Sirtuins and NAD+

Name: Sirtuins and NAD+: what has been measured in people
Creator: Aaron Cuha

Also called: SIRT1, SIRT3, NAD+

Written by Aaron CuhaReviewed Sep 2026

A family of enzymes that can only work when the cell has NAD+ to spend, which ties them to how well fed the cell is. The two most quoted results backing this field did not hold up when others repeated them.

What it is

Sirtuins are seven enzymes in mammals, SIRT1 through SIRT7. They descend from a yeast protein called Sir2. Most of them strip small chemical tags (acetyl groups) off other proteins. What makes them interesting for metabolism is what they run on. Sirtuins need NAD+, a helper molecule tied to the cell's energy state. Each time one does its job, it uses up a whole NAD+ molecule. So sirtuin activity depends on how much NAD+ the cell has, and that depends on the cell's energy state.

Not all of them strip acetyl tags. SIRT4 and SIRT6 add a different kind of tag. SIRT5 removes other small tags. SIRT6 also removes long fat-based tags.

SIRT1 works in the cell's control centre and the main body of the cell. It is the closest mammal relative of Sir2. Its targets include PGC-1 alpha, the FOXO gene switches, p53, NF-kB, and the proteins DNA is wrapped around. SIRT3 is the main tag-stripper inside mitochondria, the cell's power plants. It acts on enzymes for fat burning, energy making, waste handling, ketone making, and the antioxidant enzyme SOD2.

This page is written to be honest about a field with two serious open disputes, not to present it as settled.

How it works

NAD+ and NADH are the same molecule in two forms: charged up and used. What matters is the balance between them, not the total. The catch, which popular accounts skip, is that a fast pushes that balance in opposite directions in different parts of the same cell.

Inside the mitochondria, heavy fat burning makes lots of NADH, so the balance tips toward NADH. That is exactly why the liver turns acetoacetate into BHB during a fast: there is plenty of NADH to drive that step. In the main body of the cell, less sugar is being burned, and the balance generally moves toward more NAD+.

So the sentence "fasting raises NAD+ and so switches on sirtuins" is roughly fair for SIRT1, which works in the main body of the cell and the control centre. It is much less clean for SIRT3, which works inside mitochondria, where the balance is moving the other way at the same time.

The two best-understood effects run through PGC-1 alpha and FOXO. SIRT1 strips the tag off PGC-1 alpha, and the untagged form is the active one. In the fasting liver, it drives the genes for making new sugar. In muscle, it drives genes for mitochondria and fat burning. Note that AMPK acts on the same protein in a different way, and the two changes do not simply duplicate each other. As for FOXO, when insulin is high, it gets kept out of the control centre. Falling insulin lets it in. In the systems studied, SIRT1 then steers FOXO toward helping the cell resist stress and away from cell death. FOXO also switches on autophagy genes and the genes that break down muscle, which puts it right upstream of the recycling system. BHB also raises one FOXO gene on its own, by a separate route. So a fast reaches FOXO in at least two unrelated ways.

What switches it on

More NAD+ in the main body of the cell as sugar burning slows, plus the other changes that come with low insulin. AMPK also raises the cell's NAD+, which is one way the energy sensor feeds this second sensor.

On the timing
No study in people has measured the NAD+ to NADH balance in tissue during a fast. That is the honest answer to the timing question, and it is a real gap, not a small print note. It is hard to measure for two reasons. NAD+ and NADH switch back and forth quickly while a sample is being taken. And the balances that matter, inside mitochondria versus the rest of the cell versus the control centre, cannot be separated in a ground-up tissue sample. NAD+ in whole blood or plasma does not tell you what is happening inside the liver or muscle.

What has been measured

In people

What has been measured in living people is narrow. It covers SIRT1 gene activity in blood cells on a short eating window, SIRT1 and one related target in muscle samples in the first hours of a fast, and two studies of an NAD+ building block with muscle samples. The NAD+ to NADH balance itself, in human tissue, during a fast, has never been measured.

