Biol. Pharm. Bull. 32(3) 421—426 (2009)
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Anti-diabetic Effects of New Herbal Formula in Neonatally Streptozotocin-Induced Diabetic Rats Jung-Ok KIM,a,c Gee-Dong LEE,b Joong-Ho KWON,c and Kil-Soo KIM*,d a
Daegu Gyeongbuk Institute for Oriental Medicine Industry; 300 Sampoong-dong, Gyeongsan 712–210, Republic of Korea: b Daegu Technopark Bio Industry Center; 95 Shincheon-dong, Daegu 704–801, Republic of Korea: c Department of Food Science & Technology, Kyungpook National University; and d College of Veterinary Medicine, Kyungpook National University; 1370 Sangyeok-dong, Daegu 702–701, Republic of Korea. Received October 6, 2008; accepted December 16, 2008; published online December 25, 2008 The present study investigated the anti-diabetic effects of new herbal formula (NHF) consist of Polygonati Rhizoma, Rehmanniae Radix, Salviae miltiorrhizae Radix, Puerariae Radix, Schizandrae Fructus, Glycyrrhizae Radix in neonatal streptozotocin (nSTZ)-induced non-insulin-dependent diabetes mellitus (NIDDM) rats. Changes of food and water intakes, body weight, blood glucose, plasma insulin and immunohistochemical evaluation of insulin on pancreas, and mRNA expression of glucose transporter subtype-4 (GLUT-4) in skeletal muscle and hepatic phosphoenolpyruvate carboxykinase (PEPCK) by administration of NHF (300 mg/kg) were investigated. The nSTZ diabetic rats showed hyperglycemia, increases in food and water intake, loss of body weight gain and decrease of the number of insulin-positive cells and the size of b -cells in pancreas and mRNA of GLUT4 in soleus muscle and increase of hepatic PEPCK mRNA expression. Administration of NHF significantly decreased the blood glucose level, food and water intake and considerably increased the body weight of nSTZ diabetic rats. Also, NHF treatment significantly increased plasma insulin level and the number and size of insulinimmunoreactive cells in the pancreas of nSTZ diabetic rats. In addition, NHF treatment resulted in increased expression of the GLUT-4 mRNA in soleus muscle and in reduced expression of PEPCK mRNA in liver. These results provide possible mechanisms for the anti-diabetic effects of NHF, via a decrease of blood glucose level, an increase of insulin sensitivity, an increase of GLUT-4 gene expression and an attenuation of hepatic PEPCK gene expression. In conclusion, NHF may be useful for improving hyperglycemia and reducing the risk of diabetic complications. Key words
new herbal formula; anti-diabetic effect; neonatally streptozotocin
Diabetes mellitus (DM), which is a chronic disease caused by inherited or acquired deficiency in insulin secretion and by decreased responsiveness of the organs to insulin, is currently one of the most costly and burdensome diseases and affects about 5% of the global population.1) Insulin deficiency results in increased blood glucose level, which in turn can damage many of the body systems including blood vessels and nerves.2) DM is generally classified into two main types, namely insulin-dependent diabetes mellitus (IDDM) and noninsulin-dependent diabetes mellitus (NIDDM). IDDM is characterized by an absolute deficiency of insulin secretion, associated with auto-immune destruction of pancreatic b -cells.3) NIDDM, which accounts for more than 90% of all cases, is caused by a combination of resistance to insulin action and impaired insulin secretion.4) In NIDDM, muscle and fat cells are insulin resistant and compensatory secreted insulin is not sufficient to maintain blood glucose level within a normal physiological range.5) Commonly practiced pharmacologic treatment of DM includes oral hypoglycemic agents and insulin. There is an increasing demand by patients for the use of natural products and other dietary modulators with anti-diabetic activity. This tendency is because insulin, to date, cannot be used orally and its repeated injections had many undesirable adverse effects. In addition, certain oral hypoglycemic agents are not effective in lowering the blood glucose level in chronic diabetic patients.6) Traditional herbal medicine has been widely used for disease treatment and is recognized as an interesting alternative to conventional medicine.7—9) The uncertainty about the effi∗ To whom correspondence should be addressed.
