Nutraceutical approaches to metabolic syndrome

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1 Annals of Medicine ISSN: (Print) (Online) Journal homepage: Nutraceutical approaches to metabolic syndrome Cesare R. Sirtori, Chiara Pavanello, Laura Calabresi & Massimiliano Ruscica To cite this article: Cesare R. Sirtori, Chiara Pavanello, Laura Calabresi & Massimiliano Ruscica (2017) Nutraceutical approaches to metabolic syndrome, Annals of Medicine, 49:8, , DOI: / To link to this article: Published online: 22 Aug Submit your article to this journal Article views: 921 View related articles View Crossmark data Citing articles: 9 View citing articles Full Terms & Conditions of access and use can be found at

2 ANNALS OF MEDICINE, 2017 VOL. 49, NO. 8, REVIEW ARTICLE Nutraceutical approaches to metabolic syndrome Cesare R. Sirtori a, Chiara Pavanello b, Laura Calabresi b and Massimiliano Ruscica c a Centro Dislipidemie, A.S.S.T. Grande Ospedale Metropolitano Niguarda, Milan, Italy; b Dipartimento di Scienze Farmacologiche e Biomolecolari, Centro E. Grossi Paoletti, Universita degli Studi di Milano, Milan, Italy; c Dipartimento di Scienze Farmacologiche e Biomolecolari, Universita degli Studi di Milano, Milan, Italy ABSTRACT Metabolic Syndrome (MetS), affecting at least 30% of adults in the Western World, is characterized by three out of five variables, from high triglycerides, to elevated waist circumference and blood pressure. MetS is not characterized by elevated cholesterolemia, but is rather the consequence of a complex interaction of factors generally leading to increased insulin resistance. Drug treatments are of difficult handling, whereas well-characterized nutraceuticals may offer an effective alternative. Among these, functional foods, e.g. plant proteins, have been shown to improve insulin resistance and reduce triglyceride secretion. Pro- and pre-biotics, that are able to modify intestinal microbiome, reduce absorption of specific nutrients and improve the metabolic handling of energy-rich foods. Finally, specific nutraceuticals have proven to be of benefit, in particular, red-yeast rice, berberine, curcumin as well as vitamin D. All these can improve lipid handling by the liver as well as ameliorate insulin resistance. While lifestyle approaches, such as with the Mediterranean diet, may prove to be too complex for the single patient, better knowledge of selected nutraceuticals and more appropriate formulations leading to improved bioavailability will certainly widen the use of these agents, already in large use for the management of these very frequent patient groups. ARTICLE HISTORY Received 10 July 2017 Revised 4 August 2017 Accepted 7 August 2017 KEYWORDS Metabolic syndrome; nutraceuticals; probiotics; berberine and vitamin D KEY MESSAGES Functional foods, e.g. plant proteins, improve insulin resistance. Pro- and pre-biotics improve the metabolic handling of energy-rich foods. Nutraceutical can offer a significant help in handling MetS patients being part of lifestyle recommendations. Introduction Metabolic syndrome (MetS) is present as a common clinical condition in countries where obesity and Western unhealthy dietary patterns prevail. The incidence of MetS is increasing to epidemic proportions thus entailing substantial health care costs. Indeed, MetS associates with an elevated risk of cardiovascular disease and type 2 diabetes (T2D), as well as other conditions, such as non-alcoholic fatty liver disease. After the first formal definition of MetS introduced in 1998 by the World Health Organization (WHO), several expert panels have attempted to introduce diagnostic criteria. In 2001 the National Cholesterol Education Program (NCEP) Adult Treatment Panel III (ATP III) [1] recognized for the first time the clusterization of the metabolic risk factors, these being also cardiovascular risk factors. In 2003, the American Association of Clinical Endocrinologists modified the ATP III criteria highlighting the central role of insulin resistance in the pathogenesis of the syndrome [2]. In 2005, the International Diabetes Federation (IDF) issued a consensus document aimed at producing a clinically useful definition of Mets to identify individuals at high risk of cardiovascular disease and T2D on a worldwide basis [3]. In the same year, the American Heart Association (AHA)/National Heart, Lung and Blood Institute (NHLBI) suggested criteria for the diagnosis of MetS [3]. Finally, IDF, NHBLI, AHA, World Heart Federation and the International Association for the Study of Obesity produced a joint statement called the Harmonization definition, which, as of today, represents the most common recognized criteria for the clinical diagnosis of MetS [4], summarized in Table 1. The present review will address the management of MetS by the use of specific nutraceuticals, in particularly plant proteins, pro/prebiotics, curcumin, CONTACT Cesare R. Sirtori cesare.sirtori@icloud.com, cesare.sirtori@unimi.it Centro Dislipidemie, A.S.S.T. Grande Ospedale Metropolitano Niguarda, Milan, Italy; Massimiliano Ruscica massimiliano.ruscica@unimi.it Dipartimento di Scienze Farmacologiche e Biomolecolari, Universita degli Studi di Milano, Milan, Italy ß 2017 Informa UK Limited, trading as Taylor & Francis Group

3 ANNALS OF MEDICINE 679 Table 1. Risk factors for the clinical diagnosis of metabolic syndrome. Value Alternative indicator Waist circumference a > 94 cm in males, >80 cm in females b >102 cm in males, >88 cm in females Raised blood pressure Systolic 130 and/or diastolic 85 mm Hg Treatment of previously diagnosed hypertension Raised FPG 100 mg/dl (5.6 mmol/l) Previously diagnosed of T2DM Raised TG >150 mg/dl (1.7 mmol/l) Specific pharmacological treatment Reduced HDL-C <40 mg/dl (1.0 mmol/l) in males <50 mg/dl (1.3 mmol/l) in females Specific pharmacological treatment a Based on the International Diabetes Federation (IDF) threshold for Europid population. b Based on the AHA/NHLBI (ATP III) threshold for USA population. FPG: fasting plasma glucose; TG: triglycerides; HDL-C: high-density lipoprotein-cholesterol; T2DM: type 2 diabetes mellitus. Conversion factors: (i) mg/dl cholesterol ¼ mmol/l 38.6; (ii) mg/dl triglycerides ¼ mmol/l 88.5 and (iii) mg/dl glucose ¼ mmol/l 18. Reproduced with permission of Elsevier [64]. berberine, red yeast rice and vitamin D. The activity of these substances will be analyzed based on the described