Xanthine-based KMUP-1 improves HDL via PPARγ/SR-B1, LDL via LDLRs, and HSL via PKA/PKG for hepatic fat loss

Abstract

The phosphodiesterase inhibitor (PDEI)/eNOS enhancer KMUP-1, targeting G-protein coupled receptors (GPCRs), improves dyslipidemia. We compared its lipid-lowering effects with simvastatin and explored hormone-sensitive lipase (HSL) translocation in hepatic fat loss. KMUP-1 HCl (1, 2.5, and 5 mg/kg/day) and simvastatin (5 mg/kg/day) were administered in C57BL/6J male mice fed a high-fat diet (HFD) by gavage for 8 weeks. KMUP-1 inhibited HFD-induced plasma/liver TG, total cholesterol, and LDL; increased HDL/3-hydroxy-3-methylglutaryl-CoA reductase (HMGR)/Rho kinase II (ROCK II)/PPARγ/ABCA1; and decreased liver and body weight. KMUP-1 HCl in drinking water (2.5 mg/200 ml tap water) for 1-14 or 8-14 weeks decreased HFD-induced liver and body weight and scavenger receptor class B type I expression and increased protein kinase A (PKA)/PKG/LDLRs/HSL expression and immunoreactivity. In HepG2 cells incubated with serum or exogenous mevalonate, KMUP-1 (10(-7)∼10(-5) M) reversed HMGR expression by feedback regulation, colocalized expression of ABCA1/apolipoprotein A-I/LXRα/PPARγ, and reduced exogenous geranylgeranyl pyrophosphate/farnesyl pyrophosphate (FPP)-induced RhoA/ROCK II expression. A guanosine 3',5'-cyclic monophosphate (cGMP) antagonist reversed KMUP-1-induced ROCK II reduction, indicating cGMP/eNOS involvement. KMUP-1 inceased PKG and LDLRs surrounded by LDL and restored oxidized LDL-induced PKA expresion. Unlike simvastatin, KMUP-1 could not inhibit (14)C mevalonate formation. KMUP-1 could, but simvastatin could not, decrease ROCK II expression by exogenous FPP/CGPP. KMUP-1 improves HDL via PPARγ/LXRα/ABCA1/Apo-I expression and increases LDLRs/PKA/PKG/HSL expression and immunoreactivity, leading to TG hydrolysis to lower hepatic fat and body weight.

Keywords: 3-hydroxy-3-methylglutaryl-CoA reductase; endothelial nitric-oxide synthase/RhoA/Rho kinase II; low density lipoprotein/high density lipoprotein; protein kinase A/G; scavenger receptor class B type I/peroxisome proliferator activated receptor γ.

Fig. 1.

Effects of KMUP-1 on morphology and expression of SR-B1 and PKG/PKA in mouse livers and body weight changes accompanying a HFD. KMUP-1 HCl (2.5 mg/200 ml tap water) was drunk for 14 weeks or the last 6 weeks of a 14-week experiment and body (A) and liver (B) weights were measured. Protein expression of SR-B1 (C), PKA (D), and PKG (E) were shown by Western blotting. Arrow indicates the KMUP-1 time point at week 8. ^##P < 0.01 versus STD group; *P < 0.05, **P < 0.01 versus vehicle group (n = 6 per group).

Fig. 2.

Effects of KMUP-1 on HMGR/ROCK II/PPARγ/ABCA1 expression in mouse livers. Oral KMUP-1 (1, 2.5, and 5 mg/kg) administrated for 8 weeks by gavage reversed the downregulation of HMGR expression (A), increased PPARγ (C), and ABCA1 (D), and inactivated ROCK II (B) in the livers of mice treated with HFD for 8 weeks. ^##P < 0.01 versus STD group; *P < 0.05, **P < 0.01 versus vehicle group (n = 6 per group).

Fig. 3.

Effects of KMUP-1 and simvastatin on HMGR expression in the presence of mevalonate/serum. HepG2 cells were incubated with KMUP-1 (10⁻⁹–10⁻⁵ M) (A) or simvastatin (10⁻⁹–10⁻⁵ M) (B) and mevalonate (20–100 μM) (C) for 24 h. In addition, cells were preincubated for 1 h with mevalonate (100 μM) and then treated with KMUP-1 (10⁻⁵ M) or simvastatin (10⁻⁵ M) (D). HMGR expression was determined as described in Materials and Methods. Equal quantities of protein (20 μg) were run in each lane. Data are means ± SE of three independent experiments and expressed as relative value to control. ^##P < 0.01 versus mevalonate group; *P < 0.05, **P < 0.01 versus control or mevalonate group (n = 3 per group).

Fig. 4.

Effects of KMUP-1 and simvastatin on RhoA/ROCK II expression. HepG2 cells were incubated with KMUP-1 (10⁻⁹–10⁻⁵ M) (A, B, D) or simvastatin (10⁻⁹–10⁻⁵ M) (C) for 24 h. RhoA translocation from membrane to cytosol and ROCK II expression were determined as described in Materials and Methods. Data are means ± SE of three independent experiments and expressed as relative value to control. *P < 0.05, **P < 0.01 versus control group (n = 3 per group).

Fig. 5.

Effects of KMUP-1 and simvastatin on PPARγ and ABCA1/ApoA-I/LXRα expression. HepG2 cells were incubated with KMUP-1 (10⁻⁹–10⁻⁵ M) (A, C) or simvastatin (10⁻⁹–10⁻⁵ M) (B, D) for 24 h. PPARγ (A, B) and ABCA1(C, D) expression was determined as described in Materials and Methods. Data are means ± SE of three independent experiments and expressed as relative value to control. *P < 0.05, **P < 0.01 versus control group (n = 3 per group).

