Chinese Journal of Natural Medicines 2014, 12(4): 0259−0265
Chinese Journal of Natural Medicines
Maslinic acid modulates glycogen metabolism by enhancing the insulin signaling pathway and inhibiting glycogen phosphorylase LIU Jun1, WANG Xue1, CHEN Yu-Peng1, MAO Li-Fei1, SHANG Jing1, SUN Hong-Bin2*, ZHANG Lu-Yong 1* 1
State Key Laboratory of Natural Medicines, National Drug Screening Center, China Pharmaceutical University, Nanjing
210009, China; 2
Center for Drug Discovery, College of Pharmacy, China Pharmaceutical University, Nanjing 210009, China Available online 20 Mar. 2014
[ABSTRACT] AIM: To investigate the molecular signaling mechanism by which the plant-derived, pentacyclic triterpene maslinic acid (MA) exerts anti-diabetic effects. METHOD: HepG2 cells were stimulated with various concentrations of MA. The effects of MA on glycogen phosphorylase a (GPa) activity and the cellular glycogen content were measured. Western blot analyses were performed with anti-insulin receptor β (IRβ), protein kinase B (also known as Akt), and glycogen synthase kinase-3β (GSK3β) antibodies. Activation status of the insulin pathway was investigated using phospho-IRβ, as well as phospho-Akt, and phospho-GSK3β antibodies. The specific PI3-kinase inhibitor wortmannin was added to the cells to analyze the Akt expression. Enzyme-linked immunosorbent assay (ELISA) was used to measure the effect of MA on IRβ auto-phosphorylation. Furthermore, the effect of MA on glycogen metabolism was investigated in C57BL/6J mice fed with a high-fat diet (HFD). RESULTS: The results showed that MA exerts anti-diabetic effects by increasing glycogen content and inhibiting glycogen phosphorylase activity in HepG2 cells. Furthermore, MA was shown to induce the phosphorylation level of IRβ-subunit, Akt, and GSK3β. The MA-induced activation of Akt appeared to be specific, since it could be blocked by wortmannin. Finally, MA treatment of mice fed with a high-fat diet reduced the model-associated adiposity and insulin resistance, and increased the accumulated hepatic glycogen content. CONCLUSION: The results suggested that maslinic acid modulates glycogen metabolism by enhancing the insulin signaling pathway and inhibiting glycogen phosphorylase. [KEY WORDS] Maslinic acid; Insulin signal transduction; Glycogen phosphorylation a; Glycogen metabolism
[CLC Number] R965
[Document code] A
[Article ID] 2095-6975(2014)04-0259-07
Introduction Abnormalities in glucose metabolism have been implicated in the etiology of several of the most common diseases affecting modern-day society, including diabetes [1], ischemic heart disease [2], stroke [3], and cancer [4]. An important component of the glucose metabolism process is glycogen me[Received on] 18-Mar.-2013 [Research funding] This project is supported by the Fundamental Research Funds for the Central Universities (No. JKP2011004). * [ Corresponding author] ZHANG Lu-Yong: Prof., Tel: 86-25-83271023, Fax: 86-25-83271023, E-mail: [email protected]; SUN Hong-Bin: Prof.: Tel: 86-25-83271198, Fax: 86-25-83271198, E-mail: [email protected] These authors have no conflict of interest to declare. Published by Elsevier B.V. All rights reserved
tabolism, which itself plays a key role in several physiologic and pathologic processes. For example, under normal physiologic conditions, glycogen serves as an energy storage molecule, while dysfunctional glycogen metabolism can manifest as hyperglycemia [1], ischemic heart disease [2], and ischemic brain disease [3], much like dysfunctional glucose metabolism. Type 2 diabetes, in particular, is strongly associated with dysfunctional hepatic and peripheral glucose metabolism. The link between perturbed hepatic glycogen metabolism and diabetes involves its effects on insulin, which maintains blood glucose homeostasis [5]. The pentacyclic triterpenes have recently been recognized as bioactive compounds with therapeutic potential for a wide range of human diseases. Studies have demonstrated a variety of biological properties for these plant-derived compounds, including anti-inflammatory, hepatoprotective, gastroprotective, anti-ulcer, anti-viral (human immunodeficiency virus, HIV),
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LIU Jun, et al. / Chin J Nat Med, 2014, 12(4): 259−265
anti-cancer, anti-diabetic, hypolipidemic, anti- atherosclerotic, and immunoregulatory effects [6]. Maslinic acid (MA) is a pentacyclic triterpene acid that is abundant in olive fruit skin, and has recently attracted much research attention due to its promising anti-tumor [7], anti-HIV [8], and anti-oxidation activities [9]. This laboratory recently reported that MA and other related pentacyclic triterpenes represent a new class of inhibitors of glycogen phosphorylase, and demonstrated that their glucose-lowering activity in adrenaline-induced diabetic mice might be due, at least in part, to modulation of hepatic glycogen metabolism [10-12]. In another study, several naturally-occurring pentacyclic triterpenes were characterized as potential new multi-target drugs for diabetes [13-18]. In that survey of more than 700 pentacyclic triterpene compounds, it was shown by our group that some were effective in lowering blood glucose levels in various diabetic animal models, including a rodent model of diabetes [19-21]. The aim of the present study was to gain insight into the cellular and molecular effects of MA on hepatic glycogen metabolism and the related signaling pathways using an in vitro cell system (HepG2 cells) and an in vivo mouse model fed with a high-fat diet.
