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Cocoa, Glucose Tolerance, and Insulin Signaling: Cardiometabolic Protection Davide Grassi,* Giovambattista Desideri, Francesca Mai, Letizia Martella, Martina De Feo, Daniele Soddu, Emanuela Fellini, Mariangela Veneri, Cosimo A. Stamerra, and Claudio Ferri Department of Life, Health & Environmental Sciences, University of L’Aquila, Coppito, Italy ABSTRACT: Experimental and clinical evidence reported that some polyphenol-rich natural products may offer opportunities for the prevention and treatment of type 2 diabetes, due to their biological properties. Natural products have been suggested to modulate carbohydrate metabolism by various mechanisms, such as restoring β-cell integrity and physiology and enhancing insulin-releasing activity and glucose uptake. Endothelium is fundamental in regulating arterial function, whereas insulin resistance plays a pivotal role in pathophysiological mechanisms of prediabetic and diabetic states. Glucose and insulin actions in the skeletal muscle are improved by insulin-dependent production of nitric oxide, favoring capillary recruitment, vasodilatation, and increased blood flow. Endothelial dysfunction, with decreased nitric oxide bioavailability, is a critical step in the development of atherosclerosis. Furthermore, insulin resistance has been described, at least in part, to negatively affect endothelial function. Consistent with this, conditions of insulin resistance are usually linked to endothelial dysfunction, and the exposure of the endothelial cells to cardiovascular risk factors such as hypertension, dyslipidemia, and hyperglycemia is associated with reduced nitric oxide bioavailability, resulting in impaired endothelial-dependent vasodilatation. Moreover, endothelial dysfunction has been described as an independent predictor of cardiovascular risk and events. Cocoa and cocoa flavonoids may positively affect the pathophysiological mechanisms involved in insulin resistance and endothelial dysfunction with possible benefits in the prevention of cardiometabolic diseases. KEYWORDS: cocoa, glucose tolerance, insulin resistance, cardiometabolic protection, diabetes, endothelial dysfunction
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According to this, arterial flogosis leads to further endothelial activation/dysfunction, promotes adhesion molecule expression, and supports leukocyte−endothelial interactions. The atherogenesis is finely adjusted by the immune system and systemic inflammatory reactions. In addition, pro-inflammatory mediators are fundamental triggers for atherosclerotic plaque development and stability.4,5 The enhanced oxidative stress is responsible for a total imbalance of the nitric oxide (NO) system, with decreased NO bioavailability and a paradoxical oxidant generation by endothelial NOS. As a consequence of the antiatherogenic, antithrombotic capacity of NO4,5 and the proatherogenic prothrombotic properties of endogenous oxidants,4,5 a decreased NO bioavailability with augmented oxidative and nitrosative stress will result not only in impaired NO-dependent vasodilation and blood pressure setting but also in the advancement of the atherogenetic process and onset of acute atherothrombotic events with increased plaque vulnerability. In this context, insulin resistance and endothelial dysfunction are recognized as having a pivotal role in the onset of different cardiometabolic disorders.4−7 Accordingly, insulin resistance is well-known to be the common link between metabolic and cardiovascular disturban-
INTRODUCTION Glucose homeostasis plays a pivotal role in the regulation of metabolic stability; therefore, it is under strict hormonal control. Failure of this balance with insulin resistance might result in disturbances of energy homeostasis encompassing obesity, hyperglycemia, impaired glucose tolerance, hypertension, and dyslipidemia and also a clustering in the condition named metabolic syndrome.1−3 The most distinctive abnormality in metabolic syndrome is insulin resistance, which is the consequence of different interactions between genetic pathways and environmental conditions, including diet and lifestyle. According to this, defects in both insulin action and insulin secretion are present in the evolution from metabolic syndrome to type 2 diabetes, and their relative contributions change individually. Furthermore, postprandial glucose peaks are considered a risk factor for developing diabetes and glucose intolerance. The