Failure of white adipose tissue to appropriately store excess metabolic substrate seems to underpin obesity-associated type 2 diabetes (1,2). Encouraging ‘browning’ of white adipose has been suggested as a therapeutic strategy to dispose of excess stored lipid and ameliorate the resulting insulin resistance (3). Genetic variation at the DNA locus encoding the novel proteolipid neuronatin has been associated with obesity, and we recently observed that neuronatin expression is reduced in subcutaneous adipose tissue from obese humans (these data can be found, along with the transcript profiles, at NCBI (GSE27951)). Thus, to explore the function of neuronatin further, we used RNAi to silence expression (>90% of protein) in murine primary adipocyte cultures and examined the effects on adipocyte phenotype and glucose disposal. Adipocyte progenitors were isolated from the 129/Sv strain of mice (Harlan, UK). We found that primary adipocytes only express the longer, α-isoform, of neuronatin. Loss of neuronatin led to 10 times increased expression of uncoupling protein-1 mRNA, 2-3 fold increased expression of the mitochondrial genes peroxisome proliferator-activated receptor gamma coactivator 1α and cytochrome c oxidase (Cox) 8b mRNA, and a two-fold induction of Cox4 protein expression in primary subcutaneous white adipocytes, indicative of a ‘browning’ effect. This was accompanied by increased phosphorylation of AMP-activated protein kinase and acetyl coA carboxylase, suggestive of increased fatty acid utilisation. Similar, but less pronounced, effects of neuronatin silencing were also noted in primary brown adipocytes. In contrast, loss of neuronatin caused a two-fold reduction in both glucose uptake and glycogen synthesis without altering insulin signalling (n=4). All statistical analysis was performed using two-way ANOVA RM followed by Newman-Keuls multiple comparison tests. Thus, neuronatin appears to be a novel regulator of ‘browning’ and metabolic substrate utilisation in white adipocytes.