pharmaceuticals Article A High-Content Screen for the Identification of Plant Extracts with Insulin Secretion-Modulating Activity Roland Hager 1 , Johannes Pitsch 1,2 , Jakob Kerbl-Knapp 1, Cathrina Neuhauser 1, Nicole Ollinger 2 , Marcus Iken 3, Josef Ranner 4, Verena Mittermeier-Kleßinger 4 , Corinna Dawid 4 , Peter Lanzerstorfer 1,* and Julian Weghuber 1,2,* 1 School of Engineering, University of Applied Sciences Upper Austria, 4600 Wels, Austria; [email protected] (R.H.); [email protected] (J.P.); [email protected] (J.K.-K.); [email protected] (C.N.) 2 FFoQSI—Austrian Competence Center for Feed and Food Quality, 3430 Tulln, Austria; [email protected] 3 PM International AG, 5445 Schengen, Luxembourg; [email protected] 4 Food Chemistry and Molecular Sensory Science, Technical University of Munich, 85354 Freising, Germany; [email protected] (J.R.); [email protected] (V.M.-K.); [email protected] (C.D.) * Correspondence: [email protected] (P.L.); [email protected] (J.W.); Tel.: +43-050-804-44402 (P.L.); +43-050-804-44403 (J.W.) Abstract: Bioactive plant compounds and extracts are of special interest for the development of pharmaceuticals. Here, we describe the screening of more than 1100 aqueous plant extracts and synthetic reference compounds for their ability to stimulate or inhibit insulin secretion. To quantify Citation: Hager, R.; Pitsch, J.; insulin secretion in living MIN6 β cells, an insulin–Gaussia luciferase (Ins-GLuc) biosensor was used. Kerbl-Knapp, J.; Neuhauser, C.; Positive hits included extracts from Quillaja saponaria, Anagallis arvensis, Sapindus mukorossi, Gleditsia Ollinger, N.; Iken, M.; Ranner, J.; sinensis and Albizia julibrissin, which were identified as insulin secretion stimulators, whereas extracts Mittermeier-Kleßinger, V.; Dawid, C.; Acacia catechu Myrtus communis Actaea spicata Vaccinium vitis-idaea Calendula officinalis Lanzerstorfer, P.; et al. A of , , L., and were High-Content Screen for the found to exhibit insulin secretion inhibitory properties. Gas chromatography-mass spectrometry Identification of Plant Extracts with (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) were used to characterize several Insulin Secretion-Modulating Activity. bioactive compounds in the selected plant extracts, and these bioactives were retested for their insulin- Pharmaceuticals 2021, 14, 809. https:// modulating properties. Overall, we identified several plant extracts and some of their bioactive doi.org/10.3390/ph14080809 compounds that may be used to manipulate pancreatic insulin secretion. Academic Editors: Dejan Stojkovi´c Keywords: insulin; luciferase; natural plant extracts; bioactives; diabetes; β cells; screening; GC-MS; and Marina Sokovic LC-MS; Western blotting; natural compounds Received: 20 May 2021 Accepted: 10 August 2021 Published: 17 August 2021 1. Introduction Publisher’s Note: MDPI stays neutral Metabolic diseases are global health problems that are rapidly increasing worldwide. with regard to jurisdictional claims in In this regard, energy metabolism represents a key player that is controlled by insulin published maps and institutional affil- secretion from pancreatic β cells. Glucose-stimulated insulin secretion (GSIS) in these iations. cells is controlled by various factors [1]. When the ambient blood glucose concentration increases, glucose is transported by selective transporters into β cells. Elevated glucose levels induce intracellular energy and metabolic processes with a subsequent increase in the ATP/ADP ratio followed by the closure of ATP-triggered potassium (KATP) channels. Due to the inhibition of K channels, the exit of potassium from cells is blocked, resulting in Copyright: © 2021 by the authors. ATP 2+ Licensee MDPI, Basel, Switzerland. membrane depolarization. Voltage-dependent Ca channels (VDCCs) are thus activated, 2+ 2+ This article is an open access article allowing Ca influx; this increase in cytosolic Ca concentration then initiates GSIS. This distributed under the terms and triggering pathway is followed by a time-dependent increase in insulin secretion [2,3]. conditions of the Creative Commons A proposed simplified network of insulin exocytosis from pancreatic β cells is shown Attribution (CC BY) license (https:// in Figure1A. The intracellular network for the regulation of GSIS is very complex and creativecommons.org/licenses/by/ multifactorial. A large number of factors, including mediators of the autonomous nervous 4.0/). system, hormones and nutrients, must be considered [4,5]. Pharmaceuticals 2021, 14, 809. https://doi.org/10.3390/ph14080809 https://www.mdpi.com/journal/pharmaceuticals Pharmaceuticals 2021, 14, 809 2 of 23 Pharmaceuticals 2021, 14, 809 2 of 23 FigureFigure 1. Schematic 1. Schematic