Capsaicin Reduces Blood Glucose by Increasing Insulin Levels and

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Capsaicin Reduces Blood Glucose by Increasing Insulin Levels and Glycogen Content Better than Capsiate in Streptozotocin-induced Diabetic Rats Shiqi Zhang, Xiaohan Ma, Zhang Lei, Hui Sun, and Xiong Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00132 • Publication Date (Web): 23 Feb 2017 Downloaded from http://pubs.acs.org on February 25, 2017

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Journal of Agricultural and Food Chemistry

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Capsaicin Reduces Blood Glucose by Increasing Insulin Levels and Glycogen Content Better

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than Capsiate in Streptozotocin-induced Diabetic Rats

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Shiqi Zhang*, Xiaohan Ma*, Lei Zhang , Hui Sun*, Xiong Liu*§

‡

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*

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‡

College of Food Science, Southwest University, Tiansheng Road 2, Chongqing 400715, PR China College of Life Science, Chongqing Normal University, Chongqing 401331, PR China.

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Running title: Mechanism for Hypoglycaemic Effect of Capsaicin and Capsiate on

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Streptozotocin-induced Diabetic Rats

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§

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College of Food Science

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Southwest University

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Tiansheng Road 2, Chongqing, 400715

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PR China

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Tel.: +13996027313

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Fax: +86 023 68251947

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E-mail address: [email protected]

Corresponding author

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ACS Paragon Plus Environment


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ABSTRACT

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Chili peppers exhibit anti-obesity, anti-cancer, anti-diabetic, and pain- and itchiness-relieving

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effects on animals and humans; these effects are due to capsaicin, which is the main pungent and

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biologically active component of pepper. Capsiate, a nonpungent capsaicin analogue and is similar to

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capsaicin in terms of structure and biological activity. In this study, we investigated whether capsaicin

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and capsiate exhibit the same hypoglycemic effects on rats with type 1 diabetes (T1D). Experimental

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rats were categorized into four groups: control, model, capsaicin, and capsiate groups. The two

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treatment groups were treated orally with 6 mg/kg·bw capsaicin and capsiate daily for 28 days.

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Treatment with capsaicin and capsiate increased body weight, increased glycogen content, and

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inhibited intestinal absorption of sugar in T1D rats. Particularly, insulin levels were increased from

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14.9±0.76 mIU/L (model group) to 22.4±1.39 mIU/L (capsaicin group), but capsiate group (16.7±0.79

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mIU/L) was only increased by 12.2%. Analysis of the related genes suggested that the transient

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receptor potential vanilloid 1 (TRPV1) receptor was activated by capsaicin. Liver X receptor and

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pancreatic duodenum homeobox 1 controlled the glycometabolism balance by regulating the

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expression levels of glucose kinase, glucose transport protein 2 (GLUT2), phosphoenolpyruvate

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carboxykinase, and glucose-6-phosphatase, leading to reduced blood glucose levels in T1D rats.

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Meanwhile, hypoglycemic effect were enhanced by the down-regulated expression of sodium-glucose

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cotransporter 1, GLUT2, and GLUT5 in the intestine. The results showed that the spicy characteristics

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of capsaicin might be the root of its fall blood glucose.

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KEYWORDS: type 1 diabetes rats, capsaicin, capsiate, blood glucose, insulin secretion

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INTRODUCTION Type 1 diabetes is also called insulin-dependent diabetes, this kind of disease caused by

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disturbance of carbohydrate metabolism, because of the reduction of insulin and pancreatic

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dysfunction.1 The World Health Organization estimates that approximately 5% to 10% of individuals

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with diabetes worldwide have type 1 diabetes, and the number is still rising by 3% every year,2 and

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European diabetes prospective complications (EURODIAB) study shows that type 1 diabetes remains

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the main type in children and adolescents. With the development of the world diabetes day campaign,

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the problem about childhood diabetes has been paid more attention in recent years.

