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Ameliorating effect and potential mechanism of Rehmannia glutinosa oligosaccharides on the impaired glucose metabolism in chronic stress rats fed with high-fat diet.

ABSTRACT

The aim of this study was to determine whether the Rehmannia glutinosa oligosaccharides (ROS) ameliorate the impaired glucose metabolism and the potential mechanism in chronic stress rats fed with high-fat diet. The rats were fed by a high-fat diet and simultaneously stimulated by chronic stress over 5 weeks. Body weight, fasting plasma glucose, intraperitoneal glucose tolerance test (IPGTT), plasma lipids, gluconeogenesis test (GGT), glycogen content, and corticosterone, insulin and leptin levels were measured. The results showed that ROS administration (100, 200mg/kg, i.g.) for 5 weeks exerted the effects of increasing the organ weights of thymus and spleen, lowering the fasting plasma glucose level, improving impaired glucose tolerance, increasing the contents of liver and muscle glycogen, decreasing the gluconeogenesis ability, plasma-free fatty acid's level, as well as plasma triglyceride and total cholesterol levels in chronic stress and high-fat fed rats, especially in the group of 200 mg/kg; while the plasma corticosterone level was decreased, and plasma leptin level was increased. These results suggest that ROS exert an ameliorating effect of impaired glucose metabolism in chronic stress rats fed with high-fat diet, and the potential mechanism may be mediated through rebuilding the glucose homeostasis in the neuroendocrine immuno-modulation (N1M) network through multilinks and multitargets.

Keywords:

Rehmannia glutinosa oligosaccharides (ROS)

Chronic stress

High-fat diet

Impaired glucose metabolism

HPA axis

Introduction

Diabetes mellitus (DM), especially type 2 diabetes mellitus (T2DM), is rapidly increasing over recent years in developing countries, such as China, India, in major part as a consequence of the worldwide "epidemic" of obesity (Meddah et al., 2009). Type 2 diabetes, with the main characteristics of insulin resistance (1R), not only causes an impaired glucose metabolism, but also is the central link of a series of diseases known as the "metabolic syndrome" or "Syndrome X" (Obunai et al., 2007). Although there are many oral hypoglycemic agents used for treatment of type 2 diabetes, some potentially serious side effects have been reported with several of these compounds, especially the new agents (Philippe and Raccah, 2009). This situation has increased the research interests in traditional Chinese medicine (TCM) to find more effective herbs, plants or their extracts to treat T2DM and enhance the quality of patients' life.

Rehmannia glutinosa, a widely used traditional Chinese herb, belongs in the family of Scrophulariaceae, and it is highly esteemed in China as a high medicinal valued herb of nourishing Yin and invigorating the kidney functions in traditional Chinese medicine (Zhang et al., 2008a). Rehmannia glutinosa oligosaccharide (ROS) is the main part in water extract of fresh Rehmannia glutinosa, and among ROSs, the content of stachyose is 48.3% (Tomoda et al., 1971). In our previous studies, the experiments in vitro demonstrated that ROS not only had the promotion of the glucose consumption to improve the dexamethasone-induced IR in the 3T3-L1 adipocyte cell model (Guo et al., 2006), but also have the ability to improve the insulin resistance in HepG2 cell model (Guo et al., 2007).

In 1977, Besedovsky and Sorkin (1977) proposed the theory of neuroendocrine immuno-modulation (N1M). After more than thirty years, a series of evidence has demonstrated that the theory is reasonable (Steinman, 2004). Today, the theory is helpful to explain the pathogenesis of a variety of clinical diseases such as rheumatoid arthritis (Straub and Besedovsky, 2005), depression (Porter et al., 2004), endometriosis, metabolic syndrome (Tariverdian et al., 2007) and diabetes mellitus (Anagnostis et al., 2009). It is supposed by our research team that the regulatory function of NIM network in diabetes mellitus is usually disordered, especially the glucose homeostasis, which is regulated by two types of factors in NIM networks, including the hormone lowering blood glucose (insulin) and the counter-regulatory hormones, such as glucagon, glucocorticoids, epinephrine and growth hormone. From the point of view of NIM theory, the counter-regulatory hormones, especially the HPA axis, may play an important role in the development and progression of type 2 diabetes (Zhang et al., 2002, 2005).

The current study aimed to investigate the ameliorating effect and potential mechanism of ROS on in chronic stress rats fed with high-fat diet. A range of parameters were tested including plasma glucose level, gluconeogenesis ability test (GTT), glycogen content, corticosterone, insulin and organ index, etc., to explore the potential mechanism of improvement of ROS on impaired glucose metabolism in the rats.

