Systemic Perturbations of Key Metabolites in Type 2 Diabetic Rats

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Systemic perturbations of key metabolites in Type 2 diabetic rats treated by polyphenols extracts from Litchi chinensis seeds Shuli Man, Jiang Ma, Jingwen Yao, Jingxia Cui, Chunxia Wang, Yu Li, Long Ma, and fuping Lu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02206 • Publication Date (Web): 10 Aug 2017 Downloaded from http://pubs.acs.org on August 11, 2017

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Journal of Agricultural and Food Chemistry

Systemic perturbations of key metabolites in Type 2 diabetic rats treated by polyphenols extracts from Litchi chinensis seeds Shuli Man a, Jiang Ma a, Jingwen Yao a, Jingxia Cui a, Chunxia Wang a, Yu Li a, Long Ma a*, Fuping Lu a* a Tianjin Key Laboratory of Industry Microbiology, Key Laboratory of Industrial Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science & Technology, Tianjin, 300457, China

* Corresponding authors: Tel., 86-22-60601265; E-mail, [email protected] (Ma L), [email protected] (Lu FP).

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Abstract

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Our previous research obtained Litchi chinensis Sonn. seeds extract (LSE) which

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showed hypoglycaemic effects on type 2 diabetes (T2D) rats. In order to understand

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the detailed pathogenesis of diabetes intervened by LSE, the metabonomics strategy

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was used. As a result, LSE decreased insulin resistance index and the levels of glucose

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in urine through elevating the mRNA level of insulin, while decreasing the expression

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of glucagon to enhance the function of pancreas. Meanwhile, LSE regulated the

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glucose and fatty acid metabolisms via increasing the expression of glucose

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transporter (Glu) 2, Glu4, insulin receptor (IR) and IR substrate-2 (IRS2). LSE

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effectively restored the impairment of the IRS2/ PI3K/ Akt/ mTOR insulin signaling

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in the livers. All in all, LSE played a pivotal role in the treatment of T2D through

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regulation of broad-spectrum metabolic changes and inhibition of the glycogenesis,

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proteolysis and lipogenesis in T2D rats.

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Keywords: Litchi seeds; Type 2 diabetes; Metabolomics; Dyslipidemia

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Introduction

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Litchi chinensis Sonn. (Sapindaceae) seeds contain remarkable amount of

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phenolic compounds like protocaechuic acid, protocatechuic aldehyde, procyanidin D,

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(-)-epicatechin, cinnamtannin B1, procyanidin A1, rutin, and phlorizin, and possess

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many bioactive attributes, such as antioxidant 1, anti-inflammation 2, hepatoprotection

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chinensis Sonn. seeds extract (LSE) containing multiple polyphenols, which showed

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hypoglycaemic effects on type 2 diabetes (T2D) rats through improvement of lipid

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metabolism and inhibition of the apoptosis-induced hepatic damage and inflammation

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, anti-obesity

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and hypoglycaemic activity 5. Our previous research obtained Litchi

.

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Diabetes is the most frequent endocrine disease in developed countries

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accompanied with an increasing burden of morbidity and mortality 6. It possesses

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abnormal metabolism of carbohydrates, lipids, and proteins 7. Metabonomics as a

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powerful new approach with the use of advanced analytical apparatus holistic

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evaluate body responses in the metabolic disorders in diabetes 8.

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In this work, we employed a metabonomics strategy to discriminate the global

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profiles of diabetic, LSE-treated diabetic and normal rats. GC-MS analyses of serum

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and urine, and relative metabolic pathway have been revealed and reconstructed the

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metabolic regulatory network due to LSE intervention. The aim of the research was to

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extend the scope of our previous studies and further understand the progression of

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diabetes intervened by LSE on systemic and metabolic levels.

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Materials and Methods section

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Plant materials

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Litchi (L. chinensis Sonn.) seeds were purchased from a local herbal pharmacy in

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Yunnan. The plant was identified by professor Chunxia Wang and deposited in Tianjin

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University of Science and Technology (Tianjin, China). The dried litchi seeds (1 kg)

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were extracted with ethanol/water (95:5, 6:5, 3:7, v/v; 3L, 2 times) for 2 h at 80 ℃.

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After combination of the solutions and concentration, 184 g of litchi seed extract

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(LSE) was collected in 30-75% ethanol range in D101 macroporous resin (Nankai

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Hecheng S&T, Tianjin, China) 5.

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Vanillin-HCl assay

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The content of procyanidins was meansured by Vanillin-HCl assay 9. Briefly, the

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aliquot (100 µL) of each sample (dissolved in methanol, 1 mg/mL) was added to 600

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µL of vanillin methanol solution (4%) and 300 µL of concentrated hydrochloric acid

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orderly. Subsequently, they were placed in the dark at 30 °C ± 1 °C for 20 min.

