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Enzogenol improves diabetesrelated metabolic change in c57bl/ksjdb/db mice, a model of type 2 diabetes mellitusJournal of Pharmacy Enzogenol improves diabetes-related metabolic change in
C57BL/KsJ-db/db mice, a model of type 2 diabetes mellitus
Chae-Young Bang and Se-Young Choung Department of Preventive Pharmacy and Toxicology, College of Pharmacy, Kyung Hee University, Seoul, Republic of Korea Enzogenol; gluconeogenesis; glucosehomeostasis; insulin resistance; type 2 Objectives Dietary use of pine bark extract has been associated with reduced risk
of inflammation and diabetes. In this study, we investigated the antidiabetic effectsof enzogenol, proanthocyanidins-rich bioflavonoid extract derived from the pine bark of New Zealand Pinus radiata trees, using C57BL/KsJ-db/db mice.
Se-Young Choung, Department of Preventive Methods After 1-week acclimation period, the db/db mice were divided into
Pharmacy and Toxicology, College ofPharmacy, Kyung Hee University, 26 vehicle-treated, Enzogenol-treated (12.5, 25 and 50 mg/kg; EZ) and positive Kyunghee-daero, Dongdaemun-gu, Seoul, control (tea polyphenol 50 mg/kg; TPP) groups.
[Equiv human dose: 1, 2.1, 4.2 mg/kg] 130-701, Republic of Korea.
Key findings The administration of EZ improved the glucose tolerance and
lowered the glycosylated haemoglobin (HbA1C), insulin and glucagon levels inblood. Interestingly, EZ and TPP treatments resulted in reduced hepatic free fatty Received September 14, 2013 acid, cholesterol and triglyceride levels in db/db mice. EZ and TPP treatments sig- Accepted December 7, 2013 nificantly elevated hepatic AMPK activity, and the expression of proteins related doi: 10.1111/jphp.12211 to glucose homeostasis and lipid metabolism, such as glucokinase, peroxisome
proliferator-activated receptor (PPAR)α and long-chain acyl-CoA dehydroge-
nase protein level with a simultaneous reduction of glucose-6-phosphatase
and phosphoenolpyruvate carboxykinase protein expression. In addition, the EZ
administration groups had an increased hepatic glycogen synthase expression in
Conclusions These results suggest that EZ may be beneficial in improving insulin
resistance and hyperglycaemia in type 2 diabetic mice by enhancing the glucose
and lipids metabolism.
Maritime pine bark extract is one of the most potent natu- markers of renal and liver function, glycaemic control and ral antioxidant and anti-inflammatory properties attributed to the bioflavonoids composition of proanthocyanidins, A standardized bark extract that complies with the monomeric flavonoids and phenolic acids, and its diverse monograph of maritime pine bark extract is derived from pharmacological activity has been well documented.[1,2] Pinus pinaster, Ait. (Pycnogenol). About 65–75% of this Recently, it has been reported that dietary pine bark extract extract are procyanidins consisting of catechin and epi- reduces the development of atherosclerotic lesions in male catechin moieties of varying chain lengths. Pycnogenol apolipoprotein E-deficient mice by lowering the serum cho- (PZ) is known to possess potent antioxidant activity. It does lesterol level, suggesting its anti-atherogenic effects. More- not only scavenge the free radicals, but also enhances the over, a randomized and double-blinded clinical study has endogenous antioxidant systems. These properties have shown that the treatment of pine bark extract improved led to their long-term use in treating inflammation, dia- both cognitive and cardiovascular functions in the group of betic retinopathy and cardiovascular disease associated older adults. In the long-term study, the dietary supple- with type 2 diabetes. mentation with combined Enzogenol (EZ) and vitamin C Type 2 diabetes mellitus (T2DM) is a chronic metabo- was not associated with any adverse change in laboratory lic disorder characterized by insulin resistance in the 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
Enzogenol regulates lipids and glucose metabolism Chae-Young Bang and Se-Young Choung peripheral tissues, as well as progressive β-cell dysfunc- tion leading to hyperglycaemia. Different risk factors or events can cause T2DM, such as obesity and age. The functional disturbance of pancreatic β-cells causes hyper-glycaemia by a decrease in glucose utilization in the liver, muscle and adipose tissue, and an increase in hepatic glucose production. Liver plays an important role in the glucose homeostasis through glycolysis, glycogenesis and gluconeogenesis. Glucagon regulates blood glucose byaffecting glucose metabolism, specifically by increasing gluconeogenesis and decreasing glycolysis in vivo. The netglucose uptake by the liver depends on the activity of glucokinase (GCK) and glucose-6-phosphatase (G-6- Pase). Glucagon has been shown to increase G-6-Pase expression and activity, which is the last step of the pathway.
