Figure 4: Lactate is required for hyperproliferation.

(a) Mice were starved for 36 h and refed CE2 (orange) or ORS (blue) or CE2 with oral antibiotics (black) for 12 h. In ORS-refed mice, the number of BrdU+ cells in the colon without rectal injection (ORS), with rectal injection of control 500 mM NaCl (NaCl), fresh caecal contents from CE2-refed mice (flora-alive), killed flora (flora-killed), killed L. murinus (LM-killed), organic acid mixture (OA mix), LM-killed plus organic acid mixture (LM-killed+OA mix), 500 mM lactate (LA) or a combination of these are shown. In mice refed with CE2 and treated with oral antibiotics, 500 mM NaCl (antibiotics), lactate (LA), butyrate (BA), acetate (AA), 3-hydroxypropionate (HP), succinate (SA) or OA mixture minus lactate (OA−LA) was administered intrarectally together with antibiotics three times during refeeding. Data are shown as the average+s.d. of 3–7 mice relative to control (fed ad libitum) mice. (b) Typical patterns of metabolome analysis as relative changes from the control condition. Data are shown as the average +s.d. of three mice as the relative signal intensity to control SPF mice. (c) Relative amount of lactic acid in the caecum and colon, and glycolic acid in the colon from starvation-refed, germ-free (GF) and gnotobiotic mice monoassociated with L. murinus (GB, hatched). Data are shown as the mean+s.d. of three mice as the relative signal intensity to control SPF mice. (d) Number of BrdU+ cells in the colon of gnotobiotic mice monoassociated with lactate-producing Bifidobacterim infantis or non-lactate-producing Bacteroides thetaiotaomicron shown as % of control (fed ad libitum). Short bar indicates the mean value. (e) Gene expression in the colon. Mice were starved for 36 h and refed CE2. Some mice were treated with oral antibiotics (AB), and 500 mM lactate or butyrate was administered intrarectally together with antibiotics three times during refeeding. After 12 h of refeeding, distal 1/3 of the colon was subjected to total RNA extraction and quantitative RT–PCR for Akr1b8 and Ereg. Data were shown as relative expression (AB+refed=1). Each dot represents individual mice, and short bar indicates the mean value. In panels a, d and e, P-values were calculated using Mann–Whitney test.