Association between dietary phylloquinone intake and peripheral metabolic risk markers related to insulin resistance and diabetes in elderly subjects at high cardiovascular risk

Vitamin K (K₁ or phylloquinone and K2 or menaquinones) is recognized as an essential element in the synthesis of carboxylate clotting factors involved in prothrombotic disorders and cardiovascular disease. More recently, it has been reported that vitamin K also participates in the gamma-carboxylation reactions of other proteins such as osteocalcin, and may also exert a protective role against age-related bone loss [1, 2]. However, additional roles of vitamin K, independent of these effects have been described [3]. Thus, there is evidence that both osteocalcin and vitamin K may have a potential beneficial role in glucose metabolism, insulin sensitivity and type 2 diabetes (T2DMs) [4‐7]. Since inflammation underlies all these chronic metabolic conditions, it is plausible that the potential role of vitamin K in glucose metabolism partly occurs through the modulation of cytokines and other metabolic risk markers related to insulin resistance and diabetes.

In-vitro studies have shown an anti-inflammatory effect of vitamin K. Human macrophage THP-1 cells incubated with vitamin K reduced the interleukin-6 (IL-6) expression compared to non-incubated cells. Likewise, rats fed with a vitamin K-deficient diet showed an enhanced expression of genes involved in the acute inflammatory response [8]. In a subsample of 1,321 subjects from the Framingham Offspring Study, both plasma phylloquinone and dietary phylloquinone intake were inversely associated with peripheral concentrations of some inflammatory markers [9]. However, in a 3-year randomized clinical trial designed to assess the effect of vitamin K supplementation on bone loss, no differences were found in the plasma IL-6, C-reactive protein or osteoprotegerin concentrations of participants receiving or not a phylloquinone supplement [10].

The purposes of the present study were to assess the cross-sectional associations between dietary intake of vitamin K1 and selected adipokines or other metabolic risk markers related to inflammation, insulin resistance and diabetes; and to longitudinally analyse the associations between changes in dietary phylloquinone intake and changes in these risk markers after one-year of follow-up in a cohort of elderly subjects at high cardiovascular risk.

Of the 568 subjects consecutively recruited, 57 were excluded because they were using anti-inflammatory medication at baseline and 1 because he hd not completed the FFQ at follow-up. Table 1 summarizes the baseline characteristics of the study participants. Study subjects were 67.2±6.0 years old and 44.4% of them were male. Most of them were overweight or obese (92.2%), had hypertension (91.8%), were hypercholesterolemic (62.9%), and 55% had T2DM. Table 2 presents the baseline and 1-year dietary changes by tertiles of change in dietary phylloquinone intake. Subjects in the highest tertile of change consumed less amounts of vitamin K1 at baseline. This change after intervention was due to a higher consumption of total vegetables and, especially, leafy green vegetables, the primary dietary source of vitamin K1. In cross-sectional analyses at baseline, a negative significant association was found between dietary phylloquinone intake and PAI-1 plasma concentrations, even after adjusting for potential confounders (Table 3), but not for the rest of the metabolic risk markers. However, after 1-year of follow-up, those subjects in the upper tertile of changes in dietary phylloquinone intake showed a significant greater reduction in ghrelin (15.0%), GIP (12.9%), GLP-1 (17.6%), IL-6 (27.9%), leptin (10.3%), TNF (26.9%) and visfatin (24.9%) than those subjects in the lowest tertile (Table 4). No significant associations were found between changes in dietary phylloquinone intake and other metabolic markers of inflammation, insulin resistance and diabetes.

The results of this study show, for the first time, that an increased dietary intake of phylloquinone is associated with an improvement in inflammatory and other metabolic risk markers related to insulin resistance and diabetes, thus supporting a protective role of vitamin K on low-grade chronic inflammatory diseases.

In recent years, vitamin K has been attributed a putative role in glucose metabolism, insulin resistance and T2DM [17]. Recently, in a prospective study our group has shown that a higher dietary phylloquinone intake was associated to a lower risk of developing T2DM. However, the exact mechanisms underlying this relationship remain still unknown. One potential explanatory mechanism could be related to the role of vitamin K as a cofactor in the carboxylation of vitamin K-dependent proteins, such as osteocalcin [18], GAS6 or Protein S [19] and through their role on NF-kB [20‐22]. Additionally, some authors have suggested that inflammation may be modulated by a possible antioxidant effect of vitamin K [23].