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Different concentration of standard solution from 0-1.0 mg/mL was made up using

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procyanidin B2. The absorbance of the reaction mixture was set at 500 nm against

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methyl alcohol as a blank. Standard curve equation is y=1.2191x+ 0.0606, R2=0.9979.

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UPLC-Q/TOF-MS analysis

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Based on previous studies 5, 1 mg of LSE dissolved in 1 mL of 50% acetonitrile

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was analyzed by UPLC-Q/TOF-MS. Chromatographic separation was performed at

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30 °C on a Waters Acquity UPLC TM BEH C18 column (100 mm × 2.1 mm i.d., 1.7

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µm; Waters, Massachusetts, USA). The flow rate was 0.4 mL/min. The monitoring 4


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UV wavelength was set at 280 nm.

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Experimental animals

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Sprague-Dawley rats (100~130 g) were purchased from the Laboratory Animal

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Center of academy of Military Medical Sciences (Beijing, China quality certification

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number: SCXK (Jun) 2012-0004). This animal study was approved by the

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Institutional Animal Care and Use Committee of China. After acclimatization for one

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week, eighteen male rats were randomly allocated into 3 groups (n=6), normal control

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group (Normal), untreated diabetes (Model) and diabetes treated with litchi seeds

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extract (LSE). During the experiment, six rats as one group were allocated in a cage.

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In the first 4 weeks, rat in model and LSE groups were given a high fat diet alone

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containing 53.7% basal chow, 20% saccharose, 15% lard, 10% egg yolk powder, 1.1%

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cholesterol and 0.2% sodium cholate, which were purchased from the Experimental

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Center of Aoyide (Tianjin). While the normal group received regular chow only. On

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the beginning of the 5th week, rats fed with high fat diet received streptozotocin (STZ)

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(30 mg/kg, i.p., dissolved in 0.1 M cold citrate buffer, pH 4.5) once or twice to induce

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diabetes. Three days after STZ injection, rats with fasting blood glucose level > 11.1

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mM measured by a glucometer were confirmed diabetic. Then the LSE groups were

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administrated orally 30 mg of LSE in 10 mL of 0.9% sodium chloride per kg of body

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weight every day for six weeks. Model groups and normal groups received

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appropriate vehicles.

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Fasting blood samples from each rat were collected into heparinized tubes by the

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puncture of the retro-orbital sinus at the 10th week. Blood was centrifugated at 3000 5



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rpm for 10 min at 4 °C in order to avoid hemolysis phenomenon. Serum samples were

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separated and kept in aliquots at -20 °C for biochemical assays. The urine samples

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were collected at the 10th week over ice and then centrifuged at 3000 rpm for 10 min

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at 4 °C. The supernatant was stored at -20 °C until measurement. After the end of

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collection, all the rats were killed by neck dislocation. Portions of each tissue like

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liver, kidney and pancreas were fixed in 10% formalin (pH 7.4) at room temperature

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for histology examination, or snap frozen in liquid nitrogen for PCR and western bolt

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assays.

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Biochemical assays

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Aspartate aminotransferase (AST) and Alanine aminotransferase (ALT) were

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determined according to the commercially available kits (Nanjing Jiancheng

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Bioengineering Institute, Nanjing, China). Serum insulin was detected by

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immunoenzymatic ELISA kit (Lufeng, Shanghai, China).

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GC-MS sample collection and preparation for derivatization and GC-MS

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condition

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Referring to previous methods

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, blood and urine samples (100 µL) were

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deproteinated, derivatized and analyzed on an Agilent 7890A gas chromatography

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(Agilent Technologies Co., Ltd., Santa Clara, CA, USA) coupled to an Agilent 5975C

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mass selective detector (Agilent Technologies Co., Ltd.) performed in the electron

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ionization (EI) mode.

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Reverse transcription PCR Total RNA was isolated from rats liver and pancreatic tissues using TRIzol 6


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(Sangon Biotech Co., Shanghai, China) according to previous research 11. Polymerase

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chain reaction products were electrophorized on 3.0% agarose gel and visualized after

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ethidium bromide staining (0.5 µg/mL). ImageJ software was used to quantify the

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intensity of the bands.

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Western blot analysis of Glut 4 and FATP 4 expression in hepatic tissue

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The rat hepatic protein was separated and transferred onto PVDF membrane as

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previous reports 11. The membrane was incubated with Glut 4 (1:5000 diluted, Abcam,

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UK) and FATP 4 (1:2000 diluted, Abcam, UK) primary antibody at 4 °C for the night.

