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Research Paper
Journal of Pharmacy And Pharmacology
Chikusetsu saponin IVa regulates glucose uptake and fatty acid oxidation: implications in antihyperglycemic and hypolipidemic effects Yuwen Lia*, Tiejun Zhangb*, Jia Cuia, Na Jiaa, Yin Wua, Miaomiao Xia and Aidong Wena a Department of Pharmacy, Xijing Hospital, Fourth Military Medical University, Xi’an, China and bDepartment of Pharmacology, Medical College, Chungnam National University, Daejon, South Korea
Keywords Adenosine monophosphate activated protein kinase; Chikusetsu saponin IVa; carnitine palmitoyl transferase-1; glucose and lipid metabolism; Glucose transporter 4 Correspondence Aidong Wen, Department of Pharmacy, Xijing Hospital, Fourth Military Medical University, Changle Road, No. 127, Xi’an 710032, China. E-mail: [email protected] Miaomiao Xi, Department of Pharmacy, Xijing Hospital, Fourth Military Medical University, Changle Road, No. 127, Xi’an 710032, China. E-mail: [email protected] *These authors contribute equally to this work. Received September 29, 2014 Accepted January 1, 2015 doi: 10.1111/jphp.12392
Abstract Objectives The aim of this study is to investigate antidiabetic effects and molecular mechanisms of the chemical Chikusetsu saponin IVa (CHS) that isolated from root bark of Aralia taibaiensis, which has multiple pharmacological activity, such as relieving rheumatism, promoting blood circulation to arrest pain and antidiabetic action. Methods Rats with streptozotocin/nicotinamide-induced type 2 diabetes mellitus (T2DM) and insulin-resistant myocytes were used. Adenosine monophosphate (AMP)-activated protein kinase (AMPK) and acetyl-CoA carboxylase were quantified by immunoblotting. Assays of glucose uptake, fatty acid oxidation, glucose transporter 4 (GLUT4) translocation and carnitine palmitoyl transferase-1 (CPT-1) activity were performed. Key findings Chronic oral administration of CHS effectively decreases blood glucose, triglyceride, free fatty acid (FFA) and low density lipoprotein-cholesterol levels in T2DM rats. In both normal and insulin-resistant C2C12 myocytes, CHS activates AMPK, and increases glucose uptake or fatty acid oxidation through enhancing membrane translocation of GLUT4 or CPT-1 activity respectively. Knockdown of AMPK significantly diminishes the effects of CHS on glucose uptake and fatty acid oxidation. Conclusions CHS is a novel AMPK activator that is capable of bypassing defective insulin signalling and could be useful for the treatment of T2DM or other metabolic disorders.
Introduction Type 2 diabetes was characterized by altered lipid and glucose metabolism (fasting or postprandial hyperglycemia, dyslipidemia) that was a consequence of combined insulin resistance in skeletal muscle, liver and adipose tissue and relative defects of insulin secretion by beta cells.[1] Regulatory mechanisms of physical exercise and two major classes of anti-diabetic drugs (biguanides and thiazolidinediones) were much reported.[2,3] Novel besides, a good practice and bright future had been shown in the therapy of diabetes and its complications by natural products.[4] Exciting developments in type 2 diabetes therapy had shown that adenosine monophosphate (AMP)-activated protein kinase (AMPK) was one of the probable target of major anti-diabetic drugs (metformin and thiazolidinediones), and of insulin-
sensitizing adipokines (e.g. adiponectin).[5] For decades, the development of AMPK activators from natural products had been reported.[6] Resveratrol was a stilbene that was isolated from grapes and had potential for the treatment and prevention of type 2 diabetes. AMPK was thought to be its primary target.[7] AMPK was a phylogenetically conserved serine/threonine protein kinase, and was known to act as a key metabolic ‘master switch’ by phosphorylating target enzymes involved in lipid metabolism such as acetyl-CoA carboxylase (ACC).[8] Inhibition of ACC by AMPK activated MalonylCoA decarboxylase, resulted in reduction of malonyl CoA levels and increased fatty acid oxidation at the carnitine palmitoyl transferase-1 (CPT-1) step in liver, heart and
© 2015 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 67, pp. 997–1007
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Effects of Chikusetsu saponin IVa on T2DM
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skeletal muscle, the primary tissues that oxidize fatty acids.[9,10] Collectively, AMPK was emerged as a key player in the regulation of glucose homeostasis and lipid profile. Use of AMPK agonists could be a new therapeutic strategy for type 2 diabetes patients. Moreover diversified mechanisms by which AMPK influenced glucose and lipid metabolism in different tissues (liver, pancreas, skeletal muscle and adipocyte tissue) were already well documented.[11] Skeletal muscle was a principal site of glucose and fatty acid usage, and was one of the primary tissues responsible for insulin resistance in obesity and type 2 diabetes.[12] Stimulation of AMPK in the muscle could be an efficient method to increase glucose uptake in an insulin-independent manner, thus bypassing defective insulin signalling, such as one observed in type 2 diabetes patients.[13] Over the past few decades, many studies had shown that the anti-diabetic activity of dietary and medicinal plants were due to the presence of saponins.[14–16] Aralia taibaiensis Z.Z. Wang et H.C. Zheng (Araliaceae) was authenticated by Dr. Haifeng Tang. A voucher specimen (FMMUDP-Voucher No: SAP012) was deposited in the Herbarium of the Department of Pharmacy, Xijing Hospital, Fourth Military Medical University. It was widely distributed in the Qinba Mountains of western China. The extract of root bark of A. taibaiensis had multiple pharmacological activity, including relieving rheumatism, promoting blood circulation to arrest pain, inducing diuresis to reduce oedema and antidiabetic action.[17,18] The total saponins extracted from A. taibaiensis (sAT) had effective combined antioxidant and antiglycation activity in vitro and ex vivo.[17,19] In addition, sAT dramatically stimulated high-glucose-induced insulin secretion and its antidiabetic activity might be related to its high saponin content.[20] Previous phytochemical investigations revealed that the main active components of herb were 12 triterpenoid saponins.[21] Chikusetsu saponin IVa (CHS) was one of triterpenoid saponins and was indicated as the active insulinogenic ingredient of sAT.[21] In this study, the mechanism by which CHS effectively prevented type 2 diabetes was investigated. CHS significantly enhanced glucose uptake or fatty acid oxidation through promoting glucose transporter 4 (GLUT4) membrane translocation or increasing CPT-1 activity respectively in insulin-resistant skeletal muscle cells via an AMPKdependent pathway.
Materials and Methods Animals All animal procedures were approved by the Ethics Committee for Animal Experimentation of the Fourth Military Medical University (Xi’an, China) (ethical permission number: FMMU14005), and carried out in accordance with 998
University Ethics Guidelines for the care and use of laboratory animals. Mature male Wistar Albino rats (8–10 weeks old; 180–220 g) were used in this study and the rats were fed with standard rat pellet diet and water ad libitum.
Induction of type 2 diabetes mellitus (T2DM) Overnight fasted rats were intraperitoneally administrated with 120 mg/kg nicotinamide (NA), and T2DM was induced by a single intra-peritoneal injection of 50 mg/kg of streptozotocin (STZ) after 15 min. Hyperglycemia was confirmed by the elevated glucose levels in plasma, determined at 72 h and on day 7 after STZ and NA injection. The threshold value of fasting plasma glucose to diagnose diabetes was taken as ≥7.0 mM (World Health Organization, 2006).[22] Only rats found with T2DM were used for antidiabetic study.
Isolation of Chikusetsu saponin IVa and structure elucidation CHS was isolated from the sAT. Firstly, the dry and powdered root bark (100 g) of A. taibaiensis was extracted three times with 10-fold (v/v) 80% ethanol under reflux for 1 h. The alcoholic extracts were concentrated, suspended in distilled water and then partitioned successively with threefold (v/v) n-butanol saturated with water for three times. The n-butanol extracts were combined and evaporated using a rotary evaporator at 60°C. The yield was 15.91% (w/w). The extract (10 g) was chromatographed over silica gel, stepwise eluted with chloroform-methanol-water (from 7 : 1 : 1, lower phase, to 6.5 : 3.5 : 1, lower phase). The fraction was submitted to size exclusion chromatography on a Sephadex LH-20 column eluted with MeOH-H2O (1 : 1) to remove the pigments and carbohydrates. CHS (1 mg/ml) was analysed by using high performance liquid chromatography (HPLC) with acetonitrile and 0.2% phosphoric acid (35 : 65, v/v) at a flow rate of 1 ml/min (Figure S1) The structure of CHS was elucidated as C42H66O14 by extensive spectral analysis (IR, 1H-NMR, 13C-NMR and ESI-MS).
Acute toxicity study Acute oral toxicity study was performed as per Organization for Economic Cooperation and Development guidelines 423 (acute toxic classic method) (OECD, 2001).[23] Twenty-four male Wistar rats starved overnight were divided into four groups (n = 6) and orally fed with CHS in increasing dose levels of 25, 50, 100 and 200 mg/kg. The rats were observed individually at least once during the first 30 min, periodically during the first 24 h, with special attention given during the first 4 h, and daily thereafter, they
© 2015 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 67, pp. 997–1007
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were observed for a total of 14 days for any physical signs of toxicity such as writhing, gasping, palpitation and decreased respiratory rate or mortality. After 14 days, the lethality or death was calculated.
Experiments design Rats were randomly grouped. Body weight and food intake were monitored daily during the experimental period. Fasting blood glucose levels were monitored from vein every week using an Accu-check blood glucose meter (Roche Diagnostics, Basel, Switzerland). The serum free fatty acid (FFA), low density lipoprotein-cholesterol (LDLC), high density lipoprotein-cholesterol (HDL-C) and triglyceride (TG) levels were determined using a 7180-automatic biochemical analyser (Hitachi, Japan) at the Department of Clinical Laboratory, Xijing Hospital, Fourth Military Medical University (Xi’an, China).
