Stability and Bioavailability of Lentztrehaloses A, B, and C as

Sep 5, 2016 - Shun-ichi Wada, Ryuichi Sawa, Shun-ichi Ohba, Chigusa Hayashi, ... Manabu Kawada , Sonoko Atsumi , Shun-ichi Wada , Shuichi Sakamoto...
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The stability and bioavailability of lentztrehaloses A, B, and C as replacements for trehalose Shun-ichi Wada, Ryuichi Sawa, Shun-ichi Ohba, Chigusa Hayashi, Maya Umekita, Yuko Shibuya, Kiyoko Iijima, Fumiki Iwanami, and Masayuki Igarashi J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02782 • Publication Date (Web): 05 Sep 2016 Downloaded from http://pubs.acs.org on September 6, 2016

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Journal of Agricultural and Food Chemistry

The stability stability and bioavailability of lentztrehaloses lentztrehaloses A, B, and C as replacements for trehalose

Shun-ichi Wada*, Ryuichi Sawa, Shun-ichi Ohba, Chigusa Hayashi, Maya Umekita, Yuko Shibuya, Kiyoko Iijima, Fumiki Iwanami, and Masayuki Igarashi

Institute

of

Microbial

Chemistry

(BIKAKEN),

3-14-23,

Kamiosaki,

Shinagawa-ku, Tokyo 141-0021, Japan

* Corresponding author, (Tel.: +81-3-3441-4173; Fax: +81-3-3441-7589: E-mail: [email protected]);

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ABSTRACT

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Trehalose is widely used as a sweetener, humectant, and stabilizer, but is

3

ubiquitously degraded by the enzyme trehalase expressed in a broad variety

4

of organisms. The stability of the new trehalose analogs lentztrehaloses A,

5

B, and C in microbial and mammalian cell cultures and their

6

pharmacokinetics in mice were analyzed to evaluate their potential as

7

successors of trehalose. Among the 12 species of microbes and two cancer

8

cell lines tested, seven digested trehalose whereas no definitive digestion of

9

the lentztrehaloses was observed in any of them. When orally administered

10

to mice (0.5 g/kg), trehalose was not clearly detected in the blood and urine,

11

and only slightly detected in feces. However, lentztrehaloses were detected

12

in blood at > 1 µg/mL over several hours and were eventually excreted in

13

feces and urine. These results indicate that lentztrehaloses may potentially

14

replace trehalose as non-perishable materials and drug candidates with

15

better bioavailabilities.

16 17

Keywords: Keywords lentztrehalose, trehalose, trehalase, stability, bioavailability,

18

pharmacokinetics

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INTRODUCTION

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Trehalose is a nonreducing disaccharide formed from two molecules of

22

glucose linked by an α,α-1,1-glucoside bond. It is found in many

23

microorganisms, plants, and animals.1-3 Trehalose highly retains water, can

24

function as a chemical chaperone, and induces autophagy as a result of its

25

inhibition of glucose transporters.4-6 Since its mass production began about

26

20 years ago, trehalose has been industrially used as a sweetener,

27

humectant, and stabilizer.1,2,7 Trehalose shows therapeutic effects in mouse

28

disease models of osteoporosis,8 cancer,9 neurodegenerative diseases,10-14

29

and hepatic steatosis6 and reverses arterial aging in humans.15 Thus, it is

30

also promising as a drug candidate. However, trehalose is efficiently

31

hydrolyzed by the enzyme trehalase widely expressed in many species.16,17

32

When used industrially as a humectant, stabilizer or sweetener, trehalose

33

may contribute to product decomposition because of its digestion by bacteria

34

and fungi in the environment. It is also a concern that trehalose is

35

immediately digested by humans18 and thus, it suffers from low

36

bioavailability when used as a drug treatment. About 2–4% of trehalose in

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water was successively administered to mice in neurodegenerative disease

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experiments to observe the desired effects.10-14 The degradation product of

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trehalose, glucose, may also induce or exacerbate obesity, diabetes mellitus,

40

and vascular disorders. Identification of trehalose analogs stable to enzyme

41

hydrolysis and with similar properties as trehalose would help to overcome

42

these limitations.

