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. 2022 Aug 8;12(8):732.
doi: 10.3390/metabo12080732.

A Novel and Cross-Species Active Mammalian INDY (NaCT) Inhibitor Ameliorates Hepatic Steatosis in Mice with Diet-Induced Obesity

Grit Zahn  1 , Diana M Willmes  2   3 , Nermeen N El-Agroudy  2   3   4   5 , Christopher Yarnold  6 , Richard Jarjes-Pike  6 , Sabine Schaertl  7 , Kay Schreiter  8 , Wiebke Gehrmann  8 , Andrea Kuan Cie Wong  9 , Tommaso Zordan  9 , Jörg König  10 , Jens Jordan  11 , Andreas L Birkenfeld  2   3   4   5
Affiliations

A Novel and Cross-Species Active Mammalian INDY (NaCT) Inhibitor Ameliorates Hepatic Steatosis in Mice with Diet-Induced Obesity

Grit Zahn et al. Metabolites. .

Abstract

Mammalian INDY (mINDY, NaCT, gene symbol SLC13A5) is a potential target for the treatment of metabolically associated fatty liver disease (MAFLD). This study evaluated the effects of a selective, cross-species active, non-competitive, non-substrate-like inhibitor of NaCT. First, the small molecule inhibitor ETG-5773 was evaluated for citrate and succinate uptake and fatty acid synthesis in cell lines expressing both human NaCT and mouse Nact. Once its suitability was established, the inhibitor was evaluated in a diet-induced obesity (DIO) mouse model. DIO mice treated with 15 mg/kg compound ETG-5773 twice daily for 28 days had reduced body weight, fasting blood glucose, and insulin, and improved glucose tolerance. Liver triglycerides were significantly reduced, and body composition was improved by reducing fat mass, supported by a significant reduction in the expression of genes for lipogenesis such as SREBF1 and SCD1. Most of these effects were also evident after a seven-day treatment with the same dose. Further mechanistic investigation in the seven-day study showed increased plasma β-hydroxybutyrate and activated hepatic adenosine monophosphate-activated protein kinase (AMPK), reflecting findings from Indy (-/-) knockout mice. These results suggest that the inhibitor ETG-5773 blocked citrate uptake mediated by mouse and human NaCT to reduce liver steatosis and body fat and improve glucose regulation, proving the concept of NaCT inhibition as a future liver treatment for MAFLD.

Keywords: MAFLD; NAFLD; NASH; NaCT; SLC13A5; citrate transporter; mINDY; metabolic dysfunction.

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Conflict of interest statement

G.Z. is an employee and shareholder of Eternygen GmbH; A.L.B. and J.J. are founders and shareholders of Eternygen GmbH. All other authors declare no conflict of interest. C.Y., R.J.-P., K.S., S.S., A.K.C.W., T.Z., and W.G. are employed by Evotec (UK) Ltd., Evotec SE, Evotec International GmbH, Aptuit (Verona) Srl, an Evotec Company, and their research and authorship of this article were completed within the scope of their employment.

Figures

Figure 1
Figure 1
Molecular structure of the ETG-5773 compound.
Figure 2
Figure 2
Treatment with ETG-5773 changes metabolic parameters in obese mice. (a) Body weight of DIO mice; (b) fasting blood glucose levels; (c) fasting serum insulin levels; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; (d) glucose tolerance test, # ETG-5773 vs. vehicle, * ETG-5773 vs. vehicle pair-fed, * or # p < 0.05, ** or ## p < 0.01, ns: non-significant, ANOVA, mean SD. Animals were treated orally twice a day with 15 mg/kg ETG-5773 or with the corresponding vehicle or vehicle in the pair-fed group, n = 12 vehicle and ETG-5773, n = 8 pair-fed.
Figure 3
Figure 3
Liver triglycerides are reduced in obese mice treated with ETG-5773. Triglyceride content in the livers of DIO mice after the 28-day treatment period. Animals were treated orally twice a day with 15 mg/kg ETG-5773 or with the corresponding vehicle or vehicle in the pair-fed group ** p < 0.01, *** p < 0.001, ns: non-significant, ANOVA mean SD, n = 12 vehicle and ETG-5773, n = 8 pair-fed.
Figure 4
Figure 4
Body composition analysis in DIO mice treated with ETG-5773. The graph shows changes in absolute body weight, fat mass, and lean mass of mice after 22 days of treatment as well as relative amounts of fat and lean mass. Animals were treated orally twice a day with 15 mg/kg ETG-5773 or with the corresponding vehicle for vehicle or pair-fed groups. * p < 0.05, ** p < 0.01, **** p < 0.0001, ns: non-significant. ANOVA mean SD, n = 12 vehicle and ETG-5773, n = 8 pair-fed.
Figure 5
Figure 5
Hepatic mRNA expression levels of key genes involved in lipogenesis in DIO mice after the 28-day treatment period. Animals were treated orally twice a day with ETG-5773 at 15 mg/kg or with the corresponding vehicle or vehicle in the pair-fed group: (a) mRNA expression of sterol regulatory element-binding transcription factor 1 (Srebf1); (b) mRNA expression of the stearoyl-CoA desaturase (Scd1) gene. ** p < 0.01, *** p < 0.001, **** p < 0.0001 as indicated, ns: non-significant. ANOVA mean SD, n = 12 vehicle and ETG-5773, n = 8 pair-fed.
Figure 6
Figure 6
Body weight and glucose regulation in DIO mice treated with ETG-5773 for 7 days. (a) Body weight; (b) fasting blood glucose concentrations; (c) fasting serum insulin concentrations. Animals were treated orally twice a day with 15 mg/kg ETG-5773 or with the corresponding vehicle or vehicle in the pair-fed group. * p < 0.05, ** p < 0.01, **** p < 0.0001 as indicated, ns: non-significant. ANOVA mean SD, n = 6.
Figure 7
Figure 7
Energy metabolism biomarkers in the DIO mouse model treated with ETG-5773 for 7 days. (a) β-hydroxybutyrate (β-HBA) levels measured in an ELISA; (b) adenosine monophosphate-activated protein kinase (AMPK) activation levels measured in a Western blot and normalised for actin. Animals were treated orally twice a day with 15 mg/kg ETG-5773 or with the corresponding vehicle or vehicle in the pair-fed group. * p < 0.05, ** p < 0.01, *** p < 0.001 as indicated ns: non-significant. ANOVA mean SD, n = 6. Corresponding western blot data are shown in Figure S5.
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