Niacin muscle NAD+

This post started out exploring an NAD⁺ boosting niacin clinical trial. The thought that if some other group had shown that niacin increases NAD⁺ we don’t need to prove it. Key enzymes in the NAD salvage pathway play a role in a progressive external opthalmoplegia (PEO) as shown by a niacin clinical trial. Then a second request came in to explore telomeres and the role NAD⁺ has in controlling telomere length.

1. What is PEO and the niacin link?
   1. from ncbi conditions
   2. from the Pirinen 2020 publication
2. NAD+ and telomeres
   1. What are telomeres and telomerase?
   2. A mammalian telomere, NAD+ comes in here
   3. Back to yeast telomeres and NAD+ salvage
3. yeast telomere references

What is PEO and the niacin link?

Pirinen E, Auranen M, Khan NA, Brilhante V, Urho N, Pessia A, Hakkarainen A, Kuula J, Heinonen U, Schmidt MS, Haimilahti K, Piirilä P, Lundbom N, Taskinen MR, Brenner C, Velagapudi V, Pietiläinen KH, Suomalainen A. Niacin Cures Systemic NAD⁺ Deficiency and Improves Muscle Performance in Adult-Onset Mitochondrial Myopathy. Cell Metab. 2020 Jun 2;31(6):1078-1090.e5. PMC free article

The following are some definitions to consider before reviewing a clinical trial to improve this condition with niacin supplementation.

from ncbi conditions

“Progressive external ophthalmoplegia is a condition characterized by weakness of the eye muscles. The condition typically appears in adults between ages 18 and 40 and slowly worsens over time. The first sign of progressive external ophthalmoplegia is typically drooping eyelids (ptosis), which can affect one or both eyelids. As ptosis worsens, affected individuals may use the forehead muscles to try to lift the eyelids, or they may lift up their chin in order to see. Another characteristic feature of progressive external ophthalmoplegia is weakness or paralysis of the muscles that move the eye (ophthalmoplegia). Affected individuals have to turn their head to see in different directions, especially as the ophthalmoplegia worsens. People with progressive external ophthalmoplegia may also have general weakness of the muscles used for movement (myopathy), particularly those in the neck, arms, or legs. The weakness may be especially noticeable during exercise (exercise intolerance). Muscle weakness may also cause difficulty swallowing (dysphagia). When the muscle cells of affected individuals are stained and viewed under a microscope, these cells usually appear abnormal. These abnormal muscle cells contain an excess of cell structures called mitochondria and are known as ragged-red fibers. ”

from the Pirinen 2020 publication

Progressive External Ophthalmoplegia is generalized muscle weakness, and susceptibility to fatigue is often caused by single or multiple mitochondrial DNA (mtDNA) deletions.
A mouse model for some progressive mitochondrial myopathy manifests as NAD⁺ depletion. Intracellular NAD⁺ concentrations can be increased by various approaches, such as decreasing NAD⁺ consumption and NAD⁺ precursor supplementation: nicotinic acid (niacin), nicotinamide (NAM), and nicotinamide riboside (NR). The simplest approach would be to supplement with niacin and also examine lipid metabolic parameters that have been shown to occur with niacin supplementation and that might relate to NAD⁺

The design

This study came out of the University of Helsinki, Finland. These researchers at the University of Helsinki report that mitochondrial muscle disease leads to low NAD⁺ levels in both blood and muscle. They treatment treated niacin, a vitamin B3 form and an NAD⁺ precursor, improves NAD⁺ levels, disease signs, and muscle metabolism in patients, also improving muscle strength and performance. These results indicate that NAD⁺ depletion occurs in human diseases, and its repletion is a potential therapy for mitochondrial myopathies. Patients were given escalating doses of administered an increasing doses of the NAD⁺-booster niacin form (to 750-1,000 mg/day.

