| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1Biotech Center, 2Nutritional Sciences, Rutgers University, New Brunswick, New Jersey; and 3Pennington Biomedical Research Center, Baton Rouge, Louisiana
Submitted 3 July 2007 ; accepted in final form 24 August 2007
 |
ABSTRACT |
|---|
 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
phosphoenolpyruvate carboxykinase; polyphenols; diabetes
Normally, the increase in blood glucose levels after food intake stimulates the secretion of insulin from the pancreas. This increase in blood insulin concentration then leads to the downregulation of PEPCK gene expression and, subsequently, the cessation of gluconeogenesis by the liver (4). Insulin-resistant hepatocytes, however, are unable to effectively convey the insulin signal, leading to a decrease in PEPCK mRNA transcription. Thus, the de novo glucose synthesis persists despite a high blood glucose concentration (12). The compounds that are able to repress PEPCK expression and overcome insulin resistance could constitute a new class of glucose-lowering agents.
Plants have traditionally been a rich source of medicinal compounds for many indications, including diabetes. In fact, metformin is one of the most prescribed glucose-lowering medicines currently used and is derived from a chemical isolated from a plant (19). Recently, a number of polyphenolic substances from different plant sources were shown to decrease PEPCK expression in vitro (11), but in vivo data on the effects of these compounds have not been reported.
There are a number of reports (15) about use of plants from the genus Artemisia as a traditional treatment for diabetes. Artemisia dracunculus L. (A. dracunculus), or Russian tarragon, is a perennial herb with a long history of medicinal and culinary use. Recently, the ethanolic extract of A. dracunculus was shown (13) to significantly decrease blood glucose levels in both genetic and chemically induced murine models of diabetes. In addition, the extract significantly decreased PEPCK mRNA expression in the streptozotocin (STZ)-induced diabetic rats, suggesting a potential mode of action (13). The extract also inhibited aldose reductase, an enzyme involved in many diabetic complications (9). A phenoxychromone and dihydrochalcone were identified as the specific polyphenolics responsible for most of the activity (9).
To identify the active compounds from the Artemisia extract that are responsible for its effect on PEPCK regulation, an in vitro assay that requires only milligram quantities of test material was developed. The assay was established in an H4IIE hepatoma cell line, and real-time PCR was used to track changes in PEPCK gene expression in response to the treatment. Studies using inhibitors of specific subcellular biochemical pathways were performed to potentially elucidate a mode of action of the isolated compounds.
|
METHODS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
Plant material. The seeds of A. dracunculus were purchased from Sheffield's Seed (Locke, NY). The plants were grown in hydroponics, and the shoots were harvested and stored at –20°C.
Extraction. Two kilograms of the shoot material was heated to 80°C with 10 liters of 80% ethanol for 2 h. The extraction was continued for an additional 10 h at 20°C. The extract was filtered through a cheese cloth and evaporated to 1 liter using a rotary evaporator. The extract was then diluted with 1 liter of water and partitioned three times with 2 liters of ethyl acetate. The ethyl acetate fraction was then dried to a slurry using a rotary evaporator, followed by freeze-drying for 48 h.
Purification and isolation of bioactive compounds. Purification, isolation, and identification of bioactive compounds were done as in Longendra et al. (9). One gram of the dried extract was dissolved in 5 ml of 60% ethanol and 0.5 ml of acetonitrile and purified using a preparatory HPLC. For the initial purification, a Waters 19 x 300 mm symmetry prep C8 reverse-phase column with a particle size of 7 µm was used. The mobile phases consisted of two components: solvent A [0.5% American Chemical Society (ACS) grade acetic acid in double-distilled deionized water, pH 3–3.5] and solvent B (100% acetonitrile). For the initial separation, a gradient run of 5–95% solvent B over 35 min was used at a flow rate of 8 ml/m. Ten fractions at 5-min intervals were collected and tested for PEPCK gene expression. The fractions and subfractions that showed higher inhibition of gene expression were further purified using different conditions. Fractions 7 and 7-8 were purified by altering the gradient conditions described above, with flow rates ranging from 1 to 8 ml/min. Fraction 7-1 was purified using an isocratic condition, with a mobile phase consisting of three components: solvent A (0.5% ACS grade acetic acid in double-distilled deionized water, pH 3–3.5), solvent B (100% acetonitrile), and solvent C (100% methanol) at a ratio of 5:3:2. Purification of the bioactive subfractions 7-1 and 7-8 gave 6-demethoxycapillarisin and 2',4'-dihydroxy-4-methoxydihydrochalcone, respectively. The ultraviolet profiles were monitored at wavelengths of 210 and 290 nm.
