Nicotinamide adenine dinucleotide (NAD+) is a vital molecule with many biological functions. The roles of NAD+ are organized and coordinated by how it is subdivided into different structures within cells. The distribution of this critical compound within the cell is key to understanding the impact of NAD+ on biological processes, diseases, and aging.

Investigators from the University of Texas at Austin and the University of Texas Southwestern Medical Center published a review article in Trends in Biochemical Sciences where they reviewed the evidence supporting how differing NAD+ levels are separated into distinct structures within the cell and how the modulation of NAD+ synthesis dynamically regulates signaling by controlling subcellular NAD+ levels. They further discuss potential benefits to the cell of compartmentalizing NAD+ and methods for measuring NAD+ levels within different parts of the cell.

Every cell is made up of a menagerie of molecules generated by a complex series of reactions that convert sugars into metabolic precursors, intermediate molecules that can be either used to generate energy or to synthesize the building blocks for cells and their components, and NAD+ serves an essential role in this process. When cells are lacking NAD+, they cannot produce energy or cellular components, and even moderate decreases in NAD+ levels can limit cell functions that depend on NAD+.

The levels of NAD+ floating around freely in cells are tightly controlled to precisely modulate signaling in cells. Dividing NAD+ into different cell components helps cells to time responses, communicate status, and protect crucial NAD+ pools.

Along these lines, the regulation of NAD+ levels in cells plays an important role in regulating biological outcomes. For example, fluctuations of NAD+ levels over time regulate the rhythms of the body’s clock. Also, for the initiation of fat cell development, hormones or elevated sugar levels promote elevated amounts of an enzyme called nicotinamide nucleotide adenylyltransferase 2 (NMNAT-2), resulting in decreased NAD+ levels in the nucleus, which activates a gene program to turn precursor cells into fat cells. Finally, distributing NAD+ within cells may protect and prioritize support for particularly critical cellular processes, such as those in the mitochondria, in response to stress when other subcellular stores are depleted.

Temporal regulation of the body’s clock by NAD+ levels. The body’s clock (i.e., circadian rhythms) in mammals is coordinated with oscillating NAD+ levels. The rhythmic oscillation of NAD+ serves as a feedback ‘timer’ by controlling the activities of enzymes dependent on NAD+, helping to establish the periodicity of the cycles. This happens by modulating the levels of an enzyme critical to NAD+ synthesis called nicotinamide phosphoribosyltransferase (NAMPT) that drives the rise and fall of NAD+ levels that serve to limit the duration of activity of enzymes called sirtuins. NAM, nicotinamide; Ac, acetylation (Cambronne and Kraus Trends Biochem Sci. | 2020). 

Given the importance of how NAD+ is subdivided within cells for key biological systems, alterations to this process is a driver of disease. Although direct evidence of a role for NAD+ distribution being disrupted in certain diseases is limited, recent studies are beginning to hint at its importance. For example, the same process by which NMNAT-2 regulates partitioning of NAD+ also controls a genetic program for normal fat cell development, which is exploited by some brain cancer cells to control a genetic program for cancer cell growth. Many questions remain to be addressed to get a complete picture regarding how the distribution of NAD+ in cells is involved in cancer and aging in humans.

Determining the extent of how NAD+ levels are partitioned and dynamically modulated by NAD+ synthesis within the cell as well as their impact on signaling pathways will be a rich area for future research. “We are only beginning to elucidate how the temporal and spatial compartmentalization of NAD+ contributes to its numerous biological roles,” said the researchers.

For these reasons, biological sensors–molecules capable of detecting chemicals that can be made by genetically modified cells–for NAD+ represent a promising approach for additions to the toolbox for measurement and the study of fluctuations in NAD+ levels within cells. For example, there is a utility for different sensors that are sensitive to NAD+ at different ranges, reflecting differences in the expected concentrations in various cellular milieus, cell types, and species. Also, precise intracellular measurements for NAD+ in particular cell structures are currently lacking, and the possibility of focal regulation of NAD+ at specific sites of DNA damage has not been addressed.

Spatial Regulation of NAD+ by Dynamic Compartmentalization. Hormones that induce the development of fat cells or elevated sugar levels promote an increase in levels of an enzyme called nicotinamide nucleotide adenylyltransferase 2 (NMNAT-2), resulting in increased cytoplasmic and decreased NAD+ levels in the nucleus. During the early stages of fat cell development, a decrease in free NAD+ levels in the nucleus limits the activity of enzymes called poly(ADP-ribose) polymerases (PARPs). For example, when the activity of a PARP-1 goes down, a program to turn precursor cells into fat cells that is dependent on a protein called C/EBPβ gets activated (Cambronne and Kraus Trends Biochem Sci. | 2020).

Because NAD+ is crucial for how organisms in all domains of life depend on energy and the building blocks of cells, NAD+ regulation is integral to every discipline and field of study in biology and medicine. “Not only are its chemistry and mechanisms part of the history of biochemistry, but its broad biological impact will undoubtedly also continue to bring together expertise across multiple disciplines in the future,” said the investigators. “The next wave of discoveries in NAD+ biology will be driven by collaborations among scientists with distinct skill sets.”