Inhibitory Effect of Methyl 2-(4â²-Methoxy-4â²-oxobutanamide

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Inhibitory effect of methyl 2-(4#-methoxy-4#-oxobutanamido)benzoate from Jerusalem Artichoke (Helianthus tuberosus) on inflammatory paracrine loop between macrophages and adipocytes Yun Joo Jung, Byung Oh Kim, Jong Hwan Kwak, and Suhkneung Pyo J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03407 • Publication Date (Web): 25 Nov 2016 Downloaded from http://pubs.acs.org on December 4, 2016

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Journal of Agricultural and Food Chemistry

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Inhibitory effect of methyl 2-(4′-methoxy-4′-oxobutanamide) benzoate from Jerusalem

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Artichoke (Helianthus tuberosus) on inflammatory paracrine loop between

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macrophages and adipocytes

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Yun Joo Junga, Byung Oh Kimb, Jong Hwan Kwaka,*, Suhkneung Pyoa,*

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a

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b

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Kyungpook National University, Daegu 41566, Republic of Korea.

School of Pharmacy, Sungkyunkwan University, Suwon 16419, Republic of Korea. School of Food Sciences & Biotechnology, College of Agriculture & Life Sciences,

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* Corresponding authors. S. Pyo: Tel: +82-31-290-7713; E-mail: [email protected] and J. H. Kwak: Tel: +82-31-290-7745; Fax: +82-31-292-8800; E-mail: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT

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The interaction between macrophages and adipocytes is known to aggravate inflammation of

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the adipose tissue, leading to decreased insulin sensitivity. Hence, attenuation of the

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inflammatory paracrine loop between macrophages and adipocytes is deemed essential to

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ameliorate insulin resistance and diabetes mellitus type 2. Methyl 2-(4′-methoxy-4′-

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oxobutanamide) benzoate (compound 1), a newly isolated compound from Jerusalem

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Artichoke (JA), has not been biologically characterized yet. Here, we investigated whether

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JA-derived compound 1attenuates inflammatory cycle between Raw 264.7 macrophages and

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3T3-L1 adipocytes. Compound 1 suppressed inflammatory response of Raw264.7 cells to

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lipopolysaccharide through decreasing the secretion of IL-1β, IL-6 and TNF-α. Moreover, the

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mRNA expression of TNF-α, IL-6, IL-1β, MCP-1 and Rantes, and MAPK pathway activation

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in 3T3-L1 adipocytes, incubated in macrophage-conditioned media, were inhibited. These

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findings suggest an anti-inflammatory effect of a newly-extracted compound against adipose

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tissue inflammation and insulin resistance.

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KEYWORDS:

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lipopolysaccharides, macrophage-conditioned media, inflammation, insulin resistance,

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obesity

Helianthus

tuberosus,

Jerusalem

Artichoke,

39 40 41 42 2


3T3-L1,

Raw

264.7,

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INTRODUCTION

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Obesity is one of the biggest problems worldwide as it linked to low-grade chronic

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inflammation and is significantly more susceptible to hypertension, coronary artery disease,

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insulin resistance and diabetes type 2 (T2D).1-3 Many studies revealed that the chronic

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inflammation of adipose tissue is not only caused by the increased production of interleukin-

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1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α), but also Regulated on Activation,

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Normal T cell Expressed and Secreted (Rantes) and monocyte chemoattractant protein-1

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(MCP-1). These cytokines are responsible for the inflammation of adipocytes and adipose

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tissue and the infiltration of adipose tissue by T cells and macrophages.4-7It was also

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demonstrated that the interplay of cytokines between inflammatory macrophages and

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adipocytes aggravates adipose tissue inflammation, leading to resistance to insulin action and

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pathogenesis of T2D.8

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It is commonly accepted that inflammatory response in adipose tissue is deeply involved

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with the infiltration of macrophages and infiltrated macrophage activation, resulting in insulin

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resistance.9 Hence, suppressing the inflammatory state of adipose tissue macrophages is key

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to attenuate the vicious paracrine loop between adipose macrophages and adipocytes to

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improve insulin resistance.8 In addition, adipose tissue macrophages are not only activated by

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adipokines from surrounding adipocytes, but also by endotoxins.9 A study demonstrated that

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patients with obesity, insulin resistance and/or T2D had an increased level of plasma

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lipopolysaccharide (LPS), 4 hours after the ingestion of a high fat-diet, compared with

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healthy individuals.10 Other studies also revealed that high-fat feeding caused an increase in

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LPS-containing microbiota level in the gut and the released LPS is fused into intestinal

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capillaries via a Toll-like Receptor 4 (TLR4)-mediated process.11 Additionally, it has been 3



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documented that TLR4 activation by LPS contributes to adipose tissue inflammation, leading

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to resistance to insulin action and obesity.12, 13 Accordingly, the inflammatory response to

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LPS contributes to adipose tissue inflammation.