Fig. 6.

Effects of KMUP-1 and simvastatin on expression of ApoA-I and LXRα. HepG2 cells were incubated with KMUP-1 (10⁻⁹–10⁻⁵ M) (A, C) or simvastatin (10⁻⁹–10⁻⁵ M) (B) for 24 h. ApoA-I (A, B) and LXRα (C) expression was determined as described in Materials and Methods. Data are means ± SE of three independent experiments and expressed as relative value to control. *P < 0.05, **P < 0.01 versus control (n = 3 per group).

Fig. 7.

Effects of RhoA antagonist, ROCK inhibitor and isoprenoids on KMUP-1-induced ROCK II, PPARγ, and ABCA1 expression. HepG2 cells were incubated with KMUP-1 (10⁻⁵ M) or simvastatin (10⁻⁵ M) in the presence of RhoA antagonist C3 exoenzyme (5 ng/ml) and ROCK inhibitor Y27632 (10⁻⁵ M) for 24 h. ROCK II (A, B), PPARγ (C), and ABCA1 (D) expression was determined as described in Materials and Methods. Data are means ± SE of three independent experiments and expressed as relative value to control. ^#P < 0.05 versus vehicle group; *P < 0.05, **P < 0.01 versus control (n = 3 per group).

Fig. 8.

Effects of isoprenoids alone and with KMUP-1 or simvastatin on RhoA/ROCK II expression. HepG2 cells were incubated with GGPP (10 μM) or FPP (10 μM) alone and with GGPP or FPP with KMUP-1 (10⁻⁹–10⁻⁵ M) (A, C, D) and simvastatin (10⁻⁹–10⁻⁵ M) (B) for 24 h. RhoA translocation (A) and ROCK II expression (B, C, D) was determined as described in Materials and Methods. Data are means ± SE of three independent experiments and expressed as relative value to control. ^#P < 0.05 versus control group; ^##P < 0.01 versus control group; *P < 0.05, **P < 0.01 versus GGPP or FPP control group (n = 3 per group).

Fig. 9.

Effects of isoprenoids alone or with KMUP-1 on PPARγ and ABCA1expression. HepG2 cells were incubated with GGPP (10 μM) or FPP (10 μM) alone and with GGPP or FPP with KMUP-1 (10⁻⁹–10⁻⁵ M) for 24 h. KMUP-1 increased PPARγ (A, C) and ABCA1 (B, E) in the presence of FPP or GGPP (10 μM). However, simvastatin (10⁻⁹–10⁻⁵ M) could not increase PPARγ (D) in the presence of GGPP (10 μM), determined as described in Materials and Methods. Data are means ± SE of three independent experiments and expressed as relative value to control. ^#P < 0.05 versus control group; ^#P < 0.01 versus control group; *P < 0.05, **P < 0.01 versus GGPP or FPP group (n = 3 per group).

Fig. 10.

Effects of KMUP-1 on hepatic IHC of LDLRs and expresson of LDLRs/PKG/PKA. The IHC response to LDL receptors (LDLRs) in livers increased in the treatment and protection groups (A). In HepG2 cells, KMUP-1 increased the expression of LDLRs in the presence of LDL (500 μg/ml). These results confirmed that drinking KMUP-1 HCl (2.5 mg/200 ml during the last 6 weeks) increase LDLRs and might remove LDL in hyperlipidemic plasma. Protein expression of PKG was also significantly changed (B). In contrast, KMUP-1 could reverse oxLDL (200 μg/ml)-induced decrease of PKA expression (C). *P < 0.05 versus control group; **P < 0.01; ***P < 0.001 versus control group (n = 3 per group).

Fig. 11.

Effect of KMUP-1 on fluorescence staining of LDLRs/PKA/HSL. LDLRs/PKA/HSL (green) staining on HepG2 cells was detected with a secondary antibody conjugated to FITC (green) overnight at 4°C, followed by merging images to detect the location of LDLRs/PKA. The antibody response to LDLRs(A)/PKA(B)/HSL(C) was increased by KMUP-1 compared with simvastatin, negative control, and caffeine or theophylline in HePG2 cells. All fluorescent staining was scanned with a Nikon Eclipse TE200-S microscope (Tokyo, Japan). The results indicated that KMUP-1, simvastatin, and caffeine/theophyllin can increase PKA(A)/HSL(B) (n = 3 per group).

Fig. 11.

Effect of KMUP-1 on fluorescence staining of LDLRs/PKA/HSL. LDLRs/PKA/HSL (green) staining on HepG2 cells was detected with a secondary antibody conjugated to FITC (green) overnight at 4°C, followed by merging images to detect the location of LDLRs/PKA. The antibody response to LDLRs(A)/PKA(B)/HSL(C) was increased by KMUP-1 compared with simvastatin, negative control, and caffeine or theophylline in HePG2 cells. All fluorescent staining was scanned with a Nikon Eclipse TE200-S microscope (Tokyo, Japan). The results indicated that KMUP-1, simvastatin, and caffeine/theophyllin can increase PKA(A)/HSL(B) (n = 3 per group).

Fig. 12.

Proposed mechanism of action of KMUP-1 on hepatic lipid metabolism. KMUP-1 displays pleiotropic effects, increasing PPARγ and inhibiting SR-B1 to improve HDL, increasing LDLRs to remove LDL and activating PKA/PKG/HSL for TG hydrolysis in HFD fatty livers.