Activity of Isoliensinine in Improving the Symptoms of Type 2

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Activity of Isoliensinine in Improving the Symptoms of Type 2 Diabetic Mice via Activation of AMP-activated Kinase and Regulation of PPAR# Xinzhou Yang, Mi Huang, Jie Yang, Jialin Wang, Sijian Zheng, Xinhua Ma, Jinyan Cai, Shihao Deng, Guangwen Shu, and Guangzhong Yang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b01964 • Publication Date (Web): 26 Jul 2017 Downloaded from http://pubs.acs.org on July 27, 2017

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Journal of Agricultural and Food Chemistry

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Activity of Isoliensinine in Improving the Symptoms of Type 2

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Diabetic Mice via Activation of AMP-activated Kinase and

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Regulation of PPARγ

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Xinzhou Yanga,1, Mi Huanga,1, Jie Yanga, Jialin Wanga, Sijian Zhenga, Xinhua Maa, Jinyan

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Caib, Shihao Denga, Guangwen Shua,*, Guangzhong Yanga,*

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a

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Road, Wuhan 430074, China

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b

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*

School of Pharmaceutical Sciences, South-Central University for Nationalities, 182 Min-Zu

School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, China

Corresponding authors at: School of Pharmaceutical Sciences, South-Central University for

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Nationalities, 182 Min-Zu Road, Wuhan 430074, P.R. China. Tel.: +86 27 67841196; Fax:

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+86 27 67841196.

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*

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E-mail addresses: [email protected] (G.W. Shu); [email protected] (G.Z.

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Yang).

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1

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Running Heading: An Oral Hypoglycemic Agent

To whom correspondence can be addressed.

These authors contributed to this article equally.

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ACS Paragon Plus Environment


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ABSTRACT

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This study was designed to explore the effects and mechanism of isoliensinine (isolie) from

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embryos of Nelumbo nucifera on type 2 diabetes and dyslipidemia in vivo and in vitro. The in

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vitro study showed that isolie increased the GLUT4 translocation by 2.5 folds in L6 cells.

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Furthermore, after 4 weeks treatment, the in vivo biochemical study indices revealed that

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isolie had a positive effect on decreasing serum insulin level (42.2 ± 5.10 vs 55.7 ± 6.33

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mU/L, P < 0.05), and reducing fast blood glucose (9.4 ± 1.5 vs 18.7 ± 2.3 mmol/L, P < 0.001)

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and body weight (37.8 ± 2.9 vs 46.9 ± 5.4 g, P < 0.05) compared with the KK-Ay model mice.

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Isolie treatment led to significant increases in GLUT4 proteins (~2.7 folds in skeletal muscle

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and ~2.4 folds in WAT) and phosphorylation of AMP-activated protein kinase (~1.4 folds in

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skeletal muscle, ~3.1 folds in WAT and ~2.3 folds in liver). However, isolie caused a

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significant decrease in lipogenesis protein expressions of PPARγ and SREBP-1c, and

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decreased the activity of ACC by increasing the Phospho-ACC level. Our findings showed

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that isolie has the potential to alleviate Type 2 diabetes associated with hyperlipidemia in KK-

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Ay mice. Regulation of GLUT4, SREBP-1c, PPARγ, AMPK phosphorylation and ACC

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phosphorylation is implicated in the anti-diabetic effects of isolie.

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KEYWORDS: Isoliensinine; Type 2 diabetes; hyperlipidemia; glucose transporter 4; p-

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AMPK

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INTRODUCTION

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In 2015, according to the International Diabetes Federation (IDF) programmes, it was

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estimated that there were 415 million adult people with diabetes across the world and this

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number is expected to rise to 642 million by 2040.1 Around 97% of these diabetic patients

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will have type 2 diabetes mellitus (T2DM).2 T2DM is a progressive disease whose clinical

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manifestation is a gradual decline in glycemic control caused by β cell functional

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deterioration over time and increased associated risk of microvascular and macrovascular

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complications.3 The primary cause of T2DM is obesity-driven insulin resistance in the liver,

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white adipose tissue (WAT), and skeletal muscle, combined with insufficient secretion of

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insulin by pancreatic β cells to overcome this resistance.4 Although the strategies currently

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available for T2DM management have achieved great effects, the tolerability and significant

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mechanism-based side effects were still unignorable.5 Therefore, looking for new drugs to

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prevent and treat T2DM is becoming the urgent problem that must be solved.

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Elevating the activity of glucose transporter 4 (GLUT4) can be a promising strategy for

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controlling obesity-related insulin resistance.6,7 GLUT4 is a glucose transporter expressed

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primarily in adipose and muscle tissues.8 Reduced GLUT4 expression and abnormal GLUT4

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translocation are tightly associated with decreased glucose uptake in skeletal muscles in

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patients with insulin resistance.9,10 AMP-activated protein kinase (AMPK) is an important

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regulator of the GLUT4, metformin is a commonly used anti-diabetic drug which has been

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demontrated to increase GLUT4 expression and translocation through activating AMPK

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pathway.11,12 In this way, compounds capable of enhancing the expression or translocation of

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GLUT4 may be suitable agents for controlling T2DM.

