Cell-Specific Transport and Thyroid Hormone Receptor Isoform Selectivity Account for Hepatocyte-Targeted Thyromimetic Action of MGL-3196 - PubMed (original) (raw)
Cell-Specific Transport and Thyroid Hormone Receptor Isoform Selectivity Account for Hepatocyte-Targeted Thyromimetic Action of MGL-3196
Georg Sebastian Hönes et al. Int J Mol Sci. 2022.
Abstract
Thyroid hormones (THs) and TH receptor-beta (TRβ) reduce hepatic triglycerides, indicating a therapeutic potential for TH analogs in liver steatosis. To avoid adverse extrahepatic, especially TRα-mediated effects such as tachycardia and bone loss, TH analogs with combined TRβ and hepatocyte specificity are desired. MGL-3196 is a new TH analog that supposedly meets these criteria. Here, we characterize the thyromimetic potential of MGL-3196 in cell-based assays and address its cellular uptake requirements. We studied the contribution of liver-specific organic anion transporters (OATP)1B1 and 1B3 to MGL-3196 action. The TR isoform-specific efficacy of MGL-3196 compared with 3,5,3'-triiodothyronine (T3) was determined with luciferase assays and gene expression analysis in OATP1B1 and OATP1B3 and TRα- or TRβ-expressing cells and in primary murine hepatocytes (PMHs) from wild-type and TRβ knockout mice. We measured the oxygen consumption rate to compare the effects of MGL-3196 and T3 on mitochondrial respiration. We identified OATP1B1 as the primary transporter for MGL-3196. MGL-3196 had a high efficacy (90% that of T3) in activating TRβ, while the activation of TRα was only 25%. The treatment of PMHs with T3 and MGL-3196 at EC50 resulted in a similar induction of Dio1 and repression of Serpina7. In HEK293 cells stably expressing OATP1B1, MGL-3196 had comparable effects on mitochondrial respiration as T3. These data indicate that MGL-3196's hepatic thyromimetic action, the basis for its therapeutic use, results from a combination of hepatocyte-specific transport by OATP1B1 and the selective activation of TRβ over TRα.
Keywords: MGL-3196; OATP1B1; Resmetirom; thyroid hormone action; thyroid hormone analog; thyroid hormone transport; thyromimetic.
Conflict of interest statement
The authors declare no conflict of interest.
Figures
Figure 1
Structures of triiodothyronine (T3) and MGL-3196. In comparison to the structure of T3, the phenolic outer ring is replaced by a pyridazinone ring (R1) in MGL-3196, resulting in a heterocyclic structure. Instead of the α-amino acid group, MGL-3196 possesses an azauracil residue with a cyano group (R2) ([11]).
Figure 2
Expression of TH transporters and TRs in HEK293 cell lines: (A) RT-PCR analysis of genes encoding for TH transporters MCT8 (SLC16A2), LAT1 (SLC7A5), and LAT2 (SLC7A8) in wild-type HEK293 cells (HEK-Co) as well as in HEK-1B1 and HEK-1B3 cells stably expressing OATP1B1 (SLCO1B1) or OATP1B3 (SLCO1B3), respectively. The dotted line marks the limit of detection (l.o.d.) at Ct ≥ 35. Ct = cycle threshold; LAT = l-type amino acid transporter; MCT = monocarboxylate transporter; (B) expression of TRα and TRβ in HEK293 cells. Transient transfection led to similar expressions of both receptors. Student’s _t_-test: ns = not significant; *** p < 0.001.
Figure 3
Dose-response curve and luciferase activity: (A) EC50 values were determined by DR4 luciferase reporter assay in cells stably expressing OATP1B1 or OATP1B3 (HEK-1B1 and HEK-1B3, respectively). Additionally, the cells were transfected with plasmids either encoding for TRα (circles) or TRβ (triangles). Cells were cultured in TH-depleted serum and treated with T3 (0.1–1000 nM; red-filled triangles or circles) or MGL-3196 (10–10,000 nM; blue open triangles or circles) for 48 h; (B) inhibition of OATP1B1-dependent uptake of MGL-3196 was determined in a DR4 luciferase reporter assay with HEK-Co (control) cells and HEK-1B1 cells (stably expressing OATP1B1) and 100 µM GA (glycyrrhizinic acid) as OATP1B1 inhibitor. RLU in T3-treated cells without GA = 1.00 in each cell line. Two-way ANOVA with Tukey’s post hoc test: **** p < 0.0001.