Arm	Intervention/treatment
Experimental: Niacin in controls The arm includes healthy controls supplemented with niacin.	Dietary Supplement: Niacin The dose for a slow-released form of niacin will be 750-1000 mg/day. The daily niacin dose, 250 mg/day, is gradually escalated by 250 mg/month so that the full dose is reached after 3 months. The intervention time with the full niacin dose is 1 and 7 months for controls and patients, respectively, and subsequently total intervention time 4 and 10 months, respectively. At the end of the study, the daily dose will be decreased by 250 mg/month rate. Other Name: Nicotinic acid
Experimental: Niacin in mitochondrial myopathy patients The arm includes mitochondrial myopathy patients supplemented with niacin.	Dietary Supplement: Niacin The dose for a slow-released form of niacin will be 750-1000 mg/day. The daily niacin dose, 250 mg/day, is gradually escalated by 250 mg/month so that the full dose is reached after 3 months. The intervention time with the full niacin dose is 1 and 7 months for controls and patients, respectively, and subsequently total intervention time 4 and 10 months, respectively. At the end of the study, the daily dose will be decreased by 250 mg/month rate. Other Name: Nicotinic acid

Fig. 1 NAD⁺ chemical family changes

As a point of reference, the CopperOne clinical trial participants took 12.12mg cuprous niacin per day for 28 weeks. About 9 mg of this was niacin. The PEO clinical trial used much higher amounts of niacin.

Note that the PEO patients have lower muscle and whole blood NAD⁺ levels than the normal controls. Niacin supplementation increases the NAD⁺ in whole blood but not the muscle in the same controls. This panel is from the supplemental data section. It shows the enzymatic pathways between niacin (NA), NAD⁺, and other related small molecules. Are PEO patients the way they are because of enzyme deficiencies in these pathways?

Note that sirtuins are some of the NAD⁺ consuming enzymes. Nicotinamide riboside is not that different between PEO patients and controls. NADP and NMN are a little lower in PEO patients than the controls even after supplementation with large amounts of niacin. It is the NAM and ADPR that are much higher in the PEO patients even after supplementation.

NNMT catalyzes the reaction

S-adenosyl-L-methionine + nicotinamide ⇌ S-adenosyl-L-homocysteine + 1-methylnicotinamide.

These data are from supplemental figures of the publication. The authors were looking at gene transcripts for enzymes for proteins in NAD pathways. Note that NNMT enzyme activity is much higher in the PEO patients than the healthy controls. Not surprisingly, muscle NAM is much lower at baseline and at 4 months. Ten months of niacin supplementation seems to rectify the disparity.

Fig. 2 Lipid metabolism parameters

We became interested in the relationship between niacin and copper in fat metabolism and milk production when asked if our supplement would boost milk production in cows. See the fatty liver in cows post. While body weight was not affected, the authors saw variable decreases in fat stores. The reduction in liver fat in the PEO patients is probably most notable.

While increased muscle mass in normal and PEO patients may not be the same thing as milk production, we are extremely excited by this finding in humans. Adiponectin is a peptide hormone secreted by adipocytes. It controls fatty acid and glucose metabolism in many tissues. The energy expenditure is in units of percent of control.

Fig 3. Physical Function

Data in panels 3B- F were collected on a Good strength Metitur adjustable dynamometer chair. This is a Finnish brand. This chair measured static strength of back muscles, and dynamic strength of abdominal and shoulder muscles. FC stands for fraction of control.

Note the high variability in the PEO patients’ response to niacin. In many cases the supplemented PEO patients performed better than the controls!

The six minute walking test speaks more to the mitochondrial function that targets our customer base. As a reminder, a cartoon of pyruvate entering the NADH generating TCA cycle is shown. NADH supplies the ATP generating electron transport chain (ETC) of the mitochondria with electrons and H⁺. How much does the availability of NAD⁺/NADH + H⁺ dictate whether pyruvate goes to acetylCoA or lactate?

Note the drastic improvement in the six minute walk test in the PEO patients and the variable response in the control patients.

Fig. 4 the mitochondria

Cytochrome c oxidase (COX), respiratorycomplex IV of the electron transport chain, has some subunits encoded by mitochondrial DNA. Succinate dehydrogenase (SDH) complex II of the ETC is entirely encoded by nuclear genes. In PEO patients niacin decreased the fibers that were negative for COX yet positive for SDH. Fibers that were negative for complex I (CI) were also decreased. Mitochondrial total COX activity and mass in muscle fibers increased. Mitochondrial mass was also increased in both controls and PEO patients.

FC is fraction of control. In panels C and D we see that four months on niacin increases Cox activity and mitochondrial mass. We have to ask ourselves if increases like these can be seen with less “medicinal” levels of niacin and Cu(I).

