Part 1：Verbascoside Protects Pancreatic β-Cells Against ER-Stress

Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com

Alessandra Galli 1,+, Paola Marciani 1,+, Algerta Marku 1, Silvia Ghislanzoni 1, Federico Bertuzzi 2, Rafaella Rossi 3, Alessia Di Giancamillo 3 , Michela Castagna 1 and Carla Perego 1,*

Abstract:

Substantial epidemiological evidence indicates that a diet rich in polyphenols protects against developing type 2 diabetes. The phenylethanoid glycoside verbascoside/acteoside, a widespread polyphenolic plant compound, has several biological properties including strong antioxidant, anti-inﬂammatory and neuroprotective activities. The aim of this research was to test the possible efects of verbascoside on pancreatic β-cells, a target never tested before. Mouse and human β-cells were incubated with verbascoside (0.8–16 uM) for up to ﬁve days and a combination of biochemical and imaging techniques were used to assess the β-cell survival and function under normal or endoplasmic reticulum (ER)-stress inducing conditions. We found a dose-dependent protective efect of verbascoside against oxidative stress in clonal and human β-cells. Mechanistic studies revealed that the polyphenol protects β-cells against ER-stress mediated dysfunctions, modulating the activation of the protein kinase RNA-like endoplasmic reticulum kinase (PERK) branch of the unfolded protein response and promoting mitochondrial dynamics. As a result, increased viability, mitochondrial function and insulin content were detected in these cells. These studies provide the evidence that verbascoside boosts the ability of β-cells to cope with ER-stress, an important contributor of β-cell dysfunction and failure in diabetic conditions and support the therapeutic potential of verbascoside in diabetes.

Keywords:

verbascoside; polyphenols; insulin-producing cells; diabetes; UPR; oxidative stress; ER-stress; PERK; anti-inﬂammatory; mitochondria

1. Introduction

Diabetes is a chronic disorder affecting hundreds of million people [1]. Diferent etiology characterizes Type 1 (T1D) and Type 2 diabetes (T2D) both featuring lack of insulin [2]. Insulin regulates plasma glucose concentration stimulating glucose uptake in muscle and fat cells and modulating liver glucose metabolism. Nutrient availability, hormones, and neural inputs regulate pancreatic insulin secretion and maintain blood glucose concentrations within a physiological range [3–5]. Therefore, β-cell dysfunction leads to diabetes characterized by fasting hyperglycemia.

T2D is a progressive condition in which insulin resistance and β-cells misfunctions are linked together and recent evidence rose interest in the critical primary role of β-cells to the hyperglycemic status [6,7]. In non-diabetic obese subjects β-cells compensate insulin resistance with increased secretion of the hormone. However, in some subjects, as their conditions deteriorate, insulinemia drops and glucose level climbs to hyperglycemia [8,9].

Glucose is the most important modulator of β-cell functions. Glucose stimulation afects regulation of genes and expression of proteins involved in many cell functions such as glycolysis, insulin synthesis and secretion [10]. Glucose-induced insulin secretion relies on oxidative metabolism to produce adenosine triphosphate (ATP) and a low level of reactive oxygen species (ROS) is physiologically produced. However, β-cells are not very well equipped with scavenger enzymes and this weakness makes them very susceptible to oxidative stress [11,12]. As oxidative stress climbs β-cells insulin production declines, and β-cells produce cytokines that ignite cell damage through inﬂammation and apoptosis [13]. Interestingly, although the reduced β-cells mass has been previously attributed to cell death, recent studies suggest a major role of cell dediferentiation [14,15]. It has become clear that several stages drive to T2D. Metabolic and oxidative insults cause endoplasmic reticulum (ER) stress which leads to decline of insulin synthesis and secretion, inﬂammation processes follow the release of cytokines and β-cells loss may occur through apoptosis and dediferentiation. This knowledge is fundamental in order to develop appropriate strategies of treatment, since oxidative stress and inﬂammation can be counteracted, and the plasticity of pancreatic cells allows the possibility to revert stem cells to β-cells as proved in mice [16].