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A plethora of studies have proven that mitogen-activated protein kinases (MAPKs) and

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phosphoinositide 3-kinase (PI3k)/Protein kinase B (Akt) pathway signaling are highly

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involved in inflammation and their activation eventually causes nuclear factor-kappa B (NF-

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κB) activation.14,15 Activating NF-κB in macrophages provokes the upregulation of

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proinflammatory cytokines after exposure to endotoxin like LPS.16 Additionally, it has been

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demonstrated by several studies that AMP-activated protein kinase (AMPK) negatively

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controls inflammation. AMPK activation is brought about by IL-10 which has an anti-

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inflammatory property, whereas it is downregulated by LPS in macrophages.17,18 A well-

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known AMPK downstream is silent information regulator transcript-1 (SIRT1) which has an

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anti-inflammatory effect on macrophages in response to LPS.19-21

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Helianthus tuberosus L. is also called Jerusalem artichoke (JA) which is widely used in

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diet and is known as indigenous medicine for treatment of diabetes and rheumatism.22 Recent

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studies have indicated that JA-derived compound, germacranesesquiterpene lactones, has

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various effects, including anti-cancer, cytotoxic, anti-inflammatory and anti-microbial

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effects.23-25 Furthermore, it is shown that JA was able to prevent the onset of nonalcoholic

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fatty liver disease and T2D in vivo and both diseases are closely related with chronic

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inflammation, implicating JA is probably involved in inflammation.26 Thus, it is noteworthy

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to investigate how a JA-derived compound is responsible for its bioactive properties and

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improve one’s health state.

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Here, we demonstrated a compound, extracted from H. tuberosus is able to suppress LPS4


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induced inflammation of macrophages, hence, the level of inflammatory adipokines from

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adipocytes is reduced, highlighting an anti-obesity/inflammatory effect of the JA-derived

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compound.

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MATERIALS AND METHODS

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Experimental Process. NMR spectra were acquired on a Bruker AVANCE Ⅲ 700

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spectrometer (Ettlingen, Germany) with the usual pulse sequence, and chemical shifts(δ) are

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measured in parts per million, referenced to the solvent used. DART-MS data was obtained

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on a JMS T100LP 4G High Resolution TOF LC/MS with DART ion source (JEOL, Japan).

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Column chromatography was performed on LiChroprep RP-18 (40-63 µm, Merck, Armstadt,

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Germany) and Silica gel 60 (230-400 mesh, Merck). Thin layer chromatography (TLC) was

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carried out on TLC Silica gel 60 RP-18 F254s and TLC Silica gel 60 F254 (Merck) plates.

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HPLC was performed with a Knauer Smartline system (Knauer Advanced Scientific

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Instruments, Berlin, Germany) using a phenomenex 5µ C18 100A Kinetex column (150 X

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4.6 mm).

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Extraction and Isolation procedure. Tubers of Helianthus tuberosus were purchased from a

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market (NH NonghyupHanaro Mart, Suwon, Korea) in October 2014. The tubers of H.

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tuberosus were lyophilized at -50℃ for 30 hr. The dried tubers (1.15 kg) were percolated

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twice with methanol (MeOH) at 20℃, and once with MeOH at 60℃. All extracts were

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pooled and the solvent was vaporized under decreased pressure to generate a MeOH extract

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(MeOH Ex., 83.6 g). After MeOH extraction of the tuber, the remaining specimen was

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extracted twice with distilled water at 100℃ under reflux. The filtrate was lyophilized to

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give a water extract (H2O Ex., 518.2 g). The MeOH extract (in H2O 800 mL) was serially

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fractionated with dichloromethane, ethyl acetate, and n-butanol. Each of the fractions was

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concentrated to give ethyl acetate (EtOAc Fr., 1.25 g), dichloromethane (CH2Cl2 Fr., 3.36 g),