43

Recently, we identified a new group of trehalose analogs, lentztrehaloses A, and

C

(Figure

1)

from

an

actinomycete

B,

45

ML457-mF8.19-21 Lentztrehaloses are enzyme-resistant analogs of trehalose

46

only minimally hydrolyzed by porcine kidney trehalase.19,20 Trehalose was

47

hydrolyzed at a rate of 8.5 µM/s by one unit trehalase while lentztrehaloses

48

A, B, and C were hydrolyzed at 0.02, 0.04, and 0.05 µM/s, respectively, in

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our previous experiment.20 Likely as a result of its improved bioavailability,

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the amount of lentztrehalose A required to show comparable or higher

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activities in antitumor, bone reinforcement, and anti-obesity studies in mice

52

was one quarter to one half of that of trehalose.19 Lentztrehaloses A, B, and

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C showed comparable sweetness in a sensory test and share various other

54

properties with trehalose including the induction of autophagy.19,20 As more

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strain

Lentzea sp.

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stable analogs of trehalose, lentztrehaloses would be useful in many

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applications.

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There is the possibility that unlike porcine kidney trehalase, trehalase

58

from

other

organisms

or

non-trehalase

enzymes

may

degrade

59

lentztrehaloses. Therefore, in this study, we examined the stability of

60

lentztrehaloses in cultures of various microbes found in the environment

61

and the human intestine. We also examined the pharmacokinetics of

62

lentztrehaloses in mouse to establish their bioavailabilities.

63 64

MATERIALS AND METHODS

65 66

Chemicals

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Lentztrehaloses A, B, and C were isolated from an actinomycete strain

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Lentzea sp. ML457-mF8 as previously described.19,20 The purities of

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lentztrehaloses A, B, and C measured by quantitative NMR were

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97.08±0.61, 90.79±0.36, and 94.72±0.57%, respectively (mean±s.d., n=3).

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Other chemicals were purchased from Sigma-Aldrich (St. Louis, MO) or

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Wako Pure Chemical Industries (Osaka, Japan) unless specified otherwise.

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Microbes and human cultured cells

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Bacteroides fragilis JCM11019 and Enterococcus faecalis JCM5803 were

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obtained from the Japan Collection of Microorganisms (Tsukuba, Japan).

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Bacillus subtilis 168 and Mycobacterium smegmatis ATCC 607 were

78

purchased from the American Type Culture Collection (Manassas, VA).

79

Micrococcus luteus IFO3333 and Candida albicans 3147 were obtained from

80

the Institute of Fermentation (Osaka, Japan). Other microbial strains were

81

from the in-house collection of the Institute of Microbial Chemistry. The

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human melanoma cell line Mewo and the ovarian cancer cell line OVK18

83

were obtained from the Japanese Collection of Research Bioresources

84

(Ibaraki, Japan) and RIKEN BioResource Center (Tsukuba, Japan) cell

85

bank, respectively. The culture density, time, and media are shown in

86

Supplementary Table 1 and the cultures initially contained 500 µg each of

87

trehalose, lentztrehaloses A, B, and C. For the preparation of the sample,

88

the same volume of EtOH was added to the culture followed by

89

centrifugation at 21,000 × g for 5 min. A total of 1.5 µL 20% glycerol was

90

added to the supernatant (150 µL) as an internal standard to enable the

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detection of the remaining amounts of trehalose and lentztrehaloses.

