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Does Resveratrol Improve Insulin Signalling in HepG2 Cells?
ARTICLE IN PRESS Can J Diabetes xxx (2016) 1–6
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Original Research
Does Resveratrol Improve Insulin Signalling in HepG2 Cells? Marjan Norouzzadeh MS a, Fatemehsadat Amiri PhD b, Ali Akbar Saboor-Yaraghi a, Farnoosh Shemirani PhD a, Yas Kalikias MS a, Loghman Sharifi MS a, Monireh Sadat Seyyedsalehi MS a, Maryam Mahmoudi MD, PhD a,* a b
Department of Cellular and Molecular Nutrition, School of Nutritional Sciences and Dietetic, Tehran University of Medical Sciences, Tehran, Iran Department of Nutrition, School of Public Health, Iran University of Medical Sciences, Tehran, Iran
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 December 2015 Received in revised form 5 September 2016 Accepted 28 September 2016
Objectives: Diabetes mellitus is a common metabolic disorder with high global prevalence. It is characterized by a decrease in insulin secretion or a decrease in insulin sensitivity or both. The aim of the present study was to investigate the effects of resveratrol treatment on the expression of the genes involved in insulin signalling cascade, such as Forkhead box protein O1 (FoxO1), 3-phosphoinositide-dependent protein kinase 1 (PDPK1) and mammalian target of rapamycin (mTOR). Methods: HepG2 cells were cultured in serum-free medium with high concentrations of glucose and insulin and then were treated with resveratrol (5, 10 and 20 μM) for 24 and 48 hours. Complementary deoxyribonucleic acids (cDNAs) were synthesized followed by RNA extraction. Real-time quantitative reverse transcription polymerase chain reaction was used to analyze the expression of FoxO1, PDPK1 and mTOR. Results: Resveratrol increased the expression of PDPK1, mTOR and FoxO1. No significant difference was seen among differing dosages of resveratrol, but treatments for 48 hours exerted the greatest effectiveness. Conclusions: Our results were consistent with other studies showing the beneficial effects of resveratrol on diabetes. However, considering the effects of resveratrol in increasing FoxO1 and gluconeogenic gene expression, long-term usage of resveratrol should be investigated in greater depth in future studies. © 2016 Canadian Diabetes Association.
Keywords: FoxO1 HepG2 insulin resistance mTOR PDPK1 resveratrol
r é s u m é Mots clés : FoxO1 HepG2 insulinorésistance mTOR PDPK1 resvératrol
Objectifs : Le diabète sucré qui est une perturbation fréquente du métabolisme connaît une prévalence mondiale élevée. Il est caractérisé par une diminution de la sécrétion d’insuline ou une diminution de la sensibilité à l’insuline, ou les deux. L’objectif de la présente étude était d’examiner les effets du traitement à base de resvératrol sur l’expression des gènes impliqués dans la cascade de signalisation de l’insuline, comme la protéine FoxO1 (Forkhead box protein O1), la protéine kinase dépendante du 3-phosphoinositide (PDPK1) et la cible mammalienne de la rapamycine (mTOR). Méthodes : Les cellules HepG2 ont été mises en culture dans un milieu sans sérum à fortes concentrations de glucose et d’insuline, et ont alors été traitées par resvératrol (5, 10 et 20 μM) durant 24 et 48 heures. À la suite de la synthèse des acides désoxyribonucléiques complémentaires (ADNc), les ARN ont été extraits. La transcription inverse de la réaction en chaîne par polymérase en temps réel a été utilisée pour analyser l’expression de la protéine FoxO1, de la PDPK1 et de la mTOR. Résultats : Le resvératrol a augmenté l’expression de la PDPK1, de la mTOR et de la protéine FoxO1. Aucune différence significative n’a été observée entre les diverses posologies de resvératrol, mais les traitements de 48 h ont exercé une plus grande efficacité. Conclusions : Nos résultats ont concordé avec les autres études ayant démontré les effets bénéfiques du resvératrol sur le diabète. Toutefois, en considérant les effets du resvératrol sur l’augmentation de l’expression de la protéine FoxO1 et du gène gluconéogénique, l’usage à long terme du resvératrol devrait être examiné de manière plus approfondie au cours d’études futures. © 2016 Canadian Diabetes Association.
* Address for correspondence: Maryam Mahmoudi, MD, PhD, Department of Cellular and Molecular Nutrition, School of Nutritional Sciences and Dietetic, Tehran University of Medical Sciences, Tehran 14155-6447, Iran. E-mail address: [email protected] 1499-2671 © 2016 Canadian Diabetes Association. http://dx.doi.org/10.1016/j.jcjd.2016.09.015
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Introduction
and restrains gluconeogenesis by preventing nuclear accumulation of FoxO1. Therefore, it could be deduced that glucose and lipid homeostasis are under the control of mTORC2 via Akt. Indeed, a flaw in hepatic mTORC2 signalling may ultimately lead to diabetes (10). Both the secretion and the action of insulin could be affected by a series of plant-derived components (12). Resveratrol (RSV) (trans3,5,4’-trihydroxystilbene) belongs to the large group of polyphenols present in the skins of red grapes and in nuts and red wine (13,14). Several animal studies have demonstrated promising perspectives on the effects of RSV in preventing and/or treating metabolic disorders such as obesity and diabetes (15,16). RSV can promote the activation of Akt and PDPK1 via the deacetylation activity of sirtuin (silent mating type information regulation 2 homolog) 1(SIRT1) (17). Moreover, RSV is capable of repressing PI3K activity via its ATPbinding site (18,19). As activation of mTOR by insulin is done through the PDPK1/Akt signalling pathway, it could be hypothesized that the mechanism of the protective effect of RSV is exerted via mTOR signalling inhibition (20,21). Altogether, the present study aimed to investigate the possible mechanism via which RSV could eliminate the complications of diabetes. The insulin signalling cascade is summarized in Figure 1.