A cross-sectional study conducted in a subsample from the Framingham Offspring Study, showed an inverse association between 2-fold changes in usual dietary phylloquinone intake or plasma phylloquinone concentrations and seven of the fourteen peripheral inflammatory markers measured [9]. However, the same authors, in a cross-sectional analysis conducted on 400 healthy elderly men and women, did not find any significant association between plasma phylloquinone concentrations and C-reactive protein or IL-6 after adjusting for potential confounders [10]. Similar to the results from Shea and co-workers, we also failed to find any significant association between dietary phylloquinone intake and most of the metabolic risk markers analysed. However, our longitudinal findings are generally consistent with in-vitro studies or those of a previous epidemiological study. In-vitro studies found that the production of the proinflammatory cytokines IL-6 or TNF by human gingival fibroblast or mouse macrophage cells, respectively, decreased when cells were incubated with different vitamin K family compounds [21, 24]. Other studies have found that vitamin K suppresses inflammation by lowering the expression of genes for some proinflammatory cytokines, such as IL-6, IL-1β and TNF [8, 22]. However, only a single 3-year, double-blind, randomized controlled trial has been conducted to evaluate the effect of vitamin K supplementation on peripheral inflammatory marker concentrations. In that study, conducted with 379 healthy men and women, no significant relationship between plasma phylloquinone levels and inflammation markers was shown [10]. In our study we observed a significant improvement in the inflammatory status (leptin, IL-6, TNF) and a decrease in other metabolic risk markers related to insulin resistance and diabetes such as visfatin, ghrelin, GIP and GLP-1 among subjects who increased their dietary intake of phylloquinone after 1-year follow-up, thus contributing to extend the knowledge on the role of vitamin K in humans and to support our previous results on dietary phylloquinone intake and T2DM incidence [7]. The discrepancies between our results and those previously published could be partly explained by the differences between the populations. Our participants were elderly subjects at high cardiovascular risk, whereas, subjects in the study of Shea [9] were healthy and generally free of chronic disease. This may contribute to explain why inflammatory cytokines remained unchanged in that study. Moreover, in our study we assessed the association of inflammation with dietary phylloquinone intake instead of plasma phylloquinone as was done in the previous study. Whether circulating levels of phylloquinone are correlated to dietary phylloquinone intake remains to be elucidated.

It must be noted that leptin, IL6 and TNF are pro-inflammatory cytokines with a recognised role in the development and progression of insulin resistance, T2DM and cardiovascular disease [25, 26]. Also visfatin and ghrelin appears as important mediators of inflammation in addition to glucose-lowering and insulin-mimicking/sensitizing effects or a suppressive role of ghrelin in the release of insulin from the pancreatic islets [27‐30]. A strong inverse correlation between plasma ghrelin concentrations and insulin resistance has been observed in several studies [30‐32] and lower concentrations of ghrelin have been observed in T2DM subjects [33]. The negative relationship between changes in dietary phylloquinone intake and plasma incretin concentrations observed in our study could be explained because a higher intake of phylloquinone may promote better glycemic control thus leading less necessary the glucose and insulin regulation mediated by incretins. However, the pleiotropic role recently attributed to incretins could also contribute to explain our results. Although there is growing evidence that incretin hormones (GIP and GLP-1) simulate glucose-dependent insulin secretion and stimulate pancreatic synthesis of insulin, a novel link between inflammation and incretin hormones has been proposed. First, IL-6 increase GLP-1 production in intestinal L cells and alpha pancreatic cells improving insulin secretion and glycemia [34]. Therefore, the reduction of IL-6 levels observed in the subjects allocated in the highest tertile of change in phylloquinone intake could partly explain the reduction in GLP-1 concentrations in this group. Nie et al., have also demonstrated the capacity of GIP to activate inflammatory response and promote secretion of pro-inflammatory cytokines and chemokines in cell culture adipocytes [35]. Additionally, a potential role of GIP on adipose tissue insulin resistance mediated by osteopontin regulation has also been suggested [36]. The results from our study could be related to the potential role of incretins on adipose tissue in a fasting situation rather than to their established role on pancreatic cells in a post-prandial state. Whether or not GIP and GLP-1 need to be considered as new adipokynes or related pro-inflammatory markers could not be elucitaded from the results of our study. Specific studies are needed to get a deeper understanding of the exact role of incretins on adipose tissue and their interaction with the rest of adipokynes.

Our study has several limitations. It should be kept in mind that the subjects in our study were randomly allocated to a healthy Mediterranean diet that could partially account for the reduction in peripheral metabolic risk markers in some of them although no significant interaction between intervention group and dietary phylloquinone intake was observed for any of the outcomes. In order to minimize the potential effect of a healthy dietary pattern on inflammatory response we have adjusted the regression models for the adherence to a MedDiet. Subjects in the PREDIMED study reported a higher dietary intake of phylloquinone than in other epidemiological studies, probably because this study was conducted in a Mediterranean country where the consumption of fruit and vegetables is high. In populations with a lower consumption of phylloquinone or poor nutrition an increase in dietary vitamin K1 would probably be much more beneficial. The cohort studied was elderly and at high risk of cardiovascular disease, so our findings cannot be generalized to younger or healthier individuals. Because there is no perfect correlation between dietary phylloquinone intake and its absorption it would be interesting in the future to evaluate the associations showed in our study not only with dietary phylloquinone intake but also with a circulating marker of vitamin K status. Finally, we cannot discount a slight overestimation of dietary phylloquinone intake due to the use of FFQ and the USDA Food Database Composition. Although the FFQ used in our study was not specifically validated for phylloquinone intake, the intraclass correlation coefficient of vegetables (the main source of dietary vitamin K1) was 0.81, one of the highest coefficients obtained during the validation of the FFQ in the PREDIMED cohort [13]. Balanced against these limitations, the main strength of our study is its longitudinal design, which enables us to suggest a cause-effect relationship between changes in dietary phylloquinone intake and changes in inflammatory and related metabolic risk markers. In addition, our study was conducted in a large sample of individuals, and measured a panel of adipokines and related molecules involved in inflammation, glucose metabolism and cardiovascular risk.

In summary, our results support that an increase in dietary phylloquinone intake can lead to an improvement in inflammation and inflammatory-related molecules and also support the contention that high vitamin K1 intake has a beneficial effect on cardiovascular disease and other inflammation-related disorders.