Materials and Methods Derivation of maslinic acid Maslinic acid (Fig. 1) was derived from commercially available oleanolic acid (purity > 95%; Guangxi Changzhou Natural Products Development Co., Ltd.) using a semisynthetic process previously described [16].
Fig. 1. Structure of maslinic acid (MA).
Cell cluture HepG2 cells (American Type Culture Collection, Manassas, VA, USA) were cultured in high (4.5 mg·mL−1) glucose content Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (V/V) fetal bovine serum, 1% (W/V) glutamine, and 1% (W/V) penicillin/streptomycin (all reagents from Gibco-BRL, Invitrogen, San Diego, CA, USA) at 37°C in a humidified atmosphere of 5% CO2/95% air. Cells were passaged by trypsinization (trypsin/EDTA; Sigma Chemical Co., St. Louis, MO, USA) when 90% confluency was reached. Glycogen Phosphorylase a (GPa) activity assay HepG2 cells (on 100 mm2 plates) were used to determine the half maximal inhibitory concentration (IC50) value of MA on GPa activity. First, the HepG2 cells were gently washed three times with ice-cold phosphate-buffered saline (PBS), covered with 50 mmol·L−1 Tris-HCl buffer (500 μL), and placed on ice for 30 min to allow hypotonic lysis. Then, the entire sample
(lysed cells plus buffer) was harvested by scraping and centrifuged (12 000 g, 10 min) to obtain the GPa-containing supernatant and the total protein-containing lysate pellet. The pellet was solubilized by incubating in 1 M KOH, and total protein content was measured by the method of Lowry et al. [22]. The supernatant was used to measure GPa activity in the absence of AMP, as previously described [23]. The basic reaction mixture contained 50 mmol·L−1 KPi (pH 6.8), 0.8 mmol·L−1 EDTA, 0.4 mmol·L−1 NADP+ disodium salt, 4 μmol·L−1 glucose-1, 6-bisphosphate tetracycloammonium salt, 1.7 U·mL−1 G6-PDH, 1 U·mL−1 phosphoglucomutase, and cell homogenate. Additionally, various concentrations of MA were present. The reaction was initiated with 2 mg·mL−1 glycogen, and the activity of GPa was measured by monitoring increases in the nicotinamide adenine dinucleotide phosphate (NADPH) concentration detected at 340 nm on a spectrophotometer (Tecan, Männedorf, Switzerland). RT-qPCR Total RNA was isolated from HepG2 cells using the TRIzol Reagent (Invitrogen) and following the manufacturer's protocol. Complementary DNA was then synthesized by reverse transcription using the Superscript First-Strand Synthesis Kit (Fermentas, Vilnius, Lithuania) and used to measure the gene transcription levels of glycogen phosphorylase by qPCR. The glycogen phosphorylase and GAPDH (internal control) genes were amplified using the SYBR Green Master Mix (Qiagen, Hilden, Germany) and the following primers: glycogen phosphorylase, (forward) 5′- GAT GGT GTA GGA ACC GTG TT -3′ and (reverse) 5′ATG CGG TCG ATG TCT TTA GG - 3′; GADPH, (forward) 5′-ACC ACA GTC CAT GCC ATC AC-3′ and (reverse) 5′-TCC ACC ACC CTG TTG CTG TA-3′. The thermal cycling reaction was carried out in an iCycler IQ5 instrument (Bio-Rad, Hercules, CA, USA) using the following conditions: initial denaturation at 95 °C for 10 min, followed by 45 cycles of denaturation at 95 °C for 10 s, annealing for 10 s at 62 °C, and extension at 72 °C for 10 s. The glycogen phosphorylase data was normalized to GAPDH and the relative expression was calculated as 2−Δct × 100%, where Δct was the difference in the ct value between the target gene and GAPDH. All qPCRs were performed in triplicate using three independent samples. Glycogen content assay HepG2 cells were seeded in 6-well plates at a density of 5 × 104/mL. When the cells reached 80% confluency, MA was added (to achieve 0.1, 1, and 10 μmol·L−1 final concentrations per well) and the cells were incubated for an additional 24 h. The MA affects on glycogen content were measured as previously described [18]. Briefly, the cells were washed three times with ice-cold PBS and lysed by 0.1 mol·L−1 NaOH (400 µL). The lysates were then heated at 80°C for 1 h and the glycogen was precipitated by adding 2.5 volumes of ethanol. The samples were placed at 25 °C for 24 h and then centrifuged for 15 min at 12 000 g. The pellet was freeze-dried and re-suspended in 50 mmol·L−1 sodium acetate buffer (pH 4.8). The glycogen was digested with amyloglucosidase at 37 °C for 90 min, and the resulting glucose was
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assayed with G6-PDH, as described above. All glycogen measurements were standardized against the total protein content, which was determined in a 0.1 mol·L−1 NaOH cell solution using the Lowry method. Enzyme linked immunosorbent assay (ELISA) for whole-cell IR auto-phosphorylation ELISA detection of tyrosine phosphorylation on the insulin receptor β (IRβ) subunit was performed as described previously [24], with some modifications. After incubation with MA at 0, 0.1, 1, and 10 µmol·L−1, or 10 nmol·L−1 insulin alone as a positive control, the HepG2 cells were washed three times with ice-cold PBS and solubilized by incubating for 30 min at 4 °C in Triton X-100 lysis buffer (Invitrogen) containing 150 mmol·L−1 NaCl, 10 mmol·L−1 Hepes (pH 7.4), 1% (V/V) Triton X-100, protease inhibitors (50 μg·mL−1 