Figure 4
Gene expression analysis in HEK-1B1 cells and primary murine hepatocytes: (A) HEK-1B1 cells either expressing TRβ or TRα were treated with 10 nM T3 (dark grey) or 600 nM MGL-3196 (light grey) for 48 h and expression of TH target genes KLF9 (krüppel-like factor 9), PCK1 (pyruvate carboxylase kinase 1) and HIF2a (hypoxia-inducible factor 2a) was measured (n = 3); (B) primary murine hepatocytes of WT and TRβKO mice were treated with EC50 of T3 or MGL-3196 for 24 h and expression of hepatic TH target genes was analyzed by using RT-PCR. MGL-3196 and T3 induced similar changes in gene expression of Dio1, Serpina7, and Pck1 (n = 6). Both T3 and MGL-3196 led to an increased expression of Slco1b2, the murine orthologue of the human SLCO1B1 and SLCO1B3. One-way ANOVA with Tukey’s post hoc test: ns = not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Figure 5
MGL-3196 increases mitochondrial activity in OATP1B1-expressing cells: (A) measurement of oxygen consumption rate (OCR) in HEK-1B1 cells treated for 48 h with T3 (100 nM) or MGL-3196 (6 µM) (n = 5 biological replicates per group); (B) basal OCR ([last rate measurement before oligomycin injection] − [non-mitochondrial respiration rate]); (C) maximal respiration ([maximum rate measured after FCCP injection] − [non-mitochondrial respiration rate]); (D) ATP production ([last rate measurement before oligomycin injection] − [minimum rate measured after oligomycin injection]); (E) proton leak ([minimum rate measured after oligomycin injection] − [non-mitochondrial respiration rate]); (F) spare respiratory capacity ([maximal respiration] − [basal respiration]); (G) non-mitochondrial oxygen consumption (minimum rate measured after rotenone/antimycin A injection). One-way ANOVA with Tukey’s post hoc test: ns = not significant; * p < 0.05, ** p < 0.01. FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; rot., rotenone; AA, antimycin A.
Figure 5
MGL-3196 increases mitochondrial activity in OATP1B1-expressing cells: (A) measurement of oxygen consumption rate (OCR) in HEK-1B1 cells treated for 48 h with T3 (100 nM) or MGL-3196 (6 µM) (n = 5 biological replicates per group); (B) basal OCR ([last rate measurement before oligomycin injection] − [non-mitochondrial respiration rate]); (C) maximal respiration ([maximum rate measured after FCCP injection] − [non-mitochondrial respiration rate]); (D) ATP production ([last rate measurement before oligomycin injection] − [minimum rate measured after oligomycin injection]); (E) proton leak ([minimum rate measured after oligomycin injection] − [non-mitochondrial respiration rate]); (F) spare respiratory capacity ([maximal respiration] − [basal respiration]); (G) non-mitochondrial oxygen consumption (minimum rate measured after rotenone/antimycin A injection). One-way ANOVA with Tukey’s post hoc test: ns = not significant; * p < 0.05, ** p < 0.01. FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; rot., rotenone; AA, antimycin A.
References
- Minakhina S., Bansal S., Zhang A., Brotherton M., Janodia R., De Oliveira V., Tadepalli S., Wondisford F.E. A Direct Comparison of Thyroid Hormone Receptor Protein Levels in Mice Provides Unexpected Insights into Thyroid Hormone Action. Thyroid. 2020;30:1193–1204. doi: 10.1089/thy.2019.0763. - DOI - PMC - PubMed
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