cacy and safety of the currently available oral hypoglycemic drugs has prompted a search for safer and more effective agents in the treatment of diabetes.8) A wide variety of the traditional herbal remedies being used by diabetic patients, especially in the third world countries,9) and may, therefore, represent new avenues in the search for alternative hypoglycemic drugs. Generally, many herbal drugs are combined in the form of a multi-herbal formula to enhance their functions in the traditional medicine of Asian countries. In formulating these traditional herbal medicine recipes, special herb pairs, which are claimed to be unique combinations of traditionally defined medicinal herbal properties,10) are frequently used for achieving mutual enhancement, assistance, restraint, suppression, or antagonism.11) The herbal constituents are selected to emphasize the therapeutic actions or to reduce the toxicity or side effects of compounds from other herbal species in the mixture.12) The anti-diabetic effects of many traditional herbs or their fractions were reported but the effects of a multi-herbal formula, used practically, on experimental diabetes have rarely been studied. Therefore, a new herbal formula (NHF) has been composed based on the hypoglycemic effect of each herb including Polygonati Rhizoma (Polygonatum sibiricum REDOUTE), Rehmanniae Radix Crudus (Rhemannia glutinosa LIBOSCHITZ), Salviae miltiorrhizae Radix (Salvia miltriorrhiza BUNGE), Puerariae Radix (Pueraria lobata OHWI), Schizandrae Fructus (Schizandra chiensis BAILLON) and Glycyrrhizae Radix (Glycyrrhiza uralensis FISCHER) for the development of anti-diabetic treatment. Here the anti-hyperglycemic effects of NHF were investigated in neonatal Strep-
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tozotocin (nSTZ)-induced NIDDM rats. MATERIALS AND METHODS Preparation of the New Herbal Formula NHF is composed of six medicinal herbs. This formula was organized to maximize the pharmacological effect in the theoretical basis of traditional Oriental medicine. The ingredients of NHF include Polygonati Rhizoma, Rehmanniae Radix Crudus, Salviae miltiorrhizae Radix, Puerariae Radix, Schizandrae Fructus, Glycyrrhizae Radix at the ratio of 6 : 6 : 6 : 3 : 3 : 1.2. The mixing ratio was based on a herbalogy13) and prescription from oriental medical doctor Dam Huh of Taeeulyangsaeng oriental medicine clinic. All herbs used in this study were purchased from Omniherb Co. (Yeongcheon, Korea) and authenticated. These mixtures were boiled with distilled water (50 g/l) for 150 min and filtered. Finally, the filtered solution containing the extract was evaporated, lyophilized and powdered. The yields of NHF extract were 20.6%. The powdered NHF was stored at 20 °C until it was put to use. Induction of Diabetes Mellitus in Rats Healthy 10 heads of timed-pregnant Sprague-Dawley rats were purchased from Orient Bio (Seongnam, Korea) and kept for breeding. Animals were maintained in environmentally controlled conditions with a 12 h light/dark cycle with a temperature of 221 °C and relative humidity 505%. The animals were fed with a laboratory pellet chow (Purina Korea Inc., Korea) and water ad libitum during the experiment. To induce NIDDM, male neonatal rats were intraperitoneally injected at 2 d of age with 70 mg/kg of STZ (Sigma Chemical Co., U.S.A.). Injection of STZ at this age results in impaired insulin secretion and a condition resembling human NIDDM.14) Control littermates received an injection of citrate buffer (0.1 M, pH 4.5) alone. The animals were weaned at 21 d of age. After 7 weeks, NIDDM was confirmed by checking for 4 h fasting blood glucose levels using a glucometer (Abbott Laboratories Medisense Products, Bedford, MA, U.S.A.) and the 20 heads of male animals with blood glucose concentration more than 200 mg/dl were used for the study. All animal experimental procedures were conducted in accordance with Kyungpook National University Guidelines for the Care and Use of Laboratory Animals. Experimental Design and Tissue Collection A total of 30 male rats (10 normal control: 20 nSTZ-diabetic) were used in this study. The rats were randomly divided into the following three groups of 10 animals each: group I, normal control rats; group II, nSTZ control rats; group III, the nSTZ diabetic rats that received NHF by daily oral administration (300 mg/kg body weight) for 4 weeks. The dosage of NHF in this study was adopted based on each dosage in traditional prescriptions in Oriental medicine and blood glucose results of oral glucose tolerance test (OGTT) at doses of 0, 50, 100, 300, 500 mg/kg in the pilot test. And then, the blood glucose lowering effect of NHF was dose-dependent manner and doses of 300 and 500 mg/kg had shown the almost same hypoglycemic effect. The nSTZ control group was received an equal volume of the distilled water. The following parameters were examined in the study groups during the treatment period: weekly body weight, food and water intake, blood glucose concentration and plasma insulin level. Blood sam-