Materials and methods

Extraction and isolation of ROS

Rehmannia glutinosa was obtained from Anguo County, Hebei Province of PR China and authenticated to be Rehmannia glutinosa Libosch. by Professor Yongjian Yang, College of Pharmacy, Lanzhou University. The voucher specimen of No. 20010 was deposited in the Department of Pharmacy, Lanzhou General Hospital of Lanzhou, PLA. The fresh roots (1000g) were crushed and extracted with hot water (3000 ml) for 60 min three times. After suction filtration, all extracts were collected and concentrated to 1000 ml, then applied to a column of cation exchange resin. After washing with water, the eluates were collected and concentrated, then applied to a column of anion resin. Washing with water again, the eluates and washing with positive Molisch reaction were collected, concentrated, and vacuum dried. The yields were 201.9 g (about 20.19% of raw materials), which was further separated by charcoal column chromatography. Active charcoal was treated with hot 15% AcOH before use and then washed by distilled water. The water solution of the neutral fraction (50 g) was applied at the top of the activated charcoal column (5.4cm x 97 cm), followed by successive elution with water, 5, 10, 15, 20, and 30% ethanol, and 3.5% ammonia water. Fractions were collected at 50 ml and carbohydrates in eluates were measured by the phenol-sulfuric acid method. The eluates obtained from the column were divided among four groups: part I (3.21 g) mainly composed of monosaccharides and disaccharides: part II (13.80 g) mainly trisaccharides, part III (11.57 g) mainly tetrose, and part IV (2.91 g) is other oligosaccharide. Part III was identified by HPLC as stachyose (about 60%) (Fig. 1) and manninotriose (about 30%) and this part was applied during the experiment (Tomoda et al., 1971; Zhang et al., 2004a).

Reagents

The kits of radioimmunoassay (RIA) for 125I-insu!in and 125Icorticosterone were purchased from Tianjin Jiuding Medicine Bioengineering Co. Ltd. (Tianjin, China): 50% Glucose Injection from Hunan Middle-south Cologne Pharmaceutical Co. Ltd. (Hunan, China): L-alanine was purchased from Sigma (St. Louis, MO). The kit for blood glucose, total cholesterol (TC) and triglyceride (TG) was purchased from Sichuang Mike Technology Co. Ltd. (Sichuang, China). The kits for free fatty acids (FFA) were purchased from Nanjing Jiancheng Bioengineering Institute (Jiangsu, China). Total saponins of Panax Ginsen (TSPG) were obtained from Jiling Hongjiu Biotechnology Co. Ltd. (Jiling, China), purity > 98%. Organic solvents and other chemicals were of the highest analytical grade.

Animal used

Male Wistar rats (180-220 g) were supplied by the Animal Facility Center of Lanzhou University. The animals were allowed to acclimatize for at least 6 days before the experiments. Each experimental group was consisted of ten animals. The animals were maintained at controlled temperature (18-25)[degrees]C and relative humidity (40-60%) with a L2-h light/dark cycle. Food and water were available ad libitum. Animal care and treatment were conducted complied with the rulings of Gansu Experimental Animal Center (Gansu, China) officially approved by the Ministry of Healthy, PR China, in accordance with NIH animal care guidelines.

High-fat diet

The standard rat chow diet contains L3.2 KJ/g calories, 54% carbohydrate, 4% lipid. The high-fat diet was consisting of 40% standard diet, 20% eatable lard, 5.L% carbohydrate, 34% egg (w/w) and 0.9% NaCl, in which the calorie of diet was 22.L KJ/g, and saccharides 25%, fat 60.17%, made by the Animal Experimental Center of Lanzhou General Hospital, PLA, China (Tian et al., 2006).

Stress protocol

The stress method was improved based on the report by Zardooz et al. (2006): (1) absorbent gauze wrap secured with tape, (2) restraint in a polyvinyl chloride tube (L = 25 cm, ID = 5 cm) closed at either end, (3) immobilization on a board with tape, (4) the tail suspension. The stress protocol involved exposure, for a 2-h period between 09:00 and 11:00 am, to one of the first three restraint stressors but a half-hour between 09:00 and 09:30 am to the fourth stressor once daily. The animals were exposed in turn to one of the above stressors once a day, and were then returned to the animal facilities 15 min following stress exposure to minimize disturbance to the control group.

Experimental protocols

Induction of chronic stress rat model combined with high-fat diet: After one week acclimation period, rats were randomly divided into 5 groups (n = 10): normal control group (control group, distilled water 2ml/kg), chronic stress model group (CS group, distilled water 2 ml/kg), low-dose ROS-treated group (L-ROS group, 100mg/kg), high-dose ROS-treated group (H-ROS group, 200 mg/kg) and TSPG-treated group (TSPG group, 200 mg/kg). The rats were then treated with drugs by gavage from 8:00 to 9:00 am, but control group and CS group were only administered with the equal volume of distilled water. After drug administration, control group was fed a standard diet and did not expose to any stressors, whereas the experimental groups were fed high-fat diet and simultaneously stimulated by chronic stress for 5 weeks. Then the rats were euthanized by decapitation, and trunk blood and tissue samples were collected for subsequent analysis.

