Inhibition of human and rat sucrase and maltase activities to assess

Sep 15, 2017 - Inhibition of human and rat sucrase and maltase activities to assess anti-glycemic potential: optimisation of the assay using acarbose ...
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Inhibition of human and rat sucrase and maltase activities to assess antiglycemic potential: optimisation of the assay using acarbose and polyphenols Alison Pyner, Hilda Nyambe-Silavwe, and Gary Williamson J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03678 • Publication Date (Web): 15 Sep 2017 Downloaded from http://pubs.acs.org on September 16, 2017

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Agricultural and Food Chemistry

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Inhibition of human and rat sucrase and maltase activities to assess anti-glycemic potential:

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optimisation of the assay using acarbose and polyphenols

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Alison Pyner, Hilda Nyambe-Silavwe, Gary Williamson

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School of Food Science and Nutrition, University of Leeds, UK

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Address correspondence to G Williamson, School of Food Science and Nutrition, University

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of Leeds, Woodhouse Lane, Leeds, Yorkshire LS2 9JT, United Kingdom.

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E-mail [email protected]. Telephone: +44 (0)113 3438380.

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Fax: +44(0)113 3432982

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Reprints will not be available from the author.

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Running header: Human and rat α-glucosidases

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Abstract

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We have optimised the assays used to measure inhibition of rat and human α-glucosidases

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(sucrase and maltase activities), intestinal enzymes which catalyse the final steps of

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carbohydrate digestion. Cell-free extracts from fully differentiated intestinal Caco-2/TC7

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monolayers were shown to be a suitable source of sucrase-isomaltase, with the same

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sequence as human small intestine, and were compared to a rat intestinal extract. The kinetic

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conditions of the assay were optimised, including comparison of enzymatic and

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chromatographic methods to detect the monosaccharide products. Human sucrase activity

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was more susceptible than the rat enzyme to inhibition by acarbose (IC50 (concentration

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required for 50% inhibition) = 2.5 ± 0.5 and 12.3 ± 0.6 µM respectively), by a polyphenol-

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rich green tea extract, and by pure ( ̶ )-epigallocatechin gallate (EGCG) (IC50 = 657 ± 150 and

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950 ± 86 µM respectively). In contrast, the reverse was observed when assessing maltase

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activity (e.g. EGCG: IC50 = 677 ± 241 and 14.0 ± 2.0 µM for human and rat maltase

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respectively). 5-Caffeoylquinic acid did not significantly inhibit maltase and was only a very

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weak inhibitor of sucrase. The data show that for sucrase and maltase activities, inhibition

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patterns of rat and human enzymes are generally qualitatively similar, but can be

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quantitatively different.

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Keywords: α-glucosidase, sucrase, maltase, green tea, EGCG

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1. Introduction

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The prevalence of type 2 diabetes has doubled over the last thirty years and affects an

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estimated one in ten worldwide 1. A meta-analysis of intervention studies suggests that

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reducing the glycaemic impact of the diet can reduce the risk of developing type 2 diabetes 2.

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One current management strategy for diabetes is based on reducing the glycaemic impact

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though inhibition of α-glucosidase enzymes in the gut, and this can be achieved by regular

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consumption of the anti-hyperglycaemic drugs, acarbose, voglibose and miglitol 3. In an

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intervention study where acarbose was taken daily for 3 yr, the risk for developing type 2

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diabetes was reduced by 6% compared to the control 4. However, these drugs exhibit some

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gastro-intestinal side effects, and so natural products are sought as possible alternatives or

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adjuncts.

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Consumption of polyphenols has been associated with decreased incidence of type 2 diabetes

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and cardiovascular disease 5-7, in part owing to the potential for certain polyphenols to

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influence sugar digestion and absorption in the small intestine 8. Carbohydrate digestion can

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be attenuated by inhibition of α-glucosidases, such as sucrase and maltase, and of

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monosaccharide transport across enterocytes 6. Since polyphenols have the potential to act in

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the same way as acarbose, then this could also provide a mechanism for reduction in diabetes

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risk ascribed to polyphenol-rich diets 8. However, there are a number of limitations in the

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assays for assessing α-glucosidase activities which could lead to misleading results and poor

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predictive capacity for subsequent in vivo testing in volunteers. Although human enzymes are

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always the intended target for therapeutic interventions, many studies have used the more

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readily available rat intestinal preparations in vitro. This is coupled with a variety of detection

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methods for the products, typically enzyme-linked spectrophotometric assays based on

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glucose oxidase or hexokinase 9-12, which are susceptible to inhibition themselves by the 3 ACS Paragon Plus Environment

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tested compounds 13, confounding estimation of inhibition of the target α-glucosidase. Even

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though hexokinase was reported to be the method of choice 13, we report data here showing

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that polyphenols can substantially inhibit this enzyme.

