Primary MEM cells were obtained from the forehead mesenchyme of embryos taken from time-pregnant females at gestation day 10.5 as described31. This location and gestation time were chosen to maximize the number of cells expressing Alx3 30, 32. Briefly, the first branchial arch and the most rostroventral part of the forehead mesenchyme of each embryo was dissected and pieces from several embryos of the same genotype were pooled. Once dispersed, cells were incubated in standard DMEM containing 10% FBS and split a maximum of three times. In one set of experiments, primary MEM cells were incubated in medium containing 2.8 mM glucose overnight. After this, the medium was replaced with fresh medium containing either 2.8 mM, 16 mM or 30 mM glucose, and the incubation proceeded for another 24 hours until cells were harvested. In a different set, cells were incubated in DMEM containing 0.5% FBS and 0.5% BSA, and insulin (Human recombinant, Sigma I2643) was added to a concentration of 100 nM overnight.