Overconsumption of a maladaptive, westernized diet consisting of foods that are calorie-dense, nutritionally-poor, phytochemical-depleted, highly processed and rapidly absorbable has been shown to increase systemic inflammation and reduce insulin sensitivity [1–3]. With chronic ingestion, this dietary pattern often results in metabolic syndrome (MetS), a physiologic state encompassing a cluster of metabolic abnormalities, including dyslipidemia, central obesity, hypertension, and glucose intolerance. These are all independent risk factors for the development of type 2 diabetes and/or cardiovascular disease (CVD) [4, 5]. The NHANES 1999–2000 estimated the prevalence of MetS in the US at an unadjusted 34.5% or approximately 69 million persons [6]. As worldwide food consumption patterns shift to the aforementioned dietary pattern, MetS is becoming a significant burden in developing nations and global prevalence is growing [7, 8].

It is widely viewed that MetS results from an increasing, perpetual state of whole body insulin resistance, which is strongly associated with dietary carbohydrate [9–11] and saturated fat [12], leading to high serum triglycerides (TG) and visceral adiposity [13–15]. Acute infusion of free fatty acids leads to the accumulation of TG in skeletal muscle and evokes whole body insulin resistance with the same temporal pattern [16–19]. Metabolites of lipid metabolism such as diacylglycerol have been shown to directly induce insulin resistance by chronically activating protein kinase C (PKC). PKC activation terminates insulin signaling, preventing crucial tyrosine phosphorylation by the insulin receptor, leading to impaired insulin signaling [14]. MetS is also associated with a state of chronic inflammation. Adipocyte leakage has recently been shown to result in the recruitment of macrophages, which envelope excess lipids, form foam cells, and release inflammatory cytokines, setting up a state of systemic, chronic inflammation [20, 21]. These adipokines lead to the systemic activation of several protein kinases involved in inflammatory signal transduction, including phosphoinositide-3 kinase (PI3K), glycogen synthase kinase (GSK-3) and PKC that singly or in concert cause insulin resistance in skeletal muscle and adipose tissue [22–24]. Hence, therapies which reduce circulating lipids and reduce systemic inflammation have shown promise in the treatment of insulin resistance and MetS.

Lifestyle modifications including diet and exercise are recommended as first-line intervention for treating insulin resistance and MetS by the National Cholesterol Education Program (NCEP) and American Heart Association (AHA). The Mediterranean-style diet, high in plant foods and monounsaturated fatty acids and low in processed foods and refined carbohydrates, has been shown to reduce CVD risk factors [25–28] and inflammatory burden associated with MetS etiology [29]. Pharmacologic treatment is considered appropriate if the individual is refractory to a lifestyle approach [30, 31]. A recent review of the literature reveals that both intensive lifestyle modifications and drugs such as rimonabant or rosiglitazone may reduce the prevalence of MetS in 25 – 33% of patients [32]. While new pharmacologic approaches to MetS are under development [7], relying on drug therapy for an epidemic caused by a maladaptive diet is not as rational as realigning dietary habits [33]. These findings suggest that new lifestyle modification approaches in the treatment of MetS and its complications should be important public health priorities.

Advancing knowledge in inflammation and insulin signaling suggest that reversing the chronic imbalances of these downstream kinases provides a promising and logical strategy for reducing insulin resistance and the metabolic abnormalities of MetS. Inhibition of downstream kinases could be accomplished by using pharmaceuticals such as sunitinib and imatinib (multi-target protein kinases drugs), approved to treat cancers but which may also cause remission of diabetes [34]. However, several dietary phytochemicals, such as genistein and curcumin, have been shown to be protein kinase inhibitors [35, 36]. A recent study showed that combining additional dietary polyphenols with a Mediterranean diet could provide synergistic effects and positively impact postprandial dysmetabolism [33]. As such, an important aspect to consider in dietary recommendations for MetS is the incorporation of diverse, targeted biologically-active phytochemicals to address the multiple underlying mechanisms of MetS. In a previous study [37], we reported that the addition of soy protein and phytosterols to a Mediterranean-style, low glycemic load diet had a more favorable impact than the AHA Step 1 diet on cholesterol/HDL and TG/HDL, blood pressure (BP), and Framingham 10-year CVD risk score for coronary heart disease in overweight, postmenopausal, hypercholesterolemic women.

With the finding that soy protein and phytosterols plus a Mediterranean-style diet could favorably affect the TG/HDL, an indicator of MetS, we initiated a screening program to identify additional structurally diverse phytochemicals capable of increasing insulin sensitivity through the modulation of downstream kinases. We screened 203 botanical products in 3T3-L1 adipocytes and identified novel substituted 1, 3-cyclopentadiones as well as a number of proanthocyanidin extracts with adipogenic and anti-inflammatory activity. One of the substituted 1, 3-cyclopentadiones was rho iso-alpha acids (RIAA) derived from hops (Humulus lupulus). RIAA have been used as bitter flavoring agents in beer for decades. We found RIAA dose dependently inhibited GSK-3, PI3K, and PKCβ in cell free kinase assays (manuscript in preparation). Another bioactive material identified by our screen was the proanthocyanidin-rich extract (PAC) of Acacia nilotica. In addition to inhibiting GSK-3, PI3K, and PKC, it also inhibited IKKβ in a dose-dependent manner (manuscript in preparation). At 5:1 (RIAA:PAC), the greatest efficacy in reducing glucose and insulin in the db/db mouse model was observed (manuscript in preparation). Furthermore, in an unpublished human pilot study, MetS subjects who consumed a combination of RIAA and PAC showed greater lowering of fasting LDL, TG, and TG/HDL than the placebo group.

