Diurnal Variation in PDK4 Expression Is Associated With Plasma Free Fatty Acid Availability in People

Abstract

Context: Many biological pathways involved in regulating substrate metabolism display rhythmic oscillation patterns. In rodents, clock genes regulate circadian rhythms of metabolic genes and substrate metabolism. However, the interrelationships among substrate metabolism, metabolic genes, and clock genes have not been fully explored in people.

Objective: We tested the hypothesis that the diurnal expression pattern of pyruvate dehydrogenase kinase 4 (PDK4), a key metabolic enzyme involved in fuel switching between glucose and free fatty acids (FFAs), is associated with plasma FFA concentration and clock genes.

Design and methods: We analyzed peripheral blood mononuclear cells (PBMCs), subcutaneous adipose tissue, and plasma samples obtained serially during 24 hours from metabolically healthy women (n = 10) and evaluated the interrelationships among PDK4, plasma FFA, and clock genes. We also determined the potential mechanisms responsible for PDK4 transcriptional regulation by using primary human PBMCs and adipocytes.

Results: We found that PDK4 diurnal expression patterns were similar in PBMCs and adipose tissue (ρ = 0.84, P < 0.001). The diurnal variation in PBMC PDK4 expression correlated more strongly with plasma FFA and insulin (ρ = 0.86 and 0.63, respectively, both P < 0.001) concentrations than clock genes. Data obtained from primary culture experiments demonstrated that FFAs directly induced PDK4 gene expression, at least in part through activation of peroxisome proliferator-activated receptor α.

Conclusions: Our results suggest that plasma FFA availability is an important regulator of diurnal expression patterns of PDK4, and we identify a novel interaction between plasma FFA and cellular diurnal rhythms in regulating substrate metabolism.

Trial registration: ClinicalTrials.gov NCT02011581 NCT02093572.

Figures

Figure 1.

Diurnal expression patterns of clock genes and PDK4 in PBMCs. Gene expression of selected clock genes (CLOCK, BMAL1, PER1, PER2, and REV-ERBα) and PDK4 normalized to GAPDH expression in PBMC obtained from metabolically healthy women (n = 10). Subjects consumed identical meals at 0700 hours, 1230 hours, and 1900 hours. Data are means ± SEM. †P < 0.05, ‡P < 0.001, by one-way repeated measures ANOVA.

Figure 2.

Diurnal variation of PBMC PDK4 expression is associated with plasma FFA and insulin concentrations. Plasma FFA (A) and insulin (B) concentrations during 24 hours in metabolically healthy women (n = 10). Arrows indicate PBMC collection. (C and E) Comparisons are shown between diurnal variations in PBMC PDK4 gene expression (red line) and plasma FFA concentrations (blue line) (C) and inverse plasma insulin concentrations (E) (green line). (D and F) Relationships are shown between PBMC PDK4 gene expression and plasma FFA concentrations (D) and inverse values of plasma insulin concentrations (F). Data are means ± SEM. †P < 0.001, assessment over time by one-way repeated measures ANOVA.

Figure 3.

FFAs induce PDK4 gene expression through activation of PPAR-α in primary PBMCs. PDK4 gene expression in PBMCs was isolated from metabolically healthy people after they fasted for ∼12 hours overnight. (A and B) PBMCs were cultured and treated with FFAs (A) or insulin (B) for 3 hours (n = 8 per group). (C) Effects of insulin (10 nmol/L) or a PPAR-α antagonist (10 μmol/L GW6471) on FFA-induced PBMC PDK4 gene expression (n = 7 per group). Data are means ± SEM. *P < 0.01, by using one-way ANOVA followed by the Tukey post hoc test. N.S., not significant.

Figure 4.

Diurnal expression patterns of clock genes and PDK4 in adipose tissue. Gene expression of selected clock genes (CLOCK, BMAL1, PER1, PER2, and REV-ERBα) and PDK4 normalized to GAPDH expression in subcutaneous adipose tissue obtained from metabolically healthy women (n = 10). Data are means ± SEM. †P < 0.05, ‡P < 0.001, by using one-way repeated measures ANOVA.

Figure 5.

Adipose tissue PDK4 gene expression is associated with plasma FFA concentrations and PBMC PDK4 gene expression. (A–F) Relationship between adipose tissue PDK4 gene expression and adipose tissue PER1 (A) and PER2 (B) gene expression, plasma FFAs (C), PBMC PDK4 gene expression (D), plasma insulin (E), and cortisol concentrations (F). (G) PDK4 gene expression in differentiated human subcutaneous adipocytes treated with FFA (250 μmol/L), insulin (10 nmol/L), or both for 3 hours (n = 3 per group). Data are means ± SEM. *P < 0.05, **P < 0.001, by using one-way ANOVA followed by the Tukey post hoc test. N.S., not significant.