Inhibition of Sodium-Glucose Cotransporter-2 during Serum Deprivation Increases Hepatic Gluconeogenesis via the AMPK/AKT/FOXO Signaling Pathway

Abstract

Backgruound: Sodium-dependent glucose cotransporter 2 (SGLT2) mediates glucose reabsorption in the renal proximal tubules, and SGLT2 inhibitors are used as therapeutic agents for treating type 2 diabetes mellitus. This study aimed to elucidate the effects and mechanisms of SGLT2 inhibition on hepatic glucose metabolism in both serum deprivation and serum supplementation states.

Methods: Huh7 cells were treated with the SGLT2 inhibitors empagliflozin and dapagliflozin to examine the effect of SGLT2 on hepatic glucose uptake. To examine the modulation of glucose metabolism by SGLT2 inhibition under serum deprivation and serum supplementation conditions, HepG2 cells were transfected with SGLT2 small interfering RNA (siRNA), cultured in serum-free Dulbecco's modified Eagle's medium for 16 hours, and then cultured in media supplemented with or without 10% fetal bovine serum for 8 hours.

Results: SGLT2 inhibitors dose-dependently decreased hepatic glucose uptake. Serum deprivation increased the expression levels of the gluconeogenesis genes peroxisome proliferator-activated receptor gamma co-activator 1 alpha (PGC-1α), glucose 6-phosphatase (G6pase), and phosphoenolpyruvate carboxykinase (PEPCK), and their expression levels during serum deprivation were further increased in cells transfected with SGLT2 siRNA. SGLT2 inhibition by siRNA during serum deprivation induces nuclear localization of the transcription factor forkhead box class O 1 (FOXO1), decreases nuclear phosphorylated-AKT (p-AKT), and p-FOXO1 protein expression, and increases phosphorylated-adenosine monophosphate-activated protein kinase (p-AMPK) protein expression. However, treatment with the AMPK inhibitor, compound C, reversed the reduction in the protein expression levels of nuclear p- AKT and p-FOXO1 and decreased the protein expression levels of p-AMPK and PEPCK in cells transfected with SGLT2 siRNA during serum deprivation.

Conclusion: These data show that SGLT2 mediates glucose uptake in hepatocytes and that SGLT2 inhibition during serum deprivation increases gluconeogenesis via the AMPK/AKT/FOXO1 signaling pathway.

Keywords: AMP-activated protein kinases; FOXO1; Gluconeogenesis; Hepatocytes; Serum deprivation; Sodium-dependent glucose transporter 2.

Fig. 1.

Empagliflozin (Empa) and dapagliflozin (Dapa) inhibit hepatic glucose uptake in a sodium (Na⁺) dependent manner. Huh7 cells are cultured in sodium-free (Na⁺-free) buffer or sodium (Na⁺) buffer and treated with various concentrations of the SGLT2 inhibitors Empa and Dapa. Glucose uptake is determined using a glucose uptake assay kit. The cells were photographed using a fluorescence microscope (magnification, ×400); scale bars=20 μm. Con, control; 2-NBDG, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose. ^aP<0.05 when compared with sodium-free buffer; ^bP<0.05 and ^cP<0.01 when compared with sodium-supplemented buffer.

Fig. 2.

Sodium-dependent glucose cotransporter 2 (SGLT2) inhibition during serum deprivation increases the gluconeogenesis-associated genes’ expression in HepG2 cells. HepG2 cells pre-transfected with 50 nM SGLT2 small interfering RNA (siRNA) are cultured in serumfree Dulbecco’s modified Eagle’s medium (DMEM) (25 mmol/L glucose) for 16 hours and then in media supplemented with (serum supplementation) or without (serum deprivation) 10% fetal bovine serum for 8 hours. The mRNA expression levels of the genes encoding (A) SGLT2, (B) glucose transporter 2 (GLUT2), (C) sirtuin 1 (SIRT1), (D) peroxisome proliferator-activated receptor gamma co-activator 1 alpha (PGC-1α), (E) glucose 6-phosphatase (G6pase), and (F) phosphoenolpyruvate carboxykinase (PEPCK) are analyzed with quantitative real-time polymerase chain reaction and normalized to the β-Actin gene. Scr, scrambled. ^aP<0.05 and ^bP<0.01 when compared with the serum-free control cells; ^cP<0.01 when compared with the serum-supplemented control cells.

Fig. 3.

Sodium-dependent glucose cotransporter 2 (SGLT2) inhibition during serum deprivation increases the expression of phosphorylatedadenosine monophosphate-activated protein kinase (p-AMPK) and decreases the expression of nuclear p-AKT and phosphorylated-forkhead box class O 1 (p-FOXO1) proteins in HepG2 cells. HepG2 cells pre-transfected with 50 nM SGLT2 small interfering RNA (siRNA) are cultured in serum-free Dulbecco’s modified Eagle’s medium (25 mmol/L glucose) for 16 hours and then cultured in media supplemented with (serum supplementation) or without (serum deprivation) 10% fetal bovine serum for 8 hours. (A, B) Expression levels of p-AMPK, p-AKT, and p-FOXO1 are analyzed using Western blotting. (C) Subcellular localization of FOXO1 is determined by performing immunofluorescence staining, and the cells were photographed using a fluorescence microscope (magnification, ×400); scale bars=100 μm. Scr, scrambled; DAPI, 4′,6-diamidino-2-phenylindole; Con, control. ^aP<0.05 and ^bP<0.01 when compared with the serum-free control cells; ^cP<0.01 when compared with the serum-supplemented control cells.

Fig. 4.

Adenosine monophosphate-activated protein kinase (AMPK) inhibition in cells transfected sodium-dependent glucose cotransporter 2 (SGLT2) small interfering RNA (siRNA) during serum deprivation decreases the expression of p-AMPK and phosphoenolpyruvate carboxykinase (PEPCK) proteins and increases the expression of nuclear p-AKT and phosphorylated-forkhead box class O 1 (p-FOXO1) proteins. HepG2 cells pre-transfected with 50 nM SGLT2 siRNA in serum-free Dulbecco’s modified Eagle’s medium are treated with 10 µM of AMPK inhibitor compound C (CC). (A, B) Expression levels of p-AMPK, PEPCK, p-AKT, and p-FOXO1 are analyzed using Western blotting. (C) The presence of PEPCK is determined by performing immunofluorescence staining, and the cells were photographed using a fluorescence microscope (magnification, ×400); scale bars=100 μm. Scr, scrambled; DAPI, 4′,6-diamidino-2-phenylindole; Con, control. ^aP<0.05 and ^bP<0.01 when compared with the serum-free control cells; ^cP<0.05 and ^dP<0.01 when compared with the cells transfected with SGLT2 siRNA without CC.

Fig. 5.

Schematic diagram illustrating a possible mechanism: sodium-dependent glucose cotransporter 2 (SGLT2) inhibition during serum deprivation stimulates hepatic gluconeogenesis. Increased adenosine monophosphate-activated protein kinase (AMPK) by SGLT2 inhibition reduced the expression of nuclear p-AKT and phosphorylated-forkhead box class O 1 (p-FOXO1), consequently increasing gluconeogenesis-related gene expression. siRNA, small interfering RNA; PEPCK, phosphoenolpyruvate carboxykinase; G6pase, glucose 6-phosphatase.