J. Pineal Res. 2013; 55:207–220
© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Molecular, Biological, Physiological and Clinical Aspects of Melatonin
Doi:10.1111/jpi.12064
Journal of Pineal Research
Melatonin-mediated insulin synthesis during endoplasmic reticulum stress involves HuD expression in rat insulinoma INS-1E cells Abstract: In this study, we investigated how melatonin mediates insulin synthesis through endoplasmic reticulum (ER) via HuD expression in rat insulinoma INS-1E cells. Under ER stress condition (thapsigargin with/ without melatonin, tunicamycin with/without melatonin), phosphorylation of AMP-activated protein kinase (p-AMPK) was significantly increased when compared with only with/without melatonin (control/melatonin). Insulin receptor substrate (IRS) two protein was significantly reduced under conditions of ER stress when compared with control/melatonin, but no expression of IRS1 protein was observed. In thapsigargin treatment, melatonin (10, 50 lM) increased IRS2 protein expression in a dose-dependent manner. p-Akt (Ser473) expression significantly decreased under ER stress condition prior to control/melatonin. Melatonin (10, 50 lM) significantly reduced nuclear and cellular p85a expressions in a dose-dependent manner when compared with only thapsigargin or tunicamycin. These results indicate the activation of the aforementioned expressions under regulation of the pathway, AMPK ? IRS2 ? Akt/PKB ? PI3K (p85a). However, mammalian target of rapamycin and raptor protein, mTORC1, was found to be independent of the ER stress response. In thapsigargin treatment, melatonin increased nuclear mammalian RNA-binding protein (HuD) expression and reduced cellular HuD expression and subsequently resulted in a decrease in cellular insulin level and rise in insulin secretion in a dose-dependent manner. In tunicamycin treatment, HuD and insulin proteins showed similar expression tendencies. These results indicate that ER stress/melatonin, especially thapsigargin/ melatonin, increased nuclear HuD expression and subsequently resulted in a decrease in intracellular biosynthesis; it is hypothesized that extracellular secretion of insulin may be regulated by melatonin.
Introduction AMP-activated protein kinase (AMPK) functions as a master metabolic regulator of cellular metabolism in skeletal muscle and human cancer and is emerging as a therapeutic target for metabolic syndrome and/or abnormalities in diabetes mellitus and lipid metabolism [1–4]. The activation of AMPK improves insulin resistance by stimulating glucose uptake and suppressing blood glucose and lipid levels, but the activity of AMPK is inhibited in disorders associated with metabolic syndrome including insulin resistance, central obesity, type 2 diabetes, dyslipidemia, premature atherosclerosis, and other diseases [4, 5]. In addition, AMPK activation increases fatty acid oxidation and, conversely, decreases fatty acid and protein synthesis, which are apparently dual effects of insulin action [5]. Thus, there is a complex relationship between AMPK and insulin signaling pathways; AMPK regulates IRS1 [6–8] and Akt/PKB [9–14], whereas Akt/PKB and insulin have negative effects on AMPK activation [15–17].
Yeong-Min Yoo Department of Biomedical Engineering, College of Health Science, Yonsei University, Wonju, Korea
Key words: ER stress, HuD, insulin, melatonin, p-AMPK, rat insulinoma INS-1E cells Address reprint requests to Yeong-Min Yoo, Department of Biomedical Engineering, College of Health Science, Yonsei University, Wonju, Gangwon-do 220-710, Korea. E-mail:
[email protected] Received January 9, 2013; Accepted April 26, 2013.