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n-butanol (n-BuOH Fr., 3.91 g) and water (H2O Fr.,72.16 g) fractions. Among these fractions,

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CH2Cl2 fraction was chromatographed on Si gel column using a sequential elution with 6


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hexane-CH2Cl2 (1:1), CH2Cl2, hexane-CH2Cl2-MeOH (10:10:0.3, 10:10:1, and 10:10:2), and

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CH2Cl2-MeOH (2:1) to give nine subfractions (MC-1 to MC-9). Fraction MC-6 was

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rechromatographed over a silica gel column (hexane-EtOAc, 3:1 and 1:1), and the resulting

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fraction was applied to a RP-C18 column (60% MeOH) to obtain compound 1 (12.5 mg). The

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purity of compound 1 was determined by reversed phase C18 HPLC analysis with a gradient

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elution. The eluent was comprised of MeOH (A) and water (B). The gradient profile was: 0–5

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min, isocratic 30% A in B; 5–25 min, linear change from 30% to 60%A in B; 25–26 min,

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from 60% to 100% A in B; 26–30 min, isocratic 100% A in B. The flow rate and column

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oven temperature were set at 1 mL/min and 45°C. The UV absorption was determined at a

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wavelength of 254 nm.

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Chemicals and Reagents. Dulbecco’s Modified Eagle’s Medium (DMEM) was purchased

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from Corning Life Sciences (MA, USA). Bovine Calf Serum (BCS), Fetal Bovine Serum

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(FBS) and Penicillin/Streptomycin were purchased from Gibco (ThermoFisher scientific,

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Waltham, MA). [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT dye),

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3-isobutyl-1-methylanthine

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lipopolysaccharide (LPS) were from Sigma-Aldrich Chemical Co. (St Louis, MO).

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Antibodies for anti-mouse p-PI3k, anti-mouse Iκb-α, anti-mouse p65, anti-mouse Lamin A,

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anti-mouse α-tubulin, anti-mouse p-AMPK anti-mouse Akt, anti-mouse p-Akt and anti-mouse

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AMPK were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-mouse p-p65Ser536

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antibody was from Cell Signaling Technology (Danvers, MA). Anti-mouse SIRT1 and β-actin

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antibodies were from Abcam (Cambridge, UK). The extracts, fractions, and compound 1

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from H. tuberosus were dissolved in 0.3% DMSO, to make solutions of 1 mg/mL.

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Cell Culture and Adipocyte Differentiation. Raw 264.7 cell (ATCC cell bank, Manassas,

(IBMX),

insulin

(INS),

dexamethasone

7


(DEX)

and


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VA), a mouse macrophage cell line, was grown in DMEM with 10% FBS and 100 mg/mL

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streptomycin in a 5% CO2 humidified atmosphere at 37˚C. Murine preadipocytes, 3T3-L1,

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were also purchased from ATCC and grown in DMEM, containing 10% BCS and 100 mg/mL

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streptomycin. For adipocytic differentiation, 3T3-L1 cells were seeded in a 6-well plate and

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post-confluent 3T3-L1 cells were cultured in anadipogenic cocktail (0.5 mM of 3-isobutyl-1-

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methylxanthine, 10 µg/mL of insulin, 1 µM of dexamethasone)-containing 10% FBS DMEM

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for 48 hr. 3T3-L1 cells were then incubated in a differentiation medium, which is full DMEM

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with 10 µg/mL INS, for 2 days. 3T3-L1 cells were then incubated in the full DMEM for 4

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days with 2 day-replacement of the full DMEM.

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Measurement of Cell Proliferation. The MTT assay to measure the cell proliferation of

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Raw 264.7 was performed as described earlier.27 Raw 264.7 cells were seeded in 96-wells

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plate (5 x 104 cells per well) followed by treatment of the cells with compound 1 for 24 hr.

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The cells were treated with compound 1 (0.1, 1, 10 µg/mL) for 24 hr and a mixture of 125 µL

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of full DMEM and 25 µL of MTT solution for 2 hr was added to the cells. After aspirating

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the supernatants, 150 µL of dimethyl sulfoxide (DMSO) was added for formazan-crystal

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solubilization. The absorbance was read in a scanning multi-well plate reader at 540 nm.