Phosphoenolpyruvate carboxykinase (PEPCK) catalyses the conversion of oxaloacetate into phosphoenolpyruvate,which is the early and rate-limiting step in the pathway of hepatic gluconeogenesis by glucagon action. Also, glucagon inhibits glycogen synthesis by regulating glycogen synthase Structure of oligo- and poly-proanthocyanidins formed from in the liver. The activation of AMPK has also been shown catechin and epicatechin. Information for proanthocyanidins form of to reduce expression levels of molecules involved in Enzogenol was provided from ENZO Nutraceuticals Ltd. The pro- anthocyanidins (often referred to as OPCs = oligomeric proanthocyani- hepatocytes. Also, AMPK suppresses the expression of dins) are the most abundant group of phenolics in Enzogenol with lipogenesis-associated genes, such as fatty acid synthase, more than 80% by weight.
pyruvate kinase and ACC,[17–21] resulting in reduced malonylCoA levels and increased fatty acid oxidation. It wasrecently demonstrated that the activation of AMPK byresveratrol protected against lipid accumulation in the liver Materials and Methods
of diabetic mice. Enzogenol (EZ) is a complex mixture of plant phenolic compounds, including many different flavonoids and EZ was kindly provided by ENZO Nutraceuticals Ltd.
phenolic acid that occur naturally from the pine bark of (Auckland, New Zealand). Components information of New Zealand Pinus radiata trees. Proanthocyanidins, Enzogenol used in this study was published by Frevel the most abundant flavonoid component of pine bark extracts, are flavan-3-ols composed most commonly of proanthocyanidins 84.3% (±3.7), taxifolin 1.47% (±0.14) 2–10 units of catechin and epicatechin connected by dif- and catechin 0.67% (±0.08). Tea polyphenol, positive ferent inter-flavan linkages resulting in varying oligo- and control, was obtained from Amore Pacific Co. (Yongin-si, polymeric structures (Figure 1). In contrast to PZ, Gyeonggi-do, Korea); it contained 30% epigallocatechin which contains a wide variety of procyanidins that range gallate (EGCG). Protease and phosphatase inhibitor cock- from the monomeric catechin and taxifolin to oligomers tails were purchased from Roche (Mannheim, Germany).
with seven or more flavonoid subunits, the most Immunoblotting was performed using the following anti- abundant groups of phenolics in EZ are oligomeric bodies: anti-PEPCK (ab70358) and GCK (ab37796) from Abcam (Cambridge, UK); β-tubulin (sc-5274), G-6-Pase A number of experimental and clinical studies have (sc-134714) and AMPK (sc-25792) from Santa Cruz Bio- shown the hypolipidemic, antioxidant and other beneficial technology, Inc. (Santa Cruz, CA, USA); glycogen synthase effects of PZ in type 2 diabetes.[27,28] However, the anti- (#3893), p-AMPK (#2531S), anti-rabbit (#7074) and anti- diabetic effect of EZ is not well delineated. Here, we investi- mouse (#7076) IgG horseradish peroxidase (HRP)-linked gated the antidiabetic effect of EZ in db/db mice. Blood antibody from Cell Signaling Technology, Inc. (Beverly, glucose concentration, insulin secretion, insulin resistance MA, USA); long-chain acyl-CoA dehydrogenase (LCAD) and its effect on the expression of the glucose metabolism (17526-1-AP) from ProteinTech Group, Inc. (Chicago, IL, regulating genes in the diabetic C57BL/KsJ-db/db mice were USA); and PPAR-α (PA1-822A) from Thermo Scientific (Rockford, IL, USA).
2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
Chae-Young Bang and Se-Young Choung Enzogenol regulates lipids and glucose metabolism In-vivo experimental design
Rutherford, NJ, USA) and centrifuged (890g for 15 min at4°C) to separate the serum from blood cells. Insulin and Six-week-old male C57BL/KsJ db/db mice and non-diabetic glucagon levels in serum were measured by enzyme-linked mice (C57BL/6J) were purchased from Jackson Laboratory immunoassays. Mouse insulin ELISA kit was purchased (Bar Harbor, ME, USA). The animals were individually from Shibayagi (Shibukawa, Japan), and mouse glucagon housed in stainless steel cages, and adapted to 22 ∼ 28°C ELISA kit was purchased from USCN LIFE (Wuhan, and to the light–dark cycle (12 h–12 h). The 6-week-old db/db mice and non-diabetic mice were fed a pelletizedcommercial chow diet (Cargill Agri Purina Inc., Seongnam,Gyeonggi, Korea) for a period of 1 week after arrival and Western blotting for hepatic
then randomly divided into six groups: normal control, dia- gluconeogenesis and beta-oxidation
betes control, positive control (tea polyphenol containing Lysates were prepared using lysis buffer (20 mM Tris-HCl 70% catechins, 50 mg/kg; TPP) and EZ (12.5, 25 and (pH 7.4), 0.32 mM sucrose, protease inhibitor, 1 mM 50 mg/kg) groups (n = 8), respectively. TPP and EZ were PMSF, 0.5 M EDTA (pH 8.0), 1 mM NaF and 1 mM dissolved in distilled water and administered orally once a day for 6 weeks. The mice had free access to food and dis- 3VO4). One hundred micrograms of was separated by SDS-polyacrylamide gel (8 or 10%) tilled water. Food and water consumption and weight gain electrophoresis. Proteins were transferred onto Poly- were measured three times a week.
