A Limonoid 7-Deacetoxy-7-Oxogedunin from Andiroba, Carapa

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Bioactive Constituents, Metabolites, and Functions

A Limonoid 7-Deacetoxy-7-Oxogedunin from Andiroba, Carapa guianensis, Meliaceae Decreased Body Weight Gain, Improved Insulin Sensitivity, and Activated Brown Adipose Tissue in High-fat Diet-fed Mice Chihiro Matsumoto, Toko Maehara, Reiko Tanaka, and Ko Fujimori J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b04362 • Publication Date (Web): 21 Aug 2019 Downloaded from pubs.acs.org on August 25, 2019

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Journal of Agricultural and Food Chemistry

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A Limonoid 7-Deacetoxy-7-Oxogedunin from Andiroba, Carapa guianensis,

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Meliaceae Decreased Body Weight Gain, Improved Insulin Sensitivity, and

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Activated Brown Adipose Tissue in High-fat Diet-fed Mice

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Chihiro Matsumoto#, Toko Maehara#, Reiko Tanaka$, and Ko Fujimori#,*

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# Department

Nasahara, Takatsuki, Osaka 569-1094, Japan

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of Pathobiochemistry, Osaka University of Pharmaceutical Sciences, 4-20-1

$ Department

of Medicinal Chemistry, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan

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* To whom correspondence should be addressed: Ko Fujimori, Ph.D.

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Department of Pathobiochemistry, Osaka University of Pharmaceutical Sciences,

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4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan

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Phone and Fax: +81-72-690-1215, E-mail: [email protected]

1 ACS Paragon Plus Environment


ABSTRACT

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We examined the anti-obesity effect of a limonoid 7-deacetoxy-7-oxogedunin, named CG-1

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purified from the seeds of Carapa guianensis, Meliaceae, known as andiroba in high-fat diet

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(HFD)-fed mice. C57BL/6 mice were fed a low-fat diet or an HFD, and orally administered

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CG-1 (20 mg/kg) for 7 weeks. CG-1 lowered body weight gain and improved serum

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triglyceride level and insulin sensitivity in HFD-fed mice. The expression level of the

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adipogenesis-related genes was lowered by CG-1 in visceral white adipose tissue (vWAT).

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The mRNA expression level of the macrophage-related genes decreased in vWAT following

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administration of CG-1 to HFD-fed mice. Noteworthly, CG-1 activated brown adipose tissue

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(BAT) with enhanced expression of uncoupling protein 1 and increased rectal temperature in

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HFD-fed mice. These results indicate that a limonoid CG-1 decreased body weight gain and

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ameliorated hypertriglyceridemia and insulin resistance with activation of BAT in HFD-fed

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mice.

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Keywords: andiroba, limonoid, insulin resistance, obesity, BAT

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INTRODUCTION

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Obesity is currently growing epidemic worldwide 1 and represents the complex interaction of

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genetic, behavioral, and environmental effects 2. Obesity is closely related to the occurrence

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of a variety of metabolic diseases such as hypertension, hyperlipidemia, type 2 diabetes

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mellitus, and cardiovascular disease

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chronic inflammation state through infiltrating and activating macrophages in obese adipose

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tissue, which finally leads to insulin resistance and glucose intolerance 5-8.

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1, 3, 4.

It is also known that obesity is associated with the

These are two different types of adipose tissue: white adipose tissue (WAT) that stores 9

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energy as triglyceride (TG)

and brown adipose tissue (BAT) that burns energy for

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thermogenesis 10. Adipogenesis in WAT is regulated via a coordinated transcription cascade 11.

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Peroxisome proliferator-activated receptor (PPAR) γ, CCAAT/enhancer binding proteins

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(C/EBPs), and Krüppel-like factors (KLFs) play key roles in the control of adipogenesis

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through regulation of various adipogenesis-associated proteins. In contrast, BAT oxidizes

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intracellular lipids to produce heat through the activation of the thermogenesis-related

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proteins such as uncoupling protein 1 (UCP1) 10, 12. Thus, both adipose tissues play important

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roles in the control of energy homeostasis.