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Natural products are gaining an increasing amount of attention from pharmaceutical

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researchers and clinicians because of their efficiency, safety, and immediate availability.13,14

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Over the past 30 years, more than 70% of drugs approved by the U.S. Food and Drug



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Administration originated from natural products.15 To look for potential hypoglycemic agents

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from natural products, a cell-based GLUT4 translocation assay system using stable L6

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myotubes expressing pIRAP-mOrange cDNAs was established to screen potential antidiabetic

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plants extracts, fractions, and individual natural compounds by evaluating their effects on

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GLUT4 translocation.16,17 During the screening of a plant extract library (800 biotas) on

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GLUT4 translocation, we found that the total alkaloids from embryos of Nelumbo nucifera

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displayed a promising positive activity on GLUT4 translocation.

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N. nucifera is commonly known as lotus, and it is a well-known edible plant. Foods and

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dietary supplements derived from N. nucifera are very popular in China.18 Embryos of N.

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nucifera have been long used in Chinese herbal medicine for the prevention of arrhythmia,

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insomnia, hypertension, spermatorrhea, cardiac action, and hyperpyrexia.19,20 The main active

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components in the total alkaloids from embryos of N. nucifera are liensinine, isoliensinine

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(isolie), and neferine (Figure 1A and 1B). Alkaloids are one of the most important active

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ingredients in natural medical and edible plants. Alkaloids plays a vital role in many of the

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new drugs which being evaluated for treating cancer, metabolic disease, cardiovascular

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disease, and other conditions.21 Isolie showed the strongest activity on GLUT4 translocation

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among the three compounds (Figure 1C and S3). Previous works focusing on isolie have

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shown biological activity such as anti-tumor, and anti-pulmonary fibrosis.22,23 However, the

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effects of isolie on diabetes have not been investigated or reported until now. In this study, we

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demonstrated that isolie exhibited strongly hypoglycemic effects both in vitro and in vivo.

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Mechanistically, activating proteins related to glucose and lipid metabolism was here

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implicated in the antidiabetic activity of isolie.

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MATERIALS AND METHOD

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Chemicals. Minimum Essential Medium-ɑ (MEM-ɑ) and fetal bovine serum (FBS, and


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antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin)) were obtained from Hyclone

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(Logan, UT, USA). Compound C, Wortamannin and AICAR were purchased from Sigma-

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Aldric (St. Louis, MO, USA). The 2-NBDG assay kits were purchased from Cayman

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Chemical (Ann Arbor, Michigan USA). The insulin Elisa assay kit was obtained Jiancheng

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Bioengineering Institute (Nanjing, Jiangsu Province, PR China). TG, TC, FFA, HDLC, LDLC,

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AST, ALT kits were all purchased from Jiancheng Bioengineering Institute, Nanjing, China.

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BCA protein quantification kit was purchased from Beyotime Biotechnology (Nantong,

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Jiangsu Province, PR China). Antibodies of β-Actin, GLUT4, AMPKα, p-AMPKα (Thr172),

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ACC, p-ACC, PPARγ and the corresponding secondary antibodies were got from Cell

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Signaling Technology (Danvers, MA, USA). The insulin and SREBP-1c antibody and

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corresponding secondary antibody were obtained from Abcam (Cambridge, MA, USA). The

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enhanced chemiluminescence (ECL) kits were obtained from Amersham-Pharmacia

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(Piscataway, NJ, USA). The ECL reagent kit was from GE Healthcare BioSciences

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(Buckinghamshire, UK).

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Plant material. The embryos of Nelumbo nucifera were purchased from Jointown

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Pharmaceutical Group Co. Ltd., Wuhan, P.R. China, in September 2013, and identified by

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Prof. Dingrong Wan from School of Pharmaceutical Sciences, South-Central University for

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Nationalities (SCUN), Wuhan. The voucher specimen (SC0005) was deposited in the

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Herbarium of the School of Pharmaceutical Sciences, SCUN.

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Isolation and purification of active compounds. The total alkaloid extract and three pure

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alkaloids were isolated and purified from N. nucifera as our previous publication.22

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Cell culture. L6 cells were from rat skeletal muscle cells, and it has been investigated as a

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potential tissue culture model for the study of thyroid hormone effects on skeletal muscle

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metabolism.24 In our study, L6 cells were maintained in MEM-ɑ supplemented with 10% FBS

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and 1% antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin) at 37 oC in 5% CO2.



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L6 cells were cultured in MEM-ɑ plus 2% FBS at 37 oC in 5% CO2 to differentiate into

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myotube cells and the medium was replaced every 2 days. 7 days after the initiation of the

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differentiation, the cells were used for experiment.

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Plasmid and cell line construction. pIRAP-mOrange cDNAs were inserted into the pQCXIP

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plasmid. The retrovirus was prepared by transfecting pQCXIP-IRAP-mOrange, VSVG,

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PHIT60 with a ratio of 2: 1: 1 by lipofectamine 2000 into PLATET cells, collecting the

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cultural supernatant after 48 hours, and concentrating the viruses by supercentrifuge (50,000 g,

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30 min). L6 cells at the exponential growth phase were infected with fresh prepared viruses.

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The polybrene (Millipore, 8 µg/mL) was used to facilitate the infection efficiency. The red

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fluorescence cells were isolated by FACS and single cell was seeded into 96 well-plates.

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Finally the single clone, which had the highest increase in red fluorescence intensity

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following stimulation with insulin (100 nM), was selected.