aprotinin, 10 μg·mL−1 leupeptin, 40 μg·mL−1 pepstatin A, and 1 mmol·L−1 PMSF), and phosphatase inhibitors (400 μmol·L−1 sodium vanadate, 10 m mol·L−1 sodium fluoride, and 10 m mol·L−1 sodium pyrophosphate). The cell debris was removed by centrifugation (800 g, 30 min, 4 °C) and equal amounts of cell lysates (40 μg of total protein) were applied to 96-well Immulon-1 flat-bottomed-plates (Nalge-Nunc International, Rochester, NY, USA) that had been pre-coated with monoclonal anti-human IRβ antibody (Ab) (C-19; Santa Cruz Biotechnology, Santa Cruz, CA, USA) in carbonate/bicarbonate buffer (15 mmol·L−1 Na2CO3 and 35 mmol·L−1 NaHCO3, pH 9.6). After incubating at 4 °C overnight, the plates were washed with PBS containing 0.05% Tween 20 (PBST) and biotin-conjugated anti-pTyr Ab (4G10; Millipore, Billerica, MA, USA) was added to the wells. After incubating for 1 h at 25 °C, the wells were washed with PBST and horseradish peroxidase (HRP)-conjugated streptavidin (Invitrogen) was added. Following the addition of the peroxidase substrate, o-phenylenediamine dihydrochloride, the rate of tyrosine phosphorylation was quantified by measuring absorbance at 490 nm with a microplate reader (Tecan, Männedorf, Switzerland). Western blotting analysis Total HepG2 cell proteins were resolved by electrophoresis through 10% SDS-polyacrylamide gels and transferred onto nitrocellulose membranes. Non-specific binding sites were first blocked by incubating the membranes in Tris-buffered saline (TBS) containing 0.1% Tween 20 and 5% non-fat dried milk for 1 h at room temperature. Then, Abs against the phosphorylated proteins (pIRβ; phospho-Ser473-Akt (pAkt) or phospho-Ser9-glycogen synthase kinase 3β (pGSK3β) from Cell Signaling Technology, Beverly, MA, USA) or their non-phosphorylated forms (IRβ; Akt or GSK3β (Cell Signaling Technology)) were applied to the respective membranes and allowed to react for overnight at 4°C. After a wash with TBS + 0.1% Tween 20 (TBST), the appropriate HRP-conjugated secondary Abs (affinity-purified mouse anti-rabbit IgG or rabbit antimouse IgG from Sigma Chemical Co.) were applied and allowed to react for 1 h at room temperature. Immunoreactive signals were detected by an enhanced chemiluminescence kit (Amersham Biosciences, NJ, USA).
Animal experiments All animal experiments were carried out with approval from the Animal Ethics Committee of China Pharmaceutical University. The authors have received permission from the Animal Care and Use Committee of China Pharmaceutical University. Male C57BL/6J mice (6–8 weeks old) were housed in a temperature-controlled room [(22 ± 2) °C), with a 12 h light/dark cycle. Animals were randomly assigned to receive either standard rodent diet (control group) or a high-fat diet (HFD) containing 2% cholesterol and 10% lard to generate insulin resistance [25-26]. The ad libitum diets were maintained for a total of 12 weeks, but at week 10 the mice in the HFD group were sub-divided into three groups to assess the effects of MA. The first group remained untreated on the HFD for the final two weeks (model group). The other two groups received two weeks of MA treatment at 50 or 100 mg·kg−1·d−1. The body weight (bw) was measured every week, and the food intake was calculated everyday. All animals were sacrificed at the end of the experimental period and liver and epididymal fat tissue samples were harvested. In vivo glucose metabolism The effects of MA treatment on plasma insulin levels were determined using an ultrasensitive ELISA kit (Shanghai ExCell Biology, Inc., Shanghai, China) following the manufacturer’s instructions. In addition, hepatic glycogen content was measured in the mouse liver tissue specimens by using the previously described anthrone method [22], with some modifications. Briefly, 0.1 g liver tissue was homogenized, then was re-suspended in 30% KOH and incubated for 15 min at 95°C to extract glycogen. Then, 2% Na2SO4 was added and the glycogen was precipitated with ethanol. The glycogen precipitate was re-suspended in H2O : sulfuric acid (3 : 7.6) containing 0.15% anthrone (Sigma Chemical Co.) and heated at 95°C for 15 min. The reaction was terminated by plunging the tubes into ice, and the glycogen content was measured spectrophotometrically at 620 nm. Statistical analysis The data are expressed as x ± s, unless otherwise indicated. Intergroup differences were assessed by one-way analysis of various (ANOVA) followed by the Bonferroni post-hoc test. P-< 0.05 was considered to indicate statistical significance.
Results MA inhibits both GPa activity and mRNA expression in HepG2 cells The IC50 of MA in HepG2 cells was 6.91 μmol·L−1, which was substantially lower than the IC50 of the positive control compound (caffeine: 307 μmol·L−1; Fig. 2A). Moreover, the inhibitive effect of MA on GPa activity in the HepG2 cells was dose-dependent. The MA-treated cells also showed reduced levels of GPa mRNA (Fig. 2B) and increased levels of glycogen content (Fig. 2C). Taken together, these results suggest that MA may increase accumulated glycogen in HepG2 cells by inhibiting the activity of GPa, which is the rate-limiting enzyme in hepatic glycogen metabolism.