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Chili pepper is reported to exert anti-obesity, analgesic, anti-cancer and anti-inflammatory effects

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in animals and humans.3-6 These actions are related to a major component of red peppers, capsaicin.

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Meanwhile, experiments on mice and humans show capsaicin can stimulate insulin secretion by

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activating the transient receptor potential vanilloid subfamily member 1 (TRPV1) in islet beta cells,

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and this phenomenon leads to reduced blood glucose levels in rats,7 significantly reduced effects of

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postprandial blood glucose, and improved insulin secretion and glucose tolerance. 8 Capsaicin can also

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reduce gestational age neonatal morbidity rate and the concentration of oral glucose postprandial blood

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glucose.9-10 These results suggest that capsaicin has potential application on diabetes prevention.

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However, capsaicin has some drawbacks for treating type 1 diabetes. First, capsaicin appears to be only

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modestly effective in doses tolerable to most humans,11 especially children and adolescents. Secondly,

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capsaicin has both harmful and beneficial effects on human health, sometimes acting as a carcinogen or

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co-carcinogen, and other times as an anti-carcinogen.12 Alternatively, capsiate, a nonpungent capsaicin

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analogue found in sweet red peppers, also activates TRPV1 and exhibits potential in promoting energy

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metabolism and reducing the accumulation of body fat,13-14 but has not been investigated for possible

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anti-diabetic effects, especially the related mechanism of glucose metabolism in type 1 diabetes.

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We hypothesized that oral consumption of both capsaicin and capsiate will reduce blood glucose

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by increasing insulin levels and improving glucose metabolism. More importantly, based on insulin

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levels increase with the occurrence of insulin resistance in type 2 diabetes, but this phenomenon does

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not exist in type 1 diabetes, so the hypothesis was tested in type 1 diabetes rat model in our study,

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related biochemical analysis methods and western blot were also used to reveal the cause of

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hypoglycaemic effect of capsaicin and capsiate at the molecular level and provide evidence for the

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scientific and rational use of chili peppers. 3



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MATERIALS AND METHODS

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Materials

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Four-week-old male Sprague–Dawley (SD) rats were obtained from Chongqing Tengxin Inc.

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(Chongqing, China). Capsaicin (95% purity) was obtained from Sigma–Aldrich (St. Louis, MO).

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Capsiate (90% purity) was obtained from Wuhan Fengzhulin Chemical Technology Co., Ltd (Hubei,

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China).

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Chemicals

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Streptozotocin (STZ) was obtained from Sigma–Aldrich (St. Louis, MO). Glycogen and rats of

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insulin (INS) enzyme-linked immune detection kits were obtained from Nanjing Jiancheng

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Bioengineering Institute. (Nanjing, China). Amylase was obtained from Zhengzhou Tianle Chemical

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Products Co., Ltd. (Zhengzhou, China). All other reagents were of analytical grade and supplied by

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Dishui Chemical Co., Ltd. (Chongqing, China). Milli-Q system (Millipore Corp, USA) ultrapure water

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was used throughout this research.

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Administration in Streptozotocin-induced Diabetic Rats: Four-week-old male SD rats were

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maintained under controlled conditions (25 ± 1 °C, 55% ± 5% relative humidity, and a 12 hour

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light/dark cycle). Distilled water and commercial solid diet were given ad libitum. Rats were reared for

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1 week to adapt to the environment. Streptozotocin (STZ, 60 mg/kg) was injected into the model rats

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through the abdominal cavity, and the control group was injected with the same dose of citrate buffer.

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Rats with FBG ≥ 11.1 mmol/L were selected as diabetes model to determine fasting blood glucose

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(FBG) levels through the tail blood after 72 h.

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Experimental rats were divided into four groups (8 rats/group): control, model, capsaicin, and

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capsiate groups. The treatment groups were given with 6 mg/kg·bw capsaicin and capsiate; meanwhile,

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the control and model group were treated with the same amount of soybean oil.