Collection of blood and tissue's samples: All rats were fasted for 6 h (8:00-14:00), anesthetized slightly with diethyl ether, and blood samples of the 6h-fasted rats were collected immediately from retrobulbar venous plexus with capillary tubes under ether anesthesia and with 0.1 M EDTA as an anticoagulant, centrifuged at 2900 x g for 10min to remove the contaminations and separate the plasma. At the end of experiment, the rats were sacrificed by decapitation and trunk blood was collected, centrifuged immediately to separate the plasma for determination, or stored at -70[degrees]C. The tissues of liver, thymus, spleen, adrenal gland and kidney were removed, weighed, and stored at -70[degrees]C until assay.

Measurement of fasting plasma glucose levels: Fasting plasma glucose level was estimated by commercially available glucose kit based on the glucose oxidase method. The results were expressed as mmol/1.

IPCTT (intraperitoneal glucose tolerance test): All animals were fasted for 6h and were then injected intraperitoneally with a single dose of 50% glucose at a concentration of 2.5 g/kg body weight. The drugs were administered 60 min prior to the glucose load. The blood samples were collected from each group just before glucose administration (0 min) and at 30, 60 and 120 min after glucose administration. Plasma glucose levels were determined by the glucose oxidase method. 1PGTT was performed twice on the d14 and d28 of the experiment, respectively.

Measurement of plasma lipid levels: Plasma total cholesterol level and triglyceride level were measured by COD-CE-PAP method. The results were expressed as mmol/1.

Cluconeogenesis test (CCT): All rats were fasted for 6h (8:30-14:30), injected intraperitoneally with a single dose of L-glutamic acid at a concentration of 1.5 g/kg. The blood samples were collected from each group just before L-glutamic acid administration (Omin) and at 30, 60 and 90 min after L-glutamic acid administration. Plasma glucose level was determined by the glucose oxidase method. If there is an increase in level of blood glucose after L-glutamic acid administration, it is demonstrated that the ability of gluconeogenesis is enhanced (Tian et al., 2006).

Measurement of glycogen levels (anthrone-sulfuric acid colorimetric assay): According to the manufacturer's instruction, glycogen content of liver and skeletal muscles was measured by the previously established method (Sadasivam and Manickam, 1996). The amounts of glycogen in the tissue samples were expressed as mg of glucose/g tissue (wet weight).

Measurement of free fatty acid (FFA) levels: FFA was measured by biscydohexanoneoxaiyldihydraone coloration method using commercial assay kits according to the manufacturer's directions. The amounts of FFA were expressed as mmol/l.

Measurement of plasma insulin, corticosterone and leptin levels: plasma levels of insulin, corticosterone and leptin were estimated using commercially available radio-immunoassay kits for rat's insulin, corticosterone and leptin, according to the manufacturer's instructions. The results of plasma insulin level were expressed as [micro]lU/ml, and corticosterone and leptin levels were expressed as ng/ml.

Statistical analysis

Results of every group are expressed as mean [+ or -] SD. Comparisons between groups were made using Student's t-test. When an effect was statistically significant (p < 0.05), mean comparisons were done.

Results

Effects of ROS on body weights and organ index in chronic stress rats fed with high-fat diet

Table 1 shows that the body weights in CS group were slowly decreased from the second week during the experiment, compared with control group. After 5 weeks of ROS administration, weight gains in both of H-ROS and L-ROS groups were by 14.59% (p < 0.05) and 9.19% (p<0.05), respectively, compared with CS group. TSPG (200 mg/kg) showed the similar effect and after 5 weeks of administration, the body weight was increased by 10.27% (p<0.05).

As shown in Fig. 2, the index of thymus and spleen in CS group rats were significantly decreased whereas the adrenal gland index were slightly increased compared with the control group, the percentages of change were 33.05% (p<0.05), 27.27% (p<0.01) and 37.93% (p<0.05), respectively. In contrast, the index of thymus and spleen in TSPG group were increased by 39.24% (p<0.05) and 42.65% (p < 0.01), in H-ROS group by 51.90% (p<0.05) and 43.38% (p < 0.01), in L-ROS group by 50.63% (p < 0.05) and 58.09% (p < 0.01), respectively, compared with CS group. Besides, administrations of TSPG, H-ROS and L-ROS seem to decrease adrenal gland index, but only L-ROS showed the significance (p < 0.05).

