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Bioactive Constituents, Metabolites, and Functions

Cottonseed Extracts and Gossypol Regulate Diacylglycerol Acyltransferase Gene Expression in Mouse Macrophages Heping Cao, and Kandan Sethumadhavan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01240 • Publication Date (Web): 29 May 2018 Downloaded from http://pubs.acs.org on May 29, 2018

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Journal of Agricultural and Food Chemistry

Cottonseed Extracts and Gossypol Regulate Diacylglycerol Acyltransferase Gene Expression in Mouse Macrophages

Heping Cao*, Kandan Sethumadhavan

U.S. Department of Agriculture, Agricultural Research Service, Southern Regional Research Center, New Orleans, LA 70124, USA

* To whom correspondence should be addressed. Tel: 504-286-4351. Fax: 504-286-4367. E-mail: [email protected].

Running Title: Cottonseed extracts and gossypol regulate DGAT gene expression

Manuscript Correspondence: Heping Cao, PhD USDA-ARS-SRRC 1100 Robert E. Lee Blvd New Orleans, LA 70124 Phone: (504) 286-4351 E-mail: [email protected]

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ABSTRACT

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Plant bioactive polyphenols have been used for the prevention and treatment of various diseases

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since ancient history. Cotton (Gossypium hirsutum L.) seeds are classified as glanded or glandless

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depending on the presence or absence of pigment glands, which contain polyphenolic gossypol.

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Diacylglycerol acyltransferases (DGATs) are integral membrane proteins that catalyze the last

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step of triacylglycerol biosynthesis in eukaryotes. Understanding the regulation of DGATs will

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provide information for therapeutic intervention for obesity and related diseases. However, little

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was known if DGAT gene expression was regulated by natural products. The objective of this

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study was to investigate the effects of cottonseed extracts and gossypol on DGAT gene expression

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in mouse RAW264.7 macrophages. Mouse cells were treated with different concentrations of

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cottonseed extracts, gossypol, and lipopolysaccharides (LPS) for various times. Quantitative PCR

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assay showed that coat extract of glanded seeds had a modest effect on DGAT1 and minimal effect

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on DGAT2 mRNA levels. Kernel extract of glanded seeds had a minimal effect on DGAT1 but

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increased DGAT2 mRNA levels more than 20-fold. Coat extract of glandless seeds and LPS had

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minimal effects on DGAT mRNA levels. Kernel extract of glandless seeds did not have much

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effect on DGAT1 and slightly increased DGAT2 mRNA levels. Gossypol increased DGAT1 and

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DGAT2 mRNA levels by up to 3-fold and more than 80-fold, respectively. The coefficient

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correlation (R2) between DGAT2 mRNA levels and glanded kernel extract and gossypol

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concentrations were 0.82-0.99. This study suggests that Dgat2 is an inducible gene rapidly

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responding to stimulators such as polyphenols whose protein product DGAT2 plays an important

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role in fat biosynthesis. We conclude that gossypol and ethanol extract from glanded cottonseed

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kernel are strong stimulators of DGAT2 gene expression and that they may be novel agents for

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intervention of lipid-related dysfunction via increasing DGAT2 gene expression in target tissues.

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Keywords: Cottonseed extract, diacylglycerol acyltransferase, gene expression, gossypol,

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lipopolysaccharides, macrophages

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INTRODUCTION

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Cotton (Gossypium hirsutum L.) plant provides two economically important products: fiber and

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cottonseed. Cottonseeds account for 20% of the crop value. Cottonseeds are classified as either

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glanded or glandless seeds depending on the presence or absence of pigment glands, which contain

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toxic gossypol (Figure 1).1 Glanded cottonseeds are composed of 10% linters, 40% hulls and 50%

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kernels.2 The kernels contain about 35% oil and 40% protein.3 Commercial cottonseed meal

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contains approximately 1% gossypol after oil extraction.4 The presence of gossypol limits its use

