Pak1 mediates the stimulatory effect of insulin and curcumin on hepatic ChREBP expression

Abstract

Insulin can stimulate hepatic expression of carbohydrate-responsive element-binding protein (ChREBP). As recent studies revealed potential metabolic beneficial effects of ChREBP, we asked whether its expression can also be regulated by the dietary polyphenol curcumin. We also aimed to determine mechanisms underlying ChREBP stimulation by insulin and curcumin. The effect of insulin on ChREBP expression was assessed in mouse hepatocytes, while the effect of curcumin was assessed in mouse hepatocytes and with curcumin gavage in mice. Chemical inhibitors for insulin signaling molecules were utilized to identify involved signaling molecules, and the involvement of p21-activated protein kinase 1 (Pak1) was determined with its chemical inhibitor and Pak1-/- hepatocytes. We found that both insulin and curcumin-stimulated ChREBP expression in Akt-independent but MEK/ERK-dependent manner, involving the inactivation of the transcriptional repressor Oct-1. Aged Pak1-/- mice showed reduced body fat volume. Pak1 inhibition or its genetic deletion attenuated the stimulatory effect of insulin or curcumin on ChREBP expression. Our study hence suggests the existence of a novel signaling cascade Pak1/MEK/ERK/Oct-1 for both insulin and curcumin in exerting their glucose-lowering effect via promoting hepatic ChREBP production, supports the recognition of beneficial functions of ChREBP, and brings us a new overview on dietary polyphenols.

Keywords: Akt; ChREBP; Oct-1; Pak1; curcumin; dietary polyphenol intervention; insulin.

Figure 1

Both insulin and curcumin stimulate hepatic ChREBP expression. (A) Insulin (INS) treatment increased ChREBPα protein (~95 kDa) levels in mouse primary hepatocytes. Left panel is a representative blot (n = 3), while right panel shows the densitometric quantification results. (B–D) qRT-PCR show that insulin-stimulated ChREBP expression can be blocked by the MEK/ERK inhibitor PD98059 or the PI3K inhibitor Ly294002 but not the Akt inhibitor Akti in mouse primary hepatocytes. (E–G) Hepatic ChREBP and SREBP-1c mRNA levels were increased in C57BL/6J mice received 6 day curcumin gavage, assessed by qRT-PCR. (H and I) Curcumin treatment (0.5 or 1 μM) increased ChREBP mRNA levels in mouse hepatocytes (H) and the stimulation with 0.5 μM curcumin was not observed with the incubation time less than 4 h (I). CUR, curcumin; Pd, PD98059; Ly, Ly294002. n ≥ 3 for B–I. * P < 0.05, ^#P < 0.05 vs. the corresponding control.

Figure 2

Elevated ChREBP expression in response to curcumin treatment is associated with elevated expression of its downstream targets. (A–E) Four-hour curcumin treatment (0.5–1.0 μM) increased the expression of Lpk (A), Fas (B), Acc1 (C), Scd1 (D), and Me1 (E) levels in mouse hepatocytes. n ≥ 3 for A–E. * P < 0.05, ^#P < 0.05 vs. the corresponding control.

Figure 3

The stimulation on ChREBP expression by curcumin treatment also involves Oct-1 attenuation and it is Akt-independent. (A and B) Four-hour insulin (INS) or curcumin (CUR) treatment stimulated the activity of the 1.4 kb-ChREBPα-LUC reporter when it was transfected into mouse primary hepatocytes. (C) Curcumin-stimulated ChREBP expression in mouse primary hepatocytes was blocked by 45 min pre-incubation with the MEK inhibitor PD98059 (Pd) but not the Akt inhibitor Akti. (D) Overall organization of the mouse ChREBPα gene proximal 5′ flanking region and the positions of primers utilized for ChIP and qChIP. (E and F) ChIP (E) and qChIP (F) show that curcumin attenuated Oct-1 binding to ChREBPα promoter in mouse primary hepatocytes can be blocked by MEK/ERK or PI3K inhibition but not by Akt inhibition. (G) Increased ChREBPα levels in mouse hepatocytes in response to curcumin or insulin treatment (4 h with indicated doses) were associated with reduced Oct-1 levels. n ≥ 3 for A–C and F. * P < 0.05 vs. the corresponding control. Representative blots of three independent experiments are shown in G.

Figure 4

Pak1 is among mediators of both curcumin and insulin in stimulating ChREBP expression. (A and B) Pak1 Thr423 phosphorylation in mouse hepatocytes was stimulated by curcumin (1 μM) or insulin (10 nM) at indicated time intervals. (C–E) qRT-PCR shows that in mouse primary hepatocytes, curcumin-stimulated ChREBP (total as well as α and β isoforms) expression was blocked by the Pak inhibitor IPA3. (F and G) Western blotting show the effect of IPA3 pre-treatment on insulin (F) or curcumin (G)-stimulated ChREBP expression, Oct-1 content, Pak1 as well as ERK phosphorylation. Panels F and G are representative blot of three independent experiments. n ≥ 3 for C–E, where level means without a common letter are statistically different.

Figure 5

Curcumin-stimulated ChREBP expression is attenuated in Pak1^−/− hepatocytes. (A and B) Aged Pak1^−/− mice exhibited reduced whole-body fat. MRI was performed in aged mice (32 weeks). Adipose tissue was measured and was used to calculate whole-body fat volume. Representative serial MRI scans of wild-type and Pak1^−/− mice are shown in A. WT, n = 4. KO, n = 5. Panel B shows the quantitative analysis result of Panel A. (C) Pak1^−/− hepatocytes show attenuated response to 4 h insulin (10 nM) or curcumin (1 μM) treatment on Lpk expression. (D–G) Western blotting shows the response to insulin treatment (D) on ChREBPα protein expression, as well as Akt and Erk phosphorylation in wild-type and Pak1^−/− hepatocytes. Panels E–G are densitometric analysis data of Panel D. (H–K) Western blotting show the response to curcumin treatment (H) on ChREBPα protein expression, as well as Akt and Erk phosphorylation in wild-type and Pak1^−/− hepatocytes. Densitometric analysis data of H are shown in I–K. For B, E–G, and I–K, * P < 0.05, ^#P < 0.05 vs. the correspondent control. For Panel C, level means without a common letter are statistically different.

Figure 6

A simplified diagram shows our current understanding of the role of insulin and curcumin on hepatic lipogenesis. Both insulin and curcumin can repress gluconeogenesis via activating the Akt signaling, which inactivates FoxO1 and the hepatic gluconeogenic pathway. Curcumin shares with insulin in using the IRS1/PI3K/Akt/TSC1/2/mTORC1 signaling cascade in activating SREBP-1c, although we are still unclear whether a yet to be defined receptor mediates the function of curcumin (indicated with the question mark). Importantly, both insulin and curcumin utilize the newly defined Pak1/MEK/ERK/Oct-1 signaling cascade in up-regulating ChREBPα transcription, leading to increased hepatic lipogenesis. The stimulation of ChREBPβ expression by insulin or curcumin is likely mediated by ChREBPα. The existence of possible feedback loop between Pak1 and Akt are illustrated with dotted arrows.