A Theacrine-Based Supplement Increases Cellular NAD+ Levels and Affects Biomarkers Related to Sirtuin Activity in C2C12 Muscle Cells In Vitro

Abstract

There is evidence in rodents to suggest that theacrine-based supplements modulate tissue sirtuin activity as well as other biological processes associated with aging. Herein, we examined if a theacrine-based supplement (termed NAD3) altered sirtuin activity in vitro while also affecting markers of mitochondrial biogenesis. The murine C2C12 myoblast cell line was used for experimentation. Following 7 days of differentiation, myotubes were treated with 0.45 mg/mL of NAD3 (containing ~2 mM theacrine) for 3 and 24 h (n = 6 treatment wells per time point). Relative to control (CTL)-treated cells, NAD3 treatments increased (p < 0.05) Sirt1 mRNA levels at 3 h, as well as global sirtuin activity at 3 and 24 h. Follow-up experiments comparing 24 h NAD3 or CTL treatments indicated that NAD3 increased nicotinamide phosphoribosyltransferase (NAMPT) and SIRT1 protein levels (p < 0.05). Cellular nicotinamide adenine dinucleotide (NAD⁺) levels were also elevated nearly two-fold after 24 h of NAD3 versus CTL treatments (p < 0.001). Markers of mitochondrial biogenesis were minimally affected. Although these data are limited to select biomarkers in vitro, these preliminary findings suggest that a theacrine-based supplement can modulate select biomarkers related to NAD⁺ biogenesis and sirtuin activity. However, these changes did not drive increases in mitochondrial biogenesis. While promising, these data are limited to a rodent cell line and human muscle biopsy studies are needed to validate and elucidate the significance of these findings.

Keywords: mRNAs; mitochondria; sirtuins; theacrine.

Figure 1

Effects of experimental (NAD3) treatments on sirtuin (SIRT) markers. These data illustrate 3 h treatments on SIRT-related mRNAs (panel a), 24 h treatments on SIRT-related mRNAs (panel b), 3 h treatments on global SIRT activity (panel c), and 24 h treatments on global SIRT activity (panel d). All data are presented as the means ± standard deviation values (n = 6 treatment wells per condition), and significance was set at p < 0.05. Symbol: *, indicates more highly expressed in NAD3-treated versus control (CTL)-treated cells. Other abbreviation: OD, optical density.

Figure 2

Effects of NAD3 treatments on mitochondrial biogenesis markers. Legend: these data illustrate 3 h treatments on mRNAs related to mitochondrial biogenesis (panel a), 24 h treatments on mRNAs related to mitochondrial biogenesis (panel b), and 24 h treatments on a surrogate marker of mitochondrial volume (panel c). All data are presented as the means ± standard deviation values (n = 6 treatment wells per condition), and significance was set at p < 0.05. Symbols: *, indicates more highly expressed in NAD3-treated versus control (CTL)-treated cells; #, indicates more highly expressed in control (CTL)-treated versus NAD3-treated cells. Other abbreviation: CS, citrate synthase.

Figure 3

Effects of NAD3 treatments on select protein targets. Legend: these data illustrate 24 h NAD3 treatments that do not affect cellular morphology, while also increasing protein levels per well (a surrogate of cellular health) (panel a). Twenty-four hour NAD3 treatments also increased SIRT1 and NAMPT protein levels, while not affecting other protein targets (panel b). Panel c also illustrates representative Western blots of each assayed target. All data are presented as the means ± standard deviation values (n = 5–6 treatment wells per condition), and significance was set at p < 0.05. Symbols: *, indicates more highly expressed in NAD3-treated versus control (CTL)-treated cells.

Figure 4

Effect of 24 h NAD3 treatments on cellular NAD⁺, NADH, and NAD⁺/NADH. Legend: these data illustrate that 24 h NAD3 treatments increased cellular NAD⁺ levels (panel a), decreased NADH levels (panel b), and increased the NAD⁺/NADH ratio (panel c). (Panel d) illustrates a visual representation of assay results (SC indicates a standard curve, and NADt is NAD⁺ + NADH where NAD⁺ levels are determined by subtracting NADH levels from NADt levels). All data are presented as the means ± standard deviation values (n = 6 treatment wells per condition), and significance was set at p < 0.05. Symbols: *, indicates more highly expressed in NAD3-treated versus control (CTL)-treated cells; #, indicates more highly expressed in CTL-treated versus NAD3-treated cells.