Rethinking NAD and Sirtuins: How Flawed Assumptions Reinforce Good Research Practices

In 2022, Dr. Charles Brenner brought to light a somewhat obscured narrative in academia through a review article published in Life Metabolism. His paper scrutinizes the research community's leanings toward the notion of sirtuins as longevity drugs. While this issue primarily impacts aging biologists, Brenner's skepticism of broadly accepted "facts" offers essential guidance to readers, fostering more informed consumption of longevity research.

Science and discovery are typically slow and painstaking processes, particularly when the goal is to produce factual results. The human tendency to seek swift solutions, especially in confronting mortality and disease, can lead to hasty conclusions. One example of this phenomenon was the pervasive belief in sirtuins as conserved longevity drugs—a viewpoint that has resulted in the considerable expenditure of time and resources. It's a cautionary tale for the scientific community—reminding us that the pursuit of knowledge should prioritize accuracy over expediency.

Sirtuins are a class of proteins recognized for their role in removing modifications from specific protein sequences, causing a cascade of events that usually lead to changes in certain genes being more or less expressed at any given time.

From the perspective of a longevity researcher, sirtuins are often seen as potential "longevity proteins." They are considered pivotal regulators of lifespan and healthspan through their involvement in fundamental biological pathways, and their activity can be influenced by diet and other lifestyle factors.

Firstly, sirtuins are thought to increase the stability and health of cells by improving DNA repair and reducing DNA damage, which can lead to aging and age-related diseases. By doing so, they help to maintain the integrity of the genome, a key factor in cellular health and longevity.

Secondly, sirtuins are thought to be involved in the regulation of metabolism, particularly in response to caloric restriction, a known lifespan-extending intervention. They are thought to mediate the beneficial effects of caloric restriction on health and longevity.

Finally, sirtuins are thought to play a role in inflammation, a key factor in many age-related diseases. They can help to reduce inflammation, thereby improving health and potentially extending lifespan.

This family of enzymes has provided these functions throughout evolution, meaning that with astounding similarity from yeast to worms, flies, and humans, these proteins exist and perform similar functions.

To be clear, that does not mean these exact proteins act in the exact same context in every organism, but their base functions as gene-regulating machines are preserved across species.

Sirtuins depend on NAD to carry out their cellular functions. NAD, a vital metabolite, has been identified as a potentially beneficial supplement in various facets of human health and aging, although the precise details remain unclear.

Brenner, along with other researchers in the field, often employs well-tested and reproducible phenomena as benchmarks for the interventions they plan to implement. For instance, similar to the uniform enzymatic activity of sirtuins across various species, organisms under caloric restriction (CR) exhibit extended lifespans is one of these benchmarks.

If any eukaryotic organism—from yeast, worms, and flies to mice, rats, and monkeys—is put on a minimalist diet, most vital organs show quantifiable signs of "youthfulness."Using this reproducible phenomenon as a benchmark allows scientists to say that any intervention—a molecular compound they are testing, gene alteration, or behavioral change variable—is just as good or better than CR.

Another reproducible measuring stick is NAD supplementation. NAD supplements have garnered much interest in the past few decades and continue to be a robust general anti-aging strategy in mice. Brenner himself is the Principal Investigator on studies related to NAD and its applications to human health.

In 2016 his group published a paper documenting the crucial finding that supplements of this ilk increase the bioavailability of these molecules in humans. This may seem like a widely impactful finding, but the basis for this conclusion is riddled with many flawed assumptions—including how the results of this research apply to humans.

In proving efficacy in humans, this is the first hurdle—if a human were to consume a supplement, would it get absorbed in their bodies in a meaningful way?

Regarding NAD, the original assumption that Brenner identifies deals with Sirtuin 2, which was thought to be a huge reason why NAD supplements were so beneficial. More available NAD was thought to mean that the sirtuins were 'super active' and doing relatively unknown things that promoted longevity—the more NAD, the healthier and more active sirtuins were believed to be.