disturbance of glucose metabolism is most frequently related to the increase of abdominal fat mass or generally to the tissues where fat is not normally stored for the energy balance.1−3 Insulin resistance, visceral fat, dyslipidemia, endothelial dysfunction, arterial hypertension, and blood hypercoagulability often draw the lines of this syndrome. Furthermore, visceral adiposity and insulin resistance are linked to chronic inflammation associated with the production of abnormal adipokines [i.e., tumor necrosis factor α, interleukin-1 (IL-1), IL-6, leptin, and adiponectin].3−5 The relationship between clinical and biological phenotype of the metabolic syndrome favors the development of a pro-inflammatory setup and, furthermore, a chronic, subclinical vascular flogosis able to modulate and enhance the atherosclerotic processes. © XXXX American Chemical Society
Special Issue: ISCHOM 1st International Congress on Chocolate and Cocoa in Medicine Received: February 24, 2015 Revised: June 24, 2015 Accepted: June 30, 2015
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that insulin is able to peculiarly recruit flow to the microvasculature in skeletal muscle via a NO-dependent pathway and that this may be fundamental for insulin action in regulating glucose uptake in vivo.15,16 In keeping with this, insulin signaling pathways, parallelly involved in vascular or metabolic functions, may help to strictly link the control of vascular function with glucose metabolism playing a fundamental physiological role in coupling hemodynamic and metabolic homeostasis in healthy states.12,15,16 On the other hand, considering glucose metabolism is coupled with blood flow, we might also affirm that metabolic variations could be able to impair blood flow, and increasing flow could provide changes in metabolism.15−17 Concordantly, it has been demonstrated that increased metabolic activity needs additional blood flow to provide the required substrates.15−17 Specifically, Vincent and colleagues,17 reported that in healthy subjects glucose and insulin peaked at 30 min after a meal, whereas the blood flow in the brachial artery (p < 0.05) and the microvascular volume (p < 0.01) increased at 60 min (microvascular volume in human forearm increased by ∼45%). Moreover, after exercise, both 50 and 80% maximal handgrip increased forearm and brachial artery blood flow (p < 0.01). The arterial flow immediately rose after contractions and decreased at basal levels over 15 s. Eighty percent maximal handgrip exercise was able to increase microvascular volume and microvascular flow velocity (p < 0.05 for both). Therefore, these findings supported the concept that physical exercise and meals might favor an improved muscle microvascular volume, suggesting that endothelial cells could be able to specifically interact with different nutrient effects and that capillary recruitment should be considered a fundamental step to ameliorate nutrient/hormone delivery and physiological functions in healthy humans.17 Consistent with this, the presence of PI3K-dependent insulin signaling pathways in vascular endothelium, skeletal muscle, and adipose tissue specifically supports the hypothesis that insulin is able to set some fundamental vasodilator and metabolic effects.12,14,18 Accordingly, MAPK-dependent insulin signaling pathways seem to be involved in blood pressure regulation and pro-atherogenic actions of insulin in various tissues.12,14,18 Furthermore, the effects of insulin on NOdependent arterial responses occur in distinct stages. Baron19 reported that after the dilation of the terminal arterioles we can see a rise in perfusing capillaries within a few minutes, not paralleled by changes in total limb blood flow in humans. Therefore, the author affirmed that the vasodilation induced by insulin is the consequence of the increased capillary recruitment and elevated total blood flow.19 Indeed, Coggins et al.20 suggested that local insulin intra-arterial infusion was able to enhance capillary blood volume by 25% in the deep flexor muscles of the human forearm. In particular, in agreement with a mechanism of capillary recruitment, the authors reported that physiologic increments in plasma insulin levels were able to raise microvascular blood volume, with changes in microvascular perfusion.20 They observed that insulin increased muscle glucose uptake (180%, p < 0.05) and microvascular blood volume (54%, p < 0.01) and decreased microvascular flow velocity (−42%, p = 0.07) without changes in total forearm blood flow in healthy subjects. Therefore, in vivo experimental and human studies support that insulin at physiological concentration is able to quickly increase skeletal muscle capillary recruitment. These vascular effects are fundamental in augmenting the delivery of insulin and glucose
