Resveratrol Ameliorates Hyperglycemic Cultured Cells and Inhibits the Rheb/mTOR Interaction

Abstract

Resveratrol (RSV) is a natural polyphenol with anti-diabetic effects and has been reported to ameliorate diabetes-induced metabolic disorders through regulating activities of the mTOR signaling pathway. To delineate the effects of RSV treatment on the mTOR signaling pathway, hyperglycemic HepG2 cells were used for the following experiments. Cellular glucose uptake assays showed that high-glucose levels in the culture medium decelerate the glucose uptake of cultured cells. Co-immunoprecipitation showed that high-glucose culture promotes the interaction between mTOR and Rheb-GTP, which is the active form of Rheb. RSV treatment of the cells suppressed this interaction and accelerated the glucose uptake. Western blotting revealed that RSV down-regulated members of the mTOR signaling pathway, namely SREBP1, p70, and S6. Additionally, RSV ameliorated the metabolic disorders, including the decreased levels of AMPK, glycogen synthase, and glucose-6-phosphatase, in hyperglycemic HepG2 cells. These results indicate that RSV inhibits the Rheb/mTOR interaction and ameliorates metabolic disorders associated with high-glucose levels.

Introduction

Resveratrol (RSV, trans-3,5,4′-trihydroxystilbene) is a natural polyphenol found in some fruits and red wine. It has a wide spectrum of biological properties, including anti-oxidative, anti-inflammatory, anti-cancer, anti-obesity, neuroprotective, and cardioprotective.^1‐5 RSV has been shown to improve metabolic disorders caused by type 1 diabetes. ⁶ Numerous animal studies, including those on rats, fed a high-fat or high-fructose diet, have demonstrated that RSV strengthens insulin action and ameliorates insulin resistance. ⁷ Additionally, RSV treatment has been found to regulate energy expenditure through increased skeletal muscle SIRT1 and AMPK (AMP-activated protein kinase) expression in rats with type 1 diabetes mellitus. ⁸

mTOR is a serine/threonine protein kinase belonging to the PI3K-related kinase family, which regulates cell survival, cell proliferation, protein synthesis, tumor growth, synaptic plasticity, and gene transcription. ⁶ Various hormonal, nutritious, and environmental signals are incorporated into mTOR in several pathways associated with many diseases, including cancer, diabetes, and immune disorders. ⁹

Several studies have considered RSV as a novel therapeutic for diseases associated with hyperactivation of mTOR signaling. RSV has extensive applications, such as in suppressing seizure-induced inflammatory responses partially via the AMPK/mTOR signaling pathway, ¹⁰ inhibiting S6 phosphorylation, suppressing senescence in human cells, ¹¹ and preventing TNF-alpha–induced muscle atrophy. ¹² RSV suppresses the ERK (extracellular signal-regulated kinase) and mTOR (mammalian target of rapamycin) signaling pathways through AMPK in sensory neurons and inhibits incision-induced acute and chronic pain. ¹³ Furthermore, a recent study has shown that RSV induces autophagy through suppression of the mTOR-ULK1 pathway by docking into the ATP-binding pocket of mTOR. ¹⁴

Rheb is a GTP-binding protein belonging to the Ras GTPase superfamily, which is widely conserved in eukaryotes from yeasts to humans. It is an activator of mTOR, which plays important role in cell survival and cell metabolism. ¹⁵ The mTOR signaling pathway has been reported to regulate energy homeostasis through sensing environmental nutrients and energy levels. Dysregulated mTOR signaling has been shown to promote aging and certain diseases, including diabetes, obesity, and cancers. ¹⁶ Studies have recently concentrated on investigating factors that affect the cellular Rheb-GTP/Rheb-GDP ratio and mTOR signaling. ¹⁷

Our previous study has indicated that RSV exerts anti-diabetic effects differently from insulin. The anti-diabetic effects of RSV seem to be associated with the suppression of hyper-activated hepatic mTOR activity. Additionally, in diabetic rats, various metabolic benefits of RSV, including up-regulation of glycogen synthesis and down-regulation of protein degradation and lipogenesis, have been suggested to be mediated through the normalization of the dysregulated hepatic mTOR activity. ⁶ In a study on hepatic cytotoxicity, HepG2 hepatoma cells were used to further study the molecular mechanisms whereby RSV regulates mTOR activity in the liver. ¹⁸ This study indicates that RSV inhibits the Rheb/mTOR interaction and ameliorates metabolic disorders associated with high-glucose levels.

Experimental

Reagents

RSV, rapamycin, and 2-NBD-Glucose were purchased from Sigma-Aldrich, the reagents to measure glucose and triglyceride (TG) concentrations from Randox, and all the primary antibodies against Rheb-GTP, mTORc1, Rheb, GAPDH (glyceraldehyde 3-phosphate dehydrogenase), SREBP1 (sterol regulatory element-binding protein 1), p-SREBP1, p70, S6, p-S6, AMPK, p-AMPK, ACC (acetyl-CoA carboxylase), p-ACC, fatty acid synthase, glycogen synthase, and G6PC, as well as secondary antibodies from Abcam.