Diabetes mellitus is a common metabolic disorder that is occurring with increasing incidence worldwide (1,2). It is a chronic disorder that is characterized by a decrease in blood insulin levels or inefficient use of it, which involves metabolism of carbohydrates, proteins and lipids. Impaired insulin secretion and/or insulin resistance are the main causes, leading to hyperglycemia and the malfunction of many organs, including the liver (3). Insulin acts through the key proteins, which exert their effects via insulin receptor β(IRβ). Insulin receptor substrate (IRS) is a prominent protein that transmits signals to downstream kinases, including phosphoinositide 3-kinase (PI3K), 3‘-phosphoinositide-dependent protein kinase (PDPK1) and Akt/protein kinase B (PKB). Recruiting of the mammalian target of rapamycin complex 2 (mTORC2) and PDPK1 are needed for Akt activation (4–6). The mammalian target of rapamycin complex 1 (mTORC1) is 1 of the targets of Akt (7) that has main components named mTOR, raptor and mammalian lethal with SEC13 protein 8 (mLST8) (8). It has been shown that the mammalian target of mTORC1 exerts its effects by increasing transcription of lipogenic genes, including sterol regulatory element-binding protein 1c (SREBP1c), fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC) in mice fed high-fat diets (9). The effects of mTORC1 and mTORC2 are applied by regulation of influential kinases, including ribosomal protein S6 kinase (S6K) and Akt (7). It has been revealed that mTOR is involved in the onset and progression of diabetes, cancer and ageing (7,10). Glucose homeostasis is controlled by hepatic mTORC2 (11). mTORC2 contains mTOR, rictor, mammalian stress-activated protein kinase interacting protein 1 (mSIN1), mLST8 and prolinerich protein 5/proline rich protein 5-Like (PRR5/PRR5L) (8)
Methods Study design The current study is experimental. Each experiment was carried out in triplicate, and the data were the average results of 3 independent experiments.
Insulin
IR P IRS1/2
PI3K
PDPK1
P
Gluconeogenesis
mTORC2
P
P
FoxO1
Akt mTORC1 GSK3
Lipin1
S6K P
Insig2a
SREBP1c LXR Lipogenesis
Figure 1. Insulin signalling cascade.
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Table 1 The primer sequences and parameters used for RT-qPCR Gene
Oligo
Sequence
Length
Tm
CG %
β-actin β-actin FoxO1 FoxO1 mTOR mTOR PDPK1 PDPK1
Forward Reverse Forward Reverse Forward Reverse Forward Reverse
5’-AGCCTCGCCTTTGCCG-3’ 5’-ATCACGCCCTGGTGCCT-3’ 5’-GCTTAGACTGTGACATGGAATCC-3’ 5’-GTACTTTTAAGTGTAACCTGCTCAC-3’ 5’-GCCAGGGATCTCTTCAATGCT-3’ 5’-GTCTGTGTGACTTCAGCGATG-3’ 5’-GCAGTACATTCACGATCTGGA-3’ 5’-TGTGGTCCTTCTGTGAGCAAC-3’
16 17 23 25 21 21 22 21
55.9 57.2 62.9 62.5 61.3 61.3 62.1 61.3
68.75 64.71 47.83 40 52.38 52.38 50 52.38
CG,; tm,; RT-qPRC, real-time quantitative reverse transcription polymerase chain reaction.
Materials Resveratrol (RSV-1185247-70-4-100 mg) was purchased from Santa Cruz Biotechnology (St. Louis, Missouri, United States). Dulbecco modified Eagle medium (DMEM-F12-32500032), fetal bovine serum (FBS-10270106) and trypsin/EDTA (25200-056) were purchased from Gibco (Pittsburgh, Pennsylvania, United States) and dimethyl sulforoxide (DMSO) was purchased from SigmaAldrich (St. Louis, Missouri, United States) (#D2650). Cell lines and cell culture We obtained human hepatoma HepG2 cells from the Pasteur Institute of Iran. These cells were incubated at 37°C in a humidified atmosphere including 5% (v/v) CO2 in culture flasks containing DMEM supplemented with 10% FBS, and 100 U/mL streptomycin and 100 mg/mL penicillin. The first time, they were passaged by trypsinization after 1 week and then every 3 days. For the main experiment, these cells were seeded into 6-well plates in a concentration of 1.2×106 cells/well and maintained in serum-free DMEM overnight. Induction of insulin resistance by high-glucose and high-insulin concentrations To create an insulin-resistant model, we incubated HepG2 cells in serum-free DMEM with a high concentration of glucose (30 mM Dglucose) and insulin (1 μM) for 24 hours according to the protocol of Hu X et al (22). The effectiveness of this treatment was confirmed by the glucose GOD-PAP method(Parsazmoon, Iran) at the diabetes clinic of Tehran. As previously described (23), the supernatant was collected, and the level of glucose and hyperglycemia was measured. Treatment with resveratrol To have a stock concentration of 100 mmol/L, 22.825 mg of RSV was dissolved in 1 mL DMSO and stored at −20°C. For the experiment, this stock was diluted with DMEM in 3 aliquots of 5, 10 and 20 μM. Then the cells were treated with different doses or left untreated as control for 24 and 48 hours. The final concentration of DMSO was <0.5%. RNA extraction and real-time quantitative polymerase chain reaction Total RNA was extracted from HepG2 cells using RNeasy plus mini kit (Qiagen, Valencia, California, United States; #A-74134) according to the manufacturer’s instructions. The total RNA concentration was measured by NanoDrop 2000c Spectrophotometer (Thermo, Waltham, Massachusetts, United States). Approximately 500 ng of the total RNA was converted into cDNA using PrimeScript RT reagent kit (Takara Bio, Mountain View, California, United States; #RR037A). Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was done by using a SYBR green kit (Takara Bio; # RR820L) by 40 cycles. Each gene (β-actin, FoxO1, mTOR, PDPK1) in each cDNA sample was run in duplicate and the values of threshold cycle (ΔCT) were analyzed in comparison with the housekeeping
gene β-actin. Primer Express 3 software (Thermo) was used to design all the primers. The primers sequences are listed in Table 1.