ces.4−7 Lifestyle changes are considered the topical treatment strategies of therapy, with eventual additional pharmacologic treatments targeting the individual components of the metabolic syndrome. Thus, focusing the attention for therapies on either insulin resistance or endothelial dysfunction alone is likely to simultaneously improve both metabolic and cardiovascular pathophysiology and clinical aftermaths.4,8,9 In this regard, some authors10 supposed that high flavonoid intake and consequent NO system activation, for example, island-dwelling Kuna have a 3-fold larger urinary nitrate/nitrite ratio than do mainland dwellers,10,11 could be important in reducing ischemic heart disease, stroke, diabetes mellitus, and cancer, all NO-sensitive processes, in this particular population. The hypothesis was confirmed showing that in mainland Panama, cardiovascular disease was the leading cause of death (83.4 ± 0.70 age-adjusted deaths/100,000) and cancer was second (68.4 ± 1.6). In contrast, the rate of cardiovascular disease and cancer among island-dwelling Kuna was much lower (9.2 ± 3.1 and 4.4 ± 4.4, respectively). Similarly deaths due to diabetes mellitus were much more common in the mainland (24.1 ± 0.74) than in the San Blas (6.6 ± 1.94). This comparatively lower risk among Kuna in the San Blas possibly reflected a very high flavanol intake and sustained NO synthesis activation.10,11 In keeping with this, hemodynamic and metabolic homeostasis could be specifically related to the capacity of insulin to stimulate NO production in the vascular endothelium.5,8,12,13 Furthermore, different metabolic disturbances such as type 2 diabetes, obesity, and dyslipidemia present with a peculiar relationship between insulin resistance and endothelial dysfunction.5,8,12,13 According to this, increasing interest has been addressed to the putative role of cocoa flavanols, including their monomers and procyanidins, in conteracting the development of obesity and type 2 diabetes.
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INSULIN AND ENDOTHELIUM: FUNCTION AND DYSFUNCTION Insulin favors glucose uptake in skeletal muscle and adipose tissue by activation of the insulin receptor tyrosine kinase, subsequent phosphorylation of IRS-1, binding and activation of PI 3-kinase, and activation of the serine kinase PDK-1, which in turn phosphorylates and activates Akt and PKC-ζ, finally resulting in the expression of the GLUT-4 glucose transporter on the cellular membrane.12−14 Similarly, eNOS activity and NO production in endothelial cells are supported by a similar sequence of signals involving IRS-1, PI 3-kinase, PDK-1, and Akt able to finally phosphorylate eNOS at Ser1177.5,8,14−16 Insulin-stimulated NO synthesis brings capillary recruitment, drives vasodilatation, and increases blood flow to skeletal muscle, therefore improving the effects of glucose and insulin to skeletal muscle in vivo in male Sprague−Dawley rats.15,16 Evaluating the effects of L-nitro-L-arginine-methyl ester (LNAME), an inhibitor of eNOS, on hind limb blood flow, muscle microvascular recruitment, and hind limb glucose uptake after euglycemic hyperinsulinemia in rats, an experimental in vivo study showed that insulin significantly increased total blood flow of hind limb (p < 0.05), glucose uptake (p < 0.05), and skeletal muscle microvascular volume (p < 0.05) in male Sprague−Dawley rats.15 In contrast, addition of L-NAME to insulin (rats were infused for 2 h with either saline, insulin, LNAME, or insulin plus L-NAME) fully abolished the insulin effects on both limb flow and microvascular recruitment and reduced glucose uptake by 40% (p < 0.05), therefore suggesting B