Identification of compounds. The bioactive compounds were identified using liquid chromatography-mass spectrometry (LC-MS) and 1H-, 13C-, and 2D-NMR spectroscopic data. LC-MS system used for analysis includes the Waters (Milford, MA) LC-MS integrity system consisting of a solvent delivery system with a W616 pump and W600S controller, W717 plus autosampler, W996 photodiode array (PDA) detector, and Waters TMD Thermabeam electron impact (EI) single quadrupole mass detector with fixed ionization energy of 70 eV. Data were collected and analyzed with the Waters Millennium version 3.2 software and linked with the sixth edition of the Wiley Registry of mass spectral data, containing 229,119 EI spectra of 200,500 compounds. After the W996 PDA detector the eluent flow was split into two equal flow paths with an adjustable flow splitter (model no. 600-PO10-06; Analytical Scientific Instruments, El Sobrante, CA). One of them was to the Thermabeam EI mass detector and the other to a Varian 1200L (Varian, Palo Alto, CA) triple quadrupole mass detector with electrospray ionization interface operated in either positive or negative ionization mode. The electrospray voltage was –4.5 kV, heated capillary temperature was 240°C, and sheath gas was air for the negative mode, and electrospray voltage was 5 kV and sheath gas was nitrogen for the positive ionization mode; the mass detector was used in scanning mode from 110 to 1,400 atomic mass units. Data from the Varian 1200L mass detector were collected and compiled using Varian's MS Workstation, version 6.41, SP2. The 1H- and 13C-NMR spectra and 2D-NMR experiments were recorded using a Bruker Avance AV-300 NMR spectrometer at 300 (1H) and 75 MHz (13C). The 2D experiments 1H-1H COSY (Correlation Spectroscopy), heteronuclear multiple-bond correlation, and edited heteronuclear single quantum coherence were acquired using standard Bruker software. All compounds were measured in CD3OD.
Cell culture and treatment. The H4IIE hepatoma cells (CRL-1600; American Type Culture Collection) were plated in 24-well tissue culture plates (Greiner Bio One) and were grown to near confluence in Dulbecco's modified Eagle's medium containing 2.5% (vol/vol) newborn calf serum and 2.5% (vol/vol) fetal calf serum. Cells were treated for 8 h with 500 nM dexamethasone and 0.1 mM 8-CTP-cAMP (Dex-cAMP) to induce PEPCK gene expression together with different concentrations of test compounds, plant extract, or 10 nM insulin. The fractions were tested at 50 µg/ml medium, and the compounds were tested at doses of 2.5, 5, 10, and 25 µg/ml. Three wells were allocated for each treatment as well as for a negative control (untreated cells). For inhibitory assays, cells were pretreated with 20 µM LY-294002 for 30 min, washed with phosphate-buffered saline, and incubated for an additional 7 h with Dex-cAMP together with different concentrations of test compounds, plant extract, or 10 nM insulin.
Cell viability assay and dose range determination. Cell viability was measured by the MTT assay (10). The MTT (Sigma) tetrazolium dye assay was performed to measure cell survival in culture after incubation with treatments. MTT (100 µg/ml) was added to the medium in each well, and plates were incubated in the cell growth chamber for 5 h. Medium was then removed, and dimethyl sulfoxide (150 µl) was added to each well to solubilize the purple formazan crystals created by mitochondrial dehydrogenase reduction of MTT. After 5 min of additional incubation, absorbance was read at 550 nm on a microplate reader spectrophotometer (Molecular Devices, Sunnyvale, CA). The concentrations of test reagents that showed significant cell viability compared with that of the control (dimethyl sulfoxide, 0.1%) were further selected for in vitro gene expression assays. All treatments were performed in duplicate.