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ELISA Quantification of cytokines. Raw 264.7 cells were bedded in a 96 well microplate (6

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x 104 cells per well). Raw 264.7 were treated with compound 1 for 2 hr followed by treatment

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with LPS for 24 hr. The supernatant was then collected and used to determine the levels of

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TNF-α, IL-6 and IL-1β secretion by ELISA kits obtained from BioLegend (San Diego, CA).

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Preparation of Macrophage-Conditioned Medium. Raw 264.7 cells were bedded in 100

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mm culture plates. The cells were pre-treated with compound 1 (0.1, 1, 10 µg/mL) for 2 hr,

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prior to 1 µg/mL of LPS treatment for 4 hr. The media from each group was collected and 8


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filtered using a 0.2 µm syringe filter. The media was stored at -75˚C for further use.

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(Supporting information Fig S6).

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Western Blotting. Raw 264.7 cells were pre-treated with compound 1 (0.1, 1, 10 µg/mL) for

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2 hr, then incubated for 4 or 24 hr with 1 µg/mL of LPS. After exposure to LPS, phosphate

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buffered salin (PBS) was used to wash the macrophages. The fully differentiated adipocytes

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were kept in macrophage-conditioned media for 6 hr, followed by wash with PBS. The cells

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were scraped using scrapers, suspended in a lysis buffer, and the supernatants were

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transferred to other 1.5 ml tubes after centrifugation at 13,000 rpm for 10 min at 4˚C. Proteins

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in the samples were resolved by electrophoresis on an 8% SDS-polyacrylamide gel and

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transferred to nitrocellulose membranes or polyvinylidenedifluoride membranes, which were

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probed with the indicated antibodies. Enhanced chemiluminescence (ECL) solution was used

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to detect the protein bands.

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Separation of Nuclear Extracts and Cytosolic Extracts. Raw 264.7 cells were pre-

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incubated with of compound 1 (0.1, 1, 10 µg/mL) for 2 hr before treatment with LPS (1

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µg/mL) for 3 hr. The cells were collected and centrifuged at 13,000 rpm for 10 min. Cell

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pellets were subjected to cytosolic and nuclear extract isolation using NE-PERTM Nuclear

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and cytoplasmic extraction reagent kits obtained from ThermoFisher Scientific (MA, USA).

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RNA extraction and Quantitative Real-Time PCR (RT-PCR). Raw 264.7 cells were

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pretreated with compound 1 (0.1, 1, 10 µg/mL) for 2 hr, followed by treating with 1 µg/ml of

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LPS for 3 hr. RNA was extracted using TriZol reagent (iNtRON Biotechnology, Sungnam,

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Korea). cDNA was produced using Primescript first strand synthesis kit (TaKaRa Bio Inc,

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Shiga, Japan). Real-time PCR was carried out to investigate the expression of various

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cytokine genes using the TOPrealqPCR 2x PreMIX (Enzynomics, Daejeon, Korea) and 9



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specific mouse primers (Macrogen, Seoul, Korea). The relative mRNA levels were quantified

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using a comparative ∆∆Ct method. The primer sequences, which were used for RT-PCR,

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were listed in Supporting information Table S1.

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Statistics. The results were reported as the mean ± S.E.M. For comparisons between groups,

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GraphPad program (Software for Science) was used. Differences between groups were

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considered as significant at p < 0.05.

10


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RESULTS AND DISCUSSION

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Proinflammatory cytokines are released from macrophages in response to toxins such as

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LPS in adipose tissue. The released cytokines activates adipocytes which produce adipokines.

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The adipokines subsequently aggravate inflammation in both macrophages and adipocytes.

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This interplay of cytokines/adipokines between macrophages and adipocytes in adipose tissue

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in obese is very important in cellular resistance to insulin and obesity. Accordingly, it is

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essential to discover new compounds that may help to attenuate insulin resistance.

