an-in-ovo-model-for-testing-insulin-mimetic-compounds

An <em>In Ovo</em> Model for Testing Insulin-mimetic Compounds

Elevated blood glucose levels in type 2 diabetes mellitus (T2DM), a complex and multifactorial metabolic disease, are caused by insulin resistance and ...

University of Applied Sciences Upper Austria

The compositional characteristics of untreated pure juice prepared from 88 apple varieties grown in the region of Eferding/Upper Austria were determined. Many of the analyzed varieties are noncommercial, old varieties not present in the market. The aim of the study was to quantitate the mineral, phosphate, trace elements, and polyphenolic content in order to identify varieties that are of particular interest for a wider distribution. Great variations among the investigated varieties could be found. This holds especially true for the total polyphenolic content (TPC) ranging from 103.2 to 2,275.6 mg/L. A clear dependence of the antioxidant capacity on the TPC levels was detected. Bioinformatics was employed to find specific interrelationships, such as Mg²⁺/Mn²⁺ and PO₄³⁻/K⁺, between the analyzed bio- and phytochemical parameters. Furthermore, special attention was drawn on putative effects of grafting on the phytochemical composition of apple varieties. By grafting 27 different apple varieties on two trees grown close to each other, it could be shown that the apple fruits remain their characteristic phytochemical composition. Finally, apple juice prepared from selected varieties was further characterized by additional biochemical analysis including cytotoxicity, epidermal growth factor receptor (EGFR) inhibition, and α-amylase activity tests. Cytotoxicity and inhibition of EGFR activation were found to be dependent on the TPC, while α-amylase activity was reduced by the apple juices independent of the presence of polyphenolic substances. Taken together selected apple varieties investigated within this study might serve as preferable sources for the development of apple-based food with a strong focus on health beneficial effects.

Bioanalytical characterization of apple juice from 88 grafted and nongrafted apple varieties grown in Upper Austria.

The identification of the epidermal growth factor receptor (EGFR) as an oncogene has led to the development of several anticancer therapeutics directed against this receptor tyrosine kinase. However, drug resistance and low efficacy remain a severe challenge, and have led to a demand for novel systems for an efficient identification and characterization of new substances. Here we report on a technique which combines micro-patterned surfaces and total internal reflection fluorescence (TIRF) microscopy (μ-patterning assay) for the quantitative analysis of EGFR activity. It does not simply measure the phosphorylation of the receptor, but instead quantifies the interaction of the key signal transmitting protein Grb2 (growth factor receptor-bound protein 2) with the EGFR in a live cell context. It was possible to demonstrate an EGF dependent recruitment of Grb2 to the EGFR, which was significantly inhibited in the presence of clinically tested EGFR inhibitors, including small tyrosine kinase inhibitors and monoclonal antibodies targeting the EGF binding site. Importantly, in addition to its potential use as a screening tool, our experimental setup offers the possibility to provide insight into the molecular mechanisms of bait-prey interaction. Recruitment of the EGFR together with Grb2 to clathrin coated pits (CCPs) was found to be a key feature in our assay. Application of bleaching experiments enabled calculation of the Grb2 exchange rate, which significantly changed upon stimulation or the presence of EGFR activity inhibiting drugs. 

Quantification and kinetic analysis of Grb2-EGFR interaction on micro-patterned surfaces for the characterization of EGFR-modulating substances.

Insulin stimulates the transport of glucose in target tissues by triggering the translocation of glucose transporter 4 (GLUT4) to the plasma membrane. Resistance to insulin, the major abnormality in type 2 diabetes, results in a decreased GLUT4 translocation efficiency. Thus, special attention is being paid to search for compounds that are able to enhance this translocation process in the absence of insulin.

Identification of novel insulin mimetic drugs by quantitative total internal reflection fluorescence (TIRF) microscopy.