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Mouse experiments

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The mouse experiments were conducted in accordance with a code of

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practice established by the ethics committee of the Microbial Chemistry

96

Research Foundation (Numazu, Japan). ICR mice (4 weeks old, female)

97

were purchased from Charles River Laboratories Japan, Inc. (Yokohama,

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Japan) and kept for 5–6 weeks in an aseptic room at 23 °C. The mice were

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fasted overnight and then orally dosed with trehalose, lentztrehaloses A, B,

100

or C at 0.5 mg/10 µL saline/g body weight (n=5), which was a comparable

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amount to that a mouse ingested from a 2% solution in one hour. The urine

102

and feces were collected before (0 h) and at 2, 4, 8, 24, and 48 h after the

103

administration. The feces were dissolved in water at 200 mg/mL and

104

centrifuged at 21,000 ×g for 5 min. The urine and supernatants of the fecal

105

solution were diluted with MeOH at 1/1000 and used for the liquid

106

chromatography–mass spectrometry (LC-MS) detection. The blood samples

107

of 10–20 µL were collected from the caudal vein before (0) and at 0.5, 1, 2, 4,

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8, 24, and 48 h after administration, and added to 200 µL MeOH. After

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vortexing and centrifugation at 21,000 ×g for 5 min, the supernatant was

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diluted to 1/5 with MeOH and used for the LC-MS detection. Melezitose

111

(500 ng/mL) was added to the samples from the pharmacokinetics study as

112

an internal standard.

113 114

Statistical analysis was performed using Student's t-test. A value of p < 0.05 was considered statistically significant.

115 116

Detection of lentztrehaloses

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Trehalose and lentztrehaloses in microbial and human cancer cell culture

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extracts prepared as above were separated by HPLC (Alliance 2695, Waters,

119

Milford, MA) using a hydrophilic interaction chromatography column

120

(HILIC, XBridge Amide, Waters) and a linear gradient of 90–50%

121

acetonitrile. For cases where lentztrehaloses B and C overlapped with other

122

components in certain media, a 90% or 80% isocratic acetonitrile solvent

123

system was used to perform the separation (Supplementary Table 1).

124

Trehalose and lentztrehaloses were detected using an evaporative light

125

scattering detector (ELSD) system (ELSD 2000ES, Alltech, Deerfield, IL).

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The samples for the pharmacokinetic study were separated by HPLC

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(Accela, Thermo Fisher Scientific, Waltham, MA, USA) using a HILIC

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column (ACQUITY UPLC Ethylene Bridged Hybrid (BEH) Amide 1.7 µm,

129

2.1 × 50 mm, Waters) at 40 °C. Acetonitrile-water was used as the solvent

130

and the acetonitrile concentrations were as follows; 0–2.47 min: 90–70%,

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2.47–3.27 min: 70%, 3.27–3.36 min: 70–50% 3.36–3.96 min: 50%. The flow

132

rate was 0.5 mL/min. The positive MS of the lentztrehaloses and trehalose

133

were acquired using a LTQ Orbitrap XL (Thermo Fisher Scientific).

134 135

Blood glucose level

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Because glucose is separated into two broad peaks under our HPLC

137

conditions, it is difficult to measure the precise amount using the

138

ELSD-HPLC

139

lentztrehaloses. Therefore, glucose was measured by a hexokinase

140

assay.18,19 The MeOH extracted blood samples described above were

141

dispensed into the wells of a 96-well microplate at 5 µL/well and evaporated

142

in vacuo. The sample was dissolved with 25 µL distilled water and 75 µL of

143

glucose assay reagent was added (Sigma-Aldrich). After 1 h incubation at

144

37 °C, the absorbance at 340 nm was measured using a Cytation 5 (BioTek

and

LC-MS systems

employed

for

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detection of

Journal of Agricultural and Food Chemistry

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Instruments, Inc. Winooski, VT).