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The only intestinal enzyme able to hydrolyse sucrose is the enzyme sucrase. Sucrase

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isomaltase (SI) is a highly N- and O-glycosylated brush border type-II membrane protein

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with various α-glucosidase activities, and is trafficked to the cell surface through association

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with lipid rafts 14. SI consists of two subunits, sucrase and isomaltase, which are cleaved by

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luminal proteases, but remain associated through non-covalent interactions 15. The sucrase

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subunit has both maltase (α-1,4) and sucrase (α-1,2) activities, while the isomaltase subunit

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has maltase (α-1,4) and isomaltase (α-1,6) activities 16. SI is therefore involved in the

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digestion of both sugars and starch. Type 2 diabetic patients have increased levels of SI,

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which potentially increases the rate of sugar digestion and uptake and exacerbates the

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problem, making it an interesting target for inhibition 17. Caco-2 cells express sucrase-

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isomaltose after differentiation, and could be a suitable source of the enzyme for in vitro

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experiments. However, it is conceivable that the enzyme contains mutations in the cell line

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compared to human tissue, which, if present, could invalidate the use of the cell line as a

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source of genuine human enzyme.

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Some studies have used Caco-2 cells as a source of enzyme, where the inhibition of α-

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glucosidases by mulberry leaves was tested 18, but the method did not take into account any

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subsequent transport and metabolism of the product. Other assays have been reported 19, 20 but

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not optimised. Given the potential importance of the assay in evaluating naturally-occurring

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compounds for acarbose-like activity, the aim of this work was to optimise the assay for

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inhibition of human sucrase and maltase activities, and evaluate differences in inhibition

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between the human and rat enzymes.

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2. Materials and Methods

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2.1 Chemicals

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Acarbose, 5-caffeoylquinic acid, sodium potassium monobasic, sodium potassium dibasic,

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glucose, sucrose, maltose, hexokinase glucose assay reagent, Bradford reagent, intestinal

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acetone powder extract from rat, protease inhibitors, epigallocatechin gallate, trypsin EDTA

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solution, L-cysteine, papain from papaya latex, Dulbeccos’s modified Eagle’s Medium, non-

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essential amino acids, penicillin-streptomycin solution, phosphate buffered saline (PBS) and

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fetal bovine serum were purchased from Sigma-Aldrich, Dorset, UK. Glutamax™ was from

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Thermo Fisher Scientific, Waltham, MA, USA. Oasis MAX cartridges were purchased from

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Waters Corporation, Milford, MA, USA. Green tea water-soluble extract was from Nestlé,

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Choladi, India. The Caco2/TC7 cell line was a kind gift by Prof. Monique Rousset, Centre de

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Recherche des Cordeliers, Paris, France. The green tea extract was analysed by HPLC 21 and

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contained 199.8 mg/g (-)-epigallocatechin gallate, 124.4 mg/g (-)-epigallocatechin, 34.4 mg/g

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(-)-epicatechin gallate and 23.3 mg/g (-)-epicatechin (w/w).

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2.2 Equipment used for measurement of enzyme activity

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Absorbance measurements were performed on a Pherastar FS plate reader from BMG

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labtech, Ortenberg, Germany. High performance anion exchange chromatography with

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pulsed amperometric detection (HPAE-PAD) was performed on the ICS4000 system from

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Thermo Fisher Scientific, Waltham, MA, USA.

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2.3 Gene sequencing 6 ACS Paragon Plus Environment

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The sequencing of SI from Caco-2/TC7 cells used the following materials: RNAqueous®

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Total RNA Isolation kit from Thermo Fisher Scientific, Waltham, MA, USA. GoScript™

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Reverse Transcription System was from Promega, Madison, WI, USA. Cloneamp HiFi PCR

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premix and In-fusion cloning kit were from Takara Bio Europe, Saint-Germain-en-Laye,

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France. Primers were ordered from Sigma-Aldrich, Dorset, UK. Zymoclean™ gel extraction

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PCR recovery kit was from Zymo Research, California, USA. Human RNA as a reference

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sample for sucrase sequencing was from DV biologics, California, USA.

