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. 2021 Mar 11;23(2):41.
doi: 10.1208/s12248-021-00575-z.

Peptide Hormone Insulin Regulates Function, Expression, and SUMOylation of Organic Anion Transporter 3

Jinghui Zhang  1 , Guofeng You  2
Affiliations

Peptide Hormone Insulin Regulates Function, Expression, and SUMOylation of Organic Anion Transporter 3

Jinghui Zhang et al. AAPS J. .

Abstract

Organic anion transporter 3 (OAT3) plays an important role in the disposition of various anionic drugs which impacts the pharmacokinetics and pharmacodynamics of the therapeutics, thus influencing the pharmacological effects and toxicity of the drugs. In this study, we investigated the effect of insulin on the regulation of OAT3 function, expression, and SUMOylation. We demonstrated that insulin induced an increase in OAT3 transport activity through a dose- and time-dependent manner in COS-7 cells. The insulin-induced elevation in OAT3 function was blocked by PKA inhibitor H89, which correlated well with OAT3 protein expression. Moreover, both PKA activator Bt2-cAMP-induced increase and insulin-induced increase in OAT3 function were blocked by PKB inhibitor AKTi1/2. To further investigate the involvement of SUMOylation, we treated OAT3-expressing cells with insulin in presence or absence of H89 or AKTi1/2 followed by examining OAT3 SUMOylation. We showed that insulin enhanced OAT3 SUMOylation, and such enhancement was abrogated by H89 and AKTi1/2. Lastly, insulin increased OAT3 function and SUMOylation in rat kidney slice. In conclusion, our investigations demonstrated that insulin regulated OAT3 function, expression, and SUMOylation through PKA/PKB signaling pathway. Graphical abstract.

Keywords: Drug transport; Insulin; Organic anion transporter 3; Regulation; SUMOylation.

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Conflict of interest statement