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ples were collected via the ophthalmic venous plexus after a 4 h fast and blood glucose levels were determined by glucometer. Plasma insulin levels were assayed using an enzyme-linked immunosorbent assay kit (Mercodia, Uppsala, Sweden). At the end of the experimental period (on the 29th day), the rats were deprived of food for 6 h and blood was collected for the estimation of blood glucose, insulin levels. Plasma was separated for the estimation of insulin. Oral Glucose Tolerance Test (OGTT) OGTT were performed in all animals in each group. After 4 weeks of NHF treatment (on the 28th day), a set of blood samples, following 6 h of fasting were taken from all groups. Four more blood samples were collected at 30, 60, 90 and 120 min intervals after the oral administration of 2 g/kg glucose for the determination of glucose. Determination of Tissue Glucose Transporter-4 and Phosphoenolpyruvate Carboxykinase mRNA Expression The soleus muscle and liver from 5 animals in each experimental group were immediately removed, frozen in liquid nitrogen, and stored at 70 °C for the determination of gene transcripts. The mRNA of glucose transporter-4 (GLUT-4) in soleus muscle and phosphoenolpyruvate carboxykinase (PEPCK) in the liver were determined by reverse transcription-polymerase chain reaction (RT-PCR). The total RNA was isolated using easy-BLUE (iNtRON Biotec, Korea). An aliquot of 3 m g of total RNA from each sample was reversetranscribed to cDNA using the first-strand cDNA synthesis kit (Bioneer, Korea). PCR amplification of 1 m l of cDNA was carried out in a final volume of 20 m l with random hexamers as primer. The primer sequences used were as follows: b actin (forward: 5-AGTACCCCATTGAACGC-3 and reverse: 5-CATGGGTCCGTAACGACTGT-3), GLUT-4 (forward: 5-CAACGTGGCTGGGTAGGCA-3 reverse: 5-TGGCCGACCCGACTACACA-3) and PEPCK (forward: 5AGTTGAATGTGTGGGTGATGACA-3 reverse: 5-AAAACCGTTTTCTGGGTTGATG-3). The standard amplification program included 30—40 cycles, which involved heating the product to 94 °C with a 30 s hold, annealing to 56— 60 °C with a 30 s hold, and extending to 72 °C with a 45— 60 s and final elongation step of 72 °C for 10—15 min. The PCR products were separated by electrophoresis on 1— 1.5% agarose gel, visualized by ethidium bromide staining under ultraviolet light using Eagle eyes image analysis software (Stratagene Co., La Jolla, CA, U.S.A.). The abundance of each specific mRNA was normalized on the basis of that b -actin mRNA. Immunohistochemical Evaluation on Pancreas The pancreas was removed immediately from the animals after sacrifice and rinsed in ice-cold saline. The tissue samples were fixed in 10% neutral buffered formalin, dehydrated in a graded series of ethanol, and embedded in paraffin wax before sectioning. Paraffin block samples were sectioned with 5 m m thickness and stained with hematoxylin and eosin for histological observation. Sections were dewaxed and rehydrated. After the step of washing in phosphate-buffered saline, sections were immersed in a solution of 3% H2O2 for 10 min. The sections were then pre-incubated with nonimmune serum for 15 min and subsequently replaced with the mouse anti-insulin antibody (1 : 200, Dako, Santa Barbara, CA, U.S.A.) for incubation at 4 °C for 16 h. Biotinylated goat anti-mouse immunoglobulin was used as a secondary anti-
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423 Table 1. Effect of NHF Treatment on Food and Water Intake in nSTZInduced Diabetic Rats for 4 Weeks Groups Normal nSTZ control nSTZNHF
Food intake (g/d) 28.613.21 51.485.33* 35.832.94*,#
Water intake (ml/d) 26.382.23 41.167.64* 28.892.77*,#
Each value is the meanS.E.M. ∗ p0.05 versus normal control; nSTZ control.
#
p0.05 versus
Fig. 1. Effect of the NHF Treatment on Body Weight in nSTZ-Induced NIDDM Rats for 4 Weeks Values are meanS.E.M. from 10 animals in each group. ∗ Significantly different from normal (p0.05). # Significantly different from nSTZ control (p0.05).
body. They were labelled with streptavidin peroxidase following incubation with the secondary antibody at 37 °C for 30 min. The localization of the antigen was indicated by a brown color obtained with 3-amino-9-ethyl-carbazole (AEC) as chromogenic substrate for peroxidase activity. Statistical Analysis The data from the experiments are presented as meanS.E.M. Student’s t-test was used for statistical analyses (SAS software, SAS Institute, Cary, NC, U.S.A.). Values were considered statistically significant when p0.05.