Total RNA extraction, purification, and cDNA synthesis. Total RNA was extracted from H4IIE rat hepatoma cells using Trizol reagent (Invitrogen), following the manufacturer's instructions. RNA was quantified spectrophotometrically by absorbance measurements at 260 and 280 nm using the NanoDrop system (NanoDrop Technologies). Quality of RNA was assessed by separation in gel electrophoresis. RNA was then treated with DnaseI (Invitrogen), following the manufacturer's guidelines, to remove any traces of DNA contamination. The cDNAs were synthesized with 2.5 µg of RNA for each sample, using Stratascript reverse transcriptase (Stratagene), following the manufacturer's protocol.
Quantitative PCR and data analysis. The synthesized cDNAs were diluted fourfold. Five microliters of each of these diluted samples was used for PCR reactions of 25 µl final volume. The other components of the PCR reactions were 0.5 µl of 6 µM gene-specific primers (synthesized by IDT, Coralville, IA) and 12.5 µl of Brilliant SYBR Green PCR master mix (2x; Stratagene) containing green jump-start Taq ready mix. ROX (Stratagene) was used as a reference dye. The primers were selected using the Primer Express version 2.0 software (Applied Biosystems, Foster City, CA) as follows: β-actin: forward primer 5'-GGGAAATCGTGCGTGACATT-3', reverse primer 5'-GCGGCAGTGGCCATCTC-3'; PEPCK: forward primer 5'-GCAGAGCATAAGGGCAAGGT-3', reverse primer 5'-TTGCCGAAGTTGTAGCCAAA-3'.
β-Actin primers were selected from the RefSeq sequence with the accession no. NM_031144. Both primers reside on exon 4 of the rat β-actin gene (Rat Genomic Sequence Consortium, assembly version 3.4). These primers generated a 76-bp product from β-actin mRNA. PEPCK primers were selected from the RefSeq sequence with the accession no. NM_198780. The intron-spanning forward primer was selected to cover the exon 9–10 boundary. The reverse primer was selected from exon 10. The oligos were synthesized by IDT. These primers generated a 74-bp product from PEPCK mRNA and a 207-bp product from genomic DNA.
Quantitative PCR (qPCR) amplifications were performed on an MX3000p system (Stratagene) using one cycle at 50°C for 2 min and one cycle of 95°C for 10 min, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. The dissociation curve was completed with one cycle of 1 min at 95°C, 30 s at 55°C, and 30 s at 95°C. Non-RT control and no-template control were included in each experiment as quality control steps.
PEPCK mRNA expressions were analyzed using the 
CT method and normalized with respect to the expression of the β-actin housekeeping gene. The CT values obtained from these analyses directly reflect the relative mRNA quantities for a specific gene in response to a treatment as relative to a calibrator. The Dex-cAMP treatment (positive control) served as the calibrator sample in this study. The PECPK gene expression of the calibrator sample was assigned to a value of 1.0. A value of <1.0 indicates transcriptional downregulation (inhibition of gene expression) relative to the calibrator. Amplification of specific transcripts was further confirmed by obtaining melting curve profiles. All samples were run in duplicate.
AMPK
1 and -2 activity assay.
AMP-activated protein kinase (AMPK) activity was assayed as previously described (6). Briefly, AMPK was immunoprecipitated from 200 ug of H4IIE cell lysate using anti-AMPK1 (Upstate Biotechnology, Lake Placid, NY) or -2 (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies in 500 ul of buffer A (50 mM Tris·Hcl, pH 7.4, 150 mM NaCl, 50 mM NaF, 5 mM sodium pyrophosphate, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 5 ug/ml aproptin) at 4°C for 2 h. Immunocomplexes were washed with buffer A three times, buffer B containing 0.5 M NaCl and 62.5 mM NaF, and then reaction buffer (50 mM HEPES, pH 7.4, 1 mM dithiothreitol) three times. AMPK activity of immunocomplexes was determined by phosphorylation of SAMS peptide in reaction buffer containing 0.25 mM SAMS, 5 mM MgCl2, and 10 uCi of [r-32P]ATP for 10 min at 30°C with or without 200 µM AMP stimulation. The reaction was terminated by spotting reaction mixtures onto P81 filter paper and rinsed in 1% (vol/vol) phosphoric acid with gentle stirring to remove free ATP. Phosphorylated substrate was measured by scintillation counting.