Oral glucose tolerance test (OGTT) The oral glucose tolerance test was performed in overnight fasted (18 h) normal rats. Glucose (2 g/kg) was fed 30 min after the administration of drugs. Blood was withdrawn from the retro orbital sinus under ether inhalation at 15, 30, 60 and 120 min of glucose administration and glucose levels were estimated using a glucose oxidase–peroxidase reactive strips and the glucometer (Roche Diagnostics, Basel, Switzerland).
Cell culture, plasmid and transfection Mouse C2C12 myoblasts (ATCC, Manassas, VA, USA) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 50 U/ml penicillin and 50 μg/ml streptomycin. When cells reached confluence, the medium was switched to the differentiation medium containing DMEM and 2% horse serum, which was changed every other day. After 4 additional days, the differentiated C2C12 cells had fused into myotubes. To induce insulin resistance, C2C12 cells were cultured in DMEM containing 15 g/l of glucose, 2% horse serum, sodium pyruvate, 1 μg/ml of insulin, 100 nM dexamethasone and 1% penicillin/streptomycin for 4 days.[24] Plasmid pEGFP-C1-GLUT4 was prepared by amplifying the C2C12 cDNA library with corresponding primers. Sequences of the oligonucleotides are available upon request. For the knockdown of siRNA, scramble and siRNA specific for mouse AMPK were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Transient transfections were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol.
Effects of Chikusetsu saponin IVa on T2DM
Antibodies and regents DMEM medium, FBS, L-glutamine, penicillin, streptomycin and other cell culture reagents were obtained from Gibco (Invitrogen, Carlsbad, CA, USA). Anti-AMPK antibody, anti-phospho-Thr172 (AMPK) antibody, anti-ACC antibody, anti-phospho-Ser79 (ACC) antibody, anti-phosphoSer473 (protein kinase B (PKB)) and anti-PKB antibody were obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-β-actin antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). 2-deoxy[1-3H]-glucose, [1-14C]-palmiticacid and [γ-32P] ATP were obtained from PerkinElmer Life and Analytical Sciences (Boston, MA, USA). Insulin, glibenclamide STZ and NA were purchased from Sigma-Aldrich (St Louis, MO, USA).
AMPK activity measurement To measure isoform-specific AMPK activity in skeletal muscles, we immunoprecipitated AMPK from cell lysates (1 mg of protein) with specific antibodies against the α1 or α2 catalytic subunits bound to protein-G/sepharose beads. We measured kinase activity using synthetic ‘SAMS’ peptide and [γ-32P] ATP.
Glucose uptake determination Glucose uptake was assayed by using 2-deoxy-[1-3H]-glucose. Cells were incubated in the presence or in the absence of 1 μg/ml CHS for 15 min and then washed two times with washing buffer (20 mM HEPES (pH 7.4), 140 mM NaCl, 5 mM KCl, 2.5 mM MgSO4 and 1 mM CaCl2). Cells were then incubated in transport solution (the buffer containing 0.5 mCi 2-deoxy-[1-3H]-glucose and 10 μm 2-deoxyglucose) for 10 min. Non-specific uptake was determined with incubating the cells in the presence or in the absence of 5 mM cytochalasin B. The reaction was terminated by aspiration of the solution. Cells were then washed three times, and radioactivity associated with the cells was determined by cell lysis in 0.05M NaOH, followed by scintillation counting. Protein concentration was determined by the Bradford method. 2-Deoxyglucose uptake was expressed as pmol/minute/ milligram. Measurements were performed in duplicate and in three independent experiments.
GLUT4 translocation detection To detect GFP-GLUT4 translocation, C2C12 myocytes were grown on glass coverslips and then transfected with pEGFPC1-GLUT4. After 48 h, the cells were treated as indicated in the figure legend, and were fixed with 4% paraformaldehyde for 10 min, then were mounted with Fluoromount-G (SouthernBiotech, Birmingham, UK) and were visualized using a fluorescence microscope equipped with the appropriate filters (Olympus, Tokyo, Japan).
© 2015 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 67, pp. 997–1007
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Fatty acid oxidation determination To determine fatty acid oxidation in muscle, [1-14C]palmitic acid oxidation was measured in C2C12 myotubes using 14CO2 and 0.2 ml of benzethonium solution. Briefly, cells were mixed the assay buffer containing 1.25M NaCl, 0.1M MgSO4•7H2O, 1.2 mM KH2PO4, 0.5M KCl, 0.2M glucose, 1M NaHCO3, 0.25M CaCl2 and 1 mCi [1-14C]palmitic acid, gassed for 30 s under humidified 95/5% O2/CO2 and covered with filter paper that moistened with 0.2 ml of benzethonium solution. After incubation for 1 h at 37°C, the reaction was stopped by 6% trichloroacetic acid (TCA) solution and the radioactivity trapped in the filter paper was determined. Fatty acid β-oxidation activity was expressed as nmol/minute/milligram.
CPT-1 enzyme activity measurement The activity was assayed in these supernatants spectrophotometrically by following the release of CoA-SH from palmitoyl-CoA using the general thiol reagent 5,5dithiobis-(2-nitrobenzoic acid) (DTNB). Eight hundred forty-five microlitre Tris-HCl–DTNB (116 mM Tris, 25 mM EDTA, 2 mM DTNB, 0.2% Triton X-100, pH 8.0) was prepared and was added to semi-microcu-vettes (Greiner, Frickenhausen, Germany); 50 μg proteins were adjusted to 100 μl and were added then. After 5 min pre-incubation at 30°C, 50 μl palmitoyl-CoA (1 mM) was added. The action was then started by adding 5 μl L-carnitine solution (1.2 mM dissolved in 1M Tris, pH 8.0), immediately followed by photometric measurement (Hitachi U-2010; 30°C, 4GE nm) for 360 s. The difference between absorbance readings with or without substrates measured the release of CoA-SH.
Western blot analysis After treatment with different conditions as described in the figure legends, cells were lysed in M2 buffer (20 mM Tris at pH 7.6, 0.5% NP-40, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 2 mM DTT, 0.5 mM PMSF, 20 mM β-glycerol phosphate, 1 mM sodium vanadate, 1 μg/ml leupeptin). Fifty microgram of the cell lysates were subjected to SDSpolyacrylamide gel and blotted onto PVDF membrane (Millipore, Bedford, MA, USA). After blocking with 5% skim milk in PBS/T, the membrane was probed with the relevant antibody and visualized by enhanced chemiluminescence (Biological Industries, Beit Haemek, Israel).
Statistical analysis Data were expressed as mean ± standard deviation. Statistical analysis was performed by analysis of variance followed 1000
by least significant difference (LSD)’s test for multiple comparisons. Data analyses were performed using the SPSS 11.0 software package. Differences were considered significant at P < 0.05.
Results Acute toxicity study In rats, oral administration of CHS at four doses did not produce any drug-induced physical signs of toxicity and no death was registered in 14 days, indicating that CHS was nontoxic in rats with an oral dose of 200 mg/kg. Therefore, investigation of antihyperglycemic activity of CHS at 7.5, 15 and 30 mg/kg doses were safe and feasible.
CHS regulates glucose and lipid metabolism in T2DM rats To further evaluate effects of CHS on glucose and fatty acid metabolism, graded doses of CHS were administrated to T2DM rats daily intragastrically for 4 weeks. CHS significantly decreased body weight and fasting blood glucose level of T2DM rats (Figure 1a and 1b). In OGTT, the high dose of CHS treatment showed significant reduction of 44.2% in plasma glucose levels after 120 min of glucose administration compared with vehicle treated T2DM group (Figure 1c). The effects of CHS on serum lipid parameters were depicted (Table 1). The results showed that high dose of CHS treatment significantly countered the rise in the levels of serum TG, FFA and LDL-C in T2DM rats, and increased HDL-C level by 13.2% compared with vehicle. No significant differences in food intake were found in T2DM rats that were treated with different doses of CHS (Figure S2). These data showed that CHS effectively increased disposal of glucose and lipid from blood in T2DM rats.
CHS increased AMPK phosphorylation, glucose uptake and fatty acid oxidation in C2C12 myocytes Next, it was examined as to how CHS could modulate glucose and lipid metabolism in C2C12 myocytes. CHS increased phosphorylation of AMPK and ACC. The time of onset and the duration of these effects on ACC phosphorylation were similar to AMPK phosphorylation, but peaked at 30 min and short lived (Figure 2a). In-vitro kinase assay, AMPK activity was increased 1.3 fold by CHS (Figure 2b). After that, we examined function of CHS on glucose uptake and GLUT4 translocation. We found that glucose transport increased 30% in the presence of CHS (Figure 2c). The stimulation of glucose transport was mainly regulated by the translocation of GLUT4 to the plasma membrane in myocytes. The cytosolic
© 2015 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 67, pp. 997–1007
Yuwen Li et al.