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All animal experiments were performed in accordance to the requirements and regulations of the

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International Animal Welfare Committee. The experiment was considered ethically acceptable, and

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experimental procedures were approved by the Committee on Animal Experimentation [Experimental

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Animal License SCXK (Chongqing) 200120008].

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Sample Collection: At the end of the feeding period, fresh feces were collected from rats in each group

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on the last 2 days. Blood was collected from the neck of the rats and placed in a blood collection tube

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(Shandong Aosite Medical Instrument Factory, Shandong, China). Plasma was separated via 4


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centrifugation at 1400 rpm at 4 °C for 15 min and stored at −80 °C. Reagent kits of insulin and

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glycosylated protein were used for the biochemical detection of serum, which was isolated from the

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blood samples. Quadriceps, liver, pancreas, and small intestine were isolated, and samples were snap

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frozen and stored at −80 °C for subsequent RNA isolation and gene expression analysis.

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Apparent Absorptivity Determination of Total Sugar: Food intake was measured, and feces were

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collected from rats in each group on the 27th and 28th day of the experiment. Total sugar (TS) was

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measured in the fodder and feces through anthrone colorimetry method.15 Before the measurement,

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fodder and feces samples were pretreated by amylase hydrolysis for 2 h at 70 °C.

 %TS in fodder    %TS in feces 

Digestibility of TS (%) = 100-100× 

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Biochemical Index Determination: FBG and glycosylated serum protein (GSP) levels in the plasma

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were determined using a commercial diagnostic kit (Sichuan Maker Technology Co., Ltd, Sichuan,

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China) on the HITACHI 7020 automatic biochemistry analyzer (Hitachi High-Technologies

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Corporation, Tokyo, Japan). Oral glucose tolerance test (OGTT) began before the intake of glucose

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(2.5 g/kg), and then blood glucose levels were measured at 0, 0.5, 1, 1.5, and 2 h; the corresponding

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area under the curve (AUC) was then established. Glycogen and rats of insulin (INS) enzyme-linked

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immune detection kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) were used to

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determine plasma insulin and glycogen contents, respectively, in liver and muscle tissues in accordance

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with the manufacturer’s instructions.

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Preparation of mRNA and cDNA: The total RNA was extracted from the frozen tissues (liver,

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pancreas, and ileum) according to the method described by Yu-Ming You and Ting Ren.16 RNA

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concentration and purity were quantified using a NanoDrop 1000 spectrophotometer (Thermo

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Scientific, Delaware, USA). The integrity of RNA was verified by agarose gel electrophoresis by using

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a Gel Doc XR+ system (Bio-Rad, Hercules, CA, USA). Afterwards, 2 µg of RNA was reverse

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transcribed to cDNA by using a PrimeScript RT reagent kit (TaKaRa Bio, Otsu, Japan). The mRNA

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expression of liver X receptor (LXR), glucose transporter 2 (GLUT2), glucose transporter 5 (GLUT5),

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pancreatic duodenal homeobox-1 (PDX-1), sodium/glucose cotransporter 1 (SGLT1), glucose 6

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phosphatase (G6pase), glucokinase (GK), phosphoenolpyruvate carboxykinase (PEPCK), insulin

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receptor substrate 1 (IRS1), insulin receptor substrate 2 (IRS2), and transient receptor potential

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vanilloid 1 (TRPV1) were determined by using RT-PCR with a light cycler instrument (Roche 5



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Diagnostics, Mannheim, Germany). A total of 2 µL of cDNA and 10 µL of SYBR Premix Ex Taq II

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(Bio-Rad Corp., USA) were freshly mixed before the experiment. The polymerase activation and DNA

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were initially incubated at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 5 s, and

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then at 60 °C for 30 s. The 2−∆∆CT method was used to calculate the relative expression level of each

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gene, and the beta-actin gene was used as reference.

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Western Blot Analysis of the Target Proteins: Experimental samples of liver, pancreas, and ileum

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tissues were collected from rats in each group, homogenized in an ice-cold lysed buffer, and

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centrifuged at 14000 rpm and 4 °C for 15 min. Protein content was measured using BCA protein assay.