In terms of the initial experiment in yeast, the specifics of the initial experiments are thoroughly outlined in Brenner's review. I recommend you read Brenner's rendition for in-depth details about the flaws in experimental design and logical conclusions gleaned from the results.

The summary I will give here is that in some respects, increasing the expression of Sirtuin 2 prolonged the replicative ability of yeast similarly to caloric restriction, the widely accepted "positive control" that extends lifespan in all model organisms—it met the benchmark criteria. . However, this set of experiments could have been more robustly replicable. They were not replicable when they were tested in worms, flies, and mice. Recognizing this, we would think the initial discovery would have been more closely examined and that the pitfalls of the experiment would have come to light before anyone spent a huge amount of money following a rabbit hole that leads nowhere, right?

While we would have expected new data to change wildly held views on the role of sirtuins, that did not happen. Unfortunately, if a paper promising a pro-longevity target is published in a high-impact journal (especially early on in the aging field), people will likely accept it, cite it, and mask reasons why their incredibly expensive experiments won't replicate it. Moreover, to go against it would be suicidal to one's scientific career. So, they accepted it, warts and all.

This awful merry-go-round involving Sirtuins went on, and inconceivable amounts of money were spent (Brenner claims billions) to make these inherently flawed things make sense. Brenner does state that sirtuins are still an incredibly important enzyme, likely with large implications for certain aspects of human health, but he does not let this assuage him from outlining in great detail how the science became separated from the "hype."

The Importance of Control Groups

In his argument, Brenner asserts that certain foundational tenets are required to perform good science. Any good experimental design needs to have the proper controls. Controls are extra arms of experiments to help accurately attribute the change you are seeing to whatever intervention you are testing. Without appropriate controls, you may see a change occurring and erroneously conclude that the compound you added is the reason why.

Therefore, the more variables you introduce, the more controls you need to introduce. This creates an incredibly complex experimental design and is a major reason why science takes so long. You need to do an experiment, no matter how complicated, and add enough arms to control for all the possible things that could cause an unintended change and do it with enough numbers (known as n) to get a satisfying (statistically significant) result.

Herein lies the beauty of model organisms. A model organism is a species that has been extensively studied to understand particular biological phenomena, with the expectation that discoveries made in the model organism will provide insight into the workings of other organisms.

Model organisms are in vivo models and are widely used to research human disease when human experimentation would be unfeasible or unethical. This strategy is made possible by the common descent of all living organisms, and the conservation of metabolic and developmental pathways and genetic material throughout evolution.

We know that yeast, worms, and flies share a striking amount of homology with humans. Homology refers to shared ancestry between a pair of structures, or genes, in different species. Thus, these organisms have numerous genes and structures similar to humans due to common evolutionary origins.

We also know that the organisms used in the Sirtuin studies do not live nearly as long as humans. These model organisms also come with powerful tools to make drastic changes in a controlled population large enough to make meaningful conclusions about biology. We can, for example, tightly control their environment, diet, lifestyle, and genes in studies using model organisms like a fly. We can begin their treatment late in life or start it before they are born—this is not possible in humans.

Instead of aging humans in a box for 65 years before starting an intervention thought to reduce the risk of neurodegeneration, we can house even more worms for ~10 days or flies for ~20 days and start the treatment. Mice and other rodents are an interesting middle ground because their homology is even more similar to humans. However, with an average lifespan of 2 years, these experiments still take a long time to complete.

That is why this level of control is crucial, and we cannot waste time or resources on discoveries that are not significant. We have the tools for this level of control, so we are responsible for designing experiments with any possible caveats or confounding variables in mind. If it is not reproducible in the lab, where we can control for everything, it is unlikely to be true in the real world.

Brenner delves into an experiment where the researchers engineered a fruit fly to deliberately alter its genome, specifically to enhance the expression of Sirtuin2. They reported a lifespan extension in comparison to a normal fruit fly. However, a critical detail was overlooked. No significant change was observed when comparing the lifespan of the genetically modified fly to an identical one, but without any genomic alteration. This finding suggests that the introduction of gene-altering machinery, rather than the increased Sirtuin expression, led to the lifespan extension beyond that of a standard fruit fly. A thoroughly controlled experiment, where identical flies with and without a gene edit are compared, would have identified this discrepancy.

