PROVIT-CLOCK: A Potential Influence of Probiotics and Vitamin B7 Add-On Treatment and Metabolites on Clock Gene Expression in Major Depression

Abstract

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, 4-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 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 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 4-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.

Keywords: Circadian clock genes; Gut-brain axis; Major depressive disorder; Randomized controlled trial.

Fig. 1.

Timeline of the PROVIT study.

Fig. 2.

Group effects of CLOCK expression: qPCR expression changes (FC) of the clock gene CLOCK in fasting blood samples of study participants with MDD over time (t₀ at admission vs. t₂ after 4 weeks of intervention). The black boxes illustrate probiotics; the gray boxes illustrate placebo.

Fig. 3.

Time effects of ARNTL expression: RT-qPCR expression changes (FC) of the clock gene ARNTL in fasting blood samples of study participants with MDD over time (t₀ at admission vs. t₂ after 4 weeks of intervention). The black line illustrates probiotics; the gray line illustrates placebo.

Fig. 4.

Time effects of TIMELESS expression: qPCR expression changes (FC) of the clock gene TIMELESS in fasting blood samples of study participants with MDD over time in the Austrian PROVIT study (t₀ at admission vs. t₂ after 4 weeks of intervention). The black line illustrates probiotics; the gray line illustrates placebo.

Fig. 5.

Top 25 correlations with gene expression levels of ARNTL in stool (a), ARNTL in serum (b), CLOCK in stool (c), CLOCK in serum (d) and with the quantitatively assessed metabolites by NMR metabolomics. x-axis correlation coefficients (raw values, irrespective of p value) and on the y-axis the top 24 metabolites analyzed by NMR spectroscopy correlating with the upper most feature (r = 1).

Fig. 6.

Top 25 correlations with gene expression levels of TIMELESS in stool (a), TIMELESS in serum (b), NR1D1 in stool (c), NR1D1 in serum (d) and with the quantitatively assessed metabolites by NMR metabolomics. x-axis correlation coefficients (raw values, irrespective of p value) and on the y-axis the top 24 metabolites analyzed by NMR spectroscopy correlating with the upper most feature (r = 1).

Fig. 7.

Top 25 correlations with gene expression levels of PER3 in stool (a), PER3 in serum (b) and with the quantitatively assessed metabolites by NMR metabolomics. x-axis correlation coefficients (raw values, irrespective of p value) and on the y-axis the top 24 metabolites analyzed by NMR spectroscopy correlating with the upper most feature (r = 1).