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IRAP translocation assay. L6 cells stably expressing IRAP-mOrange (L6 IRAP-mOrange)

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were cultured in MEM-ɑ supplemented with 10% fetal bovine serum and 1% antibiotics (100

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U/mL penicillin and 100 µg/mL streptomycin) at 37 oC in 5% CO2. Before starting the

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experiment, L6 IRAP-mOrange was seeded in 48 well plates, and incubated until 100%

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confluence and then starved in serum-free MEM-ɑ for 2 h. After adding different dose of

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irritants, the cells were imaged with a laser-scanning confocal microscope LSM 510 (Carl

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Zeiss, Jena, Germany) to monitor the dynamics of IRAP-mOrange translocation. Images were

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taken after addition of test samples, using 555 nm excitation laser every 10 seconds in first 2

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minutes and then every 5 minutes in later 30 minutes.

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Assay of glucose uptake. To determine the effects of isolie in promoting the glucose uptake

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in L6 cells, fully differentiated L6 myotube cells were pre-incubated for 12 h in serum-free

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and α-MEM medium, followed by treatment with isolie, Wortmanin or Compound C in 100

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µL glucose-free α-MEM medium containing 150 µg/mL 2-NBDG for 24 h. The fluorescence


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retained in cell monolayers was measured with a fluorescence microplate reader (Thermo

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Fisher Scientific, San Jose, CA, USA), set at an excitation wavelength of 485 nm and

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emission wavelength of 535 nm.

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Experimental animals. KK-Ay mice are obtained from a cross of black KK females with

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obese yellow Ay males, which are obese, hyperglycemic, hyperinsulinemic and insulin

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resistant.25,26 In our study, we choose KK-Ay mice as a model of metabolic disorders to

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evaluate the anti-diabetic effects of isolie. The experiments were conducted under the

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guidelines of International Guidelines for Care and Use of Laboratory Animals and approved

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by the Animal Ethical Committee of the Institute for Health and Epidemic Prevention (Wuhan,

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P.R. China). 8-week-old male KK-Ay mice (n = 55) and 8-week-old male C57BL/6J mice (n

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= 10) were purchased from the Beijing HFK Bioscience Co, Ltd (Certification number:

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SCXK 2009-0015). All animals were single housed in laminar flow cabinets under specific

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pathogen-free (SPF) conditions in facilities approved for Accreditation of Laboratory Animal

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Care. KK-Ay mice were given a 45% high-fat diet purchased from Medicience Co., Ltd.,

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Yangzhou, China. The composition of the diet was as follows: protein, 225 g/kg; fat, 200 g/kg;

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carbohydrate substances, 450 g/kg; cholesterol, 12.5 g/kg; sodium cholate, 5 g/kg; energy,

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4500 kcal/kg. Four consecutive weeks (an average body weight of 43 g) feeding to KK-Ay

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mice established a type 2-like diabetic mouse model. The C57BL/6J mice were given

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standard laboratory diet. All mice were allowed free access to the diet and water. Four weeks

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later, the fasted blood glucose levels of the KK-Ay mice were tested. The fasted blood glucose

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levels ≥ 11.1 mmol/L were classified as T2DM. Successful T2DM mice (n = 47) were

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randomly divided into 4 groups: saline treatment (n = 11), isolie treatment (30 mg/Kg/day, n =

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12), isolie treatment (60 mg/Kg/day, n = 12) and metformin treatment (200 mg/Kg/day, n =

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12). C57BL/6J mice were administered saline as a normal control. Mice were orally

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administered with isolie once a day for 4 weeks. Upon completion of treatment, mice were



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anesthetized by diethyl ether inhalation after overnight fasting. Blood samples were collected

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by retro-orbital sinus puncture using capillary tubes under diethyl ether anesthesia. And the

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livers, pancreas, and other tissues were harvested. Parts of tissues were stored in 10% neutral

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buffered formalin to be fixed immediately, and rest parts were stored in liquid nitrogen tank

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for further experiments.

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Ethical statement The research project was approved by the Ethical Committee at South-

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Central University for Nationalities (SCUN) and all procedures for the use and care of

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animals for this research were carried out under the approval by the Ethical Committee of

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Experimental Animal Care at SCUN (approval no. 2016-SCUEC-AEC-0023).

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Body weight, food intake, fasted blood glucose levels, oral glucose tolerance test. During

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the period of animal studies, body weights and food intake of all groups were daily recorded

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at the same time. Fasted blood glucose levels were measured weekly by a blood glucose meter

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(OneTouch Ultra®, Lifescan Inc., Wayne, USA). An oral glucose tolerance test (OGTT) was

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performed according to previous report.27 Briefly, all mice were administrated 2.0 g/kg

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glucose orally after 12 h fasting on the 26th day of treatment. And blood glucose taken from

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the tail tip at 0, 30, 60, 90 and 150 min after glucose administration was measured and

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recorded.

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Biochemical analysis of serum and tissues. At the end of the experiment, all mice serums

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were prepared by centrifuging the blood samples at 3000 rpm for 15 min. The fasting serum

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insulin (FINS) was determined by ELISA kit. The serum triglycerides (TG), total cholesterol

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(TC), aspartate transaminase (AST), alanine aminotransferase (ALT), low density lipoprotein

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cholesterol (LDL-C), and high density lipoprotein cholesterol (HDL-C) were determined by

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automatic biochemical analyzer (Hitachi 7180+ISE, Tokyo, Japan). Serum free fatty acid

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(FFA), and tissue TC, TG, and FFA were determined by corresponding assay kits.


