From Molecule to Men: Inhibition of Intestinal Glucose Absorption by Polyphenols and Plant Extracts for Reducing the Glycemic Response
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TECHNISCHE UNIVERSITÄT MÜNCHEN Lehrstuhl für Ernährungsphysiologie From molecule to men: Inhibition of intestinal glucose absorption by polyphenols and plant extracts for reducing the glycemic response Christine Schulze Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigten Dissertation. Vorsitzender: Univ.-Prof. Dr. D. Haller Prüfer der Dissertation: 1. Univ.-Prof. Dr. H. Daniel 2. Univ.-Prof. Dr. J.J. Hauner Die Dissertation wurde am 18.02.2014 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 24.06.2014 angenommen. „ So eine Arbeit wird eigentlich nie fertig, man muss sie für fertig erklären, wenn man nach Zeit und Umständen das Mögliche getan hat.“ Johann Wolfgang von Goethe Table of Content Table of Content 1. Introduction .................................................................................................................. 1 1.1 Background and general introduction ...................................................................... 1 1.2 Intestinal glucose absorption with focus on SGLT1 ................................................. 5 1.2.1 The classical model of glucose absorption ........................................................ 5 1.2.2 Proposed alternative routes for glucose absorption ........................................... 7 1.2.3 Structure of SGLT1 ........................................................................................... 8 1.2.4 Regulation of SGLT1 and its role in postprandial hyperglycemia ....................... 9 1.3 Dietary polyphenols ................................................................................................11 1.3.1 Classification, structures and dietary sources ..................................................11 1.3.2 Bioavailability and metabolism of dietary polyphenols ......................................16 1.3.3 Polyphenols and diabetes mellitus ...................................................................18 2. Aim of the thesis ........................................................................................................ 20 3. Methods and Material ................................................................................................ 21 3.1 Methods .................................................................................................................21 3.1.1 Synthesis and analysis of the plant extracts .....................................................21 3.1.2 In vitro screening using the Xenopus laevis oocyte expression system ............21 3.1.2.1 Isolation and preparation of Xenopus laevis oocytes .................................21 3.1.2.2 cRNA microinjection in oocytes .................................................................22 3.1.2.3 Electrophysiological recordings using two-electrode voltage-clamp (TEVC) ..................................................................................................................22 3.1.2.4 Uptake of radiolabelled substrates into oocytes expressing GLUT2 ..........24 3.1.3 Screening using isolated murine small intestinal segments ..............................25 3.1.3.1 Uptake studies performed in everted gut rings ...........................................25 3.1.3.2 Uptake studies performed in everted gut sacs ...........................................26 3.1.4 In vivo screening for antihyperglycemic effects of plant extracts in mice ..........27 3.1.4.1 Experimental animals and feeding trial ......................................................27 3.1.4.2 Oral glucose tolerance test ........................................................................27 3.1.5 Antihyperglycemic effects of plant extracts in humans .....................................28 3.1.5.1 Subjects ....................................................................................................28 3.1.5.2 Study design..............................................................................................29 3.1.5.3 Blood and urine sampling ..........................................................................31 3.1.5.4 Clinical parameters in plasma and urine ....................................................31 Table of Content 3.1.6 Transport kinetics .............................................................................................32 3.1.6.1 Calculation of Imax/ Vmax, Km and IC50 value ................................................32 3.1.7 Statistical Analysis ...........................................................................................33 3.2 Material ..................................................................................................................35 3.2.1 Plant material and natural compound library ....................................................35 3.2.1.1 Plant material ............................................................................................35 3.2.1.2 Amount of plant extracts administered in the human intervention trials ......37 3.2.1.3 Natural compound library...........................................................................38 3.2.2 Buffer and solutions .........................................................................................40 3.2.3 Chemicals and reagents ..................................................................................41 3.2.4 Consumables for the human study ...................................................................42 3.2.5 Technical equipment and software ...................................................................42 4. Results ...................................................................................................................... 43 4.1 Electrophysiological experiments (TEVC) in oocytes expressing hSGLT1 ..............43 4.1.1 Concentration-dependent transport of α-MDG .................................................43 4.1.2 Transport of individual plant extracts via hSGLT1 ............................................44 4.1.3 Inhibition of hSGLT1 by plant extracts, calculation of IC50 and demonstration of reversibility of inhibition .........................................................................................45 4.1.4 Inhibition of hSGLT1 by polyphenolic compounds identified in curly kale-, onion-, grapevine- and apple extracts .......................................................................47 4.2 Uptake experiments in oocytes overexpressing hGLUT2 .......................................51 4.2.1 Studies on specifity of different glucose analogues ..........................................51 4.2.2 Inhibition of hGLUT2 by the plant extracts........................................................52 4.2.3 Inhibition of hGLUT2 by plant extract-derived polyphenols ...............................52 4.3 Uptake experiments using isolated intestinal segments from mice .........................55 4.3.1 Time and concentration dependent uptake of α-MDG into everted gut rings ....55 4.3.2 Inhibition of α-MDG uptake in everted gut rings by plant extracts .....................57 4.3.3 Inhibition of α-MDG uptake in everted gut rings by phlorizin, ε-viniferin and Q4’glc .......................................................................................................................59 4.3.4 Reversibility of SGLT1 inhibition in everted intestinal sacs ...............................60 4.3.4.1 Proof of concept ........................................................................................60 4.3.4.2 Reversibility of SGLT1 inhibition by apple extract, phlorizin and ε-viniferin 61 4.4 Antihyperglycemic effects of selected plant extracts in vivo in mice ........................63 4.4.1 Body weight and 6-h fasting blood glucose in mice fed a control or high fat diet............................................................................................................................63 4.4.2 Effects of apple and onion extracts on postprandial glycemic responses in mice fed a control diet ...............................................................................................63 Table of Content 4.4.3 Effects of apple and onion extract on postprandial glycemic responses in mice fed a high fat diet ..............................................................................................65 4.5 Antihyperglycemic effects of plant extracts in vivo in humans .................................66 4.5.1 Effects of plant extracts on glucose and insulin response in healthy young men (OGTT study) ....................................................................................................66 4.5.2 Effects of plant extracts on renal glucose excretion in healthy young men (OGTT study) ............................................................................................................69 4.5.3 Effects of plant extracts and plant extract mixtures on glucose and insulin responses in obese volunteers (white bread study) ..................................................70 5. Discussion ................................................................................................................. 74 5.1 Influence of polyphenolic plant extracts on SGLT1 and GLUT2 activity in vitro .......74 5.1.1 Transport of dietary polyphenols via SGLT1 ....................................................74 5.1.2 Inhibition