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resistance (−56%), as well as in glucose-intolerant (−85%), normotensive (−91%), and hypertensive diabetic patients (−120%).23 Thus, authors reported that progressive impairment in the endothelium-dependent coronary vasodilation may occur with increasing severity of insulin resistance and impaired glucose tolerance.23 Accordingly, supporting the fundamental role of NO bioavailability in the regulation of metabolic effects of insulin, Baron et al.24 reported that the inhibition of the eNOS by the infusion of L-N-monomethyl arginine (L-NMMA) into the femoral artery in lean humans induced a decrease of 25% in the insulin-dependent glucose uptake. Of interest, clarifying the relationship between NO and insulin sensitivity, Shankar et al.25 reported that knockout mice that are homozygous null for the eNOS gene present with an hemodynamic phenotype of increased basal blood pressure and also insulin resistance.25 Therefore, both mechanisms are coupled in such a manner that endothelial dysfunction can cause insulin resistance, and this, in a vicious circle, negatively affects endothelial function. Furthermore, it has been reported that conditions with decreased NO bioavailability (PI3K-dependent pathway) and insulin resistance are able to stimulate ET-1 production (MAPK-dependent pathway) in overweight, obese, and type 2 diabetes patients.26,27 Thus, an imbalance between the endothelial and insulin-dependent pathways (MAPK-dependent and PI3K-dependent signals) might be involved in the endothelial derangements successively participating in the metabolic disturbances with insulin resistance.5,12 These specific pathophysiological links suggest the strict relationship between metabolic and cardiovascular diseases.5,12 All of these findings and the peculiar pathophysiological mechanisms sustaining the impairment in insulin action and NO-mediated vascular responses to the metabolic derangements induced by insulin resistance are of clinical interest to better understand the interrelationship between insulin resistance and endothelial dysfunction in the initiation and progression of atherosclerosclerotic process.5,12 Because many of these early mechanisms of cardiometabolic damage are present before the development of frank disease, it is possible to consider some lifestyle changes as fundamental interventions including flavonoids from food as effective tools of prevention and cardiometabolic protection.
to skeletal muscle. Therefore, insulin presents direct effects (rising glucose availability and uptake in skeletal muscle) and indirect effects (favoring glucose disposal by increasing blood flow). This crosstalk between metabolic and vascular systems is fundamental for coupling glucose homeostasis and endothelial function.18−20 Conversely, in pathological states with increased cardiometabolic risk (glucotoxicity, lipotoxicity, and inflammation) PI3K-dependent insulin signaling pathways, concurrent with the reciprocal link between insulin resistance and endothelial dysfunction, could be specifically impaired. Particularly, genetic and environmental factors lead to insulin resistance and endothelial dysfunction, and mechanisms supporting insulin resistance also frequently favor endothelial dysfunction.18−21 Indeed, glucotoxicity induced by hyperglycemia is able to give rise to endothelial damage and aggravate metabolic disturbances.5,8,12,13,18,21 Similarly, lipotoxicity in consequence of elevated free fatty acid levels in diabetes, obesity, and dyslipidemia might prime other mechanisms of insulin resistance and endothelial dysfunction. Pro-inflammatory and oxidative states associated with metabolic and cardiovascular disease could ulteriorly impair insulin resistance and endothelial damage induced by cardiovascular and metabolic risk factors.5,8,12,13,18,21 Consistent with this, experimental and clinical studies showed that insulin resistance is generally related with endothelial dysfunction and that the exposure of vascular endothelium to high blood pressure and circulating levels of lipids and glucose is linked to decreased NO bioavailability, resulting in impaired endothelial-dependent vasodilatation.5,8,12,13,18,21 These findings suggest that endothelial dysfunction, with reduced NO bioavailability, may be considered both cause and consequence of the metabolic disturbances observed in states of insulin resistance. According to this, Chiu et al.,22 investigating the effects of insulin directly injected into the interstitial space bypassing the transendothelial insulin transport step, reported that delay in insulin action, with respect to the intravenous administration of insulin, was virtually eliminated, with immediate dose-dependent increments in hindlimb glucose uptake in dogs. Additionally, the direct injection of insulin was able to give a 4-fold greater sensitivity to insulin of in vivo muscle tissue than previously reported from intravenous insulin administration (intramuscular injections of saline or insulin were