Effects of Chikusetsu saponin IVa on T2DM
Chikusetsu saponin IVa (CHS) COOGlc HOOC HO HO
O
(b)
O OH
16
60 #
*
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*
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CHS (L)
CHS (M)
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Fasting blood glucose (mM)
Difference between final and initial body weight (g)
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** 4
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Nor
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T2DM
CHS (M)
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(c) OGTT
Plasma glucose level (mM)
25
Nor Veh CHS(L) CHS(M) T2DM CHS(H) Glib
20 15
** **
10
** ** 5
** **
0 0
30
60
120 min
Figure 1 Effects of Chikusetsu saponin IVa (CHS) on glucose metabolism in type 2 diabetes mellitus (T2DM) rats. Streptozotocin (STZ)nicotinamide (NA)-induced T2DM rats (n = 6) were daily intragastrically treated with different doses of CHS (L: 7.5 mg/kg per day; M: 15 mg/kg per day; H: 30 mg/kg per day) and glibenclamide (0.6 mg/kg per day) for 4 weeks. After treatment, all of the rats were fasted overnight. Structure of CHS was shown above. The body weight (a) and fasting blood glucose level were measured (b); the rats were orally loaded with glucose (2 g/kg). Then their blood glucose levels were determined at 0, 30, 60 and 120 min after glucose administration (c). The data were presented as the mean ± standard deviation (SD), *P < 0.05 or **P < 0.01 vs T2DM rats treated with vehicle alone.
and membrane fractions of C2C12 myocytes were subsequently prepared for Western blot analysis of GLUT4. Membrane expression of GLUT4 was significantly increased by CHS (Figure 2d). GFP-GLUT4 distributed throughout the cytoplasm in control cells treated with vehicle. CHS caused a substantial redistribution of GFP-GLUT4, such that an almost continuous line of fluorescence was observed around the plasma membrane (Figure 2e). The effect of CHS on GLUT4 translocation was in agreement with glucose uptake data. In addition, CHS strongly increased
fatty acid oxidation (Figure 2f). CPT-1 was an enzyme that catalysed the esterification of long-chain acyl-CoAs to L-carnitine for transporting into mitochondria for fatty acid oxidation, and was inhibited by ACC.[10] CHS significantly increased CPT-1 activity (Figure 2g). Our results clearly demonstrated that CHS effectively elevated glucose uptake via increasing translocation of GLUT4 to plasma membran. Moreover, activation of AMPK by CHS decreased the activity of ACC by phosphorylation (Figure 2a), led to relieving inhibitory effect on CPT-1
© 2015 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 67, pp. 997–1007
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Table 1
Yuwen Li et al.
Hypolipidemic activity of CHS in normal and T2DM rats (mean ± SD, n = 6)
Groups
FFA mg/dl
TG mg/dl
LDL-C mg/dl
HDL-C mg/dl
Normal T2DM (veh) T2DM CHS (L) T2DM CHS (M) T2DM CHS (H) T2DM Gli
12.09 ± 1.16 21.03 ± 1.20*** 19.09 ± 1.36 17.46 ± 1.15** 14.02 ± 2.31**** 12.16 ± 1.15****
79.43 ± 4.10 131.87 ± 8.65*** 123.37 ± 8.21 117.31 ± 6.45** 108.11 ± 6.36** 87.50 ± 5.27****
21.53 ± 1.17 32.55 ± 2.45*** 30.51 ± 1.22 27.51 ± 1.33** 23.29 ± 0.88**** 21.47 ± 1.26****
48.91 ± 1.35 42.64 ± 1.25*** 43.76 ± 1.54 45.29 ± 1.62** 47.75 ± 1.08**** 48.83 ± 1.56****
FFA, free fatty acid; HDL-C, high-density lipoprotein-cholesterol; LDL-C, low density lipoprotein-cholesterol; TG, triglyceride. *P < 0.05 vs normal group; **P < 0.05 vs T2DM group; ***P < 0.05 vs normal group; ****P < 0.05 vs T2DM group.
activity (Figure 2g), therefore enhanced the entry of fatty acid into the mitochondria for oxidation (Figure 2f).
CHS increased AMPK phosphorylation, glucose uptake and fatty acid oxidation in insulin-resistant C2C12 myocytes Two distinct pathways encourage glucose transport activity in skeletal muscle, that is the contraction-stimulated pathway reliant on AMPK-dependent mechanisms and an insulin-dependent pathway activated via upregulation of PKB.[25] PKB phosphorylation on Ser-473 was declined to basal level under insulin stimulation in insulin-resistant C2C12 myocytes (Figure 3a). Insulin lost its action on glucose uptake in insulin-resistant cells also (Figure 3b). After treatment with CHS, both AMPK and ACC phosphorylation were dramatically increased (Figure 3c). We next observed effects of CHS on glucose uptake and membrane translocation of GLUT4. Apparently, CHS increased glucose uptake (Figure 3d), and induced translocation of cytosolic GLUT4 to plasma membrane (Figure 3e). Moreover, CHS evidently promoted the migration of GFP-GLUT4 to plasma membrane (Figure 3f). Additionally, CHS increased fatty acid oxidation and CPT-1 activity in insulin-resistant C2C12 myocytes (Figure 3g and 3h).
AMPK was essential for CHS and sAT mediated glucose and fatty acid utilization To further assess the essentiality of AMPK in CHSstimulated glucose uptake and fatty acid oxidation, siRNAAMPK was used to ablate expression of AMPK in insulinresistant C2C12 myocytes. The effectiveness of AMPK knockdown on protein level was presented (Figure 4a). Silencing of endogenous AMPK by siRNA markedly reduced CHS-mediated glucose uptake compared with the cells transfected with scramble siRNA (Figure 4b). In consistent with glucose uptake result, CHS lost their effects on increasing expression of membrane GLUT4 in siRNAAMPK transfected cells (Figure 4c). Enhancement of fatty 1002
acid oxidation and CPT-1 activation by CHS were also diminished by siRNA-AMPK (Figure 4d and 4e). All the results confirmed that AMPK was essential for modulating CHS-mediated glucose and lipid metabolism.
Discussion CHS was a newly determined AMPK activator that prevented an increase of blood glucose and lipid levels in experiment T2DM rats. An enhancement of GLUT4 membrane translocation through AMPK was a mechanism by which CHS increases glucose uptake both in normal and insulin-resistant C2C12 myocytes. Moreover, inhibition of AMPK downstream target ACC by CHS resulted in an increase of fatty acid oxidation via relieving inhibitory effect on CPT-1 activity (Figure 5). Skeletal muscle was the main site for glucose disposal and fat catabolism. Our results clearly demonstrated that CHS effectively elevated glucose uptake and fatty acid oxidation in C2C12 cells (Figure 2). Indeed, regulation of energy metabolism through activation of AMPK in response to pharmacological regents was also observed in extramuscular tissues such as adipose tissue and liver. Activation of AMPK by CHS was observed in 3T3-L1 adipocytes also (data were not shown), which strongly suggested it could control AMPK-mediated increase of glucose uptake, inhibition of lipolysis and lipogenesis in adipose tissue.[26] Hepatic gluconeogenesis was a major cause of fasting hyperglycemia in diabetic patients and fatty liver disease. A disorder of TG, FFA and cholesterol in the liver was the critical complications of type 2 diabetes.[27] Inhibition of ACC by AMPK led to a decrease in fatty acid synthesis and an increase in fatty acid oxidation, thus reduced FFA and excessive storage of TG (Figure 1). The findings showed a promising function of CHS in lipid abnormalities in the liver. Furthermore, the importance of AMPK in the regulation of energy homeostasis provided a potentially widespread function of CHS in the treatment of a cluster of metabolic disorders. To further investigate benefits of CHS in the treatment of T2DM, insulin-resistant C2C12 myocytes was used. Upon
© 2015 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 67, pp. 997–1007
Yuwen Li et al.
Effects of Chikusetsu saponin IVa on T2DM
(a)
(c)
CHS 0.1
1
µg/mL
10
Relative AMPK activity (32p)
0
p-AMPK(Thr172) T-AMPK Actin
120
**
100 80 60 40 20 0 Veh
CHS 0
15
30
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120
min p-AMPK(Thr172) T-AMPK p-ACC(Ser79)
Relative Glucose uptake (3H)
(b)
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*
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S
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Ve h
S CH
Ve h
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GLUT4 Caveolin-1 Tubulin
(g)
(h) 120
Relative fatty acid oxidation (14C)
CPT-I activity (nmol palmitate/min/mg protein)
C2C12 myocytes
*
100 80 60 40 20 0 Veh
CHS
4
*
3 2 1 0 Veh
CHS
Figure 2 Effects of Chikusetsu saponin IVa (CHS) on AMPK activation, glucose uptake and fatty acid oxidation in C2C12 myocytes. C2C12 myocytes were treated with CHS for indicated concentrations (a), C2C12 myocytes were treated with 1 μg/ml CHS for indicated times (b) and whole cell lysates were separated by SDS-polyacrylamide gel (PAGE) and then immunoblotting was performed using indicated antibodies; the relative intensity of phosphorylated AMPK and acetyl-CoA carboxylase (ACC) were determined and normalized to total (bottom); C2C12 myocytes were treated with 1 μg/ml CHS for 15 min at followed experiments. Cell lysates were assayed with [γ-32P] ATP to measure AMPK activity (c); in-vitro glucose uptake was measured with 2-deoxy-[1-3H]-glucose (d); membrane and cytosolic proteins were isolated respectively; immunoblotting was performed using indicated antibodies. Anti-caveolin-1 or anti-tubulin antibodies were used to determine membrane or cytosol (e); C2C12 myocytes were transfected with pEGFP-C1-GLUT4 for 48 h. Then cells were treated with vehicle or CHS (1 μg/ml) for 15 min; the images were observed under a fluorescence microscope. The distributions of GFP-GLUT4 in C2C12 myocytes were shown. The scale bars represent 10 μm (f); C2C12 myocytes were treated with 1 μg/ml CHS for 15 min; fatty acid oxidation was determined by [1-14C]-palmitic acid (g); CPT-1 activity was measured and defined as nmol CoA-SH released/min/mg protein (h). The data were presented as the mean ± standard deviation (SD) from three independent experiments. *P < 0.05 or **P < 0.01 vs vehicle. © 2015 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 67, pp. 997–1007
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(a)
CHS
(c)
CON Insulin 0 15
Ins-res 0 15
0
15
30
60
Ins-res min
120
min p-AMPK(Thr172) T-AMPK
p-PKB(ser473)
p-ACC(Ser79)
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T-ACC p-PKB(Ser473) T-PKB
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Veh Ins Veh Ins CON
Ins-res
120 100 80 60 40 20 0
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100
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80 60
0 15 30
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GLUT4
40
Caveolin-1
20 0
p-AMPK/T-AMPK p-ACC/T-ACC
** **
(e)
120
Ve h in su lin CH S
Relative Glucose uptake (3H)
(d)
Actin
Ve h in su lin CH S
120 100 80 60 40 20 0
Relative intensity
Relative Glucose uptake (3H)
(b)
Tubulin Veh insulin CHS
(f) Veh
GFP-GLUT4 insulin
CHS
Insulin resistant C2C12 myocytes (h)
120
*
100 80 60 40 20 0
Veh insulin CHS Ins-res
4
CPT-1 activity (nmol palmitate/ min/mg protein)
Relative fatty acid oxidation (14C)
(g)
*
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Figure 3 Effects of Chikusetsu saponin IVa (CHS) on AMPK phosphorylation, glucose uptake and fatty acid oxidation in insulin-resistant C2C12 myocytes. Insulin-resistant C2C12 myocytes were treated with insulin (100 nM, 15 min). Whole cell extracts were subjected to immunoblotting with anti-phospho-PKB (Ser-473) and anti-PKB antibodies (a); in-vitro glucose uptake was measured with 2-deoxy-[1-3H]-glucose, *P < 0.05 vs control treated with vehicle. Insulin-resistant C2C12 myocytes were treated with vehicle, CHS (1 μg/ml) or insulin (100 nM) for 15 min (b). Whole cell lysates were separated by SDS-polyacrylamide gel (PAGE) and then immunoblotting was performed using indicated antibodies. The relative intensity of phosphorylated AMPK and ACC were determined and normalized to total (bottom) (c); glucose uptake was measured with 2-deoxy-[1-3H]-glucose (d); membrane and cytosolic proteins were isolated respectively; immunoblotting was performed using indicated antibodies. Anti-caveolin-1 or antitubulin antibodies were used to determine membrane or cytosol (e); insulin-resistant C2C12 myocytes were transfected with EGFP-C1-GLUT4 for 48 h, then cells were treated with vehicle, insulin (100 nM) or CHS (1 μg/ml) for 15 min. The images were observed under a fluorescence microscope. The distributions of GFP-GLUT4 in C2C12 myocytes were shown. The scale bars represent 10 μm (f); fatty acid oxidation was determined by [1-14C]-palmitic acid (g), and CPT-1 activity was measured and defined as nM CoA-SH released/min/mg protein (h). The data were presented as the mean ± standard deviation (SD) from three independent experiments. *P < 0.05 or **P < 0.01 vs vehicle.