The key target of Akt/PKB and insulin in a multitude of cellular events including glucose uptake, glycogen synthesis, gluconeogenesis, and lipid storage is phosphoinositide 3-kinase (PI3K), which is activated by binding of the p85 (regulatory subunit of PI3K) to specific sites on IRS1/IRS2 that are tyrosine phosphorylated by the insulin receptor [17–20]. A major down-regulator of insulin action is mTOR, which belongs to the phosphatidylinositol 3-kinase-related kinase protein family that possess exclusively protein kinase activity. mTOR functions in a mitogenic pathway downstream of PI3K and is activated by insulin and growth factors in the presence of sufficient nutrients including amino acids and glucose [21, 22]. Activated mTOR regulates protein synthesis via phosphorylation of ribosomal protein S6 kinase 1 (S6K1). Recent reports show that mTOR and S6K1 induce IRS1 phosphorylation to affect negatively insulin signaling and thus play a role in insulin resistance [23, 24]. In addition, recent studies have shown that the activation of AMPK leads to inhibition of mTOR/S6K1 [25, 26]. This occurs 207
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Fig. 1. Phosphorylation of AMPK under the condition of ER stress and/or melatonin in rat insulinoma INS-1E cells. INS-1E cells were incubated in RPMI 1640 medium supplemented with 2% FBS with/without melatonin and/or thapsigargin (1 lM) for 6 hr or tunicamycin (2 lg/mL) for 16 hr at 37°C with 5% CO2. p-AMPK expression was then analyzed by Western blot (A). The relative amount of p-AMPK (B) was quantified as described in the Materials and methods section. Data represent mean S.D. of three experiments. ***P < 0.001 versus 2% FBS; ###P < 0.001, thapsigargin or tunicamycin versus thapsigargin and melatonin or tunicamycin and melatonin; &&P < 0.01, &&& P < 0.001, control/melatonin versus thapsigargin/melatonin or tunicamycin/ melatonin.
Fig. 2. The expressions of IRS1 and IRS2 proteins under the condition of ER stress and/or melatonin in rat insulinoma INS-1E cells. INS-1E cells were incubated in RPMI 1640 medium supplemented with 2% FBS with/without melatonin and/or thapsigargin (1 lM) for 6 hr or tunicamycin (2 lg/mL) for 16 hr at 37°C with 5% CO2. p-AMPK expression was then analyzed by Western blot (A). The relative amounts of IRS2 (B) were quantified as described in the Materials and methods section. Data represent mean S.D. of three experiments. ***P < 0.001 versus 2% FBS; #P < 0.05, ##P < 0.01, ###P < 0.001, thapsigargin or tunicamycin versus thapsigargin and melatonin or tunicamycin and melatonin; &&P < 0.01, &&& P < 0.001, control/melatonin versus thapsigargin/melatonin or tunicamycin/ melatonin.
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Synthesis of insulin by melatonin involves HuD in INS-1E cells
Fig. 3. Phosphorylations of Akt (Ser473) and Akt (Thr308) proteins under the condition of ER stress and/or melatonin in rat insulinoma INS-1E cells. INS-1E cells were incubated in RPMI 1640 medium supplemented with 2% FBS with/without melatonin and/or thapsigargin (1 lM) for 6 hr or tunicamycin (2 lg/mL) for 16 hr at 37°C with 5% CO2. p-Akt expression was then analyzed by Western blot (A). The relative amounts of p-Akt (Ser473) (B) and p-Akt (Thr308) (C) were quantified as described in the Materials and methods section. Data represent mean S.D. of three experiments. **P < 0.01, ***P < 0.001 versus 2% FBS; ###P < 0.001, thapsigargin or tunicamycin versus thapsigargin and melatonin or tunicamycin and melatonin; &P < 0.05, &&&P < 0.001, control/melatonin versus thapsigargin/melatonin or tunicamycin/melatonin.
