Critique 027: Effects of resveratrol and quercetin on inflammation and insulin resistance – 20 December 2010

Chuang C-C, Martinez K, Xie G, Kennedy A, Bumrungpert A, Overman A, Jia W, McIntosh MK.  Quercetin is equally or more effective than resveratrol in attenuating tumor necrosis factor-a–mediated inflammation and insulin resistance in primary human adipocytes.  Am J Clin Nutr 2010;92:1511–1521.

Authors’ Abstract

Background: Quercetin and trans-resveratrol (trans-RSV) are plant polyphenols reported to reduce inflammation or insulin resistance associated with obesity.  Recently, we showed that grape powder extract, which contains quercetin and trans-RSV, attenuates markers of inflammation in human adipocytes and macrophages and insulin resistance in human adipocytes.  However, we do not know how quercetin and trans-RSV individually affected these outcomes.

Objective: The aim of this study was to examine the extent to which quercetin and trans-RSV prevented inflammation or insulin resistance in primary cultures of human adipocytes treated with tumor necrosis factor-a (TNF-a)—an inflammatory cytokine elevated in the plasma and adipose tissue of obese, diabetic individuals.

Design: Cultures of human adipocytes were pretreated with quercetin and trans-RSV followed by treatment with TNF-a.  Subsequently, gene and protein markers of inflammation and insulin resistance were measured.

Results: Quercetin, and to a lesser extent trans-RSV, attenuated the TNF-a–induced expression of inflammatory genes such as interleukin (IL)-6, IL-1b, IL-8, and monocyte chemoattractant protein-1 (MCP-1) and the secretion of IL-6, IL-8, and MCP-1.  Quercetin attenuated TNF-a–mediated phosphorylation of extracellular signal–related kinase and c-Jun-NH2 terminal kinase, whereas trans-RSV attenuated only c-Jun-NH2 terminal kinase phosphorylation.  Quercetin and trans-RSV attenuated TNF-a–mediated phosphorylation of c-Jun and degradation of inhibitory jB protein.  Quercetin, but not trans-RSV, decreased TNF-a–induced nuclear factor-jB transcriptional activity. Quercetin and trans-RSV attenuated the TNF-a– mediated suppression of peroxisome proliferator–activated receptor c (PPARc) and PPARc target genes and of PPARc protein concentrations and transcriptional activity.  Quercetin prevented the TNF-a– mediated serine phosphorylation of insulin receptor substrate-1 and protein tyrosine phosphatase-1B gene expression and the suppression of insulin-stimulated glucose uptake, whereas trans-RSV prevented only the TNF-a–mediated serine phosphorylation of insulin receptor substrate-1.

Authors’ Conclusion: These data suggest that quercetin is equally or more effective than trans-RSV in attenuating TNF-a–mediated inflammation and insulin resistance in primary human adipocytes.

Forum Comments

Ever since the American public first learned about the “French Paradox” almost two decades ago, the media has latched on to resveratrol as “the magic ingredient in red wine” that protects the French from coronary heart disease.  In fact, numerous pharmaceutical companies have begun to formulate pills containing high doses of resveratrol and selling them as “alternatives to red wine.”  This paper is of interest as it compares resveratrol with another constituent in wine, quercetin.

Background:  There have been hundreds of epidemiologic and basic scientific studies of the effects of alcohol and of polyphenols and other constituents in wine for their effects on cardiovascular risk.  Further, recent epidemiologic studies have reported reductions in the risk of metabolic diseases such as diabetes and metabolic syndrome from such consumption.  One constituent of red wine is quercetin; the effects of quercetin and other polyphenols on glucose uptake in adipocytes were reported by Strobel et al in 2005.¹ These investigators showed that three flavonoids, quercetin, myricetin and catechin-gallate, inhibit the uptake of methylglucose by adipocytes over the concentration range of 10-100 microM and suggested that GLUT transporters are involved in flavonoid incorporation into cells.  The present paper compares effects of quercetin and resveratrol on inflammation and insulin resistance in human adipocytes.

Comments on Study by Forum members:  The paper by Chuang is intrinsically correct and demonstrates that the two polyphenols studied do have a biological effect on these cells under those specific conditions.  On the other hand, as described below, the in vitro conditions they describe are minimally representative of an in vivo condition.  While the authors state that quercetin or resveratrol have been shown to penetrate by passive diffusion into cells, these results were only assessed in vitro.  In vivo, after consumption of quercetin or resveratrol, these compounds undergo extensive metabolism, leading to glucuronidated, sulphated or methylated compounds.  Indeed experiments carried out in rats have shown that more than 85% of the ingested quercetin was present in the gastrointestinal tract at all time points with ∼6% being absorbed and present in blood and internal organs, primarily the liver and kidneys.  More than 95% of the absorbed quercetin was in the form of > 20 different methylated glucuronated and/or sulfated quercetin conjugates.² Moreover, phenolic glycosides that are not absorbed in the small intestine pass into the colon, which contains micro-organisms, with a very high degree of biocatalytic power.