Effects of ROS on fasting plasma glucose levels in chronic stress rats fed with high-fat diet

Fasting plasma glucose level in CS group was gradually increased from the third week during the experiment (Table 2) and after 5 weeks of administration of stress and high-fat diet, fasting plasma glucose level in CS group was increased by 19.67%. Both TSPG and HROS were found to decrease the hyperglycemia by 12.33% (p < 0.05) and 10.96% (p<0.05), respectively.

Effects of ROS on IPCTT in chronic stress rats fed with high-fat diet

On the d 14 of the experiment (Table 3), IPGTT was performed in each group. Plasma blood glucose levels at 30 min and 60 min after glucose loads in CS group were significantly increased by 46.04% (p<0.05) and 40.82% (p<0.05), respectively, compared with control group. Both L-ROS and H-ROS groups showed the trends of decreasing the hyperglycemia at 30 min, but the differences were not significant (p>0.05). However, the results of IPGTT on d28 (Table 4) showed the blood glucose levels at 30 min were significantly decreased by 21.24% (p<0.05) in L-ROS group and 26.55% (p < 0.05) in H-ROS group, but at 60 min still showed the decreasing of hyperglycemia by 23.53% (p<0.05) in H-ROS group, compared with CS group. TSPG also showed the similar effects of lowering the hyperglycemia at 30 min by 24.78% (p<0.05) and at 60 min by 22.79%.

Blood of ROS on lipids levels in chronic stress rats fed with high-fat diet

Fig. 3 represents that the rats in CS group had the elevated levels of plasma TG and TC. Treatment with H-ROS reduced plasma TC and TG levels by 36.30% (p < 0.05) and 40.57% (p < 0.05), respectively, while L-ROS only decreased plasma TC levels by 24.57% (p< 0.05), compared with CS group. Though treatment with TSPG showed a trend to lower plasma levels of TG and TC, the differences remain not significant. The results suggested that H-ROS exert better improvement to the dyslipidemia in CS rats.

Effects of ROS on CCT in chronic stress rats fed with high-fat diet

Table 5 shows that plasma glucose levels after L-glutamic acid injection in CS rats gradually became higher from 30, 60 and 90 min by 25.71% (p > 0.05), 30.38% (p <0.05) and 45.46% (p < 0.01), respectively, compared with the control group. Treatment with both H-ROS and TSPG can improve the GGT in CS rats. H-ROS administration decreased the plasma glucose level at 60 min and 90 min by 18.45% (p< 0.05) and 25.00% (p<0.05), respectively; L-ROS also showed a trend to lower plasma glucose level, but the difference was not significant. TSPG had the similar effect of lowering plasma glucose levels at 60 min and 90 min by 15.53% (p < 0.05) and 28.13% (p < 0.05), respectively, compared with CS group.

Effects of ROS on the glycogen contents of liver and muscle and plasma FFA level in chronic stress rats fed with high-fat diet

As showed in Fig. 4, the glycogen contents in liver and muscle in CS group were reduced by 35.67% (p<0.01) and 19.27% (p<0.05), respectively, compared to the control group. Treatment with HROS increased the liver and muscle glycogen contents by 20.00% (p < 0.05) and 13.64%, respectively, compared with CS group. TSPG did not show a significant effect to increase the liver and muscle glycogen contents (p>0.05) compared with CS group.

Fig. 5 indicates that the plasma FFA level in CS rats was elevated by 120.68% (p<0.01) compared with control group, and administrations with H-ROS and L-ROS lowered the plasma FFA level by 35.18% (p < 0.01) and 16.49% (p < 0.05), respectively, compared with CS group. TSPG administration showed the similar effect by 22.01% (p<0.05).

Effects of ROS on plasma corticosterone, insulin and leptin levels in chronic stress rats fed with high-fat diet

Fig. 6 indicates that the plasma corticosterone level was significantly increased by 73.91% (p < 0.01) whereas the insulin level was decreased by 31.01% (p < 0.05) in CS group compared to the control group. Both H-ROS and L-ROS lowered the plasma corticosterone level by 26.88% (p<0.05) and 22.50% (p<0.05), respectively; TSPG also showed the similar effect to plasma corticosterone level by 25.00% (p < 0.05), compared with CS group. However, the effects on the plasma insulin level of both ROS and TSPG were not significant (p>0.05).

Fig. 7 shows that the leptin level in CS group was significantly decreased by 25.89% (p< 0.05) compared to the control group. H-ROS significantly improved the leptin level by 22.89% (p<0.05), L-ROS and TSPG showed the trends to increase the plasma leptin level but there is not significant difference compared with CS group.