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A total of 8% and 10% SDS-PAGE (Bio-Rad Corp., USA) were used to isolate the target protein; the

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gels were then transferred to 0.45 µm PVDF membrane (Millipore Corp., USA). The PVDF membrane

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was placed in a sealing fluid (5% defatted milk powder), sealed for 2 h, and incubated with anti-

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PEPCK, anti-G6Pase, anti-GK, anti-PDX-1, anti-GLUT2, anti-TRPV1, anti-SGLT1, anti-GLUT5, anti-

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IRS1, anti-IRS2, or anti-LXR (Abcam Inc., USA) antibodies for 1.5 h at 37 °C. The membranes were

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washed three times with TBST and incubated with secondary antibodies conjugated to the horseradish

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peroxidase enzyme (Santa Cruz, USA) at 37 °C for 1.5 h.16 After the membrane was washed, the

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proteins were visualized through ECL (Millipore Corp., USA). Protein expression level was

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determined using Quantity One Software (Bio-Rad, USA).

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Statistical Analysis

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All data were expressed as means and standard deviations (n = 8). Data were subjected to one-way

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analysis of variance using Origin 8.5 and SPSS version 19.0. The differences among groups were

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examined by Duncan’s multiple-range test. P < 0.05 was considered statistically significant.

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RESULTS

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Apparent Absorption of Total Sugar, Body Weight, and Food Intake

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The total sugar content in feces was significantly higher in the capsaicin and capsiate groups than

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that in the model group (Table 2). The apparent digestibility of total sugar in the two treatment groups

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was also reduced by 1.91% and 1.35%, respectively. Meanwhile, Table 2 also shows the effects of

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capsaicin and capsiate on body weight of diabetic rats. All of the rats were in the growth phase. The

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model group showed significantly lower weight gain than the control group. The weight of rats in the

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capsaicin and capsiate groups significantly increased by 15.83 and 6.63 g, respectively, compared with

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that in the model group. Furthermore, dietary food intake of capsaicin (32.5±2.53 g) and D−H

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(32.2±1.44 g) were obviously less than model group (39.2±3.36 g).

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Fasting Blood Glucose and Oral Glucose Tolerance Test

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The model group exhibited significantly higher Fasting Blood Glucose (FBG) than control group

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(Figure 1A). The FBG in the capsaicin group significantly decreased after 4 weeks and was 18.4%

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lower than that at 0 week. By contrast, the FBG in the capsiate group was only 4.9% lower than that at

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0 week. In oral glucose tolerance test (OGTT), the blood glucose peak in the normal control and model

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groups was obtained at 30 min, but treatment groups were delayed for 30 min (Figure 1B). Moreover,

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after the blood glucose levels of the four groups reached their peak, the falling rates of the capsaicin

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and capsiate were the fastest, and the area under the curve (AUC) for blood glucose concentration of

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these two groups showed significant differences compared with that of the model group that was

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reduced by 33.5% and15.4%, respectively ((Figure 1C).

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Determination of Glycosylated Serum Protein, Serum Insulin, and Glycogen

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T1D rats in the model group showed increased GSP level compared with those in the control

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group (Figure 2). After treatment with capsaicin and capsiate, the level of GSP in capsaicin group was

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decreased significantly compared with that in the model group; by contrast, the decrease of GSP in

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capsiate group was not significant. Furthermore, as shown in Figure 3, the model group (14.9±0.76

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mIU/L) showed significantly lower insulin levels than the control group (25.3±1.62 mIU/L) (Fig. 4).

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Compared with model group, the insulin levels of capsaicin group (22.4±1.39 mIU/L) was significantly

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increased by 50.5%, but capsiate group (16.7±0.79 mIU/L) was only increased by 12.2%.

