Flavonoids alleviating insulin resistance through inhibition of

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Flavonoids alleviating insulin resistance through inhibition of inflammatory signaling Ning Ren, Eunhye Kim, Bo Li, Haibo Pan, Tuantuan Tong, Chung S Yang, and youying Tu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05348 • Publication Date (Web): 05 Jan 2019 Downloaded from http://pubs.acs.org on January 7, 2019

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Journal of Agricultural and Food Chemistry

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Flavonoids alleviating insulin resistance through inhibition of inflammatory signaling

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Ning Ren,1,# Eunhye Kim,1,# Bo Li,1 Haibo Pan,1 Tuantuan Tong,1 Chung S. Yang, 2,* Youying

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Tu1,*

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Department of Tea Science, Zhejiang University, Hangzhou, Zhejiang 310058, P.R. China

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Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, The State

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University of New Jersey, Piscataway, NJ 08854, USA

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#The

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Corresponding Authors

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authors contributed equally.

*Telephone: +86 (136) 2571-3998. E-mail: [email protected] +1 848-445-5360. E-mail: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT: During the past 20 years, many studies have focused on polyphenol

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compounds for their potential beneficial health effects. Flavonoids represent a large class of

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phenolic compounds found in fruits, vegetables, nuts, grains, cocoa, tea, and other beverages.

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Flavonoids have shown antioxidant and anti-inflammatory activities. Given the putative

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relationship between inflammation and insulin resistance, the consumption of flavonoids or

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flavonoid-rich foods have been suggested to reduce the risk of diabetes by targeting inflammatory

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signals. This is the first comprehensive review summarizing the current research progress on the

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inhibition of inflammation and alleviation of insulin resistance by flavonoids, as well as the

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mechanistic link between these disorders. Laboratory and human studies on the activities of major

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flavonoids (flavones, isoflavones, flavonols, etc.) are discussed.

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KEYWORDS: Flavonoid； Insulin resistance； Inflammatory signaling; IRS-1; Macrophage

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INTRODUCTION

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Insulin resistance (IR), defined as a diminished ability of cells to respond to the stimulation

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of insulin, is a crucial feature of pre-diabetes and is the first detectable abnormality in type 2

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diabetes mellitus (T2DM). The prevalence of T2DM is rapidly increasing and is currently one of

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the most challenging health problems in the world.1 In the early stage of IR, normal pancreatic

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beta-cells increase the production of insulin to compensate for IR, and glucose utilization remains

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relatively normal. However, when IR continues, the beta-cells gradually fail to produce sufficient

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compensatory hyperinsulinemia, leading to insulin insufficiency, impaired glucose tolerance and

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eventually frank T2DM.2-5 IR usually occurs in peripheral tissues such as liver, adipose, and

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skeletal muscle.

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Inflammation is an immune response of tissues to deal with injuries, displayed as swelling,

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redness, pain and fever, which rely upon metabolic support and energy redistribution. Thus, the

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basic inflammatory response favors a catabolic state, and inhibits anabolic pathways such as the

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highly conserved and powerful insulin signaling pathway.1 In normal circumstances, inflammation

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is a short-term process that contributes to tissue repair. However, the long-term consequences of

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prolonged inflammation have detrimental effects.6 Intervention studies have demonstrated the

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occurrence of inflammation in T2DM pathogenesis, especially that with IR.7

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A correlation between IR and inflammation has been suggested since the 1990s.8 In the

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subsequent decades, studies demonstrated a clearer association between chronic inflammatory

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signaling and IR.9-10 Elevated levels of proinflammatory cytokines, such as tumor necrosis factor-

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(TNF-)11-14, interleukin-6 (IL-6)12, interleukin-1 (IL-1)15 and resistin16, as well as decreased

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levels of anti-inflammatory cytokines, such as interleukin-10 (IL-10)17 and adiponectin,18-19 have

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been reported in various diabetic and IR states.

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Flavonoids represent a large class of more than 6000 phenolic compounds found in fruits,

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vegetables, nuts, grains, cocoa, tea, and other beverages. Flavonoids are secondary metabolites of

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plants and fungi, and have a 15-carbon skeleton containing two phenyl rings and a heterocyclic

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ring. The basic structure of flavonoids is shown in Fig. 1. There are six major subgroups of

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flavonoids with various differences in generic structure of the C ring, functional groups on the

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rings and positions in the C ring that linked the B ring.4 A number of studies have demonstrated

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the potential beneficial effects of flavonoids against inflammation and their implication to different

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diseases20-23. For example, in a recent review by Leyva-Lopez et al.21, the modulation of cytokines

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by flavonoids were extensively discussed with an emphasis on therapeutic application against

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inflammation-related diseases. Our present review summarizes the current research progress on

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flavonoids with an emphasis on the molecular mechanisms that link the anti-inflammatory

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activities to the prevention or alleviation of IR. By assessing the biological activities of different

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flavonoids in laboratory and human studies, we believe that it is prudent to consider flavonoids as

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dietary constituents in a food-based approach for the prevention or alleviation of inflammation, IR

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and T2DM. To our knowledge, this is the first comprehensive review on this topic.

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FLAVONOIDS MODULATING INFLAMMATORY SIGNALING AND ALLEVIATING

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INSULIN RESISTANCE IN LABORATORY STUDIES

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Over the last 20 years, extensive research has focused on the health effects of plant

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phenolics.5 Flavonoids may function as antioxidants to prevent oxidative stress. Given the putative

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relationship between diabetes and inflammation,2, 24 and the potential for flavonoids to protect the 4


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body against oxidative stress and inflammation,25-26 it is plausible that flavonoids or flavonoid-

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rich foods can lower the risk for developing IR and T2DM.27-28 Some studies are discussed below,

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and possible sites of actions are shown in Fig. 2 and Fig. 3.

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Flavones

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Flavones, characterized by the backbone of 2-phenylchromen-4-one (shown in Fig. 4), are

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found mainly in celery, parsley and different herbs.20, 22 Luteolin, tangeretin, velutin, nobileten and

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apigenin are typical flavones that have been studied for their activities in the alleviation of

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inflammation and IR. Some key studies are summarized in Table 1.

