Coronarin A modulated hepatic glycogen synthesis and gluconeogenesis via inhibiting mTORC1/S6K1 signaling and ameliorated glucose homeostasis of diabetic mice

Abstract

Promotion of hepatic glycogen synthesis and inhibition of hepatic glucose production are effective strategies for controlling hyperglycemia in type 2 diabetes mellitus (T2DM), but agents with both properties were limited. Herein we report coronarin A, a natural compound isolated from rhizomes of Hedychium gardnerianum, which simultaneously stimulates glycogen synthesis and suppresses gluconeogenesis in rat primary hepatocytes. We showed that coronarin A (3, 10 μM) dose-dependently stimulated glycogen synthesis accompanied by increased Akt and GSK3β phosphorylation in rat primary hepatocytes. Pretreatment with Akt inhibitor MK-2206 (2 μM) or PI3K inhibitor LY294002 (10 μM) blocked coronarin A-induced glycogen synthesis. Meanwhile, coronarin A (10 μM) significantly suppressed gluconeogenesis accompanied by increased phosphorylation of MEK, ERK1/2, β-catenin and increased the gene expression of TCF7L2 in rat primary hepatocytes. Pretreatment with β-catenin inhibitor IWR-1-endo (10 μM) or ERK inhibitor SCH772984 (1 μM) abolished the coronarin A-suppressed gluconeogenesis. More importantly, we revealed that coronarin A activated PI3K/Akt/GSK3β and ERK/Wnt/β-catenin signaling via regulation of a key upstream molecule IRS1. Coronarin A (10, 30 μM) decreased the phosphorylation of mTOR and S6K1, the downstream target of mTORC1, which further inhibited the serine phosphorylation of IRS1, and subsequently increased the tyrosine phosphorylation of IRS1. In type 2 diabetic ob/ob mice, chronic administration of coronarin A significantly reduced the non-fasting and fasting blood glucose levels and improved glucose tolerance, accompanied by the inhibited hepatic mTOR/S6K1 signaling and activated IRS1 along with enhanced PI3K/Akt/GSK3β and ERK/Wnt/β-catenin pathways. These results demonstrate the anti-hyperglycemic effect of coronarin A with a novel mechanism by inhibiting mTORC1/S6K1 to increase IRS1 activity, and highlighted coronarin A as a valuable lead compound for the treatment of T2DM.

Keywords: IRS1; coronarin A; gluconeogenesis; glycogen synthesis; mTORC1/S6K1 pathway; type 2 diabetes mellitus.

Fig. 1. Coronarin A stimulated glycogen synthesis and inhibited gluconeogenesis in rat primary hepatocytes.

a Chemical structure of coronarin A. b and c Cell viability was measured in rat primary hepatocytes after treatment with different doses of coronarin A for 5.5 h (b) or 12 h (c). d Effect of 10 μM coronarin A on glycogen synthesis for the indicated time points. e and f Glycogen synthesis was measured in primary hepatocytes after treatment with different doses of coronarin A for 4 h (e) or 12 h (f). 100 nM insulin was set as a positive control. g Dose-dependent inhibition on gluconeogenesis by coronarin A in rat primary hepatocytes. 500 μM metformin was used as a positive control. Forskolin (20 μM) was applied to stimulate gluconeogenesis in primary hepatocytes, and the effects of coronarin A on gluconeogenesis (h), the expression levels of gluconeogenic genes Pck1 (i) and G6pc (j) were determined. All results were presented as mean ± SEM (n = 4). ^*P < 0.05, ^**P < 0.01 versus control under basal condition; ^#P < 0.05, ^##P < 0.01 versus control under forskolin-induced condition.

Fig. 2. Coronarin A stimulated glycogen synthesis through activating PI3K/Akt/GSK3β signaling in primary hepatocytes.

a–c Coronarin A increased the Akt and GSK3β phosphorylation dose-dependently. d and e Cultured hepatocytes were pre-treated with the Akt inhibitor MK-2206 (2 μM) or PI3K inhibitor LY294002 (10 μM) and then co-treated with coronarin A. The phosphorylation levels of Akt and GSK3β (d) and glycogen synthesis (e) were detected. 100 nM insulin was set as a positive control. All results were presented as mean ± SEM (n = 3). *P < 0.05, **P < 0.01 versus the corresponding control.