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Figure 4 shows that the glycogen contents (hepatic glycogen and muscle glycogen) in the model

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group significantly decreased compared with those in the control group. After treatment with capsaicin

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and capsiate, the glycogen contents of treatment groups were significantly different from that in the

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model group; on the one hand, the hepatic glycogen content significantly increased by 66.0% and

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16.4% compared with model group, respectively; on the other hand, the muscle glycogen content

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significantly increased by 69.3% and 41.9% compared with model group, respectively.

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Effects of Capsaicin and Capsiate on mRNA and Protein Expression of Key Genes for

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Glycometabolism in the Liver

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The mRNA and protein expression of GLUT2, PDX-1, and GK decreased, and those of G6pase

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and LXR increased in the model group (Figures 5A, 5B, and 5C). The mRNA and protein expression of 7



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GLUT2, PDX-1, GK, and LXR significantly increased in the capsaicin group compared with those in

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the model group. By contrast, the mRNA and protein expression of PDX-1 significantly increased and

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those of GLUT2 and GK decreased in the capsiate group. In addition, the protein level of LXR was

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unchanged in the capsiate group, although its mRNA expression was significantly increased compared

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with that in the model group.

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Figures 5A, 5B, and 5C also show that the mRNA and protein expression of PEPCK and TRPV1

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in the model group were no significant difference compared with that in the control group. However,

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after with capsaicin and capsiate, the protein level of PEPCK was significantly decreased, and that of

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TRPV1 was significantly increased in treatment groups.

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Glycometabolism in the pancreas

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The mRNA and protein expression of PDX-1, IRS1, IRS2 and GLUT2 significantly decreased in

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the model group compared with than in the normal control group (Figures 6A, 6B, and 6C). After

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treatment with capsaicin and capsiate, the mRNA and protein expression of PDX-1, IRS1, IRS2, and

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GLUT2 significantly increased in the capsaicin group compared with those in the model group.

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However, the expression of PDX-1 and IRS1 did not significantly change in the capsiate group

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compared with those in the model group. In addition, the pancreatic mRNA expression of IRS2 was

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significantly increased in the capsiate group but the corresponding protein expression showed no

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obvious changes. Furthermore, compared with those in the control group, the mRNA and protein

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expression of TRPV1 were not significantly up-regulated in the model group but significantly up-

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regulated in the capsaicin and capsiate groups.

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Glycometabolism in the ileum

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As shown in Figures 7A, 7B, and 7C, the mRNA and protein expression of SGLT1 and GLUT2

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were up-regulated in the model group compared with those in the control group, but there was no

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significant difference in protein level of GLUT5. After treatment with capsaicin and capsiate, the

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mRNA and protein expression of SGLT1, GLUT2, and GLUT5 gradually decreased in the capsaicin

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and capsiate groups compared with those in the model group.

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DISCUSSION

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Chili pepper is a traditional medicine used for treatment of diabetes in Jamaica.17 Consumption of

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chili pepper and its active principle, capsaicin, increases the plasma insulin levels and reduces blood

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glucose levels, possibly by improving pancreatic dysfunction and promoting insulin secretion.7,18,19

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Capsiate is produced by ‘CH-19 Sweet’ (Capsicum annuun L.), a nonpungent cultivar of red pepper,

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and is reported to have similar effects as capsaicin on insulin sensitivity and energy metabolism.20-21

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However, no study has researched the effect of capsiate on glycometabolism in T1D rats. In this study,

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our results showed that capsaicin and capsiate improved glucose metabolism and increased insulin

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level and glycogen content. More importantly, by determining the expression of glucose metabolism-

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related genes, we found that capsaicin and capsiate elicited different effects on the liver and pancreas of

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T1D rats. During the course of the experiment, we have encountered many problems worth our

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attention and discussion.

