Inhibition of Human and Rat Sucrase and Maltase Activities To Assess

Sep 15, 2017 - The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b03678. Primer lists a...
0 downloads 6 Views 2MB Size
Subscriber access provided by University of Sussex Library

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

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.

Page 1 of 42

Journal of Agricultural and Food Chemistry

1

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

2

optimisation of the assay using acarbose and polyphenols

3 4

Alison Pyner, Hilda Nyambe-Silavwe, Gary Williamson

5

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

6

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

7

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

8

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

9

Fax: +44(0)113 3432982

10 11

Reprints will not be available from the author.

12

Running header: Human and rat α-glucosidases

13

1 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

14

Abstract

15

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

16

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

17

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

18

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

19

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

20

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

21

chromatographic methods to detect the monosaccharide products. Human sucrase activity

22

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

23

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

24

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

25

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

26

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

27

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

28

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

29

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

30

quantitatively different.

31 32

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

2 ACS Paragon Plus Environment

Page 2 of 42

Page 3 of 42

Journal of Agricultural and Food Chemistry

33

1. Introduction

34

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

35

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

36

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

37

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

38

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

39

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

40

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

41

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

42

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

43

adjuncts.

44 45

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

46

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

47

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

48

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

49

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

50

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

51

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

52

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

53

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

54

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

55

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

56

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

57

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

Journal of Agricultural and Food Chemistry

58

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

59

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

60

that polyphenols can substantially inhibit this enzyme.

61 62

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

63

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

64

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

65

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

66

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

67

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

68

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

69

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

70

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

71

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

72

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

73

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

74

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

75

source of genuine human enzyme.

76 77

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

78

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

79

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

80

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

81

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

4 ACS Paragon Plus Environment

Page 4 of 42

Page 5 of 42

Journal of Agricultural and Food Chemistry

82

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

83

between the human and rat enzymes.

84

5 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

85

2. Materials and Methods

86 87

2.1 Chemicals

88 89

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

90

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

91

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

92

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

93

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

94

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

95

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

96

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

97

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

98

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

99

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

100

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

101 102

2.2 Equipment used for measurement of enzyme activity

103 104

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

105

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

106

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

107

Thermo Fisher Scientific, Waltham, MA, USA.

108 109

2.3 Gene sequencing 6 ACS Paragon Plus Environment

Page 6 of 42

Page 7 of 42

Journal of Agricultural and Food Chemistry

110 111

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

112

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

113

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

114

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

115

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

116

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

117

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

118 119

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

120 121

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

122

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

123

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

124

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

125

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

126

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

127

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

128

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

129

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

130

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

131

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

132

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

133

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

134

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

Journal of Agricultural and Food Chemistry

135

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

136

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

137

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

138

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

139

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

140 141

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

142

cDNA for sucrase isomaltase

143 144

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

145

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

146

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

147

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

148

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

149

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

150

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

151

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

152

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

153

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

154

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

155

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

156

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

157

sequenced as a reference using the same procedure.

158 159

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

Page 8 of 42

Page 9 of 42

Journal of Agricultural and Food Chemistry

160 161

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

162

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

163

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

164

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

165

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

166

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

167

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

168

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

169

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

170

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

171

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

172

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

173

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

174

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

175

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

176

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

177 178

2.7 Glucose detection methods – hexokinase and HPAE-PAD

179 180

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

181

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

182

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

183

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

184

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

Journal of Agricultural and Food Chemistry

185

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

186

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

187

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

188

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

189

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

190

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

191

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

192

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

193

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

194

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

195

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

196

samples without substrate containing inhibitor at the highest concentration used.

197 198

2.8 Effect of polyphenols on hexokinase assay and removal by SPE

199 200

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

201

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

202

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

203

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

204

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

205

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

206

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

207

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

208

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

209

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

Page 10 of 42

Page 11 of 42

Journal of Agricultural and Food Chemistry

210

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

211

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

212

glucose.

213 214

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

215 216

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

217

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

218

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

219

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

220 221

3.0 Data and statistical analysis

222 223

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

224

inhibitor:

225

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

226

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

227

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

228

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

229

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

230

11 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

231

3. Results

232 233

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

234

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

235 236

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

237

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

238

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

239

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

240

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

241

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

242

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

243

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

244

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

245

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

246

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

247

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

248

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

249 250

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

251

substrate concentration dependence and assay time course linearity.

252

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

253

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

254

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

255

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

Page 12 of 42

Page 13 of 42

Journal of Agricultural and Food Chemistry

256

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

257

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

258

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

259

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

260

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

261

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

262

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

263

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

264

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

265

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

266

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

267

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

268

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

269

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

270

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

271

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

272 273

3.4 Effect of papain treatment on enzyme activities

274 275

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

276

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

277

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

278

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

279

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

13 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

280

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

281

inhibition.

282 283

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

284 285

Hexokinase and glucose oxidase are commonly used enzymes for assessing glucose

286

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

287

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

288

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

289

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

290

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

291

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

292

in glucose concentration (p