The Stable Isotope-based Dynamic Metabolic Profile of Butyrate-induced HT29 Cell Differentiation

Abstract

Stable isotope-based dynamic metabolic profiling is applied in this paper to elucidate the mechanism by which butyrate induces cell differentiation in HT29 cells. We utilized butyrate-sensitive (HT29) cells incubated with [1,2-13 C 2 ]glucose or [1,2-13 C 2 ]butyrate as single tracers to observe the changes in metabolic fluxes in these cells. In HT29 cells, increasing concentrations of butyrate inhibited glucose uptake, glucose oxidation, and nucleic acid ribose synthesis in a dose-dependent fashion. Glucose carbon utilization for de novo fatty acid synthesis and tricarboxylic acid cycle flux was replaced by butyrate. We also demonstrated that these changes are not present in butyrate-resistant pancreatic adenocarcinoma MIA cells. The results suggest that the mechanism by which colon carcinoma cells acquire a differentiated phenotype is through a replacement of glucose for butyrate as the main carbon source for macromolecule biosynthesis and energy production. This provides a better understanding of cell differentiation through metabolic adaptive changes in response to butyrate in HT29 cells, demonstrating that variations in metabolic pathway substrate flow are powerful regulators of tumor cell proliferation and differentiation.Butyrate is a four-carbon short chain fatty acid produced by fermentation of fiber polysaccharides by the intestinal microflora of the human colon (1). Butyrate is utilized primarily by colon epithelial cells as a substrate for energy production (2). Previous studies demonstrated that deficiency in the availability or utilization of butyrate causes colitis and may be involved in colon carcinogenesis (3).Butyrate has been shown to induce dose dependent differentiation of various malignant cell lines (4 -6). Studies performed in recent years in colorectal cancer have shown that there is a cell cycle arrest in G 1 phase due to the activation of cyclin D3 and p21Waf1/Cip1 after incubation with butyrate (7). These studies implicate many known cell signaling events in mediating the cell-differentiating effect of butyrate, including cyclin-dependent kinase inhibitors (8), mitogen-activated phosphorylase kinases (9), down-regulation of c-myc (10), and the proinflammatory transcription factor NF-B (11, 12). Among the genes studied using gene array technology, the most significantly affected were those of transcription factors related to cell growth, apoptosis, and oxidative metabolism (13,14). How butyrate induces these specific molecular changes is mostly unknown. To assess how known genetic modifications can be translated into metabolic changes characteristic of differentiated cells, techniques allowing analysis of the levels of low molecular weight compounds are required. Stable isotopebased dynamic metabolic profiling using gas chromatography/ mass spectrometry (GC/MS) 1 is a new tool with a largely untapped potential in the field of functional genomics. In this paper we demonstrate the usefulness of this technique to elucidate the metabolic mechanism underlying butyrate induced cell different...

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