Recently, the vulnerability of β-cells to oxidative stress has successfully prompted the use of dietary antioxidants to prevent diabetes [17]. Among the most interesting antioxidant food, olive oil is known for its beneﬁcial properties since the ancient Greeks. Olive oil contains good amount of mono-unsaturated fatty acids (MUFA) and several polyphenols such as tyrosol, hydroxytyrosol, oleuropein, and verbascoside.

Verbascoside, also known as acteoside, is a phenylethanoid glycoside extracted from Olea europea, plants of the Verbascum species, and 23 other plant families [18–20]. It can also be obtained from olive oil by-products (it is enriched in olive-mill wastewater derived from olive fruit processing) or be produced by metabolic engineering and synthetic biology approaches [21].

No data have been reported about verbascoside bioavailability in humans, yet. Studies conducted on mice, SKBR3 and Caco-2 cells suggest that it may be feasible for non-metabolized verbascoside to cross the intestinal barrier, circulate in blood plasma, and exert antioxidant efects [20,22,23]. Unlike most of plant polyphenols, verbascoside mainly acts on cells through the modulation of gene transcription of a variety of enzymes and regulatory factors, with antioxidant and anti-inﬂammatory efects [24–30].

Although many beneﬁcial efects of verbascoside for human health are known, there are no data about its efect on pancreatic β-cells. Since oxidative stress and inﬂammation are at the basis of T2D pathogenesis, we investigated whether verbascoside treatment might improve β-cells viability and

function in ER-stress inducing conditions and we characterized the molecular mechanisms of its action. Our data reveal that verbascoside prevents β-cells oxidative stress and inﬂammation modulating the activation of the unfolded protein response and promoting mitochondrial dynamics, thus resulting in increased β-cell viability and insulin content.

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2. Materials and Methods

2.1. Cells Culture and Materials

Mouse βtc3 cells (kindly provided by Prof. Hanahan—Department of Biochemistry and Biophysics, University of California, San Francisco, CA [31]) were cultured in RPMI 1640 medium (Euroclone S.p.A, ECB900, Pero MI, Italy) supplemented with 10% (v/v) heat-inactivated fetal calf serum (Euroclone S.p.A, ECS0180L, Pero MI, Italy), 1% (v/v) penicillin-streptomycin (EuroClone S.p.A., ECB3001D, Pero MI, Italy) and 1% (v/v) L-glutamine (EuroClone S.p.A., ECB300D, Pero MI, Italy). Human islets of Langerhans were isolated in Milan (Niguarda Ca’ Granda) from cadaveric multiorgan donors according to the procedure described by Ricordi et al. [32]; they were cultured in RPMI culture medium containing 5.5 mmol/L glucose, 10% heat-inactivated fetal bovine serum, 0.7 mM Glutamine, 50 units/mL penicillin and 50 ug/mL streptomycin (EuroClone, S.p.A., Pero MI, Italy). Four diferent islets preparation were used, islets purity was 80 ± 10%. Islet isolation and islet studies were approved by the Ethics Committee of the Niguarda Ca’ Granda hospital in Milan (11.12.2009).βtc3 cells were treated with 0.8, 1.6, and 16 uM verbascoside (Carbosynth, OV08034, Compton, UK), cafeic acid and hydroxytyrosol (kind gift of Prof. Dell’Agli, Department of Pharmacological and Biomolecular Sciences, Universita degli Studi di Milano, Milan, Italy) in complete RPMI medium for 5 days, while human islets with 16 uM verbascoside. Methanol/ethanol treated cells were used as controls. In order to induce oxidative stress, cells were treated with H2O2 (Sigma Aldrich, H1009, St. Louis MO, USA) 500 uM in complete RPMI medium for 20 min before analysis, while treatment with tunicamycin 2 ug/mL (T7765, Sigma Aldrich) for 7 h was performed to induce ER stress.