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Luteolin, which is abundant in vegetables and fruits,29-31 was reported to alleviate IR through

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inhibiting inflammation pathways. In C57BL/6 mice fed a high-fat diet (HFD), luteolin

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supplementation suppressed inflammatory macrophage infiltration32 and mast cell infiltration33 into

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mouse epididymal adipose tissues, possibly leading to improved insulin sensitivity. The serum

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levels of inflammatory cytokines, TNF-α, IL- 6, and monocyte chemotactic protein-1 (MCP-1)

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were also decreased.32-33 In addition, luteolin alleviated neuroinflammation and ameliorated

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neuronal IR in the mouse brain by inhibiting the expression of TNF-α, IL- 1, MCP-1, resistin

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and NF-B.34 In cell models of inflammation and IR – the inflammatory macrophages and

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adipocyte 3T3-L1 stimulated with lipopolysaccharide (LPS) or palmitate (PA), luteolin inhibited

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the inflammatory polarization of macrophages via activating AMPKα1 and enhanced insulin

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signals in adipocytes.32 Luteolin also enhanced insulin sensitivity via activation of PPARγ and

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PKC transcriptional activity in inflammatory adipocytes.33, 35 In another study, using a cell model

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of PA-treated human umbilical vein endothelial cells (HUVECs) and PA-treated aortic ring, pre-

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treatment of luteolin reduced inhibitor of nuclear factor kappa-B (IKKb) phosphorylation and the 5



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expression of TNF-α and IL-6, blocked NF-kB activation (through attenuating P65 phosphorylation),

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inhibited the Ser/Thr phosphorylation of insulin receptor substrates-1 (IRS-1) and restored downstream

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Ak transforming(AKT, also known as Protein kinase B)/ endothelial nitric oxide synthase (eNOS)

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activation in endothelial cells, as well as ameliorated the insulin-mediated endothelium-dependent

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relaxation in rat aorta.36 Interestingly, in a co-culture system of adipocytes and macrophages,

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luteolin reduced the production of inflammatory mediators and inhibited the interaction between

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adipocytes and macrophages via suppressing JNK phosphorylation; both events contributed to the

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alleviation of IR.37 Luteolin was also shown to exert anti-inflammatory action by blocking LPS-

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mediated TNF-α and IL-638 production through inhibiting NF-κB activation and the MAPK

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pathway. Whether inhibition of NF-κB activation and mitogen-activated protein kinase (MAPK)

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pathway by luteolin could alleviate IR in adipocytes was not investigated in the study.38

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Tangeretin, which is prevalent in mandarin oranges and other citrus fruits, also showed anti-

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obesity and anti-diabetic effects through inhibiting inflammation. Tangeretin decreased total

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cholesterol and blood glucose, and regulated the expression of IL-6, leptin, MCP-1 and resistin in

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HFD-induced obese mice, and activated AMPKα1 and GLUT4 translocation in C2C12 cells.39

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Tangeretin also alleviated IR by regulating the secretion of insulin-sensitizing factor – adiponectin

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and insulin-resistance factor – MCP-1 in 3T3-L1 adipocytes.40 The same results were found in

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this model with nobiletin, another flavone isolated from citrus peels.40 Nobiletin was also reported

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to significantly decrease proinflammatory cytokine levels in plasma and improve insulin

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sensitivity in HFD-fed mice.41

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Velutin, a unique flavone found in the pulp of açaí fruit (Euterpe oleracea Mart.), had a higher

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activity than the three other flavones studied, luteolin, nobileten, and apigenin, in reducing TNF6


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α and IL-6 production in macrophages induced by LPS.38 Velutin also had the highest inhibitory

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activity against NF-κB activation and the degradation of NF-κB inhibitor, as well as in inhibiting

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the phosphorylation of MAPK P38 and JNK.38

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Isoflavones

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Isoflavones are commonly found in soybean and soy products (structure shown in Fig. 4).

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The common dietary isoflavones are puerarin, genistein and daidzein.20, 22 Some reported effects

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of isoflavones on inflammation and IR are summarized in Table 2. A study on soy isoflavones

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(including daidzein, genistein, and glycitein) in IR rats showed that soy isoflavone could decrease

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the body weights and improve IR through regulating the expression of adipocytokines, such as

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adiponectin, leptin, resistin and TNF-.42

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Genistein is found in legumes and the Chinese plants Genista tinctoria Linn.43 Genistein was

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reported to decrease homeostasis model assessment of IR (HOMA-IR), and to improve liver

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function and insulin sensitivity, and to ameliorate non-alcoholic fatty liver diseases in a rat model

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of IR.44 Genistein was also reported to reverse PA-induced loss of insulin-mediated vasodilation

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in rat aorta, and to inhibit IKKb and NF-кB activation associated with down-regulation of TNF-α,

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IL-6 production and expression.45 Genistein helped regulate of insulin action under IR conditions

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by inhibiting inflammation-stimulated IRS-1 serine phosphorylation in endothelial cells. These

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results demonstrated that genistein ameliorated endothelial dysfunction implicated in IR, as well

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as inhibiting inflammation.45 In this article, both positive and negative regulations of insulin action

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in endothelial cells by genistein were found. Negative regulations were shown in the inhibition of

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insulin-stimulated tyrosine phosphorylation of IRS-1 and attenuating downstream AKT by

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genistein, which leaded to a decreased nitric oxide (NO) production in endothelial cells.45 7



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Daidzein also belongs to the isoflavone subclass and is commonly found in soybeans, soy-

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based products, fruits and nuts.46 Earlier studies suggested that daidzein had anti-diabetic effects

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in improving glucose and lipid metabolism, as well as insulin sensitivity.47-49 Daidzein

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significantly inhibited the downregulation of adiponectin expression via molecular mechanisms

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different from genistein.50 In inflammatory signaling, daidzein inhibited the downregulation of

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adiponectin by restoring the TNF-α-mediated reduction of Forkhead box protein O1 (FoxO1)

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protein expression, but not involving JNK as was shown for genistein; neither genistein nor

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daidzein acted as agonist of PPAR.50 The only structural difference between these two compounds

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is the hydroxyl (genistein) or hydrogen (daidzein) moiety at the R1 position (shown in Fig. 4).

