Hexane-Isopropanolic Extract of Tungrymbai, a North-East Indian fermented soybean food prevents hepatic steatosis via regulating AMPK-mediated SREBP/FAS/ACC/HMGCR and PPARα/CPT1A/UCP2 pathways

Abstract

This study for the first time examined the prophylactic role of Tungrymbai, a well-known fermented soybean food of North-East India, against hepatic steatosis. Treatment with hexane-isopropanolic (2:1, HIET) but not hydro-alcoholic (70% ethanol, HAET) extract dose-dependently (0.1, 0.2, or 0.3 µg/mL) reduced the intracellular lipid accumulation as shown by lower triglyceride levels and both Oil Red O and Nile Red staining in palmitate (PA, 0.75 mM)-treated hepatocytes. Immunobloting, mRNA expression, and knock-down studies demonstrated the role of AMPK-mediated SREBP/FAS/ACC/HMGCR and PPARα/CPT1A/UCP2 signaling pathways in facilitating the beneficial role of HIET against lipid accumulation in PA-treated hepatocytes. Animal studies further showed a positive effect of HIET (20 µg/kg BW, 8 weeks, daily) in regulating AMPK/SREBP/PPARα signaling pathways and reducing body weight gain, plasma lipid levels, and hepatic steatosis in high fat diet (HFD)-fed mice. Histological analyses also revealed the beneficial effect of HIET in reducing hepatic fat accumulation in HFD mice. Chemical profiling (HRMS, IR, and HPLC) demonstrated the presence of menaquinone-7 (vitamin K2) as one of the bio-active principle(s) in HIET. Combining all, this study demonstrates the positive effect of HIET on reducing hepatic steatosis via regulating AMPK/SREBP/PPARα signaling pathway.

Figure 1

Effect of the different extracts of Tungrymbai, HAET (hydro-alcoholic, 70% ethanol) and HIET (hexane-isopropanol, 2:1) on intracellular lipid accumulation in palmitate (PA)-treated hepatocytes. (A) triglyceride content; (B) levels of Oil Red O staining; (C) cell viability; (D) representative photomicrographs of Oil Red O staining (10×); (E) representative photomicrographs of Nile Red and DAPI staining (40×); and (F) quantification of Nile Red/DAPI staining. Cells were pre-treated with HAET or HIET (0.1, 0.2, or 0.3 µg/mL) for 2 h followed by PA (0.75 mM) exposure for the next 20 h. Data are expressed as mean ± SE (n = 6). “*” Denotes the significant difference from untreated (*p < 0.05) and “#” denotes the significant difference from PA-treated groups (^#p < 0.05).

Figure 2

Effect of the hexane-isopropanolic extract (HIET) of Tungrymbai on the protein and mRNA expressions of different signaling molecules involved in lipid metabolism in palmitate (PA)-treated hepatocytes. (A) Phospho AMPK/AMPK protein expression; (B) SREBP1 protein expression; (C) SREBF1 mRNA; (D) SREBP2 protein expression; (E) SREBF2 mRNA; (F) FAS protein expression; (G) FAS mRNA; (H) phospho ACC/ACC protein expression; (I) ACACA mRNA; (J) phospho HMGCR/HMGCR protein expression; (K) HMGCR mRNA; (L) PPARα protein expression; (M) PPARA mRNA; (N) CPT1A protein expression; (O) CPT1A mRNA; (P) UCP2 protein expression; and (Q) UCP2 mRNA. The blots are representatives of three independent experiments. Full-length blots are presented in Supplementary Figure 2. Cells were pre-treated with HIET (0.1, 0.2, or 0.3 µg/mL) for 2 h followed by PA (0.75 mM) exposure for the next 20 h. Data are expressed as mean ± SE. “*” Denotes the significant difference from untreated (*p < 0.05) and “#” denotes the significant difference from PA-treated groups (^#p < 0.05).

Figure 3

Effect of the hexane-isopropanolic extract of Tungrymbai, HIET on the protein expressions of different signaling molecules involved in lipid metabolism in palmitate (PA)-treated hepatocytes after transfection with either AMPK or control siRNA (100 nM). (A) AMPK; (B) SREBP1; (C) SREBP2; (D) FAS; (E) phospho ACC/ACC; (F) phospho HMGCR/HMGCR; (G) PPARα; (H) CPT1A; and (I) UCP2 protein expression in AMPK siRNA transfected cells. (J) Phospho AMPK/AMPK, phospho ACC/ACC, phospho HMGCR/HMGCR, and UCP2 protein expression in control siRNA transfected cells. The blots are representatives of three independent experiments. Full-length blots are presented in Supplementary Figure 3. Cells transfected with either AMPK or control siRNA were treated with HIET (0.3 µg/mL) for 2 h followed by PA (0.75 mM) exposure for the next 20 h. Control siRNA is a scrambled nonspecific RNA duplex that shares no sequence homology with any of the genes. Data are expressed as mean ± SE. “*” Denotes the significant difference from untreated (*p < 0.05).

Figure 4

Schematic diagram of animal experiment and the effect of the hexane-isopropanolic extract of Tungrymbai, HIET on liver histology and the protein expressions of different signaling molecules in normal and high fat diet (HFD)-fed mice. Upper left panel represents the schematic diagram of animal experiment. Upper right panel represents the liver histology (40×) (arrows indicate lipid droplets). Lower panel represents the immunoblotting studies showing the protein expression of different signaling molecules including phospho AMPK/AMPK (A) SREBP1 (B) FAS (C) phospho ACC/ACC (D) phospho HMGCR/HMGCR (E) PPARα (F) CPT1A (G) and UCP2 (H). The blots are representatives of three independent experiments. Full-length blots are presented in Supplementary Figure 4. Normal mice were gavaged with HIET at a dose of 20 µg/kg BW (HIET) and HFD-mice were gavaged with HIET at a dose of 20 µg/kg BW (HFD + HIET) daily for 8 wks, each group consisting of five mice. Data are expressed as mean ± SE. “*” Denotes the significant difference from Normal (*p < 0.05) and “#” denotes the significant difference from HFD (^#p < 0.05).

Figure 5

Chemical profiling of HIET and schematic diagram of proposed mechanism underlying the beneficial role of HIET against hepatic steatosis. (A) HRMS; (B) IR; (C) HPLC; and (D) proposed mechanism.