Leucine and niacin interactions

Leucine and niacin/ nicotinic acid (NA) seem to interact with each other to promote fat metabolism. B6 might also have synergistic interaction with leucine. The leucine/NA combination is also found in dietary supplements marketed by NuBio, which initiated this exploration that led us to NuSirt exploration.

NuBio to NuTert

A request was made to check out a NuBioAge product, [1] This supplement contains four ingredients:

Ingredients

1 Sytrinol® 300mg a mixture of extracts from palm and citrus fruit.
   + Nobiletin (citrus flavenoid) : Tangerentin (specific methylated flavone) 270mg
   + Tocotrienols 30mg, part of the vitamin E family
2 Leucine 1,100mg
3 Nicotinic Acid 75mg

NuSirt, the origin of mixing leucine and nicotinic acid

The idea of mixing leucine and B vitamins came from a company called NuSirt.com. HealthJade’s post on leucine is pretty good, especially pertaining to its role in weight loss. The post is well referenced from the peer reviewed literature. .

LNA, Leucine-nicotinic acid and pyridoxal [2]

The Zemel 2020 review discussed aging and calorie restriction involving Sirt activation.

B3, aka nicotinic acid

The review [2] covers clinical trials and what appears to be basic biochemistry

The authors demonstrated that the he branched chain amino acid leucine, and its metabolites b-hydroxy-b-methylbutyrate (HMB) and a-ketoisocaproate, are direct activators of Sirt1. [2] Leucine reduces the concentration of NAD⁺ for half maximal binding by >50% (Fig. 2) such that non fasting levels of NAD⁺. Optimal activation of Sirt1 occurs when leucine is increased from fasting levels of *0.10–0.15 mM to a concentration of 0.50 mM achieved with doses of 1–1.5 g leucine.. This plasma level is less than what would come from a high protein meal. Concomitant mTOR activation that may otherwise occur at very high leucine levels (>1 mM) is avoided.

B6 aka pyridoxal

This section is somewhat of an aside to the main theme of this post. A series of trials was conducted with obese individuals who consumed leucine (1.1 g twice daily) with 15 mg pyridoxine (vitamin B6) twice daily The first was conducted in the absence of energy restriction. Body fat assessed by dual-energy X-ray absorptiometry (Fig. 3). No weight loss was observed in this group. Participants on the leucine-B6 supplement AND the calorie restricted diet lost 82% more weight (Fig. 4) and twice the fat (Fig. 5) These changes were accompanied by a reduction in the waist circumference.
These effects were accompanied by significant changes in fat content and overall weight.

This paragraph links to specific images in the semi-public access Zemel [2] publication. Images are not shown to avoid potential copy right infringements.

• FIG. 3. Effects of Leucine-Pyr versus placebo on fat loss in obese subjects subjected to weight maintenance conditions. No decrease in the control group. There were significant decreases at 12 and 24 weeks.
• FIG. 4. Effects of Leucine-Pyr versus placebo on weight loss in obese subjects subjected to a hypocaloric (-500 kcal) diet. The control group experienced weight loss as well but not as much as tLeu-Pyr group.
• FIG. 5. Effects of Leucine-Pyr versus placebo on fat loss in obese subjects subjected to a hypocaloric (-500 kcal) diet (all panels, n = 24) The Lue-Pyr group experience a larger change in the percent fat.

Leucine enhances nicotinic acid via the AMPK/Sirt axis [3]

This earlier study from the NuSirt group focused only on nicotininc acid (NA) and leucine interactions. This particular study had three arms:

1. human cell lines: myocyte, hepatocyte, and adipoyctes,
2. C elegans worms
3. mice

The mice, in groups of 12, were on these diets:

• a Western diet (WD) alone
• WD and 24 g of leucine/kg diet
• WD and 24 g of leucine/kg diet and 50 mg nicotinic acid/kg diet
• WD and 24 g of leucine/kg diet and 250 mg nicotinic acid/kg diet
• WD and 1000 mg nicotinic acid/kg diet.

1. muscle and fat cells, AMPK and Sirt

Phosphorylated AMPK is activated AMPK. We are starting to see evidence that leucine and NA act energetically. Sirt1 activity was measured in fluorescence units (FU).

Fig 1 Looking a muscle and fat cells in response to NA-LEU

NA-Leu combination increases AMPK and Sirt1 activity in muscle cells and fat cells. Differentiated 3T3L1 adipocytes and C2C12 muscle cells were treated with indicated treatments for 4 or 24 h. Protein expression of Phospho-AMPK and total AMPK (A) and Sirt1 activity in 3T3L1 and Sirt1 protein expression in C2C12 (B) were measured, as described under Methods. Quantitative data and representative blots are shown. Data are represented as mean ± SEM (n=3 to 6).