146 147 148

RESULTS AND DISCUSSION

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Herein, we describe a method to separate trehalose and lentztrehaloses A,

151

B, and C by HPLC with detection using an ELSD system (Figure 2 a). The

152

HILIC column XBridge Amide was found to be the most suitable as it

153

provided better separation than the other columns tested including an

154

octadecyl silica column and a polyamine column. Because trehalose and

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lentztrehaloses A and C do not absorb UV light, ELSD was employed for the

156

detection. Digestion or assimilation of lentztrehaloses by various microbes

157

was also evaluated using this system. A representative result for E. coli is

158

shown in Figure 2. The ELSD-HPLC pattern of the original culture medium

159

is shown in Figure 2 b. After culturing E. coli for 18 h, the HPLC pattern

160

slightly changed as a result of the consumption and production of some

161

components (Figure 2 c). When trehalose and the lentztrehaloses were

162

added to the original medium (Figure 2 d), the HPLC pattern was a

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combination of peaks resulting from all the sugar components (Figure 2 a

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and b). After culturing E. coli in the medium containing the sugars, the

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trehalose peak disappeared (Figure 2 e arrow), but the lentztrehalose peaks

166

did not noticeably change. This result indicates that E. coli can digest or

167

assimilate trehalose but not the lentztrehaloses.

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We selected 12 microbes including Gram-negative and Gram-positive

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bacteria, a mycobacterium and a fungi residing in the environment and

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human intestine, respectively, and two human cancer cell lines to examine

171

whether the organisms can digest lentztrehaloses (Figure 3). While seven of

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the microbes digested trehalose (Figure 3 a–g), P. aeruginosa (Figure 3 h), M.

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luteus (Figure 3 i), B. fragilis (Figure 3 j), yeasts (Figure 3 k and l) and

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human cells (Figure 3 m and n) did not digest it. However, lentztrehaloses A,

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B, and C were not noticeably digested by any organism or cell type tested

176

(Figure 3). Each organism was cultured in a medium suitable for growth

177

and some of the media contained high concentrations of certain components

178

(mainly glucose and NaCl) whose HPLC retention times overlapped with

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those of lentztrehaloses B and C. In these cases, the measured retention

180

times tended to vary more although the separation could be improved by

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changing the elution solvent to an isocratic condition of 80%–90%

182

acetonitrile. As a result of the variations in retention times as well as the

183

deterioration of the column and accumulation of dirt on the detector, the

184

quantification of the components using the ELSD-HPLC detection system

185

deviated by up to ± 20%. Therefore, the decrease of lentztrehalose amounts

186

up to 20% in some microbial cultures, as shown in Figure 3, are considered

187

to be within the experimental error of the system as a whole. We further

188

examined the digestibility of lentztrehaloses in the culture of four microbes

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for an extended time period and supplemented with excess amounts of

190

glucose, maltose, or sucrose in the media. No clear digestion of

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lentztrehaloses was observed under these conditions either (Supplementary

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Figures 1 and 2). Thus we concluded that the lentztrehaloses were only

193

minimally hydrolyzed by microbes and human cells.

194

We

next

examined

the

pharmacokinetics

of

trehalose

and

the

195

lentztrehaloses. Because we could not further improve the precision of the

196

ELSD-HPLC detection system, an alternative method was needed to

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enhance the detection sensitivity to minimize sample usage. An LC-MS

198

system using a smaller BEH amide column was chosen to process the

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pharmacokinetic samples. Although we did not achieve better precision

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with this method, the detection sensitivity was more than 1000 fold higher

201

compared with the ELSD-HPLC detection system. Several natural

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disaccharides with the same molecular weight as trehalose including

203

maltose and sucrose have shorter retention times using this column. The

204

tails of their peaks overlapping with the trehalose peak were counted as

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part of trehalose (Figure 4 b and c). The estimated trehalose concentration

206

in the peripheral blood before the administration of trehalose ranged from

207

200 to 400 ng/mL in five mice. After the administration of 0.5 g/kg trehalose,

208

it was not significantly increased with the exception of one mouse where it

209

increased from 100 to 200 ng/mL depending on the time point (Figure 5

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TRH p.o.). However, an increase in the glucose level was observed in the

211

blood 30 min after the administration of trehalose (Figure 6 TRH p.o.).