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Figure 4 AMPK knockdown reversed the effects of Chikusetsu saponin IVa (CHS) on glucose uptake and fatty acid oxidation in insulin-resistant C2C12 myocytes. Insulin-resistant C2C12 myocytes were transfected with either a nonspecific siRNA (si-scramble), or siRNA specific for AMPK (siAMPK) for 48 h. Cells were stimulated with 1 μg/ml CHS for 15 min; whole cell lysates were separated by SDS-polyacrylamide gel (PAGE) and then immunoblotting was performed using indicated antibodies (a). Glucose uptake was measured with 2-deoxy-[1-3H]-glucose (b); membrane proteins were isolated respectively, and anti-caveolin-1 antibody was used to determine membrane (c); fatty acid oxidation was measured with [1-14C]palmitic acid (d); CPT-1 activity was defined as nmol CoA-SH released/min/mg protein (e). The data were presented as the mean ± standard deviation (SD) from three independent experiments. *P < 0.05 vs si-scramble group.
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Blood Glucose and Fatty Acid Figure 5 Schematic model of Chikusetsu saponin IVa (CHS) in control of glucose and fatty acid metabolism. CHS promoted migration of glucose transporter 4 (GLUT4) to plasma membrane and increased carnitine palmitoyl transferase-1 (CPT-1) activity through activating AMPK and inhibiting acetyl-CoA carboxylase (ACC), thus enhanced glucose and fatty acid utilization.
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binding to its receptor, insulin activates phosphatidylinositol 3-kinase through phosphorylation of insulin receptor substrate family.[28] A critical downstream target of 3-phosphoinositides is serine/threonine kinase PKB.[28] PKB phosphorylation on Ser-473 was declined to basal level under insulin stimulation in insulin-resistant C2C12 myocytes (Figure 3). Despite insulin lost its action, CHS was still able to activate AMPK and inhibit ACC, therefore significantly improved glucose uptake and fatty acid oxidation (Figure 3). The results suggested that CHS was capable of bypassing defective insulin signalling, and it mediated insulin independent activation of AMPK. It should be noted that activation of PKB was not observed in response to CHS, which suggested that PKB-mediated signalling was not involved in CHS-modulated energy metabolism (Figure 3). Knockdown of AMPK ablated CHS-induced translocation of GLUT4 and activation of CPT-1, which confirmed that AMPK was essential for CHS-mediated glucose and lipid utilization (Figure 4).
References 1. Saltiel AR, Kahn CR. Insulin signalling and the regulation of glucose and lipid metabolism. Nature 2001; 414: 799– 806. 2. Palitzsch D, Buhrlen M. Prevention of type 2 diabetes mellitus. MMW Fortschr Med 2012; 154: 45–48. 3. Wong AK et al. The effect of metformin on insulin resistance and exercise parameters in patients with heart failure. Eur J Heart Fail 2012; 14: 1303–1310. 4. Jia W et al. Antidiabetic herbal drugs officially approved in China. Phytother Res 2003; 17: 1127–1134. 5. Viollet B et al. Targeting the AMPK pathway for the treatment of type 2 diabetes. Front Biosci 2009; 14: 3380– 3400. 6. Adachi Y et al. Petasin activates AMPactivated protein kinase and modulates glucose metabolism. J Nat Prod 2014; 77: 1262–1269. 7. Baur JA et al. Resveratrol improves health and survival of mice on a highcalorie diet. Nature 2006; 444: 337– 342. 8. Winder WW, Hardie DG. AMPactivated protein kinase, a metabolic master switch: possible roles in type 2 diabetes. Am J Physiol 1999; 277: E1–E10. 1006
Conclusions Our study demonstrated antihyperglycemic and hypolipidemic activity of CHS in T2DM rats. The beneficial action of CHS appears to be mediated by AMPK enhancement and the subsequent activation of glucose uptake and fatty acid oxidation. The findings provide the basis for the future development of CHS as a new candidate for the management of metabolic disorders.
Declarations Acknowledgements and funding This work was supported by grants from the National Natural Science Foundation of China (No. 81201985, 81373947, 81470174 and 81001673) and the National New Drug ‘R&D’ project (No. 2011ZXJ09302).
9. Abu-Elheiga L et al. Continuous fatty acid oxidation and reduced fat storage in mice lacking acetyl-CoA carboxylase 2. Science 2001; 291: 2613–2616. 10. Saha AK, Ruderman NB. MalonylCoA and AMP-activated protein kinase: an expanding partnership. Mol Cell Biochem 2003; 253: 65–70. 11. Misra P, Chakrabarti R. The role of AMP kinase in diabetes. Indian J Med Res 2007; 125: 389–398. 12. Kirwan JP. Insulin sensitivity in skeletal muscle: ‘use it or lose it, fast’. J Appl Physiol 2010; 108: 1023–1024. 13. Hardie DG. The AMP-activated protein kinase pathway – new players upstream and downstream. J Cell Sci 2004; 117: 5479–5487. 14. Abdel-Zaher AO et al. Antidiabetic activity and toxicity of Zizyphus spina-christi leaves. J Ethnopharmacol 2005; 101: 129–138. 15. Oishi Y et al. Inhibition of increases in blood glucose and serum neutral fat by Momordica charantia saponin fraction. Biosci Biotechnol Biochem 2007; 71: 735–740. 16. Lee KT et al. Hypoglycemic and hypolipidemic effects of tectorigenin and kaikasaponin III in the streptozotocin-lnduced diabetic rat and their antioxidant activity in vitro. Arch Pharm Res 2000; 23: 461–466.
17. Xi M et al. Antioxidant and antiglycation properties of triterpenoid saponins from Aralia taibaiensis traditionally used for treating diabetes mellitus. Redox Rep 2010; 15: 20–28. 18. Xi M et al. Antioxidant and antiglycation properties of total saponins extracted from traditional Chinese medicine used to treat diabetes mellitus. Phytother Res 2008; 22: 228–237. 19. Weng Y et al. Antihyperglycemic, hypolipidemic and antioxidant activities of total saponins extracted from Aralia taibaiensis in experimental type 2 diabetic rats. J Ethnopharmacol 2014; 152: 553–560. 20. Cui J et al. Insulin-secretagogue activity of eleven plant extracts and twelve pure compounds isolated from Aralia taibaiensis. Life Sci 2013; 92: 131–136. 21. Tang HF et al. Studies on the triterpenoid saponins of the root bark of Aralia taibaiensis. Yao Xue Xue Bao 1996; 31: 517–523. 22. Diabetes Research and Clinical Practice 2000; 49: 181–186. 23. Organisation for Economic Cooperation and Development (OECD). Guideline for Testing of Chemicals 423: Acute Oral Toxicity acute Toxic Class Method. 24. Koistinen HA et al. 5-aminoimidazole carboxamide riboside
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increases glucose transport and cellsurface GLUT4 content in skeletal muscle from subjects with type 2 diabetes. Diabetes 2003; 52: 1066–1072. 25. Kasuga M et al. Insulin stimulates the phosphorylation of the 95 000-dalton subunit of its own receptor. Science 1982; 215: 185–187. 26. Kapeller R, Cantley LC. Phosphatidylinositol 3-kinase. Bioessays 1994; 16: 565–576. 27. Mackenzie RW, Elliott BT. Akt/PKB activation and insulin signaling: a novel insulin signaling pathway in the
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treatment of type 2 diabetes. Diabetes Metab Syndr Obes 2014; 7: 55–64. 28. Berdichevsky A et al. Acute oxidative stress can reverse insulin resistance by inactivation of cytoplasmic JNK. J Biol Chem 2010; 285: 21581–21589.
Supporting information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Figure S1 The high performance liquid chromatography (HPLC) fingerprint of
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Chikusetsu saponin IVa (CHS). The HPLC analysis was performed on a Zorbax SB-C18 column (250 mm × 4.6 mm, 5 μm) and kept at 30°C. The mobile phase consisted of acetonitrile and 0.2% phosphoric acid (35 : 65, v/v). The flow rate was 1.0 ml/min and the chromatogram was monitored at 206 nm. Figure S2 Food intake. The food intake was recorded daily, and each group was indicated by the type of connecting line. The data were presented as the mean ± standard deviation (SD).
