Published: 2022 IF: 8.

022

Gypenoside ameliorates insulin resistance and

hyperglycemia via the AMPK-mediated signaling pathways
in the liver of type 2 diabetes mellitus mice
Mengxue Song, Dehong Tan, Bin Li, Yanqun Wang, Lin Shi

Presented by
Mohanto Nijaya
PhD Candidate
Student ID: 20217774
College of Pharmacy
Chosun University
Type-2 diabetes

 The foods provide glucose in the body, which is the main source of energy. The
pancreas produces insulin hormone that facilitates glucose to enter the cells,
produces energy, and controls the amount of glucose in the blood

 In the case of diabetes, the body cells do not respond normally to insulin, it is known
as insulin resistance (IR)

 The pancreas produces more insulin to make the cells respond. Finally, the pancreas
stops making new insulin and, as a result, the blood sugar level rises, resulting in
prediabetes or type 2 diabetes
Insulin resistance

 Insulin resistance (IR) not only causes primary type 2 diabetic mellitus (T2DM),
it is also the leading cause of nonalcoholic fatty liver disease, while
hepatocellular injury and liver fatty change would aggravate IR
 Besides, the greatest source of whole-body IR is the defection of transduction
pathways of the insulin signal in liver
 The liver is the main target organs of insulin action and plays a significant role
in glucose regulation and lipid metabolism
 T2DM causes the increase of free fatty acid and steatosis, accumulated in the
liver, and then causes lipotoxicity damage
 Therefore, improving the steatosis pathological changes in the liver is essential
for intervening T2DM IR
AMPK signaling Pathway

A target pathway to control diabetes is the 5′-adenosine

monophosphate-activated protein kinase (AMPK) signaling pathway

AMPK is a heterotrimeric protein with α, β, and γ subunits and it is an

energy sensor that is widely involved in various metabolic regulation
and plays a key role in regulating glucose homeostasis, insulin
sensitivity, and all aspects of cell function

In diabetic patients, increased activity or synthesis of key enzymes of

hepatic gluconeogenesis leads to increased hepatic glucose output,
causing the occurrence of IR, which is the primary cause of fasting
blood glucose (FBG) abnormal increase
AMPK signaling Pathway

 Furthermore, activated phosphorylation of

AMPK (p-AMPK) plays an important role in the
regulation of lipid synthesis via inhibiting
related transcription factors

 It also restrains the level of gluconeogenesis-

related key enzymes, such as PC (pyruvate
carboxylase), PEPCK (phosphoenolpyruvate
carboxykinase), G6Pase (glucose-6-
phosphatase), thereby reducing blood glucose
concentration to control blood sugar stability

Fig: Activation of AMPK signaling Pathway

AMPK increases glucose uptake in the muscle through the

regulation of glucose transporter 4(GLUT4). When AMPK is
activated, GLUT4 is translocated to the cellular membrane,
creating a doorway for glucose uptake, in muscle cells
 Natural products

 It is of great significance and value to investigate whether natural

products regulate glycolipid metabolism and improve IR through
the AMPK-mediated signaling pathway

 The research group used crude saponins of Gynostemma

pentaphyllum, also called “Southern Ginseng” which is a
traditional Asian folk medicinal plant

 Gypenosides (Gps) are the biologically active constituents of G.

pentaphyllum, which have been reported with hypoglycemic
activity

 It was reported that Gps exhibited various biological activities, such

as anti-cancer, anti-inflammation, prevention of atherosclerosis,
inhibition of apoptosis, protection of nerve, and anti-hypolipidemic
and hypoglycemic effects
Research Objectives

 In the series of studies on Gps, the research group found some active
compounds. Gp-I and Gp-II are the more abundant among them, about
0.8% and 1.0%, respectively

 As a continuation work for discovering the functional factors, in this present

study-
• Evaluated the effects of two groups (Gp-I and Gp-II) on the
hypoglycemic mechanism and liver pathology in T2DM mice
induced by high-fat and high-sugar diet (HFSD) and streptozotocin
(STZ)
Materials and methods

 Plant material and reagents: crude saponins of Gynostemma

pentaphyllum
 Sample preparation
 Cell culture
 Cytotoxicity evaluation
 Animal experimental design
 Oral glucose tolerance test (OGTT)
 Determination of metabolic parameters
 Histopathological examinations
 Western blot analysis
 Statistical analysis
Sample preparation

