Antidiabetic and antihyperlipidemic activities of Phyllanthus emblica L. extract in vitro and the regulation of Akt phosphorylation, gluconeogenesis, and peroxisome proliferator-activated receptor α in streptozotocin-induced diabetic mice

Abstract

Background: The fruits of Phyllanthus emblica L. are high in nutrients and have excellent health care function and developmental value. There are many management strategies available for diabetes and hyperlipidemia. Nevertheless, there is a lack of an effective and nontoxic drug.

Objective: The present study was designed to first screen four extracts of P. emblica L. on insulin signaling target gene expression levels, including glucose transporter 4 (GLUT4) and p-Akt/t-Akt. The ethyl acetate extract of P. emblica L. (EPE) exhibited the most efficient activity among the four extracts and was thus chosen to explore the antidiabetic and antihyperlipidemic activities in streptozotocin (STZ)-induced type 1 diabetic mice.

Design: All mice (in addition to one control (CON) group) were administered STZ injections (intraperitoneal) for 5 consecutive days, and then STZ-induced mice were administered EPE (at 100, 200, or 400 mg/kg body weight), fenofibrate (Feno) (at 250 mg/kg body weight), glibenclamide (Glib) (at 10 mg/kg body weight), or vehicle by oral gavage once daily for 4 weeks. Finally, histological examination, blood biochemical parameters, and target gene mRNA expression levels were measured, and liver tissue was analyzed for the levels of malondialdehyde (MDA), a maker of lipid peroxidation.

Results: EPE treatment resulted in decreased levels of blood glucose, HbA1C, triglycerides (TGs), and total cholesterol and increased levels of insulin compared with the vehicle-treated STZ group. EPE treatment decreased blood levels of HbA1C and MDA but increased glutathione levels in liver tissue, implying that EPE exerts antioxidant activity and could prevent oxidative stress and diabetes. The EPE-treated STZ mice displayed an improvement in the sizes and numbers of insulin-expressing β cells. EPE treatment increased the membrane expression levels of skeletal muscular GLUT4, and also reduced hepatic mRNA levels of glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase thereby inhibiting hepatic gluconeogenesis. This resulted in a net glucose lowering effect in EPE-treated STZ mice. Furthermore, EPE increased the expression levels of p-AMPK/t-AMPK in both the skeletal muscle and liver tissue compared with vehicle-treated STZ mice. EPE-treated STZ mice showed enhanced expression levels of fatty acid oxidation enzymes, including peroxisome proliferator-activated receptor α (PPARα), but reduced expression levels of lipogenic genes including fatty acid synthase, as well as decreased mRNA levels of sterol regulatory element binding protein 1c (SREBP1c), apolipoprotein-CIII (apo-CIII), and diacylglycerol acyltransferase-2 (DGAT2). This resulted in a reduction in plasma TG levels. EPE-treated STZ mice also showed reduced expression levels of PPAR γ. This resulted in decreased adipogenesis, fatty acid synthesis, and lipid accumulation within liver tissue, and consequently, lower TG levels in liver tissue and blood. Furthermore, EPE treatment not only displayed an increase in the Akt activation in liver tissue, but also in C2C12 myotube in the absence of insulin. These results implied that EPE acts as an activator of AMPK and /or as a regulator of the insulin (Akt) pathway.

Conclusions: Taken together, EPE treatment exhibited amelioration of the diabetic and hyperlipidemic state in STZ-induced diabetic mice.

Keywords: Phyllanthus emblica L; antihyperlipidemic; diabetes; gluconeogenesis; insulin-expressing β cells; streptozotocin.

Fig. 1

The fruits of Phyllanthus emblica L.

Fig. 2

Four extracts of Phyllanthus emblica L. activate Akt signaling pathways. Four extracts including (A) ethyl acetate extract of P. emblica L. (EPE), (B) butanol extract of P. emblica L. (BPE), (C) methanol extract of P. emblica L. (MPE), and (D) water extract of P. emblica L. (WPE) were analyzed via Western blotting for phospho-Akt (p-Akt) and total-Akt (t-Akt). (1) Representative images; Akt activation is assessed in C2C12 cells, and administered 40 μg/mL EPE for the individual duration (5–60 min); (2) The ratios of p-Akt to t-Akt form were expressed as Akt activation. *P < 0.05, **P < 0.01, or ***P < 0.001 compared to the 0 min group. All values are means ± SE (n = 3).