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of meals primarily to feed ruminants, which have a relatively high tolerance for the compound.5-7

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Glandless cottonseeds do not contain pigment glands and have only trace levels of gossypol,

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making them a good source of protein as a food ingredient or as a feed for non-ruminant

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animals.8-10 This development has generated considerable interest within the cotton industry.11-13

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Glanded cottonseeds contain a number of bioactive components, such as gossypol, gallic acid,

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and 3,4-dihydroxybenzoic acid.14 Gossypol is a well-studied complex polyphenol found in the

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small intercellular pigment glands in the leaves, stems, roots, and seed of cotton plants (Figure

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1).15 Gossypol and related compounds have anticancer activities against breast cancer,16 colon

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cancer,17 pancreatic cancer,18 and prostate cancer.19 It has additional bioactivities such as

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antiobesity,20 antiinflammatory,21 and antifungal activities.22 Gossypol has also been proposed as

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an antifertility agent for males.23 These discoveries have created much excitement in biomedical

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field and enormous amounts of research have been focused on understanding the medicinal

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properties of gossypol and related compounds. However, chronic consumption of

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gossypol-containing cottonseed oil can result in human male infertility.5 Therefore, gossypol is

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regarded as unsafe for most animals and humans. Significant efforts have been directed at reducing

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gossypol content in cottonseed by selecting glandless cotton varieties

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engineering of cotton to provide gossypol-free seeds.11,12

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and genetic

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Minor bioactive components have been studied in glandless cottonseed with flavonol

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glycosides (flavonoids) being the best-studied. Five flavonoids have been identified in glandless

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cottonseed.25,26 Seven flavonol glycosides have also been identified from whole cottonseed.14 It

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was reported that aqueous extracts from glandless cottonseed meal had an antidepressant effect

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due to the flavonol glycoside, quercetin.26 This compound was shown to have antidepressant

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effects in pharmacological tests27,28 with potential applications in treating anxiety, depression and

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Alzeheimer’s disease.29 Independent studies have confirmed that flavonoids could be used as a

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therapy for depression associated with diabetes30 and under other conditions.31 These studies

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suggest that bioactive components in glandless cottonseed could be value-added agricultural

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products in cottonseed with health promotion and disease prevention potentials.

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Triacylglycerols (TAGs) are the primary form of energy storage in eukaryotes. They also serve

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as a reservoir of fatty acids for membrane biogenesis of the cells and can lead to obesity when

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excessively accumulated in adipose tissues.32,33 Diacylglycerol acyltransferases (DGATs) esterify

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sn-1,2-diacylglycerol with a long-chain fatty acyl-CoA and are responsible for the final and

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rate-limiting step of TAG biosynthesis in eukaryotic organisms.34 It is generally accepted that

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DGATs are divided into DGAT1 and DGAT2 subfamilies in animals and that additional DGAT3

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subfamily are present in plants.34-37 Plants and animals deficient in DGATs accumulate less

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TAG.38-40 Animals with reduced DGAT activity are resistant to diet-induced obesity,39,41 lack milk

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production39 and maybe subject to a congenital diarrheal disorder.42 Over-expression of the DGAT

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enzymes increases TAG content in plants,43-45 animals46-48 and yeast.49 These genetic studies have

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demonstrated that DGAT isoforms have non-redundant functions in TAG biosynthesis in species

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such as mice40 and tung tree.37 Therefore, understanding the regulation of DGATs will help in the

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development of transgenic plants and microbes with value-added properties and provide

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information for therapeutic intervention for obesity and related diseases. It is well-known about

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the number of DGAT isoforms and their expression profiles in some plants and animals, however,

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little is known if DGAT gene expression is regulated by natural products.34,35

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Plant flavanol and polyphenol compounds have potential therapeutic benefits. For example,

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cinnamon polyphenols have been shown to improve lipid profiles of diabetic people.50 We

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hypothesized that cottonseed may contain novel polypehnol compounds, which may be useful for

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nutritional and/or medical purposes. The specific objective of this study was to test if extracts from

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cottonseed have the capability to regulate DGAT gene expression in mammalian cells. We

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investigated the regulation of DGAT gene expression by ethanol extracts isolated from glanded

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and glandless cottonseed and compared directly with gossypol and lipopolysaccharides (LPS)

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using cultured mouse macrophages.