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2.4 Growth conditions for Caco-2 cells as a source of human enzyme for in vitro assays

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Caco-2/TC7 cells were routinely cultured at a density of 1.2 x 106 cells per 75 cm2 culture

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flask in standard medium consisting of Dulbeccos’s modified Eagle’s Medium supplemented

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with 20% (v/v) fetal bovine serum, 2% (v/v) Glutamax™, 2% (v/v) non-essential amino

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acids, 1% penicillin-streptomycin solution. Cells were maintained in an incubator at 37°C

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with 10% CO2. Medium was replaced every 2 d and at this seeding density, 80% confluence

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was reached in 3 d at which point the cells were sub-cultured by detaching with 0.25%

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trypsin. Cells were allowed to fully differentiate for 21 d and a crude lysate was prepared.

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The cells were washed three times with cold phosphate buffered saline and cells were scraped

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with 1 mL of assay buffer (10 mM phosphate buffer, pH 7.0) containing protease inhibitors.

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The cell lysates were snap frozen in a dry ice and ethanol bath and stored at -80°C until

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required. On the day of assay, cell lysates were thawed, vortexed and then passed 10-15 times

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though a 21G needle syringe, and this preparation is referred to as a cell-free extract since it

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contains no intact cells. Papain-digested lysates were also prepared to evaluate different

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lysate preparation methods. Fully differentiated cells were washed with warm PBS and then 7 ACS Paragon Plus Environment

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incubated at 37°C for 15 min with 2 mL papain solution containing 1 mg/mL papain and 0.5

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mg/mL L-cysteine 22. After the incubation, cells were scraped and transferred into Eppendorf

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tubes and centrifuged at 210 g for 10 min at 4°C. The supernatant was used for the assay. The

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protein content of the lysate, either crude or papain-digested, was determined by Bradford

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Assay 23 and the lysate was diluted in assay buffer as required.

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2.5 Extraction of mRNA from Caco-2/TC7 cells, conversion to cDNA and sequencing of

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cDNA for sucrase isomaltase

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Total RNA was extracted using the RNAqueous® Total RNA Isolation kit according to the

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manufacturer’s protocol and then 250 ng of total RNA was converted into cDNA by reverse

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transcription using the reverse transcription kit according the manufacturer’s protocol. The

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gene was reverse transcribed in two parts with two sucrase isomaltase specific reverse

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primers A and B, and amplified using primer pairs specific to each segment (see

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supplemental table S1) using Cloneamp HiFi PCR premix. Primers contained overlapping

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ends for future cloning into a vector (underlined and in italics). The PCR products from

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segment A (bases 1-1811) and segment B (1174-5784) were separated on an agarose gel

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extracted using Zymoclean™ gel extraction PCR recovery kit and then cloned together using

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the In-fusion cloning kit. This kit uses a recombinase enzyme, which fuses the overlapping

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homologous sequences together, and resulted in the full SI coding region which was sent for

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DNA Sanger sequencing by Beckman Coulter Genetics using the primers as found in

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supplemental table S2. The RNA from a human intestinal biopsy from DV Biologics was

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sequenced as a reference using the same procedure.

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2.6 Sucrase and maltase assay using a Caco-2 TC7 cell-free extract 8 ACS Paragon Plus Environment

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Assay samples of 250 µL contained sucrose or maltose, with Caco-2/TC7 cell lysate

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preparation as the enzyme source in 0.1 M phosphate buffer pH 7.0. A time course was

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evaluated from 0 to 30 min and assays to determine the specific activity in the lysate were

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performed with a range of enzyme concentrations (total protein from 40 to 320 µg). The

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specific activity was determined as the amount of substrate consumed per minute per mg of

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total protein in the assay. The kinetic parameters Km and Vmax were determined using initial

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rates derived from the selected enzyme concentration and incubation times with substrate

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concentrations ranging from 1-60 mM. One enzyme unit (U) is equivalent to the amount of

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enzyme that catalyses the hydrolysis of 1 µmol of substrate per min. For inhibition assays,

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inhibitor was added to the reaction mixture at the indicated concentrations: Acarbose from 1

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to 50 µM, EGCG from 100 to 1500 µM, GT from 0.5 to 5 mg/mL and 5-caffeoylquinic acid

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from 100 to 500 µM. For inhibitors dissolved in DMSO to ensure solubility (EGCG and 5-

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caffeoylquinic acid), control samples were included to ensure no interference and a maximum

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of 2% of the total reaction volume was DMSO. Assay samples were incubated in a water bath

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at 37°C for 10 min and the reaction was stopped by placing in a boiling water bath for 10

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min. Samples were centrifuged at 17000 g for 10 min.

