Nutr Res Pract.  2018 Feb;12(1):20-28. 10.4162/nrp.2018.12.1.20.

Anti-hyperglycemic effects and signaling mechanism of Perilla frutescens sprout extract

• Kim DH
• Kim SJ
• Yu KY
• Jeong SI
• Kim SY

• KMID: 2429015
• DOI: http://doi.org/10.4162/nrp.2018.12.1.20

Abstract

BACKGROUND/OBJECTIVES
Perilla frutescens (L.) Britton var. (PF) sprout is a plant of the labiate family. We have previously reported the protective effects of PF sprout extract on cytokine-induced β-cell damage. However, the mechanism of action of the PF sprout extract in type 2 diabetes (T2DM) has not been investigated. The present study was designed to study the effects of PF sprout extract and signaling mechanisms in the T2DM mice model using C57BL/KsJ-db/db (db/db) mice.
MATERIALS/METHODS
Male db/db mice were orally administered PF sprout extract (100, 300, and 1,000 mg/kg of body weight) or rosiglitazone (RGZ, positive drug, 1 mg/kg of body weight) for 4 weeks. Signaling mechanisms were analyzed using liver tissues and HepG2 cells.
RESULTS
The PF sprout extract (300 and 1,000 mg/kg) significantly reduced the fasting blood glucose, serum insulin, triglyceride and total cholesterol levels in db/db mice. PF sprout extract also significantly improved glucose intolerance and insulin sensitivity, decreased hepatic gluconeogenic protein expression, and ameliorated histological alterations of the pancreas and liver. Levels of phosphorylated AMP-activated protein kinase (AMPK) protein expression also increased in the liver after treatment with the extract. In addition, an increase in the phosphorylation of AMPK and decrease in the phosphoenolpyruvate carboxykinase and glucose 6-phosphatase proteins in HepG2 cells were also observed.
CONCLUSIONS
Our results sugges that PF sprout displays beneficial effects in the prevention and treatment of type 2 diabetes via modulation of the AMPK pathway and inhibition of gluconeogenesis in the liver.

Keyword

Perilla frutescens; diabetes mellitus; gluconeogenesis

MeSH Terms

AMP-Activated Protein Kinases
Animals
Blood Glucose
Cholesterol
Diabetes Mellitus
Fasting
Gluconeogenesis
Glucose Intolerance
Glucose-6-Phosphatase
Hep G2 Cells
Humans
Insulin
Insulin Resistance
Liver
Male
Mice
Pancreas
Perilla frutescens*
Perilla*
Phosphoenolpyruvate
Phosphorylation
Plants
Triglycerides
AMP-Activated Protein Kinases
Blood Glucose
Cholesterol
Glucose-6-Phosphatase
Insulin
Phosphoenolpyruvate

Figure

• 

  Fig. 1 Chromatograms of the major compound in Perilla frutescens (L.) Britton var. (PF) sprout extract. (A) The chemical structure of rosmarinic acid (RA). (B) HPLC chromatogram of RA standard. (C) HPLC chromatogram of PF sprout extract at 325 nm.

• 

  Fig. 2 Effects of Perilla frutescens (L.) Britton var. (PF) sprout extract on body weight gain in db/db mice. (A) Non-diabetic (ND), diabetes mellites (DM), positive control (PC, rosiglitazone, 1 mg/kg), low dose (PF 100, 100 mg/kg), medium dose (PF 300, 300 mg/kg), and high dose (PF 1,000, 1,000 mg/kg) PF sprout extract treated mice groups. (B) Body weight increase for 4 weeks.

• 

  Fig. 3 Anti-diabetic effects of Perilla frutescens (L.) Britton var. (PF) sprout extract in db/db mice. (A) Fasting blood glucose. (B) Area under the curve (AUC) of fasting blood glucose in db/db mice. (C) Oral glucose tolerance test (OGTT) after 12 h of food deprivation. (D) AUC was measured over the next 120 min in db/db mice. Data are expressed as mean ± SD (n = 5-10). ***P < 0.001 vs. ND. ##P < 0.01 and ###P < 0.001 vs. DM group. ND, non-diabetic; DM, diabetes mellites; PC, positive control (1 mg/kg); PF 100, low dose (100 mg/kg) PF sprout extract treated; PF 300, medium dose (300 mg/kg) PF sprout extract treated; PF 1,000, high dose (1,000 mg/kg) PF sprout extract treated.