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Histopathological observation. At the end of the experiment, the animals were sacrificed,

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and parts of liver and white adipose tissue (WAT) were fixed in neutral buffered solution and

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embedded in paraffin. 5 µm-thick sections were cut and stained with standard hematoxylin

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and eosin (HE). The stained sections were observed and photographed through an optical

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microscope photographed. The quantification of adipocyte size was performed by an Image J-

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based Adiposoft program as described before.28

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Immunohistochemical analysis of pancreas. All procedures of immunohistochemical

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sections were conducted as previously described.29 Briefly, parts of pancreas were fixed in

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paraformaldehyde, dehydrated in a graded series of ethanol, and embedded in paraffin wax

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before sectioning. And then, 5 µm-thick pancreas sections were cut and incubed with mouse

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anti-insulin antibody for 16 h. Subsequently, biotinylated goat anti-mouse immunoglobulin

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was used as a secondary antibody. The stained pancreas sections were observed and

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photographed through an optical microscope photographed.

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Western blot analysis. To detect related proteins in L6 cells, L6 cells (5 × 105 cells) were

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subcultured into 60 mm dishes and cultured for 7 days to form myotubes in 3 mL of MEM-ɑ

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with 2% FBS. After incubation, the L6 myotubes were treated with AICAR (1 mM),

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Compound C (10 µM), isolie, or vehicle (0.1% DMSO) for 12 h. Then cells were lysed using

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a RIPA protein extraction kit after washing with ice-cold PBS. The whole cell lysate was

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centrifuged at 15,000 rpm for 15 min to remove insoluble protein. The protein concentration

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of lysates was determined using the BCA protein assay kit. To determine protein expression in

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tissues, part of tissues were homogenized with the RIPA protein extraction kit. The mixture

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was lysed for 30 min on the ice, and then the protein concentration was measured as described

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above. For immunoprecipitation, an equivalent amount of samples was mounted on 8% or

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10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The separated

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proteins were transferred onto polyvinylidene difluoride membranes. Levels of protein and



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actin were detected with a 1 : 2000 dilution of each antibody specific for β-Actin, GLUT4,

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AMPK, p-AMPK, ACC, p-ACC, PPARγ and SREBP-1c. Subsequently, protein bands were

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detected using horseradish peroxidase linked to an anti-IgG secondary antibody and incubated

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in enhanced chemiluminescence kits (Amersham-Pharmacia, Piscataway, NJ, USA). And the

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immunoreactive signals were imaged and quantified with the Gel Image system (Aplegen Inc.,

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Pleasanton, USA).

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Statistical analysis. Three parallel replications were conducted for Western blot analysis.

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One-way ANOVA was used for multiple group comparisons. Data was shown as means ±

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standard error (M ± SEM). Statistical analyses were performed using Tukey's post hoc test of

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GraphPad Prism 5.0 software package, P values < 0.05 were considered significant.

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RESULTS

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Effects of isolie on GLUT4 translocation, glucose uptake and the related protein assay in

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L6 myotubes.

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The effect of isolie on GLUT4 translocation was test in L6 cells which stably expressed

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IRAP-mOrange. As shown in Figure 2A and 2B, treatment with isolie increased fluorescence

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intensity in a time-dependent manner. And 30 minutes later, the fluorescence intensity was

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enhancing at 2.5 folds after the addition of isolie. These results showed that isolie had a strong

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effect on the translocation of GLUT4 in L6 cells. But the translocation of GLUT4 to

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membrane plasmique caused by isolie stimulation was completely inhibited when adding of

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Compound C (an inhibitor of AMPK). Besides, the addition of Wortmannin (an inhibitor of

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PI3K) had no significant effect on the IRAP trafficking response (Figure 2B).

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At the same time, the effect of isolie on glucose uptake was carried out in L6 cells. Isolie

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treatment increased glucose uptake in a concentration-dependent manner (Figure 2C). The


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AICAR (5-Aminoimidazole-4-carboxamide 1-β-D-ribofuranoside, the AMPK agonist)

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showed the most significant enhance of glucose uptake in L6 cells (reaching 2.1 folds). Isolie

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sigficantly stimulated glucose uptake at the concentration of 15 µg/mL (1.3-fold, P＜0.01 vs

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control group). But when adding Compound C, the glucose uptake was reduced significantly

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(P＜0.01 vs control group).

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The effect of isolie was further investigated on related proteins expression by performing

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western blotting assays with Compound C and AICAR in L6 myotubes. As shown in Figure

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2D and 2E, after adding of isolie, the AMPK phosphorylation was increased and presented a

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dose-dependent effect in L6 cells compared with normal control. Corresponding to the p-

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AMPK increasing, the expression of GLUT4 was enhanced more significantly (P＜0.001 vs

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control group). The p-AMPK and GLUT4 expressions in AICAR group also obtained

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remarkable enhancement. As is well known, Compound C is an AMPK inhibitor. When

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adding Compound C in L6 cells, the GLUT4 expression and AMPK phosphorylation were

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decreased significantly coinciding with our expectations. However, to our interests, when

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adding isolie accompanied with Compound C, the effects of AMPK phosphorylation and

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GLUT4 increase caused by isolie were totally repressed.

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Western blotting of GLUT4, p-AMPK (Thr172) proteins in tissues.