major MetS features. While in many cases, e.g. MetS linked to elevated blood pressure, drug treatments may be of higher efficacy, the present favourable view of nutraceuticals by patients and by the medical community, makes it mandatory to carefully analyze what these products may offer in terms of improvement of metabolic health in MetS. The major features of MetS will be hereafter outlined. Metabolic syndrome: a deep overview Body variables Abdominal obesity is a prominent feature of MetS contributing to an elevation of cardiovascular risk. Indeed, excess abdominal adipose tissue releases large amounts of FFAs via the portal system to the liver, and may interfere with hepatic insulin clearance [5], thus contributing to IR [6]. In addition, it is an active endocrine organ, secreting different adipokines, including leptin, adiponectin, resistin, interleukins (IL), and tumour necrosis factor (TNF)-a [7]. In the case of excess abdominal weight, there is evidence of macrophage infiltration into the adipose tissue [8], contributing to the inflammatory profile reported in MetS patients. As opposed to proinflammatory adipokines, adiponectin levels are instead reduced in patients with excess visceral adiposity [9]. Arbitrary cut-offs based on epidemiological studies have been adopted for an elevated waist circumference (WC) [10] rated by all as an appropriate maker of central obesity with a strong association with the metabolic components of cardiovascular disease. However, cut-offs for WC varied for sexes, ethnic groups and even countries [1,3]. The IDF proposed a WC 94 cm for men and 80 cm for women as a threshold for abdominal obesity [3]; these thresholds were, however, not found to be applicable to most Western populations. Higher thresholds are generally used to define abdominal obesity in the United States [11] and the AHA/NHLBI (ATPIII) guidelines for MetS recognize an increased cardiovascular and diabetic risk at WCs of 102 cm for men and 88 cm for women [4]. In view of the difficulty in agreeing on a common threshold, in addition to the general unavailability of an accurate WC measurement, other variables, such as BMI [12,13] and the waist-to-height ratio (WHtR) have been suggested. This latter, in addition to WC, is being adopted as a more accurate indicator of obesityrelated cardiovascular risk. The WHtR has the advantage of being similar in most populations, i.e. generally below 0.5 for both men and women, as well as for ethnical subgroups [14]. Atherogenic dyslipidemia Carriers of MetS are characterized by a triad of metabolic perturbations called atherogenic dyslipidemia. Such perturbations, frequently linked to insulin-resistance (IR), the hallmark of pre-diabetes and T2D, are comprehensive of (i) high levels of apolipoprotein B (apob)-containing lipoproteins, including verylow-density lipoprotein (VLDL) remnants, (ii) small lowdensity lipoprotein (sldl), and (iii) reduced highdensity lipoprotein (HDL) levels [15]. In particular, IR affects the activities of enzymes and transfers proteins that play a key role in lipoprotein metabolism, such as lipoprotein lipase (LPL) and cholesteryl ester transfer protein (CETP). IR may also affect hepatic lipase (HL), phospholipid transfer protein (PLTP), and endothelial lipase (EL) [16]. IR may cause these metabolic changes in the presence of suppressed hormone-sensitive lipase (HSL) in visceral adipocytes; this process raises free fatty acids (FFA) mobilization from adipose tissue, leading to hepatic triglyceride (TG) overload [17]. HSL is an intracellular neutral lipase that hydrolyzes triacylglycerols, diacylglycerols, monoacylglycerols and cholesteryl esters. The increased efflux of FFA from adipose tissue

4 680 C. R. SIRTORI ET AL. and their reduced skeletal muscle uptake raises FA flux to the liver [18], thus raising VLDL secretion and consequent plasma TG levels [19]. Insulin may further affect TG-rich lipoprotein metabolism by inhibiting the microsomal transfer protein (MTP), responsible for the lipidation of nascent apob during their translocation into the lumen of the endoplasmic reticulum [20]. Notably, TGs lipidating apob are derived from the FFA released from the adipocyte, from VLDL remnants and from de novo lipogenesis [21]. The reduced activity of LPL [22], impairing the metabolism of TG-rich lipoproteins, may lead to hypertriglyceridemia and will, in turn, increase the CETPmediated exchange of VLDL TG for HDL-CE. This step will raise the atherogenic cholesterol-rich VLDL remnant particles and the TG-rich, cholesterol-depleted HDL particles [23]. Specifically, in the condition of atherogenic dyslipidemia excess TGs in HDL become a substrate for HL and LPL, enzymes that convert HDL to smaller particles filtered by the renal glomeruli and degraded in renal tubular cells [24]. Besides the decrement in HDL concentrations, in the presence of hypertriglyceridemia, the higher activity of cholesteryl ester transfer protein (CETP) accounts for a marked reduction of cholesterol carried in LDL [25]. sldl particles are rated as being more atherogenic, easily undergoing glycation and oxidation, compared with the larger LDL. sldl (i) possess a higher capacity to penetrate the arterial wall, (ii) show prolonged plasma half-life, and (iii) lower affinity for the LDLR [26]. Thus, a point to note is that in carriers of MetS, LDL-C is not the only lipid goal: the 2016 European guidelines point out to monitor atherogenic TG-rich lipoproteins (VLDL, IDL and remnants), recommending the achievement of specific cut-offs for non-hdl-c and apob [27]. Indeed, non-hdl-c may be used to estimate the total amount of atherogenic lipoproteins in plasma, being well related to apob levels. Elevated blood pressure High blood pressure is one of the most relevant independent risk factors for cardiovascular disease; indeed, worldwide, suboptimal blood pressure accounts for around 7.6 million premature deaths annually [28]. At ages years, a reduction of 20 mmhg in systolic blood pressure (SBP), halves the risk of coronary heart diseases [29]. Based on the European guidelines, hypertension is defined as values >140mmHg SBP and/or >90mmHg DBP, whereas a SBP goal <140 mmhg is recommended in patients with diabetes. Moreover, a diastolic blood pressure (DBP) target of <90 mmhg is always recommended, except in patients with diabetes, in whom the recommended objective is reduced to <85 mmhg [30]. A