Insulin receptor substrates (IRS) are phosphorylated by activated insulin/insulin-like growth factor I receptor tyrosine kinases, with this comprising an initial key event for downstream signaling and bioactivities. Despite the structural similarities, increasing evidence shows that IRS family proteins have nonredundant functions. Although the specificity of insulin/insulin-like growth factor signaling and biological responses partly reflects which IRS proteins are dominantly phosphorylated by the receptors, the precise properties of the respective IRS interaction with the receptors remain elusive. In the present study, we utilized a technique that combines micropatterned surfaces and total internal reflection fluorescence microscopy for the quantitative analysis of the interaction between IRS proteins and insulin/insulin-like growth factor in living cells. Our experimental set-up enabled the measurement of equilibrium associations and interaction dynamics of these molecules with high specificity. We revealed that several domains of IRS including pleckstrin homology and phosphotyrosine binding domains critically determine the turnover rate of the receptors. Furthermore, we found significant differences among IRS proteins in the strength and kinetic stability of the interaction with the receptors, suggesting that these interaction properties could account for the diverse functions of IRS. In addition, our analyses using fluorescent recovery after photobleaching revealed that kinases such as c-Jun N-terminal kinase and IκB kinase β, which phosphorylate serine/threonine residues of IRS and contribute to insulin resistance, altered the interaction kinetics of IRS with insulin receptor. Collectively, our experimental set-up is a valuable system for quantitifying the physiological interaction of IRS with the receptors in insulin/insulin-like growth factor signaling. 

Analysis of insulin receptor substrate signaling dynamics on microstructured surfaces.

Polyphenols are chemical compounds of the secondary plant metabolism. High concentrations can be found in various fruits including apples, berries and grapes. Polyphenols are associated with numerous health beneficial effects including a reduced risk for cardiovascular disease or diabetes. The human body cannot synthesize or store polyphenols and relies on continuous replenishment by daily diet. Unfortunately, knowledge on absorption, metabolization and excretion is still limited. The aim of this study was to determine the pharmacokinetic fate of apple polyphenols in young healthy adults.

Differences in pharmacokinetics of apple polyphenols after standardized oral consumption of unprocessed apple juice.

Induction of GLUT4 translocation in the absence of insulin is considered a key concept to decrease elevated blood glucose levels in diabetics. Due to the lack of pharmaceuticals that specifically increase the uptake of glucose from the blood circuit, application of natural compounds might be an alternative strategy. However, the effects and mechanisms of action remain unknown for many of those substances. For this study we investigated extracts prepared from seven different plants, which have been reported to exhibit anti-diabetic effects, for their GLUT4 translocation inducing properties. Quantitation of GLUT4 translocation was determined by total internal reflection fluorescence (TIRF) microscopy in insulin sensitive CHO-K1 cells and adipocytes. Two extracts prepared from purslane (Portulaca oleracea) and tindora (Coccinia grandis) were found to induce GLUT4 translocation, accompanied by an increase of intracellular glucose concentrations. Our results indicate that the PI3K pathway is mainly responsible for the respective translocation process. Atomic force microscopy was used to prove complete plasma membrane insertion. Furthermore, this approach suggested a compound mediated distribution of GLUT4 molecules in the plasma membrane similar to insulin stimulated conditions. Utilizing a fluorescent actin marker, TIRF measurements indicated an impact of purslane and tindora on actin remodeling as observed in insulin treated cells. Finally, in-ovo experiments suggested a significant reduction of blood glucose levels under tindora and purslane treated conditions in a living organism. In conclusion, this study confirms the anti-diabetic properties of tindora and purslane, which stimulate GLUT4 translocation in an insulin-like manner. 

Biomolecular Characterization of Putative Antidiabetic Herbal Extracts.

Receptor-PI3K-mTORC1 signaling and fatty acid synthase (FASN)-regulated lipid biosynthesis harbor numerous drug targets and are molecularly connected. We hypothesize that unraveling the mechanisms of pathway cross-talk will be useful for designing novel co-targeting strategies for ovarian cancer (OC). The impact of receptor-PI3K-mTORC1 onto FASN is already well-characterized. However, reverse actions-from FASN towards receptor-PI3K-mTORC1-are still elusive. We show that FASN-blockade impairs receptor-PI3K-mTORC1 signaling at multiple levels. Thin-layer chromatography and MALDI-MS/MS reveals that FASN-inhibitors (C75, G28UCM) augment polyunsaturated fatty acids and diminish signaling lipids diacylglycerol (DAG) and phosphatidylinositol 3,4,5-trisphosphate (PIP3) in OC cells (SKOV3, OVCAR-3, A2780, HOC-7). Western blotting and micropatterning demonstrate that FASN-blockers impair phosphorylation/expression of EGF-receptor/ERBB/HER and decrease GRB2-EGF-receptor recruitment leading to PI3K-AKT suppression. FASN-inhibitors activate stress response-genes HIF-1α-REDD1 (RTP801/DIG2/DDIT4) and AMPKα causing mTORC1- and S6-repression. We conclude that FASN-inhibitor-mediated blockade of receptor-PI3K-mTORC1 occurs due to a number of distinct but cooperating processes. Moreover, decrease of PI3K-mTORC1 abolishes cross-repression of MEK-ERK causing ERK activation. Consequently, the MEK-inhibitor selumetinib/AZD6244, in contrast to the PI3K/mTOR-inhibitor dactolisib/NVP-BEZ235, increases growth inhibition when given together with a FASN-blocker. We are the first to provide deep insight on how FASN-inhibition blocks ERBB-PI3K-mTORC1 activity at multiple molecular levels. Moreover, our data encourage therapeutic approaches using FASN-antagonists together with MEK-ERK-inhibitors.