Fig. 3

Effects of ethyl acetate extract of Phyllanthus emblica L. (EPE) in streptozotocin (STZ)-induced diabetic mice on (A) final body weight, (B) relative weights of white adipose fat tissues (including EWAT, MWAT, or RWAT), (C) relative weights of spleen and pancreas, (D) blood glucose levels, (E) blood glycated hemoglobin (HbA1_C) levels, (F) triglyceride levels, (G) total cholesterol levels, (H) insulin levels, (I) adiponectin levels, (J) leptin levels, (K) lipid peroxidation (LPO), superoxide dismutase (SOD), (M) glutathione peroxidase (GSH-Px), and glutathione (GSH) contents in liver tissues. #P < 0.05, ^##P < 0.01, or ^###P < 0.001 as compared to the control (CON) group; *P < 0.05, **P < 0.01, and ***P < 0.001 as compared to the STZ plus vehicle (distilled water) (STZ) group. All values are means ± SE (n = 6 per group). EPE: EPE1: 100, EPE2: 200, EPE3: 400 mg/kg body weight; Glib: glibenclamide (10 mg/kg body weight); Feno: fenofibrate (250 mg/kg body weight). Epididymal white adipose tissue (EWAT); MWAT (mesenteric white adipose tissue); RWAT (retroperitoneal white adipose tissue).

Fig. 3

Effects of ethyl acetate extract of Phyllanthus emblica L. (EPE) in streptozotocin (STZ)-induced diabetic mice on (A) final body weight, (B) relative weights of white adipose fat tissues (including EWAT, MWAT, or RWAT), (C) relative weights of spleen and pancreas, (D) blood glucose levels, (E) blood glycated hemoglobin (HbA1_C) levels, (F) triglyceride levels, (G) total cholesterol levels, (H) insulin levels, (I) adiponectin levels, (J) leptin levels, (K) lipid peroxidation (LPO), superoxide dismutase (SOD), (M) glutathione peroxidase (GSH-Px), and glutathione (GSH) contents in liver tissues. #P < 0.05, ^##P < 0.01, or ^###P < 0.001 as compared to the control (CON) group; *P < 0.05, **P < 0.01, and ***P < 0.001 as compared to the STZ plus vehicle (distilled water) (STZ) group. All values are means ± SE (n = 6 per group). EPE: EPE1: 100, EPE2: 200, EPE3: 400 mg/kg body weight; Glib: glibenclamide (10 mg/kg body weight); Feno: fenofibrate (250 mg/kg body weight). Epididymal white adipose tissue (EWAT); MWAT (mesenteric white adipose tissue); RWAT (retroperitoneal white adipose tissue).

Fig. 3

Effects of ethyl acetate extract of Phyllanthus emblica L. (EPE) in streptozotocin (STZ)-induced diabetic mice on (A) final body weight, (B) relative weights of white adipose fat tissues (including EWAT, MWAT, or RWAT), (C) relative weights of spleen and pancreas, (D) blood glucose levels, (E) blood glycated hemoglobin (HbA1_C) levels, (F) triglyceride levels, (G) total cholesterol levels, (H) insulin levels, (I) adiponectin levels, (J) leptin levels, (K) lipid peroxidation (LPO), superoxide dismutase (SOD), (M) glutathione peroxidase (GSH-Px), and glutathione (GSH) contents in liver tissues. #P < 0.05, ^##P < 0.01, or ^###P < 0.001 as compared to the control (CON) group; *P < 0.05, **P < 0.01, and ***P < 0.001 as compared to the STZ plus vehicle (distilled water) (STZ) group. All values are means ± SE (n = 6 per group). EPE: EPE1: 100, EPE2: 200, EPE3: 400 mg/kg body weight; Glib: glibenclamide (10 mg/kg body weight); Feno: fenofibrate (250 mg/kg body weight). Epididymal white adipose tissue (EWAT); MWAT (mesenteric white adipose tissue); RWAT (retroperitoneal white adipose tissue).

Fig. 4

Representative pathogenesis photographs of (A) liver tissues and (B) pancreatic islets of Langerhans, (C) the average area of islets of Langerhans, (D) 200×, and (E) 400× immunohistochemical staining of pancreatic insulin-expressing β cells (brown) and glucagon-expressing (green) α cells of streptozotocin (STZ)-induced mice following treatment with ethyl acetate extracts of Phyllanthus emblica L. (EPE), Glib: glibenclamide (10 mg/kg body weight), or Feno: fenofibrate (250 mg/kg body weight). EPE: EPE1, EPE2, and EPE3 (100, 200, and 400 mg/kg body) by hematoxylin and eosin-staining. ^###P < 0.001 as compared to the control (CON) group; ***P < 0.001 as compared to the STZ plus vehicle (distilled water) (STZ) group.