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The apparent absorption rate and weight gain were evaluated in T1D rats. Capsaicin and capsiate

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inhibited the intestinal absorption of glucose and promoted fecal excretion of sugar in the small

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intestine of diabetic rats. These results were validated by determining the expression of intestinal genes

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(SGLT1, GLUT2, and GLUT5). Intestinal mucosa cells are generally known to contain significant

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amounts of SGLT and GLUT, which synergistically work to complete the transport of glucose in the

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body.22 GLUT2 and GLUT5 contain different subunits; that is, the former transports high

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concentrations of glucose, whereas the latter transports fructose.22-26 Intestinal mucosa cells absorb

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monosaccharides in three ways, active absorption is one of the most important way, SGLT1 and

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GLUT2 play an important role in active absorption.27 Related studies showed that the gene and protein

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expression levels of SGLT1 in patients with non-insulin-dependent diabetes mellitus were 3.0 and 4.3

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times higher than those in healthy patients, this finding is consistent with the present experimental

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results. More importantly, the SGLT family is one of the current research hotspots; Forxiga™

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(Dapaglifozin) is the first SGLT2 inhibitor approved by the European Union for treatment of type 2

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diabetes.28 This drug selectively inhibits SGLT2 expression in the kidney, resulting in removal of

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excess glucose and its associated calories through urine and decreased FBG.29 However, oral drugs can

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cause a series of adverse symptoms, such as kidney stress, thirsty, nausea, increased urea nitrogen level,

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genital fungal infection, urinary tract infection,30-31 and even bladder cancer and breast cancer.32-33

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Nonetheless, SGLT1 directly affects the small intestine, and functions with GLUT2 and GLUT5 to 9



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inhibit the intestinal absorption of glucose and excrete unnecessary sugar form the body; this

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phenomenon consequently reduce the burden of diabetic patients at risk for kidney problems and

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urinary tract infection. In our opinion, the function of capsaicin and capsiate is beneficial for people

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with diabetes. Moreover, this is a very interesting phenomenon that capsaicin and capsiate can increase

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the weight of the T1D rats. Based on capsaicin. Many studies have shown that capsaicin has a potential

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to serve as a fat-reducing drug, possibly by increasing the expression of adiponectin (ADP), thus

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reducing fat accumulation in the adipose tissue of obese mice.34 In our study, the weight of model rats

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was significantly lower than that in the normal group, and the weight of capsaicin and capsiate groups

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increased significantly (Table 2). Such weight increase may be due to improved glycometabolism and

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diabetes typical symptoms (increased food and water intake, diuresis, and decreased body weight) by

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capsaicin and capsiate, thus reducing the risk of overeating, and then resulting in increased weight of

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diabetic rats. Coincidentally, previous research has shown that the weight of T1D rats were

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significantly increased after treatment with Zanthoxylum alkylamides.16

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Some studies also showed that the hepatic glycogen content is related to the development of

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diabetes.35 Capsaicin can restrain glycogen decomposition under stressful conditions to effectively use

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fatty acids as energy source.36 In the present study, capsaicin significantly increased insulin levels and

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glycogen content, in contrast to capsiate; this finding was validated by analysis of liver-related genes.

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Two key genes, namely, LXR and PDX-1, associated with glycometabolism were abundantly

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expressed in the liver and played a crucial role in treatment of diabetes. LXR is the key gene that

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activates the inhibition of liver sugar dysplasia and reduces serum glucose levels.37 This gene also

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inhibits the expression of PEPCK and G6pase, thereby suppressing sugar dysplasia.38 Studies have

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shown that LXR can also stimulate islet cells to secrete insulin and is related to GK and GLUT2, which

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can promote insulin secretion.37 Furthermore, GK and GLUT2 are considered glucose sensors in beta

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cells and regulate insulin secretion by affecting the concentrations of blood glucose.39-40 PDX-1 can

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directly regulate not only the expression of GK and GLUT2 but also the expression of other genes

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(amylin and synaptophysin), thereby stimulating islet cells to secrete insulin.41 In addition, PDX-1 can

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induce liver cells to develop into pancreatic endocrine and exocrine cells, which induce liver cells to

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secrete insulin.42-43 Figures 6A, 6B, and 6C show that capsaicin significantly increased the expression

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of PDX-1, LXR, GLUT2, and GK, whereas capsiate increased the expression of PDX-1 only. These

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results are consistent with those of the analysis on liver glycogen, serum glycosylated protein, and

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insulin and thus verify the reliability of the experiment.