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In vivo study, we examined the expression of p-AMPK and GLUT4 in different tissues.

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As shown in Figure 3, the levels of GLUT4 and p-AMPK were lower in skeletal muscle and

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WAT in KK-Ay model mice than normal C57BL/6J mice. The isolie and metformin treated

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mice showed increased muscular GLUT4 and p-AMPK protein contents compared with the

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vehicle group. And there were significant difference between isolie-treated group and vehicle

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group. The mice in the vehicle group showed decreased phospho-AMPK in liver tissue

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compared with the mice in normal group. But the protein contents of phospho-AMPK in liver



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tissues were significantly increased in the isolie (30 mg/Kg), isolie (60 mg/Kg) and metformin

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groups (P < 0.01, P < 0.001, P < 0.001, respectively).

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Effects of isolie on body weight, food intake, blood glucose levels, OGTT.

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In Figure 4A, the body weights of metformin and isolie treated groups continuously

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declined during the treatment period (P < 0.05). And the average body weight in metformin

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group was reduced to 36.3 g. Among the isolie treated groups, the high dose group (60 mg/Kg)

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obtained better effect (average body weight decrease: 7.6 g) than the low dose group (30

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mg/Kg) on body weight decline(average body weight decrease: 5.5 g). Adversely, at the end

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of the experiment, the vehicle control group had little weight gain compared to the initial

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weight. The food intake was shown in Figure 4B. Although there were some changes of food

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intake every week in each group, there were no significant differences in food intake between

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the vehicle group and isolie groups during the 4 weeks treatment period.

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At the same time, we administered isolie orally to KK-Ay mice to measure its glucose-

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lowering effect in vivo. As shown in Figure 4C, the fasting blood glucose levels of treated

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mice were significantly lower after 4 weeks (P < 0.001) compared with vehicle group. On the

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contrary, the control mice had no significant changes in the serum glucose level.

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Oral glucose tolerance (OGTT) was also improved significantly, as evidenced by lower

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glucose levels. An OGTT was performed in mice at the 26th day of isolie treatment. As shown

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in Figure 4D, at the time point of 30 min, the blood glucose of all groups had a deep increase.

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At the time point of 60 min, the blood glucose of isolie and metformin treated groups had

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decreased significantly compared with the vehicle group (P < 0.001). At the time point of 90

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min, the blood glucose of isolie and metformin groups had a steady decrease. And at the end

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of the experiment, the blood glucose of isolie and metformin groups had decreased to the

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initial level. But the blood glucose of the vehicle group was still high compared to its initial


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level. Based on the above, significant improvements in OGTT were observed in metformin

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and isolie treated groups.

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Blood and tissue biochemical analysis.

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Significant lipid metabolism disorder was observed in KK-Ay T2DM model mice

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(Figure 5B-F), including the increase of total cholesterol (TC), triglyceride (TG), free fatty

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acid (FFA) and low density lipoprotein cholesterin (LDL-C), and decline of high density

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lipoprotein cholesterin (HDL-C) in blood serum. Increases of TC, TG, FFA were also

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observed in livers and skeletal muscle (Figure 6). However, the isolie (30 mg/Kg)-, isolie (60

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mg/Kg)- and metformin-treatment significantly lowered TC, TG, FFA, LDL-C levels both in

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blood and in tissues. And HDL-C levels were also improved by isolie and metformin

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treatment.

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Blood insulin levels were greater in the vehicle group than in the normal control group

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(Figure 5A). After treated by isolie and metformin, the insulin levels were reduced

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significantly in KK-Ay mice. Liver enzymes, such as serum ALT and AST were increased in

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KK-Ay model mice (Figure 5G and 5H). Conversely, ALT and AST were significantly

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reduced in isolie- and metformin-treated groups.

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White adipose tissue (WAT) histological changes and western blotting assay.

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Histological analysis of the WAT and quantification of adipocytes size were shown in

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Figure 7A and Figure 7B. Correlated with higher body weight, bigger adipose cell size was

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observed in the mice of vehicle group. After treatment of isolie (30 mg/Kg), isolie (60

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mg/Kg) and metformin, the mean values of adipocyte size in three treated groups were

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significantly smaller than those from the vehicle group.

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To gain some insights into the molecular mechanism for the effect of isolie on fat

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decrease in WAT, we assessed the levels of lipogenic proteins in this tissue (Figure 7C). The

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expressions of PPARγ and SREBP-1c were higher in KK-Ay model mice than in normal



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C57BL/6J mice. After treatment of isolie and metformin, the levels of PPARγ and SREBP-1c

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were decreased significantly. The relative content of p-ACC/ACC in vehicle group was lower

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than the normal control group. But the expressions of p-ACC/ACC were increased in isolie-

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and metformin- treated groups.

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Hepatic steatosis histological changes and western blotting assay.

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The results of histological analysis of liver samples were shown in Figure 8A. As the

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results shown, the hepatocytes of KK-Ay model mice had significant vacuolation, resulting

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from the massive intracellular lipid droplets. By contrast, few lipid droplets and the reduction

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of hepatic intracellular lipid load were observed under treatment with isolie and metformin.

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In further study, the expression of PPARγ, SREBP-1c and the phosphorylation of ACC

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were detected by western blotting assay. As shown in Figure 8B, compared with the normal

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control, the expressions of PPARγ and SREBP-1c were significantly increased in the vehicle

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group. But the abnormal expressions were relieved by the treatment of isolie and metformin.