Fig 1. NA-Leu combination increases AMPK and Sirt1 activity in muscle cells and fat cells. Differentiated 3T3L1 adipocytes and C2C12 muscle cells were treated with indicated treatments for 4 or 24 h. Protein expression of Phospho-AMPK and total AMPK (A) and Sirt1 activity in 3T3L1 and Sirt1 protein expression in C2C12 (B) were measured, as described under Methods. Quantitative data and representative blots are shown. Data are represented as mean ± SEM (n=3 to 6). NA and Leu work better in combination.

Figure 2 explored the increase in oxygen consumption in response to palmitate, a fatty acid in mucle and fat cells since this event is down stream of Sirt1 signalling. The combination of leucine and NA increased consumption and presumably fatty acid oxidation too.

Fig 2. Leu-NA stimulation of mitochondria in liver and muscle cells

Mitochondria, complex IV specifically, consume oxygen. The oxygen consumption rate was measured in a Seahorse repirometer. [3] These results are consistent with a leucine stimulation of Sirt1 in a follow up study from a different laboratory. [4] AUC, area under the curve, is a measure of time dependent cumulative mitochondria activity.

See Fig 6: Proposed mechanism of leucine-induced mitochondrial biogenesis in myotubules as outlined in Liang 2014 [4].

1. Leucine treatment leads to activation of Sirt1.
2. Sirt1 deacetylates and activates LKB1, a protein kinase.
3. LKB1 phosphorylates AMPK thereby activating it.
4. Activated AMPK promotes SIRT1 activation via intracellular NAD⁺ level by increasing expression and activity of Nampt, a key enzyme in the biosynthesis of NAD⁺.
5. Activated AMPK and Sirt1 further activate PGC-1α via phosphorylation and deacetylation, resulting in elevated mitochondrial biogenesis and oxidative function. [4]

Fig 3. C elegans, AMPK and Sirt

C elegans is a nematode worm and a very simply multicellular organism. Fat accumulation may be visualized with oil red. The combination of leucine and NA decreased the accumulation of fat as well as activating Sir2 and AMPK. DAF16 is the worm version of our FOXO transcription factor.

Fig 4. Making free radicals with diquat to sicken worms

In addition to being a nasty biocide, diquat is a convenient way of generating oxygen radicals. Leucine and NA alone did not do much but the combination promoted survival in the 4-14 day period.

Fig 5. Mice on the Western diet

This post is not going to suggest that humans consuming a Western diet can take any supplement and get away with it. NA and NA+Leu improved total cholesterol at four and eight weeks. The same could be said for esterified cholesterol. The NA and NA+Leu improvemetn in triglycerides was seen at four weeks but not at eight weeks.

6-7 the atherosclerosis connection in mice [3]

Fig 6 shows reduction of lipid deposits in the aorta but not synergistic Na-Leu reductions. Fig 7 shows a NA-Leu synergisic reduction in macrophage infiltration.

Fig. 8 summarizes the conclusions of the Buckbauer 2017 study. [3]

Concluding Thoughts and connection to PEMF…

The NuSirt studies focused almost entirely on fatty acid metabolism. However, the Sirt/AMPK axis seems to be central to this function. Previous posts on PIKfyve and Vps34 showed that both are regulated by lysine acetylation and AMPK phosphorylation.

References

1. Stytrinol-LNA
2. Zemel MB. Modulation of Energy Sensing by Leucine Synergy with Natural Sirtuin Activators: Effects on Health Span. J Med Food. 2020 Nov;23(11):1129-1135. SciHub free paper
3. Bruckbauer A, Banerjee J, Cao Q, Cui X, Jing J, Zha L, Li F, Xue B, Shi H, Zemel MB. Leucine-nicotinic acid synergy stimulates AMPK/Sirt1 signaling and regulates lipid metabolism and lifespan in Caenorhabditis elegans, and hyperlipidemia and atherosclerosis in mice. Am J Cardiovasc Dis. 2017 Apr 15;7(2):33-47. PMC free paper
4. Liang C, Curry BJ, Brown PL, Zemel MB. Leucine Modulates Mitochondrial Biogenesis and SIRT1-AMPK Signaling in C2C12 Myotubes. J Nutr Metab. 2014;2014:239750 PMC free article