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Trehalose was not detected in urine and feces (Figure 7). These results

213

indicate that this amount of trehalose was almost completely digested

214

within a short time and the resultant product glucose circulates in the blood.

215

The one sample where 8 µg/mL trehalose was detected in the blood at 8 h as

216

shown in Figure 5 (mouse 2) is likely an outlier and may represent the

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measurement of another disaccharide. The trehalose elution peak is located

218

at 3.00 to 3.01 min (Figure 4 a). In the HPLC samples, two large

219

disaccharide peaks presumed to be maltose appeared at 2.90 and 2.95 min.

220

The tails of these peaks occurring from 3.00 to 3.01 min containing high

221

amounts of disaccharide were counted as trehalose (Figure 4 c).

222

Lentztrehaloses are new compounds with unique molecular weights. The

223

mass signals for the lentztrehaloses were not detected in blood, urine, and

224

feces before the administration (Figures 5 and 7). After the oral

225

administration of the lentztrehaloses, they were detected in the peripheral

226

circulation within 30 min and the highest amount in some mice exceeded 10

227

µg/mL. Approximately 1 µg/mL of the administered lentztrehalose was

228

maintained for 4 to 8 h in all cases (Figure 5). The administration of

229

lentztrehaloses did not clearly increase the blood glucose level although a

230

slight increase was observed at 30 min (Figure 6). The lentztrehaloses were

231

largely excreted in the feces and to a lesser extent in the urine (Figure 7). A

232

small amount of trehalose was tentatively detected in the feces of

233

lentztrehalose A administered mice. Lentztrehalose A, C, and possibly

234

trehalose were detected in the feces and lentztrehalose C was detected in

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the urine of lentztrehalose B administered mice (Figure 7). This suggests

236

that some portion of the lentztrehaloses, especially lentztrehalose B, is

237

modified or digested in the body to form trehalose or other lentztrehaloses.

238

Nevertheless, compared with trehalose, much higher amounts of the

239

lentztrehaloses must be absorbed in their intact forms and circulate in the

240

body for some period of time. In a recent report,6 contrary to our results,

241

trehalose was detected in the serum up to 5 mM (1.7 mg/mL) at 30 min after

242

the oral administration of 3 g/kg trehalose to mice, a six-fold higher dose

243

than that described here. At this higher concentration, trehalase would not

244

be able to digest the trehalose completely and a certain amount of intact

245

trehalose would be absorbed and circulated. Considering our and others’18

246

results, such a high amount of trehalose (180 g for a 60 kg person) would

247

induce an abrupt increase of the blood glucose level and increase the risk of

248

diseases such as diabetes, vascular disorders, and cancer. It is also a

249

concern that repeated intake of trehalose will increase the trehalase

250

expression level and reduce the absorption of the intact molecule. Because

251

lentztrehaloses are much more stable in the body than trehalose, a smaller

252

amount of lentztrehaloses would induce the same or better effect than that

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of trehalose without a significant increase in the blood glucose level.

254

Therefore, lentztrehaloses are promising second generation trehalose

255

analog drug candidates with better bioavailability for the treatment of

256

diseases such as neurodegenerative disorders and hepatic steatosis.

257

In conclusion, lentztrehaloses were only minimally digested by the

258

ubiquitous microbes tested and may prove useful as a material in place of

259

trehalose. As the bioavailabilities of the lentztrehaloses were higher and the

260

increment in the blood glucose levels were lower than those of trehalose,

261

lentztrehaloses may be better candidates for the treatment of diseases

262

where trehalose is currently regarded as being effective.

263 264

AUTHOR INFORMATION

265

Corresponding Author

266

* Institute of Microbial Chemistry (BIKAKEN), 3-14-23, Kamiosaki,

267

Shinagawa-ku, Tokyo 141-0021, Japan. E-mail: [email protected] Tel.:

268

+81-3-3441-4173 Fax: +81-3-3441-7589

269 270

Notes

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The authors declare no competing financial interests.

