Introduction and objectiveCholesterol homeostasis is a culmination of cellular synthesis, efflux, and catabolism to important physiological entities where short chain fatty acid, butyrate embodied as a key player. This discourse probes the mechanistic molecular details of butyrate action in maintaining host-cholesterol balance.MethodsHepatic mir-122 being the most indispensable regulator of cholesterol metabolic enzymes, we studied upstream players of mir-122 biogenesis in the presence and absence of butyrate in Huh7 cells and mice model. We synthesized unique self-transfecting GMO (guanidinium-morpholino-oligo) linked PMO (Phosphorodiamidate-Morpholino Oligo)-based antisense cell-penetrating reagent to selectively knock down the key player in butyrate mediated cholesterol regulation.ResultsWe showed that butyrate treatment caused upregulation of RNA-binding protein, AUF1 resulting in RNase-III nuclease, Dicer1 instability, and significant diminution of mir-122. We proved the importance of AUF1 and sequential downstream players in AUF1-knock-down mice. Injection of GMO-PMO of AUF1 in mouse caused near absence of AUF1 coupled with increased Dicer1 and mir-122, and reduced serum cholesterol regardless of butyrate treatment indicating that butyrate acts through AUF1.ConclusionThe roster of intracellular players was as follows: AUF1-Dicer1-mir-122 for triggering butyrate driven hypocholesterolemia. To our knowledge this is the first report linking AUF-1 with cholesterol biogenesis.

This discourse probes the mechanistic molecular details of butyrate action in maintaining host- cholesterol balance. Hepatic miR122 being the most indispensable regulator of cholesterol metabolic enzymes, we studied upstream players of miR122 biogenesis in the presence and absence of butyrate in Huh7 cells and mice model. We showed that butyrate treatment caused upregulation of RNA-binding protein, AUF-1 resulting in RNase-III nuclease, Dicer-1 instability, and significant diminution of miR122. We proved its importance of AUF-1 and sequential downstream players in AUF-1-knock-down mice. We synthesized unique self-transfecting GMO (guanidinium-morpholino- oligonucleotides) linked PMO (Phosphorodiamidate-Morpholino Oligonucleotides)-based antisense reagent and injection of which in mouse caused near absence of AUF-1 coupled with increased Dicer- 1 and miR122, and reduced serum cholesterol regardless of butyrate treatment indicating that butyrate acts though AUF-1. The roster of intracellular players was as follows: AUF-1-Dicer-1-miR122 for triggering butyrate driven hypocholesterlaemia. To our knowledge this is the first report linking AUF-1 with cholesterol biogenesis.

The gut microbiome derived short chain fatty acids perform multitude of functions to maintain gut homeostasis. Here we studied how butyrate stymie enteric bacterial invasion in cell using a simplistic binary model. The surface of the mammalian cells is enriched with microdomains rich in cholesterol that are known as rafts and act as entry points for pathogens. We showed that sodium butyrate treated RAW264.7 cells displayed reduced membrane cholesterol and less cholera-toxin B binding coupled with increased membrane fluidity compared to untreated cells indicating that reduced membrane cholesterol caused disruption of lipid rafts. The implication of such cellular biophysical changes on the invasion of enteric pathogenic bacteria was assessed. Our study showed, in comparison to untreated cells, butyrate-treated cells significantly reduced the invasion of Shigella and Salmonella, and these effects were found to be reversed by liposomal cholesterol treatment, increasing the likelihood that the rafts' function against bacterial invasion. The credence of ex vivo studies found to be in concordance in butyrate fed mouse model as evident from the significant drift towards a protective phenotype against virulent enteric pathogen invasion as compared to untreated mice. To produce a cytokine balance towards anti-inflammation, butyrate-treated mice produced more of the gut tissue anti-inflammatory cytokine IL-10 and less of the pro-inflammatory cytokines TNF-α, IL-6, and IFN-γ. In histological studies of Shigella infected gut revealed a startling observation where number of neutrophils infiltration was noted which was correlated with the pathology and was essentially reversed by butyrate treatment. Our results ratchet up a new dimension of our understanding how butyrate imparts resistance to pathogen invasion in the gut.

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