In general, phenolic acids arise from the B ring of flavonoids and hence the type of metabolite will depend on the hydroxylation pattern.  As a specific example of microbial metabolism, quercetin 3-glucoside was transformed to 3,4-dihydroxyphenylacetic acid, acetate and butyrate by Eubacterium ramulus from human gut.³ Only 3′-methylquercetin has been detected in human plasma, present at a concentration of 0.1 to 0.2 µM after 3 h. The authors of the current paper are using concentrations up to 60 µM, concentrations which have not been found in vivo.

There are also problems with the work on cell uptake of quercetin and resveratrol.  Primary adipocytes were incubated with the polyphenols, but it is not clear whether or not the concentrations used were subtoxic.  The authors stated: “No visible signs of quercetin or trans-RSV cytotoxicity were noted (e.g., no floating cells, no visual differences in the number of attached cells or protein concentrations, and no abnormal changes in cell morphology”).  It would have been helpful if the authors presented a cell-death assay such as MTT, LDH or Alamar.  Indeed it would take at least 3-6 hours before seeing any toxicity (caspases activation, drop in GSH, activation of proteasome, etc.).  Moreover, the authors state: “It appears that quercetin and trans-RSV are rapidly taken up by adipocytes.  However, it is not possible to determine the relative rates of absorption of quercetin or trans-RSV from these data.”  And they also state, “Indeed, we detected some unknown peaks by LC-TOF-MS after treatment of quercetin or trans-RSV in primary human adipocytes.”  If they were using LC-TOF-MS, they should provide the fragmentation pattern and the m/z of the ion formed.

Difficulties in extrapolation from basic science studies to clinical medicine:  A Forum reviewer states: “Our current knowledge is limited about local concentration of the molecules we are studying in subcellular compartments, their interaction with alternative targets, and eventually their transformation into products that could be more or less active on a given specific pathway.  The real difficult and important issue is the identification of a reasonable convergence — if not agreement — between data originating from extremely distant approaches.  In this case, the notion that metabolic diseases are related to a homeostatic imbalance in adipose tissue, linked to a different redox status, linked to activation of specific pathways, and that different redox sensitive polyphenols do have a protective effect, encompasses the evidence produced by extremely distant approaches.  None of those is perfect, but the apparent convergence is per se an achievement worth of consideration.”  Another Forum reviewer adds: “I would point out that there are many articles similar to this one that are studying the ‘effects’ of the natural food components in such systems, so these authors are not singularly out of touch.”

Still another Forum reviewer states: “The paper by Chuang et al provides an example of the difficulty of drawing serious conclusions from molecular nutrition studies.  Our information ranges from epidemiological analysis to cell biology to biochemical studies at the molecular level.  Each approach has its limits and bias.  Another problem emerges from the fact that we are usually approaching nutraceuticals by using the pharmacological approach we are familiar with.  We are usually looking for a dose-effect relationship and we — usually– assume a linear relationship.  This cannot necessarily be the case for nutraceuticals that do not produce a straightforward effect, but most frequently slight metabolic shifts eventually decreasing the risk of a given pathological status.

“Let us take as a pertinent example quercetin, as in this study: we have to deal with information from epidemiology to molecular dynamics for interaction with specific targets.  It is an easy prevision that a full agreement and consistency of all the results is no more than a wishful dream and results could be confuted when seen from a different perspective.”

Wine as a “functional food”:  With the current epidemic of obesity-related diseases emerging worldwide, dietary manipulation with functional foods is becoming a potentially valuable therapeutic tool for clinicians.  Wine, as a functional food may play an important role in this regard, because it is a reliable natural source of quercetin and resveratrol.  A Forum reviewer states: “The outcome of the study is suggestive of a mechanism that could easily account for a known nutraceutical effect.  The distance between the objects of analytical observation — from molecules to populations — are too far from each other to be encompassed in univocal and unbiased conclusions.  Aiming to provide a rational nutritional recommendation, we have to consider all the aspects and to apply an unavoidable educated guess to bridge gaps that would be unrealistic hoping to unravel to the bottom.