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After being washed, the secondary antibody (combined with horseradish peroxidase)

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(1:10000 diluted, Abcam, UK) was used to detect bound primary antibody. Equal

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protein loading was checked by quantifying β-actin (1:1000 diluted, ZSGB-BIO,

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China) in the western blot. ImageJ software was used to quantify the intensity of the

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bands.

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Statistical analysis

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Data were analyzed by SPSS 17.0 for Windows (SPSS Inc.). The statistical

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significance were calculated by a one-way analysis of variance (ANOVA). The

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relative metabolic pathway revealed and reconstructed was mainly according to the

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information from the metabolic databases KEGG.

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Results

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Identification of the main oligomeric procyanidins in extracts of litchi seeds

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Litchi as a catechin-rich agent

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exhibited a potential anti-diabetic effect on

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diabetic patients

. In our previous report, there were 21 compounds including

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multiple catechin-rich compounds identified by UPLC-Q/TOF-MS. However, there

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was still some composition without identification 5. Most of these compounds

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displayed a maximum absorbance at about 280 nm according to UV-V analysis,

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corresponding to the absorbance spectrum of flavan-3-ols 14. In our study, the content

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of procyanidins was 33.8 ± 3.5% by vanillin-HCl assay. They were composed of

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flavan-3-ol monomers ((+)-catechin and (−)-epicatechin), linked mainly through

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C4-C8 or C4-C6 bonds to form B-type procyanidins, while others coexisting with

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redundant interflavan bonds by C2-O-C7 or C2-O-C5 to form A-type procyanidins. In

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our study, 15 peaks among 1.5 to 5.5 min were identified as oligomeric procyanidins

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(Table 1) including A-type and B-type procyanidins (Figure 1 and 2). The [M-H]- ion

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at m/z 289 suggested the structure of (epi)catechin in identified compounds. Adding

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288 resulting in molecular weight of 577 corresponded to procyanidin with the degree

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of polymerizations (DPs) of 2 belonging to B-type. In addition, if there were

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additional interflavan bonds in compounds, series abundant ions with m/z 575, 863,

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and 1151 corresponded to the procyanidins with the degree of DPs of 2-4 belonging to

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A-type 14-15. Furthermore, the molecular masses would decrease by 2 with the increase

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of each cycle. As Table 1 indicated, the oligomeric procyanidins in litchi seeds were

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composed primarily of A-type procyanidins dimers, trimers, and tetramers. 8


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Effects of LSE on T2D rats

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In our previous research, LSE exhibited anti-diabetic effect in T2D rats 5. With

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the similar consumption of food, the body weight decreased drastically in T2D rats.

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The levels of fasting blood glucose, urinary sugar and insulin resistance index were

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significantly elevated after STZ injection. However, after 2 weeks administration of

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LSE, water consumption, insulin resistance index and urinary sugar gradually

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decreased compared with that in the model group. As shown in Table 2,

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hyperglycaemia also evoked high levels of transaminases like ALT and AST, which

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further confirmed steatohepatitis in T2D rats. LSE lowered serum ALT and AST

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concentrations to a certain extent compared with the model group to protect against

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liver injury.

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Metabolic profiling of serum and urine from the T2D rats

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Diabetic diseases were regarded as heterogeneous metabolic disorder, for

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abnormal metabolisms of carbohydrates, lipids, and proteins 7. As shown in Table 3,

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there were more than 20 compounds detected and identified as derivatives of

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endogenous metabolites during 9 to 50 min, such as carbohydrate, organic acids,

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amino acids and so forth. GC-MS analyses in serum indicated that the concentrations

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of carbohydrate such as D-fructose, D-glucose, D-mannose and ribitol, lipids

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including octadecanoic acid, cholesterol and monostearin, and ketone body like

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3-hydroxybutyric acid were extremely elevated, while the levels of amino acids

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containing L-alanine and valine decreased. In addition, levels of malic acid,

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D-glucose, D-mannose, 3-hydroxybutyric acid and glycerol increased in T2D rats’ 9



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urine. LSE relatively increased glycine, serine, threonine metabolism, alanine

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metabolism,

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gluconeogenesis and lipid relative metabolites. Especially for D-mannose and

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glycerol, LSE significantly decreased their levels in urine. All these proved that LSE-

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treatment relatively made the levels of these metabolites close to the normal ones.

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Furthermore, all the GC-MS data obtained from serum samples were analyzed by

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PCA and PLS-DA scores plot (Figure 3), which showed an obvious discrimination to

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classify three groups.

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Relative metabolic enzymes and transporters

arginine

and

proline

metabolism,

but

decreased

glycolysis,

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In the present study, mRNA level of insulin in non-treated T2D rats was

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significantly decreased compared with normal ones (p

Systemic Perturbations of Key Metabolites in Type 2 Diabetic Rats

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