Abstract
The fat-soluble vitamin K (VK) has long been known as a requirement for blood coagulation, but like other vitamins, has been recently recognized to play further physiological roles, particularly in cell development and homeostasis. Vertebrates cannot de novo synthesize VK, which is essential, and it can only be obtained from the diet or by the activity of the gut microbiota. The IPEC-J2 cell line, obtained from porcine small intestine, which shows strong similarities to the human one, represents an excellent functional model to in vitro study the effect of compounds at the intestinal level. The acute VK treatments on the bioenergetic features of IPEC-J2 cells were evaluated by Seahorse XP Agilent technology. VK exists in different structurally related forms (vitamers), all featured by a naphtoquinone moiety, but with distinct effects on IPEC-J2 energy metabolism. The VK1, which has a long hydrocarbon chain, at both concentrations (5 and 10 μM), increases the cellular ATP production due to oxidative phosphorylation (OXPHOS) by 5% and by 30% through glycolysis. The VK2 at 5 μM only stimulates ATP production by OXPHOS. Conversely, 10 μM VK3, which lacks the long side chain, inhibits OXPHOS by 30% and glycolysis by 45%. However, even if IPEC-J2 cells mainly prefer OXPHOS to glycolysis to produce ATP, the OXPHOS/glycolysis ratio significantly decreases in VK1-treated cells, is unaffected by VK2, and only significantly increased by 10 μM VK3. VK1, at the two concentrations tested, does not affect the mitochondrial bioenergetic parameters, while 5 μM VK2 increases and 5 μM VK3 reduces the mitochondrial respiration (i.e., maximal respiration and spare respiratory capacity). Moreover, 10 μM VK3 impairs OXPHOS, as shown by the increase in the proton leak, namely the proton backward entry to the matrix space, thus pointing out mitochondrial toxicity. Furthermore, in the presence of both VK1 and VK2 concentrations, the glycolytic parameters, namely the glycolytic capacity and the glycolytic reserve, are unaltered. In contrast, the inhibition of glycoATP production by VK3 is linked to the 80% inhibition of glycolysis, resulting in a reduced glycolytic capacity and reserve. These data, which demonstrate the VK ability to differently modulate IPEC-J2 cell energy metabolism according to the different structural features of the vitamers, can mirror VK modulatory effects on the cell membrane features and, as a cascade, on the epithelial cell properties and gut functions: balance of salt and water, macromolecule cleavage, detoxification of harmful compounds, and nitrogen recycling.
Highlights
Vitamin K (VK) was originally recognized as a component in blood clotting, being a cofactor for vitamin K–dependent carboxylase, which facilitates the post-translational modification of glutamic acid to γ-carboxy-glutamic acid residues in selected proteins (Suttie, 1985)
The cellular ATP level produced by oxidative phosphorylation (OXPHOS) and glycolysis in the presence of the different VK vitamers are shown in Figure 2 by oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) values, respectively, under basal metabolic conditions
The calculation of the mitoATP and glycoATP production rate (Figure 2), obtained by injecting oligomycin to inhibit mitochondrial ATP synthesis and rotenone plus antimycin A to block mitochondrial respiration, highlight that IPEC-J2 cells are characterized by an oxidative metabolism
Summary
Vitamin K (VK) was originally recognized as a component in blood clotting, being a cofactor for vitamin K–dependent carboxylase, which facilitates the post-translational modification of glutamic acid to γ-carboxy-glutamic acid residues in selected proteins (Suttie, 1985). The intestinal absorption of VKs follows the dietary lipid pattern (Shearer et al, 2012). The VK family consists of structurally similar naphthoquinones (Figure 1), which, due to their lipophilicity, can cross cell membranes. VK naturally exists in two forms, namely, phylloquinone (VK1), which bears a phytyl side chain, mainly comes from vegetables and constitutes more than 90% of dietary VKs, and menaquinones (VK2), which exist in multiple structures, mainly come from bacterial synthesis in the gut, and occur in fermented foods (Schwalfenberg, 2017). The VK homologs are characterized by a 2-methyl1,4-naphthoquinone nucleus and a polyisoprenoid side chain at the 3-position (Figure 1). Menadione, or VK3, lacks the hydrocarbon side chain and is considered as a metabolite or a provitamin (Schwalfenberg, 2017)
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