 503: 19–34.
41. Schreiber R et al. Of mice and men: the physiological role of adipose triglyceride lipase (ATGL). Biochim Biophys Acta 2019; 1864: 880–99.
42. Zhang YX et al. The fatty acid oxidation enzyme long-chain acyl-CoA dehydrogenase can be a source of mitochondrial hydrogen peroxide. Redox Biol 2019; 26: 101253.
43. Poirier Y et al. Peroxisomal beta-oxidation–a metabolic pathway with multiple functions. Acta Mol Cell Res 2006; 1763: 1413–26.
44. Kim J-a et al. Role of mitochondrial dysfunction in insulin resistance. Circ Res 2008; 102: 401–14.
45. Schieber M, Chandel NS. ROS function in redox signaling and oxidative stress. Curr Biol 2014; 24: 453–62.