Fig. 2. Effect of the NHF Treatment on Blood Glucose Level in nSTZInduced NIDDM Rats for 4 Weeks Values are meanS.E.M. from 10 animals in each group. ∗ Significantly different from normal (p0.05). # Significantly different from nSTZ control (p0.05).
RESULTS Effects of NHF on Body Weight Change The body weights of experimental animals are shown in Fig. 1. Body weight gains of the nSTZ control group were significantly lower than those of the normal group in the corresponding time periods (p0.05). However, body weight gains of the NHF treated group significantly increased thereafter 4 weeks compared with the nSTZ control group. Intakes of Food and Water The food and water intakes in experimental animals are shown in Table 1. The nSTZ-induced NIDDM resulted in significantly elevated intakes of both food and water. The food and water intakes of NHF treated diabetic rats were significantly (p0.05) reduced compared with nSTZ control rats. Effects of NHF on Blood Glucose Levels Figure 2 shows the changes in blood glucose levels of experimental groups. The nSTZ diabetic rats showed a significant increase in the level of blood glucose compared with the normal control group. Blood glucose levels of nSTZ control rats were reduced after 1 week administration of NHF for 4 weeks. At 4 weeks after treatment, a time-dependent decrease of blood glucose was observed in NHF-treated diabetic rats. The percentage reduction in glucose levels after 4 weeks in the NHFtreated diabetic rats was 46.3%. Effect of NHF on Plasma Insulin The plasma insulin level in normal control rats was constantly maintained during the experiment and nSTZ diabetic rats produced a drop in the plasma insulin level from 1 week to 4 weeks after treatment compared with normal control (Fig. 3). The plasma insulin level increased from 0.95 to 2.03 m g/dl after 4 weeks in
Fig. 3. Effect of NHF on Plasma Insulin Level in nSTZ-Induced NIDDM Rats for 4 Weeks Values are meanS.E.M. from 10 animals in each group. ∗ Significantly different from normal (p0.05). # Significantly different from nSTZ control (p0.05).
nSTZ diabetic rats treated with NHF. Effect of NHF on Oral Glucose Tolerance Test (OGTT) Figure 4 illustrates changes in the blood glucose levels during OGTT. In the nSTZ-induced NIDDM rats treated with NHF for 4 weeks, the levels of blood glucose obtained 30, 60, 90 and 120 min after glucose intake (2 g/kg body weight) were significantly lower than those in nSTZ diabetic rats. Effects of NHF on mRNA Expression of GLUT-4 in Soleus Muscle and Hepatic PEPCK Relative changes in mRNA expression of GLUT-4 in the soleus muscle and hepatic PEPCK are illustrated in Fig. 5. The mRNA level of GLUT-4 in the soleus muscle isolated from nSTZ diabetic rats was 58% of that from normal rats. The value for GLUT-4
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mRNA in the soleus muscle isolated from nSTZ diabetic rats was approximately 76% of that for normal rats. The nSTZ diabetic rats treated with NHF resulted in an elevation of GLUT-4 mRNA level in the soleus muscle to values approaching those of normal rats. The PEPCK mRNA level in the liver, isolated from nSTZ diabetic rats, was increased approximately 4.8% of that from normal rats. After treatment with NHF, hepatic PEPCK mRNA was reduced to approximately 56% of that for normal control. Histological Changes and Immunohistochemical Evaluation on Pancreas Figure 6 demonstrates the histological and immunohistochemical appearances of pancreas in experimental rats. Tightly arranged pancreatic islet architectures were observed in normal rats (Fig. 6A). In the nSTZ diabetic group, extensive cell lysis, atrophy, decreases in numbers, and vacuolization of islets, and invasion of connective tissues in the parenchyma of pancreatic islets were detected (Fig. 6B). However, these abnormal histological appearances markedly decreased in the NHF treatment group as compared
to nSTZ control (Fig. 6C). In normal rats, the islets showed the normal structure with a large central core formed by insulin-secreting b -cells (Fig. 6D). The numbers of immunoreactive insulin-producing b -cells in nSTZ control group were remarkably reduced in restricted pancreatic islets compared to normal control (Fig. 6E). However, these diabetic-induced changes of pancreas were partially reversed by NHF treatment (Fig. 6F). DISCUSSION Diabetes mellitus is the chronic metabolic disorder and is
Fig. 5. Representative Images Indicating the mRNA Level for GLUT-4 and b -Actin in the Soleus Muscle, and the mRNA Level for PEPCK from the Liver Fig. 4. Effect of the NHF Treatment on Oral Glucose Tolerance Test (OGTT) in nSTZ-Induced NIDDM Rats for 4 Weeks OGTT was carried out at the end of 4 weeks of NHF treatment. All rats fasted for 6 h before OGTT. Blood was taken from the ophthalmic venous plexus at 0, 30, 60, 90 and 120 min after the oral glucose administration (2 g/kg body weight). Plasma glucose concentration was determined by the glucose oxidase method. Data are meansS.E.M. of 10 animals in each group. ∗ Significantly different from normal (p0.05). # Significantly different from nSTZ control (p0.05).