334

The level of p-ACC/ACC was decreased more significantly in the vehicle group than that in

335

the normal control, whereas isolie- and metformin-treated mice increased the protein content

336

in liver.

337

Immunohistochemical evaluation on pancreas.

338

As shown in Figure 9, insulin immunoreactivity of the pancreatic islets in the normal

339

control

group

showed

normal

pancreatic

structure

340

immunohistochemical staining areas of insulin immunoreactive β-cells in the pancreatic islets

341

in diabetic KK-Ay mice (vehicle group) were significantly decreased comparing with the

342

C57BL/6J normal group. Comparing with the vehicle control group, metformin-treated KK-

343

Ay mice presented bigger areas of insulin immunoreactive β-cells in the pancreatic islets. Be

344

consistent with the effects of metformin, both of the 60- and 30 mg/Kg dose Isolie treated

345

groups also increased the area of insulin immunoreactive β-cells in the pancreatic islets


and

size.

However,

the

Page 15 of 38


346

significantly. Our results suggested that isolie and metformin groups were capable of

347

protecting pancreatic β-cells in KK-Ay mice.

348 349

DISCUSSION

350

In the present study, we have evaluated the ameliatory effects of isolie on T2DM and

351

possible mechanisms. Isolie significantly increased the glucose uptake in L6 cells by 1.3-fold,

352

comparing with the control. In vivo studies showed that in response to isolie treatment, KK-

353

Ay mice became resistant to HFD-induced hyperinsulinemia. Moreover, our data also showed

354

that isolie suppressed body weight gains and abdominal white adipose tissue size. These

355

findings suggest that isolie can effectively improve insulin resistance and dyslipidemia in a

356

mouse model of type-2 diabetes and obesity.

357

Insulin resistance, defined as the reduced response of cells or tissues to physiological

358

levels of insulin, is a remarkable feature of T2DM.30 GLUT4 is a glucose transporter

359

expressed primarily in adipose and muscle tissues.31 Many reports have demonstrated that

360

abnormalities at this transporter level are a hallmark of peripheral insulin resistance.32 As an

361

important regulator of the GLUT4, AMP-activated protein kinase (AMPK) has been

362

extensively studied to investigate the GLUT4-reguated mechanism in T2DM research.33

363

AMPK is commonly known as a key regulator of energy balance expressed widely in

364

eukaryotic cells.34 In glucose metabolism, the phosphorylation and activation of AMPK leads

365

to GLUT4 translocation and eventually glucose uptake.33 In the present study, we found isolie

366

exhibited stronger stimulatory effects on GLUT4 translocation by 2.5-fold in L6 cells than on

367

control cells. However, the translocation was completely inhibited by a known AMPK

368

inhibitor, Compound C (Figure 2). This result inspired us to explore whether isolie stimulates

369

GLUT4 translocation in an AMPK-dependent manner. In the metabolism of glucose and

370

lipids, the phosphorylation and activation of AMPK leads to GLUT4 translocation and



371

eventually glucose uptake.35 As expected, in response to isolie, the phosphorylation of AMPK

372

in L6 cells increased significantly. In the animal experiments, we found that isolie-treated

373

mice manifested elevated phosphorylation of AMPK in skeletal muscle, WAT, and the liver.

374

At the same time, activating AMPK in skeletal muscle, WAT, and the liver can reduce the

375

insulin resistance states of the whole body, decrease the demand of insulin and make a

376

contribution to relief the burden in β-cell.36 Our immunohistochemical observation of

377

pancreas showed the isolie to have an inhibitive effective on protection of the pancreas

378

(Figure 9). Combining the findings concerning reduced serum insulin levels (Figure 5) and

379

the improvement of OGTT, the present results demonstrated that isolie could improve insulin

380

sensitivity in KK-Ay mice by activating AMPK and GLUT4.

381

Obesity is closely associated with insulin resistance. In fact, more than 90% of T2DM

382

patients are overweight or obese.37 Obesity plays a crucial role in inducing obesity-associated

383

metabolic disorders, including type-2 diabetes and cancer.38,39 Thus, relieving the obesity or

384

limiting the weight gain may be suitible strategy for treating T2DM. In the present study,

385

isolie significantly improved lipid metabolism in KK-Ay mice. After 4 weeks of treatment

386

with isolie, the body weight has been reduced remarkably (Figure 4A). The indexes of blood

387

lipid metabolism (such as HDL-C, LDL-C, FFA, TG and TC) had also obtained different

388

degree improvements (Figure 5). As the most common cause of the insulin resistant state,

389

body fat stores expand with calorie excess and progressive obesity, alterations in lipid

390

metabolism together with inflammation in adipose tissue and ectopic sites of fat deposition.40

391

In the current study, lipid accumulation in the liver and the adipocyte size and adipose mass

392

were significantly reduced in isolie-treated groups (Figure 7A and Figure 8A). These results

393

supported the conclusion that isolie could target the fundamental cause of insulin resistance

394

and thereby contributed to the overall improvement of metabolic features in T2DM animals.


Page 16 of 38

Page 17 of 38


395

Adipogenesis is accompanied by morphological and biochemical changes in adipose

396

tissue.41 PPARγ is the key transcription factor that regulates the numerous transcriptional

397

pathways involved in adipogenesis.42 PPARγ can induce the expression of C/EBPα, which

398

plays a critical role in SREBP-1c-dependent gene expression during adipogenesis.43 Many

399

reports have shown the importance of PPARγ and SREBP-1c in lipid metabolism.44 Wu et al.

