Ginkgo Biloba Extract (GBE) Enhances Glucose Tolerance
in Hyperinsulinism-induced Hepatic Cells
Zaiqing Yang, Tao Xia, Li Gan, Xiaodong Chen, Lei Zhou
College of Life Science and Technology, Huazhong Agricultural University, Wuhan city, Hubei
province, 430070, P.R.China firstname.lastname@example.org
Ginkgo biloba, an herbal medication, is capable of dropping glucose, fat and lipid peroxide and preventing atherosclerosis and complications in diabetic patients. In our studies, we tested the hypothesis that ginkgo biloba extract (GBE) prevents glucose intolerance induced by hyperinsulinism in hepatocytes. We investigated the effects of GBE on glucose ingestion, glucokinase activity and mRNA levels of key genes in glucose metabolism and insulin signaling pathway. To better show its efficacy, we included a control group that was treated with rosiglitazone, a kind of thiazolidinediones (TZDs). The data showed that GBE repressed glucose ingestion in normal status, whereas it dramatically improved glucose tolerance in insulin resistance status. Moreover, by analyzing gene expression, we suggested that GBE chiefly exerted its effects by stimulating IRS-2 transcription. It should be noted that, not like rosiglitazone, GBE didn’t stimulate overmuch glucose uptake in
improving glucose tolerance. It is said that GBE treatment could avoid drug-induced obesity. The data suggested that GBE had potential efficacy to prevent insulin resistance induced by hyperinsulinism. Keywords: antidiabetic drugs, diabetes prevention, insulin resistance, liver, rosiglitazone
Glucose-6-P, glucose-6-phosphate; GK, glucokinase; G-6-Pase, Abbreviations:
glucose-6-phosphatase; IRS, insulin receptor substrate; GLUT, glucose transporter; PPAR, peroxisome proliferator-activated receptor; SREBP, sterol regulatory element-binding protein. 1 INTRODUCTION
Ginkgo biloba, an herb, has been used as traditional Chinese medicine for thousands of years. Ginkgo biloba extract (GBE) is being widely studied and applied for its beneficial properties in treatment or prevention of human diseases. Ginkgo biloba trees mainly distribute in China, France, and
USA, producing a mass of dried leaves each year to meet the commercial demand of market . GBE has been reported to drop glucose, fat and lipid peroxide and prevent atherosclerosis in animal model and human. To our knowledge, no systematic study illustrates the molecular mechanism of its efficacy on improving insulin sensitivity and enhancing glucose tolerance in insulin resistance model.
In previous studies, GBE was found to have anti-inflammatory  and stimulate skin microcirculation . And it was used as a therapeutic agent for some cardiovascular and neurological disorders. People also found it could attenuate the negative effects of some drugs [4, 5]. Recently, accumulating in vitro and in vivo evidence demonstrated that it had potential efficacy in lipid metabolism, glucose metabolism and diabetes mellitus. Saponara showed that GBE inhibited cAMP phosphodiesterase in rat adipose tissue . Mario illustrated that the biflavones of ginkgo biloba
stimulated lipolysis in fully differentiated 3T3-L1 adipocytes . Boveris obtained the same results as Mario and further showed GBE inhibited lipid peroxidation . By investigating diabetic rodent model, Nian hong et al. indicated that GBE dropped the after-dinner blood-glucose, decreased the contents of
This work was supported by the High Education Doctorial Subject Research Program (No: 20010504003), the grants from the General Program (No: 30170674) and Key Program of National Natural Science Foundation (No: 30330440) and 863 Program (No: 2004AA222170) of China to Dr. Z.Q. Yang.
TC, TG and LDL, promoted SOD activity and relieved the damage of pancreatic islet . Moreover, Li et al. found GBE could prevent and treat atherosclerosis by decreasing serum lipids levels, suppressing inflammatory response and protecting endothelial cells . As we know, oxidation-modified LDL plays an important role in the pathogenesis of artherosclerosis with type II diabetes melllitus. GBE was also capable of inhibiting the oxidation of LDL [11, 12]. In addition, early detection of pathologic function of the retina plays very crucial role in monitoring of visual complications in diabetic patients. Researchers showed GBE prevented diabetic retinopathy and they thought it was a good adjuvant to patient with long lasting diabetes mellitus . All data suggested that GBE was of value in diabetic therapy.