“Phytochemicals such as those present in wine are highly complex compounds, and the in vitro studies cannot always be applied to the in vivo situation.  For instance, quercetin is a cancer producing glycoside, but when ingested, the sugar moiety is split off, and it becomes an anti-cancer chemical.  From a clinical point of view, the role of phytochemicals acting as antioxidants and anti-inflammatory agents is becoming extremely important in inflammation-associated chronic conditions such as cardiovascular disease, diabetes, and cancer.  Quercetin and resveratrol may indeed play an important role in this regard, and need to be investigated to establish the clinical importance of natural dietary compounds in the prevention of chronic degenerative conditions.”

References from Forum critique

1.  Strobel, P., Allard, C., Perez-Acle, T., Calderon, R., Aldunate, R., Leighton, F..  The effects of myricetin, quercetin and catechin-gallate on glucose uptake in isolated rat adipocytes  Biochemical Journal 2005;386:471-478.

2.  Graf BA, Mullen W, Caldwell ST, Hartley RC, Duthie GG, Lean ME, Crozier A, Edwards CA.  Disposition and metabolism of [2-14C]quercetin-4′-glucoside in rats.  Drug Metab Dispos 2005;33:1036-1043.

3.  Williamson G,  Day A-J, Plumb GW, Couteau D.  Human metabolic pathways of dietary flavonoids and cinnamates.  Biochem Soc Trans 2000;28:16-22.

Forum Summary

A study was carried out to examine the extent to which quercetin and trans-resveratrol (RSV) prevented inflammation or insulin resistance in primary cultures of human adipocytes treated with tumor necrosis factor-a (TNF-a)—an inflammatory cytokine elevated in the plasma and adipose tissue of obese, diabetic individuals.  Cultures of human adipocytes were pretreated with quercetin and trans-RSV followed by treatment with TNF-a.  Subsequently, gene and protein markers of inflammation and insulin resistance were measured.  The authors report that quercetin, and to a lesser extent trans-RSV, attenuated the TNF-a–induced expression of inflammatory genes such as interleukin (IL)-6, IL-1b, IL-8, and monocyte chemoattractant protein-1 (MCP-1) and the secretion of IL-6, IL-8, and MCP-1.

Forum members were concerned about certain aspects of the study, especially the extrapolation of in vitro results to in vivo situations.  The in vitro conditions the authors describe are minimally representative of an in vivo condition.  In vivo, after consumption of quercetin or resveratrol, these compounds undergo extensive metabolism, leading to glucuronidated, sulphated or methylated compounds.  In a previous study, quercetin 3-glucoside was transformed to 3,4-dihydroxyphenylacetic acid, acetate and butyrate in cells from human gut; only 3′-methylquercetin has been detected in human plasma, present at a concentration of 0.1 to 0.2 µM after 3 h.  The authors of the current paper are using concentrations up to 60 µM, concentrations which have not been found in vivo.

There were also concerns with the work on cell uptake of quercetin and resveratrol.  Primary adipocytes were incubated with the polyphenols, but it is not clear whether or not the concentrations used were subtoxic.  Our current knowledge is limited about local concentration of the molecules we are studying in subcellular compartments, their interaction with alternative targets, and eventually their transformation into products that could be more or less active on a given specific pathway.  The real difficult and important issue is the identification of a reasonable convergence — if not agreement — between data originating from extremely distant approaches.  In this case, the notion that metabolic diseases are related to a homeostatic imbalance in adipose tissue, linked to a different redox status, linked to activation of specific pathways, and that different redox sensitive polyphenols do have a protective effect, encompasses the evidence produced by extremely distant approaches.

From a clinical point of view, the role of phytochemicals acting as antioxidants and anti-inflammatory agents could be extremely important in inflammation-associated chronic conditions such as cardiovascular disease, diabetes, and cancer.  Quercetin and resveratrol may indeed play an important role in this regard, and need to be investigated further to establish the clinical importance of natural dietary compounds in the prevention of chronic degenerative conditions.

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Comments included in this critique by the International Scientific Forum on Alcohol Research were provided by the following:

David Vauzour, PhD, Dept. of Food and Nutritional Sciences, The University of Reading, UK

Fulvio Ursini, MD, Dept. of Biological Chemistry, University of Padova, Padova, Italy

David Van Velden, MD, Dept. of Pathology, Stellenbosch University, Stellenbosch, South Africa

Andrew L. Waterhouse, PhD, Marvin Sands Professor, Department of Viticulture and Enology, University of California, Davis

Federico Leighton, MD, Laboratorio de Nutricion Molecular, Facultad de Ciencias Biologicas, Universidad Catolica de Chile, Santiago, Chile

R. Curtis Ellison, MD, Section of Preventive Medicine & Epidemiology, Boston University School of Medicine, Boston, MA, USA

Harvey Finkel, MD, Hematology/Oncology, Boston University Medical Center, Boston, MA, USA