272 273

ACKNOWLEDGEMENTS

274

We acknowledge the assistance of Ms. Yumiko Kubota, Dr. K Yamazaki. Dr.

275

Y Takahashi and the members of the Biology Division at the Institute of

276

Microbial Chemistry for their support of this study and helpful discussions.

277 278

SUPPORTING INFORMATION

279

Culture conditions of microbes and cancer cells. (Supplementary Table 1)

280

Stability

281

(Supplementary Figure 1)

282

Digestion

283

(Supplementary Figure 2)

284

Captions to the supplementary figures.

285

(PDF)

of

lentztrehaloses

of

trehalose

and

A,

B,

other

and

C

in

microbial

cultures.

sugars

in

microbial

cultures.

286 287

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(14) Sarkar, S.; Chigurupati, S.; Raymick, J.; Mann, D.; Bowyer, J. F.;

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Schmitt, T.; Beger, R. D.; Hanig, J. P.; Schmued, L. C.; Paule, M. G.;

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Neuroprotective effect of the chemical chaperone, trehalose in a chronic

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MPTP-induced Parkinson’s disease mouse model. Neurotoxicology. 2014, 2014 44,

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250–262.

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(15) Kaplon, R. E.; Hill, S. D.; Bispham, N. Z.; Santos-Parker, J. R.; Nowlan,

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M. J.; Snyder, L. L.; Chonchol, M.; LaRocca, T. J.; McQueen, M. B.; Seals, D.

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R. Oral trehalose supplementation improves resistance artery endothelial

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function in healthy middle-aged and older adults. Aging. 2016, 2016 8 (Epub

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ahead of print; http://www.impactaging.com/papers/v8/n6/full/100962.html)

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(16) Jorge, J. A.; Polizeli, M. L.; Thevelein, J. M.; Terenzi, H. F. Trehalases

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and trehalose hydrolysis in fungi. FEMS Microbiol Lett. 1997, 1997 154, 165-171.

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(17) Shukla, E.; Thorat, L. J.; Nath, B. B.; Gaikwad, S. M. Insect trehalase:

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physiological significance and potential applications. Glycobiology. 2015, 2015 25,

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357–367.

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(18) Oku, T.; Nakamura, S. Estimation of intestinal trehalase activity from

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a laxative threshold of trehalose and lactulose on healthy female subjects.

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Eur. J. Clin. Nutr. 2000, 2000 54, 783–788.

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(19) Wada, S.; Ohba S.; Someno T.; Hatano M.; Nomoto A. Structure and

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biological properties of lentztrehalose: a novel trehalose analog. J. Antibiot.

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2014, 2014 67, 319–322.

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(20) Wada S.; Kubota Y.; Sawa R.; Umekita M.; Hatano M.; Ohba S.;

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Hayashi

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lentztrehaloses A, B and C. J Antibiot. 2015, 2015 68, 521–529.

C.;

Igarashi

M.;

Nomoto

A.

Novel

autophagy

inducers

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(21) Zhang M.; Wada S.; Amemiya F.; Watanabe T.; Shibasaki M. Synthesis

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and Determination of Absolute Configuration of Lentztrehalose A. Chem

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Pharm Bull. 2015, 2015 63, 961–966.

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Funding

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This study was supported by the Japan Society for the Promotion of Science

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(15K08013 and 26450107).

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FIGURE CAPTIONS

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Figure 1. Structures of trehalose and lentztrehaloses A, B, and C.

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Figure 2. Evaluation of the digestion of trehalose and lentztrehaloses in

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Escherichia coli. A standard mixture of lentztrehaloses A, B, C, (LTA, B, C)

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and trehalose (TRH) at 500 µg/mL each (a) and ethanol extracts of the

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culture media before (b and d) and after (c and e) culturing E. coli for 18 h

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were separated by HPLC using a HILIC column and detected with an ELSD

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system. Lentztrehaloses A, B, C, and trehalose were added to the media in

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(d) and (e) at 500 µg/mL each and the arrow in (e) indicates the digestion of

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trehalose by E. coli after the culture. Glycerol was added to each sample at

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0.2% just before the measurement as an internal standard.