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Puerarin is extracted from Pueraria lobata (Wild.) Ohwi. It has been reported to have

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pharmacological action in the treatment of diabetes51 and cardiovascular diseases.52-53 In high

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glucose induced IR endothelial cells, puerarin potentiated insulin-induced differentiation of pre-

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adipocytes to increase glucose uptake by adipocytes, and this was associated with elevated PPAR

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expression.54 In addition, puerarin was shown to inhibit IKKb/NF-κB activation, decrease TNF-α

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and IL-6 production, attenuate PA-induced serine phosphorylation of IRS-1, and effectively

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restore insulin-mediated tyrosine phosphorylation of IRS-1.55

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Flavonols

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Flavonols, including the commonly consumed quercetin, kaempferol, isorhamnetin, fisetin,

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and myricetin from vegetables and fruits, are the most abundant flavonoids in our diet.20, 22 Their

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structures are shown in Fig. 5. Some studies about the effects of flavonols on inflammation and IR

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are summarized in Table 3.

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Quercetin is one of the most common flavonoid in the human diet,20, 22 found in various fruits,

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flowers, and tea (Camellia sinensis). The anti-obesity and anti-diabetic properties of quercetin

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have been shown in many studies.56-60 In a HFD model of rats, dietary consumption of quercetin

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reduced the abdominal fat, decreased oxidative stress and suppressed inflammation, suggesting

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that the attenuation of metabolic syndrome by quercetin is associated with the anti-inflammatory

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effect in adipose tissue.61 In mice fed a HFD, supplementation with 0.08% quercetin for 10 weeks

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ameliorated IR associated with decreased expression of TNF- and MCP-1, together with

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increased expression of adiponectin in serum.56 Quercetin treatment improved whole body insulin

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sensitivity by increasing GLUT4 expression and decreasing JNK phosphorylation, as well as by

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decreasing TNF- and iNOS expression in skeletal muscle in obese ob/ob mice.62 Quercetin also

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prevented the downregulation of insulin-induced glucose uptake and AKT serine phosphorylation

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by TNF-α, and reduced nuclear accumulation of NF-κB in L6 myotubes.62 In another study,

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treatment with different concentrations of quercetin in three experimental systems, namely co-

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culture of macrophages and adipocytes (6.25, 12.5 or 25 µM), zebrafish (6.25, 12.5 or 25 µM) and

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mice (25, 50 or 100 mg/kg), improved IR; this effect was associated with decreased expression of

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TNF- and MCP-1 as well as increased expression of adiponectin in serum.17 The secretion of the

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inflammatory cytokines IL-1β and IL-6 was decreased and the anti-inflammatory IL-10 was

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increased.17 In a study with TNF--induced primary human adipocytes, quercetin treatment

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inhibited the activation of inflammatory cascades (JNK, NF-B) and the expression of

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inflammatory cytokines IL-6, IL-1 and MCP-1. The treatment also attenuated the suppression of

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PPAR and insulin-stimulated glucose uptake, as well as decreased the serine phosphorylation of

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IRS-1 and expression of protein tyrosine phosphatase 1B (PTP-1B).63 Moreover, quercetin 9



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improved glucose uptake and insulin sensitivity in skeletal muscle cells via AKT and AMPK

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signaling pathways.64

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Myricetin is abundant in our diet. There are several publications describing its anti-

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inflammatory and anti-diabetic properties.65-67 An earlier report indicated that injection of

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myricetin to rats on a high fructose diet significantly improved insulin sensitivity, decreased serum

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glucose and insulin levels, and reversed the defective expression of regulatory subunit of

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phosphoinositide (PI) 3-kinase.67 A recent study in db/db mice showed that myricetin treatment

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alleviated obesity and IR by activating brown adipose tissue, recruiting beige cells, increasing

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adiponectin secretion in brown adipose tissue, and decreasing plasma TNF concentration in db/db

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mice.68 Another study in mice fed a high-fat, high-sucrose diet showed that myricetin, given at

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0.12% in the diet significantly reduced body weight gain and epidydimal white adipose tissue

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weight, alleviated hypertriglyceridemia and hypercholesterolemia, ameliorated IR and reduced

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serum proinflammatory cytokine (TNF-α, IL-6) levels.69

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Kaempferol is abundant in different kinds of fruits and vegetables, such as apple, grape,

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spinach, broccoli and tea.70-71 Several studies reported that kaempferol could alleviate diabetes and

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obesity,72-73 possibly by inhibiting inflammation74-75 and improving IR via inhibiting hepatic

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IKK/NF-κB signaling.72, 76-77 As shown in Table 3, when Type 2 diabetic rats (induced by HFD

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for 6 weeks and then an injection of streptozotocin), were treated with kaempferol orally for 10

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weeks, the blood lipids and insulin levels were ameliorated in a dose-dependent manner.77

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Kaempferol also effectively improved glucose catabolism, inhibited the serine phosphorylation of

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IRS-1, decreased the expression of IKKα and IKKβ, reduced the nuclear and cytosol levels of NF-

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κB, and decreased the serum levels of TNF-α and IL-6.77 Another study showed that kaempferol 10


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and quercetin could improve insulin-stimulated glucose uptake in mature 3T3-L1 adipocytes,

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inhibit NO production significantly in LPS treated macrophage cells in which the PPARγ was

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overexpressed, and compete with rosiglitazone for the same binding site of PPARγ in CV-1 cells.78

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In this case, kaempferol and quercetin showed no difference in their mechanisms of action.

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Other flavonoids

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Other flavonoids, such as catechins from tea, flavon-3-ols and amentoflavone, have been

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studied for alleviation of IR. Structures of these compounds are shown in Fig. 5 and their effects

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on inflammation and IR are summarized in Table 4.