vinylidene fluoride membranes in transfer buffer (25 mM All of the experiments were performed in accordance Tris-HCl (pH 7.4), 192 mM glycine and 20% v/v metha- with protocols approved by the Institutional Animal Care nol). The transferred membranes were incubated for 2 h in and Use Committees (Approval No. KHP-2010-04-21).
blocking solution (5% dried milk in Tris-buffered salinecontaining 0.1% Tween-20) at room temperature. Blots Blood and tissue sampling
were incubated with the appropriate primary antibodiesat a dilution of 1 : 1000, and then further incubated with At the end of treatment, animals were fasted overnight.
HRP-conjugated secondary antibody at a dilution of 1 : Blood samples were drawn from the tail vein and the infe- 5000. Bound antibodies were detected using enhanced rior vena cava to determine serum biomarkers. After col- chemiluminescence plus kits (Amersham International, lecting blood samples, the liver was immediately removed Little Chalfont, UK). The PEPCK, GCK, G-6-Pase, and stored at −80°C for subsequent determination of lipid AMPK, p-AMPK, glycogen synthase, LCAD and PPAR-α parameters and protein levels.
bands were detected with rabbit polyclonal anti-antibody(1 : 1000), respectively. The β-tubulin band was detected Blood glucose and HbA1C concentrations
Blood glucose concentration was measured using gluco- meter (GlucoDr; All Medicus Co., Anyang-si, Gyeonggi-do,Korea). The blood HbA1C concentration was measured Measurements of hepatic lipid levels
using EASY A1CTM (Infopia Co., Anyang-si, Gyeonggi-do,Korea).
Hepatic lipids were extracted by Folch method. Thelevels of triglyceride (TG), total cholesterol (TC) and HDLcholesterol (HDL-C) were determined by enzymatic Oral glucose tolerance test
method (Asan Pharm. Co. Ltd, Whaseong-si, Gyeonggi- At the end of EZ treatment, oral glucose tolerance test di, Korea), and the free fatty acid (FFA) was determined (OGTT) was performed after an overnight fast. The animals using Labassay NEFA (Wako Chemicals, Richmond, VA, were fed glucose (3.0 g/kg of body weight) solution by oral administration. Blood samples were collected from the tailvein before, and 30, 60, 90 and 120 min after, glucose administration. Blood glucose levels were measured usingGlucoDr (All Medicus Co.).
The results were presented as mean ± SE. Statistically sig-nificant differences between the groups were determinedby statistical package for social sciences (SPSS; Chicago, Measurements of serum insulin and
IL, USA) using one-way analysis of variance. Multiple comparisons were performed with Tukey's test as described.
Blood samples were collected from the interior vena The data were considered significantly different at cava, drawn into Vacutainer (Becton Dickinson & Co., P < 0.05 ∼ 0.001.
2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
Enzogenol regulates lipids and glucose metabolism Chae-Young Bang and Se-Young Choung EZ treatment (40.6%) reduced blood glucose level moreeffectively than TPP treatment (31.2%). To further deter- Body weight, food and water intake
mine the effect of EZ on blood glucose level, we performed The increase of body weight during the 6-week experimen- OGTT, which measures the ability to clear the circulating tal period in the diabetic control group was significantly blood glucose (Figure 2b, Table 3). After glucose adminis- higher as compared with that of control group, as expected, tration, the rate of blood glucose removal change in non- although the average body weight in the db/db mice group diabetic group significantly higher than all experimental was higher than that in the non-diabetic control group groups showed only 10% increased blood glucose level. In (Table 1). EZ medium- and high-dose or TPP-treated group contrast, a rapid increase of blood glucose level was showed decreased body weight gain compared with the dia- observed as early as 30 min after glucose administration betic control group, although they were not statistically sig- and remained elevated for at least 120 min in db/db mice, nificant. During the experimental period, the food intake in suggesting that diabetic control mice have declined glucose- db/db mice group was approximately 2.2-fold higher than handling ability. Interestingly, EZ treatment caused a rapid that in non-diabetic group. EZ medium and high-dose or removal of blood glucose compared with diabetic control TPP administration did not exhibit a significant change of group (P < 0.05). As shown in Figure 2c, the glucose incre- cumulative food intake. The food efficiency ratios of the mental area under the curve was significantly induced in EZ- (medium and high-dose) or TPP-treated groups were diabetic control than that in non-diabetic control group.
lower than that of the diabetic control group; however, EZ But EZ medium- and high or TPP-treated group signifi- low-dose-treated group was highest among the groups. The cantly reduced compared with diabetic control group.
