Verbascoside Protects Pancreatic β-Cells against ER-Stress

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 effects of verbascoside on pancreatic β-cells, a target never tested before. Mouse and human β-cells were incubated with verbascoside (0.8–16 µM) 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 effect 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.


Introduction
Diabetes is a chronic disorder affecting hundreds of million people [1]. Different 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][4][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

Detection of Cell Death by Flow Cytometry
Five days after incubation with 16 µM verbascoside, the β-cells were detached by 7 min incubation with trypsin/EDTA, collected and centrifuged; the pellet was gently resuspended in phosphate buffer saline low salts. Cells were stained with Muse TM 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.
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 (µm 2 ), 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.

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).

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.

Verbascoside Improves β-Cells Viability
Since there were no data on verbascoside effect 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 µM concentration that statistically was proven effective to enhance β-cells viability.

Verbascoside Improves β-Cells Viability
Since there were no data on verbascoside effect 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 µM concentration that statistically was proven effective 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 µM H2O2. In the basal condition, verbascoside did not affect 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 µM 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 effect, βtc3 cells were treated with hydroxytyrosol and caffeic acids (0.8 and 16 µM) for 5 days. Both metabolites did 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 µM H 2 O 2 . In the basal condition, verbascoside did not affect cell survival, suggesting that the increased β-cell viability observed in the MTT assay may be due to improved mitochondrial activity. Whereas, after H 2 O 2 exposure, 16 µM 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 effect, βtc3 cells were treated with hydroxytyrosol and caffeic acids (0.8 and 16 µM) for 5 days. Both metabolites did not enhance cell viability, on the contrary a cytotoxic effect, more relevant after H 2 O 2 exposure, was detected for the 16 µM concentration ( Figure S1). This finding proves that verbascoside and not its metabolites exerts the protective effect to H 2 O 2 treatment.

Verbascoside Modulates the Redox Homeostasis and Exerts an Anti-Inflammatory Effect in β-Cells
We first confirmed in βtc3 cells the anti-inflammatory and anti-oxidant effects 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 different mechanisms, directly as a ROS scavenger and indirectly by inducing the expression of antioxidant enzymes ( Figure 2B,C).
The anti-inflammatory effect of verbascoside on β-cells was assessed by evaluating the activation of the NFκB 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 (IκBα) and nuclear factor kappa-light-chain-enhancer (NFκB), and a significant decrease of NFκB phosphorylation in pre-treated cells, backing the hypothesis of the verbascoside effectiveness to reduce cellular inflammation ( Figure 2D,E).

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 effector, the eukaryotic translation initiation factor 2 (eIF2α), were detected. of superoxide dismutase (SOD1) expression, suggesting that verbascoside exerts its antioxidant activity probably with two different mechanisms, directly as a ROS scavenger and indirectly by inducing the expression of antioxidant enzymes ( Figure 2B,C). The anti-inflammatory effect of verbascoside on β-cells was assessed by evaluating the activation of the NFκB 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 (IκBα) and nuclear factor kappa-light-chain-enhancer (NFκB), and a significant decrease of NFκB phosphorylation in pre-treated cells, backing the hypothesis of the verbascoside effectiveness to reduce cellular inflammation ( Figure 2D,E).

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 effector, the eukaryotic translation initiation factor 2 (eIF2α), were detected. Taken together, these data suggest that verbascoside protects β-cell from dysfunctions associated with ER-stress and acts by modulating the PERK branch of the UPR, a pathway deregulated in diabetes [41]. To confirm this hypothesis, the cells were treated with tunicamycin, an inhibitor of N-linked glycosylation and a potent ER-stress inducer ( Figure 3C). As expected, a sevenhour incubation with 2 µg/mL tunicamycin increased the activation of the PERK pathway ( Figure S2) and significantly reduced β-cell viability ( Figure 3B). Interestingly, verbascoside pre-treatment (16 µM for 72 h) downregulated the expression of P-eIF2α and partially reverted the action of tunicamycin on β-cell viability. Taken together, these data suggest that verbascoside protects β-cell from dysfunctions associated with ER-stress and acts by modulating the PERK branch of the UPR, a pathway deregulated in diabetes [41]. To confirm this hypothesis, the cells were treated with tunicamycin, an inhibitor of N-linked glycosylation and a potent ER-stress inducer ( Figure 3C). As expected, a seven-hour incubation with 2 µg/mL tunicamycin increased the activation of the PERK pathway ( Figure S2) and significantly reduced β-cell viability ( Figure 3B). Interestingly, verbascoside pre-treatment (16 µM for Taken together these data strongly support a role of verbascoside in protecting β-cells by attenuation of ER-stress response.

