Abstract
As a physiological small molecular product from the microbial fermentation of dietary fibers, butyrate plays an important role in maintaining intestinal health. Our previous works have proved that the effect of sodium butyrate (NaB) on the intestinal barrier function is mediated by activation of AMP-activated protein kinase (AMPK). However, the detailed pathway involved remains unknown. Using the calcium switch assay in the Caco-2 cell monolayer model, we found here that NaB activated AMPK mainly by increasing the calcium level, but not the ATP concentration, via promoting store-operated calcium entry (SOCE). Upon the activation of AMPK, NaB promoted the reassembly of tight junctions (TJs) based on reducing the phosphorylation of myosin II regulatory light chain (MLC2) at Ser19 and increasing phosphorylation of protein kinase C β2 (PKCβ2) at Ser660. Inhibiting (protein kinase C β) PKCβ blocked the reassembly of TJs induced by NaB in the barrier monolayer model. These results indicated that NaB could activate the calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ) pathway to mediate AMPK phosphorylating, which then inhibited the phosphorylation of MLC2 and promoted the phosphorylation of PKCβ2, respectively, so that the downstream molecules of AMPK coordinately contributed to the reassembly of TJs in the Caco-2 barrier model. These results suggested a potential mechanism of butyrate for intestine homeostasis and protection.
Highlights
Butyric acid, sourced from degradable fiber and degradable starch, is one of the major short-chain fatty acids (SCFA) derived from the fermentation of bacterial carbohydrates [1,2]
Our previous research showed that butyrate might improve the barrier function of gastrointestinal epithelia by increasing the transepithelial electrical resistance (TER) of the Caco-2 monolayer when treated with 2 mmol/L sodium butyrate [7], and this effect was attributed to the reassembly of the tight junctions (TJs) through activating AMP-activated protein kinase (AMPK)
We found that NaB can activate calmodulin-dependent protein kinase kinase β (CaMKKβ) in ways other than changing the ATP concentration to mediate the activity of AMPK [13], suggesting sodium butyrate (NaB) may regulate the calcium concentration in Caco-2 cells
Summary
Butyric acid, sourced from degradable fiber and degradable starch, is one of the major short-chain fatty acids (SCFA) derived from the fermentation of bacterial carbohydrates [1,2]. The increase in ADP and AMP relative to ATP ratios is a signal to turn on AMPK in its energy-sensing role by promoting the phosphorylation at Thr172 [10,11], and calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ) mediates another activation mechanism of AMPK through reacting to a rise in intracellular Ca2+ [12]. We found that NaB can activate CaMKKβ in ways other than changing the ATP concentration to mediate the activity of AMPK [13], suggesting sodium butyrate (NaB) may regulate the calcium concentration in Caco-2 cells. NaB regulates biological activities in cells in two ways: inhibiting histone deacetylase to impact gene expression or activating G-protein coupled receptors such as GPR43 [15] and GPR109A [16]. GPR109A is a main receptor of butyrate in colonic epithelial cells and mediates the protective effect against colitis [17]
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