administered directly into the vastus medialis of anesthetized dogs).22 The described delay in the action of insulin was related to the protracted time for insulin to pass through the capillary endothelium, therefore suggesting defects in insulin transport across the vascular endothelium of skeletal muscle may contribute to insulin resistance.22 Thus, the reported mechanisms with the onset of endothelial dysfunction, particularly in the arterioles and capillaries, ulteriorly damaging the effects of insulin on metabolism and glucose uptake, may initiate a vicious self-perpetuating negative feedback cycle.5,8,12,13,18,21 On the other hand, an increase of NO bioavailability with an improved endothelium-dependent vasodilation may be related to a specific decrease in insulin resistance and vice versa.5,8,12,13,18,21 Of clinical interest, Prior and colleagues23 indicated that total vasodilator capacity was similar in normoglycemic subjects, whith significant differences in normotensive (−17%) and hypertensive (−34%) patients with diabetes. With respect to insulin sensitivity, endothelium-dependent coronary vasodilation was significantly reduced in the presence of insulin
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INSULIN RESISTANCE AND ENDOTHELIAL DYSFUNCTION: COCOA AND CARDIOMETABOLIC PROTECTION Experimental Findings. Epidemiological and clinical studies revealed high-flavonoid diet or isolated flavanols such as (−)-epicatechin are able to improve the function of the vascular endothelium, as assessed by flow-mediated dilation, through elevation of NO bioavailability and bioactivity.4,8,28 Cocoa and chocolate received attention because of their high content in flavonoids.4,8,28 Flavonoids are structured as a common carbon skeleton of diphenylpropanes, two benzene rings (rings A and B) joined by a linear three-carbon chain (C6−C3−C6) usually forming an oxygenated heterocycle nucleus, the flavan nucleus (ring C). Depending on the structural complexity of flavonoids, particularly on the oxidation state of the central ring C, flavonoids are themselves subclassified as flavonols, flavones, flavanones, flavanols, or flavan-3-ols (catechins and their oligomers, proanthocyanidins), isoflavones, and anthocyanins.8,28 Differences in the chemical structures of these C
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substantially similar to those of mice with exercise.41 Furthermore, the same authors showed an additive effect in stimulating myocardial angiogenesis by the combination of epicatechin and exercise.35 Clinical Evidence. With regard to clinical studies, growing evidence supports that flavonoids and flavonoid-rich food intake can improve NO bioavailability in humans as suggested by a significant improvement in endothelial-dependent flowmediated dilation (FMD). Consistent with this, Fisher et al.42,43 showed that the intake of a cocoa beverage rich in flavonoids in healthy young and older subjects was able to improve endothelial-dependent vasodilation. Of interest, these vascular benefits were abolished by the infusion of an inhibitor of eNOS.42,43 According to this, an Australian study,44 aiming to test the effects of cocoa flavanols and regular exercise on cardiometabolic function and body composition in obese individuals, suggested that, compared to low-flavanol (36 mg flavanols), high-flavanol (902 mg flavanols) cocoa acutely increased NOdependent FMD by 2.4% (p < 0.01) and chronically (over 12 weeks; p < 0.01) by 1.6%, reducing insulin resistance by 0.31% (p < 0.05), independent of exercise. Supporting this, we reported that the intake of dark chocolate rich in flavonoids significantly increased the NO-dependent FMD, also positively affecting additional cardiovascular risk factors in healthy subjects45 as well as in hypertensive patients with and without glucose intolerance.46,47 Thus, supporting the hypothesis that flavanols are able to not just ameliorate the function of a normal endothelium but also restore endothelial dysfunction of an abnormal endothelium.46,47 In agreement with our results obtained in patients with metabolic disturbances, Balzer and colleagues48 showed that a single dose of flavanol-rich cocoa was able to dose-dependently and acutely raise FMD (at 2 h, from 3.7 ± 0.2 to 5.5 ± 0.4%, p < 0.001) and consequently increase circulating levels of flavanols in patients with medicated type 2 diabetes. Moreover, evaluating longer term effects, the same authors reported that administration of cocoa three times a day