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Epicatechin occurs widely in plants, especially in tea plants (Camellia sinensis) as both (+)-

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catechin and (-)-epicatechin forms. The capacity of (-)-epicatechin to prevent TNF--induced

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activation of pro-inflammatory signaling and IR has been reported.79 In another study, it was found

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that (-)-epicatechin suppressed the expression of NF-κB-regulated proinflammatory cytokines and

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chemokines that contributed to IR in rats fed a high fructose diet.80 Epigallocatechin gallate

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(EGCG) is a characteristic and major catechin in tea plants (Camellia sinensis). Its activities have

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been extensively studied81 and are covered in other articles in this volume. In HFD-fed rats, EGCG

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ameliorated IR, while inhibiting the expression of inflammatory cytokines and infiltration of

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macrophages in adipose tissue.82 In PA and glucose pretreated HepG2 cells, EGCG was reported

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to markedly decrease levels of inflammatory and oxidative stress factors, including NF-κB, TNF-

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α, IL-6, as well as improve insulin sensitivity.83 Theaflavins, the products of oxidative

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condensation of catechins in the manufacture of black tea, also had the ability to improve insulin

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sensitivity.84

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Silymarin, an extract of milk thistle seed (Silybum marianum) and containing silybin,

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silydianin and silychrisin, was found to decrease body weight, plasma lipids, inflammation, liver

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injuries and IR induced by HFD in mice.85

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Amentoflavone, at concentration of 13.94 µM, was reported to reduce the expression of

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inflammatory cytokines, such as TNF-α, IL-6, and IL-8 in insulin pre-treated HepG2 cells.86 The

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mechanisms of action may involve inhibition of PI3K-AKT signaling and the modulation of

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glucose metabolism.

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HUMAN STUDIES

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In comparison to the laboratory studies described above, there are few studies on the effects

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of flavonoids in humans. In a 2005 review on the bioavailability and bio-efficiency of

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polyphenols in humans, 93 intervention studies were included.87 The results showed that

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monomeric catechins (found at high concentrations in tea) increased plasma antioxidant

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biomarkers, energy metabolism and vascular dilation. Procyanidins (oligomeric catechins

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found in red wine, grapes, cacao, etc) had beneficial effects on the vascular system. The effects

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of polyphenols in vivo were generally weaker than those observed in vitro. Effects on

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inflammatory markers and IR were not observed or not investigated in these studies on

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polyphenols.87 In a double-blind RCT for one year on 93 postmenopausal women with T2DM

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(on medication), ingestion of flavon-3-ols (enriched in chocolate) and isoflavones improved

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insulin sensitivity and lipoprotein status. The combined flavon-3-ol and isoflavone intervention

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resulted in a significant reduction in estimated peripheral IR (HOMA-IR) and improvement in 12


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insulin sensitivity (quantitative insulin sensitivity index).88 In a prospective study of 38,018 US

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middle-aged and older women in a follow-up of 8.8 years (332,905 person-years), the results did

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not support the thesis that higher consumption of flavones and flavonols was associated with lower

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risk for T2DM; however, consumption of apples and tea might be associated with decreased risk

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for the disease.89 In a cross section study of 1,997 females, intake of total flavonols and their

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subclasses were calculated from food frequency questionnaires. Higher anthocyanins and

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flavone intake was associated with significantly lower peripheral IR. Higher anthocyanin

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intake was also associated with lower levels of high-sensitivity C-reactive protein.90 In another

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cohort study involving 124,086 men and women, subjects self-reported change in weight over

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multiple four-year time intervals between 1986 and 2011. Higher consumption of most flavonoids

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including anthocyanins, flavonoid polymers, flavonols and flavan-3-ols (mostly from apples,

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pears, berries, peppers, etc), was inversely associated with body weight change over four-year time

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intervals.91 The weight reduction could alleviate IR.

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MECHANISTIC CONSIDERATIONS

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Obesity is a major cause of IR and T2DM. It has been shown that chronic, low-grade

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inflammation associated with obesity is a major cause of IR. In recent decades, many studies and

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reviews discussed the mechanistic links among obesity, inflammation and IR.65-67 There is strong

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evidence that T2DM is closely related to inﬂammation. Flavonoids and related compounds have

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become a group of important agents for research in this area. Epidemiological and human

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intervention studies have provided evidence supporting the protective role of polyphenol-rich

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foods against chronic diseases including diabetes, cardiovascular diseases, cancer and 13



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neurodegenerative diseases.22 In this review, we paid special attention to the effects of flavonoids

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on the signaling pathways that link inflammation and IR in diabetes or pre-diabetes models. To

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our knowledge, this is the first comprehensive review on this topic.

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Flavonoids are known to be absorbed in the small intestine, with different bioavailabities

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dependent on the structure, and converted to methylated, glucuronidated and sulfated

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metabolites.81, 92-94 In addition, they can be degraded by intestinal microbes. For example, EGCG

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and other catechins can be degraded to 5-(3', 4', 5'-trihydroxyphenyl)-γ-valerolactone and 5-(3', 4'-

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dihydroxyphenyl)-γ-valerolactone, and these metabolites generally have lower biological

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activities than the parent compounds.94-96 Trans-resveratrol sulfates show similar anti-

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inflammatory activities compared to their parent compounds.97 Daidzein and genistein are

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converted to equol and 5-hydroxy-equal by gut microbes.98 Equol is known to have stronger

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estrogenic activity than daidzein99; however, the formation of equol has no effect on the plasma

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level of insulin-like growth factor 1.98 Thus, flavonoid metabolites may have lower, the same, or

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higher biological activity than the parent compounds. In the studies reviewed herein, the

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biotransformation issue was mostly not seriously considered. The activities were assumed to be

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mainly due to the parent compounds and the contribution by their metabolites are not clear.