recent meta-analysis of randomized trials of blood pressurelowering agents in >100,000 patients with T2D confirmed that lowering blood pressure reduces the risk of cardiovascular events, coronary artery disease, stroke as well as of all-cause mortality [31]. Most recently there has been a heated debate on the blood pressure target values. The SPRINT study on drug treated hypertensives indicated that an SBP below 120 mmhg should be the appropriate target for an optimal risk reduction [32]. This was not accepted by other investigators [33], but a most recent largescale study indicated that outcomes after therapy may differ according to the achieved blood pressure targets in specific patient subgroups [34] and the lowest targets may not always be the most appropriate. In MetS, blood pressure elevation is linked to IR, possibly to the elevated WC. It is a common practice to note that blood pressure reduction occurs in the presence of reduced body weight and improved glucose control [35]. Elevated glycaemia Elevated plasma glucose as well as increased immunoreactive insulin are in general the major features of what was earlier called chemical diabetes [36]. Increased insulin response to an elevated glucose has been, from then on, generally linked to a higher cardiovascular risk, in particular hyperinsulinemia being found in the majority of hypertensives and constituting a common feature of the triad: obesity/glucoseintolerance/hypertension [37]. Hyperglycemia per se leads to an increased risk of reduced fibrinolysis and T2D [38]; eventually leading to a definition of elevated fasting glucose as a risk factor in the Framingham OFFSPRING Study [39]. In view of the poor practicality of evaluating IR or even insulin levels per se, investigators aimed to define an optimal correlation between glycaemia and IR to obtain a reliable indicator of the presence of MetS. Initially, high significance was given to the condition of impaired fasting glucose (IFG), i.e. a fasting glycaemia above 110 mg/dl [40] and the best threshold for fasting glucose based on correlations between glucose and insulin was mg/dl [41]. Later, it was suggested that this fell short of properly diagnosing a syndrome characterized by hyperinsulinaemia, and an adequate information could be provided by a fasting glycaemia above 100 mg/dl, resulting in the

5 ANNALS OF MEDICINE 681 classification of 20.4% of patients in their study group as IR, with a predictive value of 63% [42]. A fasting glucose 100 mg/dl has thus been selected as the basis for the diagnosis of MetS. The possible overlap between IFG and MetS has been clearly outlined: in a non-diabetic population 50 years of age, about twice as many individuals have IFG and MetS versus those with IFG alone Grundy [43]. A sizable portion of non-diabetics have MetS without IFG and vice versa. With these considerations, it appears that the MetS is a pre-diabetic state and MetS treatment of hyperglycaemia should aim to the prevention of microvascular disease [44]. In the absence of specific treatments aimed only to glucose reduction, priority must be given to reduce IR. The efficacy of this approach may be lifestyle intervention, weight reduction and increased physical activity as well established by the results of the DPP trial [45]. Nutraceutical approach to metabolic syndrome Functional foods: plant proteins Functional foods are characterized by the presence of components with a clear activity on metabolite levels that may be associated to specific clinical syndromes [46]. In the case of MetS, plant proteins deserve a special consideration. Indeed, protein-enriched diets have become a popular strategy to enhance weight management and weight loss [47]. Increasing protein intake may also have a potential for the prevention of T2D [48]. Although optimal amount and quality of proteins for these indications are still controversial, there are strong indications that, e.g. high intake of red meat is associated with an increased risk of T2D [49], whereas plant proteins are generally associated with some improvement in diabetes risk and also in the general management of hyperlipidaemia. Very recently, Virtanen et al. [50] in a follow up of the Kuopio Ischemic Heart Disease Risk Factor Study clearly indicated that plant proteins are associated with a decreased risk of T2D. Data on MetS are less extensive but two types of proteins, i.e. lupin and soy, have been studied more in details in patients with MetS providing clinical evidence of potential benefit [51]. In addition, other plant proteins, in particular pea and wheat protein, have shown some potential benefit. Lupin Lupin is a protein-rich grain legume. It commonly represents four domestic species, i.e. Lupinus albus (white lupin), L. luteus (yellow lupin), L. mutabilis (pearl lupin) and L. angustifolius (sweet leaf lupin). In Europe, both L. angustifolius, available as a protein concentrate, and L. albus are widely available. Lupin seeds have the interesting feature of a limited, close to nil, content of antinutritional factors such as phytate, tannins, lectins, protease inhibitors and oligosaccarides [52]. Lupin seeds contain two classes of proteins, i.e. the 7S and 11S globulins, (also conglutins a and b) in addition to two minor distinct proteins, conglutin c and d.. Lupin proteins have been studied for a number of years mainly for their activity on plasma cholesterol reduction, attributable in large part to an LDL-receptor (LDL-R) activating mechanism [53]. Moreover, conglutin c has been shown to reduce glycemia both in animal models and, moderately, in humans, particularly in the postprandial condition [54]. In animal models, similar to soy proteins, lupin proteins have displayed hypolipidemic and a remarkable antiatherosclerotic effect [55]. In order to investigate the mechanism/s of the possible activity on plasma glucose, recently different soy and lupin proteins were hydrolyzed by pepsin and trypsin and the resulting peptides separated and screened for their capacity to inhibit dipeptidyl peptidase IV (DPP-IV). This new molecular target is correlated to the development of T2D; a number of pharmacological inhibitors have been developed and are currently being used [56]. In the post-hydrolysis study [57], two small peptides were isolated and identified as Soy 1 and Lup 1, both with efficient inhibitory capacity on DPP-IV, with IC 50 s equal to 106 and 228 