These experiments, despite their erroneous conclusions, often gain traction due to their promising proposals for robust lifespan extension, resulting in significant "hype." Brenner's usage of the term "hype" adeptly encompasses the broad attention these findings attract from a diverse audience. As a consequence, this hype—originating from inadequately analyzed or controlled experiments—may inadvertently guide other researchers into comparable logical pitfalls, further perpetuating the prevailing dogma.

Regrettably, it's commonplace for data to be manipulated to align with an accepted narrative or an optimistic hypothesis. This practice triggers a cycle of citing flawed data and subsequently publishing even more unreliable findings. The most glaring version of this is the conclusions about the longevity compound resveratrol.

Resveratrol is a natural component of red wine and had been previously studied before its tie to the fabled family of proteins. However, it really came into the limelight when attempting to explain "The French Paradox".The French Paradox is an interesting observation that the French have very low incidence of coronary disease, despite a diet high in saturated fats. Naturally, speculation arose that their above-average consumption of good quality red wine could be a factor.

Designing a controlled experiment to test this hypothesis would be challenging. Regardless, the idea was captivating enough to spark hype and subsequent scientific exploration of resveratrol's relationship with sirtuins.

As the narrative unfolds, it is reported that resveratrol increased yeast lifespan in a specific assay measuring yeast longevity and activated Sirt1 in a manner similar to caloric restriction—meeting our benchmark criteria. Conducting biochemical interaction studies is a highly complex endeavor. Asserting that molecule' x' interacts with protein 'y' to produce effect 'z' demands unequivocal evidence—an element conspicuously missing from this study. Brenner refers to this oversight as a "clear failure of peer review."

To illustrate the hype that surrounded this discovery, Brenner included several New York Times headlines. Seeing them compiled as a list truly emphasizes their collective impact:

• "Fighting the effects of fat: Pass the pinot (Nov 1, 2006)"

• "Yes, Red Wine Holds Answer. Check Dosage (Nov 2, 2006)"

• "Aging Drugs: Hardest Test Is Still Ahead (Nov 7, 2006)"

• "An Age-Defying Quest (Red Wine Included) (Jul 8, 2007)"

• "Glaxo to buy Sirtris, Maker of a Drug Based on Red Wine (Apr 8, 2008)"

• "Hoping Two Drugs Carry a Side Effect: Longer Life (Jul 22, 2008)"

• "Quest for a Long Life Gains Scientific Respect (Sep 28, 2009)."

Revisiting Sirtuins

To round out his review, Brenner puts forth similar effort into breaking down the papers and data that publish positive longevity yields about the family of sirtuins. The research he mentions clearly has issues and certainly does not move the needle on sirtuins as the holy grail of longevity targets (especially in reference to caloric restriction and NAD supplementation), but they do point to the sirtuins being incredibly important molecular regulators with targets in many different contexts.

These contexts become muddy very quickly, and studying the nuance of sirtuin targets is not trivial. Often when attempting to publish a story that you've already spent a huge amount of money on, you're willing to handwave some things that shouldn't be glossed over. For all of these reasons, Brenner rejects the paradigm put forth by his peer sirtuin researchers and claims, instead, that hype and mediocre science took the sirtuins much farther than they had any business going.

As I mentioned in the beginning, the scientific content of this review was relevant to reassert robust science standards, especially for people looking to read primary literature and understand why studying human biology can be so complicated. Brenner makes a perfectly good case to reject the titular findings that sirtuins are conserved longevity genes but also digs into the failings of many highly cited publications that led to their fame in the first place. In doing so, he provides a guidebook for experimental design, unbiased conclusion drawing, and logical thought. Practicing good science is slow, deliberate, and expensive, but avoiding the pitfalls outlined here is vital to ensure that the statements you publish are robust because it is far more expensive and time-consuming to follow false leads.