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Research Paper
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Chikusetsu saponin IVa regulates glucose uptake and fatty acid oxidation: implications in antihyperglycemic and hypolipidemic effects Yuwen Lia*, Tiejun Zhangb*, Jia Cuia, Na Jiaa, Yin Wua, Miaomiao Xia and Aidong Wena a Department of Pharmacy, Xijing Hospital, Fourth Military Medical University, Xi’an, China and bDepartment of Pharmacology, Medical College, Chungnam National University, Daejon, South Korea
Keywords Adenosine monophosphate activated protein kinase; Chikusetsu saponin IVa; carnitine palmitoyl transferase-1; glucose and lipid metabolism; Glucose transporter 4 Correspondence Aidong Wen, Department of Pharmacy, Xijing Hospital, Fourth Military Medical University, Changle Road, No. 127, Xi’an 710032, China. E-mail: [email protected] Miaomiao Xi, Department of Pharmacy, Xijing Hospital, Fourth Military Medical University, Changle Road, No. 127, Xi’an 710032, China. E-mail: [email protected] *These authors contribute equally to this work. Received September 29, 2014 Accepted January 1, 2015 doi: 10.1111/jphp.12392
Abstract Objectives The aim of this study is to investigate antidiabetic effects and molecular mechanisms of the chemical Chikusetsu saponin IVa (CHS) that isolated from root bark of Aralia taibaiensis, which has multiple pharmacological activity, such as relieving rheumatism, promoting blood circulation to arrest pain and antidiabetic action. Methods Rats with streptozotocin/nicotinamide-induced type 2 diabetes mellitus (T2DM) and insulin-resistant myocytes were used. Adenosine monophosphate (AMP)-activated protein kinase (AMPK) and acetyl-CoA carboxylase were quantified by immunoblotting. Assays of glucose uptake, fatty acid oxidation, glucose transporter 4 (GLUT4) translocation and carnitine palmitoyl transferase-1 (CPT-1) activity were performed. Key findings Chronic oral administration of CHS effectively decreases blood glucose, triglyceride, free fatty acid (FFA) and low density lipoprotein-cholesterol levels in T2DM rats. In both normal and insulin-resistant C2C12 myocytes, CHS activates AMPK, and increases glucose uptake or fatty acid oxidation through enhancing membrane translocation of GLUT4 or CPT-1 activity respectively. Knockdown of AMPK significantly diminishes the effects of CHS on glucose uptake and fatty acid oxidation. Conclusions CHS is a novel AMPK activator that is capable of bypassing defective insulin signalling and could be useful for the treatment of T2DM or other metabolic disorders.
Introduction Type 2 diabetes was characterized by altered lipid and glucose metabolism (fasting or postprandial hyperglycemia, dyslipidemia) that was a consequence of combined insulin resistance in skeletal muscle, liver and adipose tissue and relative defects of insulin secretion by beta cells.[1] Regulatory mechanisms of physical exercise and two major classes of anti-diabetic drugs (biguanides and thiazolidinediones) were much reported.[2,3] Novel besides, a good practice and bright future had been shown in the therapy of diabetes and its complications by natural products.[4] Exciting developments in type 2 diabetes therapy had shown that adenosine monophosphate (AMP)-activated protein kinase (AMPK) was one of the probable target of major anti-diabetic drugs (metformin and thiazolidinediones), and of insulin-
sensitizing adipokines (e.g. adiponectin).[5] For decades, the development of AMPK activators from natural products had been reported.[6] Resveratrol was a stilbene that was isolated from grapes and had potential for the treatment and prevention of type 2 diabetes. AMPK was thought to be its primary target.[7] AMPK was a phylogenetically conserved serine/threonine protein kinase, and was known to act as a key metabolic ‘master switch’ by phosphorylating target enzymes involved in lipid metabolism such as acetyl-CoA carboxylase (ACC).[8] Inhibition of ACC by AMPK activated MalonylCoA decarboxylase, resulted in reduction of malonyl CoA levels and increased fatty acid oxidation at the carnitine palmitoyl transferase-1 (CPT-1) step in liver, heart and
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skeletal muscle, the primary tissues that oxidize fatty acids.[9,10] Collectively, AMPK was emerged as a key player in the regulation of glucose homeostasis and lipid profile. Use of AMPK agonists could be a new therapeutic strategy for type 2 diabetes patients. Moreover diversified mechanisms by which AMPK influenced glucose and lipid metabolism in different tissues (liver, pancreas, skeletal muscle and adipocyte tissue) were already well documented.[11] Skeletal muscle was a principal site of glucose and fatty acid usage, and was one of the primary tissues responsible for insulin resistance in obesity and type 2 diabetes.[12] Stimulation of AMPK in the muscle could be an efficient method to increase glucose uptake in an insulin-independent manner, thus bypassing defective insulin signalling, such as one observed in type 2 diabetes patients.[13] Over the past few decades, many studies had shown that the anti-diabetic activity of dietary and medicinal plants were due to the presence of saponins.[14–16] Aralia taibaiensis Z.Z. Wang et H.C. Zheng (Araliaceae) was authenticated by Dr. Haifeng Tang. A voucher specimen (FMMUDP-Voucher No: SAP012) was deposited in the Herbarium of the Department of Pharmacy, Xijing Hospital, Fourth Military Medical University. It was widely distributed in the Qinba Mountains of western China. The extract of root bark of A. taibaiensis had multiple pharmacological activity, including relieving rheumatism, promoting blood circulation to arrest pain, inducing diuresis to reduce oedema and antidiabetic action.[17,18] The total saponins extracted from A. taibaiensis (sAT) had effective combined antioxidant and antiglycation activity in vitro and ex vivo.[17,19] In addition, sAT dramatically stimulated high-glucose-induced insulin secretion and its antidiabetic activity might be related to its high saponin content.[20] Previous phytochemical investigations revealed that the main active components of herb were 12 triterpenoid saponins.[21] Chikusetsu saponin IVa (CHS) was one of triterpenoid saponins and was indicated as the active insulinogenic ingredient of sAT.[21] In this study, the mechanism by which CHS effectively prevented type 2 diabetes was investigated. CHS significantly enhanced glucose uptake or fatty acid oxidation through promoting glucose transporter 4 (GLUT4) membrane translocation or increasing CPT-1 activity respectively in insulin-resistant skeletal muscle cells via an AMPKdependent pathway.
Materials and Methods Animals All animal procedures were approved by the Ethics Committee for Animal Experimentation of the Fourth Military Medical University (Xi’an, China) (ethical permission number: FMMU14005), and carried out in accordance with 998
University Ethics Guidelines for the care and use of laboratory animals. Mature male Wistar Albino rats (8–10 weeks old; 180–220 g) were used in this study and the rats were fed with standard rat pellet diet and water ad libitum.
Induction of type 2 diabetes mellitus (T2DM) Overnight fasted rats were intraperitoneally administrated with 120 mg/kg nicotinamide (NA), and T2DM was induced by a single intra-peritoneal injection of 50 mg/kg of streptozotocin (STZ) after 15 min. Hyperglycemia was confirmed by the elevated glucose levels in plasma, determined at 72 h and on day 7 after STZ and NA injection. The threshold value of fasting plasma glucose to diagnose diabetes was taken as ≥7.0 mM (World Health Organization, 2006).[22] Only rats found with T2DM were used for antidiabetic study.
Isolation of Chikusetsu saponin IVa and structure elucidation CHS was isolated from the sAT. Firstly, the dry and powdered root bark (100 g) of A. taibaiensis was extracted three times with 10-fold (v/v) 80% ethanol under reflux for 1 h. The alcoholic extracts were concentrated, suspended in distilled water and then partitioned successively with threefold (v/v) n-butanol saturated with water for three times. The n-butanol extracts were combined and evaporated using a rotary evaporator at 60°C. The yield was 15.91% (w/w). The extract (10 g) was chromatographed over silica gel, stepwise eluted with chloroform-methanol-water (from 7 : 1 : 1, lower phase, to 6.5 : 3.5 : 1, lower phase). The fraction was submitted to size exclusion chromatography on a Sephadex LH-20 column eluted with MeOH-H2O (1 : 1) to remove the pigments and carbohydrates. CHS (1 mg/ml) was analysed by using high performance liquid chromatography (HPLC) with acetonitrile and 0.2% phosphoric acid (35 : 65, v/v) at a flow rate of 1 ml/min (Figure S1) The structure of CHS was elucidated as C42H66O14 by extensive spectral analysis (IR, 1H-NMR, 13C-NMR and ESI-MS).
Acute toxicity study Acute oral toxicity study was performed as per Organization for Economic Cooperation and Development guidelines 423 (acute toxic classic method) (OECD, 2001).[23] Twenty-four male Wistar rats starved overnight were divided into four groups (n = 6) and orally fed with CHS in increasing dose levels of 25, 50, 100 and 200 mg/kg. The rats were observed individually at least once during the first 30 min, periodically during the first 24 h, with special attention given during the first 4 h, and daily thereafter, they
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were observed for a total of 14 days for any physical signs of toxicity such as writhing, gasping, palpitation and decreased respiratory rate or mortality. After 14 days, the lethality or death was calculated.
Experiments design Rats were randomly grouped. Body weight and food intake were monitored daily during the experimental period. Fasting blood glucose levels were monitored from vein every week using an Accu-check blood glucose meter (Roche Diagnostics, Basel, Switzerland). The serum free fatty acid (FFA), low density lipoprotein-cholesterol (LDLC), high density lipoprotein-cholesterol (HDL-C) and triglyceride (TG) levels were determined using a 7180-automatic biochemical analyser (Hitachi, Japan) at the Department of Clinical Laboratory, Xijing Hospital, Fourth Military Medical University (Xi’an, China).