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As summarized in Tables 1-4, inhibition of inflammation related cytokines and signaling

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pathways by most of the flavonoids are correlated with the impediment of IR development. This

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is consistent with the well-established link between inflammation and the development of IR.1, 9-

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10

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discussed below.

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Intercellular mechanisms

The possible mechanisms of flavonoids inhibiting inflammation signaling and alleviating IR are

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It is known that in overfed and overweight animals, excess caloric intake results in enlarged

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adipocytes filled with triacylglycerol. The enlarged adipocytes in a state of inflammation produce

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MCP-1 to attract macrophages. In response to MCP-1, macrophages infiltrate adipose tissue.

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Macrophages in inflamed adipose tissue of the M1 type, produce TNF-α, which triggers lipid

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breakdown and fatty acids are released into the blood9-10, 100 (Fig. 2). The fatty acids released from

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adipose tissue enter muscle cells, where they accumulate in small lipid droplets. This ectopic

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storage of lipids causes IR, possibly by triggering lipid-activated protein kinases that inactivate

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some element in the insulin-signaling pathway, causing glucose transporter GLUT4 to leave the

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muscle cell surface, preventing glucose entry into muscle, and the myocytes now become IR. Some

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of the possible actions of flavonoids are depicted in Fig.4. For example, luteolin has been shown

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to inhibit MCP-1 and the infiltration of macrophages into adipocytes, as well as the formation of

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macrophage M1. It can also upregulate PPAR and adiponectin, which would favor the formation

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of macrophage M2.33, 35, 42, 69 Apparently, luteolin has been shown to have more activities than

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other flavonoids. It is unclear whether this is because luteolin is more commonly occurring or more

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active than other flavonoids, or it just has been used more commonly in studies.

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Intracellular mechanisms

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Binding of insulin to its receptor initiates the intracellular insulin action by activating the

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tyrosine kinase on the β-subunit of insulin receptor to phosphorylate the tyrosine of IRS-1, which

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serves as a docking protein for various effector molecules.1 However, when IRS-1 is alternatively

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phosphorylated on serine 307, it blocks the tyrosine phosphorylation on IRS-1 (Fig.3). Since

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some inflammatory cytokines can induce serine phosphorylation on IRS-1, this is one of the

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most important mechanisms linking inflammatory cytokines to IR in adipocytes.1, 101-102 The main 15



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inflammatory cytokines and pathways, involving TNF-, IL-6, NF-B and JNK, could be

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inhibited by flavonoids to prevent IR.23 Flavonoids have shown activities to inhibit serine

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phosphorylation of IRS-1, while decreasing the expression of inflammatory cytokines.35,

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Some possible sites of activation are depicted in Fig.3.

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Structure-activity relationship

80, 82

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Several studies compared the activities, mechanisms of action and effective dosages of

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different flavonoids,38, 50, 64, 103 or between flavonoids and other polyphenols or natural products,63

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to elucidate the structure-activity relationship. For example, as discussed previously, two

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structurally related soy isoflavones, genistein and daidzein (Fig. 4), were shown to significantly

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inhibit the downregulation of adiponectin expression via different molecular mechanisms.50

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Another study showed that kaempferol and quercetin competed with rosiglitazone for the same

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binding pocket of PPARγ.78 Quercetin was reported to be equally or more effective than trans-

317

resveratrol in inhibiting TNF-α–mediated inflammation and IR in primary human

318

adipocytes.63 Four different flavones: velutin, luteolin, chrysoeriol, and apigenin were used in

319

a study to investigate the structure-activity relationship in LPS-stimulated macrophages from

320

HFD-fed mice.38 The results indicated that velutin, whose structure is similar to luteolin but

321

bears two methoxy groups at 7- and 3′-positions, had the strongest inhibitory effect among

322

the four flavones, and chrysoeriol was the most ineffective one. A study that screened six

323

flavonoids, 14 flavonoid metabolites and their various combinations (0.1–10M), for ability to

324

inhibit LPS-induced TNF- secretion in THP-1 monocytes, but these authors did not screen for

325

the ability to improve insulin sensitivity of these flavonoids.104 16


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326

However, it is unclear how so many compounds with polyphenolic structures can affect the

327

similar groups of cytokines and signaling pathways as reviewed above. Even though structure-

328

activity relationships have been explored in some studies, clear structure-related activity

329

relationship and dose-response relationships have not been fully established. It is possible that,

330

because most of these actions are linked to the inflammation process, most of these changes in

331

signaling pathways are subsequent events of the suppression of inflammation by flavonoids. ROS

332

is known to promote the inflammatory process, and the antioxidant activity of flavonoids may be

333

a key contributor to most of the activities observed in the studies reviewed herein. The possibility

334

that these phenolic compounds bind directly to specific cytokines, transcription factors and

335

receptors is also possible, as has been proposed in some of the studies reviewed herein and in other

336

reviews.4, 23, 105-106 However, clues on this information derived from in vitro studies needs to be

337

interpreted with caution because of non-specific binding activities of flavonoids and the

338

bioavailability issues. The body weight-lowering effects of some flavonoids could also contribute

339

to the lowering of inflammation and IR, especially in animals fed a HFD.105

340

Because of the wide occurrence of flavonoids in fruits and vegetables, and the similar

341

activities of many of the compounds, it is likely that these flavonoids, in combination, contribute

342

to a healthy diet that impedes inflammation and the development of IR. For example, a study on

343

soy isoflavones (including daidzein, genistein, and glycitein) in IR rats showed that these

344

isoflavones could decrease the body weights and improve IR through regulating the expression of

345

adipocytokines.42 Human studies as described above also showed that dietary flavonoids, exist as

346

mixtures of different compounds, have beneficial health effects. Therefore, it is prudent to study

347

flavanoids as food constituents, rather than leads for developing pharmaceutical agents to treat 17



Page 18 of 41

348

inflammation and IR, even though the later possibility cannot be excluded. For future research, we

349

would like to suggest the following:

350 351 352

1. The molecular mechanisms by which different flavonoids exert their anti-inflammatory effects, including structure-function relationship, need to be further investigated. 2. It is important to determine whether the cytokines and signaling pathways that are

353

affected by treatments with flavonoids are direct targets of action or are subsequent events of the

354

inhibition of an upstream event.