Data analysis and statistical analysis β-actin was used as a reference gene. LinRegPCR software (Heart Failure Research Center, Amsterdam, The Netherlands) was used to adjust the differences in efficiency between PCR reactions for each sample. The threshold cycle (Ct) values were entered into the Relative Expression Software Tool (REST) (Qiagen) and were analyzed for significant differences (p<0.05) by the pairwise fixed reallocation randomization test using the following formula for calculating expression ratio:
Expression ratio =
(Etarget )ΔCttarget(MEANcontrol−MEANsample) (Eref )ΔCtref (MEANcontrol−MEANsample)
where E is efficiency of the PCR reaction and ΔCt the crossing point difference of an unknown sample (treated) vs. a control (untreated). Analysis of REST supplied ratios of expression, statistical analysis and confidence intervals. All p values less than 0.05 were considered statistically significant.
Results The effect of differing doses of resveratrol on the expression of FoxO1 in HepG2 cells After induction of insulin resistance by incubation of HepG2 cells with 1 μM insulin and 30 mM glucose for 24 hours, HepG2 cells were treated with RSV. In the qRT-PCR results, we unexpectedly observed that FoxO1 expression was decreased in insulin-resistant cells while increased followed by RSV treatment in all doses that were timedependent and in doses of 5 and 20 μM. Our data showed that the greatest effect of RSV was achieved in 5 μM of concentration in a time-dependent manner. Our data did not show any significant dosedependent effect of RSV. Figure 2 shows the expression ratio of differing treatments.
The effect of differing doses of resveratrol on the expression of mTOR in HepG2 cells As seen in Figure 3, downregulation of mTOR was observed in our insulin-resistant model (p=0.001). At 24 hours it was deregulated upon treatment by RSV, but a significant increase was observed in all doses at 48 hours compared to 24 hours (p=0.001). There was a dose-dependent downregulation at 48 hours and a time-dependent upregulation for all doses. The results showed that the most effective dose of RSV for upregulation of mTOR was 5 μM.
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Figure 2. The effect of insulin (Ins)+glucose (Glc) and resveratrol on FoxO1 expression in comparison to control group. Comparison of FoxO1 expression after 24 h and 48 h of treatment with 5, 10 and 20 μM of resveratrol and Ins+Glc, analyzed by quantitative real-time PCR and graphed as 2Log of expression ratios (y-axis). shows significant differences.
Figure 3. The effect of insulin (Ins)+glucose (Glc) and resveratrol on mTOR expression in comparison to control group. Comparison of mTOR expression after 24 h and 48 h of treatment with 5, 10 and 20 μM of resveratrol and Ins+Glc, analyzed by quantitative real-time PCR and graphed as 2Log of expression ratios (y-axis). shows significant differences.
The effect of differing doses of resveratrol on the expression of PDPK1 in HepG2 cells The expression of PDPK1 was downregulated upon high doses of glucose and insulin in our insulin-resistant model, as demonstrated in Figure 4, which was significantly counteracted upon treatment with RSV (p=0.001). According to our data, there was no dose-dependent effect of RSV, whereas a time-dependent effect was observed in all doses. The 10 mM concentration of RSV meaningfully upregulated PDPK1 after 48 hours more than after 24 hours (p=0.001). The data of the qRT-PCR analysis are summarized in Table 2.
Discussion Diabetes mellitus is considered a metabolic disorder that is affected by a number of factors, including diet, stress and lack of physical activity (24). In the present study, we used q-RT-PCR to
detect the expression of genes involved in insulin signalling among them. Akt plays a central role, which is activated by PDPK1 and mTORC2 (25). There are many studies demonstrating beneficial effects of RSV on insulin secretion (26), reduction of glucose level (27,28), homeostasis model assessment of insulin resistance (HOMAIR) index (27), increase in the uptake of glucose, glucose transporter 4 translocation to the cell membrane in muscle cells and protection of beta cells (16), and improving insulin sensitivity (29), although some of them demonstrate no significant changes in insulin sensitivity (30). In this experiment, we initially showed that PDPK1 is significantly upregulated upon RSV at 48 hours, which is consistent with other studies. Hong et al showed that RSV normalizes the decreased phosphorylation levels of Akt, PI3K, PDPK1, insulin receptor substrate-1 and glycogen synthase kinase-3 (GSK-3) in mice exposed to high-fat diets (31). Another study demonstrated that SIRT1 increased the phosphorylation of Akt and PDPK1 in insulinresistant cells and enhanced insulin sensitivity (32). A recent study demonstrated that insulin-signalling components in the hepatic tissues of rats with diabetes are recovered by
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Figure 4. The effect of insulin (Ins)+glucose (Glc) and resveratrol on PDPK1 expression in comparison to control group. Comparison of PDPK1 expression after 24 h and 48 h of treatment with 5, 10 and 20 μM of resveratrol and Ins+Glc, analyzed by quantitative real-time PCR and graphed as 2Log of expression ratios (y-axis). shows significant differences.