Cell Culture

The human hepatoma cell line HepG2 was purchased from the Food Industry Research and Development Institute, Taiwan. The cells were cultured in the basal medium (DMEM/F-12 at 3:1 v/v; Invitrogen) with 10% fetal bovine serum (Invitrogen) in a humidified incubator with 5% CO₂/95% air at 37 °C, as previously described. ¹⁹ For the high-glucose–treated group, HepG2 cells were pretreated with a high-glucose medium (64 mM) for 3 days alongside the control group, whose medium contained 24 mM glucose. After the pretreatment, 5 nM rapamycin and 10 µM RSV were added to the culture medium where indicated, and the cells were then cultured for 24 h.

Cellular Glucose Uptake Assay

HepG2 cells were cultured in 96-well black plates to 70%-80% confluence before starting the experiments. 2-NBD-Glucose was added to the culture to reach a final concentration of 100 µM, and the cells were then cultured for 1 h. Afterward, the cells were washed once with phosphate-buffered saline (PBS), and then the relative rates of cellular glucose uptake were measured using a fluorescence reader (excitation/emission = 480/520 nm).

Cell Counting Kit-8 Assay

HepG2 cells were cultured in 96-well plates with a clear flat bottom to 70%-80% confluence before starting the experiments. About 10 µL of WST-8 solution was added to each well. The plates were protected from light and incubated for 1 h at 37 °C and then their viability and proliferation were evaluated according to the manual provided by the manufacturer (Abcam).

Glucose and Triglyceride Quantitation

HepG2 cells were cultured in a 96-well plate with a clear flat bottom to 70%-80% confluence before starting the experiments. Glucose and TG concentrations were determined using assay kits (colorimetric method) according to the instructions of the manufacturer (Sangon Biotech).

Co-Immunoprecipitation

HepG2 cells cultured and treated in 100 mm dishes were lysed. Subsequently, cell lysate with 1 mg of total protein was incubated with 1 µg anti–Rheb-GTP antibody overnight at 4 °C. Then, 20 µL protein A-agarose (Upstate) was added, and the mixture was shaken at 4 °C for 1 h. Afterward, the agarose beads were pelleted by centrifugation at 7000 g at 4 °C for 2 min. The pellet was washed 3 times with ice-cold wash buffer (20 mM Tris-HCl [pH 8.9], 1 mM EDTA/100 mM NaCl, and 0.5% (v/v) Nonidet P-40) and then resuspended in 100 µL sample buffer (Bio-Rad). The COIPed proteins were separated from the beads by boiling the samples for 5 min. The proteins were then fractionated by 10% SDS-PAGE, and then Western blot analysis was carried out as described below.

Western Blotting

HepG2 cells were cultured in 100 mm dishes to 70%-80% confluence. The cells were then washed twice with ice-cold PBS and lysed in Mammalian Protein Extraction Reagent (M-PER; Pierce). The cell lysate was cleared of debris by centrifugation at 15 000 g at 4 °C for 2 min. The total protein concentration of the lysate was determined using a BCA protein assay kit (Pierce). Afterward, the protein samples were fractionated by 10% SDS-PAGE and then blotted onto Immobilon-P membranes (Millipore). Subsequently, the blots were incubated with 1:1000-diluted primary antibodies and 1:5000-diluted secondary antibodies. Immunoreactive protein bands were visualized by enhanced chemiluminescence (Amersham Biosciences) and recorded using radiography films in a dark room.

Statistical Analysis

The data were expressed as the mean ± SD from 6 independent experiments. All the data were analyzed using the statistical software SPSS 19.0 (IBM Inc., Armonk, NY, USA). The differences between any 2 groups were analyzed using a two-tailed Student's t-test, and a P-value <0.05 was considered to indicate the statistical difference.

Results

Resveratrol Facilitated Cellular Glucose Uptake Under Hyperglycemic Conditions

To delineate the effects of high-glucose culture and RSV on cellular glucose uptake, HepG2 cells were pretreated with high-glucose media, and then their rate of glucose uptake was measured. As shown in Figure 1A, the rate of cellular glucose uptake was significantly reduced in the hyperglycemic culture in comparison to the normoglycemic culture. RSV treatment (10 µM) of the cells ameliorated the hyperglycemia-induced reduction in glucose uptake. However, increasing the concentration of RSV (20 µM) did not significantly increase the rate of glucose uptake. Additionally, the Cell Counting Kit-8 (CCK-8) assay was performed to confirm that cell viability was not affected under the experimental conditions (Figure 1A).

Figure 1.