lm, respectively, thus indicating that both proteins may be sources of DPP-IV inhibitory peptides. Clinically, lupin proteins have been tested in different conditions, predominantly in hypercholesterolemia. Within this context, possibly due to the mechanism exerted on the LDL-R, a positive effect on LDL-C and on the LDL:HDL cholesterol ratio was reported in two studies, i.e. one with supplementation [58] and another with a mixed diet enriched with lupin [59]. Our group showed instead that lupin protein combinations with cellulose led to a remarkable hypocholesterolemic effect; a similar range of cholesterolaemia reduction was noted after a pea protein/fibre combination [60]. A TG reduction was noted in rodent studies, apparently associated with reduced fatty acid synthesis and increased TG hydrolysis [61]. This was not noted in studies in hypercholesterolemic individuals. However, in a randomized study in patients predominantly with MetS, Pavanello et al. [62] reported that a lupin protein concentrate, compared with a lactose-free milk powder diet, led to a reduction, in addition to the LDL cholesterol ( 8.0%), also of non-hdl cholesterol ( 7.5%). This study also reported that lupin proteins,

6 682 C. R. SIRTORI ET AL. in men, lead to a 12.7% reduction of proprotein convertase subtilisin/kexin type 9 (PCSK9), the key regulator of LDLR [63,64]. A direct modulation of the protein protein interaction of PCSK9 with the LDL-R has been shown [65]. Further, lupin proteins can provide a potential T2D treatment, possibly also due to the recently described modulatory effect of conglutyn b on the insulin signaling pathways [66]. It can be, thus, concluded that lupin protein supplemented diet or a well-designed whole diet with lupin proteins can provide an effective dietary tool for the management of the biochemical alterations characteristic of MetS. Soy proteins Soy proteins are the most widely evaluated dietary proteins for metabolic control. Proteins from Glycine max are the prototype plant proteins and, as such, have reached the attention, as reported in the recent systematic review on the effects of plant versus animal protein sources on features of MetS [51]. In addition to proteins, glycine max provides components such as isoflavones or phytoestrogens, with a potential estrogenic activity and possibly antiatherosclerotic effects [67]. Isoflavones may be of potential risk for, particularly growing children, but, so far, there is no clear evidence of a significant activity on the major components of MetS [68]. Indeed, careful extraction of isoflavones from soy did not change their lipid lowering and LDL-R stimulating activity in an experimental animal model [69]. While there is clear evidence that soy proteins per se can raise the activity of LDL-R in different animal models and in familial hypercholesterolemic patients [70], decreased triglyceridaemia and changes in other MetS-associated variables have been reported in a lower number of studies. Some reports have shown an improvement of glucose homeostasis after soy proteins. In post-menopausal women with MetS, soy proteins and soy nuts were compared (nuts have somewhat reduced protein and slightly higher phytoestrogen content). The soy nut regimen reduced fasting glucose more significantly than the soy protein, in addition to an improved LDLcholesterol reduction ( 5.0 ± 0.6% versus protein and 9.5%±0.6% versus control diet, both p <.01) [71]. The HOMA index was also reduced, somewhat more, by soy nuts ( 7.4 ± 0.8% versus protein and 12.9 ± 0.9% versus control diet, both p <.01). Changes of triglyceridaemia were reported in a number of studies. In individuals with mildly elevated plasma TG, the reduction was in the range of 15 20%, independent of isoflavone content [72]. In a detailed study in patients with moderate hypercholesterolemia treated with the bile acid binding resin colesevelam, 25 g/d of an insoluble fraction of partially hydrolyzed soy proteins were compared with a similar intake of milk proteins [73]. There was a clear benefit of soy proteins on LDL-cholesterol (10.9 ± 1.8 versus 5.9 ± 2% with milk proteins, p <.01) and a somewhat higher rise of HDL-cholesterol. The difference in TG levels was modest, but non-hdl-cholesterol, was reduced to a highly significant extent with soy proteins ( 10.8 ± 1.7% versus 3.9 ± 1.8% with milk protein, p <.01). There was also a highly significant difference in apo B levels, whereas faecal sterol excretion was reduced by milk proteins and significantly raised by soy proteins. In a single study, a direct comparison of the acute intake of soy versus lupin protein on glycaemia was evaluated [74] in T2D patients. A soy or lupin-based beverage containing 50 g glucose significantly reduced glycaemia for 4 h post- beverage, with no significant difference between lupin and soy. The insulin response was somewhat higher for lupin and soy, compared with controls. While blood pressure changes following plant protein sources, compared with animal proteins, have been mainly inconsistent, body changes have been generally characterized by some weight reduction [51]. Animal protein intake was positively associated with BMI and WC but plant proteins were instead inversely associated with BMI only in males and with WC in both genders [75]. In the recent longitudinal EPIC- PANACE study [76], increasing dietary plant source proteins by 5% at the expense of animal-source proteins in an isoenergetic diet reduced weight gain by nearly 1 kg per year in men over a 5-year period, not in women. In another study, in children, an increase in weight and BMI was noted after consumption of animal proteins, especially dairy (for periods of up to 7 years) versus vegetable proteins [77]. A study by our group specifically investigated patients with clinical features of MetS; they received 30 g/d soy proteins in parallel group versus animal food. A significant reduction of body weight ( 1.5% and BMI 1.5%) was observed [78]. The expected reductions of total and LDL-cholesterol as well as of non-hdl-c ( 7.14%) versus milk proteins were found (Figure 1). Thirteen out of the 26 participating subjects had a reduction of MetS indexes, bringing them to normality. Other plant protein sources (without isoflavones) have been tested in individuals with MetS. Among these, pea, fava bean proteins and wheat gluten did not show any further beneficial effect on fasting lipemia compared with proteins of animal sources. However, wheat gluten ingestion resulted in a larger