Fig. 4

Representative pathogenesis photographs of (A) liver tissues and (B) pancreatic islets of Langerhans, (C) the average area of islets of Langerhans, (D) 200×, and (E) 400× immunohistochemical staining of pancreatic insulin-expressing β cells (brown) and glucagon-expressing (green) α cells of streptozotocin (STZ)-induced mice following treatment with ethyl acetate extracts of Phyllanthus emblica L. (EPE), Glib: glibenclamide (10 mg/kg body weight), or Feno: fenofibrate (250 mg/kg body weight). EPE: EPE1, EPE2, and EPE3 (100, 200, and 400 mg/kg body) by hematoxylin and eosin-staining. ^###P < 0.001 as compared to the control (CON) group; ***P < 0.001 as compared to the STZ plus vehicle (distilled water) (STZ) group.

Fig. 5

The hepatic target gene mRNA levels in streptozotocin (STZ)-induced mice following treatment with ethyl acetate extract of Phyllanthus emblica L. (EPE), glibenclamide (Glib; 10 mg/kg body weight), or fenofibrate (Feno; 250 mg/kg body weight) by semiquantitative reverse transcription polymerase chain reaction (RT-PCR) analysis in STZ-induced mice. EPE: EPE1, EPE2, and EPE3 (100, 200, and 400 mg/kg body). (A) Representative image; (B) and (C) quantification of the ratio of target gene to β-actin mRNA expression. Total RNA isolated from the livers and then reverse transcripted by MMLV-RT, and followed by10 μL of RT products were administered as a template for PCR. The mRNA levels of G6Pase, PEPCK, apo C-III, SREBP1c, DGAT2, and SREBP2 were assessed and quantified by image analysis. Values were normalized to β-actin mRNA expression. All values are means ± SE (n = 6 per group). ^###P < 0.001 as compared to the control (CON) group; *P < 0.05, **P < 0.01, ***P < 0.001 as compared to the STZ plus vehicle (distilled water) (STZ) group.

Fig. 6

The skeletal muscular target gene expression levels in streptozotocin (STZ)-induced mice following treatment with ethyl acetate extract of Phyllanthus emblica L. (EPE), glibenclamide (Glib, 10 mg/kg body weight); fenofibrate (Feno; 250 mg/kg body weight) by Western blotting analysis on membrane GLUT4, p-AMPK (Thr¹⁷²) / t-AMPK and p-Akt (Ser⁴⁷³) /t-Akt (Ser⁴⁷³). (A) Representative image; (B) quantification of the p-AMPK to t-AMPK and p-Akt (Ser⁴⁷³) /t- Akt (Ser⁴⁷³). Protein was separated by 12% SDS-PAGE. ^###P < 0.001 as compared to the control (CON) group; ***P < 0.001 compared to the STZ plus vehicle (distilled water) (STZ) group. All values are means ± SE (n = 6 per group). EPE: EPE1, EPE2, and EPE3 (100, 200, and 400 mg/kg body).

Fig. 7

The hepatic target gene expression levels in streptozotocin (STZ)-induced mice following treatment with ethyl acetate extract of Phyllanthus emblica L. (EPE), glibenclamide (Glib, 10 mg/kg body weight), or fenofibrate (Feno; 250 mg/kg body weight) by Western blotting analysis. EPE: EPE1, EPE2, and EPE3 (100, 200, and 400 mg/kg body weight, respectively). (A) and (B) p-Akt (Ser⁴⁷³) / t-Akt (Ser⁴⁷³) and p-FoxO1 (Ser²⁵⁶) / t-FoxO1 (Ser²⁵⁶), (C) and (D) p-AMPK (Thr¹⁷²) / t-AMPK, PPARα, FAS, and PPARγ. (A) and (C) Representative image; (B) and (D) quantification of the p-AMPK to t-AMPK, PPARα, FAS, and PPARγ. Protein was separated by 12% SDS-PAGE assessed by Western blot. All values are means ± SE (n = 6 per group). ^#P < 0.05, ^###P < 0.001 as compared to the control (CON) group; *P < 0.05, **P < 0.01, ***P < 0.001 as compared to the STZ plus vehicle (distilled water) (STZ) group.

Fig. 8

High-performance liquid chromatography analysis of (A) 5000 ppm (24.6 mg/5 mL) ethyl acetate of Phyllanthus emblica L., (B) 1000 ppm ethyl acetate of P. emblica L. + standard 50 ppm gallic acid + standard 50 ppm chebulagic acid + standard 50 ppm ellagic acid.

Fig. 9

The graphic abstract of ethyl acetate extract of Phyllanthus emblica L. (EPE) in streptozotocin (STZ)-induced T1DM mice.