Liver is an important organ for glucose metabolism and energy homeostasis. And hepatic insulin resistance is an important component in the development of type II diabetes mellitus. In this process, PPARs, GLUT, G6Pase, IRS and SREBPs play crucial roles. GLUT2 is the primary
glucose-transporter isoform in liver and plays a key role in glucose homeostasis by mediating bidirectional transport of glucose . PPARs and SREBPs are characterized well transcription factors. PPAR； was deemed the main isoform in adipocytes before, but now people found it might also mediate lipid metabolism and energy homeostasis by changing its expression in liver . SREBP1c is crucial for the regulation of lipogenic gene. And recent studies also found that it was interrelated with insulin action . G6Pase as the last enzyme in hepatic glucogenesis, is an important determinant of hepatic glucose fluxes. Moreover, IRS-2 was main isoform in liver. It compensated for the lack of IRS-1 in IRS-1-/- model . So hepatic insulin signaling was mediated mainly through IRS-2, rather than IRS-1 .
In this study, we tested the hypothesis that GBE involves in modulation of insulin action and enhances glucose tolerance. To better interpret its molecular mechanism, we assayed above gene expressions and glucokinase activity.
2 MATERIALS and METHODS
The powder form of GBE was purchased from Greensky Biological Tech Co., Ltd. (Hangzhou, China). The GBE contained 24？ flavonoids, 6？ terpenes and less than 1 ppm of ginkgolic acid. The
composition of the flavonoids and terpenes in GBE was similar to that of EGb 761 used in European countries. TRIzol was obtained from Sangon Co., Ltd. (Shanghai, China). Mammalian Cell Protein Extraction kit was purchased from Shenergy Biocolor BioScience & Technology Co., Ltd. (Shanghai, China). Glucose Assay Kit was obtained from Shenergy-diasys Diagnostic Technology Co., Ltd. (Shanghai, China).
2.2 Cell culture and treatment.
L-02 cell line was derived from normal adult liver . They were grown in DMEM supplemented with 10% fetal bovine serum at 5% CO2 and 37?C. For the relevant experiments, the density of cells
was about 5，105 cells/well in 24-well culture plates for RNA extraction or 5；， 106 cells/dish in 60-mm
Petri dishes for metabolite concentration assay. There were two group cells in experiments, namely A and B. A mimicked normal physiological status and included NC, NRT and NGT. All cells were treated with 10nM insulin. NC stood for Normal Control (NC); NRT was given 10；M rosiglitazone
that was a kind of TZDs and stood for Normal Rosiglitazone Treatment (NRT); NGT was given 10mg/l GBE and stood for Normal GBE Treatment (NGT). B mimicked insulin resistance status by hyperinsulinaemic and hyperglycaemic treatment in vitro [20, 21]. There were three kinds of B,
http:///week114 designated AC, ART, AGT. AC, ART and AGT were given 100nM insulin and AC stood for Abnormal Control (AC). ART was given 10；M rosiglitazone and stood for Abnormal Rosiglitazone Treatment (ART). AGT was given GBE and stood for Abnormal GBE Treatment (AGT). All treatments are listed in table 1.
Table 1. The description of cell treatment.；， indicates that groups include relevant agents.
Group A B
Agent NC NRT NGT AC ART AGT
2.3 RNA isolation.
Total RNAs were isolated from L-02 cells using TRIzol after culturing the cells for 36h. All of the RNA samples were treated with Dnase I to digest the genomic DNA and stored at -80?C. 2.4 Semi-quantitative RT-PCR.
Semi-quantitative RT-PCR with glyseraldehyde- 3-phosphate dehydrogenase (GAPDH) as an internal control was performed to determine the levels of PPAR；, IRS-2, GLUT2, SREBP1c and G6Pase
mRNA in L-02 cells. A 4μl RNA sample was reverse transcribed with oligo(dT)18. cDNA (2μl) was
used for PCR amplification with 1U Taq DNA polymerase. The PCR products were run on a 1% agarose gel containing ethidium bromide and viewed under UV light. All primers are listed in table 2. Preliminary experiments were carried out with various amounts of PCR cycles to determine the linear range of amplification for all of the studied genes. The results for the expression of studied gene mRNAs are always presented relative to the expression of GAPDH.
Table 2. The primers use for semi-quantitative RT-PCR.
Forward and Reverse primer (5！-3