7 ANNALS OF MEDICINE 683 Figure 1. Percentage median changes of non-hdl-c. Subjects randomly assigned to receive the experimental diet, containing whole soy foods corresponding to 30 g/day soy protein in substitution of animal foods containing the same amount of protein or the control diet containing the animal foods, for 12 weeks [with permission of Elsevier [78]]. postprandial TG response compared with wheat proteins [79]. In general, studies comparing diets from plant versus animal sources with a positive metabolic effect had a longer duration [51]. Probiotics, prebiotics and synbiotics In humans, microbial density comprises a biomass of kg and increases from the proximal to the distal end of the intestine; in the body, the ratio of bacteria to human cells is close to 1:1 [80]. Recently, numerous studies in humans and in animal models have reported how gut microbiota, established within the first 3 years of life, converts dietary and endogenous molecules into metabolites, that allow communication with peripheral organs and tissues in the host [81]. Among microbial metabolites, short-chain fatty acids, i.e. acetate, butyrate and propionate, are known to exert several health benefits, including reduction of the luminal ph, inhibition of enteropathogens, increased absorption of some nutrients and maintenance of gut barrier function [82]. Conversely, a rise in the absorption of branched-chain amino acids, as in the case of alterations in the intestinal barrier (e.g. high-fat diets), is associated with a higher risk of T2D [83,84]. Indeed, dysbiosis, i.e. the alteration of gut homeostasis, is associated to obesity, T2D and IR, all traits of MetS that can lead to atherosclerosis and heart failure [85]. It has been, further, proposed an Interaction Model, demonstrating that, at least in permissive genetic backgrounds, gut microbiota plays a significant role in modulating key metabolic traits, such as obesity, diabetes and IR [86] (Figure 2). Figure 2. The development of obesity, insulin resistance, type 2 diabetes and metabolic syndrome in general are the consequence of a complex multidirectional interaction between host genetics, environment, diet and the gut microbiota [with permission of Elsevier [86]]. Since diet shapes the composition of gut microbiota, restoring its diversity and activity, nutrients and particularly nutraceuticals can represent a promising approach to improve metabolic features of MetS [81]. Moreover, in older people, in which microbiota is susceptible to a larger inter-individual variation, dietary supplements with defined food ingredients promoting specific components of microbiota may be useful in maintaining the best health status [87]. Consumption of some nutraceuticals i.e. probiotics, prebiotics and synbiotics should be of primary consideration. Probiotics are living micro-organisms that when administered in adequate amounts confers beneficial health effects to the host (i.e. Lactobacillus rhamnosus GG, L. reuteri, Bifidobacteria certain strains of L. casei or the L. acidophilus group) [88]. Prebiotics are defined as selectively non-digestible plant-derived carbohydrates, allowing specific changes, both in the composition and in the activity of gastrointestinal microflora; indeed, these food ingredients (i.e., inulin and its hydrolysis product oligofructose, and (trans)galactooligosaccharides) confer health benefits by stimulating growth and/or activity of a limited number of bacteria within the colon [89]. The definition of prebiotic should include (a) resistance to gastric acidity, hydrolysis by mammalian enzymes and gastrointestinal absorption; (b) fermentation by intestinal microflora; (c) selective stimulation of the growth and/or activity of intestinal bacteria associated with health and wellbeing [89]. Synbiotics are a synergistic combination of pro- and prebiotics [90]. A detailed summary of data from randomized controlled studies (RCTs) related to the use of these

8 684 C. R. SIRTORI ET AL. nutraceuticals should also consider that results may be affected by different biases, since the majority of the RCTs (i) did not follow the same design, (ii) were small in terms of participants, (ii) were of short duration and (iii) used probiotics from multiple strains as well as different delivery methods (i.e. yogurt, cheese or capsules) [91]. Thus, RCTs studies with a variety of sources, daily doses and different durations of intervention are required to confirm health benefits and specific roles of probiotics, prebiotics and synbiotics on the metabolic components of the MetS. Probiotics and traits of metabolic syndrome Weight and BMI Data from a meta-analysis of 15 RCTs reported, in obese, that the administration of Colony Forming Units (CFU)/day of Lactobacilli probiotics for 2 3 months reduced body weight (kg) by 0.54 (95% CI 0.83, 0.25; p <.001); when BMI (kg/m 2 ) was instead considered, reduction in the Lactobacilli arms was 0.43 (95% CI 0.67, 0.20; p ¼.005). Conversely, upon probiotic supplementation, a slight increment in body weight was observed in children (þ0.20 kg; 95% CI 0.04, 0.36) and infants (þ0.30 kg; 95% CI 0.01, 0.62; p ¼ 0.05) [92]. Another meta-analysis, on four RCTs, showed no significant effects of probiotics on body weight, BMI and visceral fat of obese subjects [93]. In line with the above-described findings, reporting a weak (<3%) or absent effect of probiotics on body components [94], in obese with elevated cardiovascular risk, probiotic supplementation reduced BMI by 0.52 kg/m 2 (95% CI 0.81, 0.25; p <.001) and waist circumference by 2.11 cm (95% CI 3.54, 0.68; p ¼.004) [95]. A further contribution to the understanding of this complex issue has come from a metaregression analysis reporting that the effects of probiotics on BMI depend on the duration of intervention ( 8 weeks), number of species of probiotics and baseline BMI 25 kg/m 2, thus highlighting the effectiveness of probiotics in reducing BMI, especially in overweight or obese subjects [96]. A possible mechanism explaining the effects on weight loss are the improvement of the intestinal barrier function that may reduce metabolic endotoxemia [97], thus ameliorating levels of lipopolysaccharides (LPS). In the adipose tissue, endotoxins from LPS trigger systemic and local inflammation with an increase in reactive