Protective Effects of Gomisin N against Hepatic Cannabinoid Type 1 Receptor-Induced Insulin Resistance and Gluconeogenesis

Abstract

Activation of the hepatic cannabinoid type 1 receptor (CB1R) induces insulin resistance and gluconeogenesis via endoplasmic reticulum (ER) stress, thereby contributing to hyperglycemia. Gomisin N (GN) is a phytochemical derived from Schisandra chinensis. In the current study, we investigated the inhibitory effects of GN on hepatic CB1R-mediated insulin resistance and gluconeogenesis in 2-arachidonoylglycerol (AG; an agonist of CB1R)-treated HepG2 cells and in high-fat diet (HFD)-induced obese mice. Treatment with 2-AG induced the expression of ER stress markers, serine/threonine phosphatase PHLPP1, Lipin1, and ceramide synthesis genes, but reduced the expression of ceramide degradation genes in HepG2 cells. However, GN reversed 2-AG-mediated effects and improved the 2-AG-mediated impairment of insulin signaling. Furthermore, GN inhibited 2-AG-induced intracellular triglyceride accumulation and glucose production in HepG2 cells by downregulation of lipogenesis and gluconeogenesis genes, respectively. In vivo, GN administration to HFD obese mice reduced the HFD-induced increase in fasting blood glucose and insulin levels, which was accompanied with downregulation of HFD-induced expression of CB1R, ER stress markers, ceramide synthesis gene, and gluconeogenesis genes in the livers of HFD obese mice. These findings demonstrate that GN protects against hepatic CB1-mediated impairment of insulin signaling and gluconeogenesis, thereby contributing to the amelioration of hyperglycemia.

Keywords: cannabinoid type 1 receptor; endoplasmic reticulum stress; gluconeogenesis; gomisin N; insulin resistance; lipogenesis.

Figure 1

Gomisin N (GN) inhibited 2-AG-induced endoplasmic reticulum (ER) stress in HepG2 cells. HepG2 cells were incubated with 2-AG in the absence or presence of GN (50 or 100 µM) for 12 h. (A) qPCR analysis of GRP78, CHOP, and XBP1c; (B) qPCR analysis of CB1R; (C) Western blot analysis of GRP78, CHOP, and XBP1c; (D) HepG2 cells were incubated with tunicamycin (Tu) in the absence or presence of GN (50 or 100 µM) for 12 h. qPCR analysis of GRP78, CHOP, and XBP1c. Values are expressed as mean ± SEM (n = 3 independent experiments). ^## p < 0.01 vs. untreated control. * p < 0.05, ** p < 0.01 vs. 2-AG or tunicamycin-treated control.

Figure 2

GN reversed 2-AG-mediated expression of insulin resistance-associated genes in HepG2 cells. HepG2 cells were incubated with 2-AG in the absence or presence of GN (50 or 100 µM) for 12 h. qPCR analysis of PHLPP1 (A), CREBH (B), Lipin1 (C), Cer6 and SPTLC3 (D), and Asah1 and SPK1 (E). Values are expressed as mean ± SEM (n = 3 independent experiments). ^# p < 0.05, ^## p < 0.01 vs. untreated control. * p < 0.05 vs. 2-AG-treated control.

Figure 3

GN improved 2-AG-mediated inhibition of insulin signaling in HepG2 cells. (A) HepG2 cells were incubated with 2-AG in the absence or presence of GN (50 or 100 µM) for 3, 12 and 24 h. Serine-307 phosphorylation of IRS1 was detected by western blot analysis; (B) HepG2 cells were incubated with 2-AG in the absence or presence of GN (50 or 100 µM) for 12 h, and then incubated with insulin (10 nM) for 30 min. The phosphorylation of IRS1 (tyrosine-895) and AKT (serine-473) was detected by western blot analysis.

Figure 4

GN inhibited 2-AG-induced lipogenesis in HepG2 cells. HepG2 cells were incubated with 2-AG in the absence or presence of GN (50 or 100 µM) for 24 or 48 h. (A) qPCR and western blot analysis of SREBP1c; (B) qPCR analysis of FSA, SCD1, and ACC; (C) Intracellular TG levels were measured by TG measurement and ORO staining. Values are expressed as mean ± SEM (n = 3 independent experiments). ^# p < 0.05, ^## p < 0.01 vs. untreated control. * p < 0.05, ** p < 0.01 vs. 2-AG-treated control.

Figure 5

GN inhibited 2-AG-induced gluconeogenesis in HepG2 cells. (A) HepG2 cells were incubated with 2-AG in the absence or presence of GN (50 or 100 µM) for 12 h. qPCR analysis of PEPCK and G6Pase; (B) Measurement of glucose production; (C) HepG2 cells were incubated with tunicamycin (Tu) in the absence or presence of GN (50 or 100 µM) for 12 h. qPCR analysis of CREBH, PEPCK, and G6Pase. Values are expressed as mean ± SEM (n = 3 independent experiments). ^# p < 0.05, ^## p < 0.01 vs. untreated control. * p < 0.05, ** p < 0.01 vs. 2-AG or Tu-treated control.

Figure 6

GN ameliorated high-fat diet (HFD)-induced hyperglycemia through inhibition of hepatic CB1R signaling. C57BL6 mice were fed HFD for 12 weeks and orally administered GN for 6 weeks. (A) Fasting levels of glucose and insulin. qPCR analysis of CB1R (B), GRP78, CHOP, and XBP1c (C), PHLPP1 (D), Lipin1 (E), CerS6 (F), and PEPCK and G6Pase (G). The values are expressed as mean ± SEM (n = 5 mice per group). ^# p < 0.05, ^## p < 0.01 vs. ND mice. * p < 0.05, ** p < 0.01 vs. HFD-induced obese mice control. ND; normal diet.

Figure 7

GN reversed HFD-mediated inhibition of insulin signaling in the liver of HFD obese mice. C57BL6 mice were fed HFD for 12 weeks and orally administered GN for 6 weeks. The liver homogenates were subjected to western blot using indicated antibody. (A) Representative western blotting. (B) Densitometric results. The values are expressed as mean ± SEM (n = 5 mice per group). ^# p < 0.05 vs. ND mice. * p < 0.05 vs. HFD-induced obese mice control. ND; normal diet.