Oral glucose tolerance test (OGTT) The oral glucose tolerance test was performed in overnight fasted (18 h) normal rats. Glucose (2 g/kg) was fed 30 min after the administration of drugs. Blood was withdrawn from the retro orbital sinus under ether inhalation at 15, 30, 60 and 120 min of glucose administration and glucose levels were estimated using a glucose oxidase–peroxidase reactive strips and the glucometer (Roche Diagnostics, Basel, Switzerland).
Cell culture, plasmid and transfection Mouse C2C12 myoblasts (ATCC, Manassas, VA, USA) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 50 U/ml penicillin and 50 μg/ml streptomycin. When cells reached confluence, the medium was switched to the differentiation medium containing DMEM and 2% horse serum, which was changed every other day. After 4 additional days, the differentiated C2C12 cells had fused into myotubes. To induce insulin resistance, C2C12 cells were cultured in DMEM containing 15 g/l of glucose, 2% horse serum, sodium pyruvate, 1 μg/ml of insulin, 100 nM dexamethasone and 1% penicillin/streptomycin for 4 days.[24] Plasmid pEGFP-C1-GLUT4 was prepared by amplifying the C2C12 cDNA library with corresponding primers. Sequences of the oligonucleotides are available upon request. For the knockdown of siRNA, scramble and siRNA specific for mouse AMPK were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Transient transfections were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol.
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Antibodies and regents DMEM medium, FBS, L-glutamine, penicillin, streptomycin and other cell culture reagents were obtained from Gibco (Invitrogen, Carlsbad, CA, USA). Anti-AMPK antibody, anti-phospho-Thr172 (AMPK) antibody, anti-ACC antibody, anti-phospho-Ser79 (ACC) antibody, anti-phosphoSer473 (protein kinase B (PKB)) and anti-PKB antibody were obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-β-actin antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). 2-deoxy[1-3H]-glucose, [1-14C]-palmiticacid and [γ-32P] ATP were obtained from PerkinElmer Life and Analytical Sciences (Boston, MA, USA). Insulin, glibenclamide STZ and NA were purchased from Sigma-Aldrich (St Louis, MO, USA).
AMPK activity measurement To measure isoform-specific AMPK activity in skeletal muscles, we immunoprecipitated AMPK from cell lysates (1 mg of protein) with specific antibodies against the α1 or α2 catalytic subunits bound to protein-G/sepharose beads. We measured kinase activity using synthetic ‘SAMS’ peptide and [γ-32P] ATP.
Glucose uptake determination Glucose uptake was assayed by using 2-deoxy-[1-3H]-glucose. Cells were incubated in the presence or in the absence of 1 μg/ml CHS for 15 min and then washed two times with washing buffer (20 mM HEPES (pH 7.4), 140 mM NaCl, 5 mM KCl, 2.5 mM MgSO4 and 1 mM CaCl2). Cells were then incubated in transport solution (the buffer containing 0.5 mCi 2-deoxy-[1-3H]-glucose and 10 μm 2-deoxyglucose) for 10 min. Non-specific uptake was determined with incubating the cells in the presence or in the absence of 5 mM cytochalasin B. The reaction was terminated by aspiration of the solution. Cells were then washed three times, and radioactivity associated with the cells was determined by cell lysis in 0.05M NaOH, followed by scintillation counting. Protein concentration was determined by the Bradford method. 2-Deoxyglucose uptake was expressed as pmol/minute/ milligram. Measurements were performed in duplicate and in three independent experiments.
GLUT4 translocation detection To detect GFP-GLUT4 translocation, C2C12 myocytes were grown on glass coverslips and then transfected with pEGFPC1-GLUT4. After 48 h, the cells were treated as indicated in the figure legend, and were fixed with 4% paraformaldehyde for 10 min, then were mounted with Fluoromount-G (SouthernBiotech, Birmingham, UK) and were visualized using a fluorescence microscope equipped with the appropriate filters (Olympus, Tokyo, Japan).
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Fatty acid oxidation determination To determine fatty acid oxidation in muscle, [1-14C]palmitic acid oxidation was measured in C2C12 myotubes using 14CO2 and 0.2 ml of benzethonium solution. Briefly, cells were mixed the assay buffer containing 1.25M NaCl, 0.1M MgSO4•7H2O, 1.2 mM KH2PO4, 0.5M KCl, 0.2M glucose, 1M NaHCO3, 0.25M CaCl2 and 1 mCi [1-14C]palmitic acid, gassed for 30 s under humidified 95/5% O2/CO2 and covered with filter paper that moistened with 0.2 ml of benzethonium solution. After incubation for 1 h at 37°C, the reaction was stopped by 6% trichloroacetic acid (TCA) solution and the radioactivity trapped in the filter paper was determined. Fatty acid β-oxidation activity was expressed as nmol/minute/milligram.
CPT-1 enzyme activity measurement The activity was assayed in these supernatants spectrophotometrically by following the release of CoA-SH from palmitoyl-CoA using the general thiol reagent 5,5dithiobis-(2-nitrobenzoic acid) (DTNB). Eight hundred forty-five microlitre Tris-HCl–DTNB (116 mM Tris, 25 mM EDTA, 2 mM DTNB, 0.2% Triton X-100, pH 8.0) was prepared and was added to semi-microcu-vettes (Greiner, Frickenhausen, Germany); 50 μg proteins were adjusted to 100 μl and were added then. After 5 min pre-incubation at 30°C, 50 μl palmitoyl-CoA (1 mM) was added. The action was then started by adding 5 μl L-carnitine solution (1.2 mM dissolved in 1M Tris, pH 8.0), immediately followed by photometric measurement (Hitachi U-2010; 30°C, 4GE nm) for 360 s. The difference between absorbance readings with or without substrates measured the release of CoA-SH.
Western blot analysis After treatment with different conditions as described in the figure legends, cells were lysed in M2 buffer (20 mM Tris at pH 7.6, 0.5% NP-40, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 2 mM DTT, 0.5 mM PMSF, 20 mM β-glycerol phosphate, 1 mM sodium vanadate, 1 μg/ml leupeptin). Fifty microgram of the cell lysates were subjected to SDSpolyacrylamide gel and blotted onto PVDF membrane (Millipore, Bedford, MA, USA). After blocking with 5% skim milk in PBS/T, the membrane was probed with the relevant antibody and visualized by enhanced chemiluminescence (Biological Industries, Beit Haemek, Israel).
Statistical analysis Data were expressed as mean ± standard deviation. Statistical analysis was performed by analysis of variance followed 1000
by least significant difference (LSD)’s test for multiple comparisons. Data analyses were performed using the SPSS 11.0 software package. Differences were considered significant at P < 0.05.
Results Acute toxicity study In rats, oral administration of CHS at four doses did not produce any drug-induced physical signs of toxicity and no death was registered in 14 days, indicating that CHS was nontoxic in rats with an oral dose of 200 mg/kg. Therefore, investigation of antihyperglycemic activity of CHS at 7.5, 15 and 30 mg/kg doses were safe and feasible.
CHS regulates glucose and lipid metabolism in T2DM rats To further evaluate effects of CHS on glucose and fatty acid metabolism, graded doses of CHS were administrated to T2DM rats daily intragastrically for 4 weeks. CHS significantly decreased body weight and fasting blood glucose level of T2DM rats (Figure 1a and 1b). In OGTT, the high dose of CHS treatment showed significant reduction of 44.2% in plasma glucose levels after 120 min of glucose administration compared with vehicle treated T2DM group (Figure 1c). The effects of CHS on serum lipid parameters were depicted (Table 1). The results showed that high dose of CHS treatment significantly countered the rise in the levels of serum TG, FFA and LDL-C in T2DM rats, and increased HDL-C level by 13.2% compared with vehicle. No significant differences in food intake were found in T2DM rats that were treated with different doses of CHS (Figure S2). These data showed that CHS effectively increased disposal of glucose and lipid from blood in T2DM rats.
CHS increased AMPK phosphorylation, glucose uptake and fatty acid oxidation in C2C12 myocytes Next, it was examined as to how CHS could modulate glucose and lipid metabolism in C2C12 myocytes. CHS increased phosphorylation of AMPK and ACC. The time of onset and the duration of these effects on ACC phosphorylation were similar to AMPK phosphorylation, but peaked at 30 min and short lived (Figure 2a). In-vitro kinase assay, AMPK activity was increased 1.3 fold by CHS (Figure 2b). After that, we examined function of CHS on glucose uptake and GLUT4 translocation. We found that glucose transport increased 30% in the presence of CHS (Figure 2c). The stimulation of glucose transport was mainly regulated by the translocation of GLUT4 to the plasma membrane in myocytes. The cytosolic
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Effects of Chikusetsu saponin IVa on T2DM
Chikusetsu saponin IVa (CHS) COOGlc HOOC HO HO
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120 min
Figure 1 Effects of Chikusetsu saponin IVa (CHS) on glucose metabolism in type 2 diabetes mellitus (T2DM) rats. Streptozotocin (STZ)nicotinamide (NA)-induced T2DM rats (n = 6) were daily intragastrically treated with different doses of CHS (L: 7.5 mg/kg per day; M: 15 mg/kg per day; H: 30 mg/kg per day) and glibenclamide (0.6 mg/kg per day) for 4 weeks. After treatment, all of the rats were fasted overnight. Structure of CHS was shown above. The body weight (a) and fasting blood glucose level were measured (b); the rats were orally loaded with glucose (2 g/kg). Then their blood glucose levels were determined at 0, 30, 60 and 120 min after glucose administration (c). The data were presented as the mean ± standard deviation (SD), *P < 0.05 or **P < 0.01 vs T2DM rats treated with vehicle alone.
