Ginsenoside Rb1 and compound K improve insulin signaling and inhibit ER stress-associated NLRP3 inflammasome activation in adipose tissue

Abstract

Background: This study was designed to investigate whether ginsenoside Rb1 (Rb1) and compound K (CK) ameliorated insulin resistance by suppressing endoplasmic reticulum (ER) stress-induced inflammation in adipose tissue.

Methods: To induce ER stress, epididymal adipose tissue from mice or differentiated 3T3 adipocytes were exposed to high glucose. The effects of Rb1 and CK on reactive oxygen species production, ER stress, TXNIP/NLRP3 inflammasome activation, inflammation, insulin signaling activation, and glucose uptake were detected by western blot, emzyme-linked immunosorbent assay, or fluorometry.

Results: Rb1 and CK suppressed ER stress by dephosphorylation of IRE1α and PERK, thereby reducing TXNIP-associated NLRP3 inflammasome activation in adipose tissue. As a result, Rb1 and CK inhibited IL-1β maturation and downstream inflammatory factor IL-6 secretion. Inflammatory molecules induced insulin resistance by upregulating phosphorylation of insulin receptor substrate-1 at serine residues and impairing insulin PI3K/Akt signaling, leading to decreased glucose uptake by adipocytes. Rb1 and CK reversed these changes by inhibiting ER stress-induced inflammation and ameliorating insulin resistance, thereby improving the insulin IRS-1/PI3K/Akt-signaling pathway in adipose tissue.

Conclusion: Rb1 and CK inhibited inflammation and improved insulin signaling in adipose tissue by suppressing ER stress-associated NLRP3 inflammation activation. These findings offered novel insight into the mechanism by which Rb1 and CK ameliorate insulin resistance in adipose tissue.

Keywords: NLRP3 inflammasome; compound k; endoplasmic reticulum stress; ginsenoside Rb1; insulin resistance.

Fig. 1

Structures of ginsenoside Rb1 and compound K. Ginsenoside CK is generated from ginsenoside Rb1 by eliminating the C-20 and two C-3 sugar chains following hydrolysis by intestinal bacteria after oral administration. CK, ginsenoside compound K.

Fig. 2

Rb1 and CK inhibit ROS production in adipose tissue. Epididymal adipose tissue was separated from mice, cultured with Rb1 (10μM), CK (10μM), or TUDCA (200μM), and then stimulated with high glucose (33mM) for 24 h. ROS production in the tissue was determined with an ELISA kit. Data were expression as mean ± SD from three independent experiments. *p < 0.05 versus high glucose-only treatment. CK, ginsenoside compound K; ROS, reactive oxygen species, SD, standard deviation.

Fig. 3

Rb1 and CK inhibit ER stress in adipose tissue. Mice were sacrificed by cervical dislocation, and epididymal adipose tissue was separated. The tissue was pretreated with Rb1 (10μM), CK (10μM), or TUDCA (200μM), and then cultured with high glucose (33mM) for 24 h. IRE1α (A) and PERK (B) phosphorylation was determined by western blot. The results were expressed as the mean ± SD of three independent experiments. *p < 0.05 versus high glucose-only treatment. CK, ginsenoside compound K; ER, endoplasmic reticulum; SD, standard deviation; TUDCA, tauroursodeoxycholic acid.

Fig. 4

Rb1 and CK attenuate TXNIP expression in adipose tissue. Epididymal adipose tissue was separated after the mice were sacrificed. The tissue was pretreated with Rb1, CK, or TUDCA at given the concentrations, and exposed to high glucose (33mM) for 24 h. The expression of TXNIP was determined by western blot. Data were expressed as mean ± SD from three independent experiments. *p < 0.05 versus high glucose-only treatment. CK, ginsenoside compound K; SD, standard deviation; TUDCA, tauroursodeoxycholic acid.