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Presently, overeating, especially intake of fats, is a critical problem worldwide, because

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it promotes obesity and the occurrence of the obesity-triggered metabolic diseases. A variety

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of therapeutic methods for the prevention and treatment of obesity such as diet modification,

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exercise, surgery, and pharmacotherapy are now available 13. Although the pharmacotherapy

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is more common, some synthetic medicines fail to produce the desired effects or show

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unexpected side effects

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natural products are used for treatment of obesity by preventing weight gain or promoting

14, 15.

Some traditional medicines and constituents derived from



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weight loss 16, 17. One of the clinical benefit of natural products is that the effects are generally

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mild, but they show fewer side effects 17.

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Limonoids are phytochemicals of the triterpenoids that are abundant in the Rutaceae

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and Meliaceae plant families 18-20. Limonin and nomilin are limonoids and contained plenty in

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citrus that is a member of the Rutaceae family

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abilities such as anti-cancer, anti-malarial, and anti-microbial activities

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several limonoids such as nomilin 21, obacunone 22, ceramicine B 23, and kihadanin B 24 have

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anti-adipogenic or anti-obesity effects. However, there are few in vivo analyses of the

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biological effects of limonoids on metabolic diseases.

18-20.

Limonoids have several biological 18-20.

Moreover,

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Carapa guianensis known as andiroba is a large neotropical tree, belonging to the

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Meliaceae family. Andiroba is grown in the north of South America, Central America,

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Sub-Saharan Africa, and the Amazon region in Brazil 25, 26. The seed oil of C. guianensis has

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been used as a natural medicine for treating allergies

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Andiroba seeds contain abundant limonoids

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CG-1 as the major limonoid 30. We recently found that a limonoid CG-1 has anti-adipogenic

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effects in 3T3-L1 adipose cells 31. In the present study, we examined an anti-obesity effect of

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a limonoid CG-1 of andiroba in high-fat diet (HFD) -fed obese mice.

29,

27,

cancer

28,

and inflammation

27.

including 7-deacetoxy-7-oxogedunin, named

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MATERIALS AND METHODS

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Purification of A Limonoid CG-1 from Seeds of Andiroba

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A limonoid CG-1 (Figure 1, inset) was purified from andiroba seeds in previous study

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Prior to the initiation of this study, we confirmed the purity of CG-1 using HPLC (Figure 1;

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JASCO, Tokyo, Japan) with acetonitrile:H2O (60:40) as the mobile phase and the

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COSMOSIL 5C18-MS column (Nacalai Tesque, Kyoto, Japan). The purity of CG-1 was

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calculated using a JASCO 807-IT integrator (JASCO). Furthermore, the chemical structure of

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CG-1 was determined using nuclear magnetic resonance (data not shown).

32.

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Animals

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Mice (C57BL/6J, male, 6-week-old; Japan SLC, Shizuoka, Japan) were maintained in

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temperature (24 °C)-

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randomly divided into four groups [n=10 for each HFD group and n=10 for each low-fat diet

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(LFD) group]. These groups were orally administered CG-1 (20 mg/kg) that was suspended in

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0.5%(w/v) methyl cellulose 400 (FUJIFILM Wako Pure Chemicals, Osaka, Japan) or

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0.5%(w/v) methyl cellulose 400 alone (vehicle), respectively, each other day. They were

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designated as CG-1/LFD, CG-1/HFD, vehicle/LFD, and vehicle/HFD groups, respectively

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(n=10). Each group was fed either an LFD (FR-2, 4.8% fat; Funabashi Farm, Chiba, Japan) or

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HFD (D12492; 35% fat; Research Diet, New Brunswick, NJ, USA). The formulation of LFD

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and HFD was shown in Supplemental Table 1. Body weight was measured once a week. The

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mice were given free access to food and water. All animal studies were performed according