Verbascoside Modulates β-Cells Mitochondrial Activity and Dynamics
MTT viability assays suggest a possible impact of verbascoside directly on mitochondria which ensure the coupling of insulin secretion to the nutritional state and the cell survival in this cell type. Shape and membrane potential are markers of the mitochondrial health [42]. We first evaluated mitochondrial membrane potential by labelling the cells with MitoSpy™ Orange CMTMRos, a permeable dye whose concentration depends on the inner mitochondrial membrane potential, while MitoSpy™ Green FM was used to normalize data to the mitochondrial mass ( Figure 4A,B). Membrane potential of mitochondria in verbascoside-treated cells was increased compared to control in both basal and stress conditions. Interestingly, verbascoside metabolites exerted different effects. Caffeic acid did not improve the mitochondrial potential, whereas hydroxytyrosol caused a dose-dependent impairment of mitochondrial function, in agreement with data on cell viability, such an effect was more evident after H 2 O 2 exposure ( Figure S3). Taken together these data strongly support a role of verbascoside in protecting β-cells by attenuation of ER-stress response.

Verbascoside Modulates β-Cells Mitochondrial Activity and Dynamics
MTT viability assays suggest a possible impact of verbascoside directly on mitochondria which ensure the coupling of insulin secretion to the nutritional state and the cell survival in this cell type. Shape and membrane potential are markers of the mitochondrial health [42]. We first evaluated mitochondrial membrane potential by labelling the cells with MitoSpy™ Orange CMTMRos, a permeable dye whose concentration depends on the inner mitochondrial membrane potential, while MitoSpy™ Green FM was used to normalize data to the mitochondrial mass ( Figure 4A,B). Membrane potential of mitochondria in verbascoside-treated cells was increased compared to control in both basal and stress conditions. Interestingly, verbascoside metabolites exerted different effects. Caffeic acid did not improve the mitochondrial potential, whereas hydroxytyrosol caused a dosedependent impairment of mitochondrial function, in agreement with data on cell viability, such an effect was more evident after H2O2 exposure ( Figure S3).
Since function is strictly related to morphology, we measured the Feret's diameter, area, and circularity of the mitochondria. Acute oxidative stress promotes an extensive mitochondrial fission, thus resulting in smaller, circular mitochondria [42]. Under H2O2 stress condition, mitochondria of verbascoside-treated cells were more elongated and showed increased surface than control ones ( Figure 4C,D and Figure S4). Mitochondrial circularity was lower in verbascoside-treated cells than in controls and differences were more evident in H2O2 treated cells ( Figure S3), again confirming the protective role of the polyphenol.  Since function is strictly related to morphology, we measured the Feret's diameter, area, and circularity of the mitochondria. Acute oxidative stress promotes an extensive mitochondrial fission, thus resulting in smaller, circular mitochondria [42]. Under H 2 O 2 stress condition, mitochondria of verbascoside-treated cells were more elongated and showed increased surface than control ones ( Figure 4C,D and Figure S4). Mitochondrial circularity was lower in verbascoside-treated cells than in controls and differences were more evident in H 2 O 2 treated cells ( Figure S3), again confirming the protective role of the polyphenol.
The ability of these organelles to modify their shape in response to nutritional states or stressful conditions is under the control of fusion and fission events and requires mitochondrial motility [39]. Tracking of mitochondria movements led us to calculate the cumulative distance travelled and the analysis revealed a significant increase of mitochondria dynamics in the verbascoside treated cells when compared to control, already in basal conditions ( Figure 4E,F and Videos S1-S4). The effect was more pronounced after H 2 O 2 treatment, indeed verbascoside pre-treatment almost completely reverted the impact of oxidative stress on mitochondrial dynamics.
Taken together these data strongly support a key role of verbascoside in ensuring mitochondria dynamics which is crucial to promote the rapid adaptation of this organelle to stress conditions.