Fig. 5

NLRP3 inflammasome activation in adipose tissue was prevented by Rb1 and CK. Epididymal adipose tissue was pretreated with Rb1 (10μM), CK (10μM), or TUDCA (200μM), and then stimulated with high glucose (33mM) for 24 h. NLRP3 (A) expression and cleaved caspase-1 (B) protein levels were determined by western blot. The results were expressed as the mean ± SD of three independent experiments. *p < 0.05 versus high glucose-only treatment. CK, ginsenoside compound K; SD, standard deviation; TUDCA, tauroursodeoxycholic acid.

Fig. 6

Rb1 and CK inhibit inflammation in adipose tissue. Epididymal adipose tissue was separated and stimulated with glucose in the presence of Rb1, CK, or TUDCA. IL-1β (A) and IL-6 (B) in the supernatant were measured with ELISA kits. The results were expressed as the mean ± SD (n = 4). *p < 0.05 versus high glucose-only treatment. CK, ginsenoside compound K; ELISA, enzyme-linked immunosorbent assay; SD, standard deviation; TUDCA, tauroursodeoxycholic acid.

Fig. 7

Rb1 and CK modulate IRS-1 phosphorylation in the presence of high glucose concentrations. Epididymal adipose tissue was separated after the mice were scarified. The tissue was pretreated with Rb1, CK, or TUDCA at given concentrations, followed by stimulation with high glucose (33mM) for 24 h with or without insulin treatment for an additional 30 min. (A,B) Serine phosphorylation of IRS-1 (S307) and tyrosine phosphorylation IRS-1 (PY99), respectively, were determined by western blot. (C) The level of PI3K in the supernatant of lysed tissue was assayed with an ELISA kit. All results were derived from three independent experiments for the western blot or expressed as the mean ± SD (n = 4) for ELISA. *p < 0.05 versus high glucose-only treatment; **p < 0.05 versus the indicated treatment. CK, ginsenoside compound K; ELISA, enzyme-linked immunosorbent assay; SD, standard deviation; TUDCA, tauroursodeoxycholic acid.

Fig. 8

Rb1 and CK enhance Akt phosphorylation and promote insulin-mediated glucose uptake in 3T3-L1 adipocytes. (A) Epididymal adipose tissue was separated and treated with Rb1, CK, or TUDCA, and cultured with high glucose concentrations with or without insulin treatment. Akt phosphorylation was detected by western blot. (B) After treatment with Rb1 and CK, 3T3-L1 adipocytes were incubated with 2-NDBG (0.5mM) and insulin (0.1μM), and fluorescence in cells was viewed by fluorescence microscope. Bar, 1000 μm. The results were expressed as the mean ± SD of three independent experiments. *p < 0.05 versus high glucose-only treatment; ** p < 0.05 versus the indicated treatment. CK, ginsenoside compound K; SD, standard deviation; TUDCA, tauroursodeoxycholic acid.

Fig. 9

The combination of Rb1 and CK improved insulin signaling. Epididymal adipose tissue was separated from mice, cultured with Rb1 (10μM), CK (10μM), Rb1 (5μM)+CK (5μM), DPI (1μM), or anti-IL-1β antibody (0.06 μg/mL), and then stimulated with TG (1μM) with or without insulin treatment. (A) ROS production in the tissue was determined by ROS-specific fluorescent-probe dye DCFH-DA by a microplate reader. (B) IL-1β in the supernatant was measured with an ELISA kit. (C) Akt phosphorylation was detected by western blot. Data were expressed as the mean ± SD from three independent experiments or as the mean ± SD (n = 4) for ELISA. *p < 0.05 versus TG-only treatment; ** p < 0.05 versus the indicated treatment. CK, ginsenoside compound K; DCFH-DA, dichloro-dihydro-fluorescein diacetate; DPI, diphenyleneiodonium chloride; ELISA, enzyme-linked immunosorbent assay; ROS, reactive oxygen species; SD, standard deviation; TUDCA, tauroursodeoxycholic acid.