400

have reported that the decrease in PPARγ and SREBP-1c limits the availability of hepatic

401

lipids.45 In this study, we found that isolie decreased the expression of PPARγ and SREBP-1c

402

in both livers and adipose tissues. These observations implicated that inhibiting the expression

403

of PPARγ and SREBP-1c was in the anti-obesity effects of isolie. These data were supported

404

by a previous study showing that the inhibition of the PPARγ and SREBP-1c reduced the lipid

405

accumulation in WAT, thereby decreasing the bodyweight gain in diabetic, obese mice.46

406

In the fatty acid metabolism, acetyl-CoA carboxylase (ACC) is a critical precursor in the

407

biosynthesis of fatty acids and a potent inhibitor of mitochondrial fatty acid oxidation in

408

controlling lipid metabolism.47 ACC is inhibited by AMPK through phosphorylation, which

409

leads to a decrease in malonyl-CoA content and an intercurrent decrease in triglyceride

410

synthesis concomitant with β-oxidation increasing.48 In the current study, we found that isolie

411

increased p-ACC and suppressed its activity, suppressing the de novo synthesis of free fatty

412

acid. Consistently, the concentrations of TG, TC, and FFA in liver were also reduced by isolie

413

treatment (Figure 6A-C). This suggested that isolie may limit hepatic lipid availability by

414

inhibiting lipogenesis, thereby down-regulating hepatic lipotoxicity markers (ALT and AST

415

decreasing in blood shown in Figure 5G and 5H). In accordance with these results in the liver,

416

the protein levels of PPARγ, SREBP1-c were decreased, and levels of phosphorylated-ACC

417

were elevated in WAT.

418

In conclusion, isolie, a purified natural benzylisoquinoline alkaloid derived from an edible

419

plant N. nucifera, effectively inhibited the hyperglycemia and hyperlipidemia in T2DM KK-



420

Ay mice. In this study, we compared our results between isolie-treated HFD induced KK-Ay

421

mice and KK-Ay mice fed with HFD without any positive drugs. And we illustrated that isolie

422

has the potential to alleviate Type 2 diabetes associated with hyperlipidemia in KK-Ay mice.

423

Isolie activated AMPK signal pathway, and enhanced the GLUT4 expression and

424

translocation in vitro and in vivo. At the same time, isolie suppressed adipogenesis by

425

inhibiting the expression of PPARγ and SREBP-1c. Isolie also decreased the de novo

426

synthesis of fatty acid through inhibiting the activity of ACC. In the previous publications,

427

isolie showed various biological activity including inducing cancer cell apoptosis.49 But in our

428

present study, no toxic effects were found in isolie-treated KK-Ay mice. In order to get more

429

evidences about the toxicity of isolie, we have performed acute toxicity of isolie in KM mice.

430

We found that the LD50 of isolie was reaching 391.5 mg/kg in KM mice (S11 in the

431

supplemental information). According to a common dosage conversion between human and

432

mice, the LD50 of isolie for KM mice was approaching 390~980 folds as same as clinical

433

daily dosage. Therefore, we can get the preliminary conclusion that isolie has no toxicity at its

434

clinical dosages. Our studies provide a novel idea that it is reasonable to consider this type of

435

natural products as a potential resource soil of anti-T2DM agents.These findings indicated

436

that, isolie has the potential to become an effective and safe agent in therapy for T2DM.

437

In addition, there are still some limitations in our present study. Although isolie exhibited

438

a distinct effect on reducing glucose level in vivo, its ability to promote glucose uptake in L6

439

cells was not significant. This nonidentity seemed to be interesting and explorable. And we

440

have investigated whether isolie takes effect on KK-Ay mice by targeting other targets. We

441

found isolie showed strong inhibitory activities of DPP-4 and α-glucosidase in vitro (Figure

442

S9). This results illustrate isolie may display significant anti-diabetic effects by targeting

443

multi-targets, and more evidences need us to explore to support this speculation.

444


Page 18 of 38

Page 19 of 38


445

ASSOCIATED CONTENT

446

Supporting Information

447

Supporting Information associated with this article includes Supplementary Materials and

448

Methods, Supplementary Figures and their captions. This material is available free of charge

449

via the Internet at http://pbs.acs.org.

450

451

AUTHOR INFORMATION

452

Corresponding Author

453

Tel./Fax: + 86 27 6784 1196. E-mail: [email protected] (Guangwen Shu);

454

[email protected] (G.Z. Yang).

455

Funding

456

This work is supported by the Natural Science Foundation of China (81573561, 81102798 and

457

31301147).

458

Notes

459

The authors declare no conflicts of interest.