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The functions of pancreas in T1D rats should not be neglected. Current studies suggest that insulin

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receptor substrate family are closely related to incidence of type 2 diabetes mellitus, knockout of the

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IRS1 in rats revealed insulin resistance,44 and knockout of the IRS2 in rats revealed severe diabetes

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symptoms.45 However, the therapeutic mechanism of IRS1/2 for T1D is not yet clear. In addition, some

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studies show that capsaicin activated TRPV1 ion channels and stimulated islet beta cells to secrete

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insulin, thereby reducing the blood glucose levels in rats.7 However, no study has researched the effect

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of capsiate on insulin secretion in T1D rats. The results showed that capsaicin significantly increased

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the expression levels of PDX-1, IRS1, IRS2, and GLUT2 in the pancreas of T1D rats (Figures 6A, 6B,

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and 6C); by contrast, capsiate only up-regulated the expression of GLIU2. According to the

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experimental results, three ways for the suppression of elevation of blood glucose level after capsaicin

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treatment were proposed, one is the stimulation of insulin secretion; second is the amelioration of

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pancreatic dysfunction, three is the improvement of glycometabolism. However, capsiate did not

293

exhibit such significant effects.

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TRPV1 receptors play a crucial role in the development of T1D and are considered the main

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channel through which capsaicin elicits its physiological functions in the body.46 The expression of

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TRPV1 was significantly increased in the liver after treatment with capsaicin and capsiate (Figures 5A,

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5B, and 5C), it was also found that the protein expression of TRPV1 in capsaicin group was as 1.56

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times (pancreas) as that in capsiate group. Moreover, related studies showed that capsaicin dose-

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dependently increases insulin secretion and plasma insulin concentrations in TRPV1 expressing islet

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cells, and this effect is inhibited by the TRPV1 inhibitor capsazepine.7 In addition, different TRPV1-

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mediated brain responses to intragastric infusion of capsaicin and capsiate,47 this phenomenon also

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suggested the uniqueness of capsaicin in activating TRPV1 receptors. Our findings are consistent with

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those in previous studies. Comprehensive the above results, we infer that the pungent ingredient of chili

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peppers is mainly responsible for activation of TRPV1 receptors and elicits hypoglycemic effects; on

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the contrary, the nonpungent ingredient of red pepper, capsiate, its hypoglycemic effect is not so

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satisfactory. More importantly, we found that the expression levels of GLUT2, IRS1, and IRS2 were

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associated with TRPV1 expression level. This findings were verified by the interesting phenomenon,

308

wherein the expression of TRPV1 was up-regulated by capsiate but the expression level was low, and 11



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the expression levels of GLUT2, IRS1, and IRS2 were also down-regulated (Figures 6A, 6B, and 6C).

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Basing on the above analysis and experimental results, we could speculate that the signaling pathway

311

TRPV1–(PDX-1)–(GLUT2/GK) or TRPV1–(PDX-1)–(IRS1/2) may exist in the liver or pancreas.

312

In general, the hypoglycemic effect of pepper could be due to the synergistic effect of several

313

factors. This study indicates that capsaicin activated the TRPV1 ion channel in the liver and increased

314

the expression of TRPV1, LXR, and PDX-1. LXR and PDX-1 controlled glycometabolism balance by

315

regulating the expression levels of GK, GLUT2, PEPCK, and G6pase, thereby inhibiting

316

gluconeogenesis and promoting glycogen synthesis; in the pancreas, the TRPV1 ion channel was

317

activated by capsaicin and capsiate. The up-regulated expression of PDX-1 controlled insulin secretion

318

by regulating the expression levels of GLUT2, and cooperate with IRS1/2 in improving glucose