Discussion

Recent awareness of therapeutic potential of several traditionally used plants has opened a new dimension to the study and research on medicinal plants. In traditional medicine, several medicinal plants or their extracts are widely used in many countries for the treatment of diabetes mellitus (Akhtar and Ali, 1984). Rehmannia glutinosa Libosch. is a kind of Chinese traditional plant which is widely distributed throughout China. Traditionally, its root has been used to replenish vitality, strengthen the liver, kidney, and heart, and for treatment of a variety of ailments like diabetes, anemia, and urinary tract problems in China (Zhang et al., 2008a). It is usually used as the form of prescriptions in TCM, in which more than 46% of them use Rehmannia glutinosa as a principal herb in current times (Ye, 2001). Reports have shown that catalpol (Kitagawa et al., 1971; Huang et al., 2010) and polysaccharides (Kiho et al., 1992) were the main active components with the hypoglycemic effect, and our previous research showed that Rehmannia glutinosa oligosaccharide (ROS) exerted a significant hypoglycemic effect in normal and alloxan-induced diabetic rats, and its regulatory mechanism on glucose metabolism was adrenal dependent and had a close relation with the neuroendocrine system (Zhang et al., 2004b).

It is well known that high stress levels and high caloric intake are the important causes of type 2 diabetes and the metabolic syndrome in the developed countries and the modern society. In addition to persistent (chronic) stress caused by high-intensity work and life pressure, the improvement of living standards and improper food structure (high calorie, high fat food) are also the basic characteristics of the modern society. Plus less exercises, the above factors easily lead to obesity, which is associated with insulin resistance. More and more reported studies (Bjorntorp and Rosmond, 1999; Anagnostis et al., 2009; Kyrou and Tsigos, 2009) demonstrated there is a relationship among obesity, insulin resistance and HPA axis and the occurrence and development of diabetes are closely related to the disorders of neuroendocrine-immune modulation (NIM), especially the hyperactivity of HPA axis. Reports also demonstrated that basal HPA axis function is up regulated in uncontrolled or poorly controlled diabetes (Cameron et al., 1987; Roy et al., 1990; Chan et al., 2001, 2005). After many years of study, we found that there was an HPA axis dysfunction in chronic stress-induced diabetic rats with the decreased glucose tolerance, while after ROS administration, either the function of HPA axis or the plasma glucose levels were improved in diabetic rats induced by high-fat diet and low-dose streptozotocin (STZ) (Zhang et al., 2008b; Wang et al., 2010).

Based on the above points, the main aim of this study is to set up the chronic stress rat model combined with a high-fat diet, study the relationship between glucose metabolism and the functional changes of HPA axis and observe the pharmacological changes after ROS administration. TSPG was used as a positive control drug in this study, which has been demonstrated to have hypoglycemic effects and improvement of impaired HPA axis function induced by stress (Xie et al., 2005; Kim et al., 1999, 2003).

Firstly, we observed the changes in body weight and organ index caused by chronic stress and high-fat diet. The results showed that during 5 weeks of the experiment, the body weights of rats in CS group remained almost unchanged (8.82%), but that in normal control group were increased by 47.37%, administration of H-ROS can make 14.59% of body weight increase, and TSPG administration by 10.27%. Meanwhile, the organ index of immune organs such as thymus and spleen were also increased, but the weight of the adrenal gland was significantly increased only in CS group, which may be due to the provocation of stressors, was partially improved after ROS administration.

What more important is that after 5 weeks administration of chronic stress and high-fat diet, the glucose tolerance was significantly reduced in CS model rats, the change became obvious from the third week and in the fifth week, the fasting plasma glucose level increased by 19.67%, compared to normal control rats. Meanwhile, the intraperitoneal glucose tolerance test (IPGTT) was significantly decreased, with the increased capacity of gluconeogenesis. However, plasma insulin level was decreased, which was increased in other reports (Finger et al., 2012). With decreased liver and muscle glycogen content, we speculate that the lower plasma insulin may be due to impaired islet function rather than the structural damage because of long-term exposure of chronic stress and high fat diet, but it still needs to be further confirmed by islet pathological biopsy.

One of the interesting findings in our experiment is that H-ROS administration in CS rats showed a decreased fasting blood glucose level by 12.33%, and the increased gluconeogenesis was significantly inhibited. Furthermore, the contents of liver glycogen and muscle glycogen were also significantly increased by ROS treatment; the reason may be the stimulation of chronic stress in rats needs more energy supply, and ROS administration promoted the transportation of blood glucose to the liver glycogen, which, in fact, regulated the distribution of glucose in the blood and increased the storage of the glucose. The positive control drug, TSPG, also showed the similar regulatory effects on the glucose metabolism but with less effect on the liver and muscle glycogen.