overview overview of the of GSIS the pathwayGSIS pathway from pancreatic from pancreaticβ cells that β cells produce that andproduce secrete and secrete insulininsulin in response in response to changes to changes in ambient in ambient blood glucoseblood glucose concentrations. concentrations. Glucose entersGlucose the enters cell via the cell via thethe glucose glucose transporter transporter GLUT2 GLUT2 and is and metabolized is metabolized to pyruvate to pyruvate and ATP. Theand generatedATP. The ATP generated binds to ATP binds to and closes ATP-dependent potassium channels (KATP channels). Due to channel closure, potas- and closes ATP-dependent potassium channels (KATP channels). Due to channel closure, potassium exitsium is blocked, exit is resultingblocked, inresulting depolarization in depolarization of the cell membrane. of the cell membrane. Voltage-gated Voltage calcium-gated channels calcium chan- arenels thus ar triggered,e thus triggered, and an influx and of an calcium influx occurs.of calcium The elevatedoccurs. cytoplasmicThe elevated calcium cytoplasmic concentration calcium concen- triggerstration the triggers release of the insulin release and of C-peptide insulin inand equimolar C-peptide amounts in equimolar (A). Insulin amounts secretion (A depending). Insulin secretion depending on different glucose concentrations in MIN6 β cells and in response to 35 mM KCl. Fold on different glucose concentrations in MIN6 β cells and in response to 35 mM KCl. Fold changes changes in the secreted luciferase activity expressing Ins-GLuc normalized to the activity of 0 mM in the secreted luciferase activity expressing Ins-GLuc normalized to the activity of 0 mM glucose glucose and expressed as fold change ± SEM. Data are the average of at least three independent and expressed as fold change ± SEM. Data are the average of at least three independent experiments experiments with a minimum of 17 replicates in total (B). Schematic overview of the insulin secre- with a minimum of 17 replicates in total (B). Schematic overview of the insulin secretion stimulation tion stimulation and suppression assay (C). MIN6 β cells were cultured in flasks or dishes, tryp- and suppression assay (C). MIN6 β cells were cultured in flasks or dishes, trypsinized, counted sinized, counted and diluted in cell culture media (1). Cells (200 µL) were aliquoted into wells of a and diluted in cell culture media (1). Cells (200 µL) were aliquoted into wells of a 96-well plate 96-well plate and cultured before washing and starving in KRPH buffer and incubation with plant and cultured before washing and starving in KRPH buffer and incubation with plant extracts (2). extracts (2). Fifty microliters of supernatant were removed, pipetted into a white 96-well plate and Fiftymixed microliters with working of supernatant solution were (3). removed, Luminescence pipetted was into measured a white 96-well immediately plate and after mixed pipetting with (4). To workingtest the solution suppression (3). Luminescence of insulin secretion was measured of the immediatelyplant extracts, after 10 pipetting mM glucose (4). Towas test added the (5) after suppressionincubation of insulinwith different secretion plant of the extracts plant extracts, (2). Assay 10 mM preparation glucose was and added measurements (5) after incubation (6, 7) were per- withformed different as described plant extracts previously (2). Assay (3, preparation4). and measurements (6, 7) were performed as described previously (3, 4). Currently,Currently, there there are numerous are numerous antidiabetic antidiabetic agents agents available available for the treatmentfor the treatment of dia- of dia- betesbetes mellitus mellitus (DM), (DM), which which target target different different receptors receptors [6]. The [6] most. The important most important classes of classes of antidiabeticantidiabetic oral oral medicines medicines include include biguanides, biguanides, such assuch metformin, as metformin, sulfonylureas, sulfonylureas, megli- megliti- tinide,nide, thiazolidinedione, thiazolidinedione, dipeptidyl dipeptidyl peptidase peptidase 4 inhibitors, 4 inhibitors, sodium sodium glucose cotransporterglucose cotransporter (SGLT2)(SGLT2) inhibitors inhibitors and αand-glucosidase α-glucosidase inhibitors inhibitors [7,8]. Sulfonylureas [7,8]. Sulfonylureas increase increase insulin se- insulin se- cretioncretion by blockingby blocking KATP KATPchannels channels and and therefore therefore lower lower blood blood glucose glucose levels. levels. They areThey are di- dividedvided into into first-generation first-generation agents, agents, such such as tolbutamide, as tolbutamide, chlorpropamide, chlorpropamide, acetohexamide, acetohexamide,