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Fig. 2. Effect of MA on glycogen phosphorylase in HepG2 cells (mean ± SD, n = 4). (A) Inhibitive effects of MA and caffeine (control) on GPa activity, as measured by inhibition rate. (B) Inhibitive effects of MA on GPa gene transcription, as mRNA determined by RT-qPCR detection of mRNA (normalized to GAPDH). (C) Effects of MA on intercellular glycogen content. HepG2 cells were left untreated (control) or treated with 0.1, 1, or 10 μmol·L−1 MA for 24 h. *P < 0.05, **P < 0.01 vs control group
MA stimulates IRβ auto-phosphorylation in HepG2 cells Tyrosine phosphorylation of IRβ in HepG2 cells was increased in response to MA treatment, and the effects were dose-dependent, as shown by Western blotting analysis with anti-pTyr (Fig. 3A). Moreover, the MA treatment also led to increased IRβ auto-phosphorylation, as shown by ELISA (Fig. 3B). This result suggested that the MA-induced IR pTyr signal from HepG2 cells might be due to an increase in the number of phosphorylated IR molecules. MA induces phosphorylation of Akt and GSK3β in HepG2 cells Akt Ser473 phosphorylation is an important node in the insulin signal transduction pathway, and it reflects receipt and acceptance of an upstream signal for downstream transmission that can modulate the processes of glucose uptake and glycogen accumulation. In HepG2 cells, MA-induced activation of IRβ led to increased phosphorylation of Akt, as shown by Western blotting with anti-pAkt (Fig. 4). Specifically, Akt phosphorylation was remarkably increased in HepG2 cells treated with 1 µmol·L−1 and 10 µmol·L−1 MA (Fig. 4A). The MA-induced Akt phosphorylation response was completely inhibited by pre-treatment of cells with the PI-3K inhibitor, wortmannin (Fig. 4B). GSK3β is a well-known downstream target of Akt that plays an important role in regulating cellular glucose metabolism. Analysis of MA effects on the phosphorylation of GSK-3β indicated that MA treatment led to increased GSK3β phosphorylation at Ser9 in HepG2 cells (Fig. 4C).
Fig. 3. Effect of MA on IRβ and IRβ auto-phosphorylation (mean ± SD, n = 3). (A) Cells were starved for 8 h with serum-free medium, and treated with 0, 0.1, 1, and 10 μmol·L−1 MA for 30 min. Equal amounts of soluble proteins were used to detect with Abs specific for the phosphorylated forms of IRβ. Blots were stripped and re-probed with Abs directed against total cellular forms of IRβ. A single Western blot representative of three independent experiments is shown. (B) ELISA was used to detect IRβ auto-phosphorylation. Cells were incubated in the presence of MA at 0, 0.1, 1, and 10 µmol·L−1 for 30 min. Cells treated with insulin alone were used as the positive control. IRβ auto-phosphorylation was measured by ELISA as described in the Experimental section. * P < 0.05, **P < 0.01, ***P < 0.001 vs control group
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Fig. 4. Effect of MA on Akt and GSK3β phosphorylation in HepG2 cells. MA treatment induced Akt phosphorylation in HepG2 cells in a (A) dose-dependent manner. (B) Wortmannin treatment blocked MA-induced Akt phosphorylation. After 8 h starvation in serum-free DMEM medium, cells were pre-incubated with 100 nmol·L−1 Wortmannin for 30 min, and then changed to serum-free medium containing 10 μmol·L−1 MA for 2h, and 0.2% DMSO alone used as the negative control. The immunoblots shown (a-c) are representative of three independent experiments. (C) Dose-dependent course of MA on GSK3β phosphorylation in HepG2 cells
treated HFD mice at both the low concentration (50 mg·kg−1·d−1) and high concentration (100 mg·kg−1·d−1) showed markedly reduced blood glucose levels. MA at the high concentration (100 mg·kg−1·d−1) also significantly improved the hyperinsulinemic condition (Table 1), markedly reduced adiposity (Fig. 5A), and increased the accumulated hepatic glycogen (Fig. 5B). Collectively, these results suggested that MA may modulate glycogen metabolism and improve HFD-induced hyperinsulinemia and hyperglycemia by enhancing the accumulation of hepatic glycogen and reducing insulin levels. Thus, the hypothesis that MA can modulate glycogen metabolism in vivo appears to be valid.