water intake in diabetic control group was significantlyhigher than that in the non-diabetic control and sample treated group. EZ treatment reduced water intake in db/db C, serum insulin and glucagon levels
To further understand the antidiabetic effect of EZ, weexamined the HbA1c, insulin and glucagon levels inblood. Blood HbA1c concentrations of the diabetic control Fasting blood glucose, oral glucose
group were 2.6-fold (P < 0.001) higher than that of non- tolerance test and the area under curve of
diabetic group. Medium (25 mg/kg) and high dose of EZ oral glucose tolerance test
(50 mg/kg), and TPP (50 mg/kg), treatment significantly To assess the effect of EZ in glucose level, we treated db/db decreased the blood HbA1c level by 35.2%, 57.8% and mice with various doses of EZ and examined the blood 36.3%, respectively. The serum insulin and glucagon con- glucose levels. The administration of EZ or TPP tended to centrations in the diabetic control group were approxi- lower the blood glucose level compared with the diabetic mately 7- and 15-fold higher than that in the non-diabetic control group during the experimental period in a dose- group, respectively). However, both levels were signifi- dependent manner (Figure 2a, Table 2). The treatment of cantly decreased by medium and high dose (25 and EZ or TPP decreased blood glucose level by 10.1% (EZ, 50 mg/kg) administration of EZ. The serum glucagon 12.5 mg/kg), 19.6% (EZ, 25 mg/kg) and 40.6% (EZ, 50 mg/ level in low-dose (12.5 mg/kg) treated group significantly kg), respectively (Figure 2a, Table 2). In addition, 50 mg/kg The effect of EZ administration for 6 weeks on body weight, food intake and water intake in C57BL/6J and db/db micea db/db (mg/kg) Body weight (g)Initial Water intake (ml) EZ, Enzogenol; FER, food efficiency ratio; TPP, tea polyphenol. aThe values are expressed as mean ± SD (n = 8). bFER (%) = (body weight gain/foodintake) × 100. cP < 0.001 vs C57BL/6J group based on Tukey's test. #P < 0.05 and ##P < 0.01 vs db/db group based on Tukey's test.
2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
Chae-Young Bang and Se-Young Choung Enzogenol regulates lipids and glucose metabolism Blood glucose (mg/d ) AUC (mg·h/d ) 300 Blood glucose (% of zero time) Blood glucose and area under curve levels in db/db mice. (a) Fasting glucose levels were measured after fasting for 6 h. Blood samples were collected via the tail vein. (b) The effect of administration of Enzogenol on glucose tolerance in db/db and C57BL/6J mice. After a 12 h fast,male mice were orally administered with glucose (3 g/kg). The blood glucose concentration was measured at the indicated time points. (c) The areaunder curve levels in blood glucose of oral glucose tolerance test. The values are expressed as mean ± SE (n = 5). ***P < 0.001 vs NC group;#P < 0.05, ##P < 0.01 vs DC group based on Tukey's test. NC, C57BL/6J mice (normal control); DC, db/db mice (diabetic control); TPP-50, teapolyphenol 50 mg/kg.
Blood glucose levels during the 6-week experimental period in C57BL/6J and db/db micea db/db (mg/kg) TPP, tea polyphenol. aThe values are expressed as mean ± SE (n = 5). ***P < 0.001 vs C57BL/6J group based on Tukey's test. #P < 0.05 and##P < 0.01 vs db/db group based on Tukey's test.
Expression of hepatic glucose-regulating
hepatic glycogen synthase protein level in diabetic control enzyme and glycogen synthase
group was lower than that in the non-diabetic group(Figure 3). The expression of hepatic glycogen synthase in To investigate the roles of EZ in hepatic glucose-regulating medium and high dose of EZ-treated groups was increased proteins, we examined the expression of enzymes involved in a dose-dependent manner in db/db mice. TPP group also in glucose metabolism and glycogen synthesis. The diabetic significantly increased in the expression of hepatic glycogen control group showed reduced hepatic GCK protein level.
In contrast, the expression of hepatic gluconeogenicenzymes, such as G-6-Pase and PEPCK, was higher than Hepatic glucose homeostasis and lipid
that of the non-diabetic control group (Figure 3). The reduced GCK expression was rescued by EZ treatment.
Conversely, the increased G-6-Pase and PEPCK expression Recent studies demonstrated that the activity of hepatic were down-regulated by EZ or TPP administration. The AMPK, which is determined by phosphorylation of AMPK, 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
Enzogenol regulates lipids and glucose metabolism Chae-Young Bang and Se-Young Choung Oral glucose tolerance test in C57BL/6J and db/db micea EZ, Enzogenol; TPP, tea polyphenol. aThe values are expressed as mean ± SE (n = 5). *P < 0.05 and **P < 0.01 vs C57BL/6J group based on Tukey'stest. #P < 0.05 and ##P < 0.01 vs db/db group based on Tukey's test.
