Glucose induced the expression of Alx3 in wild type primary MEM cells (Fig. 5A), confirming our initial results in embryos. Glucose also induced the expression of Foxo1 and Foxo4 in these cells (Fig. 5B). In contrast, in cells from Alx3-deficient embryos the induction of Foxo1 expression by glucose was inhibited, whereas that of Foxo4 was unaffected (Fig. 5B). In addition, in serum starved conditions expression of Foxo1 was significantly decreased in Alx3-deficient MEM cells, but addition of insulin did not result in significant modification of Foxo1 expression regardless of whether ALX3 was present or not (Fig. 5C). Thus, these experiments indicate that ALX3 regulates the Foxo1 gene and is specifically required for glucose induced Foxo1 expression.Figure 5Impaired response to glucose in Alx3-deficient cells. (A) Relative levels of Alx3 mRNA in primary MEM cells obtained from wild type mice cultured in the presence of the indicated concentrations of glucose (n = 5). (B) Relative levels of Foxo1 and Foxo4 mRNA in primary MEM cells obtained from wild type (white bars) or Alx3-deficient (black bars) embryos cultured in the presence of the indicated glucose concentrations (n = 7 per group). (C) Relative levels of Foxo1 mRNA in primary MEM cells from wild type (white bars) or Alx3-deficient (black bars) embryos cultured in the presence of 0.5% FBS without (−) or with (+) insulin (100 nM) (n = 5 per group). (D) Relative levels of MnSOD, catalase and Gpx1 mRNA in primary MEM cells from wild type (white bars) or Alx3-deficient (black bars) embryos cultured in the indicated glucose concentrations (n = 7 per group). All data obtained by quantitative RT-PCR. In all cases, values represent mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mathop{+}\limits_{-}$$\end{document}+− s.e.m. *p < 0.05, **p < 0.01, Student’s t-test.