MA treatment of mice does not modulate body weight or the amount of daily food intake When mice fed with HFD were treated with MA for two weeks, they did not experience any significant changes in body weight or daily food intake (vs normal diet control and untreated HFD model control mice; Table 1). MA modulates glycogen metabolism in vivo by improving insulin resistance and increasing the hepatic glycogen accumulation To gain more insight into the effect of MA on glycogen metabolism, HFD-induced mice were treated with MA for two weeks and the effects on blood glucose and insulin levels were assessed. Compared to the untreated HFD model mice, the MA-
Table 1 Effects of MA on body weight, food intake, serum glucose, and serum insulin (mean ± SD, n = 10) Group
Dose/(mg·kg−1·d−1)
Body weight/g
Food intake/(g/25 g/d)
cBg/(mmol·L−1)
cSi/(µg·L−1)
Control
−
27.5 ± 1.4
3.36 ± 0.31
7.32 ± 0.15
1.58 ± 0.67
Model
−
27.2 ± 1.3
2.84 ± 0.42
8.66 ± 1.36
MA
100
25.9 ± 2.8
2.82 ± 0.23
6.75 ± 0.58*
5.59 ± 2.19*
MA
50
26.7 ± 1.4
2.76 ± 0.54
6.78 ± 1.22*
8.66 ± 0.20
#
8.33 ± 2.11###
cBg: concentration of blood glucose; cSi: concentration of serum insulin ### P < 0.001, #P
Chinese Journal of Natural Medicines
Maslinic acid modulates glycogen metabolism by enhancing the insulin signaling pathway and inhibiting glycogen phosphorylase LIU Jun1, WANG Xue1, CHEN Yu-Peng1, MAO Li-Fei1, SHANG Jing1, SUN Hong-Bin2*, ZHANG Lu-Yong 1* 1
State Key Laboratory of Natural Medicines, National Drug Screening Center, China Pharmaceutical University, Nanjing
210009, China; 2
Center for Drug Discovery, College of Pharmacy, China Pharmaceutical University, Nanjing 210009, China Available online 20 Mar. 2014
[ABSTRACT] AIM: To investigate the molecular signaling mechanism by which the plant-derived, pentacyclic triterpene maslinic acid (MA) exerts anti-diabetic effects. METHOD: HepG2 cells were stimulated with various concentrations of MA. The effects of MA on glycogen phosphorylase a (GPa) activity and the cellular glycogen content were measured. Western blot analyses were performed with anti-insulin receptor β (IRβ), protein kinase B (also known as Akt), and glycogen synthase kinase-3β (GSK3β) antibodies. Activation status of the insulin pathway was investigated using phospho-IRβ, as well as phospho-Akt, and phospho-GSK3β antibodies. The specific PI3-kinase inhibitor wortmannin was added to the cells to analyze the Akt expression. Enzyme-linked immunosorbent assay (ELISA) was used to measure the effect of MA on IRβ auto-phosphorylation. Furthermore, the effect of MA on glycogen metabolism was investigated in C57BL/6J mice fed with a high-fat diet (HFD). RESULTS: The results showed that MA exerts anti-diabetic effects by increasing glycogen content and inhibiting glycogen phosphorylase activity in HepG2 cells. Furthermore, MA was shown to induce the phosphorylation level of IRβ-subunit, Akt, and GSK3β. The MA-induced activation of Akt appeared to be specific, since it could be blocked by wortmannin. Finally, MA treatment of mice fed with a high-fat diet reduced the model-associated adiposity and insulin resistance, and increased the accumulated hepatic glycogen content. CONCLUSION: The results suggested that maslinic acid modulates glycogen metabolism by enhancing the insulin signaling pathway and inhibiting glycogen phosphorylase. [KEY WORDS] Maslinic acid; Insulin signal transduction; Glycogen phosphorylation a; Glycogen metabolism
[CLC Number] R965
[Document code] A
[Article ID] 2095-6975(2014)04-0259-07
Introduction Abnormalities in glucose metabolism have been implicated in the etiology of several of the most common diseases affecting modern-day society, including diabetes [1], ischemic heart disease [2], stroke [3], and cancer [4]. An important component of the glucose metabolism process is glycogen me[Received on] 18-Mar.-2013 [Research funding] This project is supported by the Fundamental Research Funds for the Central Universities (No. JKP2011004). * [ Corresponding author] ZHANG Lu-Yong: Prof., Tel: 86-25-83271023, Fax: 86-25-83271023, E-mail: [email protected]; SUN Hong-Bin: Prof.: Tel: 86-25-83271198, Fax: 86-25-83271198, E-mail: [email protected] These authors have no conflict of interest to declare. Published by Elsevier B.V. All rights reserved
tabolism, which itself plays a key role in several physiologic and pathologic processes. For example, under normal physiologic conditions, glycogen serves as an energy storage molecule, while dysfunctional glycogen metabolism can manifest as hyperglycemia [1], ischemic heart disease [2], and ischemic brain disease [3], much like dysfunctional glucose metabolism. Type 2 diabetes, in particular, is strongly associated with dysfunctional hepatic and peripheral glucose metabolism. The link between perturbed hepatic glycogen metabolism and diabetes involves its effects on insulin, which maintains blood glucose homeostasis [5]. The pentacyclic triterpenes have recently been recognized as bioactive compounds with therapeutic potential for a wide range of human diseases. Studies have demonstrated a variety of biological properties for these plant-derived compounds, including anti-inflammatory, hepatoprotective, gastroprotective, anti-ulcer, anti-viral (human immunodeficiency virus, HIV),
– 259 –
LIU Jun, et al. / Chin J Nat Med, 2014, 12(4): 259−265
anti-cancer, anti-diabetic, hypolipidemic, anti- atherosclerotic, and immunoregulatory effects [6]. Maslinic acid (MA) is a pentacyclic triterpene acid that is abundant in olive fruit skin, and has recently attracted much research attention due to its promising anti-tumor [7], anti-HIV [8], and anti-oxidation activities [9]. This laboratory recently reported that MA and other related pentacyclic triterpenes represent a new class of inhibitors of glycogen phosphorylase, and demonstrated that their glucose-lowering activity in adrenaline-induced diabetic mice might be due, at least in part, to modulation of hepatic glycogen metabolism [10-12]. In another study, several naturally-occurring pentacyclic triterpenes were characterized as potential new multi-target drugs for diabetes [13-18]. In that survey of more than 700 pentacyclic triterpene compounds, it was shown by our group that some were effective in lowering blood glucose levels in various diabetic animal models, including a rodent model of diabetes [19-21]. The aim of the present study was to gain insight into the cellular and molecular effects of MA on hepatic glycogen metabolism and the related signaling pathways using an in vitro cell system (HepG2 cells) and an in vivo mouse model fed with a high-fat diet.
