Part 2：Verbascoside Protects Pancreatic β-Cells Against ER-Stress
Mar 05, 2022
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,*
3.4 Verbascoside Modulates β-Cells Mitochondrial Activity and Dynamics
MTT viability assays suggest a possible impact of verbascoside directly on mitochondria which ensures the coupling of insulin secretion to the nutritional state and the cell survival in this cell type. Shape and membrane potential are markers of mitochondrial health [42]. We first evaluated mitochondrial membrane potential by labeling the cells with MitoSpyw Orange CMTMRos, a permeable dye whose concentration depends on the inner mitochondrial membrane potential, while MitoSpyw Green FM was used to normalize data to the mitochondrial mass (Figure 4A, B). The 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. Cafe 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 H2O2 exposure (Figure S3).
Since the 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 conditions, 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.
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 traveled 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 H2O2 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.
3.5 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 uM 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 H2O2 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 uM 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).
4. 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, which 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 the blood plasma of rats fed with Lippia citriodora extract together with some metabolites [20]. In addition, cell accumulation of verbascoside and is verbascoside 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 uM 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 the intestinal barrier, circulate in blood plasma, and exert antioxidant efects on endocrine β-cells of the pancreas.
We found a dose-dependent (0.8–16 uM) 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 caffein acid and hydroxytyrosol, which actually appear cytotoxic at the 16 uM 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 the 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 IKBα-NFKB inflammatory pathway was also detected in our system in the presence of verbascoside. NFKB 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 NFKB 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 NFKB, 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 regulate the mitochondrial number, distribution, morphology and 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 caffeine 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 afects 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 differs from original cells in terms of metabolism and ROS production. We here provide evidence that verbascoside exerts a protective efect 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 efect on insulin content reflects the compound's 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.
5. 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 a 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 conditions by using natural compounds.
Supplementary Materials: The following are available online at http://www.mdpi.com/2227-9059/8/12/582/s1. Figure S1: Effects of caffein acid and hydroxytyrosol on β-cell viability. Figure S2: Effects of verbascoside on tunicamycin-induced ER stress. Figure S3: Efects of caffein acid and hydroxytyrosol on mitochondrial membrane potential. Figure S4: Quantitative image analyses of mitochondria in cells treated with caffein acid and hydroxytyrosol. Video S1, S2 Videos: Mitochondria dynamics recorded under basal conditions, in the absence (S1) or presence (S2) of verbascoside. Video S3, S4: Mitochondria dynamics recorded under oxidative stress conditions, in the absence (S3) or presence (S4) of verbascoside.
Author Contributions: Conceptualization, A.G., P.M. and C.P.; data curation, A.G. and P.M.; formal analysis, A.G. and P.M.; funding acquisition, C.P.; investigation, M.C.; methodology, A.M. and S.G.; resources, F.B., R.R. and A.D.G.; supervision, C.P.; validation, A.M., S.G. and M.C.; visualization, A.G. and A.M.; writing—original draft, A.G.; writing—review and editing, P.M., A.M., F.B., R.R., A.D.G., M.C. and C.P. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Acknowledgments: We would like to thank Carlo Corino and Mario Dell’Agli for the reagents and helpful discussion.
Conflicts of Interest: The authors declare no conflict of interest.
References
1. Cho, N.H.; Shaw, J.E.; Karuranga, S.; Huang, Y.; da Rocha Fernandes, J.D.; Ohlrogge, A.W.; Malanda, B. IDF Diabetes Atlas: Global estimates of diabetes prevalence for 2017 and projections for 2045. Diabetes Res. Clin. Pract. 2018, 138, 271–281. [CrossRef]
2. Leslie, R.D.; Palmer, J.; Schlott, N.C.; Lernmark, A. Diabetes at the crossroads: Relevance of disease classification to pathophysiology and treatment. Diabetologia 2016, 59, 13–20. [CrossRef] [PubMed]
3. Tokarz, V.L.; MacDonald, P.E.; Klip, A. The cell biology of systemic insulin function. J. Cell Biol. 2018, 217, 2273–2289. [CrossRef] [PubMed]
4. Perego, C.; Da Dalt, L.; Pirillo, A.; Galli, A.; Catapano, A.L.; Norata, G.D. Cholesterol metabolism, pancreatic β-cell function and diabetes. Biochim. et Biophys. Acta (BBA) Mol. Basis Dis. 2019, 1865, 2149–2156. [CrossRef] [PubMed]
5. Galli, A.; Algeria, M.; Marciani, P.; Schulte, C.; Leonardi, C.; Milani, P.; Mafoli, E.; Tedeschi, G.; Perego, C. Shaping Pancreatic β-Cell Differentiation and Functioning: The Influence of Mechanotransduction. Cells 2020, 9, 413. [CrossRef]
6. Ashcroft, F.M.; Rorsman, P. Diabetes Mellitus and the β Cell: The Last Ten Years. Cell 2012, 148, 1160–1171. [CrossRef] [PubMed]
7. Rosengren, A.H.; Braun, M.; Mahdi, T.; Andersson, S.A.; Travers, M.E.; Shigeto, M.; Zhang, E.; Almgren, P.; Ladenvall, C.; Axelsson, A.S.; et al. Reduced Insulin Exocytosis in Human Pancreatic -Cells With Gene Variants Linked to Type 2 Diabetes. Diabetes 2012, 61, 1726–1733. [CrossRef] [PubMed]
8. Polonsky, K.S. Dynamics of insulin secretion in obesity and diabetes. Int. J. Obes. 2000, 24, S29–S31. [CrossRef]
9. Roden, M.; Shulman, G.I. The integrative biology of type 2 diabetes. Nature 2019, 576, 51–60. [CrossRef]