Verbascoside Impact on Human of Langerhans Survival and Function
Considering the translational potential of verbascoside application in human health, we verified its impact on human isolated islets of Langerhans, a more relevant model for physiopathological and pharmacological studies on β-cells. Isolated islets were treated with 16 µM verbascoside for 5 days and the mitochondrial membrane potential was evaluated. As reported in Figure 5A, while organelle potential was compromised in control human islets after H 2 O 2 exposure, the inner membrane potential of mitochondria in verbascoside pre-treated islets did not decrease significantly, supporting the hypothesis of a protective role of verbascoside against ROS-induced β-cell dysfunction. Analysis of ER-stress markers expression showed an important reduction of P-eIF2α fraction in the two distinct islet preparations maintained in 16 µM verbascoside, confirming the downregulation of this pathway also in human samples ( Figure 5D). The beneficial effects of the polyphenol treatment were corroborated by the increased insulin content observed in verbascoside-treated islets when compared to controls ( Figure 5B). Data dispersion in our experiments did not allow to demonstrate any significant difference of glucose-stimulated insulin secretion (GSIS) in verbascoside-treated islets, although a positive trend seems apparent under both basal and stress conditions ( Figure 5C). pathway also in human samples ( Figure 5D). The beneficial effects of the polyphenol treatment were corroborated by the increased insulin content observed in verbascoside-treated islets when compared to controls ( Figure 5B). Data dispersion in our experiments did not allow to demonstrate any significant difference of glucose-stimulated insulin secretion (GSIS) in verbascoside-treated islets, although a positive trend seems apparent under both basal and stress conditions ( Figure 5C).