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Since it was discovered that the paracrine action between macrophages and adipocytes is

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important in insulin resistance, various naturally occurring botanical components have been

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proposed to exert anti-inflammatory effects on macrophages and adipocytes.28-30 Compound 1

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is derived from JA which is already known to be involved in chronic inflammation. There are

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few studies in which compounds with the same or similar functional groups to compound 1

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are used. First of all, compound 1 had inhibitory effect on chymotrypsin and antibacterial

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activities,25 but it was not examined for anti-inflammatory activity so far. 2-[(3-Carboxy-1-

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oxopropyl) amino]-benzoic acid (2) did not possess significant cytotoxicity on HeLa cell

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line31 and the docking of 2 at the active site of acetylcholine esterase and butyrylcholine

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esterase were also studied.32 However, the biological activity of 2-[(4-methoxy-1,4-

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dioxobulyl) amino]-benzoic acid (3) has not been reported. Lastly, 2-[(3-carboxy-1-

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oxopropyl) amino ]-benzoic acid,1-methyl ester (4) showed weal anti-oxidant activity.33 The

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structural difference between compound 1 and these compounds is illustrated in Supporting

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Information Fig. S8.

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We, first, demonstrated new JA-derived compound attenuates the vicious inflammatory

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paracrine loop between macrophages and adipocytes. 11



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Isolation of Compound 1

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First, we isolated and identified compound 1 from H. tuberosus MeOH Extract. Freeze-

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dried tubers of H. tuberosus were extracted consecutively with methanol and H2O. The

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methanol extract was applied to solvent fractionation to give CH2Cl2, EtOAc, n-BuOH, and

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H2O fractions (Fig. 2A). Compound 1 was isolated from the CH2Cl2 fraction, which exhibited

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the strongest inhibition of IL-6 and TNF-α production in LPS-stimulated Raw 264.7 cells, by

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means of activity-oriented chromatographic separation (Fig. 2B, Supporting information Fig

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S5). The 1H NMR spectrum of Compound 1 revealed signals for DART-MS at m/z 266

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[M+H]+, for 1H NMR at (700 MHz, CDCl3) δ 11.15 (1H, br s, NH), 8.68 (1H, dd, J = 8.5, 0.9

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Hz, H-3), 8.02 (1H, dd, J = 8.0, 1.6 Hz, H-6), 7.53 (1H, td, J = 8.5, 1.6 Hz, H-4), 7.08 (1H, td,

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J = 7.6, 1.1 Hz, H-5), 3.93 (3H, s, 7-OCH3), 3.71 (3H, s, 4′-OCH3), 2.77 (4H, m, H-2′ and 3′)

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and for 13C NMR at (176 MHz, CDCl3) δ 173.1 (C-4′), 170.3 (C-1′), 168.7 (C-7), 141.4 (C-2),

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134.7 (C-4), 130.8 (C-6), 122.5 (C-5), 120.4 (C-3), 114.9 (C-1), 52.4 (OCH3), 51.9 (OCH3),

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32.7 (C-2′ or C-3′), 29.0 (C-3′ or C-2′). Thus, compound 1 was identified as methyl2-(4′-

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methoxy-4′-oxobutanamide) benzoate. Methyl 2-(4′-methoxy-4′-oxobutanamide)benzoate (1)

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was previously reported from a dark brown alga, Jolynalaminarioides.25 However, it was

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isolated for the first time from H. tuberosus. The purity of 1 was determined to be more than

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97% by reversed phase C18 HPLC analysis under the 254 nm UV detection (Supporting

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information Fig S4).

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Anti-inflammatory Effect of Compound 1 in Macrophages

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Raw 264.7 cell proliferation was not affected by up to 100 µg/mL of compound 1 (Fig. 3A).

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Compound 1 effectively reduced the expression of MCP-1 and Rantes mRNA (Fig.3B),

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resulting in less infiltration of the immune cells. Furthermore, the release of IL-1β, IL-6 and 12


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TNF-α was significantly decreased by the pretreatment of compound 1, followed by 4 or 24

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hr of LPS stimulation in the macrophages (Fig. 3C). The mRNA levels of the three cytokines

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at 4 or 24 hr of LPS stimulation were markedly decreased by increasing concentrations of

239

compound 1 (Fig. 3C). This indicates that compound 1 inhibits the inflammatory cytokines at

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the transcriptional level.