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Figure 3. Digestion of trehalose and lentztrehaloses in bacteria, fungi, and

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human cell cultures. Lentztrehaloses A, B, C, and trehalose were added to

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the media at 500 µg/mL and the organisms were cultured for 18–72 h. The

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entire culture (bacteria and fungi) and the supernatant (cancer cell lines)

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were analyzed by the ELSD-HPLC system using a HILIC column. Glycerol

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or uridine was added at 0.2% as the internal standard. Organisms tested

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were as follows: (a) Escherichia coli K-12, (b) Serratia marcescens B-0524,

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(c) Enterococcus faecalis JCM5803, (d) Aspergillus niger F16, (e) Salmonella

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enteritidis 1891, (f) Mycobacterium smegmatis ATCC 607, (g) Bacillus

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subtilis 168, (h) Pseudomonas aeruginosa A3, (i) Micrococcus luteus

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IFO3333, (j) Bacteroides fragilis JCM11019, (k) Saccharomyces cerevisiae

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F-7, (l) Candida albicans 3147, (m) Mewo human melanoma cells, (n)

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OVK18 human ovarian cancer cells. Detailed culturing and detection

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conditions are shown in Supplementary Table 1.

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Figure 4. Detection of trehalose by LC-MS. Three representative

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chromatograms of trehalose detection by high resolution mass-based

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quantitation are shown. To minimize quantification of other disaccharides,

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only the portion of the peak centered around the retention time of trehalose

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in the extracted ion chromatograph (m/z 365.1054 ± 5 ppm for the sodium

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adduct of trehalose, the darker area in each peak indicated with arrows)

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was counted as trehalose. (a) The detection pattern of 1 µg/mL trehalose

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standard. The retention time (RT) of trehalose is 3.00–3.01 min. AA

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represents the automatically calculated area of the ion peaks, indicating the

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absolute intensity of the mass signal in the sample. (b) A typical blood

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sample pattern showing the broad peak area of disaccharides before the

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retention time of trehalose. The blood sample was collected from the caudal

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vein of the mouse (no. 1) 30 min after the oral administration of 0.5 g/kg

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trehalose. (c) The sample containing an exceptionally high amount of other

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disaccharides (RT 2.90 and 2.95). The blood sample was collected from the

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mouse (no. 2) 8 h after the administration of trehalose.

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Figure 5. Concentrations of trehalose and lentztrehaloses in the circulating

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blood in the mouse. Fasted ICR mice (9–10 weeks old, female, 27–32 g, n=5)

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were orally treated with trehalose (TRH), lentztrehalose A (LTA), B (LTB),

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or C (LTC) at 0.5 g/kg body weight. The blood samples were collected from

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the caudal vein at 0 (before the administration), 0.5, 1, 2, 4, 8, 24, and 48 h

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after the administration. The concentrations of lentztrehaloses and

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trehalose were analyzed by LC-MS.

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Figure 6. Blood glucose levels from mice administered trehalose or

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lentztrehaloses. The blood samples were collected from the caudal vein at 0

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(before the treatment), 0.5, 1, 2, 4, 24, and 48 h after the oral administration

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of trehalose (TRH), lentztrehalose A (LTA), B (LTB), or C (LTC). The

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peripheral blood glucose concentrations were measured by a hexokinase

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assay. The data shown are the mean±s.d. n=5. * p < 0.05 compared with the

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0 h value.

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Figure 7. Excretion of trehalose and lentztrehaloses in urine and feces. The

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urine and feces were collected before (0 h) and 2, 4, 8, 24, and 48 h after the

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oral administration of trehalose (TRH), lentztrehalose A (LTA), B (LTB), or

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C (LTC). Cumulative amounts excreted in the urine and feces are shown.

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The analogs other than the one administered are also shown when they

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were detected. Mean±s.d., n=3.

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