Considering blood sample's limitation, OGTT test was performed twice times in our experiment, on the d14 and d28, respectively. After four points of blood samples were collected each time, the animals were given two weeks' break for the recovery of the whole blood cells. The results of OGTT showed that the changes of the d28 were more significant than that of the d 14, and the glucose tolerance in CS rats was significantly impaired, but treatment with ROS made a better improvement of IPGTT in CS rats. Our results also suggested that the change of plasma glucose in IPGTT, which came earlier than that of fasting plasma glucose, is a better index for the ethnopharmacological studies of anti-diabetic plants. Additionally, because of the association of lipid metabolism with glucose metabolism, plasma GT and GC levels were accordingly increased in CS rats and were improved by the administration of ROS.

The gluconeogenesis, which is a metabolic pathway that results in the generation of glucose from non-carbohydrate carbon substrates such as lactate, glycerol, and glucogenic amino acids, was determined by using L-glutamic acid administration in our experiment. The results showed that the gluconeogenesis was significantly increased in CS rats and can be inhibited by high-dose ROS. These results indicate that the hypoglycemic effects of ROS may be mediated through increasing the content of liver and muscle glycogen and decreasing the gluconeogenesis ability in CS rats.

Reports showed that insulin resistance usually associated with HPA axis dysfunction, which causes a lot of corticotropin-releasing factor (CRF) releases in the hypothalamus and then a massive release of corticosterone in adrenal. Our data showed that the plasma corticosterone level in CS model rats was increased by 73.9% (p<0.01), compared with the control group, suggesting that there is an HPA axis dysfunction in CS model rats, which can be improved by 5 weeks of ROS treatment with 16.9% decrease of the corticosterone levels (p<0.05) in L-ROS group and 21.5% (p< 0.05) in H-ROS group. Plasma insulin levels both in H-ROS and L-ROS groups were increased by 17.4% and 5.8%, respectively, indicating that the impaired islet function was also recovered by ROS administration.

Reports also showed that high FFA level can cause an increase insulin resistance in a variety of ways. Increased FFA level inhibits the glucose-stimulated insulin secretion and the biological activity of insulin in muscle and liver. Furthermore, FFA can inhibit the insulin-induced decrease of glucose uptake through its affecting on glucose transport and/or glucose phosphorylation, insulin-induced glycogen synthesis and glucose oxidation, and increase the hepatic glucose output and finally results in insulin resistance (Boden et al., 2002). Our results showed that the administrations of H-ROS and L-ROS can significantly decrease plasma FFA levels by 35.19% (p<0.01) and 16.5% (p< 0.05), respectively. Both the high and low doses of ROS, compared with CS group, decreased plasma FFA levels, suggesting it may be one of the mechanisms of ROS on impaired glucose metabolism in CS rats.

Put the above results together, it is supposed that the ameliorating effect of ROS on glucose and lipid's metabolism may be achieved by the regulations of the hyperactivity of the HPA axis and the disorder of NIM network in chronic stress rats fed with high-fat diet. Firstly, ROS exerted a direct effect to decrease the increased plasma corticosterone level in CS rats, which is helpful for the recovering of the hyperactivity of HPA axis; Secondly, the increased plasma corticosterone, the counter-regulatory hormone of insulin, has the anti-inhibition of liver glucose production caused by insulin secretion in CS rats, and leads to the increase of gluconeogenesis and glycogenolysis to increase the blood glucose level. The balance between insulin and corticosterone was out of control, which results in an impaired glucose tolerance and high plasma glucose level. Therefore, ROS exerts its regulatory effects of glycogen, gluconeogenesis through the modulation of the function of HPA axis. Finally, according to our data obtained, ROS expressed the comprehensive regulatory effects on the NIM network, such as immune organs, plasma leptin level, and plasma FFA level, and achieved its regulatory effects on glucose homeostasis by multilinks and multitargets in NIM network, which lead to the improvement of impaired glucose metabolism in chronic stress rats fed with high-fat diet.

Abbreviations: CS, chronic stress; DM, diabetes mellitus; FFA, free fatty acids; GGT, gluconeogenesis ability test; HPA, hypothalamic-pituitary-adrenal; IR, insulin resistance; N1M, neuroendocrine immuno-modulation; IPGTT, intraperitoneal glucose tolerance test; ROS, Rehmannia glutinosa oligosaccharide; T2DM, type 2 diabetes mellitus; TC, total cholesterol; TCM, traditional Chinese medicine; TG, triglyceride; TSPG, total saponins of Panax Ginsen.

http://dx.doi.org/10.1016/j.phymed.2013.11.008

ARTICLE INFO

Article history:

Received 4 April 2013

Received in revised form 13 October 2013

Accepted 30 November 2013

Acknowledgements

The authors would like to thank their laboratory members for valuable suggestions and technical assistance. This research was supported by the National Nature Science Foundation of China (grants 30772773 to Z.Rx. and 81173620 to Z.Rx.).