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Effect of Compound 1 on Signaling Pathways

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Next, we found that the molecular mechanism of compound 1 involves the PI3k/Akt and

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AMPK/SIRT1 signaling pathways. We first investigated whether compound 1 reduces the

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phosphorylation of MAPKs, which is the most well-known inflammatory signaling, but

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compound 1 did not reduce the activation of MAPKs (Data not Shown). Hence, we analyzed

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the inhibitory effect of compound 1 on the inflammatory PI3k/Akt pathway which is widely-

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known for induction of inflammation in macrophages.34 Compound 1 markedly

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downregulated the activation of PI3k and Akt in response to LPS (Fig. 4A). It is known that

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the inhibition of PI3k/Akt blocks the activation of IκB kinase which leads to IκB-α

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degradation.35As expected from the decreased activation of PI3k/Akt, the suppression of IκB-

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α degradation was observed with pretreatment of Compound1 (Fig. 4C). Additionally, it is

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known that SIRT1 which deacetylates p65, a NF-κB subunit, at lysine 310 inhibits the nuclear

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translocation of p65.36 In this study, activation of AMPK and SIRT1 was elevated by

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pretreatment of compound 1 (Fig. 4B). SIRT1 expression was also increased by treatment of

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LPS alone. This phenomenon may be because the elevated SIRT1 expression by compound 1

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seems to be an adaptive homeostasis mechanism to maintain the normal condition.37

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The activation of p65 and its nuclear translocation have been known to induce production

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of inflammatory cytokines.38 Therefore, we examined the activation and translocation of p65 13



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in the nucleus in LPS-treated RAW234. 7 cells. By Western blot observation, the activation of

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p65 and nuclear translocation of p65 were suppressed by compound 1 (Fig. 4D), presumably

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due to combined effects of inhibition of PI3k/Akt and activation of AMPK/SIRT1 signaling.

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Collectively, these data indicate that compound 1 effectively inhibits PI3k/Akt pathway and

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activates AMPK/SIRT1 pathway, followed by decreased translocation and activation of p65.

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Effect of Macrophage-Conditioned Media on Adipocytes

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It is broadly known that inflammatory macrophages interact with adipocytes within the

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adipose tissue, hence, adipocytes under inflammatory conditions to produce various

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adipokines.39 Especially, macrophages-produced cytokines ( IL-6 and TNF-α) can facilitate to

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the development of adipose tissue inflammation and insulin resistance.40, 41 Therefore, we

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evaluated whether compound 1 attenuates the paracrine loop between macrophages and

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adipocytes. Compound 1-pretreated macrophage derived-conditioned media was prepared

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and differentiated adipocytes were incubated in CM (Supporting information Fig S6). The

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elevated levels of IL-1β, IL-6 and TNF-α, Rantes and MCP-1 expression have been known to

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provoke adipocyte and macrophage activation and macrophages, as well as, T-cell

274

infiltration.30, 42 In this study, compound 1 concentration-dependently suppressed the released

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protein level of IL-6 and TNF-α and the mRNA levels of IL-1β, IL-6, TNF-α MCP-1 and

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Rantes of CM-incubated 3T3-L1 adipocytes (Fig. 5). Recently, it has been known that

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adiponectin is a typical anti-inflammatory adipokine to negatively regulate the production of

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TNF-α and the function of T cells and is production is also reduced in obesity and adipocyte

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inflammation.43 The level of adiponectin mRNA was depleted when 3T3-L1 adipocytes were

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incubated in LPS-only stimulated macrophage-derived CM, but the mRNA level of

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adiponectin was recovered with compound 1-pretreated macrophage-derived CM (Fig. 5). 14


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The results imply that compound 1 ameliorates macrophage-derived inflammatory cytokine-

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induced adipocyte inflammation.

284

Previous work has indicated that persistent activation of pro-inflammatory signaling

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pathways to produce TNF-α, IL-6 and IL-1β within adipose tissue could cause obesity-

286

mediated insulin resistance.44, 45 Consistent with these evidence, it is necessary to find out the

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mechanisms of increased levels of the proinflammatory cytokines in CM-incubated

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adipocytes. Early work reported that MAPKs and NF-κB play a central role in obesity and

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insulin resistance.46.47Another study demonstrated that MAPKs/NF-κB activation and

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chemokine production in adipocytes are induced by macrophage-conditioned media.48 In this

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study, we clearly observed a decrease in MAPKs/NF-κB activation in CM-incubated 3T3-L1

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adipocytes (Fig. 6A and B). The suppression of MAPKs/NF-κB activation is likely

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accompanied by a decreased level of inflammatory cytokines from LPS-stimulated

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macrophages. Furthermore, Insulin Receptor Substrate-1(IRS-1)/Akt signaling is known to

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modulate insulin sensitivity.46 IRS-1/Akt signaling pathway activation causes insulin

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resistance in various tissues in response to prolonged presence of insulin. In addition, there is

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increasing evidence that IRS-1/Akt signaling is activated by JNK.49-51 The activation of Akt

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was reduced by compound 1 in CM-incubated adipocytes, as shown in Supporting

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information Fig. S9, which could be due to the decreased activation of JNK in CM-incubated

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adipocytes. Although we did not observe changes in IRS-1 activation, it is plausible that

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compound 1 inhibited IRS-1 activation in CM-incubated adipocytes.