and membrane fractions of C2C12 myocytes were subsequently prepared for Western blot analysis of GLUT4. Membrane expression of GLUT4 was significantly increased by CHS (Figure 2d). GFP-GLUT4 distributed throughout the cytoplasm in control cells treated with vehicle. CHS caused a substantial redistribution of GFP-GLUT4, such that an almost continuous line of fluorescence was observed around the plasma membrane (Figure 2e). The effect of CHS on GLUT4 translocation was in agreement with glucose uptake data. In addition, CHS strongly increased
fatty acid oxidation (Figure 2f). CPT-1 was an enzyme that catalysed the esterification of long-chain acyl-CoAs to L-carnitine for transporting into mitochondria for fatty acid oxidation, and was inhibited by ACC.[10] CHS significantly increased CPT-1 activity (Figure 2g). Our results clearly demonstrated that CHS effectively elevated glucose uptake via increasing translocation of GLUT4 to plasma membran. Moreover, activation of AMPK by CHS decreased the activity of ACC by phosphorylation (Figure 2a), led to relieving inhibitory effect on CPT-1
© 2015 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 67, pp. 997–1007
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Table 1
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Hypolipidemic activity of CHS in normal and T2DM rats (mean ± SD, n = 6)
Groups
FFA mg/dl
TG mg/dl
LDL-C mg/dl
HDL-C mg/dl
Normal T2DM (veh) T2DM CHS (L) T2DM CHS (M) T2DM CHS (H) T2DM Gli
12.09 ± 1.16 21.03 ± 1.20*** 19.09 ± 1.36 17.46 ± 1.15** 14.02 ± 2.31**** 12.16 ± 1.15****
79.43 ± 4.10 131.87 ± 8.65*** 123.37 ± 8.21 117.31 ± 6.45** 108.11 ± 6.36** 87.50 ± 5.27****
21.53 ± 1.17 32.55 ± 2.45*** 30.51 ± 1.22 27.51 ± 1.33** 23.29 ± 0.88**** 21.47 ± 1.26****
48.91 ± 1.35 42.64 ± 1.25*** 43.76 ± 1.54 45.29 ± 1.62** 47.75 ± 1.08**** 48.83 ± 1.56****
FFA, free fatty acid; HDL-C, high-density lipoprotein-cholesterol; LDL-C, low density lipoprotein-cholesterol; TG, triglyceride. *P < 0.05 vs normal group; **P < 0.05 vs T2DM group; ***P < 0.05 vs normal group; ****P < 0.05 vs T2DM group.
activity (Figure 2g), therefore enhanced the entry of fatty acid into the mitochondria for oxidation (Figure 2f).
CHS increased AMPK phosphorylation, glucose uptake and fatty acid oxidation in insulin-resistant C2C12 myocytes Two distinct pathways encourage glucose transport activity in skeletal muscle, that is the contraction-stimulated pathway reliant on AMPK-dependent mechanisms and an insulin-dependent pathway activated via upregulation of PKB.[25] PKB phosphorylation on Ser-473 was declined to basal level under insulin stimulation in insulin-resistant C2C12 myocytes (Figure 3a). Insulin lost its action on glucose uptake in insulin-resistant cells also (Figure 3b). After treatment with CHS, both AMPK and ACC phosphorylation were dramatically increased (Figure 3c). We next observed effects of CHS on glucose uptake and membrane translocation of GLUT4. Apparently, CHS increased glucose uptake (Figure 3d), and induced translocation of cytosolic GLUT4 to plasma membrane (Figure 3e). Moreover, CHS evidently promoted the migration of GFP-GLUT4 to plasma membrane (Figure 3f). Additionally, CHS increased fatty acid oxidation and CPT-1 activity in insulin-resistant C2C12 myocytes (Figure 3g and 3h).
AMPK was essential for CHS and sAT mediated glucose and fatty acid utilization To further assess the essentiality of AMPK in CHSstimulated glucose uptake and fatty acid oxidation, siRNAAMPK was used to ablate expression of AMPK in insulinresistant C2C12 myocytes. The effectiveness of AMPK knockdown on protein level was presented (Figure 4a). Silencing of endogenous AMPK by siRNA markedly reduced CHS-mediated glucose uptake compared with the cells transfected with scramble siRNA (Figure 4b). In consistent with glucose uptake result, CHS lost their effects on increasing expression of membrane GLUT4 in siRNAAMPK transfected cells (Figure 4c). Enhancement of fatty 1002
acid oxidation and CPT-1 activation by CHS were also diminished by siRNA-AMPK (Figure 4d and 4e). All the results confirmed that AMPK was essential for modulating CHS-mediated glucose and lipid metabolism.
Discussion CHS was a newly determined AMPK activator that prevented an increase of blood glucose and lipid levels in experiment T2DM rats. An enhancement of GLUT4 membrane translocation through AMPK was a mechanism by which CHS increases glucose uptake both in normal and insulin-resistant C2C12 myocytes. Moreover, inhibition of AMPK downstream target ACC by CHS resulted in an increase of fatty acid oxidation via relieving inhibitory effect on CPT-1 activity (Figure 5). Skeletal muscle was the main site for glucose disposal and fat catabolism. Our results clearly demonstrated that CHS effectively elevated glucose uptake and fatty acid oxidation in C2C12 cells (Figure 2). Indeed, regulation of energy metabolism through activation of AMPK in response to pharmacological regents was also observed in extramuscular tissues such as adipose tissue and liver. Activation of AMPK by CHS was observed in 3T3-L1 adipocytes also (data were not shown), which strongly suggested it could control AMPK-mediated increase of glucose uptake, inhibition of lipolysis and lipogenesis in adipose tissue.[26] Hepatic gluconeogenesis was a major cause of fasting hyperglycemia in diabetic patients and fatty liver disease. A disorder of TG, FFA and cholesterol in the liver was the critical complications of type 2 diabetes.[27] Inhibition of ACC by AMPK led to a decrease in fatty acid synthesis and an increase in fatty acid oxidation, thus reduced FFA and excessive storage of TG (Figure 1). The findings showed a promising function of CHS in lipid abnormalities in the liver. Furthermore, the importance of AMPK in the regulation of energy homeostasis provided a potentially widespread function of CHS in the treatment of a cluster of metabolic disorders. To further investigate benefits of CHS in the treatment of T2DM, insulin-resistant C2C12 myocytes was used. Upon
© 2015 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 67, pp. 997–1007
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Effects of Chikusetsu saponin IVa on T2DM
(a)
(c)
CHS 0.1
1
µg/mL
10
Relative AMPK activity (32p)
0
p-AMPK(Thr172) T-AMPK Actin
120
**
100 80 60 40 20 0 Veh
CHS 0
15
30
(d) 60
120
min p-AMPK(Thr172) T-AMPK p-ACC(Ser79)
Relative Glucose uptake (3H)
(b)
T-ACC
CHS
120
*
100 80 60 40 20 0
Actin
Veh
CHS
GFP-GLUT4 (e)
Cytosolic
(f)
Membrane
S
CHS
CH
Ve h
S CH
Ve h
Veh
GLUT4 Caveolin-1 Tubulin
(g)
(h) 120
Relative fatty acid oxidation (14C)
CPT-I activity (nmol palmitate/min/mg protein)
C2C12 myocytes
*
100 80 60 40 20 0 Veh
CHS
4
*
3 2 1 0 Veh
CHS
Figure 2 Effects of Chikusetsu saponin IVa (CHS) on AMPK activation, glucose uptake and fatty acid oxidation in C2C12 myocytes. C2C12 myocytes were treated with CHS for indicated concentrations (a), C2C12 myocytes were treated with 1 μg/ml CHS for indicated times (b) and whole cell lysates were separated by SDS-polyacrylamide gel (PAGE) and then immunoblotting was performed using indicated antibodies; the relative intensity of phosphorylated AMPK and acetyl-CoA carboxylase (ACC) were determined and normalized to total (bottom); C2C12 myocytes were treated with 1 μg/ml CHS for 15 min at followed experiments. Cell lysates were assayed with [γ-32P] ATP to measure AMPK activity (c); in-vitro glucose uptake was measured with 2-deoxy-[1-3H]-glucose (d); membrane and cytosolic proteins were isolated respectively; immunoblotting was performed using indicated antibodies. Anti-caveolin-1 or anti-tubulin antibodies were used to determine membrane or cytosol (e); C2C12 myocytes were transfected with pEGFP-C1-GLUT4 for 48 h. Then cells were treated with vehicle or CHS (1 μg/ml) for 15 min; the images were observed under a fluorescence microscope. The distributions of GFP-GLUT4 in C2C12 myocytes were shown. The scale bars represent 10 μm (f); C2C12 myocytes were treated with 1 μg/ml CHS for 15 min; fatty acid oxidation was determined by [1-14C]-palmitic acid (g); CPT-1 activity was measured and defined as nmol CoA-SH released/min/mg protein (h). The data were presented as the mean ± standard deviation (SD) from three independent experiments. *P < 0.05 or **P < 0.01 vs vehicle. © 2015 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 67, pp. 997–1007
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Yuwen Li et al.