Materials and Methods Derivation of maslinic acid Maslinic acid (Fig. 1) was derived from commercially available oleanolic acid (purity > 95%; Guangxi Changzhou Natural Products Development Co., Ltd.) using a semisynthetic process previously described [16].
Fig. 1. Structure of maslinic acid (MA).
Cell cluture HepG2 cells (American Type Culture Collection, Manassas, VA, USA) were cultured in high (4.5 mg·mL−1) glucose content Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (V/V) fetal bovine serum, 1% (W/V) glutamine, and 1% (W/V) penicillin/streptomycin (all reagents from Gibco-BRL, Invitrogen, San Diego, CA, USA) at 37°C in a humidified atmosphere of 5% CO2/95% air. Cells were passaged by trypsinization (trypsin/EDTA; Sigma Chemical Co., St. Louis, MO, USA) when 90% confluency was reached. Glycogen Phosphorylase a (GPa) activity assay HepG2 cells (on 100 mm2 plates) were used to determine the half maximal inhibitory concentration (IC50) value of MA on GPa activity. First, the HepG2 cells were gently washed three times with ice-cold phosphate-buffered saline (PBS), covered with 50 mmol·L−1 Tris-HCl buffer (500 μL), and placed on ice for 30 min to allow hypotonic lysis. Then, the entire sample
(lysed cells plus buffer) was harvested by scraping and centrifuged (12 000 g, 10 min) to obtain the GPa-containing supernatant and the total protein-containing lysate pellet. The pellet was solubilized by incubating in 1 M KOH, and total protein content was measured by the method of Lowry et al. [22]. The supernatant was used to measure GPa activity in the absence of AMP, as previously described [23]. The basic reaction mixture contained 50 mmol·L−1 KPi (pH 6.8), 0.8 mmol·L−1 EDTA, 0.4 mmol·L−1 NADP+ disodium salt, 4 μmol·L−1 glucose-1, 6-bisphosphate tetracycloammonium salt, 1.7 U·mL−1 G6-PDH, 1 U·mL−1 phosphoglucomutase, and cell homogenate. Additionally, various concentrations of MA were present. The reaction was initiated with 2 mg·mL−1 glycogen, and the activity of GPa was measured by monitoring increases in the nicotinamide adenine dinucleotide phosphate (NADPH) concentration detected at 340 nm on a spectrophotometer (Tecan, Männedorf, Switzerland). RT-qPCR Total RNA was isolated from HepG2 cells using the TRIzol Reagent (Invitrogen) and following the manufacturer's protocol. Complementary DNA was then synthesized by reverse transcription using the Superscript First-Strand Synthesis Kit (Fermentas, Vilnius, Lithuania) and used to measure the gene transcription levels of glycogen phosphorylase by qPCR. The glycogen phosphorylase and GAPDH (internal control) genes were amplified using the SYBR Green Master Mix (Qiagen, Hilden, Germany) and the following primers: glycogen phosphorylase, (forward) 5′- GAT GGT GTA GGA ACC GTG TT -3′ and (reverse) 5′ATG CGG TCG ATG TCT TTA GG - 3′; GADPH, (forward) 5′-ACC ACA GTC CAT GCC ATC AC-3′ and (reverse) 5′-TCC ACC ACC CTG TTG CTG TA-3′. The thermal cycling reaction was carried out in an iCycler IQ5 instrument (Bio-Rad, Hercules, CA, USA) using the following conditions: initial denaturation at 95 °C for 10 min, followed by 45 cycles of denaturation at 95 °C for 10 s, annealing for 10 s at 62 °C, and extension at 72 °C for 10 s. The glycogen phosphorylase data was normalized to GAPDH and the relative expression was calculated as 2−Δct × 100%, where Δct was the difference in the ct value between the target gene and GAPDH. All qPCRs were performed in triplicate using three independent samples. Glycogen content assay HepG2 cells were seeded in 6-well plates at a density of 5 × 104/mL. When the cells reached 80% confluency, MA was added (to achieve 0.1, 1, and 10 μmol·L−1 final concentrations per well) and the cells were incubated for an additional 24 h. The MA affects on glycogen content were measured as previously described [18]. Briefly, the cells were washed three times with ice-cold PBS and lysed by 0.1 mol·L−1 NaOH (400 µL). The lysates were then heated at 80°C for 1 h and the glycogen was precipitated by adding 2.5 volumes of ethanol. The samples were placed at 25 °C for 24 h and then centrifuged for 15 min at 12 000 g. The pellet was freeze-dried and re-suspended in 50 mmol·L−1 sodium acetate buffer (pH 4.8). The glycogen was digested with amyloglucosidase at 37 °C for 90 min, and the resulting glucose was
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assayed with G6-PDH, as described above. All glycogen measurements were standardized against the total protein content, which was determined in a 0.1 mol·L−1 NaOH cell solution using the Lowry method. Enzyme linked immunosorbent assay (ELISA) for whole-cell IR auto-phosphorylation ELISA detection of tyrosine phosphorylation on the insulin receptor β (IRβ) subunit was performed as described previously [24], with some modifications. After incubation with MA at 0, 0.1, 1, and 10 µmol·L−1, or 10 nmol·L−1 insulin alone as a positive control, the HepG2 cells were washed three times with ice-cold PBS and solubilized by incubating for 30 min at 4 °C in Triton X-100 lysis buffer (Invitrogen) containing 150 mmol·L−1 NaCl, 10 mmol·L−1 Hepes (pH 7.4), 1% (V/V) Triton X-100, protease inhibitors (50 μg·mL−1 