What Are NAD and FAD and Their Role in Cellular Function?

NAD (nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide) are two small molecules that serve as electron carriers in nearly every cell of your body, shuttling high-energy electrons from the food you eat into the machinery that produces usable energy. They participate in hundreds of chemical reactions, and without them, cells would have no way to extract energy from nutrients or defend themselves against damage. But their roles extend well beyond energy production into territory that touches aging, DNA repair, your body clock, and even how your gut bacteria communicate with the rest of your body.

How NAD and FAD Power Your Cells

When you eat carbohydrates, fats, or proteins, your cells break those nutrients down through a series of chemical reactions. At multiple steps along the way, high-energy electrons are stripped off the nutrient molecules and handed to carrier molecules. NAD picks up two electrons and a hydrogen ion to become NADH. FAD picks up two electrons and two hydrogen ions to become FADH2. These loaded carriers then deliver their cargo to the electron transport chain inside mitochondria, the structures often called the cell’s power plants. There, the electrons flow through a series of protein complexes embedded in the inner mitochondrial membrane, and the energy released at each step is used to pump protons across that membrane. The resulting proton gradient drives the production of ATP, the universal energy currency that powers everything from muscle contraction to brain signaling.

NADH typically feeds its electrons into the chain at Complex I, while FADH2 is linked to Complex II through an enzyme called succinate dehydrogenase. Because FADH2 enters at a later step, it generates slightly less ATP per molecule than NADH does. Both carriers, once they have dropped off their electrons, return to their oxidized forms (NAD+ and FAD) ready to pick up more electrons. This constant cycling between loaded and empty states is what makes them so useful: they are reusable molecular taxis.

A Widespread Textbook Error About FADH2

Many biology textbooks and diagrams show FADH2 floating freely in the mitochondrial matrix, arriving at Complex II as though it were a soluble substrate like NADH arriving at Complex I. This picture is misleading. FAD in Complex II is not free-floating. It is covalently attached to the SDHA subunit of the enzyme succinate dehydrogenase, and it gets reduced to FADH2 right there, in place, when succinate is oxidized during the citric acid cycle.¹ The electrons never leave the enzyme complex to wander around the matrix.

This matters when people try to trace where electrons from fat breakdown end up. Fatty acid oxidation does produce reduced flavin carriers, but those electrons are channeled to the same pool of mobile electron carriers (called ubiquinone, or coenzyme Q) via their own dedicated dehydrogenases, not through Complex II.¹ If you have ever been confused about how fat-derived electrons enter the chain, this common diagram error is likely the reason.

Moving Electrons Across the Mitochondrial Membrane

Here is a practical problem cells face: a lot of NADH gets produced in the cytoplasm during glucose breakdown, but the inner mitochondrial membrane will not let NADH pass through. The cell needs shuttle systems to get those electrons from the cytoplasm into the mitochondria where they can be used to make ATP.²

Two main shuttles handle this job. The malate-aspartate shuttle essentially converts cytoplasmic NADH into a small molecule (malate) that can cross the membrane, then regenerates NADH on the mitochondrial side. This shuttle preserves the full energy content of the original NADH.³ The glycerol phosphate shuttle takes a different approach: it transfers the electrons to FAD-linked enzymes on the mitochondrial membrane, feeding them into the chain at the ubiquinone level. This route is faster but yields slightly less ATP because of where the electrons enter. In some cell types, the glycerol phosphate shuttle can account for a large share of shuttle activity when glycolytic intermediates are available.⁴

Maintaining a healthy ratio of NAD+ to NADH in the cytoplasm is essential for glucose metabolism and, in the brain, for making neurotransmitters.² When these shuttles malfunction, the consequences can be severe. Inherited defects in the malate-aspartate shuttle are linked to metabolic disorders that typically present early in life.³

NADPH and the Antioxidant System

NAD has a close chemical cousin: NADP, which is NAD with an extra phosphate group. Its reduced form, NADPH, has a fundamentally different job from NADH. While NADH mainly feeds into energy-producing breakdown reactions, NADPH powers building reactions and antioxidant defense.⁵ Your cells use NADPH to synthesize fatty acids, cholesterol, and other molecules. Crucially, NADPH also keeps the antioxidant molecule glutathione in its active, reduced state, which helps neutralize the reactive oxygen species that mitochondria inevitably produce as a byproduct of energy generation.⁶

The distinction between NADH and NADPH is easy to overlook because the names look almost identical, but functionally they occupy different lanes. NADH is primarily catabolic (breaking things down for energy), while NADPH is anabolic and protective (building things up and cleaning up damage). Cells keep the two pools separate and tightly regulated.

