Influences of sterol regulatory element binding protein-1c silencing on glucose production in HepG2 cells treated with free fatty acid

Abstract

Background: Elevation of exogenous free fatty acid (FFA) level leads to insulin resistance (IR) in liver, IR is manifested by elevated hepatic glucose production. We aim to study whether inhibition of endogenous fatty acid synthesis could decrease hepatic glucose production.

Methods: Low-passage HepG2 cells derived from human liver tissue were cultured in medium supplemented with FFA to induce IR, the influences of sterol regulatory element binding protein-1c (SREBP-1c) silencing on glucose production of HepG2 cells were investigated, and genes responsible for fatty acid and glucose metabolism were detected by real-time PCR.

Results: Compared with HepG2 cells cultured in normal growth medium, glucose production of HepG2 cells treated by FFA was significantly increased {[(0.28 ± 0.01) vs (0.83 ± 0.02)] umol.ug^{- 1} protein, n = 6 wells, P < 0.01}; the mRNA expression of phosphoenolpyruvate carboxylase kinase (PEPCK) and glucose-6-phosphatase (G6PC) in HepG2 cells increased by more than 5-fold and 3-fold, respectively; the mRNA expression of fatty acid synthase (FAS) and stearoyl-CoA desaturase-1 (SCD1) increased by approximately 4-fold and 1.1-fold, respectively; the mRNA expression of carnitine palmitoyltransferase-1 (CPT-1) changed slightly. Compared with the scrambled siRNA control, glucose production of HepG2 cells treated by FFA significantly increased after SREBP-1c silencing {[(0.018 ± 0.001) vs (0.028 ± 0.002)] umol.ug^{- 1} protein, n = 6 wells, P < 0.01}; the mRNA expression of PEPCK and G6PC increased by approximately 1.5-fold and 5-fold, respectively, but the mRNA expression of FAS, SCD1 and CPT-1 changed slightly.

Conclusions: SREBP-1c silencing further augmented glucose production of HepG2 cells treated by FFA significantly, genes responsible for fatty acid synthesis and gluconeogenesis played an important role in this process. SREBP-1c functions not only as a lipid regulator but also plays an important role in regulation of glucose metabolism.

Keywords: Free fatty acid; Glucose production; HepG2 cells; SREBP-1c silencing.

Fig. 1

HepG2 cells transfected with scrambled siRNA (fluorescein conjugated control siRNA) (A1, Image observed under bright-field; A2, Image observed under dark-field) and HepG2 cells without treatment (B1, Image observed under bright-field; B2, Image observed under dark-field) were harvested 24 h later, and examined for fluorescein expression by fluorescent microscopy (Magnification× 6.3). The transfection rate was approximately 70–80% by viewing representative nuclei

Fig. 2

The mRNA and protein expression of SREBP-1c after SREBP-1c Silencing in HepG2 cells. a SREBP-1c mRNA expression in HepG2 cells treated with SREBP-1c siRNA for 24 h, 48 h, and 72 h, respectively. White squares, 0 h; wide diagonal, 24 h; black squares, 48 h; dark diamond, 72 h. b SREBP-1c mRNA expression in HepG2 cells treated with scramble siRNA for 24 h, 48 h, and 72 h, respectively. White squares, 0 h; wide diagonal, 24 h; black squares, 48 h; dark diamond, 72 h. c SREBP-1c protein expression in HepG2 cells treated with SREBP-1c siRNA and scramble siRNA for 24 h, 48 h, and 72 h, respectively. GAPDH is an internal housekeeping control. Compared with the scrambled siRNA control, SREBP-1c silencing caused the mRNA expression of SREBP-1c in HepG2 cells to decrease by approximately 75, 71 and 68% after 24 h, 48 h and 72 h, respectively. We were also able to use siRNA for 24–72 h to silence SREBP-1c protein production by more than 90% for the 125 kDa band

Fig. 3

Influence of SREBP-1c silencing on glucose production in HepG2 cells. Cells were washed three times with PBS to remove glucose, then incubated in glucose production medium, the medium was left on the cells for 10 h before being replaced with identical medium, and left overnight (10 h) for the glucose production assay. a HepG2 cells treated by FFA (normal growth medium supplemented with 0.5 mM palmitate) vs normal growth medium (DMEM supplemented with 4.5 mmol/L glucose). b SREBP-1c siRNA vs scrambled siRNA in HepG2 cells cultured in normal growth medium (DMEM supplemented with 4.5 mmol/L glucose); c SREBP-1c siRNA vs scrambled siRNA in HepG2 cells treated by FFA (normal growth medium supplemented with 0.5 mM palmitate) (n = 6 wells, ^† P < 0.01 vs control). a white squares, normal growth medium; black square, FFA supplemented medium; b white squares, scrambled siRNA in HepG2 cells cultured in normal growth medium; white background on the diagonal width, SREBP-1c siRNA in HepG2 cells cultured in normal growth medium; c black square, scrambled siRNA in HepG2 cells treated by FFA, black scottish squares, SREBP-1c siRNA in HepG2 cells treated by FFA

Fig. 4

Influence of SREBP-1c silencing on PEPCK mRNA expression of HepG2 cells. a HepG2 cells treated by FFA (normal growth medium supplemented with 0.5 mM palmitate) vs normal growth medium (DMEM supplemented with 4.5 mmol/L glucose); b SREBP-1c siRNA vs scrambled siRNA in HepG2 cells cultured in normal growth medium (DMEM supplemented with 4.5 mmol/L glucose); c SREBP-1c siRNA vs scrambled siRNA in HepG2 cells treated by FFA (normal growth medium supplemented with 0.5 mM palmitate) (n = 6 wells, ^† P < 0.01 vs control). a white squares, normal growth medium; black square, FFA supplemented medium; b white squares, scrambled siRNA in HepG2 cells cultured in normal growth medium; white background on the diagonal width, SREBP-1c siRNA in HepG2 cells cultured in normal growth medium; c black square, scrambled siRNA in HepG2 cells treated by FFA, black scottish squares, SREBP-1c siRNA in HepG2 cells treated by FFA