The biggest study: 40 men with obesity and insulin resistance took nicotinamide riboside, an NAD+ building block, at 1000 mg twice a day for 12 weeks. Muscle samples were taken before and after. It did not change NAD levels in muscle. It did not change how well the mitochondria worked, how many there were, or their shape. It lowered one protein in muscle (NAMPT). This was the first long-term study in people to report on these things (Journal of Physiology, 2020).
A smaller study of the same compound disagrees on the key point. 13 overweight adults, or adults with obesity, took nicotinamide riboside at 1000 mg daily for 6 weeks. Each person also had a comparison period without it. Muscle samples showed markers of more NAD+ being made, so the compound reached the muscle and did something there. Their share of lean mass rose (62.65% against 61.32%). Energy burned during sleep rose. A fat-burning marker in muscle, acetylcarnitine, rose too. Insulin response was measured with a clamp test, and the paper's summary does not report it as improved (American Journal of Clinical Nutrition, 2020).
SIRT1 in blood cells. In a four-day study, 11 overweight adults tried two eating windows, 8am to 2pm and 8am to 8pm. Each person tried both. The early window raised SIRT1 gene activity in blood cells in the morning before breakfast, along with the autophagy gene LC3A. It also raised MTOR gene activity in the evening. That is gene activity in blood cells over four days, not enzyme activity in tissue (Nutrients, 2019).
SIRT1 in muscle during the first 8 hours of a fast, in 10 healthy men, with samples at 4 and 8 hours. The active tag on SIRT1 drifted down a little, though the change was too small to be sure of, and so did the tag on ACC. The tag on p53, one of the proteins sirtuins strip, went up during the fast. That is the opposite of what a simple "fasting switches on sirtuins" story predicts for that target (Pflugers Archiv, 2021).
Put together: two well-run studies of the same compound in people. One found NAD-related markers in muscle went up, and one found NAD levels in muscle did not change. Neither found the gains in mitochondria that animal and lab research predicted. That is where the evidence in people stands.

In animals and in cells in a dish

The basic chemistry is not in dispute. It was worked out in yeast and with purified enzymes. SIRT1's role in the fasting liver is backed by mouse work. What is in dispute is the jump from "sirtuins respond to food supply" to "raising sirtuins extends lifespan". The worm and fly evidence first used to make that jump did not hold up when repeated.

In yeast and with purified enzymes: Sir2 was shown to strip tags off DNA's packing proteins using NAD+, leaving a unique by-product (Nature, 2000). This is the finding that makes the whole family interesting for metabolism, and nobody disputes it.
In mouse liver and liver cells, and cells in a dish: SIRT1 teams up with PGC-1 alpha to control how the liver handles sugar and fat (Nature, 2005). In mice, liver SIRT1 is needed for the normal fasting response in sugar and fat (Proceedings of the National Academy of Sciences, 2007).
Dispute one, lifespan in yeast, worms and flies. The first claims were that extra Sir2 extends lifespan in yeast, in the worm C. elegans, and in fruit flies. The fly claim was framed as working through a pathway linked to eating less (Proceedings of the National Academy of Sciences, 2004). In 2011, these were checked again with close attention to the animals' genetic background. Once the background was matched and proper comparison animals were used, the effects vanished in both worms and flies. In worms, breeding out the rest of the background removed the longer life while keeping the extra Sir2. The longer life turned out to travel with a separate mutation affecting sense nerves. In flies, a strain with extra Sir2 did live longer than normal flies, as first reported, but not longer than proper comparison flies. A new line with even more Sir2 did not live longer either (Nature, 2011).
Dispute two, resveratrol. The claim that resveratrol directly switches on SIRT1 came from lab tests in a dish using a test protein with a glowing tag attached. Resveratrol's effect turned out to depend on that glowing tag. With a natural test protein and no glowing tag, the effect did not show up (Journal of Biological Chemistry, 2005). That makes it a quirk of the lab test. It applies to a whole generation of so-called sirtuin boosters found with the same test.
What holds up on lifespan: in mice, extra SIRT6 does extend lifespan. In the first report, the effect was in males only (Nature, 2012).

Why it matters

Sirtuins are the process most often used to link fasting to ageing, and the link is usually presented as settled. It is not, and the reasons are specific and checkable, not vague.

What is solid: sirtuins run on NAD+, they respond to food supply, and in mice SIRT1 plays a proven role in the liver's fasting response through PGC-1 alpha.