2.2. Detection of Cell Viability by MTT Test

Cells were plated in 96 multi-well plates, and ﬁve days after verbascoside treatment they were incubated with 0.5 mg/mL MTT (3-(4,5-dimethyltiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Sigma-Aldrich, M5655, St. Louis, MO, USA) for 4 h in humidiﬁed atmosphere containing 5% of CO2 at 37 oC. After incubation, cells were gently resuspended in 100 uL DMSO (Euro-Clone S.p.A., BK12611S, Pero MI, Italy) and the absorbance at 540 nm was detected with a microplate reader (Benchmark, microplate reader, Bio-Rad Laboratories, Hercules CA, USA) [33]. Experiments were performed in triplicate and data were expressed as fold increase over control samples.

2.3. Detection of Cell Death by Flow Cytometry

Five days after incubation with 16 uM verbascoside, the β-cells were detached by 7 min incubation with trypsin/EDTA, collected and centrifuged; the pellet was gently resuspended in phosphate bufer saline low salts. Cells were stained with MuseTM count and viability reagent (Millipore, MCH100102, Burlington MA, USA) following the manufacturer’s protocol and analyzed through ﬂow cytometry. Experiments were performed in triplicate and data were expressed as percentage of dead cells over the total.

2.4. ROS Generation

Intracellular ROS were evaluated with DCFDA (2/,7/-dichlorofuorescein diacetate) (Sigma Aldrich, D6883, St. Louis, MO, USA), a membrane permeable probe that becomes ﬂuorescent when tied to ROS [34]. βtc3 cells were pre-loaded with 15 uM DCFDA in Krebs–Ringer bufer (125 mM NaCl, 5 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25 mM HEPES-NaOH pH 7.4 and 2 mM CaCl2)

supplemented with 11 mM glucose for 1 h at 37 oC. The ROS content was detected for 30 min both in basal and stress conditions with a microplate reader (485/528 nm Ex/Em) (TECAN Inﬁnite⑧ F500, Tecan Group Ltd. Männedorf, Switzerland). Mean values and standard deviations were based on three independent experiments.

2.5. Western Blotting

βtc3 cells were collected and solubilized in RIPA bufer (150 mM NaCl, 50 mM Tris HCl pH 7.6, 1 mM EDTA, 1% TERGITOLw NP40, 0.5% deoxycholate) added with aprotinin (Sigma Aldrich, A4529, St. Louis, MO, USA), PMSF (Sigma Aldrich, 10837091001, St. Louis, MO, USA) and Roche inhibitors (Sigma Aldrich, 5892953001, St. Louis, MO, USA) for 40 min at 4 oC. Protein concentration was determined by Bradford assay [35] by using Bradford Reagent (Sigma Aldrich, B6916, St. Louis, MO, USA), 30 ug of proteins were resolved by 10% SDS-PAGE and transferred onto nitrocellulose membranes (Millipore, Burlington MA, USA). Primary antibodies were applied for 2 h in blocking bufer with 5% non-fat milk or 5% BSA solutions; the following primary antibodies were used: mouse anti-β-actin (Novus International Inc., NB600501, St. Louis, MO, USA), mouse anti-acrolein (Abcam, ab48501,Cambridge, UK), mouse anti-BIP (kind gift of Prof. Borgese Nica, Institute of Neuroscience, CNR, Milan, Italy), rabbit anti-HNE (α-diagnostic International Inc., HNE11S, San Antonio, TX, USA), mouse anti-HSP70 (Enzo Life Sciences Inc., C92F3A-5, Farmingdale, NY, USA), rabbit anti-phospho-IKBα (Ser32) (Cell Signaling Technology Inc., 3033, Danvers, MA, USA), mouse anti-IKBα (Cell Signaling Technology Inc., 4814, Danvers, MA, USA), rabbit anti-phospho-NFKB p65 (Ser 536) (Cell Signaling Technology Inc., 3033, Danvers, MA, USA), rabbit anti-NFkB p65 (Cell Signaling Technology Inc., 8242, Danvers MA, USA), sheep anti-SOD1 (Merck, KGaA, Darmstadt, Germania), rabbit anti-PERK (Cell Signaling Technology Inc., 3192, Danvers, MA, USA), rabbit anti-eIF2α (Cell Signaling Technology Inc., 5324, Danvers, MA, USA) and rabbit anti-P-eIF2α (Ser 51) (Cell Signaling Technology Inc., 3597, Danvers, MA, USA). The secondary antibodies HRP-conjugated (Dako Agilent, Santa Clara, CA, USA) were used at 1:5000 dilution. Proteins were detected by using the ECL detection system (Euro-Clone S.p.A., Pero MI, Italy) by using Odyssey Fc Image system (LI-COR Biotechnology GmbH, Bad Homburg, Germany) and band density was quantiﬁed by Image Studiow Lite software (LI-COR Biosciences, Lincoln, NE, USA) [36]. Experiments were performed in triplicate and data were expressed as fold increase over control samples.

