Curcumin prevents leptin raising glucose levels in hepatic stellate cells by blocking translocation of glucose transporter-4 and increasing glucokinase

Abstract

Background and purpose: Hyperleptinemia is commonly found in obese patients, associated with non-alcoholic steatohepatitis and hepatic fibrosis. Hepatic stellate cells (HSCs) are the most relevant effectors during hepatic fibrogenesis. We recently reported that leptin stimulated HSC activation, which was eliminated by curcumin, a phytochemical from turmeric. This study was designed to explore the underlying mechanisms, focusing on their effects on intracellular glucose in HSCs. We hypothesized that leptin stimulated HSC activation by elevating the level of intracellular glucose, which was eliminated by curcumin by inhibiting the membrane translocation of glucose transporter-4 (GLUT4) and inducing the conversion of glucose to glucose-6-phosphate (G-6-P).

Experimental approach: Levels of intracellular glucose were measured in rat HSCs and immortalized human hepatocytes. Contents of GLUT4 in cell fractions were analysed by Western blotting analyses. Activation of signalling pathways was assessed by comparing phosphorylation levels of protein kinases.

Key results: Leptin elevated the level of intracellular glucose in cultured HSCs, which was diminished by curcumin. Curcumin suppressed the leptin-induced membrane translocation of GLUT4 by interrupting the insulin receptor substrates/phosphatidyl inositol 3-kinase/AKT signalling pathway. Furthermore, curcumin stimulated glucokinase activity, increasing conversion of glucose to G-6-P.

Conclusions and implications: Curcumin prevented leptin from elevating levels of intracellular glucose in activated HSCs in vitro by inhibiting the membrane translocation of GLUT4 and stimulating glucose conversion, leading to the inhibition of HSC activation. Our results provide novel insights into mechanisms of curcumin in inhibiting leptin-induced HSC activation.

Figure 1

Leptin caused a time- and concentration-dependent increase in the level of intracellular glucose in cultured HSCs, which was attenuated by curcumin. Analyses of levels of intracellular glucose. Values were expressed as pmol glucose·µg⁻¹ protein and presented as means ± SD (n = 3). *P < 0.05 versus the untreated control cells; ‡P < 0.05 versus cells treated with leptin alone at 100 ng·mL⁻¹. Percentages were calculated by the following formula: [Glu in target HSCs – Glu in compared HSCs)/Glu in compared HSCs] × 100% (n = 3). (A) Serum-starved HSCs were treated with leptin at 100 ng·mL⁻¹ in serum-free DMEM for indicated time-points. (B) Serum-starved HSCs were pretreated with curcumin at 0–30 µM for 1 h prior to the stimulation with or without leptin at indicated concentrations in serum-free media for additional 30 min.

Figure 2

Curcumin interrupted the leptin-activated IRS/PI3K/AKT signalling pathway in cultured HSCs, leading to inhibition of the GLUT4 membrane translocation and to reduction of intracellular glucose. Serum-starved HSCs were pretreated with or without curcumin at indicated concentrations for 1 h prior to the stimulation with or without leptin (100 ng·mL⁻¹) in serum-free media for additional 30 min. Western blotting analyses were conducted (A,B). Representative blots are shown from three independent experiments. Italic numbers beneath blots are fold changes in the densities of the bands compared to the control without treatment in the blot (n = 3), after normalization with the internal invariable control. Because of the limited space, standard deviations are not presented. (A) Detection of phosphorylated IRS1/2, PI3K and AKT. The total protein for each corresponding phospho-protein was used as an internal control for equal loading. (B) Evaluation of the abundance of GLUT2 and GLUT4, respectively, in the fractions of whole cells, membrane and cytoplasm of HSCs. (C) Serum-starved HSCs were pretreated with or without the PI3K/AKT inhibitor LY294002 at indicated concentrations, or with curcumin (20 µM), for 1 h prior to the stimulation with or without leptin (100 ng·mL⁻¹) in serum-free media for additional 30 min. Levels of intracellular glucose were determined. Values were expressed as pmol glucose·µg⁻¹ protein and presented as means ± SD. *P < 0.05 versus the untreated control; ‡P < 0.05 versus cells treated with leptin alone.