via mTOR Complex 1 (mTORC1), which is composed of rapamycin-sensitive adaptor protein of mTOR (raptor), G-protein b-subunit-like protein (GbL), and the Akt/ PKB substrate 40 kDa (PRAS40) [27, 28]. The mTORC1 is activated by insulin, growth factors, serum, phosphatidic acid, amino acids (particularly leucine), and oxidative stress [27, 29]. The mTORC1 pathway also senses energy through AMPK [30]. mTOR Complex 2 (mTORC2) is composed of mTOR, rapamycin-insensitive companion of mTOR (rictor), GbL, and mammalian stress-activated protein kinase interacting protein 1 (mSIN1) [31, 32]. mTORC2 phosphorylates the Akt/PKB at serine 473 residue. Phosphorylation of serine stimulates Akt phosphorylation at threonine 308 residue by PDK1 and leads to full Akt activation [33, 34]. It has been reported that mTORC2 is regulated by insulin, growth factors, serum, and nutrient levels [31]. In eukaryotic cells, ER stress triggers a cellular stress response including the unfolded protein response (UPR) associated with cell protection against pathologic conditions [35–37]. Three major signal proteins on the ER membrane are responsible for monitoring ER stress and initiating UPR: IRE1, PERK, and activating transcription factor-6 (ATF6). Under normal conditions, glucoseregulated protein 78 (GRP78)/Bip initiates binding to these three key proteins at the ER membrane to form the inactive complex. However, the activation mechanisms
under these ER stress-related conditions are not fully known, and the precise dissociation between GRP78/Bip and three signal molecules seems to be required for the signal activation. The activation of the UPR results in an increased ER protein folding pathway including GRP78/Bip, a global decrease in protein synthesis, and increased protein degradation. Eventually, the cell might continue to support normal ER homeostasis under ER stress and then regulate cell death such as autophagy or apoptosis [38, 39]. Much evidence shows that ER stress is one of the main causes of pancreatic b-cell dysfunction and death [40–43]. In pancreatic b-cells, ER is the crucial site for insulin biosynthesis, where the protein folding machinery for secretory proteins is localized. Perturbations in ER function of the b-cell caused by high levels of free fatty acid and insulin resistance can lead to an imbalance in protein homeostasis and ER stress, which has been recognized as an important mechanism for type 2 diabetes. Lipson et al. [44, 45] have demonstrated that IRE1a, an ER-resident protein kinase, has a crucial function in insulin biosynthesis. IRE1a functions in the regulation of insulin biosynthesis by IRE1a phosphorylation or the degradation of insulin mRNA in pancreatic b-cells. Dames et al. [46] identified a potential link between the nuclear protein Eny2 (the mammalian ortholog of yeast Sus1 and drosophila E(y)2) and insulin secretion in 209
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Fig. 4. The expressions of nuclear and cellular p85a proteins under the condition of ER stress and/or melatonin in rat insulinoma INS1E cells. INS-1E cells were incubated in RPMI 1640 medium supplemented with 2% FBS with/without melatonin and/or thapsigargin (1 lM) for 6 hr or tunicamycin (2 lg/mL) for 16 hr at 37°C with 5% CO2. Nuclear and cellular p85a proteins were then analyzed by Western blot (A). The relative amounts of nuclear p85a (B) and cellular p85a (C) proteins were quantified as described in the Materials and methods section (B). Data represent mean S.D. of three experiments. **P < 0.01, ***P < 0.001 versus 2% FBS; ###P < 0.001, thapsigargin or tunicamycin versus thapsigargin and melatonin or tunicamycin and melatonin; &P < 0.05, &&&P < 0.001, control/melatonin versus thapsigargin/melatonin or tunicamycin/melatonin.
INS-1E cells. Sirt1 (homolog of the yeast silencing information regulator2) positively regulates insulin secretion [47], and Sirt4 down-regulates insulin secretion by pancreatic b-cells in response to amino acids [48]. A recent study demonstrated binding of RNA-binding protein human antigen D (HuD) to insulin mRNA 5′ untranslated region with subsequent repression of insulin translation in pancreatic b-cells [49]. Peschke and many researchers have reported that melatonin influences insulin secretion [50–58]. However, it is not known how melatonin directly influences intracellular biosynthesis and extracellular secretion of insulin under ER stress via HuD expression in rat insulinoma INS-1E cells.