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2.7 Glucose detection methods – hexokinase and HPAE-PAD

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The glucose produced in the enzyme reaction was quantified enzymatically using hexokinase

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according to the manufacturer’s protocol, or chromatographically with HPAE-PAD, which

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has been reported previously to measure glucose 24, 25. Using HPAE-PAD, glucose was

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chromatographically separated from fructose and sucrose, and could be quantified between 1

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and 50 µM. Before HPAE-PAD analysis, protein in the samples was precipitated by the 9 ACS Paragon Plus Environment

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addition of acetonitrile (1:1 v/v) and samples were vortexed and centrifuged at 17000 g for 10

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min. The resulting supernatant was diluted with deionised water to fall within the calibration

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range, filtered through a 0.2 µM PTFE filter and maintained at 4°C in the autosampler.

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Capillary separation of carbohydrates was done using the capillary CarboPac PA20 column,

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0.4 x 150 mm with CarboPac PA20 Guard, 0.4 x 35 mm with the column at 30°C and

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compartment at 15°C. The detection was performed using a gold working electrode and

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palladium reference electrode with a collection rate of 2.00 Hz using the waveform “Gold,

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Carbo, Quad” waveform. The analysis method starts at 25 mM NaOH eluent for 10 min

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followed by a wash at 100 mM for 15 min and then re-equilibration at 25 mM from 25 to 38

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min. The flow rate was 0.008 mL/min with 0.04 µL injection volume and 20x capillary

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overfill. Inhibitors were evaluated for potential interference by preparing and analysing assay

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samples without substrate containing inhibitor at the highest concentration used.

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2.8 Effect of polyphenols on hexokinase assay and removal by SPE

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To evaluate which polyphenols or polyphenol-rich extracts interfere with the hexokinase

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assay, samples (250 µL) containing 5 mM glucose were prepared in assay buffer with and

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without polyphenols or extracts at their highest tested concentration (see section 2.6 above).

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The assay was performed using hexokinase to measure the amount of glucose, and the

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samples were compared to the control. The independent sample t-test was performed to

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determine if the inhibitors had significant effect on the assay. Any inhibitors which interfered

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significantly were removed by SPE prior to analysis in all subsequent assays as follows:

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Oasis MAX 3 cc cartridges were preconditioned with 1 mL of deionised water and 1 mL of

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methanol then dried under vacuum for 5 min. The sample collection tube was placed under

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the cartridge and 200 µL of test samples and controls were passed through and collected. A 10 ACS Paragon Plus Environment

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fixed volume was then used in the hexokinase assay as indicated above. Removal of any

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interference by SPE was confirmed by evaluating separate polyphenol samples spiked with

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glucose.

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2.9 Sucrase and maltase assay using a rat intestinal acetone-protein extract

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The enzyme assay was also optimised for incubation time, substrate concentration and linear

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range using an enzyme preparation from powdered acetone protein extract from rat

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intestine21. For maltase, 4 mg solid/mL was used with 3 mM maltose and 20 min incubation

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time. For sucrase, 20 mg solid/mL was used with 16 mM sucrose and 20 min incubation time.

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3.0 Data and statistical analysis

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The percent inhibition was determined by the following formula where the control is without

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inhibitor:

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%=([Glucose]Control-[Glucose]inhibitor)/[Glucose]control x 100

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Statistics were performed by one-way analysis of variance using SPSS Statistics version 24

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and statistical significance was determined by Tukey-Kramer multiple comparison test (p

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≤0.05). The data are presented as mean ± SEM with minimum of n = 3. Cell lysates were

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prepared from three biological passages of cells and used for IC50 determinations.