Glycogen Synthase Expression of proteins related to hepatic glucose-regulating enzyme and glycogen synthesis. The values are expressed as mean ± SE (n = 5). *P < 0.05 and ***P < 0.001 vs NC group; #P < 0.05, ##P < 0.01 and ###P < 0.001 vs DC group based on Tukey's test. NC, C57BL/6J mice(normal control); DC, db/db mice (diabetic control); TPP-50, tea polyphenol 50 mg/kg.
is dramatically reduced in db/db mice compared with the activity was reduced in diabetic control group compared non-diabetic. To explore more details about the effect of with the non-diabetic control group. EZ administration EZ on hepatic glucose homeostasis and beta-oxidation, we stimulated AMPK phosphorylation dramatically and in a examined the activity of AMPK. There was no change of dose-dependent manner (Figure 4). PPAR-α and LCAD AMPK protein levels between groups. However, AMPK protein expressions were slightly lower in the diabetic 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
Chae-Young Bang and Se-Young Choung Enzogenol regulates lipids and glucose metabolism P < 0.001 AMPK phosphorylation- and beta-oxidation-related protein expressions in liver tissue. The values are expressed as mean ± SE (n = 5).
#P < 0.05 and ##P < 0.01 vs DC group based on Tukey's test. NC, C57BL/6J mice (normal control); DC, db/db mice (diabetic control); TPP-50, teapolyphenol 50 mg/kg.
control group compared with the non-diabetic control min and thiazolidinediones (rosiglitazone and pioglita- group (Figure 4). The reduced hepatic PPAR-α and LCAD zone), counter insulin resistance. Thiazolinediones are protein levels in db/db mice were rescued by EZ or TPP one of the most frequently prescribed drugs to improve glycaemic control and increase insulin sensitivity in patientswith type 2 diabetes. However, they have serious side effects, Hepatic lipid profiles
including hypoglycaemia, oedema, hypertension and weightgain. Metformin is a synthetic biguanide and is the most As EZ treatment was found to be critical for the regulation widely prescribed medication for type 2 diabetes. This drug of enzymatic activity and expression of lipid metabolic pro- reduces hepatic glucose production, and increases periph- teins, we also examined the hepatic lipid levels in EZ-treated eral glucose utilization[33,34] and insulin sensitivity. Usual db/db mice (Table 3). EZ treatment resulted in significant side effects of metformin treatment are gastrointestinal, decreases of hepatic FFA, TG and TC levels in a dose- including nausea, vomiting, diarrhoea, abdominal discom- dependent manner in db/db mice. Also, EZ treatment sig- fort and flatulence, which become tolerable over time and nificantly and dose-dependently increased HDL-C and the can be decreased by administering the drug with food. ratio of HDL-C to TC that were lowered in db/db mice.
Currently, there is growing interest in natural products These results are consistent with the effect of EZ on the for the treatment of diabetes mellitus. Indeed, it has been enzymes related to hepatic beta-oxidation, such as AMPK, shown that several natural products, such as Ecklonia PPAR-α and LCAD.
cava, Galega officinalis and Momordica charantia, have significant antidiabetic effect in animal model. More-over, recent studies have indicated that blood glucose level, Insulin resistance is an early and sustained feature of type 2 hepatic lipid accumulation, adipose tissue weight and diabetes; two classes of oral antidiabetic agents, metfor- adipocyte size were significantly decreased by polyphenols 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
Enzogenol regulates lipids and glucose metabolism Chae-Young Bang and Se-Young Choung Effects of EZ on the levels of glycosylated haemoglobin (HbA1C), serum insulin and glucagona db/db (mg/kg) EZ, Enzogenol; TPP, tea polyphenol. aThe values are expressed as mean ± SE (n = 5∼8). bP < 0.001 vs normal group based on Tukey's test. #P < 0.05and ###P < 0.001 vs db/db group based on Tukey's test.
Effects of EZ on the levels of hepatic FFA, TG, TC and HDL-Ca db/db (mg/kg) Free fatty acid (FFA) 0.039 ± 0.004### 0.032 ± 0.007### Triglyceride (TG) Total cholesterol (TC) 53.70 ± 12.78### EZ, Enzogenol; TPP, tea polyphenol. aThe values are expressed as mean ± SE (n = 6 ∼ 8). bHigh-density lipoprotein cholesterol. cHDL cholesterol tototal cholesterol ratio (HTR) % = (HDL cholesterol (in mg/dℓ) = total cholesterol (in mg/dℓ)) × 100. *P < 0.01 and ***P < 0.001 vs normal group;##P < 0.01 and ###P < 0.001 vs db/db group based on Tukey's test.