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MATERIALS AND METHODS

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Cottonseed. Glanded and glandless cottonseeds were collected from variety “DP1321” and

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“NuMex 15 GLS”, respectively.13 The long fibers in the cotton bolls were separated from the seeds

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by ginning. The short fibers on the seeds (also called linters) were removed from the seeds with

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concentrated sulfuric acid (Sigma, St. Louis, MO). The seeds were washed with distilled water

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extensively followed by neutralizing any residual acid with sodium bicarbonate solution (Sigma).

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Glanded seeds were smaller than glandless seeds and contained numerous dark-colored

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gossypol-rich glands in the kernel (see Figure 1A).

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Cottonseed Extracts. Ethanol extracts were isolated from the coat and kernel of glanded and

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glandless cottonseeds according to a protocol for enriching polyphenolic compounds from

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cinnamon bark.51,52 The protocol consisted of three steps for seed kernel extraction (fractionation,

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defatting, and ethanol extraction) and four steps for seed coat extraction (fractionation, defatting,

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acetic acid extraction, and ethanol extraction). Briefly, glanded and glandless cottonseeds were

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fractionated into seed coat and kernel by dry grinding and then in a homogenization buffer (50 mM

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Tris-HCl and 150 mM NaCl, pH 7.4). The kernel fractions were defatted by stirring in an equal

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volume of chloroform (Sigma) followed by centrifugation.14 An equal volume of hexane (Sigma)

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was added to the upper (aqueous) layer of chloroform extraction followed by mixing and

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centrifugation. The lower (aqueous) layer of hexane extraction was used for ethanol extraction.

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The seed coat fraction was suspended in acetic acid (Sigma), autoclaved and centrifuged before

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ethanol extraction.52 Four volumes of ethanol (Sigma) were added to the kernel and coat fractions

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followed by centrifugation and drying under rotoevaporation (Integrated SpeedVac System,

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Thermo Fisher) until all ethanol was evaporated. The seed kernel extract was much darker in color

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than the seed coat extract (see Figure 1B).

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Cell Culture. Mouse RAW264.7 macrophages were purchased from American Type Culture

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Collection (Manassas, VA). The basic cell culture protocol followed previous procedures using a

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water jacket CO2 incubator (Forma Series II, Model 3100 Series, Thermo Fisher) and in a Logic+

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A2 hood (Labconco, Kansas City, MO).52,53 Mouse RAW264.7 macrophages were maintained as

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described54 in 75 cm2 polystyrene tissue culture flasks at 37 °C with 5% CO2 in Dulbecco’s

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modified Eagle’s medium (DMEM) containing 4.5 mg/ml (25 mM) glucose supplemented with

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10% (v:v) fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM

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L-glutamine (Gibco BRL, Thermo Fisher). RAW macrophages were mechanically dissociated

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from the T-75 flask with a cell scraper, stained with equal volume of 0.4% trypsin blue dye before

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counting the number of live cells with a TC20 Automatic Cell Counter (Bio-Rad, Hercules, CA).

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RAW macrophages were routinely observed before and during treatment with a Zoe Florescent

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Cell Imager (Bio-Rad).

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Chemical Treatment. Mouse RAW264.7 macrophages were sub-cultured at approximately 1

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x 105 cells/ml density in 24-well tissue culture plates (CytoOne, USA Scientific, Ocala, FL) (0.5

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ml/well). These cells were treated with 0, 10, 20, 30, 40, 50, and 100 µg/ml of ethanol extracts

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from cottonseed, 0, 0.5, 1, 5, 10, 50, and 100 µg/ml of gossypol (Sigma), and 0, 10, 20, 50, 100,

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500, and 1000 ng/ml of LPS (Sigma). The treatments lasted for 2 h, 8 h and 24 h (“0” treatment

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corresponding to 1% DMSO) in the culture medium, the same DMSO concentration used to

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suspend the chemicals followed by RNA extraction.