355

3.

The dose-response relationship, the bioavailability issue and the activity due to

356

metabolites need to be studied. It is also interesting to study whether the actions of different

357

flavonoids are additive.

358

4.

The possible toxicity due to high-level intake of flavonoids needs to be studied,

359

especially when considering the development of these compounds into dietary supplements. This

360

point is well illustrated in the use of green tea extract as dietary supplements.107 Additive effects

361

between flavonoids on toxicity need to be considered.

362

ABBREVIATIONS USED

363

AKT , Ak transforming, also known as Protein kinase B; AMPKα1, AMP-activated protein

364

kinase α1; AP-1, activator protein 1; ATMs, adipose tissue macrophages; EAT, epididymis adipose

365

tissues; EGCG, Epigallocatechin gallate; eNOS, endothelial nitric oxide synthase; FoxO1,

366

Forkhead box protein O1；HFD, high-fat diet; HUVECs, human umbilical vein endothelial

367

cells; HOMA-IR, homeostasis model assessment of IR; GAL-3, Galectin-3；IR, insulin

368

resistance; GLUT4,glucose transporter 4; GTT, glucose tolerance test; IKKb, inhibitor of nuclear 18


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369

factor kappa-B; IL-1, interleukin-1; IL-6,interleukin-6; IL-10, interleukin-10; IRS-1, insulin

370

receptor substrates-1; ISI, insulin sensitive index; ITT, insulin tolerance test; JNK, c-Jun N-

371

terminal kinase; LPS, lipopolysaccharide; MCP-1, monocyte chemotactic protein-1; MAPK,

372

mitogen-activated protein kinase; NF-B, nuclear factor kappa B; NO, nitric oxide ; PA,

373

palmitate; PCM, peritoneal cavity resident macrophage ; PI3K, Phosphatidylinositol-4,5-

374

bisphosphate 3-kinase; PIG, Palmitate insulin and glucose; PKC, Protein kinase C; PPAR,

375

peroxisome proliferator-activated receptor; PTP-1B, protein tyrosine phosphatase 1B ; ROS,

376

reactive oxygen species; SOD1, superoxide dismutase 1; SOD2, Superoxide dismutase 2;T2DM,

377

type 2 diabetes mellitus; TNF- ,tumor necrosis factor-;

378

ACKNOWLEDGEMENT

379

This research was supported by key research and development projects in Zhejiang

380

Province “Industrialization model projects on exploring functional components and related

381

products from tea flowers and fruits”（2018C02012）and National key research and

382

development plan “Processing of tea products, key technology of quality control during storage

383

and equipment development” ( 2017YFD0400803) and National Natural Science Foundation of

384

China (grant no. 31501474).The research was also supported by 2016 Project of Funding

385

Municipal Project “Study on transformation of health benefits and its mechanism during white

386

tea storage”, the Fundamental Research Funds for the Central Universities, and the Fujian

387

Province “2011 Collaborative Innovation Center” Chinese Oolong Tea Project (Fujian

388

Educational Department,[2015] No.75). We thank Ms. Vi Dan of Rutgers University for her

389

assistance in the preparation of this manuscript.

19



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699 700 701 702 703 704 705 706 707 708


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709

27



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Figures captions

710

711

Figure 1. Basic structures of flavonoid subclasses

712

Figure 2. Mechanisms linking inflammation to insulin resistance in adipose tissue and

713

possible effects of flavonoids. Adipose tissue is infiltrated with increased numbers of

714

macrophages. These adipose tissue macrophages (ATMs) are a major source of cytokines. There

715

are two types of ATMs, M1 cells are defined as classically activated, proinflammatory

716

macrophages and M2 cells comprise an alternatively activated, anti-inflammatory macrophage

717

population. The formation of M1 and M2 depends on the expression of cytokines in adipose

718

tissue. (A) Signal transduction pathways in obesity that lead to chronic inflammation and IR,

719

and possible effects of flavonoids; (B) Signal transduction pathways that are modulated by

720

flavonoids to increase the population of M2 macrophages and increase insulin sensitivity

721

(Modified from Figure 2 of the review by Olefsky, et al.9). *Abbreviation used: GAL-3,

722

Galectin-3.

723

Figure 3. Intracellular mechanisms linking inflammation to insulin resistance and possible

724

effects of flavonoids. Activation of the insulin receptor by insulin leads to tyrosine

725

phosphorylation of IRS-1 thereby initiating signal transduction. When IRS-1 is alternatively

726

phosphorylated on serine 307, its downstream signaling ability is diminished. The activation

727

of inflammatory pathways, such as those involving NF-B and JNK, by inflammatory

728

cytokines, such as TNF- and IL-6, triggers phosphorylation on the serine 307 of IRS-1 to 28


Page 29 of 41


729

increase IR and further increase the expression of inflammatory cytokines, which sets up a

730

feed-forward process that further activate the inflammatory pathway. The sites of action by

731

flavonoids are shown by “

732

Luca, et al. 102).

” and “

”. (Modified from Figure 1 of the review by Carl de

733

Figure 4. Structures of flavones and isoflavones.