• 

  Fig. 4 Insulin sensitivity in Perilla frutescens (L.) Britton var. (PF) sprout extract treated-db/db mice. (A) Serum insulin levels at 4 weeks after PF sprout extract treatment. (B) Intraperitoneal insulin tolerance test (IPITT) after 4 h fasting, in db/db mice. (C) AUC value for IPITT. (D) Effect of PF sprout extract on pancreatic morphological changes in db/db mice (n = 6-10). (E) Effect of PF sprout extract on pancreas weight (PW) change. (F) The ratio of PW and body weight (BW). Data are expressed as mean ± SD (n = 5-10). ***P < 0.001 vs. ND. #P < 0.05 and ##P < 0.01 vs. DM group. ND, non-diabetic; DM, diabetes mellites; PC, positive control (1 mg/kg); PF 100, low dose (100 mg/kg) PF sprout extract treated; PF 300, medium dose (300 mg/kg) PF sprout extract treated; PF 1,000, high dose (1,000 mg/kg) PF sprout extract treated.

• 

  Fig. 5 Comparison of liver damage after Perilla frutescens (L.) Britton var. (PF) sprout extract treatment in db/db mice. (A) Representative phenotype of whole liver in each group after PF sprout treatments. (B) Histological comparison after H & E staining of each group (n = 5-10). Serum concentrations of TG, TC, AST (GOT), and ALT (GPT). At the end of the PF sprout extract treatment, whole blood of each mouse was collected for investigating the levels of (C) TG, (D) TC, (E) GOT, and (F) GPT. Data are expressed as mean ± SD (n = 5-10). ***P < 0.001 vs. ND. #P < 0.05 and ##P < 0.01 vs. DM group. ND, non-diabetic; DM, diabetes mellites; PC, positive control (1 mg/kg); PF 100, low dose (100 mg/kg) PF sprout extract treated; PF 300, medium dose (300 mg/kg) PF sprout extract treated; PF 1,000, high dose (1,000 mg/kg) PF sprout extract treated.

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  Fig. 6 Effects of Perilla frutescens (L.) Britton var. (PF) sprout extract on AMPK phosphorylation and gluconeogenic protein expression in db/db mice. Western blot analysis for (A) AMPK phosphorylation and (B) G6Pase and PEPCK in db/db mice liver lysates. These data are representative of three independent experiments. Results were normalized by total AMPK and β-actin. Data are expressed as mean ± SD (n = 5-10). **P < 0.01 and ***P < 0.001 vs. ND. ##P < 0.01, and ###P < 0.001 vs. DM group. ND, non-diabetic; DM, diabetes mellites; PF, Perilla frutescens; PC, positive control; p-AMPK, phosphorylated-AMPK; AMPK, AMP-activated protein kinase; G6Pase, glucose-6-phosphatase; PEPCK, phosphoenolpyruvate carboxykinase.

• 

  Fig. 7 Perilla frutescens (L.) Britton var. (PF) sprout extract activates AMPK and inhibit gluconeogenesis in HepG2 cells. (A) HepG2 cells were pretreated with PF sprout extract or metformin (positive control) in the presence of glucose production medium (PM) in glucose free media. Levels of p-AMPK, G6Pase, and PEPCK were measured by immunoblotting with specific antibodies as indicated. (B) PF sprout extract inhibits glucose production in HepG2 cells. HepG2 cells were similarly pretreated with PF sprout extract, and then stimulated by cAMP/dexamethasone in the presence of PF sprout extract or metformin, as indicated. These data were representative of three independent experiments. Data are expressed as mean ± SD. *P < 0.05 and **P < 0.01 vs. PM. p-AMPK, phosphorylated-AMPK; AMPK, AMP-activated protein kinase; G6Pase, glucose-6-phosphatase; PEPCK, phosphoenolpyruvate carboxykinase.

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