An active metabolite of oltipraz (M2) increases mitochondrial fuel oxidation and inhibits lipogenesis in the liver by dually activating AMPK

Abstract

Background and purpose: Oltipraz, a cancer chemopreventive agent, has an anti-steatotic effect via liver X receptor-α (LXRα) inhibition. Here we have assessed the biological activity of a major metabolite of oltipraz (M2) against liver steatosis and steatohepatitis and the underlying mechanism(s).

Experimental approach: Blood biochemistry and histopathology were assessed in high-fat diet (HFD)-fed mice treated with M2. An in vitroHepG2 cell model was used to study the mechanism of action. Immunoblotting, real-time PCR and luciferase reporter assays were performed to measure target protein or gene expression levels.

Key results: M2 treatment inhibited HFD-induced steatohepatitis and diminished oxidative stress in liver. It increased expression of genes encoding proteins involved in mitochondrial fuel oxidation. Mitochondrial DNA content and oxygen consumption rate were enhanced. Moreover, M2 treatment repressed activity of LXRα and induction of its target genes, indicating anti-lipogenic effects. M2 activated AMP-activated protein kinase (AMPK). Inhibition of AMPK by over-expression of dominant negative AMPK (DN-AMPK) or by Compound C prevented M2 from inducing genes for fatty acid oxidation and repressed sterol regulatory element binding protein-1c (SREBP-1c) expression. M2 activated liver kinase B1 (LKB1) and increased the AMP/ATP ratio. LKB1 knockdown failed to reverse target protein modulations or AMPK activation by M2, supporting the proposal that both LKB1 and increased AMP/ATP ratio contribute to its anti-steatotic effect.

Conclusion and implications: M2 inhibited liver steatosis and steatohepatitis by enhancing mitochondrial fuel oxidation and inhibiting lipogenesis. These effects reflected activation of AMPK elicited by increases in LKB1 activity and AMP/ATP ratio.

Figure 1

M2 inhibition of hepatic fat accumulation and body weight gain. (A) Oil Red O staining. Male C57BL/6 mice were fed on either a normal diet (ND) or high-fat diet (HFD) for 8 weeks. M2 (10 or 30 mg·kg⁻¹·day⁻¹) was orally administered to mice four times per week during the last 4 weeks of the diet feeding. Control animals received vehicle only. The representative pictures show Oil Red O staining of the liver sections (magnification 100×). (B) Hepatic TG contents (n = 5–9 animals per group). (C) Body weight gains. At the end of HFD feeding, mice were fasted overnight to measure fasting plasma glucose levels. Fasting caused decreases in body weight at the eighth week. (D) Plasma TG, total cholesterol and fasting glucose contents. For B, C and D, **P < 0.01, significantly different from ND alone; ^#P < 0.05, ^##P < 0.01, significantly different from HFD alone. For C and D, n = 7–12 animals per group.

Figure 2

Inhibition of liver injury, inflammation and oxidative stress by M2. (A) H&E staining of the liver sections with lower (100×) and higher (400×) magnifications. Animals were treated as described in the legend of Figure 1A (M2, 10 or 30 mg·kg⁻¹·day⁻¹). In A, with higher magnification, Vs represent lipid droplet formation; white arrowheads indicate fatty degeneration of hepatocyte, whereas the black arrowhead denotes infiltration of inflammatory cells. (B) Plasma ALT activity (n = 7–12 animals per group). (C) qRT-PCR assays. The levels of CD68, TNFα and iNOS mRNAs were measured in liver tissue. The levels of GAPDH mRNA were used as a reference for data normalization. (D) Immunoblotting for iNOS and nitrotyrosinylated proteins in the liver homogenates. (E) Hepatic GSH contents. Reduced GSH levels were measured on the liver homogenates. For B–E, *P < 0.05, **P < 0.01, significantly different from ND alone; ^#P < 0.05, ^##P < 0.01, significantly different from HFD alone. For C–E, n = 3–11 animals per group.

Figure 3

The effects of M2 on the expression of proteins associated with mitochondrial fuel oxidation and oxygen consumption. (A) qRT-PCR assays for CPT-1, PPARα and PGC-1α mRNA. HepG2 cells were treated with 30 μM M2 for the indicated times. (B) Time-course effects of M2 on CPT-1, PPARα and PGC-1α expression. Proteins of interest were immunoblotted in lysates of HepG2 cells treated with 30 μM M2 for the indicated times. (C) Concentration-dependent increases in CPT-1, PPARα and PGC-1α levels. HepG2 cells were treated with M2 for 12 h. For A–C, *P < 0.05,**P < 0.01, significantly different from vehicle. (D) Immunoblottings for CPT-1 in the liver homogenates. Mice were treated as described in the legend of Figure 1A. **P < 0.01, significantly different from ND alone; ^##P < 0.01, significantly different from HFD alone. (n = 6–9 animals per group) (NS, not significant). (E) qRT-PCR assays for mtDNA. Total DNA was isolated from the liver tissue of mice fed a HFD with or without M2 treatment. For cell-based assays, HepG2 cells were treated with M2 for 6 h. The samples were subjected to qRT-PCR assays using primers for the mtDNA region COX II. Nuclear DNA-encoded gene RIP140 was amplified as a reference for data normalization. **P < 0.01, significantly different from HFD alone (left; n = 4–8 animals per group) or between M2 treatment and control (right). (F) Mitochondrial oxygen consumption rate in the liver. Mitochondria were isolated from the liver of mice fed HFD with or without M2. **P < 0.01, significantly different from ND alone; ^#P < 0.05, significantly different from HFD alone.(n = 3). (G) Oxygen consumption rate in HepG2 cells. HepG2 cells were treated with M2 alone (left), or in combination with CCCP (10 μM) for 12 h (right). *P < 0.05, **P < 0.01, significantly different from vehicle; ^#P < 0.05, significantly different from M2 alone.