derived from natural product in type 2 diabetic animals sible for the resultant suppression of levels of plasma without altering body weight.[40–42] glucose and insulin, EGCG decreases glucose production In the current study, we investigated the effect of EZ on of H4IIE rat hepatoma cells; furthermore, EGCG mimics insulin resistance and glucose homeostasis in C57BL/KsJ- insulin, increases tyrosine phosphorylation of the insulin db/db mice, an animal model for type 2 diabetes. The blood receptor and the insulin receptor substrate, and reduces glucose (Figure 2a and 2c, Tables 4 and 5), serum insulin gene expression of the PEPCK, and suppresses cytokine- and glucagon levels (Table 2) in EZ-treated groups were induced pancreatic beta-cell damage in vitro. improved after EZ treatment for 6 weeks, which suggests an To gain insight into the molecular mechanisms underly- enhanced rate of glucose disposal in peripheral tissues, ing the effects described above, we investigated genes that such as the liver, muscle and adipose tissue. Glucagon plays are likely to be involved in glucose homeostasis and lipid a key role in glucose metabolism in vivo. Consistent with its metabolism. Hepatic GCK plays a major role in controlling role as a counter-regulatory hormone of insulin, glucagon blood glucose homeostasis, and its activity is low in dia- raises plasma glucose levels in response to insulin-induced betes. G-6-Pase is a key enzyme controlling hepatic hypoglycaemia. Glucagon induces glycogen synthase gluconeogenesis and glucose output in liver and is normally phosphorylation and inhibits glycogen synthase activity in suppressed by the action of insulin. PEPCK catalyses the the liver.[44–46] We also observed that EZ administration conversion of oxaloacetate into phosphoenolpyruvate, an improved the glucose tolerance in db/db mice. EZ treatment early and rate-limiting step in the pathway of hepatic resulted in a rapid removal of blood glucose in diabetic gluconeogenesis. Due to their strategic positions in liver control group, suggesting that EZ treatment enhances glucose metabolism, both of these enzymes are supposed insulin sensitivity (Figure 2b).
to be the target of important regulatory mechanisms of TPP, used as a positive control in this study, contains hepatic glucose production. Our studies demonstrated approximately 30% EGCG, which is known to have benefi- that EZ administration showed hypoglycaemic effect by cial effect on type 2 diabetes. In animal and cell culture stimulating GCK activity and inhibiting G-6-Pase and experiments, catechins have demonstrated several poten- PEPCK activity, which are hepatic glucose-regulating tially therapeutic effects as follows: the inhibition of enzymes in the liver. The expression of hepatic GCK and α-amylase in the intestine, which was presumably respon- glycogen synthase was increased remarkably, whereas those 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
Chae-Young Bang and Se-Young Choung Enzogenol regulates lipids and glucose metabolism of G-6-Pase and PEPCK were significantly decreased in a body weight, which is observed in some synthetic anti- dose-dependent manner in the EZ-treated groups in db/db diabetic drug-treated patients. Compared with non- mice (Figure 3). Thus, it appears that EZ improves hepatic treated control group, we found decrease of body weight glucose metabolism through an increase in GCK and glyco- gain in EZ-treated db/db mice (Table 1), although it was not gen synthase activity, and a decrease in gluconeogenic statistically significant (Table 1). To determine whether EZ enzyme activity (i.e. G-6-Pase and PEPCK).
has pharmaceutical possibility, it is necessary to examine We showed that EZ administration promotes activation whether other organs, such as white adipose tissue and skel- of AMPK, equivalent to TPP administration (Figure 4).
etal muscle, regulate lipids or insulin signal pathway.
AMPK activation serves to (1) inhibit hepatic fatty acid andcholesterol synthesis, and (2) stimulate fatty acid oxidationin the liver and muscle.[53,54] Our studies also demonstrated that EZ and TPP treatment increased the protein expression In this study, EZ treatment significantly lowered the levels of of LCAD (Figure 4). The peroxisome proliferator-activated glucose, insulin and glucagon in serum, and improved receptor (PPAR)α is a nuclear receptor mainly involved in glucose tolerance. EZ appears to have hypoglycaemic effects the regulation of lipid levels. PPAR-α agonists is known to by modulating the expression of hepatic glucose-regulating reduce both intrahepatic and intramuscular TG content and enzymes, GCK, G-6-Pase and PEPCK, and enzymatic activ- improve insulin sensitivity in rodents.[55–57] ity of AMPK in the liver. These results demonstrate that As shown in Figure 4, PPAR-α expression, which was EZ supplementation could exert beneficial effects on type 2 reduced in diabetic control, was rescued by EZ treatment.
These results are consistent with the decrease of hepatic TGsand FFA contents in EZ-treated group (Table 3). In addi-tion, as shown in Table 3, TC level in diabetic control group was significantly decreased by EZ administration in a dose-dependent manner. The HDL-C/TC ratio was also sig-nificantly improved in the EZ-treated group (Table 3).
This work was supported by the R&D program of Korea Noticeably, EZ treatment did not induce further increase of Evaluation Institute of Industrial Technology (10033818).
endothelial function and biochemical insulin action. Nat Rev Mol Cell Biol 2006; 7: 85–96.
1. Chayasirisobhon S. Use of a pine inflammation in chronic smokers. Free 12. Kahn SE. The relative contributions bark extract and antioxidant vitamin Radic Res 2006; 40: 85–94.
of insulin resistance and beta-cell combination product as therapy for 6. Rohdewald P. Pycnogenol, French dysfunction to the pathophysiology of migraine in patients refractory to maritime pine bark extract. In: Ency- type 2 diabetes. Diabetologia 2003; 46: pharmacologic medication. Headache clopedia of Dietary Supplements. New 2006; 46: 788–793.