Table 2 The analysis of the results of real-time quantitative reverse transcription polymerase chain reaction Ins+Glc
FoxO1 mTOR PDPK1
24 h 48 h 24 h 48 h 24 h 48 h
RSV 5 μM
RSV 10 μM
RSV 20 μM
2Log (expression ratio ± SEM)
p values
2Log (expression ratio ± SEM)
p values
2Log (expression ratio ± SEM)
p values
2Log (expression ratio ± SEM)
p values
−1.242±0.06606 −2.714±0.00978 −0.031±0.00801 −3.443±0.0043 −1.071±0.05339 −2.132±0.01604
0.178 0.001* 0.647 0.001* 0.001* 0.153
1.036±0.22007 1.909±0.08338 0.301±0.03776 2.524±0.11268 1.711±0.27248 2.075±0.08988
0.178 0.001* 0.178 0.001* 0.001* 0.001*
0.802±0.18305 0.321±0.04627 −0.171±0.02251 1.863±0.22018 1.098±0.19517 2.634±0.31226
0.001* 0.500 0.178 0.001* 0.178 0.001*
1.131±0.26288 1.764±0.1472 −0.159±0.01209 1.699±0.14939 1.296±0.205 2.308±0.2333
0.178 0.001* 0.325 0.001* 0.178 0.001*
Notes: Expression ratios and p values of insulin signalling cascade genes upon treatment with differing doses of resveratrol (5, 10, 20 μM) for 24 and 48 h. The insulin+glc treatment groups were compared with the control group, and resveratrol treatment groups were compared with the insulin+glc group. For detecting significant differences (p<0.05), data were analyzed by pairwise fixed reallocation randomization test using the Relative Expression Software Tool (REST). * Shows significant difference.
RSV supplementation, and the expression levels of insulin receptor substrate-1 (IRS-1), PI3K, Akt and mTOR were increased (33). Improvement of insulin resistance by means of short-term RSV supplementation has been shown by a number of studies (31,34). mTORC1 activates SREBP1c and promotes hepatic lipogenesis. FoxO1 and gluconeogenesis are suppressed by mTORC2-Akt. High activity of hepatic mTORC1 is seen in obesity and overfeeding, which results in hepatic insulin resistance, gluconeogenesis and lipogenesis (35). A number of studies have claimed that mTORC1 and S6K1 activity are increased in the adipose, muscle and liver tissues of insulin-resistant rodents, which supports the idea that mTOR signalling affects insulin sensitivity (36,37). In addition, the elevated phosphorylation level of mTOR in obese mice was decreased by RSV (31). However, rapamycine induces insulin resistance by mTOR inhibition and obstructs mTORC2’s inhibitory effect on hepatic gluconeogenesis (38). It has been shown that high doses of RSV (10 to 100 μM) suppresses the mTOR pathway (39–42). However, in another study, the total amount of mTORC2 components (mTOR, Rictor and mSIN1) and the protein levels of those components did not change in SIRT1-deleted mice, but the complex arrangement of them was impressive (43). Our results showed that the mTOR expression ratio was decreased in insulin-resistant HepG2 cells while increased following RSV treatment. According to the results of our study and some other studies, it could be concluded that mTOR is deregulated in insulin-resistance conditions, which be regulated under treatment with RSV. FoxO1 is another gene in the insulin signalling cascade that regulates gluconeogenesis (44). RSV deacetylates the FoxO1 via SIRT1
in the nucleus and independent of SIRT1 in the cytosol, therefore increasing gluconeogenic gene expression, such as phosphophenol pyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase) (45). In contrast, another study has shown that the phosphorylation levels of FoxO1A were decreased by RSV in mice exposed to high-fat diets (31). In our experiment, FoxO1 was upregulated by RSV, which may result in insulin resistance. However, there are studies showing that RSV decreases SREBP1c expression via the SIRT1-FoxO1 pathway and protects the body against nonalcoholic fatty liver disease (NAFLD) (46,47). But the surprising result was related to the effect of insulin and glucose on FoxO1 expression, which is downregulated, although several studies have demonstrated the opposite effect (48,49). The results of several studies have demonstrated that FoxO1 is downregulated in cancer cells such as HepG2 (50), and because insulin facilitates the PI3K-Akt pathway, it could promote proliferation (51). Our data showed that although all of the doses of RSV were effective to some extent, it seems that the optimum impact was achieved after 48 hours. One of the limitations of this study was determining only the gene expression, but in some situations, the expression of genes is affected without any change in their protein content (33). Using immunoblotting (Western blotting) analysis is recommended for future experiments. Also, examination of the levels of protein phosphorylation is good advice for ensuring the treatment effect. However, we carried out qRT-PCR in duplicate and reached the same result. There was a question about why the expression ratio of RSV10
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point in the 48-hour treatment in FOXO expression was low compared to RSV5 and RSV20. This should be examined more closely in future studies. In all, the results of the present study showed that treatment by RSV enhances the expression of PDPK1, mTOR and FoxO1 in insulin-resistant HepG2 cells. Elevation of PDPK1 and mTOR ameliorates insulin resistance, but FoxO1 upregulation results in the increase of gluconeogenic gene expression. Hence, in the context of diabetes, it seems that RSV has a bilateral effect in controlling the complications of diabetes and, thus, necessitates caution in longterm usage of RSV for ameliorating the conditions involved in diabetes.
Acknowledgements This research was supported by Tehran University of Medical Sciences and Health Services, grant number 93-04-159-27489.