The total Gynostemma pentaphyllum saponins (100 g) were chromatographed

repeatedly over silica gel with CH2Cl2-MeOH-H2O (7:2:1, 7:3:1, 7:4:1)

Six fractions, A‒F were obtained

Fraction E was separated into five fractions, Ea-Ee, by ODS (Octadecyl-silica)

column (85% MeOH)

Fraction Ec was purified by preparative HPLC using MeOH-H2O (75:25) as eluent to

afford Gp-I (810 mg, tR = 33 min)

Fraction D was purified on preparative HPLC (82% MeOH) to yield Gp-II (1020 mg,
tR = 17 min)
 Results and discussion
Table 1

Structural identification of compounds Gp-I

and Gp-II:
Isolated and identified two gypenosides
(Table 1) via Preparative HPLC and NMR
Spectra respectively

Cytotoxicity assays on liver cells:

Using the HepG2 cell line, the antitumor
activities of 2 Gps were evaluated in vitro. The
CC50 values of them were (125.76 ± 2.47),
(169.62 ± 3.09) μg/mL, respectively. It was
clear that the cell viabilities were 109.13%
and 113.27% at 50 μg/mL

Notes: Cells were treated with various concentrations of

compounds. CC50 values (μg/mL) of compounds for 72 h
were calculated. The values were expressed as means ± S.D.
of three independent experiments. Each data was obtained
from triplicate experiments
Animal experimental design
After 1 week, the mice with fasting blood
Four-week-old male Kunming mice (18± 2) g glucose (FBG) levels ≥ 16.7 mmol/L were
considered successfully induced to T2DM and
selected for further treatment

Normal control Experimental Diabetic mice were randomly divided into 4

group (Normal group (high-fat and groups (n = 8):
pellet diet) high-sugar diet) 1. diabetic model group (DM) (Untreated)
2. positive control group (MH, administered
Four weeks later, Four weeks later, with metformin hydrochloride tablet, 50
treated with intraperitoneal injected mg/kg bw)
equal amounts of with STZ (60 mg/kg bw),
dissolved in citric acid- 3. Gp-I group
citric acid-sodium
citrate buffer sodium citrate buffer, pH 4. Gp-II group
4.4) after being fasted for 12 Administration:
h to induce type 2 diabetes
Experimental: Intragastric (50 mg/kg bw)
Normal control & Diabetic model: equal
volume of normal saline for 4 weeks

All animals were sacrificed 24 h after the last Gps treatment with phenobarbital sodium (i.p., 100 mg/kg)
anesthesia, and blood and liver samples were taken for assays
Mice body weight, liver index, food
intake and water consumption

 The initial body weight (0 week) of diabetic

mice was less than the NC group, which was
attributed to STZ injection

 The weight of mice in the NC group showed

an increasing trend, which was significantly
different from the DM group, while the other
groups were not

 The liver index of diabetic mice was

significantly higher than that in the NC group,
and no clear difference was observed in
experimental groups except the MH group

 The Gp-I and Gp-II groups showed lower liver

index than that of the DM group, but with no
significant difference, it may be related to
weight loss, polydipsia, and polyphagia in
diabetes with impaired energy metabolism
Fig. 1 The effect of Gps on (A) body weight,
(B) liver index in diabetic mice
Mice body weight, liver index, food
intake and water consumption

 The food intake of the DM group

mice increased from 3.19 g/10 g
bw to 4.41 g/10 g bw (Fig. 1C), and
the water consumption increased
from 10.81 mL/10 g bw to 13.17
mL/10 g bw (Fig. 1D)

 From the data, the treatment of

Gp-I and Gp-II alleviated the thirst
of diabetic mice and improved
their consumption of food

Fig. 1 The effect of Gps on (C) food intake and

(D) water consumption in diabetic mice
Effects of Gp on FBG and OGTT in T2DM mice

**The FBG data of mice in each group were measured weekly **

 At 0 week, the fasting blood glucose of

the model group (24.07 mmol/L) was
significantly higher than that of the NC
group, suggested that the T2DM model
was successfully constructed

 Gp-I and Gp-II markedly reduced the

levels of FBG and alleviated the IR in
T2DM mice induced by HFSD and STZ
after 4 weeks of treatment