The objective of this study was to investigate whether by enhancing a modified Mediterranean-style, low glycemic load diet with specific phytochemical supplementation we could improve cardiometabolic outcomes in subjects with MetS. Targeted supplementation included soy protein, phytosterols, hops RIAA and acacia PAC. The same diet was used in both arms and incorporated a spectrum of unprocessed, modified Mediterranean-style foods with an overall glycemic load less than 65.

Due to the complex mechanistic underpinnings of MetS, strategies for reducing its incidence and consequences need to be similarly comprehensive and multi-factorial. Targeting multiple, chronically dysregulated signaling pathways at play in insulin resistance is likely to yield the greatest benefit [45] in the treatment of MetS. To our knowledge, this is the first intervention study to successfully demonstrate the concept of modifying cardiometabolic risk factors by combining a modified Mediterranean-style, low glycemic load dietary pattern, and regular exercise, with diverse phytochemicals targeting multiple signaling abnormalities of MetS. A greater than 2-fold improvement in total cholesterol, non-HDL cholesterol, TG, cholesterol/HDL, and TG/HDL were observed with the addition of soy protein, phytosterols, hops RIAA, and acacia PAC to a modified Mediterranean-style low glycemic load diet and exercise program. Increase in HDL and decrease in LDL and VLDL particle numbers were seen only in the PED arm. Also, diastolic BP fell only in the PED arm. Furthermore, by 12 weeks, almost double the number of subjects experienced a net resolution of MetS in the PED group compared with MED group, and the PED group experienced almost twice the reduction in the Framingham 10-year CVD risk score as the MED group. Overall, CVD risk was reduced to a greater degree in the subjects supplemented with phytochemicals relative to the subjects on MED alone.

Previously [37], we compared a calorically-restricted version of the diet used in this study supplemented with soy protein and phytosterols to the frequently-studied AHA Step I low-fat diet in postmenopausal hypercholesterolemic women. We demonstrated that this targeted phytochemical, supplemented diet produced greater benefits than the AHA diet alone. The protocol for the current trial differed from that of our previous study in 2 key aspects: (i) subjects in both study arms were instructed to consume their diet until satisfied, and (ii) 2 additional supplemental phytochemicals were provided to target the underlying inflammatory mechanisms of MetS. The modified Mediterranean-style, low glycemic load diet by itself is an effective approach to weight loss and CVD risk reduction [46]. Our findings suggest that the addition of the soy protein, phytosterols, in combination with targeted phytochemicals was responsible for the more favorable CVD risk profile outcome in the PED group. This observation could not be attributed to greater weight loss in the PED group, since the extent of weight loss was similar on both arms. A recent review by Volek and Feinman reported that dietary carbohydrate may affect various cardiometabolic factors [10]. In our analysis, we adjusted for calorie and carbohydrate intake, and body weight change, and found that the observed differences between arms remained unaffected (Table 3). Likewise, the glycemic loads of the diets and the levels of exercise did not differ between arms. Although provision of the powdered beverage may have simplified meal planning, and in turn, enhanced dietary compliance and convenience, an effect similar to that reported by Noakes et al. [47], again weight loss did not differ. Finally, although only subjects in the PED arm experienced a decrease in hunger levels in the period between the evening meal and bedtime, which might have reduced excessive, nighttime snacking, differences in intake should have been reflected in differential body weight loss and recorded caloric intake, neither of which occurred.