York: Marcel Dekker, 2005: 545–553.
13. Ferre T et al. Correction of diabetic 2. Frevel MA et al. Production, com- 7. Nelson AB et al. Pycnogenol inhibits alterations by glucokinase. Proc Natl position and toxicology studies of macrophage oxidative burst, lipopro- Acad Sci U S A 1996; 93: 7225–7230.
tein oxidation, and hydroxyl radical- 14. Striffler JS et al. Effects of glucagon extract. Food Chem Toxicol 2012; 50: induced DNA damage. Drug Dev Ind on hepatic microsomal glucose-6- Pharm 1998; 24: 139–144.
phosphatase in vivo. Diabete Metab 3. Sato M et al. Dietary pine bark extract 8. Bartlett HE, Eperjesi F. Nutritional 1984; 10: 91–97.
reduces atherosclerotic lesion develop- supplementation for type 2 diabetes: a 15. Jiang G, Zhang BB. Glucagon and ment in male ApoE-deficient mice by systematic review. Ophthalmic Physiol regulation of glucose metabolism. Am lowering the serum cholesterol level.
Opt 2008; 28: 503–523.
J Physiol Endocrinol Metab 2003; 284: Biosci Biotechnol Biochem 2009; 73: 9. Zibadi S et al. Reduction of cardiovas- cular risk factors in subjects with type 16. Yamauchi T et al. Adiponectin stimu- 4. Pipingas A et al. Improved cognitive 2 diabetes by Pycnogenol supplemen- lates glucose utilization and fatty-acid performance after dietary supple- tation. Nutr Res 2008; 28: 315–320.
oxidation by activating AMP-activated mentation with a Pinus radiata bark 10. Ziyadeh FN, Wolf G. Pathogenesis of protein kinase. Nat Med 2002; 8: extract formulation. Phytother Res the podocytopathy and proteinuria in 2008; 22: 1168–1174.
diabetic glomerulopathy. Curr Diabe- 17. Foretz M et al. Short-term overex- 5. Young JM et al. Comparative effects of tes Rev 2008; 4: 39–45.
pression of a constitutively active form Enzogenol® and vitamin C supple- 11. Taniguchi CM et al. Critical nodes in of AMP-activated protein kinase in mentation versus vitamin C alone on the liver leads to mild hypoglycemia 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
Enzogenol regulates lipids and glucose metabolism Chae-Young Bang and Se-Young Choung and fatty liver. Diabetes 2005; 54: conduction velocity and markers of ginseng. Nutr Metab Cardiovasc Dis oxidative stress in mild type diabetes 2005; 15: 149–160.
18. Leclerc I et al. Hepatocyte nuclear in rats. Phytother Res 2009; 23: 1169– 39. Hazarika R et al. Binding energy cal- factor-4alpha involved in type 1 culation of GSK-3 protein of human maturity-onset diabetes of the young 28. Parveen K et al. Protective effects is a novel target of AMP-activated of Pycnogenol on hyperglycemia- pounds of Momordica charantia linn protein kinase. Diabetes 2001; 50: induced oxidative damage in the liver (Bitter melon). Bioinformation 2012; of type 2 diabetic rats. Chem Biol 8: 251–254.
19. Woods A et al. Characterization of the Interact 2010; 186: 219–227.
40. Ae Park S et al. Genistein and daidzein role of AMP-activated protein kinase 29. Folch J et al. A simple method for the modulate hepatic glucose and lipid in the regulation of glucose-activated isolation and purification of total regulating enzyme activities in C57BL/ gene expression using constitutively lipides from animal tissues. J Biol KsJ-db/db mice. Life Sci 2006; 79: active and dominant negative forms Chem 1957; 226: 497–509.
of the kinase. Mol Cell Biol 2000; 20: 30. Bhalla K et al. Metformin prevents 41. Jung UJ et al. Effects of the ethanol extract of the roots of Brassica rapa 20. Foretz M et al. AMP-activated protein pathways driving hepatic lipogenesis.
on glucose and lipid metabolism in kinase inhibits the glucose-activated Cancer Prev Res (Phila) 2012; 5: 544– C57BL/KsJ-db/db mice. Clin Nutr expression of fatty acid synthase gene 2008; 27: 158–167.
in rat hepatocytes. J Biol Chem 1998; et al.
cemic therapy for type 2 diabetes: hypolipidemic action of Du-zhong 21. Leclerc I et al. The 5′-AMP-activated scientific review. JAMA 2002; 287: (Eucommia ulmoides Oliver) leaves protein kinase inhibits the transcrip- water extract in C57BL/KsJ-db/db mice.
tional stimulation by glucose in liver 32. Kim KR et al. KR-62980: a novel J Ethnopharmacol 2006; 107: 412–417.
cells, acting through the glucose peroxisome proliferator-activated re- 43. Zhou G et al. Role of AMP-activated response complex. FEBS Lett 1998; ceptor gamma agonist with weak protein kinase in mechanism of 431: 180–184.
adipogenic effects. Biochem Pharmacol metformin action. J Clin Invest 2001; 22. Assifi MM et al. AMP-activated pro- 2006; 72: 446–454.
tein kinase and coordination of hepa- 33. Stephenne X et al. Metformin acti- 44. Akatsuka A et al. Glucagon-stimulated tic fatty acid metabolism of starved/ vates AMP-activated protein kinase phosphorylation of rat liver glycogen .carbohydrate-refed rats. Am J Physiol in primary human hepatocytes by synthase in isolated hepatocytes. J Biol Endocrinol Metab 2005; 289: E794– Chem 1985; 260: 3239–3242.
