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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

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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

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