Sub cutaneous adipose biopsies were taken around the umbilical region. This tight clin ical setup allowed us to control for variables that might affect gene expression changes not due to the main pertur bations. The time of first day and patient to patient variation had the most profound effect on gene expression, inde pendent of day of biopsy or treatments. To evaluate which factor had the greatest effect on the gene expression observed in the adipose samples, we first performed ANOVA analysis for patient, time and drug treatment on each gene that passed a variance threshold. Among 20,000 genes that passed the variance criteria, 5,194 had p values 0. 01 for diurnal variation, 6,097 had p values 0. 01 for inter patient variation, and 180 had p values 0. 01 for treatment.

The large but expected inter patient variability reflects the power of study and the quality of the expression profiles. In addition to the uni variate analysis above, we also performed Principal Com ponent Analysis and found the first principal component to be most significantly associated with time of day. Both the univariate and multivariate analyses showed that time of biopsy had a significant effect on gene expression with approximately 5,000 transcripts regulated in a diurnal fashion. Overall, the most profound changes occurred from the morning to the afternoon and the gene expres sion changes were smaller from the afternoon to the evening. The circadian gene, PER1, was prominent among the genes with significantly higher expression in the morning versus the afternoon or evening, with up to a 10 fold change in some patients for PER1 mRNA expression.

Known clock genes, including CLOCK, CRY2, BHLHB2 and others, were diurnally regulated in the human adipose. Approximately 5,000 genes were significantly correlated with PER1 mRNA levels. As expected, significant overlap was observed between the diurnal output gene set from the ANOVA analysis and the PER1 correlated gene set. Genes that were positively correlated with PER1 mRNA levels included those involved in fructose and mannose metab olism and glycolysis. Con versely, such genes involved in inflammatory pathways as the cytokines, glucose transporters, cholesterol biosynthesis genes, the low density lipoprotein receptor Carfilzomib and genes that control response to free radicals and hypoxia were significantly, but negatively, correlated with PER1 mRNA.

Transcripts that were most strongly correlated to PER1 were ZNF145, METRS and IL6. Genes previously shown to be diurnally regulated, such as SERPINE1, were also negatively correlated to PER1 mRNA expression in this dataset. Pathways that were enriched in the diurnal signature selleck chemicals llc included inflammatory pathways and the NFKb pathway. Supple mental literature mining processes showed that IL 10, IL 6, p38MAPK and PPAR signaling pathways were also enriched.