Western blot analysis. Cells were cultured as described above, and whole cell extracts were prepared in ice-cold lysis buffer [62.5 mM Tris·HCl (pH 6.8), 2% wt/vol SDS, 10% glycerol, 50 mM DTT, 0.01% wt/vol bromophenol blue] and centrifuged at 12 000 g for 20 min at 4°C. Equal amounts of protein (50 ug) from the supernatants were separated on SDS 10% polyacrylamide gels and blotted onto nitrocellulose membrane. Western blot analysis was performed with monoclonal phospho-Akt (Ser473) antibodies according to the manufacturer's instructions (Cell Signaling Technology, Danvers, MA). After being washed, the blots were incubated with an anti-rabbit peroxidase-labeled secondary antibody and visualized using ECL Western Blotting Detection Reagent (GE Healthcare, Piscataway, NJ). After being stripped, the blots were probed with Akt2 (5B5) antibodies.
Glucose production assay. H4IIE rat hepatoma cells were treated with Dex-cAMP in the presence or absence of 5 nM insulin (Sigma) or test compound (6-demethoxycapillarisin or 2',4'-dihydroxy-4-methoxydihydrochalcone) for 5 h at 37°C. Cells were incubated for an additional 3 h in glucose production buffer (glucose-free Dulbecco's modified essential medium, pH 7.4, containing 20 mM sodium lactate and 2 mM sodium pyruvate without phenol red) with dexamethasone and 0.1 mM 8-CPT-cAMP in the presence or absence of 5 nM insulin or test compound. At the end of this incubation, 0.5 ml of medium was taken to measure the glucose concentration of the culture medium using the Amplex Red glucose assay kit (Invitrogen). Corrections for cell number were made on the basis of the protein concentration measured by the Bradford method.
Statistical analysis. Experimental observations are expressed as means ± SE. One-way ANOVA was used to determine the significance of treatments. Tukey's multiple means comparison test was performed to determine the significance of the difference between the control and treatments. Treatments were considered significantly different if P < 0.05.
|
RESULTS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
|
|
|
2 catalytic subunit by 150%, whereas both compounds increased the basal activity of the AMPK1 subunuit within the same cells (Fig. 6, A and B).
|
|
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
mice by 20 and 24%, respectively, after 7 days of treatment at 500 mg·kg–1·day–1. The extract was also shown (13) to decrease liver PEPCK mRNA expression by 28% in rats made diabetic with the STZ treatment. PEPCK is a key enzyme modulating hepatic gluconeogenesis, and its activity is closely correlated with hepatic glucose output (5). PEPCK gene expression is upregulated by glucocorticoids and glucagon (through cAMP) during times of stress and fasting, when blood glucose concentrations need to be increased. Alternatively, PEPCK gene expression is downregulated by the increase in circulating blood insulin resulting from postprandial hyperglycemia. The impaired insulin response in the liver caused by insulin resistance secondary to type 2 diabetes, however, results in a continuous elevated expression of PEPCK due to the unopposed action of glucagon. This permits continuous hepatic glucose output, thereby contributing significantly to basal and fasting hyperglycemia and the complications associated with diabetes. A number of drugs currently in use, such as metformin and thiazolidinediones, are able to repress basal PEPCK expression but not hormone-induced PEPCK expression, thus limiting their effectiveness for improving insulin sensitivity (2, 20).
The activity-guided fractionation of the A. dracunculus extract led to the identification of its major components responsible for the inhibition of hormone-induced PEPCK mRNA transcription. Two polyphenolic substances, 6-demethoxycapillarisin and 2',4'-dihydroxy-4-methoxydihydrochalcone, were isolated from active fraction 7 of the extract and shown to be the primary factors responsible for the downregulation of PEPCK gene expression. With the IC50 values equal to 43 and 61 µM, respectively, these compounds are more potent inhibitors of PEPCK mRNA transcription than metformin or thiazolidinediones, which have active doses ranging from 100 to 5,000 µM in hepatoma cells (2, 20). Although these identified compounds are primarily responsible for the PEPCK inhibitory activity of the total extract, partial inhibitory activity of other fractions and subfractions suggests that other inhibitors may also be present in the extract.