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to the guideline of the Animal committee of Osaka University of Pharmaceutical Sciences.

and light (12 h light-12 h dark cycle)-controlled room. The mice were

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Measurement of mRNA Expression Level

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RNA extraction of and first-strand cDNA synthesis were done as described previously

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Messenger RNA levels were measured using quantitative PCR (qPCR) using Power SYBR

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Green PCR Master Mix (Thermo Fischer Scientific, Waltham, MA, USA) and gene-specific

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primers (Table 1) in an ABI 7500 Real-Time PCR System (Thermo Fischer Scientific), The

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expression level was analyzed using the 2-ΔΔCt method and normalized to TATA-binding

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protein (TBP) mRNA.

33.

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Western Blot Analysis

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Protein was isolated from tissues as described previously 34. A Pierce BCA Protein Assay Kit

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(Thermo Fisher Scientific) was used to determine protein concentrations. Proteins were

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separated on SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Immobilon;

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Merck Millipore, Whitehouse Station, NJ, USA). The membranes were then blocked using

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Blocking One (Nacalai Tesque), and subsequently incubated with the following primary

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antibodies: anti-uncoupling protein (UCP) 1 (U6382) and anti-glucose transporter (GLUT) 4

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(G4048) polyclonal antibodies and anti-β-actin monoclonal antibody (AC-15; Sigma, St.

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Louis, MO, USA), anti-CCAAT/enhancer-binding protein (C/EBP) α polyclonal antibody

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(#2295; Cell Signaling, Danvers, MA, USA), anti-fatty acid synthase (FAS; H-300), and

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anti-stearoyl-CoA desaturase (SCD) polyclonal antibodies (Santa Cruz Biotechnology, Dallas,

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TX,

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horseradish-peroxidase-conjugated secondary antibodies (Santa Cruz Biotech., Dallas, TX,

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USA), immunoreactive signals were visualized by an ECL Prime Western Blotting Detection

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Reagent (GE Healthcare, Buckinghamshire, UK) and an LAS-3000 Lumino Image Analyzer

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(FUJIFILM, Tokyo, Japan), and analyzed using a Multi-Gauge software (FUJIFILM).

USA).

After

incubation

of

the

membranes


with

the

respective

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Serum Biochemical Parameter

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Mice were fasted for 16 h, and blood samples were collected from the abdominal aorta.

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Serum insulin, triglyceride (TG), glucose, total cholesterol (CHO), low-density lipoprotein

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(LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, and non-esterified fatty acid

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(NEFA) levels were measured using the respective LBIS Mouse Insulin ELISA KIT (RTU;

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FUJIFILM Wako Shibayagi, Gunma, Japan), L-Type TG M, GLUCOSE, CHO M, L-Type

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LDL-C, L-Type HDL-C, and NEFA Kits (FUJIFILM Wako Pure Chemicals).

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Insulin Tolerance Test (ITT)

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Mice were fasted for 16 h, and then intraperitoneally injected HUMULIN® (0.75 IU/kg body

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weight; Eli Lilly, Indianapolis, IN, USA). The measurement of glucose levels were carried

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out using blood samples that prepared from tail vein at 0, 15, 30, 60, and 120 min after the

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injection by the use of a MEDISAFE MINI Blood Glucose Monitoring System (Terumo,

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Tokyo, Japan).

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Oral Glucose Tolerance Test (OGTT)

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Mice were fasted for 16 h before orally administrating glucose (2 g/kg body weight). Glucose

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level in blood samples from the tail vein was measured with a MEDISAFE MINI Blood

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Glucose Monitoring System (Terumo) at 0, 15, 30, 60, and 120 min after administration.

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OGTT was carried out using the same mice as those used in ITT in different weeks.

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Statistical Analysis

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Dara were shown as means ± S.D. or S.E. Results were analyzed using one way ANOVA, and



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significance was analyzed by Tukey’s test. p

A Limonoid 7-Deacetoxy-7-Oxogedunin from Andiroba, Carapa

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