RIAA is a modified hop extract that has been used in beer for flavoring, foam stability and bittering for decades, and is prized for its chemical stability. RIAA is derived from hop cones in a process that first involves extraction of whole hops with supercritical CO₂, yielding an extract containing a mixture of alpha acids, beta acids and hop oils [48, 49]. The alpha acids are differentially isolated, isomerized, and reduced to form RIAA. The Acacia nilotica tree grows naturally in many arid climates in Australia, Africa, and India and the young trees are a food source for many animals including cattle [50–52]. The young leaves have been fried and consumed like other leafy vegetables. The bark is ingested traditionally as a hot beverage and in a more concentrated form as a folk medicine for various ailments including colds, diarrhea, tuberculosis and leprosy [53]. The rationale for inclusion of the RIAA/PAC tablet was based on results of in vitro screening studies employing insulin resistant 3T3-L1 adipocytes, db/db mouse diabetes studies, and a pilot clinical trial (unpublished results); all consistently showing improvement in insulin sensitivity. Briefly, the RIAA/PAC combination displayed lipogenic and anti-inflammatory activity in murine 3T3-L1 adipocytes stimulated with TNFα. Both agents inhibited TNFα-stimulated IL-6 secretion and improved adiponectin secretion. Individually and in various combinations these compounds also demonstrated favorable modulation of the activity of proteins and kinases implicated in insulin signaling such as PI3K, GSK-3, AKT, PKC and c-Jun N-terminal kinase (manuscript in preparation). Other groups have reported that feeding of isohumulone, which is structurally similar to RIAA, to C57BL/6N mice reduced plasma TG and free fatty acid levels [54]. Additional animal and clinical studies with isohumulone have revealed that: (i) diabetic KK-Ay mice had reduced plasma glucose, TG, and free fatty acid levels, 65.3, 62.6, and 73.1%, respectively; (ii) C57BL/6N mice fed a high fat diet showed improved glucose tolerance and reduced insulin resistance; and (iii) a double-blind, placebo-controlled pilot study on diabetic subjects suggested that isohumulones decreased blood glucose and HbA1c levels by 10.1 and 6.4%, respectively, after 8 weeks [55]. We chose RIAA over IAA due to RIAA's greater chemical stability [48], potent in vitro anti-inflammatory activity in 3T3-L1 adipocytes. In our pilot clinical trial a combination of RIAA and PAC in tablet form significantly improved LDL, TG and TG/HDL in subjects with MetS.

The results of the present study, in agreement with similar studies reported by others [25–28], confirm the benefits of lifestyle intervention consisting of a phytochemical rich, low glycemic load diet and a moderate aerobic exercise regimen in subjects with MetS. For example, Jenkins et al. [56] found reductions in lipid variables with an ad libitum low glycemic load diet ("portfolio diet") in subjects with high TG at baseline. The average reductions reported by Jenkins et al. in total cholesterol, LDL and TG over one month were almost identical to those noted in the MED arm of the present study over 8 weeks (8.8% vs. 8.7%; 9.1% vs. 9.8%; and 19.3% vs. 22.3%, respectively). Also, similar to the results reported by Jenkins et al. [56], no changes in HDL were noted in MED subjects in the present study. Lastly, in both arms of the present trial, we found that subjects' food cravings fell and satiety increased with institution of the low glycemic load diet and energy intake fell despite the lack of instructions to limit caloric intake. Esposito et al. reported that 48% of subjects with MetS instructed to follow a Mediterranean-style (not necessarily low glycemic) diet over 2 years no longer met 3 or more criteria for MetS [57]. In the Diabetes Prevention Program Trial, lifestyle modification led to 38% resolution of MetS after 3 years [58]. In contrast, only minimal resolution of MetS was seen in the Diabetes Prevention Program Trial after one year with either metformin or lifestyle management [58]. Our study suggests that by complementing a therapeutic lifestyle program with a soy- and phytosterol-based powdered beverage and tablets containing hops RIAA and acacia PAC, subjects can improve to a similar magnitude (43%) attained at 2 and 3 years in just 12 weeks.

Most therapeutic treatments for hypercholesterolemia focus on achieving LDL goals recommended by NCEP. However, the NHANES 2003–2004 showed that despite better control of LDL, other lipid risk factors remained suboptimal in many US adults, particularly among those with CVD, diabetes, or MetS [59]. Non-HDL cholesterol, a stronger predictor of CVD and mortality risk than LDL [60–62], has now been added by the NCEP Adult Treatment Panel III as a secondary target of therapy [63]. In addition, because apo B indicates the total number of atherogenic lipoprotein particles and apo A-I, a major lipoprotein in HDL, has a critical role in reverse cholesterol transport, the apo B/apo A-I (as well as apo B concentration) has been proposed as a risk factor for CVD. Increasing evidence from multiple studies has repeatedly shown that the apo B/apo A-I predicts cardiovascular risk – the lower the ratio, the lower is the risk – and is a better marker than LDL and lipid ratios [64–71]. In the InterHeart study, the apo B/apo A-I was the strongest determinant of MI risk, even higher than smoking; the OR of top vs. lowest decile was 4.73 [72]. The authors state the apo B/apo A-I might be the best marker of the balance of atherogenic and antiatherogenic particles. Subjects in both PED and MED arms showed significant reduction in non-HDL cholesterol and apo B/apo A-I at 8 weeks compared to baseline, but only the PED arm showed continued reduction in the ratio at 12 weeks. These data suggest further cardiovascular benefit from the added phytochemicals.

The worldwide prevalence and multi-factorial nature of MetS do not reasonably support a pharmacologic approach for treatment or prevention. Fortunately, lifestyle modification including dietary alteration has demonstrated success in correcting metabolic abnormalities associated with the development of type 2 diabetes and CVD. The present study provides evidence that supplementation of a modified Mediterranean-style, low glycemic load diet with a combination of phytochemicals addressing multiple inflammatory and insulin signaling pathways simultaneously may be a novel, effective means to managing MetS. This comprehensive, supplemented lifestyle program represents a potentially powerful approach to the management of at risk individuals with MetS and hypercholesterolemia.