2.6. Mitochondrial Membrane Potential

βtc3 cells and human islets of Langerhans were incubated with 100 nM MitoSpyw Orange CMTMRos (BioLegend, 424803, Campoverde Srl, Milan, Italy) or 100 uM MitoSpyw Green FM (BioLegend, 424805, Campoverde Srl, Milan, Italy) for 30 min at 37 oC; ﬂuorescence intensities were detected with the microplate reader TECAN Inﬁnite⑧ F500 (551/576 nm Ex/Em for MitoSpyw Orange CMTMRos; 490/516 nm Ex/Em for MitoSpyw Green) (TECAN Inﬁnite⑧ F500, Tecan Group Ltd. Männedorf, Switzerland). Cells were incubated with 500 uM H2O2 for 20 min and ﬂuorescence intensity was detected as previously described. Mean values and standard deviations were based on three diferent experiments.

2.7. Mitochondrial Morphology and Dynamics

βtc3 cells were pre-loaded with 100 nM MitoSpyw Orange CMTMRos (BioLegend, 424803, Campoverde Srl, Milan, Italy) in 11 mM glucose Krebs–Ringer bufer at 37 oC for 30 min. Samples were positioned in an imaging chamber and random ﬁelds were imaged by using the rhodan ﬁlter of the Axio Observer Z1 microscope (Zeiss, Oberkochen Germany). To evaluate mitochondrial morphology, the following parameters were analyzed by using the ImageJ particle analyzer software: area (um2), circularity (4mArea2/Perimeter2) and Feret’s maximum diameter (um) [37].

For time-lapse experiments, single-cell imaging was carried out at 1 frame per second for 30 s under control or oxidative stress conditions. To measure the mitochondrial cumulative distance (um2), images were ﬁrst corrected for photo bleaching, then videos were analyzed by using an existing Image-Pro Plus Plug-in (object tracking) (Media Cybernetics, Rockville, MD, USA). Up to twelve

cells were imaged in three independent experiments and data were presented as mean values and standard deviations.

2.8. Insulin Secretion

Human isolated islets of Langerhans were seeded in 96-well plate at a density of 20 islets per well and, after 5 days of treatment, insulin content and secretion were measured in basal (3.3 mM glucose) and stimulated (16.7 mM glucose) conditions by means of an ELISA immunoassay (Mercodia, 10-1113-01, Uppsala, Sweden).

2.9. Statistical Analyses

All statistical analyses were performed with GraphPad Prism 8.0 on independent biological replicates. Means between two groups were evaluated by using the two-tailed Student’s t-test and a p-value < 0.05 was taken as evidence of statistical signiﬁcance. Means among three or more groups were compared by analysis of variance (ANOVA), followed by multiple post-hoc (Tukey’s) comparison test. The statistical test used, exact P values and the number of replica (n) are indicated in the individual ﬁgure legends. Error bars in the ﬁgures display the mean ± S.D. or the mean ± S.E., as indicated.