Multi-level suppression of receptor-PI3K-mTORC1 by fatty acid synthase inhibitors is crucial for their efficacy against ovarian cancer cells.

Characterization, especially quantification, of protein interactions in live cells is usually not an easy endeavor. Here, we describe a straightforward method to identify and quantify the interaction of a membrane protein ("bait") and a fluorescently labeled interaction partner ("prey") (membrane-bound or cytosolic) in live cells using Total Internal Reflection Fluorescence microscopy. The bait protein is immobilized within patterns in the plasma membrane (e.g., via an antibody); the bait-prey interaction strength can be quantified by determining the prey bulk fluorescence intensity with respect to the bait patterns. This method is particularly suitable also for the analysis of weak, transient interactions that are not easily accessible with other methods.

Protein Micropatterning Assay: Quantitative Analysis of Protein-Protein Interactions.

Insulin-like growth factor-I receptor (IGF-IR) preferentially regulates the long-term IGF activities including growth and metabolism. Kinetics of ligand-dependent IGF-IR endocytosis determines how IGF induces such downstream signaling outputs. Here, we find that the insulin receptor substrate (IRS)-1 modulates how long ligand-activated IGF-IR remains at the cell surface before undergoing endocytosis in mammalian cells. IRS-1 interacts with the clathrin adaptor complex AP2. IRS-1, but not an AP2-binding-deficient mutant, delays AP2-mediated IGF-IR endocytosis after the ligand stimulation. Mechanistically, IRS-1 inhibits the recruitment of IGF-IR into clathrin-coated structures; for this reason, IGF-IR avoids rapid endocytosis and prolongs its activity on the cell surface. Accelerating IGF-IR endocytosis via IRS-1 depletion induces the shift from sustained to transient Akt activation and augments FoxO-mediated transcription. Our study establishes a new role for IRS-1 as an endocytic regulator of IGF-IR that ensures sustained IGF bioactivity, independent of its classic role as an adaptor in IGF-IR signaling.

Article	Year
Bioanalytical characterization of apple juice from 88 grafted and nongrafted apple varieties grown in Upper Austria. Journal of agricultural and food chemistry\| PubMed ID: 24410208	2014
Quantification and kinetic analysis of Grb2-EGFR interaction on micro-patterned surfaces for the characterization of EGFR-modulating substances. PloS one\| PubMed ID: 24658383	2014
Identification of novel insulin mimetic drugs by quantitative total internal reflection fluorescence (TIRF) microscopy. British journal of pharmacology\| PubMed ID: 25039620	2014
Analysis of insulin receptor substrate signaling dynamics on microstructured surfaces. The FEBS journal\| PubMed ID: 25627174	2015
Differences in pharmacokinetics of apple polyphenols after standardized oral consumption of unprocessed apple juice. Nutrition journal\| PubMed ID: 25890155	2015
Biomolecular Characterization of Putative Antidiabetic Herbal Extracts. PloS one\| PubMed ID: 26820984	2016
Multi-level suppression of receptor-PI3K-mTORC1 by fatty acid synthase inhibitors is crucial for their efficacy against ovarian cancer cells. Oncotarget\| PubMed ID: 28086243	2017
Protein Micropatterning Assay: Quantitative Analysis of Protein-Protein Interactions. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 28188535	2017
IRS-1 acts as an endocytic regulator of IGF-I receptor to facilitate sustained IGF signaling. eLife\| PubMed ID: 29661273	2018

Peter Lanzerstorfer

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