319

metabolism; in the intestine, the expression of SGTL1, GLUT2, and GLUT5 were inhibited by

320

capsaicin and capsiate, leading to apparent reduction in the absorption rate of total sugar and increase

321

in the excretion of sugar in the feces. These results proved that capsaicin exhibited a certain function in

322

treatment of diabetes. Although capsiate contained similar biologically active components to those of

323

capsaicin and improved the symptoms of diabetes in some ways, the former was less efficient in

324

activating TRPV1 receptors and lacked the spicy ingredient. Therefore, the results suggest that

325

capsaicin has better anti-diabetic actions than capsiate in T1D. However, for diabetics (70kg), about

326

consumption of 54-80g red pepper (dry weight) every day can achieve the effect of the treatment,48-50

327

our body's systems will be damaged by excessive consumption of chili pepper, so more safe and

328

effective methods worthy of further study.

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CONFLICT OF INTEREST

The authors declare that they have no conflict of interest.

ACKNOWLEDGEMENT

This work was supported by the National Natural Science Foundation of China (NSFC31471581),

National Natural Science Foundation of Chongqing (cstc2014jcyjA10063), and Scientific and

Technological Research Program of Chongqing Municipal Education Commission (KJ1500323).

13



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Figure caption Figure 1: Effect of capsaicin and capsiate on the blood glucose level of T1D rats (A). Effect of capsaicin and capsiate on the oral glucose tolerance test (OGTT) (B) and area under curve (AUC) (C) in T1D rats. All data are expressed as means ± SD, n=8. *Means are significant difference compared with the model group (P < 0.05). Means with different superscript letters are significantly different among the diabetes groups (P < 0.05). Figure 2: Effect of capsaicin and capsiate on the Glycosylated Serum Protein in T1D rats. All data are expressed as means ± SD, n=8. *Means are significant difference compared with the model group (P < 0.05). Means with different superscript letters are significantly different among the diabetes groups (P < 0.05). Figure 3: Effect of capsaicin and capsiate on the serum insulin in T1D rats. All data are expressed as means ± SD, n=8. *Means are significant difference compared with the model group (P < 0.05). Means with different superscript letters are significantly different among the diabetes groups (P < 0.05). Figure 4: Effect of capsaicin and capsiate on hepatic glycogen and muscle glycogen in T1D rats. All data are expressed as means ± SD, n=8. *Means are significant difference compared with the model group (P < 0.05). Means with different superscript letters are significantly different among the diabetes groups (P < 0.05). Figure 5: Effects of capsaicin and capsiate on protein (A and B) and mRNA (C) expression levels of key genes for glycometabolism in liver of T1D rats. *Means are significant difference compared with the model group (P < 0.05). Means with different superscript letters are significantly different among the diabetes groups (P < 0.05). G6pase: glucose 6 phosphatase; GLUT2: glucose transporter 2; PEPCK: phosphoenolpyruvate carboxykinase; TRPV1: transient receptor potential cation channel subfamily V member 1; LXR: liver X receptor; PDX-1: pancreatic duodenal homeobox-1; GK: glucokinase. Figure 6: Effects of capsaicin and capsiate on protein (A and B) and mRNA (C) expression levels of key genes for glycometabolism in pancreas of T1D rats. *Means are significant difference compared with the model group (P < 0.05). Means with different superscript letters are significantly different among the diabetes groups (P < 0.05). TRPV1: transient receptor potential cation channel subfamily V member 1; PDX-1: pancreatic duodenal homeobox-1; IRS1: insulin receptor substrate 1; IRS2: insulin receptor substrate 2; GLUT2: glucose transporter 2.

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Figure 7: Effects of capsaicin and capsiate on protein (A and B) and mRNA (C) expression levels of key genes for glycometabolism in ileum of T1D rats. *Means are significant difference compared with the model group (P < 0.05). Means with different superscript letters are significantly different among the diabetes groups (P

Capsaicin Reduces Blood Glucose by Increasing Insulin Levels and

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