The Hepatic Plasma Membrane Citrate Transporter NaCT (SLC13A5) as a Molecular Target for Metformin

Jonathan Kopel,Vadivel Ganapathy,Sabarish Ramachandran,Bojana Ristic,Toshihiro Sato,Kei Higuchi

doi:10.1038/s41598-020-65621-w

Jonathan Kopel, Vadivel Ganapathy + Show 4 more

Open Access

https://doi.org/10.1038/s41598-020-65621-w

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Abstract

Metformin is the first-line treatment for type 2 diabetes. Inhibition of hepatic gluconeogenesis is the primary contributor to its anti-diabetic effect. Metformin inhibits complex I and α-glycerophosphate shuttle, and the resultant increase in cytoplasmic NADH/NAD+ ratio diverts glucose precursors away from gluconeogenesis. These actions depend on metformin-mediated activation of AMP kinase (AMPK). Here we report on a hitherto unknown mechanism. Metformin inhibits the expression of the plasma membrane citrate transporter NaCT in HepG2 cells and decreases cellular levels of citrate. 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR), an AMPK activator, elicits a similar effect. The process involves a decrease in maximal velocity with no change in substrate affinity. The decrease in NaCT expression is associated with decreased mRNA levels. AMPK inhibits mTOR, and the mTOR inhibitor rapamycin also decreases NaCT expression. The transcription factor downstream of AMPK that is relevant to cAMP signaling is CREB; decreased levels of phospho-CREB seem to mediate the observed effects of metformin on NaCT. Citrate is known to suppress glycolysis by inhibiting phosphofructokinase-1 and activate gluconeogenesis by stimulating fructose-1,6-bisphophatase; therefore, the decrease in cellular levels of citrate would stimulate glycolysis and inhibit gluconeogenesis. These studies uncover a novel mechanism for the anti-diabetic actions of metformin.

Highlights

To confirm directly the activation of AMPK in HepG2 cells treated with metformin or Aminoimidazole-4-carboxamide ribonucleotide (AICAR), we monitored the phosphorylation status of Ser-2448 in mTOR, which is one of the sites for AMPK-dependent phosphorylation[27]
Given that metformin and AICAR activate AMPK, we investigated whether mTOR influences the function and expression of NaCT
The most critical step in glycolysis and gluconeogenesis is the conversion of fructose-6-phosphate to fructose-1,6-bisphosphate and vice versa

Summary

Introduction

To confirm directly the activation of AMPK in HepG2 cells treated with metformin or AICAR, we monitored the phosphorylation status of Ser-2448 in mTOR, which is one of the sites for AMPK-dependent phosphorylation[27]. Www.nature.com/scientificreports mTOR inhibition by rapamycin mimics the effects of metformin and AICAR on NaCT expression and function. We treated HepG2 cells with 20 μM rapamycin, a well-known mTOR inhibitor[26], for 24 h in high-glucose (20 mM) medium and monitored the expression and activity of NaCT.

Results

Conclusion