significantly increased baseline FMD by 30% (p < 0.0001) during a 30-day period. Moreover, acute increases of FMD upon ingestion of flavanol-rich cocoa continued to be expressed throughout the study.48 Additionally, in overweight adults Faridi et al.49 reported that, compared with cocoa-free placebo, solid dark chocolate and liquid cocoa (with 22 g cocoa powder) intake improved endothelial function (dark chocolate, 4.3 ± 3.4 vs −1.8 ± 3.3%; p < 0.001; sugar-free and sugared cocoa, 5.7 ± 2.6 and 2.0 ± 1.8% vs −1.5 ± 2.8%; p < 0.001). Endothelial function was increased significantly more after sugar-free than after regular cocoa ingestion (5.7 ± 2.6 vs 2.0 ± 1.8%; p < 0.001). This also supports the idea that sugar content might contrast the healthy advantages of flavonoid intake, whereas sugar-free preparations may raise their benefits.49 Exactly consequent of this evidence, with respect to flavanolfree white chocolate, flavanol-rich dark chocolate administration was able to improve FMD (p = 0.03), arterial stiffness, ET1, and 8-iso-PGF(2α) circulation levels (p < 0.05) in healthy subjects.50 In particular, after flavanol-free white chocolate ingestion, at 1 and 2 h from an oral glucose tolerance test (OGTT), FMD decreased from 7.88 ± 0.68 to 6.07 ± 0.76 (p = 0.027) to 6.74 ± 0.51 (p = 0.046), respectively.50 Always after white chocolate but not after dark chocolate, OGTT impaired wave reflections, blood pressure, and ET-1 and 8-iso-PGF(2α) levels.50 Thus, OGTT produced an acute, transient impairment of NO-dependent vasodilation with concomitant increase in
subclasses influence both their biological efficacy and bioavailability.8,28 In this regard, several lines of evidence suggest that flavanols may be a causal relationship between flavanol consumption and improvements in cardiovascular mortality and morbidity.28−31 Some authors also demonstrated that procyanidins were able to increase aortic ring (from New Zealand White rabbits) relaxation and eNOS activity.32 Accordingly, cocoa contains high levels of flavonoids, which have been demonstrated to enhance tyrosine phosphorylation in an insulin-like manner, by the activation of the PI3K/Akt and AMPK pathways in the vascular endothelium with a rise in NO availability. 33 Supporting the fundamental role of flavanols positively affecting endothelium/NO mechanisms involved in the control of the arterial basal tone, we recently showed that epigallocatechin-3gallate and (−)-epicatechin induced a dose-dependent vasodilation, in phenylephrine precontracted endothelium-intact preparations of rat-isolated aortic rings.34 Furthermore, we also reported that epigallocatechin-3-gallate and (−)-epicatechin did not significantly induce vasorelaxation in precontracted endothelium-denuded aortic rings. However, in endotheliumintact precontracted preparations, the inhibitor of eNOS activity Nω-nitro-L-arginine (L-NNA) abolished the vasorelaxant effects of epigallocatechin-3-gallate and (−)-epicatechin. At high concentrations, epigallocatechin-3-gallate and (−)-epicatechin elicited a marked relaxation. This was significantly larger in the presence than in the absence of endothelium or in the presence of L-NNA.34 Consistent with this, Ramirez-Sanchez et al.35 reported that epicatechin phosphorylates and activates eNOS through Akt and a complex with HSP90 in human coronary artery endothelial cells, whereas Persson et al.36 reported a significant and dose-dependent inhibition of angiotensin-converting enzyme (ACE) activity in cultured human umbilical vein endothelial cells after incubation with (−)-epicatechin, (−)-epigallocatechin, (−)-epicatechin gallate, and (−)-epigallocatechin gallate. This effect was combined to yield a significant dose-dependent increase in NO production. In this regard, supporting our data, Tomaru et al.37 suggested dietary supplementation with cocoa liquor procyanidins dose-dependently prevented the development of hyperglycemia in diabetic obese mice. With respect to a control diet without cocoa liquor, a diet containing 0.5 or 1.0% cocoa liquor proanthocyanidins decreased the blood glucose and fructosamine concentrations, with no significant changes reported on body weight or food consumption.37 Furthermore, a recent study38 suggested that oligomeric cocoa procyanidins prevented the development of obesity, insulin resistance, and impaired glucose tolerance during high-fat feeding in mice. Similarly, Cordero-Herrera et al.39 showed that (−)-epicatechin and cocoa polyphenolic extract improved