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Canadian Journal of Diabetes journal homepage: w w w. c a n a d i a n j o u r n a l o f d i a b e t e s . c o m
Original Research
Does Resveratrol Improve Insulin Signalling in HepG2 Cells? Marjan Norouzzadeh MS a, Fatemehsadat Amiri PhD b, Ali Akbar Saboor-Yaraghi a, Farnoosh Shemirani PhD a, Yas Kalikias MS a, Loghman Sharifi MS a, Monireh Sadat Seyyedsalehi MS a, Maryam Mahmoudi MD, PhD a,* a b
Department of Cellular and Molecular Nutrition, School of Nutritional Sciences and Dietetic, Tehran University of Medical Sciences, Tehran, Iran Department of Nutrition, School of Public Health, Iran University of Medical Sciences, Tehran, Iran
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 December 2015 Received in revised form 5 September 2016 Accepted 28 September 2016
Objectives: Diabetes mellitus is a common metabolic disorder with high global prevalence. It is characterized by a decrease in insulin secretion or a decrease in insulin sensitivity or both. The aim of the present study was to investigate the effects of resveratrol treatment on the expression of the genes involved in insulin signalling cascade, such as Forkhead box protein O1 (FoxO1), 3-phosphoinositide-dependent protein kinase 1 (PDPK1) and mammalian target of rapamycin (mTOR). Methods: HepG2 cells were cultured in serum-free medium with high concentrations of glucose and insulin and then were treated with resveratrol (5, 10 and 20 μM) for 24 and 48 hours. Complementary deoxyribonucleic acids (cDNAs) were synthesized followed by RNA extraction. Real-time quantitative reverse transcription polymerase chain reaction was used to analyze the expression of FoxO1, PDPK1 and mTOR. Results: Resveratrol increased the expression of PDPK1, mTOR and FoxO1. No significant difference was seen among differing dosages of resveratrol, but treatments for 48 hours exerted the greatest effectiveness. Conclusions: Our results were consistent with other studies showing the beneficial effects of resveratrol on diabetes. However, considering the effects of resveratrol in increasing FoxO1 and gluconeogenic gene expression, long-term usage of resveratrol should be investigated in greater depth in future studies. © 2016 Canadian Diabetes Association.
Keywords: FoxO1 HepG2 insulin resistance mTOR PDPK1 resveratrol
r é s u m é Mots clés : FoxO1 HepG2 insulinorésistance mTOR PDPK1 resvératrol
Objectifs : Le diabète sucré qui est une perturbation fréquente du métabolisme connaît une prévalence mondiale élevée. Il est caractérisé par une diminution de la sécrétion d’insuline ou une diminution de la sensibilité à l’insuline, ou les deux. L’objectif de la présente étude était d’examiner les effets du traitement à base de resvératrol sur l’expression des gènes impliqués dans la cascade de signalisation de l’insuline, comme la protéine FoxO1 (Forkhead box protein O1), la protéine kinase dépendante du 3-phosphoinositide (PDPK1) et la cible mammalienne de la rapamycine (mTOR). Méthodes : Les cellules HepG2 ont été mises en culture dans un milieu sans sérum à fortes concentrations de glucose et d’insuline, et ont alors été traitées par resvératrol (5, 10 et 20 μM) durant 24 et 48 heures. À la suite de la synthèse des acides désoxyribonucléiques complémentaires (ADNc), les ARN ont été extraits. La transcription inverse de la réaction en chaîne par polymérase en temps réel a été utilisée pour analyser l’expression de la protéine FoxO1, de la PDPK1 et de la mTOR. Résultats : Le resvératrol a augmenté l’expression de la PDPK1, de la mTOR et de la protéine FoxO1. Aucune différence significative n’a été observée entre les diverses posologies de resvératrol, mais les traitements de 48 h ont exercé une plus grande efficacité. Conclusions : Nos résultats ont concordé avec les autres études ayant démontré les effets bénéfiques du resvératrol sur le diabète. Toutefois, en considérant les effets du resvératrol sur l’augmentation de l’expression de la protéine FoxO1 et du gène gluconéogénique, l’usage à long terme du resvératrol devrait être examiné de manière plus approfondie au cours d’études futures. © 2016 Canadian Diabetes Association.
* Address for correspondence: Maryam Mahmoudi, MD, PhD, Department of Cellular and Molecular Nutrition, School of Nutritional Sciences and Dietetic, Tehran University of Medical Sciences, Tehran 14155-6447, Iran. E-mail address: [email protected] 1499-2671 © 2016 Canadian Diabetes Association. http://dx.doi.org/10.1016/j.jcjd.2016.09.015
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Introduction
and restrains gluconeogenesis by preventing nuclear accumulation of FoxO1. Therefore, it could be deduced that glucose and lipid homeostasis are under the control of mTORC2 via Akt. Indeed, a flaw in hepatic mTORC2 signalling may ultimately lead to diabetes (10). Both the secretion and the action of insulin could be affected by a series of plant-derived components (12). Resveratrol (RSV) (trans3,5,4’-trihydroxystilbene) belongs to the large group of polyphenols present in the skins of red grapes and in nuts and red wine (13,14). Several animal studies have demonstrated promising perspectives on the effects of RSV in preventing and/or treating metabolic disorders such as obesity and diabetes (15,16). RSV can promote the activation of Akt and PDPK1 via the deacetylation activity of sirtuin (silent mating type information regulation 2 homolog) 1(SIRT1) (17). Moreover, RSV is capable of repressing PI3K activity via its ATPbinding site (18,19). As activation of mTOR by insulin is done through the PDPK1/Akt signalling pathway, it could be hypothesized that the mechanism of the protective effect of RSV is exerted via mTOR signalling inhibition (20,21). Altogether, the present study aimed to investigate the possible mechanism via which RSV could eliminate the complications of diabetes. The insulin signalling cascade is summarized in Figure 1.