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3. Results

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3.1 Sucrase isomaltase from Caco-2/TC7 cells and from human small intestine has the same

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sequence, showing that the former is a suitable source of enzyme for in vitro assays

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One of the goals was to optimise an assay for assessing inhibition using human differentiated

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Caco-2/TC7 cell extracts as a source of the sucrase-isomaltase. Only the sucrase sequence

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from Caco-2 cells, and not from Caco-2/TC7 cells, has been reported 26, and since even point

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mutations may under some circumstances lead to alteration of enzyme activity 14, it was

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important to establish that the sucrase sequence of the Caco-2/TC7 line was the same as the

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human small intestinal enzyme. Based on sequencing of sucrase from the Caco-2/TC7 clone,

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we can confirm that it has an identical sequence to the human intestine (sequencing results

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from Caco-2/TC7 are shown in Supplemental information S3). The reported Caco-2 cell

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sucrase sequence 26, the reference human biopsy control sample from DV Biologics, and the

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BC132860.1 sequence from the National Institutes of Health Mammalian Gene Collection

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(MGC) Program 27, all reported the same sequence as in the Caco-2/TC7 cells. However, it

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should be noted that there is one base different at position 4632 (GA) compared to one of

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the sequences in the NCBI database (NM_001041.3).

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3.2 Optimisation of human sucrase-isomaltase assay: Determination of kinetic parameters,

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substrate concentration dependence and assay time course linearity.

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A cell-free extract from Caco-2/TC7 cells was evaluated as a source of sucrase and maltase

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for use in assays. A time course was determined using 10 mM sucrose and maltose, and

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maltase exhibited a linear production of glucose up to 15 min (Figure 1A), with sucrase linear

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up to 40 min (Figure 1B). On the basis of this, an assay time of 10 min was selected for both 12 ACS Paragon Plus Environment

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activities. The specific activity of maltase was 725 ± 36 mU/mg (Figure 1C) and for sucrase

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was 130 ± 4 mU/mg (Figure 1D), and was proportional to total protein up to 1.28 and 0.64

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mg/mL respectively. The amount of protein chosen for future assays was selected to ensure a

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linear initial rate: 0.05 and 0.02 U per assay for maltase and sucrase respectively. Using the

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conditions established, the apparent Km was 9.5 mM and Vmax = 0.053 µmol/min for sucrose,

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and apparent Km was 7.5 mM and Vmax = 0.379 µmol/min for maltose (Figure 1E and F). The

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sucrase and maltase apparent Km values measured here were within the reported range for

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human sucrase 19, 20, 28 and for human maltase 20. Optimising the assay is extremely

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important in order to correctly evaluate and identify inhibitors. Using the optimised assay

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conditions, acarbose inhibited maltase activity (Figure 2) with an IC50 value of 5.7 ± 1.4 µM.

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When more enzyme was used (0.2 U/assay), the reaction rate became non-linear and the IC50

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value for acarbose was estimated at 22.1 ± 2.9 µM (Figure 2), which was 4-fold higher.

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Varying the substrate concentration had a less dramatic effect on the measured IC50 value.

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For sucrase, the measured IC50 value for acarbose was unchanged over a 100-fold variation in

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substrate concentration (Figure 3A). For maltose, there was a small effect on the measured

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IC50 value when the substrate concentration was greater than 10x the Km (Figure 3B).

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3.4 Effect of papain treatment on enzyme activities

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Sucrase-isomaltase is attached, via a protein tail, to the small intestine brush border

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membrane. To determine if there could be an advantage in removing it from the cellular

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membrane, we used papain to release the enzyme from the brush border. Although papain

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treatment gave an increase the specific activity of both sucrase and maltase (Figure 4A and

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B), the inhibition observed was unchanged (Figure 4C and D). It was therefore concluded that

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the papain step did not offer a significant advantage to the overall method for determining

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inhibition.

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3.5 Determination of possible interference in the hexokinase-linked estimation of glucose

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Hexokinase and glucose oxidase are commonly used enzymes for assessing glucose

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concentration and both are commercially available in the form of kits. Glucose oxidase is

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highly susceptible to inhibition by polyphenols and other compounds 13, 29, and although

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hexokinase is less susceptible, it is still reported to be inhibited 13. All compounds tested here

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were evaluated here for direct inhibition of hexokinase. Glucose standards at 1 mM were

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prepared in presence and absence of each inhibitor and the concentration of glucose was

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determined using the hexokinase assay. EGCG at 1000 µM led to an apparent 23% increase

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in glucose concentration (p