Fig. 5

Influence of SREBP-1c silencing on G6PC mRNA expression of HepG2 cells. a HepG2 cells treated by FFA (normal growth medium supplemented with 0.5 mM palmitate) vs normal growth medium (DMEM supplemented with 4.5 mmol/L glucose); b SREBP-1c siRNA vs scrambled siRNA in HepG2 cells cultured in normal growth medium (DMEM supplemented with 4.5 mmol/L glucose); c SREBP-1c siRNA vs scrambled siRNA in HepG2 cells treated by FFA (normal growth medium supplemented with 0.5 mM palmitate) (n = 6 wells, ^† P < 0.01 vs control). a white squares, normal growth medium; black square, FFA supplemented medium; b white squares, scrambled siRNA in HepG2 cells cultured in normal growth medium; white background on the diagonal width, SREBP-1c siRNA in HepG2 cells cultured in normal growth medium; c black square, scrambled siRNA in HepG2 cells treated by FFA, black scottish squares, SREBP-1c siRNA in HepG2 cells treated by FFA

Fig. 6

Influence of SREBP-1c silencing on FAS mRNA expression of HepG2 cells. a HepG2 cells treated by FFA (normal growth medium supplemented with 0.5 mM palmitate) vs normal growth medium (DMEM supplemented with 4.5 mmol/L glucose); b SREBP-1c siRNA vs scrambled siRNA in HepG2 cells cultured in normal growth medium (DMEM supplemented with 4.5 mmol/L glucose); c SREBP-1c siRNA vs scrambled siRNA in HepG2 cells treated by FFA (normal growth medium supplemented with 0.5 mM palmitate) (n = 6 wells, ^† P < 0.01 vs control). a white squares, normal growth medium; black square, FFA supplemented medium; b white squares, scrambled siRNA in HepG2 cells cultured in normal growth medium; white background on the diagonal width, SREBP-1c siRNA in HepG2 cells cultured in normal growth medium; c black square, scrambled siRNA in HepG2 cells treated by FFA, black scottish squares, SREBP-1c siRNA in HepG2 cells treated by FFA

Fig. 7

Influence of SREBP-1c silencing on SCD1 mRNA expression of HepG2 cells. a HepG2 cells treated by FFA (normal growth medium supplemented with 0.5 mM palmitate) vs normal growth medium (DMEM supplemented with 4.5 mmol/L glucose); b SREBP-1c siRNA vs scrambled siRNA in HepG2 cells cultured in normal growth medium (DMEM supplemented with 4.5 mmol/L glucose); c SREBP-1c siRNA vs scrambled siRNA in HepG2 cells treated by FFA (normal growth medium supplemented with 0.5 mM palmitate) (n = 6 wells, ^† P < 0.01 vs control). a white squares, normal growth medium; black square, FFA supplemented medium; b white squares, scrambled siRNA in HepG2 cells cultured in normal growth medium; white background on the diagonal width, SREBP-1c siRNA in HepG2 cells cultured in normal growth medium; c black square, scrambled siRNA in HepG2 cells treated by FFA, black scottish squares, SREBP-1c siRNA in HepG2 cells treated by FFA

Fig. 8

Influence of SREBP-1c silencing on CPT-1 mRNA expression of HepG2 cells. a HepG2 cells treated by FFA (normal growth medium supplemented with 0.5 mM palmitate) vs normal growth medium (DMEM supplemented with 4.5 mmol/L glucose); b SREBP-1c siRNA vs scrambled siRNA in HepG2 cells cultured in normal growth medium (DMEM supplemented with 4.5 mmol/L glucose); c SREBP-1c siRNA vs scrambled siRNA in HepG2 cells treated by FFA (normal growth medium supplemented with 0.5 mM palmitate) (n = 6 wells, † P < 0.01 vs control). a white squares, normal growth medium; black square, FFA supplemented medium; b white squares, scrambled siRNA in HepG2 cells cultured in normal growth medium; white background on the diagonal width, SREBP-1c siRNA in HepG2 cells cultured in normal growth medium; c black square, scrambled siRNA in HepG2 cells treated by FFA, black scottish squares, SREBP-1c siRNA in HepG2 cells treated by FFA

Fig. 9

Immunoblotting of total Akt and p-Akt^s473 in HepG2 cells in different groups. a Comparison of protein expression of total Akt and p-Akt ^S473 in HepG2 cells cultured in normal growth medium and treated with FFA; b Influence of SREBP-1c silencing on the protein expression of total Akt and p-Akt ^S473 in HepG2 cells cultured in normal growth medium; c Influence of SREBP-1c silencing on the protein expression of total Akt and p-Akt ^S473 in HepG2 cells treated with FFA. a white squares, normal growth medium; black square, FFA supplemented medium; b white squares, scrambled siRNA in HepG2 cells cultured in normal growth medium; white background on the diagonal width, SREBP-1c siRNA in HepG2 cells cultured in normal growth medium; c black square, scrambled siRNA in HepG2 cells treated by FFA, black scottish squares, SREBP-1c siRNA in HepG2 cells treated by FFA. Relative level of each protein was normalized to GAPDH, an internal housekeeping control, and the control group was set to 1 (n = 4 wells/treatment, the data is representative of duplicate independent protein expression experiments). Values are presented as mean ± SD; †P < 0.01 vs control. p-Akt^s473 is the activation of Akt, Akt proteins become phosphorylated and activated by phosphorylation of ser 473