insulin sensitivity of human HepG2 cells exposed to high glucose levels, preventing or counteracting a potential hepatic dysfunction via the attenuation of the insulin signaling blockade and the modulation of glucose uptake and production. The same research group also showed that a cocoa phenolic extract rich in flavonoids was able to protect against the oxidative stress induced by tert-butylhydroperoxide on pancreatic β cells.40 Of note, Nogueira et al.41 reported that epicatechin intake ameliorated exercise performance and resistance to muscle fatigue in male mice. Of particular interest, the structural and metabolic changes in skeletal and cardiac muscle of mice treated with epicatechin alone (without exercise) were D
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and, thus, a significant improvement was not expected. Our findings also supplied evidence on dose-dependent effects with respect to the epicatechin content. Indeed, independent of the flavonoid concentration, the cocoa we used in the study was similar in color, taste, flavor, and all possible vasoactive contents. Starting from these findings, the European Food Safety Authority (EFSA) for the first time claimed that “a cause-and-effect relationship may be established between the consumption of cocoa flavanols and maintenance of normal endothelium-dependent vasodilation” and clearly stated that “in order to obtain the claimed effect, 200 mg of cocoa flavanols should be consumed daily. This amount could be provided by 2.5 g of high-flavanol cocoa powder or 10 g of high-flavanol dark chocolate. These amounts of cocoa powder or dark chocolate can be consumed in the context of a balanced diet. The target population is the general population.”53 Of interest, Almoosawi et al.54 observed that the effects, in overweight and obese subjects, in reducing fasting blood glucose levels, systolic blood pressure, and diastolic blood pressure of dark chocolate containing 500 mg of polyphenols were not different from those of the 1000 mg polyphenol chocolate. These results suggest a feasible saturation effect might occur with increasing dose of polyphenols.54 Moreover, of extreme interest, Curtis and colleagues55 in a parallel-design, placebo-controlled trial55 reported that, compared with the placebo, consumption of flavonoid-rich chocolate [850 mg of flavanols (90 mg of epicatechin)] for 1 year resulted in a significant reduction in estimated peripheral insulin resistance (HOMA, −0.3 ± 0.2; p = 0.004) and gain in insulin sensitivity (QUICKI, 0.003 ± 0.00; p = 0.04), particularly as a consequence of reduced insulin levels (−0.8 ± 0.5 mU/L; p = 0.02) in medicated postmenopausal women with type 2 diabetes. Consistent with these findings on metabolism, we also reported that cocoa flavanols might be effective in improving cognitive function in elderly subjects with mild cognitive impairment, a well-known condition related to insulin resistance and diabetes.56,57 In particular, compared with lowflavanol cocoa, volunteers assigned to high and intermediate flavanol cocoa treatments presented with significant lower time required to complete Trail Making Test A and Trail Making Test B (p < 0.05).56 Verbal fluency test score was significantly (p < 0.05) better in the groups assigned to high flavanols in comparison with those assigned to low flavanols (27.50 ± 6.75 versus 22.30 ± 8.09 words per 60 s).56 Of note, insulin resistance, blood pressure, and lipid peroxidation also decreased among subjects in the high-flavanol and intermediate-flavanol groups. Nevertheless, particular interest derived from the evidence that changes of insulin resistance explained ≈40% of composite z-score variability through the study period (p < 0.0001).56 Therefore, these data suggest that regular consumption of cocoa flavanols might be effective in improving cognitive function in elderly subjects with mild cognitive impairment and this might be mediated, at least in part, by an amelioration of vascular function and insulin sensitivity.56 Concordant with this, a systematic review and meta-analysis by Shrime et al.58 suggested that short-term cocoa consumption is able to significantly improve cardiovascular health, blood pressure, LDL-cholesterol levels, insulin resistance, and FMD. Specifically, with regard to insulin resistance, the authors showed that HOMA-IR was significantly decreased (p < 0.001) by flavonoid-rich cocoa intake.58 Furthermore, flavonoid-rich cocoa consumption was described to significantly raise ISI (p