Diabetologia 2011; 54: 3101–3110.
45. Ciudad C et al. Control of glycogen 23. Zang M et al. Polyphenols stimulate 34. El-Mir M-Y et al. Dimethylbiguanide synthase phosphorylation in isolated AMP-activated protein kinase, lower inhibits cell respiration via an indirect rat hepatocytes by epinephrine, vaso- lipids, and inhibit accelerated athero- effect targeted on the respiratory pressin and glucagon. Eur J Biochem sclerosis in diabetic LDL receptor- chain complex I. J Biol Chem 2000; 1984; 142: 511–520.
deficient mice. Diabetes 2006; 55: 275: 223–228.
46. Ramachandran C et al. Hormonal 35. Gunton JE et al. Metformin rapidly regulation of the phosphorylation of 24. Gilmour I, Duncan K. Fighting Free increases insulin receptor activation in glycogen synthase in perfused rat Radicals. Auckland New Zealand: The human liver and signals preferentially heart. Effects of insulin, catechola- Pacific Scientific Press, 1998.
through insulin-receptor substrate-2.
mines, and glucagon. J Biol Chem 25. Rohdewald P. A review of the French J Clin Endocrinol Metab 2003; 88: 1983; 258: 13377–13383.
maritime pine bark extract (Pycno- 47. Matsumoto N et al. Reduction of genol), a herbal medication with a 36. JanumetTM (sitagliptin/metformin HCl) blood glucose levels by tea catechin.
diverse clinical pharmacology. Int J Biosci Biotechnol Biochem 1993; 57: Clin Pharmacol Ther 2002; 40: 158– house Station, NJ: Merck Pharmaceu- ticals, Inc, 2012.
26. Peng Z et al. Quantitative analysis 37. Wijesekara I et al. Phlorotannins from gallate supplementation alleviates dia- of polymeric procyanidins (tannins) Ecklonia cava (Phaeophyceae): bio- betes in rodents. J Nutr 2006; 136: from grape (Vitis vinifera) seeds by logical activities and potential health reverse phase high-performance liquid benefits. Biofactors 2010; 36: 408– 49. Han MK. Epigallocatechin gallate, a chromatography. J Agric Food Chem constituent of green tea, suppresses 2001; 49: 26–31.
38. Vuksan V, Sievenpiper JL. Herbal rem- cytokine-induced pancreatic beta-cell 27. Jankyova S et al. Pycnogenol® effi- edies in the management of diabetes: damage. Exp Mol Med 2003; 35: 136– ciency on glycaemia, motor nerve lessons learned from the study of 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
Chae-Young Bang and Se-Young Choung Enzogenol regulates lipids and glucose metabolism et al.
53. Hawkins M et al. Contribution of 56. Zhang BB et al. AMPK: an emerging glucokinase in glucose homeostasis elevated free fatty acid levels to the drug target for diabetes and the meta- as determined by liver and pancreatic lack of glucose effectiveness in type 2 bolic syndrome. Cell Metab 2009; 9: beta cell-specific gene knock-outs diabetes. Diabetes 2003; 52: 2748– using Cre recombinase. J Biol Chem 57. Chou CJ et al. WY14,643, a per- 1999; 274: 305–315.
54. Ye JM et al. Peroxisome proliferator- 51. Nordlie RC et al. Recent advances in activated receptor (PPAR)-alpha acti- ceptor alpha (PPARalpha) agonist, hepatic glucose 6-phosphatase regula- improves hepatic and muscle steatosis tion and function. Proc Soc Exp Biol improves insulin sensitivity in high and reverses insulin resistance in Med 1993; 203: 274–285.
fat-fed rats: comparison with PPAR- lipoatrophic A-ZIP/F-1 mice. J Biol 52. Mithieux G. New knowledge regard- gamma activation. Diabetes 2001; 50: Chem 2002; 277: 24484–24489.
ing glucose-6 phosphatase gene and 58. Safavi M et al. The importance of syn- 55. Hardie DG. AMPK: a key regula- thetic drugs for type 2 diabetes drug regulation of glucose metabolism.
tor of energy balance in the single cell discovery. Expert Opin Drug Discov Eur J Endocrinol 1997; 136: 137– and the whole organism. Int J Obes 2013; 8: 1339–1363.
(Lond) 2008; 32(Suppl. 4): S7–S12.
2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
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