734

Figure 5. Structures of flavones and catechins

29



Page 30 of 41

Table 1. Flavones inhibit inflammatory signaling and alleviate insulin resistance Model

Insulin sensitivity and resistance

Ref

↑Insulin sensitivity (GTT&ITT)

32

↑Insulin sensitivity (GTT&ITT), ↑GLUT4 translocation

33

Luteolin 10 mg/kg ↓TNF-α，↓IL-1, ↓NF-κB (in the cortex and in diet for 16 20 weeks ↑Adiponectin, weeks and i.g. for hippocampus) ↓Resistin 4 weeks ↓Adiponectin, Tangeretin 0.02% 8 weeks ↓Leptin, ↓Resistin, ↑AMPKα1 in diet ↓IL-6, ↓MCP-1

↓HOMA-IR, ↑Plasma insulin ↑Plasma glucose, ↑Insulin sensitivity

34

↑Insulin sensitivity (GTT&ITT)

39

Mice on HFD

Nobiletin 0.02% in diet

↑Insulin sensitivity (GTT&ITT), ↓Plasma insulin, ↓Free fatty acids, 41 ↓ Total cholesterols

LPS-stimulated bone marrow mast cells from mice

Luteolin (20 μM ) 12 h

↓TNF-,↓IL-6,

PPAR,PKC related

/

33

LPS/PIGLuteolin (20 μM ) 12 h stimulated RAW264.7/PCM

↓TNF-,↓IL-6, ↓MCP-1

↑AMPKα1

/

32

Mice on HFD Mice on HFD Mice on HFD Mice on HFD

Agent and dosage Duration Cytokines Signaling/Cellular changes Luteolin 0.01% in 20 weeks ↓TNF-，↓IL-6， ↑PI3K-AKT, ↑AMPKα1, diet ↓MCP-1 ↓M1/MMe polarisation cell infiltration to Luteolin 0.01% in 12 weeks ↓TNF-，↓IL-6， ↓Mast EAT, diet ↓MCP-1 ↑PI3K-AKT

16 weeks ↓TNF-，↓IL-6, ↓IL-1

3T3-L1 adipocytes

Luteolin (20 μM) 24 h

PA-treated HUVECs

Luteolin (10 μM) 30 min

PA-treated aortic ring

0.1% w/v

Co-culture of hypertrophied 3T3-L1 and RAW 264.7

Luteolin (20 μM) 24 h

30 min

↑Leptin, ↑Adiponectin, ↓TNF-α，↓IL-6, ↓MCP-1 ↓TNF-α，↓IL-6, ↑NO /

/

↑PPAR, ↑AKT2

increase the response of glucose uptake to insulin stimulation

↓IKKb. ↓NF-κB, ↑AKT/eNOS,

↓IRS-1 serine phosphorylation

/

↓TNF-α， ↓MCP-1,↓NO

↓NF-κB, ↓JNK


35

restore PA-induced insulin-mediated 36 endothelium-dependent damaged relaxation /

37

30

Page 31 of 41

Model LPS-stimulated macrophages from HFD-fed mice

Agent and dosage Luteolin (10 µM) Apigenin (10 µM) Velutin (2.5 µM) Nobiletin(10 µM) (100 C2C12 myotube Tangeretin µM) Tangeretin (64/128μM) 3T3-L1 adipocytes Nobiletin (64/128μM)


Duration Cytokines

Signaling/Cellular changes

1h

↓MAP3K-MAP2K, ↓NF-κB

3h 4 days

↓TNF-α，↓IL-6 /

↑AMPKα1

↑Adiponectin， ↓MCP-1 ↑Adiponectin， ↓MCP-1, ↓Resistin

/

Insulin sensitivity and resistance / ↑GLUT4 translocation ↓Triglyceride accumulation /

Ref 38 39 40

*Abbreviations used: AKT , Ak transforming, also known as Protein kinase B; AMPKα1, AMP-activated protein kinase α1; EAT, epididymis adipose tissues; eNOS, endothelial nitric oxide synthase; GLUT4, glucose transporter 4; GTT, glucose tolerance test; HFD, high-fat diet; HOMA-IR, homeostasis model assessment of IR; HUVECs, human umbilical vein endothelial cells; IL-1β, interleukin-1β; IL-6,interleukin-6; IRS-1, insulin receptor substrates-1; ITT, insulin tolerance test; JNK, c-Jun N-terminal kinase; LPS, lipopolysaccharide; MCP-1, monocyte chemotactic protein-1; NF-κB, nuclear factor kappa B; NO, nitric oxide ; PCM, peritoneal cavity resident macrophage ;PKC, protein kinase C; PI3K, phosphatidylinositol-4,5-bisphosphate 3-kinase;PIG, palmitate insulin and glucose; PPARγ, peroxisome proliferator-activated receptor γ; TNF-α,tumor necrosis factor-α; abbreviations are similar hereinafter.


31


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Table 2. Isoflavones inhibit inflammatory signaling and alleviate insulin resistance Model

Agent and dosage

Duration Cytokines

Insulin-resistant rat Insulin-resistant male rat

Soy isoflavones 450 30 days mg/kg i.g. daily Genistein 1 mg/kg 44 days i.g daily.

↑Adiponectin, ↓Resistin, ↓TNF-α ↓TNF-α，↓IL-6

PA-treated HUVECs

Genistein (10 μM)

↓TNF-α，↓IL-6

30 min

Signaling/Cellular changes / /

↓IKKb/NF-κB,

PA-treated aortic ring in vitro preadipocyte/adipocyte Puerarin (72M)

48 h

HUVECs PA-induced HUVECs

30 min

↓TNF-α，↓IL-6

↓IKKb/NF-kB,

24 h

↑Adiponectin

↓JNK, not PPARγ; ↑FoxO1 (not JNK, not PPAR)

Puerarin (10 μM)

TNF-α-induced 3T3-L1 Genistein (50 μM) Daidzein (50 μM)

/

/

/

↑PPARγ

Insulin sensitivity and resistance /

Ref.

↓Accumulation of lipids, ↓depletion of antioxidants, ↓HOMA-IR ↓IRS-1 serine phosphorylation restore the lost vasodilation ↑Preadipocyte differentiation, ↑Glucose-uptake / ↓IRS-1 serine phosphorylation / /

44

42

45

54

55 50

*Abbreviations used: PA, palmitate; FoxO1, Forkhead box protein O1; abbreviations are similar hereinafter.