Conflict of interest

The authors have declared that there is no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Insulin increases OAT3 transport activity. (a). Dose dependence. OAT3-expressing cells were treated for 6 h with insulin at various doses (0–1 μM). 4-min uptake of [3H]-ES (300 nM) was then measured. Uptake activity was expressed as folds of the uptake measured in control cells. The data represent uptake into OAT3-expressing cells minus uptake into mock cells (parental cells). Values are mean ± S.E. (n = 3). *P < 0.05. (b). Time dependence. OAT3-expressing cells were treated with insulin (100 nM) for 0–6 h. 4-min uptake of [3H]-ES (300 nM) was then measured. Uptake activity was expressed as folds of the uptake measured in control cells. The data represent uptake into OAT3-expressing cells minus uptake into mock cells (parental cells). Values are mean ± S.E. (n = 3). *P < 0.05. Statistical analysis was performed using one-way ANOVA (GraphPad Software Inc., San Diego, CA, USA).
Fig. 2.
Fig. 2.
Insulin alters the kinetics of OAT3-mediated estrone sulfate transport. OAT3-expressing cells were treated with the insulin (100 nM, 6h), and initial uptake (4 min) of [3H]-estrone sulfate was measured at the concentration of 0.1–10 μM. The data represent uptake into OAT3-expressing cells minus uptake into mock cells (parental COS-7 cells). Values are means ± SD (n = 3). V, velocity; S, substrate concentration. Transport kinetic values were calculated using the Eadie–Hofstee transformation.
Fig. 3.
Fig. 3.
The effects of PKA inhibitor H89 on insulin stimulation of OAT3-mediated transport. OAT3-expressing cells were treated with insulin (100 nM, 6h) with or without H89 (10 μM) followed by measuring the uptake of [3H]-ES (4 min, 300 nM). Uptake activity was expressed as a percentage of the uptake determined in control cells. The data represent uptake into OAT3-expressing cells minus uptake into mock cells. Values are mean ± SD (n = 3). *P<0.05. Statistical analysis was performed using one-way ANOVA (GraphPad Software Inc., San Diego, CA, USA).
Fig. 4.
Fig. 4.
Insulin regulates OAT3 cell surface expression through PKA pathway. (a) Top panel: OAT3-expressing cells were treated with insulin (100 nM) with or without PKA inhibitor H89 (10 μM) for 6 h. Cells were labeled with membrane impermeable biotin. Biotinylated cell surface proteins were separated with streptavidin beads, followed by immunoblotting (IB) with anti-myc antibody (OAT3 was tagged with epitope myc for immunodetection). Bottom panel: The identical blot of the top panel was re-probed with anti-E-cadherin antibody. E-cadherin is a marker for cell membrane proteins. (b) Densitometry analysis of blot results from Fig. 4a top panel as well as from other experiments. The values are mean ± SD (n = 3); *P < 0.05. Statistical analysis was performed using one-way ANOVA (GraphPad Software Inc., San Diego, CA, USA).
Fig. 5.
Fig. 5.
Insulin regulates OAT3 total expression through PKA pathway. (a). Top panel: OAT3-expressing cells were treated with insulin (100 nM) with or without H89 (10 μM) for 6 h. The cells were collected and lysed, followed by immunoblotting (IB) with anti-myc antibody (OAT3 was tagged with epitope myc for immunodetection). Bottom panel: The identical blot of the top panel was re-probed with anti-GAPDH antibody. GAPDH is a marker for total cell proteins. (b). Densitometry analyses of blot results from Fig. 5a top panel as well as from other experiments. The values are mean ± SD (n = 3); *P < 0.05. Statistical analysis was performed using one-way ANOVA (GraphPad Software Inc., San Diego, CA, USA).
Fig. 6.
Fig. 6.
Insulin regulates OAT3 transport activity through PKA/PKB pathway. (a). OAT3-expressing cells were treated with insulin (100 nM, 6h) with or without AKTi1/2 (5 μM, PKB inhibitor) followed by measuring the uptake of [3H]-ES (4 min, 300 nM). Uptake activity was expressed as a percentage of the uptake determined in control cells. The data represent uptake into OAT3-expressing cells minus uptake into mock cells. Values are mean ± SD (n = 3). *P<0.05. (b). OAT3-expressing cells were treated with Bt2-cAMP (20 μM, 2h) with or without AKTi1/2 (5 μM, PKB inhibitor) followed by measuring the uptake of [3H]-ES (4 min, 300 nM). Uptake activity was expressed as a percentage of the uptake determined in control cells. The data represent uptake into OAT3-expressing cells minus uptake into mock cells. Values are mean ± SD (n = 3). *P<0.05. Statistical analysis was performed using one-way ANOVA (GraphPad Software Inc., San Diego, CA, USA).
Fig. 7.
Fig. 7.
Insulin stimulated OAT3 SUMOylation through PKA/PKB pathway. (a). OAT3-expressing cells were transfected with HA-SUMO2 for 48h, then treated with the insulin (100 nM, 6 h) in the presence and absence of PKA inhibitor H89 (10 μM). The treated cells were lysed. OAT3 was immunoprecipitated by anti-myc antibody, followed by immunoblotting (IB) with anti-HA antibody. (b) Densitometry plot of results from Fig. 7a, as well as from other experiments. Values are mean ± SD (n = 3). (c). OAT3-expressing cells were transfected with HA-SUMO2 for 48h, then treated with the insulin (100 nM, 6 h) in the presence and absence of PKA inhibitor AKTi1/2 (5 μM). The treated cells were lysed. OAT3 was immunoprecipitated by anti-myc antibody, followed by immunoblotting (IB) with anti-HA antibody. (d) Densitometry plot of results from Fig. 7c, as well as from other experiments. Values are mean ± SD (n = 3). Statistical analysis was performed using one-way ANOVA (GraphPad Software Inc., San Diego, CA, USA).
Fig. 8.
Fig. 8.
Insulin stimulated OAT3 transport activity and SUMOylation in rat kidney slice. (a). Kidney slices were treated with the insulin (8 μM, 1h) followed by measuring the uptake of [3H]-ES (25 min, 300 nM). The data represent uptake into kidney slice minus background value. Values are means ± SD (n = 3). (b). Kidney slices were treated with the insulin (8 μM, 1h), and then the slices were lysed. SUMO2/3 was immunoprecipitated by anti-SUMO2/3 antibody, followed by immunoblotting (IB) with anti-OAT3 antibody. (c) Densitometry plot of results from Fig. 8b, as well as from other experiments. Values are mean ± SD (n = 3). Statistical analysis was performed using Student’s paired t-tests.
Fig. 9.
Fig. 9.
Insulin regulated OAT3 function, expression, and SUMOylation through PKA/PKB signaling pathway.
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References

    1. You G. Structure, function, and regulation of renal organic anion transporters. Med Res Rev. 2002;22(6):602–16. doi: 10.1002/med.10019. - DOI - PubMed
    1. Dantzler WH, Wright SH. The molecular and cellular physiology of basolateral organic anion transport in mammalian renal tubules. Biochim Biophys Acta. 2003;1618(2):185–93. doi: 10.1016/j.bbamem.2003.08.015. - DOI - PubMed
    1. Srimaroeng C, Perry JL, Pritchard JB. Physiology, structure, and regulation of the cloned organic anion transporters. Xenobiotica. 2008;38(7–8):889–935. doi: 10.1080/00498250801927435. - DOI - PMC - PubMed
    1. VanWert AL, Gionfriddo MR, Sweet DH. Organic anion transporters: discovery, pharmacology, regulation and roles in pathophysiology. Biopharm Drug Dispos. 2010;31(1):1–71. doi: 10.1002/bdd.693. - DOI - PubMed
    1. Ahn SY, Nigam SK. Toward a systems level understanding of organic anion and other multispecific drug transporters: a remote sensing and signaling hypothesis. Mol Pharmacol. 2009;76(3):481–90. doi: 10.1124/mol.109.056564. - DOI - PMC - PubMed

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