What is not: that raising sirtuin activity extends lifespan. The worm and fly lifespan results that were the most quoted support for that claim were rerun with proper genetic controls, and they did not hold. The most famous sirtuin-boosting compound turned out to depend on a glowing tag stuck to the lab test protein. Neither of those setbacks wiped out the field, and neither should be read as showing sirtuins do nothing. They removed the specific evidence that was doing the convincing.

And for a fasting page, the key gap is the simplest one. Nobody has measured the NAD+ to NADH balance in human tissue during a fast. The central number in the whole story has never been measured in people.

A claim you will see repeated

"Fasting raises NAD+, which switches on sirtuins, which is how fasting slows ageing." There are three problems, in order. The NAD+ part depends on where in the cell you look, and it moves in opposite directions at once during a fast. Inside mitochondria, the balance tips toward NADH, while the rest of the cell moves the other way. It has never been measured in human tissue during a fast, anywhere in the cell. The switch-on part has one muscle measurement in people in the right time window, and it went the other way. The SIRT1 tag drifted slightly down, and the p53 tag went up. The lifespan part rests on worm and fly results that did not survive proper comparison animals. It also rests on a class of booster compounds found with a lab test whose result depended on a glowing tag stuck to the test protein.

Sources

Study in people2019
Early Time-Restricted Feeding Improves 24-Hour Glucose Levels and Affects Markers of the Circadian Clock, Aging, and Autophagy in Humans
Nutrients
11 overweight adults each tried two eating windows for 4 days: 8am to 2pm and 8am to 8pm. The early window raised SIRT1 and an autophagy gene (LC3A) in blood cells before breakfast. It also raised the MTOR gene in the evening. Average blood sugar over 24 hours was 4 mg/dL lower.
11 overweight adults, 4-day randomised crossover of an 8am to 2pm eating window versus 8am to 8pm. The early window raised whole blood cell expression of SIRT1 and of the autophagy gene LC3A before breakfast (p < 0.04), raised MTOR expression in the evening (p = 0.007), and lowered mean 24-hour glucose by 4 mg/dL.
Study in people2021
Increasing whole-body energetic stress does not augment fasting-induced changes in human skeletal muscle
Pflugers Archiv, European Journal of Physiology
10 healthy men gave thigh muscle samples at the start, 4 hours and 8 hours of a supervised fast. A SIRT1 signal dipped slightly, but not clearly. Another energy signal (ACC) fell. A tag on the protein p53 rose over the fast, in both test conditions.
10 healthy males, vastus lateralis biopsies at baseline, 4 and 8 hours of a supervised fast. SIRT1 phosphorylation was non-significantly decreased (P < 0.06) alongside decreased ACC phosphorylation, while p53 acetylation increased over the fast in both conditions (P < 0.01).
Study in people2020
Nicotinamide riboside supplementation alters body composition and skeletal muscle acetylcarnitine concentrations in healthy obese humans
American Journal of Clinical Nutrition
13 overweight or obese adults took 1,000 mg of nicotinamide riboside a day for 6 weeks, and also spent a period without it. Muscle samples showed signs of more NAD+ being made. Lean mass went from 61.32% to 62.65% of the body, and calories burned during sleep went up. A fat-burning marker in muscle rose. Insulin response was tested, but the summary does not report it as better.
13 overweight or obese adults, crossover, 6 weeks at 1000 mg nicotinamide riboside daily, with muscle biopsies. Markers of increased NAD+ synthesis, nicotinic acid adenine dinucleotide and methylnicotinamide, were elevated in skeletal muscle. Fat free mass increased (62.65 versus 61.32%, change 1.34%, P = 0.02), sleeping metabolic rate increased, and skeletal muscle acetylcarnitine increased (4558 versus 3025 pmol per mg dry weight, P = 0.04). Insulin sensitivity by clamp was among the outcomes and is not reported as improved in the abstract.
Study in people2020
Nicotinamide riboside does not alter mitochondrial respiration, content or morphology in skeletal muscle from obese and insulin-resistant men
Journal of Physiology
40 obese men with insulin resistance took 1,000 mg of nicotinamide riboside twice a day for 12 weeks. Muscle samples were taken before and after. NAD levels in muscle did not change. Power-plant function, amount and shape in muscle did not change either. One enzyme that makes NAD, called NAMPT, went down. It was the first long human trial to report these results.