32

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Table 3. Flavonols inhibit inflammatory signaling and alleviate insulin resistance Model Mice on HFD

Agent and dosage Quercetin 0.08% in diet

ob/ob mice

Quercetin 30 mg/kg i.p. every other day Quercetin 100 mg/kg in diet

Mice on HFD

Duration Cytokines Signaling/Cellular changes 10 weeks ↓TNF-α, ↓MCP-1, / ↑Adiponectin 10 weeks ↓TNF-α ↓JNK,↓iNOS

10 weeks ↓TNF-α, ↓IL-6， ↓JNK, ↓MAPK ↓IL-1,↓MCP-1, ↑IL-10 Leptin receptor- Myricetin 400 mg/kg i.g. 14 weeks ↑Adiponectin, / deficient db/db daily ↓TNF-α mice Mice on high- Myricetin 0.12% in diet 12 weeks ↑Adiponectin, / fat-high-sucrose ↓TNF-α, ↓IL-6, diet ↓Leptin Rats on high- Myricetin 1 mg/kg i.v., 3 14 days / ↑PI3K-AKT fructose diet times daily Rats on HFD with an injection of streptozotocin Zebrafish 3T3-L1 adipocytes and RAW264.7 cells

15 days 8 days

TNF-α treated Quercetin (25 μM) L6 myotube Primary human Quercetin (60 μM) adipocytes TNF-α treated Quercetin (20 μM) C2C12 cells

20 h

3T3-L1 adipocytes

3 days

Quercetin (50 μM), Kaempferol(50 μM)

1h 20 h

Ref 56

↑GLUT4, ↑Insulin sensitivity

62

↓HOMA-IR,

17

↑Insulin sensitivity;

68

↓Serum glucose, ↓serum insulin, 69 ↓HOMA-IR ↑Insulin sensitivity; ↑GLUT4 translocation; ↓Serum glucose, ↓Serum Insulin, ↓HOMA-IR; ↓IRS-1 serine phosphorylation; ↓Blood lipids and insulin; ↑Glucose-uptake, ↓IRS-1 serine phosphorylation

67

/ ↓NO generation ↓TNF-α, ↓NO, ↓NF-κB, ↓JNK, ↓MAPK ↓IL-6，↓IL-1β, ↓MCP-1, ↑IL-10, ↑Adiponectin / ↓IKKb/NF-κB, ↑AKT

↓Lipid accumulation /

17 17

↑Glucose uptake

62

↓IL-6，↓IL-1, ↓MCP-1 ↓NO

↓IRS-1 serine phosphorylation, 63 ↑Glucose uptake ↑Glucose uptake(both basal and 64 insulin stimulated glucose uptake)

Kaempferol 150 mg/kg i.g. 10 weeks ↓TNF-α，↓IL-6 daily Quercetin (25 μM) Quercetin (25 μM)

Insulin sensitivity and resistance ↓ HOMA-IR

/

↓IKKb/NF-κB,

↓NF-κB, ↓JNK, ↓MAPK, ↑PPARγ, ↓PTP-1B ↑AKT、↑AMPK, ↓ iNOS, ↓NF-κB /


77

↑Glucose uptake ( insulin 78 stimulated glucose uptake but not basal glucose uptake) 33


Model Agent and dosage LPS treated peritoneal Quercetin (50 μM), macrophage Kaempferol(50 μM) CV-1(a monkey kidney cell line)

Page 34 of 41

Duration Cytokines 24 h ↓NO

Signaling/Cellular changes /

Insulin sensitivity and resistance /

24 h

↑PPARγ

↓3T3-L1 differentiation

/

Ref 78

*Abbreviations used: MAPK, mitogen-activated protein kinase; IL-10, interleukin-10; PTP-1B, protein tyrosine phosphatase 1B; abbreviations are similar hereinafter.


34

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Table 4. Other flavonoids inhibit inflammatory signaling and alleviate insulin resistance Model

Agent and dosage

Duration

Cytokines

Signaling/Cellular changes

Rats on Highfructose diet

(-)-Epicatechin 20 mg/kg in diet

8 weeks

↓TNF-α,↓MCP-1

Rats on HFD

(-)-Epigallocatechin 16 weeks gallate 3.2 g/kg in diet

↓TNF-α, ↓IL-6,

Rats on HFD

Theaflavins 100 mg/kg i.g. daily

↓Leptin

↓PTP-1B ↑PI3K-AKT; ↓IKKb/NF-κB; ↓JNK ↓Infiltration of macrophages in adipose tissue, ↑PI3KAKT, ↓IKKb/NF-κB, /

Mice on HFD

Silymarin 60 mg/kg 18 days in diet

↓TNF-α，↓IL-6， ↓IL-1

TNF- induced 3T3-L1 cells

(-)-Epicatechin (10 μM)

↓TNF-α, ↓Resistin, ↓NF-κB , ↓MAPK, ↓IL-6, ↓MCP-1 ↓AP-1, ↑PPARγ, ↓PTP-1B, ↓JNK ↓TNF-α, /

30 days

4h

LPS-induced RAW (-)-Epicatechin 4h 267.4 cells (10 μM) HepG 2 cells (-)-Epigallocatechin 24 h gallate (50μM) HepG 2 cells Amentoflavone 36 h (13.94 μM)

/

↓TNF-α，↓IL-6， ↓NF-κB , ↓TNF-α，↓IL-6， ↓PI3K-AKT ↓IL-8

Insulin sensitivity and resistance ↓IRS-1 serine phosphorylation;

Ref

↓ HOMA-IR, ↓IRS-1 serine phosphorylation, ↑GLUT4

82

↑Insulin sensitivity (ISI)

84

↑Insulin sensitivity;

85

80

/ /

79

↑SOD1,↑SOD2, ↓ROS, 83 ↑glucose uptake, ↑GLUT2 ↑Glucose oxygenolysis, 86 glycogen synthesis, and gluconeogenesis

*Abbreviations used: AP-1, activator protein 1; ISI, insulin sensitive index; SOD1, superoxide dismutase 1; SOD2, Superoxide dismutase 2.


35


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Figures Figure 1

36


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Figure 2

37



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Figure 3

38


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Figure 4

39



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Figure 5

40


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Graphic for Table of Contents


41

Flavonoids alleviating insulin resistance through inhibition of

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