Materials and methods Cell culture The INS-1E cells, a clonal pancreatic b-cell line received from Prof. Claes B. Wollheim, were cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) containing 11 mM glucose supplemented with 10 mM HEPES (pH 7.3), 10% heat-inactivated fetal bovine serum (FBS; Invitrogen), 50 lM b-mercaptoethanol, 1 mM sodium pyruvate, 50 lg/ mL penicillin, and 100 lg/mL streptomycin at 37°C with 5% CO2 in a humidified incubator. 210
ER stress The INS-1E cells were cultured in RPMI 1640 mediumplus 2% heat-inactivated FBS with/without melatonin and/or thapsigargin (1 lM) (Calbiochem, San Diego, MO, USA) for 6 hr or tunicamycin (2 lg/mL) (Calbiochem) for 16 hr in a 37°C and 5% CO2 incubator. Western blot analysis Cells were harvested, washed twice with ice-cold PBS, and then resuspended in 20 mM Tris-HCl buffer (pH 7.4) containing protease inhibitors (0.1 mM phenylmethylsulfonyl fluoride, 5 lg/mL aprotinin, 5 lg/mL pepstatin A, and 1 lg/mL chymostatin) and phosphatase inhibitors (5 mM Na3VO4 and 5 mM NaF). Whole cell lysate was prepared using 20 strokes of a Dounce homogenizer, followed by centrifugation at 13,000 9 g for 20 min at 4°C. Protein concentration was determined using the BCA assay (Sigma, St Louis, MO, USA). Proteins (40 lg) or media (20 lL) were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) and then transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was incubated with antibodies directed against the following proteins, as indicated: p-AMPK and AMPK (1:500) (Santa Cruz
Synthesis of insulin by melatonin involves HuD in INS-1E cells
Fig. 5. The expressions of nuclear and cellular p85b proteins under the condition of ER stress and/or melatonin in rat insulinoma INS1E cells. INS-1E cells were incubated in RPMI 1640 medium supplemented with 2% FBS with/without melatonin and/or thapsigargin (1 lM) for 6 hr or tunicamycin (2 lg/mL) for 16 hr at 37°C with 5% CO2. Nuclear and cellular p85b proteins were then analyzed by Western blot (A). The relative amounts of nuclear p85b (B) and cellular p85b (C) proteins were quantified as described in the Materials and methods section (B). Data represent mean S.D. of three experiments. *P < 0.05, **P < 0.01, ***P < 0.001 versus 2% FBS; ##P < 0.01, ### P < 0.001, thapsigargin or tunicamycin versus thapsigargin and melatonin or tunicamycin and melatonin; &P < 0.05, &&&P < 0.001, control/melatonin versus thapsigargin/melatonin or tunicamycin/melatonin.
Biotechnology, Santa Cruz, CA, USA); IRS1 (1:500) (Santa Cruz Biotechnology); IRS2 (1:1000 dilution) (Cell Signaling Technology, Beverly, MA, USA); p-Akt (T308, S473) and Akt (1:1000 dilution) (Cell Signaling Technology); p-mTOR (Ser2448, 2481) and mTOR (1:1000 dilution) (Cell Signaling Technology); raptor and rictor (1:1000 dilution) (Cell Signaling Technology); IRE1 (1:1000 dilution) (Cell Signaling Technology); p-PERK (1:1000 dilution) (Cell Signaling Technology); GRP78/ Bip (1:1000 dilution) (Cell Signaling Technology); HuD (1:500) (Santa Cruz Biotechnology); insulin (1:500) (Santa Cruz Biotechnology); and GAPDH (1:1000) (Assay Designs, Ann Arbor, MI, USA). The membrane was incubated with anti-rabbit- and anti-mouse IgG–conjugated horseradish peroxidase secondary antibodies (Santa Cruz Biotechnology) and then with ECL Western blotting reagents (Pierce Biotechnology, Rockford, IL, USA). Immunoreactive proteins were visualized by exposure to X-ray film. Protein bands were visualized by image scanning, and optical density was measured using ImageJ analysis software (version 1.37; Wayne Rasband, NIH, Bethesda, MD, USA), after the data were corrected by background subtraction and normalized by including GAPDH as an internal control.
Isolation of cytoplasmic and nuclear proteins Cytoplasmic and nuclear extracts were separated using cytoplasmic extraction and NE-PER nuclear reagents (Pierce Biotechnology) according to manufacturer’s instructions. Cells were harvested using trypsin-EDTA and subsequently washed with chilled PBS. Cytosolic proteins were first extracted with cytoplasmic extraction reagent, followed by centrifugation at 16,000 9 g for 5 min at 4°C. Intact nuclei were washed by cold PBS and then lysed with high salt NE-PER buffer, followed by centrifugation at 16,000 9 g for 10 min at 4°C. The protein was calculated according to the BCA assay (Sigma) and analyzed by Western blot analysis. Statistical analysis Significant differences were detected by ANOVA, followed by Tukey’s test for multiple comparisons. Analysis was performed using the Prism Graph Pad v4.0 (Graph Pad Software Inc., San Diego, CA, USA). Values are expressed as means S.D. of at least three separated experiments, in which case a representative experiment is depicted in the figures. P-values