aprotinin, 10 μg·mL−1 leupeptin, 40 μg·mL−1 pepstatin A, and 1 mmol·L−1 PMSF), and phosphatase inhibitors (400 μmol·L−1 sodium vanadate, 10 m mol·L−1 sodium fluoride, and 10 m mol·L−1 sodium pyrophosphate). The cell debris was removed by centrifugation (800 g, 30 min, 4 °C) and equal amounts of cell lysates (40 μg of total protein) were applied to 96-well Immulon-1 flat-bottomed-plates (Nalge-Nunc International, Rochester, NY, USA) that had been pre-coated with monoclonal anti-human IRβ antibody (Ab) (C-19; Santa Cruz Biotechnology, Santa Cruz, CA, USA) in carbonate/bicarbonate buffer (15 mmol·L−1 Na2CO3 and 35 mmol·L−1 NaHCO3, pH 9.6). After incubating at 4 °C overnight, the plates were washed with PBS containing 0.05% Tween 20 (PBST) and biotin-conjugated anti-pTyr Ab (4G10; Millipore, Billerica, MA, USA) was added to the wells. After incubating for 1 h at 25 °C, the wells were washed with PBST and horseradish peroxidase (HRP)-conjugated streptavidin (Invitrogen) was added. Following the addition of the peroxidase substrate, o-phenylenediamine dihydrochloride, the rate of tyrosine phosphorylation was quantified by measuring absorbance at 490 nm with a microplate reader (Tecan, Männedorf, Switzerland). Western blotting analysis Total HepG2 cell proteins were resolved by electrophoresis through 10% SDS-polyacrylamide gels and transferred onto nitrocellulose membranes. Non-specific binding sites were first blocked by incubating the membranes in Tris-buffered saline (TBS) containing 0.1% Tween 20 and 5% non-fat dried milk for 1 h at room temperature. Then, Abs against the phosphorylated proteins (pIRβ; phospho-Ser473-Akt (pAkt) or phospho-Ser9-glycogen synthase kinase 3β (pGSK3β) from Cell Signaling Technology, Beverly, MA, USA) or their non-phosphorylated forms (IRβ; Akt or GSK3β (Cell Signaling Technology)) were applied to the respective membranes and allowed to react for overnight at 4°C. After a wash with TBS + 0.1% Tween 20 (TBST), the appropriate HRP-conjugated secondary Abs (affinity-purified mouse anti-rabbit IgG or rabbit antimouse IgG from Sigma Chemical Co.) were applied and allowed to react for 1 h at room temperature. Immunoreactive signals were detected by an enhanced chemiluminescence kit (Amersham Biosciences, NJ, USA).
Animal experiments All animal experiments were carried out with approval from the Animal Ethics Committee of China Pharmaceutical University. The authors have received permission from the Animal Care and Use Committee of China Pharmaceutical University. Male C57BL/6J mice (6–8 weeks old) were housed in a temperature-controlled room [(22 ± 2) °C), with a 12 h light/dark cycle. Animals were randomly assigned to receive either standard rodent diet (control group) or a high-fat diet (HFD) containing 2% cholesterol and 10% lard to generate insulin resistance [25-26]. The ad libitum diets were maintained for a total of 12 weeks, but at week 10 the mice in the HFD group were sub-divided into three groups to assess the effects of MA. The first group remained untreated on the HFD for the final two weeks (model group). The other two groups received two weeks of MA treatment at 50 or 100 mg·kg−1·d−1. The body weight (bw) was measured every week, and the food intake was calculated everyday. All animals were sacrificed at the end of the experimental period and liver and epididymal fat tissue samples were harvested. In vivo glucose metabolism The effects of MA treatment on plasma insulin levels were determined using an ultrasensitive ELISA kit (Shanghai ExCell Biology, Inc., Shanghai, China) following the manufacturer’s instructions. In addition, hepatic glycogen content was measured in the mouse liver tissue specimens by using the previously described anthrone method [22], with some modifications. Briefly, 0.1 g liver tissue was homogenized, then was re-suspended in 30% KOH and incubated for 15 min at 95°C to extract glycogen. Then, 2% Na2SO4 was added and the glycogen was precipitated with ethanol. The glycogen precipitate was re-suspended in H2O : sulfuric acid (3 : 7.6) containing 0.15% anthrone (Sigma Chemical Co.) and heated at 95°C for 15 min. The reaction was terminated by plunging the tubes into ice, and the glycogen content was measured spectrophotometrically at 620 nm. Statistical analysis The data are expressed as x ± s, unless otherwise indicated. Intergroup differences were assessed by one-way analysis of various (ANOVA) followed by the Bonferroni post-hoc test. P-< 0.05 was considered to indicate statistical significance.
Results MA inhibits both GPa activity and mRNA expression in HepG2 cells The IC50 of MA in HepG2 cells was 6.91 μmol·L−1, which was substantially lower than the IC50 of the positive control compound (caffeine: 307 μmol·L−1; Fig. 2A). Moreover, the inhibitive effect of MA on GPa activity in the HepG2 cells was dose-dependent. The MA-treated cells also showed reduced levels of GPa mRNA (Fig. 2B) and increased levels of glycogen content (Fig. 2C). Taken together, these results suggest that MA may increase accumulated glycogen in HepG2 cells by inhibiting the activity of GPa, which is the rate-limiting enzyme in hepatic glycogen metabolism.