No further comments will be presented on these SEM images on this post.

Fig 5. Metabolites

This post is going to skip the Principle Component Analysis of panel 5A. In short the authors were trying to establish which metabolites separate the patients before and after and the controls before treatment. This post will present Panel 5E which summarizes the methyl transferring pathways that predominate the data

This post is going to skip presenting Panel 5D that presented amino acid metabolite changes that tended to be around 2x the baseline values. We may need to come back to it when there’s a better understanding of the salvage pathways in yeast and humans in telomeres.

Fig. 6 Pathways

The reviewing this section note will be made when the pathways intersect that of copper and copper cofactor enzymes

This will be an awkward transition from the salvage pathway, and niacin supplementation to the salvage pathway in telomeres in yeast. Three references were found from the JS Smith Lab that increased my understanding of the role of niacin in telomeres.

NAD⁺ and telomeres

The Sir2 enzyme is an NAD-dependent protein deacetylase that is required for turning off the silent mating-type loci, telomeres, and the ribosomal DNA (rDNA). Mutations in the NAD salvage gene NPT1 weaken all three forms of silencing and also cause a reduction in the intracellular NAD level. [1]

What are telomeres and telomerase?

Telomerase is a ribonucleoprotein that adds a species-specific telomere repeating sequences to the 3′ end of telomeres. A telomere is a region of repetitive sequences at each end of the chromosomes of most eukaryotes that function to prevent fusion with other chromosomes and DNA damage.

In higher eukaryotes, telomere length plays a major role in regulating cellular senescence. This is especially true in primary fibroblasts that do not express telomerase. Telomeres become shorter during each cell division. The Hayflick limit is reached when telomeres become short enough to senesce.

Yeast telomerase is constantly expressed with no progressive shortening in mitotic daughter cells. Mutations in telemerase can mimic mammalian aging with progressive shortening of telomeres. Telomerase deficient est2Δ rad52Δ mutants can’t repair their telomeres. Deleting SIR2 further accelerates senescence within the population. [3]

In yeast the lack of Sir2 results loss of telomeric silencing, which increases the transcription of non-coding TElomeric Repeat containing RNAs (TERRA) Wikipedia has an excellent page on these controllers of telomerase activity. Telomeric silencing refers to the observation that protein coding genes located close to telomers tend to be transcribed less.

A mammalian telomere, NAD⁺ comes in here

1. Rap1p binds to the telomeric repeats.
2. Sir3p/Sir4p/Sir2p complex is recruited to Rap1p. Enter NAD⁺
3. Sir2p deacetylates histones in the adjacent nucleosome.
4. Deacetylated histones recruit more Sir3p/Sir4p/Sir2p complex. Enter NAD⁺
5. The cascade of deacetylation spreads away from the telomeres.
6. Spreading of deacetylation is countered by Histone Acetyl Transferases (HATs)

Back to yeast telomeres and NAD⁺ salvage

Transcriptional repression can be highly localized and transient, such as at the promoters of specific genes, or more widespread across large regions of the genome that remain in a repressive and condensed state for extended periods. These latter domains tend to be heterochromatic and stable, sometimes even through multiple generations. In budding yeast, HML, HMR, and telomeres are generally considered to be the heterochromatin equivalents in this organism. [3]

The authors discussed nutrient sensing ensembles of proteins that also control aging. These include sensors of mitochondrial function that is not just NAD⁺ production but also the ADP to ATP ratio. This is where we think that Cu⁺ and mitochondrial cytochrome C oxidase may synergize with agents that boost NAD⁺.

yeast telomere references

1. Sandmeier JJ, Celic I, Boeke JD, Smith JS. Telomeric and rDNA silencing in Saccharomyces cerevisiae are dependent on a nuclear NAD(+) salvage pathway. Genetics. 2002 Mar;160(3):877-89. PMC free article
2. Gallo CM, Smith DL Jr, Smith JS. Nicotinamide clearance by Pnc1 directly regulates Sir2-mediated silencing and longevity. Mol Cell Biol. 2004 Feb;24(3):1301-12. PMC free article
3. Wierman MB, Smith JS. Yeast sirtuins and the regulation of aging. FEMS Yeast Res. 2014 Feb;14(1):73-88. PMC free article