Resveratrol (RSV) promotes cellular glucose uptake and inhibits hyperglycemia-induced Rheb-GTP/mTOR interaction. (A) The rates of cellular glucose uptake were measured, and the relative cell viability was assessed using the CCK-8 assay. Statistical results are presented as the mean ± SD of 6 independent analyses. P-value was determined using Student's t-test; the asterisk indicates a P-value ≤0.05. (B) Co-immunoprecipitation (COIP) assay was performed using an anti–Rheb-GTP antibody, and then Western blotting was performed using anti–mTOR and –Rheb antibodies. (C) COIP was performed using an anti–Rheb-GTP antibody. GDP (1 µg) and GTP (1 µg) were added before the COIP assays as negative and positive controls, respectively. The subsequent Western blot assays were performed using anti–mTOR and –Rheb antibodies. Input: total lysate (without COIP). GAPDH was used as an internal control in the Western blot assays.

Hyperglycemia Promoted the Interaction Between Rheb-GTP and mTOR

Rheb is a GTP-binding protein that is largely involved in the regulation of the mTOR signaling pathway. ¹⁷ To delineate whether hyperglycemia promotes the interaction between Rheb-GTP (the active form of Rheb) and mTOR, co-immunoprecipitation (COIP) assays were performed using anti–Rheb-GTP and –mTOR antibodies. As shown in Figure 1B, the binding of mTOR to Rheb-GTP was enhanced (Glucose group) under hyperglycemia versus normoglycemia, but the mTOR inhibitor rapamycin (Glucose + Rapamycin) inhibited this interaction. Western blotting for Rheb revealed that the cellular Rheb-GTP level was higher in the hyperglycemic group (Glucose group) than in the control, and the rapamycin treatment (Glucose + Rapamycin) did not have a significant influence on Rheb-GTP level. Western blot analysis of the total lysate (input) showed that rapamycin down-regulated mTOR and hyperglycemia did not have any influence on mTOR levels. These results indicated that the interaction between Rheb-GTP and mTOR was significantly increased under hyperglycemic conditions in comparison to normoglycemic conditions, but the level of total Rheb was not changed.

Resveratrol Inhibited Hyperglycemia-Induced Rheb-GTP/mTOR Interaction

To delineate whether RSV can influence the hyperglycemia-induced Rheb-GTP/mTOR interaction, HepG2 cells were treated with high-glucose media and then cultured with or without RSV. As shown in Figure 1C, hyperglycemia up-regulated Rheb-GTP and promoted the interaction of Rheb-GTP with mTOR. The RSV treatment down-regulated Rheb-GTP and inhibited the Rheb-GTP/mTOR interaction under hyperglycemia (Glucose + RSV). Either GDP- or GTP-treated cells were used to shift the transition of Rheb between the GDP- and GTP-bound states, representing the negative and positive control groups, respectively. Western blot analysis of the total lysate (input) showed that RSV significantly decreased the level of total Rheb (RSV and Glucose + RSV), but the level of mTOR was not affected.

Resveratrol Suppressed mTOR Signaling and Ameliorated Hyperglycemia-Associated Metabolic Disorders

SREBPs are mTOR-regulated transcription factors related to cholesterol and fatty acid biosyntheses. ²⁰ To examine whether RSV influences the activity of SREBPs, Western blotting for the active (68 kDa) and inactive (phospho-SREBP1 [p-SREBP1], 125 kDa) forms of SREBP1 was performed, as shown in Figure 2A; hyperglycemia (Glucose) significantly up-regulated SREBP1 and down-regulated p-SREBP1, and RSV suppressed this effect, indicating that RSV inhibits the activation of SREBP1.

Figure 2.

Resveratrol (RSV) corrects dysregulated mTOR signaling and ameliorates hyperglycemia-associated metabolic disorders. The totals of the groups indicated in Figure 1B and C were harvested. (A) Western blot assays were performed using anti–SREBP1, –p-SREBP1, –p70, –S6, –p-S6, and –GAPDH antibodies. (B) Western blot assays were performed using anti–AMPK, –p-AMPK, –ACC, –p-ACC, –fatty acid synthase, –glycogen synthase, –G6PC, and –GAPDH antibodies. (C) Relative cellular triglyceride (TG) concentrations were evaluated. Statistical results are presented as the mean ± SD of 6 independent analyses. P-value was determined using Student's t-test; the asterisk indicates a P-value ≤0.05.

The mTOR/p70 (S6 Kinase)/S6 pathway has been reported to serve as a key regulator of metabolic homeostasis, and increased mTOR/P70/S6 activity has been shown to enhance cellular protein synthesis and survival. ²¹ We observed that the RSV treatment decreased the protein levels of p70 and p-S6 (Figure 2A), and the level of total S6 was not changed by the experimental conditions.