NAD as a Signaling Molecule

One of the more surprising discoveries about NAD+ is that it does far more than carry electrons. Several families of enzymes consume NAD+ as a raw material rather than just borrowing its electrons, and these enzymes regulate some of the most important processes in the cell.

Sirtuins are a family of proteins found in organisms from bacteria to humans that remove chemical tags (acetyl groups) from other proteins, altering their activity. Sirtuins cannot work without NAD+; it is a required ingredient, not just a helper.⁷ Through this mechanism, sirtuins influence gene silencing, cell division, stress responses, and metabolism.⁸ When NAD+ levels drop, sirtuin activity drops too, which has downstream effects on how well cells handle stress and maintain their functions.

PARPs (poly-ADP-ribose polymerases) are another major class of NAD+ consumers. PARP1, the most active member, sits in the nucleus and acts as a first responder when DNA gets damaged. It uses NAD+ to build chains of ADP-ribose on proteins near the damage site, flagging the area for repair crews.⁹ The problem is that heavy DNA damage can trigger so much PARP1 activity that it rapidly drains the cell’s NAD+ supply, forcing a metabolic crisis. Researchers have observed that this NAD+ depletion triggers a cell-wide shift in metabolism as the cell scrambles to compensate.¹⁰

A third NAD+ consumer is CD38, an enzyme found on the surface and inside of many cell types. CD38 breaks down NAD+ and its phosphorylated cousin NADP+ to generate signaling molecules, including cyclic ADP-ribose, which triggers calcium release inside cells.¹¹ Since calcium signaling controls everything from muscle contraction to hormone secretion, CD38 links NAD+ metabolism to a vast network of cellular functions.¹² CD38 is expressed in most tissues and, as we will see, plays a starring role in the age-related decline of NAD+.

Why NAD Levels Drop With Age

Tissue NAD+ concentrations decline as animals age, and this decline has been linked to many hallmarks of aging, from mitochondrial dysfunction to inflammation.¹³ The causes could theoretically be reduced production of NAD+, increased consumption, or both. Researchers have now identified CD38 as a major culprit. In aged mice, CD38 protein levels increased two- to threefold across the liver, fat tissue, spleen, and skeletal muscle, while PARP1 and sirtuin levels actually decreased, ruling them out as the primary drivers of the decline.¹⁴

More recent work has traced the chain of events further. As organisms age, senescent cells (cells that have stopped dividing and started secreting inflammatory signals) accumulate in fat and liver tissue. The inflammatory molecules they release cause resident immune cells called macrophages to ramp up their expression of CD38, turning those macrophages into NAD+-consuming machines.¹⁵ This creates a feedback loop: chronic low-grade inflammation drives up CD38, which depletes NAD+, which impairs the sirtuin-mediated stress responses that might otherwise keep inflammation in check.

Boosting NAD Through Supplements and Exercise

The discovery of age-related NAD+ decline has spawned a supplement industry built around NAD+ precursors, primarily nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR). The idea is straightforward: give the body more raw materials, and it will make more NAD+. In a randomized, placebo-controlled clinical trial of healthy middle-aged adults, oral NMN supplementation at doses of 300, 600, and 900 mg per day raised blood NAD concentrations significantly compared to placebo by day 30 and day 60, with the 600 mg and 900 mg doses producing the highest levels. Participants in all three dose groups also walked farther in a six-minute walking test than those on placebo, and the supplement was well tolerated with no safety concerns up to 900 mg daily.¹⁶

Exercise appears to work through a different mechanism. Both aerobic and resistance training increase levels of the enzyme NAMPT in skeletal muscle, the rate-limiting step in the main pathway cells use to recycle NAD+. In one study, aerobic exercise boosted muscle NAMPT levels by roughly 12 percent in younger adults and about 28 percent in older adults, while resistance training raised it around 25 to 30 percent regardless of age.¹⁷ Regular exercise training is also associated with maintained muscle NAD content over time, suggesting that consistent physical activity can offset some of the age-related decline.¹⁸

These two strategies, supplements and exercise, are not mutually exclusive, but the evidence base is at different stages. Supplement trials are still relatively small and short-term, and whether raising blood NAD concentrations translates into meaningful long-term health outcomes remains an open question. Exercise, on the other hand, comes with decades of evidence for broad health benefits, even if the NAD-specific data is newer.