(a)
CHS
(c)
CON Insulin 0 15
Ins-res 0 15
0
15
30
60
Ins-res min
120
min p-AMPK(Thr172) T-AMPK
p-PKB(ser473)
p-ACC(Ser79)
T-PKB
T-ACC p-PKB(Ser473) T-PKB
*
Veh Ins Veh Ins CON
Ins-res
120 100 80 60 40 20 0
**
100
Cytosolic
80 60
0 15 30
60
120 min
Membrane
GLUT4
40
Caveolin-1
20 0
p-AMPK/T-AMPK p-ACC/T-ACC
** **
(e)
120
Ve h in su lin CH S
Relative Glucose uptake (3H)
(d)
Actin
Ve h in su lin CH S
120 100 80 60 40 20 0
Relative intensity
Relative Glucose uptake (3H)
(b)
Tubulin Veh insulin CHS
(f) Veh
GFP-GLUT4 insulin
CHS
Insulin resistant C2C12 myocytes (h)
120
*
100 80 60 40 20 0
Veh insulin CHS Ins-res
4
CPT-1 activity (nmol palmitate/ min/mg protein)
Relative fatty acid oxidation (14C)
(g)
*
3 2 1 0
Veh insulin CHS Ins-res
Figure 3 Effects of Chikusetsu saponin IVa (CHS) on AMPK phosphorylation, glucose uptake and fatty acid oxidation in insulin-resistant C2C12 myocytes. Insulin-resistant C2C12 myocytes were treated with insulin (100 nM, 15 min). Whole cell extracts were subjected to immunoblotting with anti-phospho-PKB (Ser-473) and anti-PKB antibodies (a); in-vitro glucose uptake was measured with 2-deoxy-[1-3H]-glucose, *P < 0.05 vs control treated with vehicle. Insulin-resistant C2C12 myocytes were treated with vehicle, CHS (1 μg/ml) or insulin (100 nM) for 15 min (b). Whole cell lysates were separated by SDS-polyacrylamide gel (PAGE) and then immunoblotting was performed using indicated antibodies. The relative intensity of phosphorylated AMPK and ACC were determined and normalized to total (bottom) (c); glucose uptake was measured with 2-deoxy-[1-3H]-glucose (d); membrane and cytosolic proteins were isolated respectively; immunoblotting was performed using indicated antibodies. Anti-caveolin-1 or antitubulin antibodies were used to determine membrane or cytosol (e); insulin-resistant C2C12 myocytes were transfected with EGFP-C1-GLUT4 for 48 h, then cells were treated with vehicle, insulin (100 nM) or CHS (1 μg/ml) for 15 min. The images were observed under a fluorescence microscope. The distributions of GFP-GLUT4 in C2C12 myocytes were shown. The scale bars represent 10 μm (f); fatty acid oxidation was determined by [1-14C]-palmitic acid (g), and CPT-1 activity was measured and defined as nM CoA-SH released/min/mg protein (h). The data were presented as the mean ± standard deviation (SD) from three independent experiments. *P < 0.05 or **P < 0.01 vs vehicle.
1004
© 2015 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 67, pp. 997–1007
Yuwen Li et al.
Effects of Chikusetsu saponin IVa on T2DM
(a) (b)
(c)
T-AMPK p-ACC(Ser79) T-ACC
100
Membrane si-scramble
*
80 60 40
GLUT4
20 Caveolin-1
0 Veh
Actin
Relative fatty acid oxidation (14C)
120 100
(e)
3
si-scramble si-AMPK
*
80 60 40 20 0
CHS
Veh
CPT-1 activity (nmol palmitate/ min/mg protein)
(d)
si-AMPK S
p-AMPK(Thr172)
si-scramble si-AMPK
CH
120
Ve h
CHS
S
Veh
CH
CHS
Ve h
Veh
si-AMPK
Relative Glucose uptake (3H)
si-scramble
2
*
1
0
CHS
si-scramble si-AMPK
Veh
CHS
Figure 4 AMPK knockdown reversed the effects of Chikusetsu saponin IVa (CHS) on glucose uptake and fatty acid oxidation in insulin-resistant C2C12 myocytes. Insulin-resistant C2C12 myocytes were transfected with either a nonspecific siRNA (si-scramble), or siRNA specific for AMPK (siAMPK) for 48 h. Cells were stimulated with 1 μg/ml CHS for 15 min; whole cell lysates were separated by SDS-polyacrylamide gel (PAGE) and then immunoblotting was performed using indicated antibodies (a). Glucose uptake was measured with 2-deoxy-[1-3H]-glucose (b); membrane proteins were isolated respectively, and anti-caveolin-1 antibody was used to determine membrane (c); fatty acid oxidation was measured with [1-14C]palmitic acid (d); CPT-1 activity was defined as nmol CoA-SH released/min/mg protein (e). The data were presented as the mean ± standard deviation (SD) from three independent experiments. *P < 0.05 vs si-scramble group.
G GLUT4
Glucose uptake
G
GLUT4 COOGlc O
AMPK O
Fatty Acid oxidation
OH
Chikusetsu saponin Iva (CHS)
F ACC
CPT-I
HOOC HO HO
Blood Glucose and Fatty Acid Figure 5 Schematic model of Chikusetsu saponin IVa (CHS) in control of glucose and fatty acid metabolism. CHS promoted migration of glucose transporter 4 (GLUT4) to plasma membrane and increased carnitine palmitoyl transferase-1 (CPT-1) activity through activating AMPK and inhibiting acetyl-CoA carboxylase (ACC), thus enhanced glucose and fatty acid utilization.
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binding to its receptor, insulin activates phosphatidylinositol 3-kinase through phosphorylation of insulin receptor substrate family.[28] A critical downstream target of 3-phosphoinositides is serine/threonine kinase PKB.[28] PKB phosphorylation on Ser-473 was declined to basal level under insulin stimulation in insulin-resistant C2C12 myocytes (Figure 3). Despite insulin lost its action, CHS was still able to activate AMPK and inhibit ACC, therefore significantly improved glucose uptake and fatty acid oxidation (Figure 3). The results suggested that CHS was capable of bypassing defective insulin signalling, and it mediated insulin independent activation of AMPK. It should be noted that activation of PKB was not observed in response to CHS, which suggested that PKB-mediated signalling was not involved in CHS-modulated energy metabolism (Figure 3). Knockdown of AMPK ablated CHS-induced translocation of GLUT4 and activation of CPT-1, which confirmed that AMPK was essential for CHS-mediated glucose and lipid utilization (Figure 4).
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Conclusions Our study demonstrated antihyperglycemic and hypolipidemic activity of CHS in T2DM rats. The beneficial action of CHS appears to be mediated by AMPK enhancement and the subsequent activation of glucose uptake and fatty acid oxidation. The findings provide the basis for the future development of CHS as a new candidate for the management of metabolic disorders.
Declarations Acknowledgements and funding This work was supported by grants from the National Natural Science Foundation of China (No. 81201985, 81373947, 81470174 and 81001673) and the National New Drug ‘R&D’ project (No. 2011ZXJ09302).
9. Abu-Elheiga L et al. Continuous fatty acid oxidation and reduced fat storage in mice lacking acetyl-CoA carboxylase 2. Science 2001; 291: 2613–2616. 10. Saha AK, Ruderman NB. MalonylCoA and AMP-activated protein kinase: an expanding partnership. Mol Cell Biochem 2003; 253: 65–70. 11. Misra P, Chakrabarti R. The role of AMP kinase in diabetes. Indian J Med Res 2007; 125: 389–398. 12. Kirwan JP. Insulin sensitivity in skeletal muscle: ‘use it or lose it, fast’. J Appl Physiol 2010; 108: 1023–1024. 13. Hardie DG. The AMP-activated protein kinase pathway – new players upstream and downstream. J Cell Sci 2004; 117: 5479–5487. 14. Abdel-Zaher AO et al. Antidiabetic activity and toxicity of Zizyphus spina-christi leaves. J Ethnopharmacol 2005; 101: 129–138. 15. Oishi Y et al. Inhibition of increases in blood glucose and serum neutral fat by Momordica charantia saponin fraction. Biosci Biotechnol Biochem 2007; 71: 735–740. 16. Lee KT et al. Hypoglycemic and hypolipidemic effects of tectorigenin and kaikasaponin III in the streptozotocin-lnduced diabetic rat and their antioxidant activity in vitro. Arch Pharm Res 2000; 23: 461–466.
17. Xi M et al. Antioxidant and antiglycation properties of triterpenoid saponins from Aralia taibaiensis traditionally used for treating diabetes mellitus. Redox Rep 2010; 15: 20–28. 18. Xi M et al. Antioxidant and antiglycation properties of total saponins extracted from traditional Chinese medicine used to treat diabetes mellitus. Phytother Res 2008; 22: 228–237. 19. Weng Y et al. Antihyperglycemic, hypolipidemic and antioxidant activities of total saponins extracted from Aralia taibaiensis in experimental type 2 diabetic rats. J Ethnopharmacol 2014; 152: 553–560. 20. Cui J et al. Insulin-secretagogue activity of eleven plant extracts and twelve pure compounds isolated from Aralia taibaiensis. Life Sci 2013; 92: 131–136. 21. Tang HF et al. Studies on the triterpenoid saponins of the root bark of Aralia taibaiensis. Yao Xue Xue Bao 1996; 31: 517–523. 22. Diabetes Research and Clinical Practice 2000; 49: 181–186. 23. Organisation for Economic Cooperation and Development (OECD). Guideline for Testing of Chemicals 423: Acute Oral Toxicity acute Toxic Class Method. 24. Koistinen HA et al. 5-aminoimidazole carboxamide riboside
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increases glucose transport and cellsurface GLUT4 content in skeletal muscle from subjects with type 2 diabetes. Diabetes 2003; 52: 1066–1072. 25. Kasuga M et al. Insulin stimulates the phosphorylation of the 95 000-dalton subunit of its own receptor. Science 1982; 215: 185–187. 26. Kapeller R, Cantley LC. Phosphatidylinositol 3-kinase. Bioessays 1994; 16: 565–576. 27. Mackenzie RW, Elliott BT. Akt/PKB activation and insulin signaling: a novel insulin signaling pathway in the
Effects of Chikusetsu saponin IVa on T2DM
treatment of type 2 diabetes. Diabetes Metab Syndr Obes 2014; 7: 55–64. 28. Berdichevsky A et al. Acute oxidative stress can reverse insulin resistance by inactivation of cytoplasmic JNK. J Biol Chem 2010; 285: 21581–21589.
Supporting information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Figure S1 The high performance liquid chromatography (HPLC) fingerprint of
© 2015 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 67, pp. 997–1007
Chikusetsu saponin IVa (CHS). The HPLC analysis was performed on a Zorbax SB-C18 column (250 mm × 4.6 mm, 5 μm) and kept at 30°C. The mobile phase consisted of acetonitrile and 0.2% phosphoric acid (35 : 65, v/v). The flow rate was 1.0 ml/min and the chromatogram was monitored at 206 nm. Figure S2 Food intake. The food intake was recorded daily, and each group was indicated by the type of connecting line. The data were presented as the mean ± standard deviation (SD).
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