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3. Results

3.1. Verbascoside Improves β-Cells Viability

Since there were no data on verbascoside efect on β-cells, we performed MTT test to evaluate cell viability and as shown in Figure 1A, a positive dose-dependent trend was observed. Hence, for further studies, we selected the 16 uM concentration that statistically was proven efective to enhance β-cells viability.By ﬂow cytometry we studied the impact of ﬁve days incubation of verbascoside on β-cell survival in basal conditions and after 20 min of pre-treatment with 500 uM H2O2. In the basal condition, verbascoside did not afect cell survival, suggesting that the increased β-cell viability observed in the MTT assay may be due to improved mitochondrial activity. Whereas, after H2O2 exposure, 16 uM verbascoside pre-treatment signiﬁcantly reduced oxidative stress-induced β-cell death (Figure 1B,C).Verbascoside is a complex molecule that can be modiﬁed by hydrolyzing enzymes [18]. To test whether verbascoside metabolites could be responsible for the observed protective efect, βtc3 cells were treated with hydroxytyrosol and cafeic acids (0.8 and 16 uM) for 5 days. Both metabolites did not enhance cell viability, on the contrary a cytotoxic efect, more relevant after H2O2 exposure, was detected for the 16 uM concentration (Figure S1). This ﬁnding proves that verbascoside and not its metabolites exerts the protective efect to H2O2 treatment.

3.2. Verbascoside Modulates the Redox Homeostasis and Exerts an Anti-Inﬂammatory Efect in β-Cells

We ﬁrst conﬁrmed in βtc3 cells the anti-inﬂammatory and anti-oxidant efects of verbascoside observed in other cell types [24,29,34]. The verbascoside ROS scavenging activity was evaluated with βtc3 cells labelled with the ROS speciﬁc DCFDA (2/,7/-dichlorofuorescein diacetate) cellular permeable probe. As shown in Figure 2A, a signiﬁcant decrease of ROS content was detected after verbascoside pre-treatment, both under basal and oxidative stress conditions.

Western blot analyses revealed a signiﬁcant decrease of oxidative stress markers acrolein and 4-hydroxynonenal (HNE) expression in verbascoside-treated cells. In addition, we found an increase of superoxide dismutase (SOD1) expression, suggesting that verbascoside exerts its antioxidant activity probably with two diferent mechanisms, directly as a ROS scavenger and indirectly by inducing the expression of antioxidant enzymes (Figure 2B,C).

The anti-inﬂammatory efect of verbascoside on β-cells was assessed by evaluating the activation of the NFKB pathway, the most important pro-inﬂammatory pathway in these cells [38]. By western blot analysis we found a reduced expression of inhibitor of nuclear factor kappa B (IKBα) and nuclear factor kappa-light-chain-enhancer (NFKB), and a signiﬁcant decrease of NFKB phosphorylation in pre-treated cells, backing the hypothesis of the verbascoside efectiveness to reduce cellular inﬂammation (Figure 2D,E).

3.3. Verbascoside Modulates the Unfolded Protein Response of β-Cells

We then analyzed in depth the molecular mechanism by which verbascoside exerts antioxidant and anti-inﬂammatory roles, focusing on the endoplasmic reticulum which is emerging as a key sensor of metabolic and stress signals in β-cells. Under stress conditions, the organelle mounts a homeostatic response, known as the unfolded protein response (UPR), aimed at recovering the ER function; however, the excessive activation of this pathway results in apoptosis [39,40].

Markers of increased ER stress are the upregulation of chaperones proteins and activation of the UPR response. Western blotting analysis of cells treated with verbascoside revealed decreased levels of the two chaperonins heat shock protein 70 (HSP70) and binding immunoglobulin protein (BIP) (Figure 3A,B). Furthermore, a signiﬁcant reduction of protein kinase RNA-like ER kinase (PERK) expression and decreased phosphorylation of its downstream efector, the eukaryotic translation initiation factor 2 (eIF2α), were detected.

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