Diabetes mellitus is a common metabolic disorder that is occurring with increasing incidence worldwide (1,2). It is a chronic disorder that is characterized by a decrease in blood insulin levels or inefficient use of it, which involves metabolism of carbohydrates, proteins and lipids. Impaired insulin secretion and/or insulin resistance are the main causes, leading to hyperglycemia and the malfunction of many organs, including the liver (3). Insulin acts through the key proteins, which exert their effects via insulin receptor β(IRβ). Insulin receptor substrate (IRS) is a prominent protein that transmits signals to downstream kinases, including phosphoinositide 3-kinase (PI3K), 3‘-phosphoinositide-dependent protein kinase (PDPK1) and Akt/protein kinase B (PKB). Recruiting of the mammalian target of rapamycin complex 2 (mTORC2) and PDPK1 are needed for Akt activation (4–6). The mammalian target of rapamycin complex 1 (mTORC1) is 1 of the targets of Akt (7) that has main components named mTOR, raptor and mammalian lethal with SEC13 protein 8 (mLST8) (8). It has been shown that the mammalian target of mTORC1 exerts its effects by increasing transcription of lipogenic genes, including sterol regulatory element-binding protein 1c (SREBP1c), fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC) in mice fed high-fat diets (9). The effects of mTORC1 and mTORC2 are applied by regulation of influential kinases, including ribosomal protein S6 kinase (S6K) and Akt (7). It has been revealed that mTOR is involved in the onset and progression of diabetes, cancer and ageing (7,10). Glucose homeostasis is controlled by hepatic mTORC2 (11). mTORC2 contains mTOR, rictor, mammalian stress-activated protein kinase interacting protein 1 (mSIN1), mLST8 and prolinerich protein 5/proline rich protein 5-Like (PRR5/PRR5L) (8)
Methods Study design The current study is experimental. Each experiment was carried out in triplicate, and the data were the average results of 3 independent experiments.
Insulin
IR P IRS1/2
PI3K
PDPK1
P
Gluconeogenesis
mTORC2
P
P
FoxO1
Akt mTORC1 GSK3
Lipin1
S6K P
Insig2a
SREBP1c LXR Lipogenesis
Figure 1. Insulin signalling cascade.
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Table 1 The primer sequences and parameters used for RT-qPCR Gene
Oligo
Sequence
Length
Tm
CG %
β-actin β-actin FoxO1 FoxO1 mTOR mTOR PDPK1 PDPK1
Forward Reverse Forward Reverse Forward Reverse Forward Reverse
5’-AGCCTCGCCTTTGCCG-3’ 5’-ATCACGCCCTGGTGCCT-3’ 5’-GCTTAGACTGTGACATGGAATCC-3’ 5’-GTACTTTTAAGTGTAACCTGCTCAC-3’ 5’-GCCAGGGATCTCTTCAATGCT-3’ 5’-GTCTGTGTGACTTCAGCGATG-3’ 5’-GCAGTACATTCACGATCTGGA-3’ 5’-TGTGGTCCTTCTGTGAGCAAC-3’
16 17 23 25 21 21 22 21
55.9 57.2 62.9 62.5 61.3 61.3 62.1 61.3
68.75 64.71 47.83 40 52.38 52.38 50 52.38
CG,; tm,; RT-qPRC, real-time quantitative reverse transcription polymerase chain reaction.
Materials Resveratrol (RSV-1185247-70-4-100 mg) was purchased from Santa Cruz Biotechnology (St. Louis, Missouri, United States). Dulbecco modified Eagle medium (DMEM-F12-32500032), fetal bovine serum (FBS-10270106) and trypsin/EDTA (25200-056) were purchased from Gibco (Pittsburgh, Pennsylvania, United States) and dimethyl sulforoxide (DMSO) was purchased from SigmaAldrich (St. Louis, Missouri, United States) (#D2650). Cell lines and cell culture We obtained human hepatoma HepG2 cells from the Pasteur Institute of Iran. These cells were incubated at 37°C in a humidified atmosphere including 5% (v/v) CO2 in culture flasks containing DMEM supplemented with 10% FBS, and 100 U/mL streptomycin and 100 mg/mL penicillin. The first time, they were passaged by trypsinization after 1 week and then every 3 days. For the main experiment, these cells were seeded into 6-well plates in a concentration of 1.2×106 cells/well and maintained in serum-free DMEM overnight. Induction of insulin resistance by high-glucose and high-insulin concentrations To create an insulin-resistant model, we incubated HepG2 cells in serum-free DMEM with a high concentration of glucose (30 mM Dglucose) and insulin (1 μM) for 24 hours according to the protocol of Hu X et al (22). The effectiveness of this treatment was confirmed by the glucose GOD-PAP method(Parsazmoon, Iran) at the diabetes clinic of Tehran. As previously described (23), the supernatant was collected, and the level of glucose and hyperglycemia was measured. Treatment with resveratrol To have a stock concentration of 100 mmol/L, 22.825 mg of RSV was dissolved in 1 mL DMSO and stored at −20°C. For the experiment, this stock was diluted with DMEM in 3 aliquots of 5, 10 and 20 μM. Then the cells were treated with different doses or left untreated as control for 24 and 48 hours. The final concentration of DMSO was <0.5%. RNA extraction and real-time quantitative polymerase chain reaction Total RNA was extracted from HepG2 cells using RNeasy plus mini kit (Qiagen, Valencia, California, United States; #A-74134) according to the manufacturer’s instructions. The total RNA concentration was measured by NanoDrop 2000c Spectrophotometer (Thermo, Waltham, Massachusetts, United States). Approximately 500 ng of the total RNA was converted into cDNA using PrimeScript RT reagent kit (Takara Bio, Mountain View, California, United States; #RR037A). Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was done by using a SYBR green kit (Takara Bio; # RR820L) by 40 cycles. Each gene (β-actin, FoxO1, mTOR, PDPK1) in each cDNA sample was run in duplicate and the values of threshold cycle (ΔCT) were analyzed in comparison with the housekeeping
gene β-actin. Primer Express 3 software (Thermo) was used to design all the primers. The primers sequences are listed in Table 1.