Where FAD Comes From and What Happens When It Is Missing

Unlike NAD, which cells can build from the amino acid tryptophan or recycle from breakdown products, FAD starts with riboflavin, also known as vitamin B2. Inside cells, riboflavin is first converted to FMN (flavin mononucleotide) by an enzyme called riboflavin kinase, and then FMN is converted to FAD by FAD synthetase.¹⁹ This means your FAD supply depends directly on your riboflavin intake. You cannot make riboflavin from scratch; it must come from food or gut bacteria.

FAD and FMN serve as essential partners for a large family of enzymes called flavoproteins, which participate in energy production, antioxidant defense, DNA repair, and protein folding, among other processes. When riboflavin metabolism is disrupted, the consequences can be widespread. Genetic defects in riboflavin transporters or FAD-synthesizing enzymes can cause conditions ranging from neuromuscular disease and anemia to cardiovascular problems and developmental abnormalities.²⁰ One well-characterized example is multiple acyl-CoA dehydrogenase deficiency, a disorder of fat metabolism caused by impaired flavoprotein function.²¹ Some patients with these genetic conditions respond to high-dose riboflavin supplementation, which helps by pushing more substrate through the compromised pathway.

NAD, Your Body Clock, and the Gut

NAD+ levels do not stay constant throughout the day. They oscillate in a circadian pattern, driven in part by rhythmic expression of NAMPT, the enzyme that recycles NAD+ through the salvage pathway.²² These daily NAD+ swings feed back into the clock itself, partly through sirtuin activity, creating a loop where metabolism and circadian rhythm continuously influence each other.²³ Disruption of this loop is being investigated as a potential contributor to cognitive decline and dementia, where both sleep disturbances and metabolic dysfunction are common.²⁴

The gut microbiome adds another layer to NAD metabolism that is often overlooked. Gut bacteria convert nicotinamide (a breakdown product of NAD+ that the body excretes into the intestine) into nicotinic acid, which the host then uses as raw material for NAD synthesis. This microbial conversion is significant enough that it maintains circulating nicotinic acid levels even when none is present in the diet.²⁵ Perhaps more surprising, the main route by which oral nicotinamide riboside (a popular supplement) actually boosts host NAD+ appears to run through the gut microbiome: bacteria convert the supplement to nicotinic acid, and that nicotinic acid then drives NAD synthesis in tissues. This finding suggests that gut health and antibiotic use could influence how effectively NAD precursor supplements work.

Seeing NAD and FAD Glow

Both NADH and FAD naturally fluoresce when hit with the right wavelength of light. NADH glows blue, and FAD glows green. Researchers have turned this quirk of physics into a powerful imaging tool. By measuring the ratio of NADH to FAD fluorescence in living cells or tissues, scientists can assess metabolic state in real time without adding any dyes or labels.²⁶ A cell that relies heavily on oxidative phosphorylation looks different under these techniques than one that relies on glycolysis.

This approach, sometimes called optical metabolic imaging, has become increasingly important in cancer research, where tumor cells famously shift their metabolic strategy. It is also being used to monitor wound healing, assess organ transplant viability, and study how stem cells differentiate. The advantage is that it is noninvasive: you can watch metabolism change in living tissue over time without destroying the sample.²⁷ The fact that NAD and FAD are not just metabolically important but also optically cooperative has opened a window into cellular biology that would otherwise require grinding up tissue and measuring chemicals in a test tube.

Ancient Molecules in Modern Cells

NAD and FAD are so fundamental to life that their origins almost certainly predate the emergence of protein-based enzymes. Laboratory experiments have shown that RNA molecules (ribozymes) can catalyze the synthesis of NAD, FAD, and coenzyme A from simpler precursors, supporting the idea that these coenzymes were already present in an “RNA world” before modern cellular life evolved.²⁸ Both molecules contain a nucleotide component (adenine linked to ribose and phosphate) that looks like it was borrowed from the RNA toolkit, which makes chemical sense: early life likely co-opted existing molecular parts for new functions rather than inventing everything from scratch.

This deep evolutionary history helps explain why NAD and FAD are so universal. They are not just shared among animals; they appear across bacteria, archaea, fungi, and plants. The basic electron-carrying chemistry that NADH and FADH2 perform is billions of years old, and life has never found a reason to replace it. Instead, organisms have layered additional roles on top: signaling, gene regulation, calcium mobilization, circadian timing. The electron-carrying function came first. Everything else was built around molecules that were already indispensable.