Butyrate can act as a stimulator of growth or inducer of apoptosis in human colonic epithelial cell lines depending on the presence of alternative energy sources.

Baldev Singh 1 Andrew P.Halestrap 0 Christos Paraskeva 1 0 Biochemistry, School of Medical Sciences, University of Bristol , Bristol BS8 1TD, UK 1 Pathology and Microbiology 3To whom correspondence should be addressed - In vivo, butyrate is a major energy source for the colonic epithelium and is thought to stimulate proliferation. In contrast, butyrate in vitro has been shown to inhibit proliferation and induce differentiation and apoptosis in colonic epithelial cells. Most colon cell cultures are grown in medium containing high concentrations of glucose, whereas in vivo, the main energy source used by the colon cells is butyrate. The aim of this study was to determine whether the apparent contrasting roles of butyrate in vivo and in vitro could be as a consequence of differences in glucose availability. The sensitivity of two human colorectal tumour cell lines, one adenoma (S/RG/C2) and one carcinoma (HT29) to butyrate-induced growth inhibition and apoptosis was investigated to determine whether these cellular effects were altered under glucose depleted culture conditions. Glucose depletion resulted in increased apoptosis in both cell lines in the absence of butyrate. Butyrate in standard culture conditions (containing 25 mM glucose and 1 mM pyruvate) inhibited growth and induced apoptosis in both cell lines. However, low concentrations of butyrate in glucose depleted culture conditions (i.e. standard growth medium without glucose and pyruvate supplements) were found to reduce apoptosis induced by glucose deprivation and increase cell yield in both cell lines. The results show that in glucose depleted culture conditions, butyrate at low concentrations (0.5 mM for S/RG/C2, and 0.5 and 2 mM for HT29 cells) was found to be growth stimulatory whereas in the presence of glucose, these same concentrations of butyrate induced apoptosis. Thus, whether butyrate is growth stimulatory or growth inhibitory may depend on the availability of other energy sources. These observations may, in part, provide an explanation for the apparent opposite effects of butyrate on proliferation reported in vivo and in vitro. Colorectal cancer is a common malignancy of western society and this high incidence is thought to be largely associated with dietary factors. A low fibre diet was proposed to contribute to an increased risk of colorectal cancer (1), a view supported by more recent epidemiological studies, which have provided strong support for this association (2). Carbohydrates which escape enzymatic digestion in the human small intestine *Abbreviations: DMEM, Dulbeccos modified Eagle medium; SCFA, shortchain fatty acids; Glu1Pyr1, medium supplemented with both glucose and pyruvate; Glu2Pyr2, medium deficient in both glucose and pyruvate; S/RG/ C2, human colonic adenoma cell line; HT29, human colonic carcinoma cell line. undergo anaerobic fermentation in the colon. A major component of this class of carbohydrates is the non-starch polysaccharides (also known as dietary fibre) which is subjected to fermentation by the microflora of the large intestine, resulting in the production of acetic, propionic and butyric acid as major by-products (3). These short chain fatty acids (SCFA*), which occur in millimolar amounts, are rapidly absorbed in the colon, providing important energy supplies for the colorectal epithelium and maintaining the cellular electrolyte balance (4). Out of three SCFA, butyrate is considered to be the preferred energy source and it accounts for ~70% of the total energy consumed in rat colonocytes (4,5) and in vivo is reported to stimulate proliferation (6). This stimulation of proliferation is regarded as a physiological response rather than a pre-neoplastic associated phenomenon, since the dividing cells do not extend to the top of the crypt (7). Conditions which reduce the concentration of luminal SCFA have been shown to result in decreased epithelial cell proliferation. These conditions include colonic bypass surgery (8), feeding of a substrate-free diet (9) and germ-free conditions (10). The reduced level of SCFA results in metabolic starvation, accompanied by mucosal surface degeneration and acute inflammation of the colonic epithelium (11). Colonic instillation of SCFA has been shown to be beneficial in reducing this inflammation (12) and these studies have concluded that in vivo, butyrate is growth stimulatory and beneficial. In contrast, butyrate in vitro has been shown to inhibit proliferation in a number of colorectal tumour cell lines (13,14). In addition, treatment of the colon carcinoma cell line HT29 with butyrate led to a permanently differentiated cell line (14). More recently, butyrate, propionate and acetate have been shown to induce apoptosis in a number of colonic tumour cell lines, with butyrate being the most effective inducer of apoptosis (1517). These in vitro observations suggest a possible protective role for short-chain fatty acids in colorectal carcinogenesis and may explain, in part, the apparent protective role of dietary fibre. However, the effects of butyrate on proliferation, based on in vivo and in vitro studies, appear to give contradictory results. The preferred energy source for the colonic epithelium in vivo is butyrate (4,5), whereas most cell cultures are grown in tissue culture medium containing high levels of glucose for their energy source. Hence, differences in energy availability may, in part, provide an explanation for the observation that in vivo butyrate is reported to be growth stimulatory, whereas in vitro it is growth inhibitory. In this study, since butyrate is the preferred energy source of the three SCFA present in the colon, we investigated the effects of butyrate on cell growth and apoptosis under two growth conditions: standard culture medium with high concentrations of glucose and culture medium without glucose supplementation. We asked whether under glucose-depleted culture conditions, butyrate may stimulate proliferation (as observed in vivo), and therefore whether the availability of alternative energy sources could alter the growth response of colonic cells to butyrate. Two human colonic tumour cell lines, an adenoma cell line, S/RG/C2 (18), and the carcinoma cell line, HT29, were routinely grown on tissue culture plastic in Dulbeccos modified Eagle medium (DMEM; Life Technologies, Inc.) with 20 and 10% fetal bovine serum (batch selected), respectively, as described previously (19). Cells for the experiments were grown in either normal growth medium (DMEM), which had D(1) glucose (25 mM, Sigma) and sodium pyruvate (1 mM, Gibco BRL), referred to as Glu1Pyr1, or in DMEM medium which did not have added glucose and pyruvate. This was defined as glucose and pyruvate deficient medium referred to as GluPyr in the text. In these growth conditions, the other main potential energy sources available to the cells are glutamine and amino acids present in the medium and also the fetal bovine serum supplemented to the med (...truncated)