Figure 3

Curcumin eliminated the inhibitory effect of leptin on the activity of GK and increased intracellular G-6-P in activated HSCs in vitro. (A,B) Serum-starved HSCs were pretreated with curcumin at indicated concentrations for 1 h prior to the stimulation with or without leptin (100 ng·mL⁻¹) in serum-free media for additional 30 min. (A) Analyses of GK activities. Values were expressed as fold changes (means ± SD), compared with the untreated control (n = 3). *P < 0.05 versus the untreated control; ‡P < 0.05 versus cells treated with leptin at 100 ng·mL⁻¹ alone. (B) Analyses of levels of intracellular G-6-P. Values were expressed as pmol G-6-P·µg⁻¹ protein (means ± SD) (n = 3). *P < 0.05 versus the untreated control; ‡P < 0.05 versus cells treated with leptin at 100 ng·mL⁻¹ alone. (C) Western blotting analyses of αI(I)procollagen (procol) and α-SMA in HSCs treated with G-6-P at different concentrations as indicated in serum-depleted media for 24 h. β-Tubulin was used as an invariant control for equal loading. Representatives were presented from three independent experiments. (D) The Western blotting analyses were summarized after normalization with β-tubulin. Variations in the band density were expressed as fold changes compared to the untreated control in the blot (means ± SD., n = 3). *P < 0.05 versus the untreated control.

Figure 4

Curcumin inhibited PKA activity, which facilitated the increase in the activity of GK and the reduction in intracellular glucose in cultured HSCs. (A) Serum-starved HSCs were pretreated with curcumin at indicated concentrations for 1 h prior to the stimulation with or without leptin (100 ng·mL⁻¹) in serum-free media for additional 30 min. Levels of p-PKA were evaluated by Western blotting analyses. Total PKA was used as an invariant control for equal loading. Representative blots are presented from three independent experiments. Italic numbers beneath blots were fold changes in the densities of the bands compared to the control without treatment in the blot (n = 3), after normalization with the internal invariable control. (B,C) Serum-starved HSCs were pretreated with the PKA inhibitor H89 at indicated concentrations, or with curcumin (20 µM), in serum-free media for 1 h prior to the addition of leptin (100 ng·mL⁻¹) for additional 30 min. *P < 0.05 versus the untreated control (the second column). (B) Analyses of GK activities. Values were expressed as fold changes compared with the untreated control (means ± SD) (n = 3). (C) Determination of levels of intracellular glucose. Values were expressed as pmol glucose·µg⁻¹ protein (means ± SD) (n = 3).

Figure 5

Curcumin activated AMPK activity, which resulted in the increase in the activity of GK and in the reduction in the level of intracellular glucose in cultured HSCs. (A,B) Serum-starved HSCs were pretreated with or without curcumin (0–30 µM) for 1 h prior to the stimulation with leptin at indicated concentrations in serum-free media for additional 30 min. Levels of p-AMPK were evaluated by Western blotting analyses. Total AMPK was used as an invariant control for equal loading. Representative blots are presented from three independent experiments. Italic numbers beneath blots were fold changes in the densities of the bands compared to the control without treatment in the blot (n = 3), after normalization with total AMPK. (A) Cells were stimulated with leptin only. (B) Cells were pretreated with curcumin prior to the stimulation with leptin (100 ng·mL⁻¹). (C,D) Serum-starved HSCs were pretreated with or without curcumin at 20 µM for 1 h prior to the exposure to the AMPK inhibitor compd C at various concentrations in serum-depleted media for additional 30 min. (C) Analyses of GK activities. Values were expressed as fold changes compared with the untreated control (means ± SD) (n = 3). *P < 0.05 versus the untreated control; ‡P < 0.05 versus cells treated with curcumin alone. (D) Analyses of levels of intracellular glucose. Values were expressed as pmol glucose·µg⁻¹ protein (means ± SD) (n = 3). *P < 0.05 versus the untreated control; ‡P < 0.05 versus cells treated with curcumin alone. (E) HSCs were treated with the AMPK activator AICAR at various concentrations, or with curcumin at 20 µM, for 1.5 h. Levels of intracellular glucose were determined. Values were expressed as pmol glucose·µg⁻¹ protein (means ± SD) (n = 3). *P < 0.05 versus the untreated control (the second column).

Figure 6

The roles of curcumin in altering the levels of intracellular glucose and G-6-P in HSCs in vitro were dependent on leptin receptor. Serum-starved HSCs from natural leptin receptor-deficient Zucker (fa/fa) rats and HSCs from lean wild-type Zucker (Fa/Fa) rats were pretreated with or without curcumin (20 µM) for 1 h prior to the addition of leptin (100 ng·mL⁻¹) for additional 30 min in serum-free DMEM. Levels of intracellular glucose and G-6-P were analysed. Values were expressed as pmol glucose or G-6-P·µg⁻¹ protein, and presented as means ± SD (n = 3). *P < 0.05 versus the untreated control cells; ‡P < 0.05 versus cells treated with leptin alone. (A) Analyses of levels of intracellular glucose. (B) Analyses of levels of intracellular G-6-P.

Figure 7

A simplified model for explaining the roles of curcumin in eliminating the stimulatory effects of leptin on the activation of HSCs, focusing on the level of intracellular glucose. ‘↑’ indicates the actions of leptin, while ‘▴’ represents the effects of curcumin.