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Fig. 2. Effect of MA on glycogen phosphorylase in HepG2 cells (mean ± SD, n = 4). (A) Inhibitive effects of MA and caffeine (control) on GPa activity, as measured by inhibition rate. (B) Inhibitive effects of MA on GPa gene transcription, as mRNA determined by RT-qPCR detection of mRNA (normalized to GAPDH). (C) Effects of MA on intercellular glycogen content. HepG2 cells were left untreated (control) or treated with 0.1, 1, or 10 μmol·L−1 MA for 24 h. *P < 0.05, **P < 0.01 vs control group
MA stimulates IRβ auto-phosphorylation in HepG2 cells Tyrosine phosphorylation of IRβ in HepG2 cells was increased in response to MA treatment, and the effects were dose-dependent, as shown by Western blotting analysis with anti-pTyr (Fig. 3A). Moreover, the MA treatment also led to increased IRβ auto-phosphorylation, as shown by ELISA (Fig. 3B). This result suggested that the MA-induced IR pTyr signal from HepG2 cells might be due to an increase in the number of phosphorylated IR molecules. MA induces phosphorylation of Akt and GSK3β in HepG2 cells Akt Ser473 phosphorylation is an important node in the insulin signal transduction pathway, and it reflects receipt and acceptance of an upstream signal for downstream transmission that can modulate the processes of glucose uptake and glycogen accumulation. In HepG2 cells, MA-induced activation of IRβ led to increased phosphorylation of Akt, as shown by Western blotting with anti-pAkt (Fig. 4). Specifically, Akt phosphorylation was remarkably increased in HepG2 cells treated with 1 µmol·L−1 and 10 µmol·L−1 MA (Fig. 4A). The MA-induced Akt phosphorylation response was completely inhibited by pre-treatment of cells with the PI-3K inhibitor, wortmannin (Fig. 4B). GSK3β is a well-known downstream target of Akt that plays an important role in regulating cellular glucose metabolism. Analysis of MA effects on the phosphorylation of GSK-3β indicated that MA treatment led to increased GSK3β phosphorylation at Ser9 in HepG2 cells (Fig. 4C).
Fig. 3. Effect of MA on IRβ and IRβ auto-phosphorylation (mean ± SD, n = 3). (A) Cells were starved for 8 h with serum-free medium, and treated with 0, 0.1, 1, and 10 μmol·L−1 MA for 30 min. Equal amounts of soluble proteins were used to detect with Abs specific for the phosphorylated forms of IRβ. Blots were stripped and re-probed with Abs directed against total cellular forms of IRβ. A single Western blot representative of three independent experiments is shown. (B) ELISA was used to detect IRβ auto-phosphorylation. Cells were incubated in the presence of MA at 0, 0.1, 1, and 10 µmol·L−1 for 30 min. Cells treated with insulin alone were used as the positive control. IRβ auto-phosphorylation was measured by ELISA as described in the Experimental section. * P < 0.05, **P < 0.01, ***P < 0.001 vs control group
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Fig. 4. Effect of MA on Akt and GSK3β phosphorylation in HepG2 cells. MA treatment induced Akt phosphorylation in HepG2 cells in a (A) dose-dependent manner. (B) Wortmannin treatment blocked MA-induced Akt phosphorylation. After 8 h starvation in serum-free DMEM medium, cells were pre-incubated with 100 nmol·L−1 Wortmannin for 30 min, and then changed to serum-free medium containing 10 μmol·L−1 MA for 2h, and 0.2% DMSO alone used as the negative control. The immunoblots shown (a-c) are representative of three independent experiments. (C) Dose-dependent course of MA on GSK3β phosphorylation in HepG2 cells
treated HFD mice at both the low concentration (50 mg·kg−1·d−1) and high concentration (100 mg·kg−1·d−1) showed markedly reduced blood glucose levels. MA at the high concentration (100 mg·kg−1·d−1) also significantly improved the hyperinsulinemic condition (Table 1), markedly reduced adiposity (Fig. 5A), and increased the accumulated hepatic glycogen (Fig. 5B). Collectively, these results suggested that MA may modulate glycogen metabolism and improve HFD-induced hyperinsulinemia and hyperglycemia by enhancing the accumulation of hepatic glycogen and reducing insulin levels. Thus, the hypothesis that MA can modulate glycogen metabolism in vivo appears to be valid.
MA treatment of mice does not modulate body weight or the amount of daily food intake When mice fed with HFD were treated with MA for two weeks, they did not experience any significant changes in body weight or daily food intake (vs normal diet control and untreated HFD model control mice; Table 1). MA modulates glycogen metabolism in vivo by improving insulin resistance and increasing the hepatic glycogen accumulation To gain more insight into the effect of MA on glycogen metabolism, HFD-induced mice were treated with MA for two weeks and the effects on blood glucose and insulin levels were assessed. Compared to the untreated HFD model mice, the MA-
Table 1 Effects of MA on body weight, food intake, serum glucose, and serum insulin (mean ± SD, n = 10) Group
Dose/(mg·kg−1·d−1)
Body weight/g
Food intake/(g/25 g/d)
cBg/(mmol·L−1)
cSi/(µg·L−1)
Control
−
27.5 ± 1.4
3.36 ± 0.31
7.32 ± 0.15
1.58 ± 0.67
Model
−
27.2 ± 1.3
2.84 ± 0.42
8.66 ± 1.36
MA
100
25.9 ± 2.8
2.82 ± 0.23
6.75 ± 0.58*
5.59 ± 2.19*
MA
50
26.7 ± 1.4
2.76 ± 0.54
6.78 ± 1.22*
8.66 ± 0.20
#
8.33 ± 2.11###
cBg: concentration of blood glucose; cSi: concentration of serum insulin ### P < 0.001, #P