Data analysis and statistical analysis β-actin was used as a reference gene. LinRegPCR software (Heart Failure Research Center, Amsterdam, The Netherlands) was used to adjust the differences in efficiency between PCR reactions for each sample. The threshold cycle (Ct) values were entered into the Relative Expression Software Tool (REST) (Qiagen) and were analyzed for significant differences (p<0.05) by the pairwise fixed reallocation randomization test using the following formula for calculating expression ratio:
Expression ratio =
(Etarget )ΔCttarget(MEANcontrol−MEANsample) (Eref )ΔCtref (MEANcontrol−MEANsample)
where E is efficiency of the PCR reaction and ΔCt the crossing point difference of an unknown sample (treated) vs. a control (untreated). Analysis of REST supplied ratios of expression, statistical analysis and confidence intervals. All p values less than 0.05 were considered statistically significant.
Results The effect of differing doses of resveratrol on the expression of FoxO1 in HepG2 cells After induction of insulin resistance by incubation of HepG2 cells with 1 μM insulin and 30 mM glucose for 24 hours, HepG2 cells were treated with RSV. In the qRT-PCR results, we unexpectedly observed that FoxO1 expression was decreased in insulin-resistant cells while increased followed by RSV treatment in all doses that were timedependent and in doses of 5 and 20 μM. Our data showed that the greatest effect of RSV was achieved in 5 μM of concentration in a time-dependent manner. Our data did not show any significant dosedependent effect of RSV. Figure 2 shows the expression ratio of differing treatments.
The effect of differing doses of resveratrol on the expression of mTOR in HepG2 cells As seen in Figure 3, downregulation of mTOR was observed in our insulin-resistant model (p=0.001). At 24 hours it was deregulated upon treatment by RSV, but a significant increase was observed in all doses at 48 hours compared to 24 hours (p=0.001). There was a dose-dependent downregulation at 48 hours and a time-dependent upregulation for all doses. The results showed that the most effective dose of RSV for upregulation of mTOR was 5 μM.
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Figure 2. The effect of insulin (Ins)+glucose (Glc) and resveratrol on FoxO1 expression in comparison to control group. Comparison of FoxO1 expression after 24 h and 48 h of treatment with 5, 10 and 20 μM of resveratrol and Ins+Glc, analyzed by quantitative real-time PCR and graphed as 2Log of expression ratios (y-axis). shows significant differences.
Figure 3. The effect of insulin (Ins)+glucose (Glc) and resveratrol on mTOR expression in comparison to control group. Comparison of mTOR expression after 24 h and 48 h of treatment with 5, 10 and 20 μM of resveratrol and Ins+Glc, analyzed by quantitative real-time PCR and graphed as 2Log of expression ratios (y-axis). shows significant differences.
The effect of differing doses of resveratrol on the expression of PDPK1 in HepG2 cells The expression of PDPK1 was downregulated upon high doses of glucose and insulin in our insulin-resistant model, as demonstrated in Figure 4, which was significantly counteracted upon treatment with RSV (p=0.001). According to our data, there was no dose-dependent effect of RSV, whereas a time-dependent effect was observed in all doses. The 10 mM concentration of RSV meaningfully upregulated PDPK1 after 48 hours more than after 24 hours (p=0.001). The data of the qRT-PCR analysis are summarized in Table 2.
Discussion Diabetes mellitus is considered a metabolic disorder that is affected by a number of factors, including diet, stress and lack of physical activity (24). In the present study, we used q-RT-PCR to
detect the expression of genes involved in insulin signalling among them. Akt plays a central role, which is activated by PDPK1 and mTORC2 (25). There are many studies demonstrating beneficial effects of RSV on insulin secretion (26), reduction of glucose level (27,28), homeostasis model assessment of insulin resistance (HOMAIR) index (27), increase in the uptake of glucose, glucose transporter 4 translocation to the cell membrane in muscle cells and protection of beta cells (16), and improving insulin sensitivity (29), although some of them demonstrate no significant changes in insulin sensitivity (30). In this experiment, we initially showed that PDPK1 is significantly upregulated upon RSV at 48 hours, which is consistent with other studies. Hong et al showed that RSV normalizes the decreased phosphorylation levels of Akt, PI3K, PDPK1, insulin receptor substrate-1 and glycogen synthase kinase-3 (GSK-3) in mice exposed to high-fat diets (31). Another study demonstrated that SIRT1 increased the phosphorylation of Akt and PDPK1 in insulinresistant cells and enhanced insulin sensitivity (32). A recent study demonstrated that insulin-signalling components in the hepatic tissues of rats with diabetes are recovered by
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Figure 4. The effect of insulin (Ins)+glucose (Glc) and resveratrol on PDPK1 expression in comparison to control group. Comparison of PDPK1 expression after 24 h and 48 h of treatment with 5, 10 and 20 μM of resveratrol and Ins+Glc, analyzed by quantitative real-time PCR and graphed as 2Log of expression ratios (y-axis). shows significant differences.