Figure 4

The anti-lipogenic effect of M2. (A) LXRE reporter activity and qRT-PCR assays for LXRα mRNA. After LXRE transfection, HepG2 cells were treated with vehicle or M2 for 1 h, and continuously incubated with 10 μM T090 for 12 h. LXRα mRNA levels were measured in HepG2 cells similarly treated without transfection. (B) qRT-PCR assays for lipogenic gene transcripts. HepG2 cells were treated with vehicle or M2 for 1 h, and continuously incubated with 10 μM T090 for 12 h. (C) Immunoblotting for SREBP-1c in HepG2 cells. Premature and mature forms of SREBP-1c were immunoblotted in whole cell lysates or nuclear fraction of HepG2 cells treated as described in the legend of Figure 4A. WCL, whole cell lysate; NF, nuclear fraction. (D) Immunoblotting for SREBP-1c in HepG2 cells. HepG2 cells were treated with vehicle, M2, GW3965 (10 μM), or M2 + GW3965. For (A)–(D), **P < 0.01, significantly different from vehicle; ^#P < 0.05, ^##P < 0.01, significantly different from T090/GW3965 alone. (E) Relative intracellular TG levels. TG levels were measured in HepG2 cells treated with 0.5 mM palmitate or palmitate + M2 for 24 h. **P < 0.01, significantly different from BSA; ^##P < 0.01, significantly different from palmitate alone. (F) qRT-PCR assays for lipogenic gene transcripts in the liver. **P < 0.01, significantly different from ND alone; ^#P < 0.05, significantly different from HFD alone (n = 3–8 animals per group). (G) Immunoblotting for LXRα or SREBP-1c in the liver homogenates.

Figure 5

The role of AMPK activation by M2 in the induction of proteins associated with fuel oxidation or in the inhibition of lipogenesis. (A) The activation of AMPK by M2. Phosphorylated and total forms of AMPK or ACC were immunoblotted in lysates of HepG2 cells treated with M2 for the indicated times. *P < 0.05, **P < 0.01, significantly different from vehicle. (B) The effect of Compound C treatment on lipogenic gene transcript levels. qRT-PCR assays were done on HepG2 cells treated with 3 μM Compound C for 1 h and continuously incubated M2 for 12 h. *P < 0.05, **P < 0.01, significantly different from vehicle; ^#P < 0.05, ^##P < 0.01, significantly different from M2 alone. (C) The effect of AMPK inhibition on SREBP-1c repression by M2. After DN-AMPK transfection (24 h) or Compound C treatment (1 h), HepG2 cells were treated with vehicle or M2 for 1 h, and continuously incubated with 10 μM T090 for 12 h. SREBP-1c was immunoblotted on the whole cell lysates. *P < 0.05, **P < 0.01, significantly different from vehicle; ^#P < 0.05, significantly different from T090 alone. NS, not significant.

Figure 6

LKB1 activation and increased AMP/ATP ratio induced by M2. (A) The activation of LKB1 by M2. Phosphorylated or total LKB1 was immunoblotted in lysates of HepG2 cells treated with M2 for the indicated times. *P < 0.05, **P < 0.01, significantly different from vehicle. (B) The effect of LKB1 knockdown on CPT-1, PPARα and PGC-1α induction by M2. HepG2 cells were treated with M2 for 12 h following control knockdown (siCon) or LKB1 knockdown (siLKB1). (C) The effect of LKB1 knockdown on SREBP-1c repression by M2. Cells were treated with vehicle or M2 for 1 h, and continuously incubated with 10 μM T090 for 12 h following LKB1 knockdown. *P < 0.05, **P < 0.01, significantly different from vehicle; ^#P < 0.05, significantly different from T090 alone. WCL, whole cell lysate. (D) The effect of LKB1 knockdown on AMPK activation by M2. For B and D, *P < 0.05, **P < 0.01, significantly different from vehicle. (E) Increase in cellular AMP/ATP ratio by M2. AMP and ATP contents were measured using HPLC assays in HepG2 cells treated with M2 for 1 h. **P < 0.01, M2 treatment significantly different from control.

Figure 7

A schematic diagram illustrating the proposed mechanism by which M2 inhibits liver steatosis and steatohepatitis. Inhibition of liver steatosis and steatohepatitis by M2 may result from the activation of AMPK elicited by increases in LKB1 activity and the AMP/ATP ratio.