40 obese insulin-resistant men, 12 weeks at 1000 mg nicotinamide riboside twice daily, muscle biopsies before and after. Nicotinamide riboside did not affect NAD metabolite concentrations in skeletal muscle. Mitochondrial respiration by high resolution respirometry on single fibres, mitochondrial content and mitochondrial morphology were all unaffected. NAMPT protein abundance in muscle decreased. The first long-term human clinical trial to report these outcomes.
Lab (cells in a dish)2000
Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase
Nature
In yeast and in a dish. The yeast longevity protein Sir2 strips tags off DNA-packaging proteins, and it uses up NAD+ each time. That tie to NAD+ supply is why this protein family draws so much interest, and it is not in dispute.
Yeast and purified enzymes. Sir2 established as an NAD+ dependent histone deacetylase, with the reaction consuming NAD+ stoichiometrically and producing nicotinamide plus 2'-O-acetyl-ADP-ribose. This coupling of enzyme activity to NAD+ availability is what makes the family metabolically interesting, and it is not in dispute.
Animal study2005
Nutrient control of glucose homeostasis through a complex of PGC-1alpha and SIRT1
Nature
In mice and in a dish. SIRT1 pairs up with PGC-1 alpha in the liver. Together they control how the liver handles sugar and fat during fasting. PGC-1 alpha is active once SIRT1 has removed its tags.
Mouse liver, hepatocytes and cell culture. SIRT1 forms a complex with PGC-1 alpha, and this controls the hepatic glucose and lipid response to fasting. Deacetylated PGC-1 alpha is the transcriptionally active form.
Animal study2007
Fasting-dependent glucose and lipid metabolic response through hepatic sirtuin 1
Proceedings of the National Academy of Sciences
In mice. Mice missing liver SIRT1 showed it is needed for the liver's normal sugar and fat response to fasting.
Mouse loss-of-function work confirming that hepatic SIRT1 is required for the fasting-dependent glucose and lipid metabolic response.
Animal study2004
Sir2 mediates longevity in the fly through a pathway related to calorie restriction
Proceedings of the National Academy of Sciences
In fruit flies. This is the original claim that extra Sir2 makes flies live longer, through a path linked to eating less. The result did not hold up when retested with proper comparison flies.
Drosophila. The original claim that Sir2 overexpression extends lifespan through a pathway related to calorie restriction. This result did not survive re-examination with appropriate transgenic controls.
Animal study2011
Absence of effects of Sir2 overexpression on lifespan in C. elegans and Drosophila
Nature
In worms and fruit flies. When researchers matched genetic backgrounds and used proper comparison strains, the longer lives from extra Sir2 went away. In worms, the long life came from a separate gene change affecting sensing nerves. In flies, the Sir2 strain lived longer than normal flies but not longer than proper comparison flies, and a stronger Sir2 strain did not live longer.
Standardising genetic background and using appropriate transgenic controls abolished the apparent lifespan effects in both C. elegans and Drosophila. In the worm, outcrossing a high-overexpression sir-2.1 line removed the longevity increase while leaving overexpression intact, and the longevity co-segregated with a second-site mutation affecting sensory neurons. In the fly, the overexpressing strain was long-lived versus wild type but not versus appropriate transgenic controls, and a more strongly overexpressing line was not long-lived either.
Lab (cells in a dish)2005
Substrate-specific activation of sirtuins by resveratrol
Journal of Biological Chemistry
In a dish. Resveratrol seemed to switch on sirtuins only because of a glowing tag stuck on the test protein. With a natural test protein without the tag, the effect did not show up.
In vitro enzyme assays with tagged and untagged peptide substrates. Sirtuin activation by resveratrol is substrate specific and dependent on the covalently attached fluorophore in the assay peptide; with a native substrate lacking the fluorophore, the activation was not observed.
Animal study2012
The sirtuin SIRT6 regulates lifespan in male mice
Nature
In mice. Extra SIRT6 made mice live longer. In the first report, this only worked in males.
SIRT6 overexpression extends lifespan in mice, and in the original report the effect was male specific.