460

ABBREVIATIONS USED

461

Isolie, Isoliensinine; T2DM, type 2 diabetes mellitus; GLUT4, glucose transporter 4; NPs,

462

Natural products; AMPKα, Adenosine Monophosphate Activated Protein Kinase α; p-AMPKα,

463

the Phosphorylation of AMPKα; ACC, acetyl-CoA carboxylase; p-ACC, the Phosphorylation

464

of ACC; PPARγ, peroxisome proliferator-activated receptor gamma; SREBP-1c, sterol

465

regulatory element binding protein-1c; IRAP, insulin regulation of aminopeptidase; FACS,



Page 20 of 38

466

Fluorescence Activated Cell Sorter; AICAR, 5-Aminoimidazole-4-carboxamide 1-β-D-

467

ribofuranoside;

Compound

C,

468

ylpyrazolo[1,5-a]

pyrimidinean;

Wortmannin,

469

1,6b,7,8,9a,10,11,11b-octahydro-1-(methoxy-methyl)-9a,11b-dimethyl-(3H-Furo[4,3,2-de]

470

indeno[4,5-h]-2-benzopyran-3,6,9-trione; DMSO, dimethylsulfoxide; OGTT, oral glucose

471

tolerance test; FINS, fasting serum insulin; TG, triglycerides; TC, total cholesterol; AST,

472

aspartate transaminase; ALT, alanine aminotransferase; LDL-C, low density lipoprotein

473

cholesterol; HDL-C, high density lipoprotein cholesterol; FFA, free fatty acid; WAT, white

474

adipose

tissue;

HE,

6-[4-(2-Piperidin-1-ylethoxy)phenyl]-3-pyridin-4((1S,6bR,9aS,11R,11bR)-11-(acetyloxy)-

hematoxylin


and

eosin

Page 21 of 38


475

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Page 26 of 38

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624

Figure captions

625

Figure 1 Major constituents of total alkaloids from Embryos of N. nucifera. (A) HPLC

626

chromatogram of total alkaloids from Embryos of N. nucifera (peak 1, liensinine; peak 2,

627

isoliensinine; peak 3, neferine); (B) Structure of liensinine, isolie and neferine; (C) The effects

628

of liensine, isolie and neferine on stimulating the GLUT4 translocation in vitro.

629

Figure 2 Effects of isolie in vitro. (A) Imagines of isolie stimulating the GLUT4 translocation

630

in L6 cells; (B) Date represent the fold increase in fluorescence induced by isolie with

631

inhabitors between 0 and 30 min (*P < 0.05,

632

C group); (C) Effects of isolie on increasing glucose uptake in L6 cells (++P < 0.01 compared

633

with normal control, **P < 0.01,

634

isolie on GLUT4 and p-AMPK expression in L6 cells (**P < 0.01,

635

with normal control); (E) Inhibitors change the effects of isolie on GLUT4 and p-AMPK

636

expression in L6 cells (###P < 0.001).

637

Figure 3 Investigated the molecular mechanism of isolie on improving T2DM symptom. (A)

638

Western blotting assay for the expression of GLUT4 and p-AMPK in different tissues; (B)

639

Relative intensity of GLUT4 and p-AMPK in different tissues (+++P < 0.001 compared with

640

normal control, *P < 0.05, **P < 0.01, ***P < 0.001 compared with vehicle control).

641

Figure 4 Effects of isolie on body weight, serum glucose level, OGTT and food intake. (A)

642

Effects of isolie on body weight (*P < 0.05 compared with normal control); (B) Effects of

643

isolie on food intake; (C) Effects of isolie on serum glucose level (+++P < 0.01 compared with

644

normal control, *P < 0.05,

645

Effects of isolie on OGTT (+++P < 0.001 compared with normal control, *P < 0.05, **P < 0.01,

646

***

647

Figure 5 Effects of isolie on serum biochemistryparameters. (A) Effects of isolie on serum

648

insulin level. (B-H) Effects of isolie on TC, HDL-C, FFA, TG, LDL-C, ALT and AST levels in

**

***

P < 0.001 compared with isolie + Compound

***

P < 0.001 compared with normal control); (D) Effects of

P < 0.01,

***

P < 0.001 compared

***

P < 0.001 compared with vehicle control); (D)

P < 0.001 compared with vehicle control).



Page 28 of 38

649

the serum. (+++P < 0.001 compared with normal control, *P < 0.05, **P < 0.01,

***

650

compared with vehicle control)

651

Figure 6 Effects of isolie on liver and skeletal muscle lipid levels. (A-C) Effects of isolie on

652

TC, TG and FFA levels in liver. (D-F) Effects of isolie on TC, TG and FFA levels in skeletal

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muscle. (+++P < 0.001, ++P < 0.01 compared with normal control, *P < 0.05, **P < 0.01, ***P

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< 0.001 compared with vehicle control)

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Figure 7 Chronic oral treatment of isolie decreased white adipose tissue mass (WAT) and

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adipocyte size in KK-Ay mice, and investigations on related protein expression in WAT. (A)

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Representative pictures of HE-stained WAT from each group mice; (B) Size of adipocytes

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from WAT presented as mean value (black dot) and distribution of section area of each

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individual adipocyte from HE-stained histological picture (+++P < 0.001 compared with

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normal control,

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SREBP-1c, p-ACC expression in WAT; (D) Relative band intensity of PPARγ, SREBP-1c and

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p-ACC (+++P < 0.001 compared with normal control, *P < 0.05,

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vehicle control).

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Figure 8 Chronic oral treatment of isolie prevented hepatic steatosis in KK-Ay mice, and

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investigations on related protein expression in liver. (A) Representative pictures of HE-stained

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liver from each group mice; (B) Effects of isolie on PPARγ, SREBP-1c and p-ACC

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expression in liver; (C) Relative band intensity of PPARγ, SREBP-1c and p-ACC (+++P

Activity of Isoliensinine in Improving the Symptoms of Type 2

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