Supplementary Material for: PROVIT-CLOCK - A potential influence of probiotics and Vitamin B7 add-on treatment and metabolites on clock gene expression in major depression

Introduction An increasing body of evidence suggests a strong relationship between gut health and mental state. Lately, a connection between butyrate-producing bacteria and sleep quality has been discussed. The PROVIT study, as a randomized, double-blind, four-week, multispecies probiotic intervention study, aims at elucidating the potential interconnection between the gut’s metabolome and the molecular clock in individuals with major depressive disorder (MDD). Methods The aim of the PROVIT-CLOCK study was to analyze changes in core clock gene expression during treatment with probiotic intervention versus placebo in fasting blood and the connection with the serum- and stool-metabolome in patients with MDD (n= 53). In addition to clinical assessments in the PROVIT study, metabolomics analyses with 1H Nuclear Magnetic Resonance (NMR) Spectroscopy (stool and serum) and gene expression (RT-qPCR) analysis of the core clock genes ARNTL, PER3, CLOCK, TIMELESS, NR1D1 in peripheral blood mononuclear cells (PBMCs) of fasting blood, were performed. Results The gene expression levels of the clock gene CLOCK were significantly altered only in individuals receiving probiotic add-on treatment. TIMELESS and ARNTL gene expression changed significantly over the four-week intervention period in both groups. Various positive and negative correlations between metabolites in serum/stool and core clock gene expression levels were observed. Conclusion Changing the gut microbiome by probiotic treatment, potentially influences CLOCK gene expression. The preliminary results of the PROVIT-CLOCK study indicate a possible interconnection between the gut microbiome and circadian rhythm potentially orchestrated by metabolites.