Discussion
Oxidative stress and inflammation are the basis of β-cell dysfunction occurring during T2D [43,44] as such, functional food, nutraceuticals, and phytochemicals have been investigated as tools to prevent type 2 diabetes [45]. In this work, we focused on verbascoside, that is known to exert anti-inflammatory and anti-oxidant activities in neurons [18]. Despite the high structural complexity, verbascoside and isoverbascoside have been found in blood plasma of rats fed with Lippia citriodora extract together with some metabolites [20]. In addition, cell accumulation of verbascoside and isoverbascoside has been detected in a breast cancer cell line SKBR3 [23] and studies conducted on Caco-2 cells demonstrate that moderate amounts of verbascoside and isoverbascoside remain intact and bioaccessible after in vitro digestive process. Furthermore, Caco-2 can uptake both molecules through a rapid and linear transport in 10 to 100 µM range [22]. No data have been reported about verbascoside bioavailability in humans, and yet the above-mentioned studies suggest that it may be feasible for non-metabolized verbascoside to cross intestinal barrier, circulate in blood plasma, and exert antioxidant effects on endocrine β-cells of the pancreas.
We found a dose-dependent (0.8-16 µM) protective role of verbascoside on clonal and human β-cells, both under basal and stress conditions. This effect is due to verbascoside itself and not to its metabolites caffeic acid and hydroxytyrosol, which actually appear cytotoxic at the 16 µM concentration. The concentrations proven active in our experiments are in line with those described in animals fed with verbascoside [20].
Though we do not know whether verbascoside acts extracellularly by binding to a membrane receptor or is internalized by endocytosis and works intracellularly, our data confirm that verbascoside has antioxidant and anti-inflammatory properties, and protects β-cells against ER-stress associated dysfunctions by reducing the UPR and promoting mitochondrial dynamics.
Indeed, we detected a significant decrease of ROS content in verbascoside treated cells which is further confirmed by the reduction of lipid peroxidation measured through HNE and acrolein expressions. Unlike most of plant polyphenols, verbascoside ROS scavenging activity mainly follows indirect (via upregulation of ROS removing enzymes) rather than direct pathways. In fact, it increases the gene transcription of some antioxidant enzymes through the activation of Nrf2 (NF-E2-related factor 2) pathway and via AhR (aryl hydrocarbon receptor)-dependent mechanism [24,29]. As such, we found increased expression of SOD1 in verbascoside treated cells.
ROS homeostasis is extremely relevant in β-cells pathology. Due to the shortage of antioxidant enzymes, elevated production of ROS cannot be neutralized and ER stress promoting protein misfolding is induced. ER stress leads to a reduction of insulin transcription and translation through the activation of the UPR [44,46]. This pathway has the main purpose of recovering the ER function through the reprogramming of gene expression, however when overstimulated, it triggers apoptosis. Our data suggest that an important action of verbascoside is to mitigate the UPR, allowing the modifications necessary to repair ER dysfunction, without causing apoptosis. According to this possibility, the expression of BIP, HSP70 and PERK proteins was decreased in verbascoside treated β-cells, and the activation of PERK pathway was attenuated in response to tunicamycin-induced ER stress. A similar action was reported for tyrosol in the insulinoma NIT-1 cell line [47].
Particularly interesting is the ability of verbascoside to modulate the activation of the PERK branch of the UPR. In β-cells this pathway is required to maintain the basal secretory homeostasis and β-cell survival, and it is severely deregulated in diabetes [41,48]. Even more intriguing, common variants at PERK contribute to the risk of prediabetes and recessive mutations in the EIF2AK3 gene (encoding PERK) underlie susceptibility to the Wolcott-Rallison syndrome characterized by permanent neonatal insulin dependent diabetes [49,50].
Downregulation of the IκBα-NFκB inflammatory pathway was also detected in our system in the presence of verbascoside. NFκB represents the main inflammatory pathway in β-cells and its sustained activation initiates a cascade of events culminating in β-cell death. In stressed β-cells, the reduction of NFκB expression protects pancreatic β-cells from diabetogenic agents [27,51], further supporting the verbascoside application in diabetes prevention. Interestingly, PERK signaling activates the transcription factors NFR2 (implicated in the redox homeostasis) and NFκB, thus explaining the anti-oxidant and anti-inflammatory effects of verbascoside [40,52].
Our data also reveal an important role of verbascoside on mitochondrial activity and dynamics. Marker of mitochondrial operational quality is their dynamic, a process characterized by coordinated cycles of fusion and fission events that regulates mitochondrial number, distribution, morphology and their membrane potential [39]. In line with this possibility, verbascoside improves βtc3 mitochondrial membrane potential both under basal and stress conditions. Again, the effect is due to verbascoside and not to its metabolites, as caffeic acid does not improve mitochondrial function and hydroxytyrosol significantly decreases the organelle potential. Increased mitochondrial dynamics and changes in their morphology were observed by tracking mitochondrial movements, thus suggesting that verbascoside promotes a mito-morphosis program. Reshaping of the cellular mitochondrial network affects the assembly of the respiratory chain super-complexes, thus altering not only the cell metabolism but also its redox state and enabling cells to better counteract oxidative stress and inflammation [5,37,39].
Although the molecular mechanism is still unknown, we can hypothesize that verbascoside, as other polyphenols, protects mitochondrial DNA by reducing ROS concentration [53] and prevents the opening of mitochondrial permeability transition pore [54], ensuring the physiological mitochondrial activity. Another intriguing possibility is that modification of mitochondria dynamics and activity is again mediated by the action of verbascoside on the UPR. Indeed, a recent research in the mitochondrial field indicates that sites of ER-mitochondria interaction play a key role in the control of mitochondrial dynamics and activity in response to oxidative stress and PERK is involved in the phenomenon [55].
Most in vitro studies with polyphenols were performed in cell lines, often of tumoral origin, which differ from original cells in terms of metabolism and ROS production. We here provide evidence that verbascoside exerts a protective effect also on human isolated islets of Langerhans, the final target of intervention in diabetes. Interestingly, the polyphenol mitigates the PERK signaling activation already under basal conditions, thus increasing the insulin content and preventing oxidative stress-mediated mitochondrial dysfunctions. It remains to be elucidated whether the polyphenol effect on insulin content reflects the compound ability to prevent β-cell death, to mitigate ER stress or to directly control the insulin gene expression, as shown for other polyphenolic compounds [56]. These data are particularly relevant considering the possible use of verbascoside as a complementary therapy in diabetes treatment.

Conclusions
The in vitro studies reported here on clonal and human β-cells, indicate that verbascoside exerts protective effects against ER-stress associated dysfunctions, mitigating the activation of the PERK branch of the UPR and improving mitochondria dynamics. As disruption of ER homeostasis triggers β-cells damage and diabetes, these data provide a rationale for the possible use of verbascoside as nutraceutical in disease prevention and treatment. Yet, many issues need to be resolved for an effective clinical application of verbascoside. Despite the wealth of laboratory studies, reliable clinical studies confirming the health effects of verbascoside in vivo are limited. Furthermore, increasing its stability and bioavailability is mandatory for the future application of this compound in human health. From this point, verbascoside is an interesting molecule because its scaffold has different reactive sites that can be modified by combinatory chemistry. We expect that the information resulting from these studies will open avenues for therapeutic modulation of oxidative stress and inflammation in pathological condition by using natural compounds.