oxygen species (ROS) [97]. As a possible associated event, bacterial fragments can increase the number of preadipocytes [98]. Among other proposed mechanisms linking probiotics and body weight reduction there are the ability (i) of the microbioma to extract energy from the diet [99] and (ii) to increase the secretion of the gut hormone glucagon-like peptide (GLP-1) enhancing satiety and reducing energy intake [100]. Blood pressure A meta-analysis of 9 RTCs relative to the administration of probiotics from different strains (L. reuteri, L. plantarum, L. helveticus, Enterococcus faecium and Streptococcus thermophilus), with a total daily consumption between 10 9 and CFU, with a duration of 3 9 weeks, showed an overall improvement in blood pressure. A reduction of 3.58 mmhg (95% CI 6.46, 0.66; p <.00001) and 2.38 mmhg (95% CI 3.84, 0.93; p <.0001) was recorded for SPB and DBP, respectively. Interestingly, both SBP and DBP fell further when a sub-analysis was conducted taking into consideration: (i) duration of treatment 8 weeks, (ii) BMI 30 kg/m 2, (iii) dairy source of probiotics (versus capsules), (iv) consumption of multiple probiotic species, (v) daily dose consumption CFU and (vi) blood pressure at baseline 135/85 mmhg [101]. The anti-hypertensive effects of probiotic supplementation were also confirmed in subjects with T2D; reductions of 3.28 mmhg and of mmhg in SBP and DBP, respectively, have been reported. These effects were inversely related to BMI (> 29 kg/m 2 ) and age [102]. The antihypertensive activity of probiotics has been also attributed to the reduction of inflammation-mediated endothelial dysfunction, nitric oxide synthase downregulation [103], and release of angiotensin-converting enzyme inhibitory peptides. Among these, the tripeptides Val-Pro-Pro and Ile-Pro-Pro have been the most extensively investigated [104]. As previously reviewed, two major commercial products, based on sour milk, i.e. the Finnish EvolusVR and the Japanese CalpisVR are presently available [105]. However, due to the skepticism regarding their cost and possible effectiveness, they have not reached a wide distribution outside these countries. Fasting plasma glucose The effect of probiotic supplementation on glucose homeostasis is influenced by the basal levels of FPG ( 126 mg/dl), length of intervention ( 8 weeks), baseline BMI values ( 30 kg/m 2 ), administration of multiple species of probiotics, daily dose consumption CFU and the selected sources, i.e. supplements and foods instead of fermented milk or yogurt. Indeed, the presence of lactose in fermented milk may raise FPG. Conversely, if the overall population is considered, a non-statistical FBG reduction of 0.17 mmol/l is observed [106].

9 ANNALS OF MEDICINE 685 In studies conducted in cohorts of T2D patients, mean reductions of 0.61 mmol/l [107] or 0.98 mmol/l were reported [108]. Interestingly, the higher the number of bacterial species is, the more significant the FPG reduction is [107,109]. Contrasting and inconclusive data have been instead reported on the effectiveness of probiotic supplementation on HOMA-IR and glycosylated hemoglobin (HbA1C) levels, two hallmarks of IR [ ]. The reduction of low-grade inflammation promoted by gut-derived LPS and metabolic endotoxemia has been proposed as mechanisms linking probiotics and glucose metabolism [110]. Indeed, serum endotoxin levels are correlated with fasting insulin [111] and are two-fold higher in T2D patients compared with non-diabetics [112]. HDL and TG A recent meta-analysis of 30 RCTs, investigating the effect of different strains of probiotics (L. acidophilus, L. plantarum and L. helveticus) on lipid parameters, reported no changes in HDL-C ( 0.24 mg/dl; 95% CI 1.6, 1.1) and TG ( 5.3 mg/dl; 95%CI 11.1, 0.4) levels [91], between subjects taking probiotics and controls. This has been confirmed in mild-to-moderate hypercholesterolemic individuals in whom probiotic supplementation was ineffective in improving HDL-C (0.01 mg/dl; 95%Cl 0.02, 0.03) and TG (0.01 mg/dl; 95%Cl 0.08, 0.09) levels; these findings were not modified upon correction for baseline BMI (25 kg/ m 2 ), duration (4 weeks), age ( 45 years), preparation (fermented milk products versus probiotic preparations) and bacterial strains [113]. Considering that probiotics exert a hypocholesterolemic effect by reducing the intestinal absorption of cholesterol or by enhancing the bile salt hydrolase activity, a lack of efficacy in modulating HDL-C and TG, both linked to the atherogenic dyslipidemia, is expectable [114]. Notably, if only T2D patients are considered, probiotic consumption reduced TG levels by mg/dl (95%Cl 33.7, 11.2) with no HDL-C changes [102]. HDL-C may be raised by 16 mg/dl if a longer duration of probiotic administration (>8 versus 8 weeks) is considered [107]. Prebiotics, synbiotics and the major traits of metabolic syndrome The effects of prebiotics on the metabolic traits of MetS have been evaluated in a limited number of studies investigating comparable outcomes. Their small sample sizes, heterogeneity among trial subjects, disease conditions, prebiotic supplements, intervention duration and outcomes need thus to be carefully considered. Among the different outcomes evaluated in trials investigating the effects of prebiotic supplementation on metabolic traits, i.e. body weight, glucose homeostasis and TG, evidence indicates that dietary supplementation impacts only on post-prandial glucose ( 0.76 mmol/l (95%Cl 1.41, 0.12)) and insulin ( 0.77 UI/l (95%Cl 1.50, 0.04)) levels leaving the other parameters unaffected. Interestingly, although self-reported, an improvement in satiety was found upon prebiotic intake [115]. Not surprisingly, prebiotics have been related to a decrement in Ghrelin levels and to an increment in peptide YY and GLP-1 secretion, all involved in appetite regulation [116,117]. In subjects with a BMI 25 kg/m 2 and carriers of diabetes, evaluation of the effects of prebiotics compared with placebo showed an improvement in TG and HDL-C levels, 0.72 mmol/l and þ0.49 mmol/l, respectively [118]. The same authors reported that a synbiotic supplementation decreases plasma fasting insulin and TG levels by 0.39 UI/l (95%Cl 0.75, 0.02) and 0.43 mmol/l (95%Cl 0.70, 0.15), respectively. No changes in FPG were noted [118], similar to what reported in a very recent meta-analysis [106]. Vitamin D Vitamin D has generally been shown to