Fig. 6.
The mRNA level of GLUT-4 in the soleus muscle of nSTZ diabetic rats was significantly decreased compared with normal rats and that of NHF-treated nSTZ diabetic rats was significantly higher than nSTZ diabetic control rats. The hepatic PEPCK mRNA level in nSTZ diabetic rats was not significantly different from that of normal rats. After treatment with NHF, hepatic PEPCK mRNA was significantly reduced to approximately 56% of that for normal control. The mRNA levels were analyzed in 5 animals in each group. Lane 1: normal. Lane 2: nSTZ control. Lane 3: NHF treated nSTZ diabetic group. * Significantly different from normal (p0.05). # Significantly different from nSTZ control (p0.05).
Histological Observations and Immunohistochemical Staining for Insulin of Pancreas in NHF Treated nSTZ-Induced NIDDM Rats
Normal (A, D), nSTZ control (B, E), NHF treated nSTZ diabetic group (C, F). Atrophy and reduction of islet cell numbers were showed in nSTZ control group (Fig. 6B). However, the NHF treatment reduced these diabetic changes compared to nSTZ control (Fig. 6C). Hematoxylin and eosin stain; original magnification, 400. There was condense cords of insulin positive b -cells in normal control group (Fig. 6D). However, only a few insulin-producing islet cells were restrictively distributed in the pancreas of nSTZ diabetic rats (Fig. 6E). In the NHF-treated rats, restored density of insulin-positive cells in pancreatic islets was observed compared to nSTZ control group. Scale bars, 100 m m.
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characterized by high blood glucose levels.15) Worldwide, the number of patients is rapidly growing with an increase in obesity and aging in the general population.16) The best way to control postprandial plasma glucose levels is with medication in combination with dietary restriction and an exercise program.17) NHF is consisted of Polygonati Rhizoma, Rehmanniae Radix Crudus, Salviae miltiorrhizae Radix, Puerariae Radix, Schizandrae Fructus, and Glycyrrhizae Radix. In traditional Oriental medicine, Polygonati Rhizoma,18) Rehmanniae Radix Crudus,19) Puerariae Radix20) and Schizandrae Fructus21) have been used mainly for the decrease of blood glucose. Traditionally, Salviae miltiorrhizae Radix has been widely used for the treatment of coronary heart disease, cerebrovascular disease, hepatitis, hepatocirrhosis, chronic renal failure, dysmenorrheal and neurasthetic insomnia.22) Glycyrrhizae Radix is one of the oldest and most frequently used botanicals in Oriental medicine. Glycyrrhizae Radix extract is recommended for life-enhancing properties and cure of injury or swelling as well as for detoxification.23) It was hypothesized that NHF might have antidiabetic activity based on traditional use in hyperglycemia. Thus, the effects of NHF in neonatally streptozotocin-induced diabetic rats were investigated. This NIDDM model serves as a nonsevere diabetic state and it constitutes a valuable pharmacological tool for antidiabetic drug research, because most of the diabetic patients are non-insulin dependent in nature.24) In nSTZ diabetic rats, insulin secretion is markedly abnormal at an adult age despite only mild or moderate hyperglycemia25) and adult rats become b -cell deficient and show hypoinsulinemia with relatively mild diabetes.26) During treatment with NHF in nSTZ diabetic rats, body weight gain of the NHF-treated group significantly increased thereafter 4 weeks compared with the nSTZ control group. Also the food and water intakes of the NHF-treated diabetic rats were significantly reduced compared with the nSTZ control rats. These results indicated that NHF may have a metabolic promotion effect on body tissue and improvement on polyphagia and polydipsia. In addition, prolonged administration of NHF at a dose of 300 mg/kg for 4 weeks caused a significant decrease in the level of blood glucose, and also showed a significant recovery effect on the plasma insulin level compared to the nSTZ control group. These results support that NHF might improve diabetes by normalizing the postprandial plasma glucose level as well as fasting blood glucose level. And this, in turn, suggests that in conditions like hyperinsulinemia, NHF increases the insulin sensitivity for effective