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Ruxue Zhang (a) *, Jun Zhou (a), Maoxing Li (a), Haigang Ma (a,b), Jianguo Qiu (a), Xiaohong Luo (c), Zhengping Jia (a) *

(a) Department of Pharmacy, Lanzhou General Hospital of PLA, Lanzhou, China

(b) College of Life Sciences, Lanzhou University, Lanzhou, China

(c) Department of Endocrinology. Lanzhou General Hospital of PLA. Lanzhou, China

* Corresponding author. Tel.: +86 931 8994658; fax: +86 931 8994003.

E-mail addresses: ruxuezh@hotmail.com (R. Zhang), jiazpl66@sina.com (Z.Jia).

Table 1
Effect of ROS on body weight (g) in chronic stress rats fed with
high-fat diet ([bar.x] [+ or -] SD).

Group     Dose (mg/kg/d)     0 week                1 week

Control   --                 171 [+ or -] 10       188 [+ or -] 10
CS        --                 170 [+ or -] 12       175 [+ or -] 12
TSPG      200                170 [+ or -] 15       172 [+ or -] 13
H-ROS     200                170 [+ or -] 11       177 [+ or -] 11
L-ROS     too                170 [+ or -] 13       170 [+ or -] 15

Group     2 week                3 week

Control   206 [+ or -] 11       218 [+ or -] 13
CS        172 [+ or -] 13 *     178 [+ or -] 13 *
TSPG      177 [+ or -] 12       184 [+ or -] 13
H-ROS     183 [+ or -] 12       190 [+ or -] 13
L-ROS     182 [+ or -] 12       189 [+ or -] 13

Group     4 week                5 week

Control   237 [+ or -] 15       252 [+ or -] 16
CS        180 [+ or -] 14 **    185 [+ or -] 15 **
TSPG      195 [+ or -] 14       204 [+ or -] 15 (#)
H-ROS     200 [+ or -] 12 (#)   212 [+ or -] 13 (#)
L-ROS     194 [+ or -] 14       202 [+ or -] 15 (#)

Values are expressed as means [+ or -] SD. Numbers of rats in each
group was 10.

* p < 0.05, compared with control group.

** p < 0.01, compared with control group.

(#) p <0.05, compared with CS group.

CS, chronic stress: TSPG, total saponins of Panax Ginsen: ROS,
Rehmannia glutinosa oligosaccharides.

Table 2
Effect of ROS on fasting plasma glucose level (mmol/l) in chronic
stress rats fed with high-fat diet ([bar.x] [+ or -] SD).

Group     Dose (mg/kg/d)     0 week               1 week

Control   --                 5.8 [+ or -] 0.4     6.0 [+ or -] 0.3
CS        --                 5.8 [+ or -] 0.3     6.2 [+ or -] 0.5
TSPG      200                5.9 [+ or -] 0.2     6.3 [+ or -] 0.6
H-ROS     200                5.8 [+ or -] 0.4     6.1 [+ or -] 0.5
L-ROS     100                5.9 [+ or -] 0.4     6.2 [+ or -] 0.5

Group     2 week               3 week

Control   6.1 [+ or -] 0.4     5.9 [+ or -] 0.5
CS        6.5 [+ or -] 0.6     7.0 [+ or -] 0.7 *
TSPG      6.4 [+ or -] 0.4     6.6 [+ or -] 0.7
H-ROS     6.5 [+ or -] 0.7     6.5 [+ or -] 0.7
L-ROS     6.5 [+ or -] 0.6     6.8 [+ or -] 1.0

Group     4 week                 5 week

Control   6.2 [+ or -] 0.5       6.1 [+ or -] 0.6
CS        7.2 [+ or -] 0.6 *     7.3 [+ or -] 0.7 *
TSPG      6.5 [+ or -] 0.6 (#)   6.4 [+ or -] 0.5 (#)
H-ROS     6.7 [+ or -] 0.6       6.5 [+ or -] 0.5 (#)
L-ROS     6.9 [+ or -] 0.6       6.7 [+ or -] 0.9

Values are expressed as means [+ or -] SD. Numbers of rats in each
group was 10.

* p<0.05, compared with control group.

(#) p < 0.05, compared with CS group.

CS, chronic stress; TSPG, total saponins of Panax Ginsen; ROS,
Rehmannia glutinosa oligosaccharides.

Table 3
Effect of ROS on IPGTT of d14 in chronic stress rats fed with
high-fat diet ([bar.x] [+ or -] SD).