exert a minor role in the control of MetS. However, recent data have indicated vitamin D levels as an important variable, underlying, in some cases, MetS, in addition to being a possibly significant determinant of cardiovascular risk. Vitamin D has a well-known primary physiological role in regulating calcium homeostasis. However, besides bone metabolism, growing evidence has described the involvement of vitamin D on glucose metabolism, blood pressure and other metabolic traits related to MetS [119]. Being mainly synthesized in the skin upon ultraviolet-b radiation, vitamin D is metabolized by the hepatic 25-hydroxylase enzyme to 25-hydroxyvitamin D [25(OH)D] and by a renal 1a-hydroxylase to the vitamin D hormone calcitriol, also called 1,25-(OH)2 vitamin D; the active form of vitamin D [120]. Since 1,25- (OH)2 vitamin D is active for a maximum of 27 h, 25(OH)D levels are the landmarks for the assessment of vitamin D status [121,122]. Specifically, when circulating levels of 25(OH)D are 30 nmol/l, this is diagnosed as a condition of vitamin D deficiency; inadequacy is when 25(OH)D levels are between 30 and 50 nmol/l and sufficiency when concentrations are in the range of nmol/l [123]. The relationship between 25(OH)D levels and risk of MetS has been recently reviewed; a dose response

10 686 C. R. SIRTORI ET AL. meta-analysis of 16 cross-sectional studies showed a linear and inverse association between the two variables. Moreover, the risk-ratios of MetS, per 25 nmol/l, increment in the serum/plasma 25(OH)D levels, were 0.87 (95%Cl ). Albeit, somewhat attenuated, this effect was maintained upon correction for sex, age (middle versus elderly), latitude (high versus low), assay detection method, criteria of MetS definition [124]. The same results were obtained when the relative risk for MetS was assessed comparing individuals in the top versus bottom tertiles of baseline 25(OH)D levels [119]. In the context of MetS, obese individuals show a decrement in circulating 25(OH)D, i.e. of 0.27 ng/ml in vitamin D3 for each 1 kg/m 2 increment in BMI [125]. Among the different suggested hypotheses [126], the concept of reverse causality is to be taken into consideration. Indeed, if an inverse correlation between obesity and vitamin D levels is true, on the other hand, vitamin D may be sequestered or stored in the adipose tissue leading to lower 25(OH)D serum levels. Thus, obesity may be a cause and not a consequence of vitamin D deficiency [127]. An inverse association between baseline 25(OH)D levels and incidence of T2D has been also reported. Comparing the highest to the lowest cutoffs of 25(OH)D levels, a relative risk of 0.62 (95%Cl ) emerged from a pooled analysis of 21 prospective studies. This finding was not affected by sex, duration of follow-up, sample size, diabetes or 25(OH)D assay method [128]. The same conclusions were reached by Khan et al. showing how individuals in the top versus bottom tertile of baseline 25(OH)D blood levels had a relative risk of 0.81 (95%Cl ) for T2D incidence [119]. Conversely, when vitamin D supplementation has been taken into consideration, data from RCTs did not show any effect on fasting glucose, HbA1C, insulin resistance or incidence of diabetes [129]. In general, vitamin D may have a direct effect on beta-cell function through the activation of the vitamin D receptor expressed in beta-cells; lack of a functional vitamin D receptor leads to an impairment in glucose metabolism, although a vitamin D-mediated beta-cell survival should not be excluded [129]. The earliest evidence linking vitamin D to blood pressure came from the observation that low UVB exposure was associated with an increased hypertension risk [130]. Among the 12,644 participants of a cross-sectional survey, mean SBP and DBP were 3.0 mmhg and 1.6 mmhg lower for people in the highest 25(OH)D quintile ( 85.7 nmol/l) compared with the lowest (<40.4 nmol/l) [131]. This finding has been confirmed by showing an improvement in blood pressure directly related to the increment in serum 25(OH)D levels; upon correction for sex, age, BMI and physical activity, the differences in SBP and DBP between the lowest (<41.4 nmol/l) and highest (>62.6 nmol/l) serum 25(OH)D quartiles were 3.6 and 1.0 mmhg, respectively [132]. A meta-analysis on 283,537 subjects, examining the association between vitamin D status and hypertension risk, showed that individuals in the top 25(OH)D tertile have a 30% lower risk of developing hypertension compared with those in the lowest tertile [0.70 (95%Cl )]. Notably, a 10 ng/ml increment in baseline vitamin D levels corresponded to a 12% reduction in risk [0.88 (95%Cl 0.81, 0.97)] [133]. In this type of correlations, the importance of baseline 25(OH)D levels are confirmed also for very low reference values; subjects with circulating levels <15 ng/ml compared with those with levels 30 ng/ml have a 3.18 (95%Cl ) higher relative risk of hypertension development [134]. Overall, the antihypertensive effects of vitamin D may depend on a direct activation of the vitamin D receptor on vascular endothelial and smooth muscle cells or on a modulation of the renin angiotensin aldosterone system [135,136]. Conversely, different conclusions were reached when the vitamin D supplementation was used as a blood pressure-lowering agent. Indeed, no evidence of blood pressure reduction upon vitamin D supplementation was seen on either SBP (0.0 mmhg (95%Cl 0.8 to 0.8)) or DBP (0.1 mmhg (95%Cl 0.6 to 0.5) [137]. Similar results were obtained after a sub-analysis for baseline SBP >140 mmhg or 140 mmhg and for baseline 25(OH)D values >20 ng/ml or 20 ng/ml. Supplementation with vitamin D (50,000 IU/week) in MetS patients for 16 weeks significantly reduced levels of the vascular adhesion molecule-1 and of E selectin. However, there was no significant activity on high-sensitivity C-reactive protein and carotid intima media thickness [138]. Curcumin Curcuminoids are polyphenolic compounds derived from the dried rhizomes of Curcuma longa L. (turmeric) and are responsible for the orange-yellow colour [139]. Curcumin is the major component, i.e. constituting about 5% of turmeric, with the structural analogues demethoxycurcumin and bis demethoxycurcumin. Unstable at physiological ph, it rapidly degrades to oxidized products, which mediate topoisomerase poisoning as well as other antioxidant, anti-inflammatory and lipid lowering effects [140].