 It demonstrated that Gp-I and Gp-II

both have hypoglycemic effect similar
Fig. 2 (A) Changes of FBG (fasting blood to metformin hydrochloride
glucose) in different groups
Effects of Gp on FBG and OGTT in T2DM mice

Fig. 2 (B) blood glucose curves of different groups during OGTT and (C) the AUC of blood glucose curves

 The highest glucose level occurred at 0.5 h  The AUC of the Gp-I and Gp-II groups was
after giving 2 g/kg glucose and it began to significantly lower than that of the DM group,
decline at 1 h and both were 30% lower than the DM group
 At 2 h, the blood glucose of Gp-I and Gp-II  These results suggested that the
groups returned to the initial blood glucose hypoglycemic effects of Gp-I and Gp-II on
level like MH and NC group T2DM mice are similar to those of metformin
hydrochloride tablets
Analysis of insulin level
and insulin sensitivity
*Fins= Fasting serum insulin
*HOMA-IR= Homeostasis model assessment of insulin resistance
*HOMA-β= Homeostasis model assessment of β-cell function
*QUICKI= Quantitative insulin sensitivity index
*ISI= Insulin sensitivity index

 The results suggested that the intervention of Gp-I and Gp-II reduced islet β-
cell damage and improved insulin sensitivity, and significantly improved IR
caused by diabetes

Effects of Gps on serum lipids
*TC= Total cholesterol
*TG= Triglyceride
*LDL-C= Low-density lipoprotein cholesterol
*HDL-C= High-density lipoprotein cholesterol
*NEFA= Non-esterified fatty acid

 Gp-I and Gp-II significantly decreased the levels of TG, LDL-C and NEFA and
increased TC/HDL-C levels in serum of T2DM mice, which was attributed to the
positive effects of Gps on blood glucose levels and insulin secretion probably

 Oral administration of Gps ameliorated serum lipid metabolism in T2DM mice

effectively
Effects of Gp on liver glycogen content
and liver tissue histopathological

 Compared with the DM group, the liver

glycogen content of the Gp-I and Gp-II
groups increased significantly

 The levels of AST and ALT in serum,

which reflect the liver damage severity

 Gps significantly reduced the

concentration of AST and ALT in the
serum of T2DM mice

Fig. 3 (A) Liver glycogen content. (B) AST, ALT

concentrations in serum
Effects of Gp on liver glycogen content
and liver tissue histopathological

Fig. 3 (C) Histopathological change of the liver in different groups, hypertrophy assessed by H&E

 NC group- no histological changes

 DM group- extensive liver injury, disordered arrangement and cellular swelling of the
hepatocytes
 Gp-I & Gp-II groups- less edema, the structure basically returned to normal, and the
treatment effects were similar to that of the MH group

Consistent with the results obtained by H&E staining, it is suggested that Gps
regulates blood glucose levels by protecting T2DM mice from liver damage and
promoting the production of liver glycogen content
 Effects of Gp on AMPK protein
signaling pathway in T2DM mice

 Expression of total AMPK

protein in mice of each group
had no significant difference

 Lower level of AMPK

phosphorylation in diabetic
mice was restored in various
degrees in Gp-I and Gp-II
groups

Fig. 4 Relative expression of key proteins of mice with type 2 diabetes, as

tested by (A) Western blot analysis, (B) AMPK, (C) p-AMPK
 Effects of Gp on AMPK protein
signaling pathway in T2DM mice

Fig. 4 Relative expression of key proteins of mice with type 2 diabetes, as tested by (D) PC,
(E) G6Pase and (F) PEPCK

 DM group- PC, PEPCK and G6Pase were significantly increased

 Gp-I & Gp-II groups- reduced the expression of the PEPCK and G6Pase in the liver

Gps treatment was associated with enhanced AMPK phosphorylation levels along with
decreased key enzyme levels of gluconeogenesis, suggesting that reduced hepatic
gluconeogenesis is responsible for the hypoglycemic effects of Gps
Conclusions

 In this study, it can be concluded that Gp-I and Gp-II have the potential to
treat hyperglycemia by improving IR and inhibiting gluconeogenesis
through AMPK-mediated signaling pathway

 Gp-I was a little more effective for than the other one, suggesting that the
number of sugar moieties, and the kind of side chain may affect their
biological activity

 If these findings can be translated to patients, this studies may make an

opportunity to develop Gps as potential anti-diabetic functional factors