Group     Dose (mg/kg/d)           Blood glucose level (mmol/l)

                             Omin                 30 min

Control   --                 6.1 [+ or -] 0.4     13.9 [+ or -] 3.3
CS        --                 6.5 [+ or -] 0.6     20.3 [+ or -] 4.7 *
TSPG      200                6.4 [+ or -] 0.4     16.7 [+ or -] 3.7
H-ROS     200                6.5 [+ or -] 0.7     17.4 [+ or -] 4.5
L-ROS     100                6.5 [+ or -] 0.6     18.7 [+ or -] 3.0

Group                    Blood glucose level (mmol/l)

          60 min                90 min               120 min

Control    9.8 [+ or -] 2.0      8.8 [+ or -] 1.3    7.8 [+ or -] 1.2
CS        13.8 [+ or -] 3.7 *   10.2 [+ or -] 0.9    8.9 [+ or -] 1.1
TSPG      11.2 [+ or -] 1.9      9.6 [+ or -] 0.9    8.3 [+ or -] 0.8
H-ROS     11.6 [+ or -] 2.9      9.9 [+ or -] 1.2    8.2 [+ or -] 1.0
L-ROS     11.6 [+ or -] 1.4      9.6 [+ or -] 1.1    7.7 [+ or -] 0.7

Values are expressed as means [+ or -] SD. Numbers of rats in each
group was 10.

* p < 0.05, compared with control group.

CS, chronic stress; TSPG, total saponins of Panax Ginsen; ROS,
Rehmannia glutinosa oligosaccharides.

Table 4
Effect of ROS on IPGTT of d28 in chronic stress rats fed with
high-fat diet ([bar.x] [+ or -] SD).

Group     Dose (mg/kg/d)             Blood glucose level (mmol/l)

                            Omin                 30 min

Control   --                6.2 [+ or -] 0.5     13.7 [+ or -] 2.7
CS        --                7.2 [+ or -] 0.6     22.6 [+ or -] 4.6 **
TSPG      200               6.5 [+ or -] 0.6     17.0 [+ or -] 2.6 (#)
H-ROS     200               6.7 [+ or -] 0.6     16.6 [+ or -] 3.1 (#)
L-ROS     100               6.9 [+ or -] 0.6     17.8 [+ or -] 3.0 (#)

Group                     Blood glucose level (mmol/l)

          60 min                  90 min              120 min

Control    9.6 [+ or -] 1.4        8.9 [+ or -] 1.2   8.1 [+ or -] 1.1
CS        13.6 [+ or -] 2.3 *     10.9 [+ or -] 1.5   8.2 [+ or -] 0.8
TSPG      10.5 [+ or -] 1.5 (#)    9.3 [+ or -] 1.2   7.9 [+ or -] 1.3
H-ROS     10.4 [+ or -] 2.3 (#)    9.2 [+ or -] 0.9   7.6 [+ or -] 0.8
L-ROS     11.1 [+ or -] 0.7        9.8 [+ or -] 1.1   8.2 [+ or -] 0.9

Values are expressed as means [+ or -] SD. Numbers of rats in each
group was 10.

* p<0.05.

** p < 0.01, compared with control group.

(#) p <0.05, compared with CS group.

CS, chronic stress; TSPG, total saponins of Panax Ginsen; ROS,
Rehmannia glutinosa oligosaccharides.

Table 5
Effects on GGT in chronic stress rats fed with high-fat diet
([bar.x] [+ or -] SD).

Group      Dose (mg/kg/d)             Blood glucose (mmol/l)

                             Omin                 30 min

Control    --                6.1 [+ or -] 0.6     7.0 [+ or -] 0.8
CS         --                7.3 [+ or -] 0.7     8.8 [+ or -] 1.2
TSPG       200               6.4 [+ or -] 0.5     7.6 [+ or -] 0.8
H-ROS      200               6.5 [+ or -] 0.5     7.4 [+ or -] 0.6
L-ROS      100               6.7 [+ or -] 0.9     7.9 [+ or -] 1.1

Group              Blood glucose (mmol/l)

          60 min                90 min

Control    7.9 [+ or -] 1.3      8.8 [+ or -] 1.0
CS        10.3 [+ or -] 1.7 *   12.8 [+ or -] 1.9 **
TSPG       8.7 [+ or -] 1.4 #    9.2 [+ or -] 1.6 #
H-ROS      8.4 [+ or -] 0.8 #    9.6 [+ or -] 2.9 #
L-ROS      9.2 [+ or -] 1.2     10.2 [+ or -] 1.4

Values are expressed as means [+ or -] SD. Numbers of rats in each
group was 10.

* < 0.05, compared with control group.

** < 0.01, compared with control group.

# p < 0.05, compared with IR group.

CS, chronic stress; TSPG, total saponins of Panax Ginsen; ROS,
Rehmannia glutinosa oligosaccharides.
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Author:Zhang, Ruxue; Zhou, Jun; Li, Maoxing; Ma, Haigang; Qiu, Jianguo; Luo, Xiaohong; Jia, Zhengping
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:Apr 15, 2014
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