11 ANNALS OF MEDICINE 687 Among the multiple pharmacodynamic effects of curcumin are the well-established antioxidant and anti-inflammatory properties, demonstrated by in vitro and in vivo studies. Indeed, this compound proved to increase serum total antioxidant capacity and superoxide dismutase, together with an increase in glutathione concentrations and reduction in lipid peroxides [141,142]. Interestingly, curcumin can also regulate cytokines, protein kinase, adhesion molecules and other enzymes (TNF-a, IL-1, IL-6, TGF-b, MCP-1, etc.) [139,143,144]. Supplementation with curcuminoids was associated with a statistically significant reduction in circulating high-sensitivity (hs)-crp levels in a metaanalysis (weighed mean difference: 6.44 mg/l; 95% CI: to 2.11) [145]. A small trial on 14 males showed a significant increase in postprandial flowmediated dilatation (FMD) (from 5.2% to 6.6%) after a single consumption of curry meal containing curcumin [146], suggesting a potential role in preventing endothelial dysfunction. Among the multiple properties on a wide range of diseases, curcumin proved to modify almost all features of MetS [147]. The main metabolic effect of curcumin seems to be related to an activity as insulin sensitizer [148,149], well demonstrated in animal models of diabetes [148], and confirmed in human studies. In a double-blind randomized trial, pre-diabetic patients achieved an improvement of all markers of insulin sensitivity (C-peptide, HOMA-IR and HOMA for b cells) after 9 months with 1500 mg of curcumin compared with placebo [150]. The curcumin extract in the same study also prevented T2D development (0% of curcumintreated developed DM versus 16.4% of placebo-treated patients). A þ22.5% increase in adiponectin levels was also detected, later confirmed in a further meta-analysis reporting a 77% rise (95% CI: ) together with a 26% decrease in leptin (95% CI: ) [142]; both adiponectin and leptin have been linked to cardiovascular diseases [7]. A shorter treatment period provided no comparable benefit on glucose homeostasis [151], with, however, a significant improvement of HDLcholesterol (þ6.8%) and a reduction of TG ( 28.8) and of non-hdl-c ( 13.6%). A larger study on 117 subjects with MetS provided confirmatory findings, indicating, in addition a significant antioxidant and anti-inflammatory activity with a marked reduction ( 2.2 mg/l) of CRP concentrations [152]. The lipid-lowering mechanisms of curcuminoids seem to reside in their ability to increase cholesterol efflux via ABCA1 and APOA-I expression [153,154] and to inhibit the expression of Niemann Pick C1-Like 1 (NPC1L1) via the Sterol regulatory element-binding protein 2 (SREBP2) transcription factor [155]. Curcumin also downregulates PCSK9 mrna levels by up to 31 48% in different cell lines, thus promoting LDL-R expression on the cell surface and LDL-C uptake [156]. The mechanism of down-regulation of PCSK9 appears to be linked to the inhibition of the HNF-1a transcription factor: this may lead to an additional anti-inflammatory activity [157]. Curcumin further demonstrated protection against atherosclerosis in LDLR / mice [154], by modifying peroxisome proliferator-activated receptors (PPAR)-a and c, CETP and LPL expression, therefore, affecting synthesis and catabolism of fatty acids [154,158]. Limitation to a wider use of curcumin is the poor oral bioavailability, representing a major obstacle to clinical development. Although usually well tolerated and safe, curcumin has a low hydrosolubility and undergoes a rapid gastric and intestinal metabolism. A number of new formulations have been described. A formulation with a hydrophilic carrier, cellulose base and natural antioxidants [159] proved to be absorbed at least 30 times better than curcumin base. Recently, a formulation of curcumin encapsulated in milk exosomes proved to have improved resistance to human digestion and improved cell permeability [160]. A formulation with piperine, an inhibitor of hepatic and intestinal glucoronidation is in advanced development, and the initial clinical tests have given a positive outcome [161]. Development of a curcumin product with an improved bioavailability will probably lead to a better activity on the blood lipid profile, as shown in a study investigating in parallel lipid responses and blood levels [162]. Berberine It is a natural plant alkaloid, isolated from the Chinese herb Coptis kinensis (Huanglian). In the Far East, it is commonly used for diarrhoea but a potential glucose lowering effect was noted by Chinese medicine way back in the 80s. The breakthrough finding was the description of a powerful cholesterol lowering activity (berberine 500 mg bid for 3 months) in hypercholesterolemic patients [163]. This activity was associated with an elevation of the LDL-R expression, independent of the sterol regulatory element binding proteins (SRBPs), but consequent to the activation of an extra signal regulated kinase (ERK). In a later study, Lee et al. [164] reported that berberine, in addition to stabilizing hepatic LDL-R mrna in an ERK-dependent way, could increase the transcriptional activity of LDL-R promoter by activating the JNK pathway (Figure 3). Moreover, in endothelial cells, berberine has been reported to counteract pro-atherogenic and inflammatory stimuli