We also analyzed metabolic regulators whose levels may change under hyperglycemic conditions. As shown in Figure 2B, the hyperglycemic group (Glucose) exhibited decreased levels of p-AMPK, glycogen synthase, and G6PC, and RSV treatment suppressed these effects of hyperglycemia. The levels of total AMPK, total ACC, p-ACC, and fatty acid synthase were not affected by hyperglycemia. In addition, the cellular TG was up-regulated under hyperglycemic conditions in comparison to normoglycemic conditions (Figure 2C).

Discussion

Rheb-GTP has been found to bind to the TOR complex in vivo and in vitro. ¹⁷ In the present work, we demonstrated that hyperglycemic culture conditions promote the interaction between Rheb-GTP and mTOR. This protein–protein interaction increased along with the Rheb-GTP/Rheb-GDP ratio, even though the total level of Rheb remained unaffected (Figure 1B). This result echoes a previous study that reported that mTOR shows higher kinase activity when it is bound to a Rheb mutant that is highly charged with GTP (Gln64Leu) than when bound to the wild-type Rheb. ²² Increased interaction between Rheb-GTP and mTOR might be associated with reduced cellular glucose uptake rate (Figure 1A). However, the most suitable RSV concentration may need further confirmation. The downstream members of the mTOR signaling pathway, including SREBP1 and S6, were up-regulated under hyperglycemic conditions (Figure 2A). Furthermore, levels of certain metabolic regulators, including AMPK, glycogen synthase, and G6PC, were perturbed under hyperglycemic conditions. mTOR has been reported to be important for regulating SREBP1 activity both in vivo and in vitro. ²³ Also, SREBP-1 has been reported to play a key role in hepatic induction of lipogenesis. ²⁴ This may explain the results that the cellular TG was also up-regulated under hyperglycemic conditions (Figure 2B and C). Our results suggested that hyperglycemia activates the mTOR signaling pathway and promotes metabolic disorders associated with increased Rheb-GTP/mTOR interaction.

RSV has been reported to promote autophagy by inhibiting Akt/mTOR signaling.^25,26 RSV also inhibits hypertrophic scar formation by activating autophagy through the down-regulation of Rheb. ²⁷ One of our principal purposes in the presented study was to delineate whether RSV ameliorates metabolic disorders by affecting the Rheb-GTP/mTOR interaction. We first observed that RSV inhibited this interaction under hyperglycemic conditions (Figure 1C). Interestingly, this inhibition was correlated with reduction in the levels of Rheb-GTP and total Rheb, but not with the Rheb-GTP/Rheb-GDP ratio. Additionally, RSV ameliorated the activation of SREPB1 under hyperglycemic conditions (Figure 2A). The mTOR/SREBP1 signaling pathway is well known to regulate lipid biosynthesis and stimulate diabetes-induced lipogenesis.^20,28 We observed that RSV inhibited hyperglycemia-induced activation of p70 and S6 (Figure 2A). The mTOR/p70/S6 pathway exerts anti-apoptotic effects, promotes protein translation, and thereby contributes to cell survival. Additionally, abnormal mTOR/p70/S6 signaling has been implicated in the pathogenesis of certain diseases.^29,30

The serine/threonine kinase AMPK regulates the homeostasis between cellular anabolism and catabolism. It is an energy switch that regulates numerous cellular processes, including metabolism, cell survival, and mitochondrial homeostasis.^31,32 Our results indicated that RSV suppresses hyperglycemia-associated AMPK inactivation (Figure 2B). Furthermore, RSV was found to ameliorate hyperglycemia-induced down-regulation of glycogen synthase and G6PC, which are key enzymes in regulating glucose homeostasis. G6PC is a highly hydrophobic glycoprotein that catalyzes the hydrolysis of glucose-6-phosphate to glucose and phosphate in the terminal steps of gluconeogenesis and glycogenolysis. ³³ Our results indicated that RSV ameliorates hyperglycemia-induced metabolic disorders by inhibiting the Rheb-GTP/mTOR signaling pathway.

A recent report has indicated that RSV stimulates cellular glucose uptake and ameliorates muscle insulin resistance by activation of AMPK and inhibition of the mTOR/p70/S6K pathway. ³⁴ Our results concluded that RSV ameliorates high-glucose–induced metabolic disorders. Such disorders involve altered activities of the mTOR signaling and some crucial metabolic regulators. However, we also found that enzymes related to the mTOR pathway, such as ACC and fatty acid synthase, were not changed by RSV treatment. Pathway changes caused by RSV treatment still need further confirmation. In summary, we showed that RSV down-regulates Rheb and thereby suppresses the Rheb-GTP/mTOR interaction, consequently ameliorating hyperglycemia-induced metabolic disorders. Additionally, our results demonstrated the mechanism whereby RSV modulates mTOR activity and the roles of RSV in maintaining metabolic homeostasis.