Polyphenols of plant origin, such as (–)-epigallocatechin gallate, were recently shown to mimic the effect of insulin on PEPCK gene expression by increasing PI3K, MAPK, and p70S6k activities (17). The PI3K pathway is a primary component of the insulin-signaling cascade in hepatocytes (1). The effect of insulin or (–)-epigallocatechin gallate on the expression of its target genes, including PEPCK, can be reversed by the administration of specific inhibitors of PI3K, such as LY-294002. LY-294002 is a potent and selective cell-permeable inhibitor of PI3K (16) shown to abolish the insulin inhibition of Dex-cAMP-induced PEPCK gene transcription in hepatoma cells (1). The inhibitory effect of 6-demethoxycapillarisin on PEPCK mRNA was significantly lower in the presence of this specific PI3K inhibitor, suggesting that it may suppress PEPCK gene expression by affecting the upstream components of the insulin-signaling pathway, such as PI3K. The effect of 2',4'-dihydroxy-4-methoxydihydrochalcone, to the contrary, was not significantly influenced by the LY-294002 inhibitor and, therefore, is not likely to involve the PI3K insulin-like pathway. 6-Demethoxycapillarisin, like insulin, increases phosphorylation of Akt2 protein kinase at Ser473. LY-294002 significantly inhibits Akt2 phosphorylation induced by 6-demethoxycapillarisin or insulin (Fig. 4C). The effect of Artemisia polyphenols on PEPCK gene expression was not affected by the inhibition of the the p70S6k pathway with rapamycin (Fig. 5B). Likewise, although activated Ras can downregulate cAMP-induced PEPCK gene expression in an insulin-independent manner through MAPK pathway (1), pretreatment with the MAPK inhibitor PD-184352 did not affect the PEPCK downregulation caused by Artemisia active compounds (Fig. 5A).
The biguanidine drugs, such as metformin, and thiazolidinediones such as troglitazone were shown to exert a PI3K-independent downregulation of basal PEPCK gene expression in the liver of insulin-resistant patients (2, 20) through an AMPK-dependent mechanism. This pathway leads to the insulin-independent suppression of hepatic gluconeogenesis by phosphorylation and cytoplasmic sequestration of the mammalian target of rapamycin complex 2 transcriptional coactivator (8). Both active compounds from the A. dracunculus extract are capable of increasing basal activity of ubiquitously expressed AMPK1 catalytic subunit, but only dihydroxy-4-methoxydihydrochalcone significantly increases AMP-stimulated activity of the liver-specific AMPK2 catalytic subunit. Moreover, downregulation of PEPCK mRNA expression by the dihydrochalcone is completely reversed by AMPK inhibitor compound C (Fig. 6).
In conclusion, 6-demethoxycapillarisin and 2',4'-dihydroxy-4-methoxydihydrochalcone from an A. dracunculus extract were identified as active compounds responsible for decreasing PEPCK overexpression in the liver cells of diabetic rodents. These compounds may act through insulin-like and insulin-independent pathways to achieve the suppression of both PEPCK gene expression and gluconeogenesis in the hepatocytes. Preliminary clinical (14) testing also suggested that the extract attenuated hyperinsulinemia in mildly diabetic patients. Thus, the A. dracunculus extract, or the compounds contained therein, may be useful for the prevention and treatment of diabetes and related disorders.
|
GRANTS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
|
REFERENCES |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  P. Kizelsztein, D. Govorko, S. Komarnytsky, A. Evans, Z. Wang, W. T. Cefalu, and I. Raskin 20-Hydroxyecdysone decreases weight and hyperglycemia in a diet-induced obesity mice model Am J Physiol Endocrinol Metab, March 1, 2009; 296(3): E433 - E439. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