Fig. 3. Coronarin A inhibited gluconeogenesis by activating ERK/-dependent Wnt/β-catenin/TCF7L2 pathway in primary hepatocytes.

a and b The inhibition on gluconeogenesis by coronarin A could not be attenuated by PI3K inhibitor LY294002 (a) and the Akt inhibitor MK2206 (b) in rat primary hepatocytes. c and d The mRNA expression level of TCF7L2 and β-catenin/TCF targeted genes Axin2, c-Myc and Ccnd1 were measured in primary hepatocytes after coronarin A treatment. e–g Effects of coronarin A on β-catenin, ERK and MEK phosphorylation were detected using Western blot analysis and quantitated. h and i Cultured hepatocytes were pre-treated with β-catenin inhibitor IWR-1-endo (10 μM) or ERK inhibitor SCH772984 (1 μM) and then co-treated with coronarin A. The ERK/β-catenin pathway (h) and gluconeogenesis (i) were detected. All results were presented as mean ± SEM (n = 3). ^*P < 0.05, ^**P < 0.01 versus control.

Fig. 4. Coronarin A increased tyrosine phosphorylation of IRS1 through inhibiting mTOR/S6K1 signaling.

a and b Coronarin A dose-dependently enhanced the phosphorylation of IRS1 at Tyr1222 in rat primary hepatocytes, and decreased the serine phosphorylation of IRS1 at Ser1101. c Coronarin A showed no influence on the tyrosine phosphorylation of IR in rat primary hepatocytes. d–f Coronarin A dose-dependently decreased the phosphorylation of mTOR, S6K1 and S6 in rat primary hepatocytes. g–i Primary rat hepatocytes were pre-treated with IRS1 siRNA or non-target siRNA (NC) for 48 h and the IRS1 protein (g) was detected; or the hepatocytes were followed with or without coronarin A treatment for further 4 h for glycogen synthesis (h) or 5.5 h for gluconeogenesis (i) measurement. Results were presented as mean ± SEM (n = 3–4). ^*P < 0.05, ^**P < 0.01 versus control.

Fig. 5. Intraperitoneal injection of coronarin A ameliorated hyperglycemia and insulin resistance in ob/ob mice.

Intraperitoneal injection of coronarin A (30 mg/kg) improved both non-fasting blood glucose (a) and fasting blood glucose (b) during the 22 days’ treatment. Oral glucose tolerance test (OGTT, 1.5 g/kg) was determined on day 19. The blood glucose (c) and serum insulin (d) were detected at the indicated time points. e HOMA-IR was calculated. Food intake accumulation (f) and body weight (g) were monitored during treatment. h Liver glycogen content was detected. i The expression levels of hepatic gluconeogenic genes Pck1 and G6pc were detected by real-time PCR. All results were presented as mean ± SEM (n = 8). ^*P < 0.05, ^**P < 0.01 versus vehicle mice.

Fig. 6. Effects of coronarin A on hyperglycemic, hepatic glycogen content and gluconeogenic genes expression by gavage to ob/ob mice.

Oral administration of coronarin A (100 mg/kg) also improved both non-fasting blood glucose (a) and fasting blood glucose (b) during the 22 days’ treatment. Oral glucose tolerance test was determined on day 19. The blood glucose (c) and serum insulin (d) were detected at the indicated time points. e HOMA-IR was calculated. Food intake (f) and body weight (g) were monitored during treatment. h Liver glycogen content was detected. i The expression of hepatic gluconeogenic genes Pck1 and G6pc were detected by real-time PCR. All results were presented as mean ± SEM (n = 8). ^*P < 0.05, ^**P < 0.01 versus vehicle mice.

Fig. 7. Effects of coronarin A on mTOR/S6K1/IRS1 signaling and the downstream Akt/GSK3β and ERK/β-catenin/TCFL2 pathways in livers of ob/ob mice.

a After coronarin A (100 mg/kg, once daily, p.o.) treated for 22 days, livers of ob/ob mice were collected and phosphorylations of mTOR at Ser2448, S6K1 at Thr389 and S6 at Ser235/236 were detected by Western blot analysis. Phosphorylation of IRS1 at Ser1101 (b) and Tyr895 (c) were determined. d Phosphorylation of Akt at Ser473 and Thr308, and GSK3β at Ser9 were increased by coronarin A treatment. e Phosphorylations of ERK1/2 at Thr202/Tyr204 and β-catenin at Ser675 were also significantly increased. The gene expression of TCF7L2 (f) and Wnt target genes Axin2, c-Myc and Ccnd1 (g) were stimulated by coronarin A treatment. All results were presented as mean ± SEM (n = 8). *P < 0.05, **P < 0.01 versus vehicle mice.

Fig. 8. Mechanism of coronarin A on regulating glycogen synthesis and gluconeogenesis to improve glucose homeostasis.

Coronarin A reduced the serine phosphorylation of IRS1 by inhibiting mTOR/S6K1, which led to enhancement of IRS1 tyrosine phosphorylation, and subsequently activated the PI3K/Akt/GSK3β and ERK/Wnt/β-catenin pathway to stimulate hepatic glycogen synthesis and suppress gluconeogenesis, respectively.