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Next, it is questionable whether the changes in the inflammatory status of 3T3-L1

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adipocytes in LPS-only stimulated macrophage-derived CM are due to the presence of LPS in

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CM. As shown in Supporting information Fig S7A, TNF-α and IL-6 release from adipocytes 15



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in LPS-stimulated non-CM was significantly higher than those in LPS-stimulated CM. The

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elevated cytokine production from CM-incubated adipocytes was reduced by compound 1.

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Moreover, the mRNA levels of TNF-α, IL6, IL-1β, MCP-1 and Rantes in adipocytes in LPS-

308

containing CM were distinctly higher than those in LPS-containing non-CM. The mRNA

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level of adiponectin in 3T3-L1 adipocytes in LPS-containing CM was significantly lower

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than those in LPS-treated non-CM (Supporting information Fig S7B). Consistent with

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previous studies,29 the present findings showed that the inflammation of 3T3-L1 adipocyte is

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induced not only by LPS in CM but also various inflammatory cytokines from LPS-

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stimulated macrophages.

314

To conclude, compound 1 primarily attenuates macrophage inflammation by suppressing

315

inflammatory protein production. As a consequence, CM-incubated 3T3-L1 adipocytes

316

showed a depletion of inflammatory genes, TNF-α, IL-6, IL-1β, MCP-1 and Rantes.

317

Furthermore, decreased activation of MAPKs and NF-κB was observed, meaning

318

inflammation and insulin resistance was disrupted. Altogether, compound 1 primarily

319

mitigates the inflammatory status of macrophages which attenuates the vicious paracrine loop

320

between macrophages and adipocytes, resulting in attenuation of inflammation and insulin

321

resistance.

322

16


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323

ASSOCIATED CONTENT

324

ABBREVIATIONS USED

325

JA, Jerusalem Artichoke; CM, macrophage-conditioned media; ELISA, enzyme-linked

326

immunosorbent assay

327 328

SUPPORTING INFORMATION

329

DART-MS, 1H NMR and 13C NMR spectra, and HPLC chromatogram of Compound1; Effect

330

of various extracts and fractions from Helianthus tuberosus on cell proliferation of Raw

331

264.7 cells; Preparation of macrophage-conditioned media; Primer sequences used for RT-

332

PCR.

333 334

FUNDING

335

This research was funded with internal resources.

336 337

CONFLICT OF INTEREST

338

The authors have declared that no conflict of interests.

17



339

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487

FIGURE CAPTIONS

488

Figure 1. Structure of Compound1

489

Figure 2.Effect of the extracts and solvent fractions of Helianthus tuberosus on the

490

production of TNF-α and IL-6 in LPS-stimulated Raw 264.7 cells. (A) Extraction and

491

fractionation scheme of H. tuberosus. (B) Raw 264.7 cells were pretreated with various

492

extracts and fractions for 2 hr, followed by 1 µg/mL of LPS stimulation for 24 hr. The

493

supernatants were collected for ELISA.

494

Figure 3. (A) Raw 264.7 cells were incubated with the stated concentrations of compound 1

495

for 24 hr. (B) Raw 264.7 cells were pretreated with the indicated concentrations of 1 for 2 hr,

496

prior to LPS stimulation for 4 hr. The cells were harvested with TriZol for RNA isolation. (C)

497

Raw 264.7 cells were pretreated with the stated concentrations of compound 1 for 2 hr,

498

followed by 1 µg/mL of LPS for either 4 or 24 hr. The supernatant was collected and used for

499

ELISA of TNF-α, IL-6 and IL-1β. Raw 264.7 cells were pretreated with the indicated

500

concentrations of compound 1 for 2 hr, prior to LPS stimulation for 4 hr. The cells were

501

harvested with TriZol for RNA isolation. Data are presented as the mean ± S.E.M (n=3).

502

*p

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