Table 2 The analysis of the results of real-time quantitative reverse transcription polymerase chain reaction Ins+Glc
FoxO1 mTOR PDPK1
24 h 48 h 24 h 48 h 24 h 48 h
RSV 5 μM
RSV 10 μM
RSV 20 μM
2Log (expression ratio ± SEM)
p values
2Log (expression ratio ± SEM)
p values
2Log (expression ratio ± SEM)
p values
2Log (expression ratio ± SEM)
p values
−1.242±0.06606 −2.714±0.00978 −0.031±0.00801 −3.443±0.0043 −1.071±0.05339 −2.132±0.01604
0.178 0.001* 0.647 0.001* 0.001* 0.153
1.036±0.22007 1.909±0.08338 0.301±0.03776 2.524±0.11268 1.711±0.27248 2.075±0.08988
0.178 0.001* 0.178 0.001* 0.001* 0.001*
0.802±0.18305 0.321±0.04627 −0.171±0.02251 1.863±0.22018 1.098±0.19517 2.634±0.31226
0.001* 0.500 0.178 0.001* 0.178 0.001*
1.131±0.26288 1.764±0.1472 −0.159±0.01209 1.699±0.14939 1.296±0.205 2.308±0.2333
0.178 0.001* 0.325 0.001* 0.178 0.001*
Notes: Expression ratios and p values of insulin signalling cascade genes upon treatment with differing doses of resveratrol (5, 10, 20 μM) for 24 and 48 h. The insulin+glc treatment groups were compared with the control group, and resveratrol treatment groups were compared with the insulin+glc group. For detecting significant differences (p<0.05), data were analyzed by pairwise fixed reallocation randomization test using the Relative Expression Software Tool (REST). * Shows significant difference.
RSV supplementation, and the expression levels of insulin receptor substrate-1 (IRS-1), PI3K, Akt and mTOR were increased (33). Improvement of insulin resistance by means of short-term RSV supplementation has been shown by a number of studies (31,34). mTORC1 activates SREBP1c and promotes hepatic lipogenesis. FoxO1 and gluconeogenesis are suppressed by mTORC2-Akt. High activity of hepatic mTORC1 is seen in obesity and overfeeding, which results in hepatic insulin resistance, gluconeogenesis and lipogenesis (35). A number of studies have claimed that mTORC1 and S6K1 activity are increased in the adipose, muscle and liver tissues of insulin-resistant rodents, which supports the idea that mTOR signalling affects insulin sensitivity (36,37). In addition, the elevated phosphorylation level of mTOR in obese mice was decreased by RSV (31). However, rapamycine induces insulin resistance by mTOR inhibition and obstructs mTORC2’s inhibitory effect on hepatic gluconeogenesis (38). It has been shown that high doses of RSV (10 to 100 μM) suppresses the mTOR pathway (39–42). However, in another study, the total amount of mTORC2 components (mTOR, Rictor and mSIN1) and the protein levels of those components did not change in SIRT1-deleted mice, but the complex arrangement of them was impressive (43). Our results showed that the mTOR expression ratio was decreased in insulin-resistant HepG2 cells while increased following RSV treatment. According to the results of our study and some other studies, it could be concluded that mTOR is deregulated in insulin-resistance conditions, which be regulated under treatment with RSV. FoxO1 is another gene in the insulin signalling cascade that regulates gluconeogenesis (44). RSV deacetylates the FoxO1 via SIRT1
in the nucleus and independent of SIRT1 in the cytosol, therefore increasing gluconeogenic gene expression, such as phosphophenol pyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase) (45). In contrast, another study has shown that the phosphorylation levels of FoxO1A were decreased by RSV in mice exposed to high-fat diets (31). In our experiment, FoxO1 was upregulated by RSV, which may result in insulin resistance. However, there are studies showing that RSV decreases SREBP1c expression via the SIRT1-FoxO1 pathway and protects the body against nonalcoholic fatty liver disease (NAFLD) (46,47). But the surprising result was related to the effect of insulin and glucose on FoxO1 expression, which is downregulated, although several studies have demonstrated the opposite effect (48,49). The results of several studies have demonstrated that FoxO1 is downregulated in cancer cells such as HepG2 (50), and because insulin facilitates the PI3K-Akt pathway, it could promote proliferation (51). Our data showed that although all of the doses of RSV were effective to some extent, it seems that the optimum impact was achieved after 48 hours. One of the limitations of this study was determining only the gene expression, but in some situations, the expression of genes is affected without any change in their protein content (33). Using immunoblotting (Western blotting) analysis is recommended for future experiments. Also, examination of the levels of protein phosphorylation is good advice for ensuring the treatment effect. However, we carried out qRT-PCR in duplicate and reached the same result. There was a question about why the expression ratio of RSV10
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point in the 48-hour treatment in FOXO expression was low compared to RSV5 and RSV20. This should be examined more closely in future studies. In all, the results of the present study showed that treatment by RSV enhances the expression of PDPK1, mTOR and FoxO1 in insulin-resistant HepG2 cells. Elevation of PDPK1 and mTOR ameliorates insulin resistance, but FoxO1 upregulation results in the increase of gluconeogenic gene expression. Hence, in the context of diabetes, it seems that RSV has a bilateral effect in controlling the complications of diabetes and, thus, necessitates caution in longterm usage of RSV for ameliorating the conditions involved in diabetes.
Acknowledgements This research was supported by Tehran University of Medical Sciences and Health Services, grant number 93-04-159-27489.
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