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RNA Extraction. Mouse cells in 24-well plates were washed twice with 1 ml of 0.9% NaCl

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and lysed directly with 1 ml of TRIZOL reagent (Invitrogen). RNA was isolated according to the

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manufacturer’s instructions. RNA concentrations and integrity were determined using RNA 6000

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Nano Assay Kit and the Bioanalyzer 2100 according to the manufacturer’s instructions (Agilent

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Technologies, Palo Alto, CA, USA) with RNA 6000 Ladder as the standard (Ambion, Inc., Austin,

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TX, USA)55. RNA concentrations were also quantified with an Implen NanoPhotometer

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(Munchen, Germany).

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Quantitative Real-time PCR Analysis. The basic procedure of SYBR Green qPCR assay

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was according to the MIQE guidelines: minimum information for publication of quantitative

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real-time PCR experiments.56 PCR primers and TaqMan probes (Biosearch Technologies, Inc.,

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Petaluma, CA) were designed using Primer Express software (Applied Biosystems, Foster City,

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CA). The Tms of the primers were variable to a few degrees but the Tms for the probes were

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generally 10 degrees higher than the corresponding primers. The amplicon sizes and the nucleotide

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sequences (5' to 3') of the forward primers, TaqMan probes (TET – BHQ1) and reverse primers,

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respectively, are described in Table 1. The cDNAs were synthesized from total RNA using

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SuperScript II reverse transcriptase (Life Technologies). The cDNA synthesis mixture (20 µl)

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contained 2.5 µg total RNA, 2.4 µg oligo(dT)12-18 primer, 0.1 µg random primers, 500 µM dNTPs,

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10 mM DTT, 40 units RNaseOUT and 200 units SuperScript II reverse transcriptase in 1X

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first-strand synthesis buffer (Life Technologies). The cDNA synthesis reactions were incubated at

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42°C for 50 min. The cDNA samples were stored in -80°C freezer and diluted with water to 1

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ng/µl before qPCR analyses.

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SYBR Green qPCR reaction mixtures contained 5 ng of total RNA-derived cDNAs, forward

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primer, reverse primer and 1X iQ SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA).

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The thermal cycle conditions were as follows: 3 min at 95°C, followed by 40 cycles at 95°C for 10

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s, 65°C for 30 s and 72°C for 30 s. The qPCR reactions were performed in 96-well clear plates

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sealed by adhesives with a CFX96 real-time system-C1000 Thermal Cycler (Bio-Rad

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Laboratories).

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Data Analysis. The ∆∆CT method of relative quantification was used to determine the fold

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change in gene expression (refer to Table 2 as an example of calculation).57 First, the cycle of

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threshold (CT) was obtained from three independent samples. Second, the first delta CT value

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(∆CT) was obtained by subtracting the CT values of the internal reference control, mouse 60S

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ribosome protein 32 (Rpl32) from the CT values of the target mRNA (∆CT = CTTarget - CTref). Third,

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the second delta CT value (∆∆CT) was obtained by subtracting the ∆CT of the calibrator (DMSO

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control) from the ∆CT of the target mRNAs (∆∆CT= ∆CTTarget - ∆CTcal). Finally, the fold change in

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expression was obtained using the equation 2-∆∆CT.

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Statistics.

The data in the figures represent the mean and standard deviation of three

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independent samples. They were analyzed by statistical analysis using ANOVA with SigmaStat

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3.1 software (Systat Software). Multiple comparisons among the treatments with different time or

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concentrations

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Student-Newman-Keuls Method.52 Different lower case letters displayed above each of the

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treatment concentration on the figures are significantly different between the treatment times at p

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