glucose disposal. OGTT is one of the most critical criteria for evaluating the effectiveness of hypoglycemic drugs.27) NHF might enhance glucose utilization because they significantly reduced the blood glucose level in nSTZ diabetic rats. In glucose-fed diabetic rats, the elevated blood glucose levels remained higher after 120 min. NHF significantly inhibited increases in the blood glucose level during the OGTT in nSTZ diabetic rats. NHF significantly improved glucose tolerance and this fact could be attributed to the potentiation of the insulin effect of plasma by increasing the pancreatic secretion of insulin from existing b -cells or its release from bound insulin. Glucose transport is the rate-limiting step in carbohydrate metabolism.28) A family of glucose transporters (GLUT) me-
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diates glucose transport across the cell membrane, and the GLUT-4 is the insulin sensitive glucose transporter. Reduction in insulin-mediated glucose uptake into skeletal muscle caused by decreasing GLUT-4 protein in DM, especially NIDDM, was observed.29,30) Thus, compounds that facilitate GLUT-4 translocation can be potentially beneficial for the treatment of DM. The decrease in GLUT4 expression due to induction of nSTZ-induced NIDDM was found to be increased by NHF. An increase of GLUT-4 mRNA expression may contribute to plasma glucose regulation in NHF-treated nSTZ diabetic rats. PEPCK, which catalyzes a regulatory step in gluconeogenesis, is one of the key enzymes of hepatic carbohydrate metabolism and insulin deficiency is clearly associated with changes in hepatic metabolism including increased expression of PEPCK.31) In this study, NHF treatment the reversed increase of hepatic PEPCK mRNA expression in nSTZ diabetic rats and this attenuation of the hepatic PEPCK mRNA expression was associated with plasma glucose-lowering activity of NHF. These findings suggest that NHF exerts its glucose-lowering effect mainly through an enhancement of glucose utilization of skeletal muscle and a reduction of hepatic gluconeogenesis, although the detailed action mechanism needs further investigation. In DM, hyperglycemia is held to be the consequence of increased hepatic glucose output in concert with reduced peripheral glucose utilization.31) It has been reported that the antidiabetic effect of some drugs such as metformin and myricetin via normalizations of hepatic PEPCK and muscle GLUT-4 mRNA expressions in nSTZ diabetic rats might be mediated through an activation of opioid receptors. It is likely, therefore, that the normalizations of muscle GLUT-4 and hepatic PEPCK mRNA expressions in nSTZ diabetic rats following NHF treatments might both be mediated through an activation of opioid receptors. Therefore, further investigations involving gluconeogenesis and glucose utilization need to be clarified in the future. STZ selectively destroys pancreatic b -cells, inhibits the synthesis and release of insulin, and causes the onset of diabetes mellitus.32) In the nSTZ-induced NIDDM rats at adult ages, inflammatory changes, atrophy, decrease in numbers and destruction of pancreatic insulin-positive islets cells were observed. However, after the treatment of NHF, the insulin immunoreactivity was improved and an increase in the number of immunoreactive b -cells was observed in comparison to the nSTZ control. The inhibition of histological and immunohistological changes in the pancreas are considered to be direct evidence that NHF improves DM. A decrease of the blood glucose level and an increase of the serum insulin concentration and b -cell mass were significantly time-dependent on NHF treatment compared to nSTZ control. These results indicated that the NHF treatment was effective in controlling hyperglycemia in the nSTZ-induced NIDDM model for 4 weeks. NHF is composed of six herbs which are used with the food materials. Thus, NHF may be useful for improving overall glycemic control and reducing the risk of diabetic complications. Acknowledgements This work was supported by a grant (Technical Development Project No. 10018069) from Regional Industrial Promotion Projects by Ministry of Knowledge Economy, Republic of Korea.
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