Metadichol® treatment of NHDF (Normal Human Dermal Fibroblast) cells induced increased expression of all sirtuin family genes and the closely related genes KL, TERT, FOXO1, and TP53 (Fig. 5). Fold increases in expression ranged from 3- to 15-fold after treatment with various concentrations of Metadichol® ranging from 1 pg to 100 ng. SIRT3 and FOXO1 exhibited a 15-fold increase in expression, whereas SIRT1 was increased 7-fold. The experimental procedure does not involve ause of viruses and or other gene insertion techniques. These results indicate that treatment of HDFC with Metadichol® can induce gene expression of all sirtuin family members (SIRT1–7), despite the differences between sirtuin isoforms. Given their different structures, it is improbable that Metadichol® is binding directly to DNA elements of the gene to activate them. It is far more likely that Metadichol® is activating upstream regulatory genes in the pathway, leading to the cascade of genes being activated downstream. Sirtuins are NAD+-dependent enzymes. Several nuclear receptors activate NAD+ and thus may be necessary for regulating sirtuin function. We have recently shown that Metadichol® treatment increases nuclear receptor expression (28) in stem cells and fibroblasts (Table 4).
Table 4
; Nuclear receptor expression induced by Metadichol treatment of HMSC highlighted in yellow are the highest foil increases for each of the nuclear receptors.
Nuclear receptor | Common name | Control | 1 picogram | 100 picogram | 1 nanogram | 100 ng |
NR1D1 | Rev-ErbAα | 1 | 1.5 | 0.64 | 0.07 | 0.75 |
NR1D2 | Rev-ErbAβ | 1 | 2.19 | 1.18 | 1.8 | 1.82 |
NR1H2 | LXRa | 1 | 4.95 | 1.03 | 0.03 | 1.71 |
NR1H3 | LXRB | 1 | 1.63 | 1.79 | 3.84 | 0.71 |
NR2E1 | TLX | 1 | 3.38 | 1.49 | 1.69 | 0.88 |
NR5A1 | SF1 | 1 | 3.2 | 2.56 | 1.92 | 5.09 |
NR3B1 | Err alpha | 1 | 10.31 | 8.3 | 6.58 | 11.1 |
NR1C1 | PPAR alpha | 1 | 1.9 | 1.24 | 0.72 | 1.8 |
We further analyzed the expression patterns of these 11 genes (SIRT1-7, KL, FOXO1, TERT, and TP53)) using gene network analysis. We utilized Pathway Studio software (29,30) to perform Gene Set Enrichment Analysis (GSEA) against Elsevier's proprietary pathway collection. Figure 6 shows all relations between 11 genes available in the Elsevier Biology Knowledge graph.
All the 11 genes regulate aging, metabolism, and DNA repair (31). In addition, all have been linked to age-related diseases such as cancer, cardiovascular disease, and diabetes (Table 4). These genes are essential for maintaining the health and proper functioning of cells and tissues, and their dysregulation has been associated with various age-related pathologies.
The following is a summary of the functions and pathways associated with each gene provided by Pathway Studio software.
Table.4. List of top 10 significantly enriched pathways (Fisher exact test p-value < 0.05).
A complete GSEA is available as a supplementary file.
Name | Overlap | Percent Overlap | p-value | Genes from the list |
Deacetylases Activation in Histone Deacetylation in Cancer | 7 | 10 | 6.32E-14 | SIRT7; SIRT6; SIRT5; SIRT4; SIRT3; SIRT1; SIRT2 |
Werner Syndrome (Adult Progeria) | 4 | 1 | 3.45E-06 | TP53; TERT; SIRT4; SIRT1 |
Mitochondrial Fusion and Fission | 3 | 5 | 2.36E-05 | TP53; SIRT3; SIRT1 |
MTOR/TP53 Regulates Cell Metabolism | 3 | 3 | 3.2E-05 | TP53; SIRT6; FOXO1 |
Mitophagy | 3 | 4 | 4.67E-05 | SIRT6; SIRT4; SIRT3 |
SIRT7 Signaling in Aging | 3 | 2 | 5.12E-05 | TP53; SIRT7; FOXO1 |
Alveolar Epithelial Cell Dysfunction in COPD | 3 | 1 | 5.6E-05 | TP53; SIRT6; SIRT1 |
MTOR/TP53 Regulates Cell Metabolism | 3 | 3 | 7.85E-05 | TP53; SIRT6; FOXO1 |
SIRT1 Signaling in Aging | 3 | 1 | 0.000183 | TP53; FOXO1; SIRT1 |
SIRT1 and P53
SIRT1 has been shown to interact with P53 in regulating DNA repair and apoptosis. SIRT1 can deacetylate TP53, enhancing p53 protein-mediated transcriptional activity and increasing apoptosis.
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SIRT6 and TP53: SIRT6 has also been shown to interact with TP53 in regulating DNA repair and apoptosis. SIRT6 can deacetylate TP53, reducing P53 protein-mediated transcriptional activity and decreasing apoptosis.
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SIRT3 and SIRT5: SIRT3 and SIRT5 are both involved in regulating cellular metabolism and mitochondrial function. SIRT3 has been shown to deacetylate and deactivate SIRT5, and SIRT5 protein can deacetylate and activate enzymes involved in fatty acid metabolism.
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TERT and P53: P53 has been shown to transcriptionally repress TERT expression, which is involved in maintaining the length of telomeres. In addition, TERT can interact with p53 to regulate apoptosis and DNA damage response.
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FOXO1 and SIRT1: FOXO1 can be deacetylated and activated by SIRT1, leading to increased gene expression in metabolism and cellular stress response.
These interactions suggest that the different Sirtuin family members work together to regulate various cellular processes and maintain cellular homeostasis. A detailed analysis of the protein–protein network of interactions of the Sirtuin family is available (see ref 9) They showed that the network covers 25% of the entire human interaction. Their analyses support many of the known metabolic involvements of Sirtuins in humans and open a window on the complex molecular mechanisms of action of Sirtuins that are still poorly understood.
SItt3 overexpressed by 15 fold. SIRT3 overexpression protects cochlear hair cells against noise-induced hearing loss by reducing oxidative stress and apoptosis, disrupting mitochondrial proteostasis, and influencing cell cycle progression by altering the acetylation levels of mitochondrial proteins and inducing DNA damage (32,33). SIRT3 also antagonizes high-glucose-accelerated cellular senescence by deacetylating and activating FOXO1, which influences the expression of antioxidant enzymes and reduces oxidative stress. This process can protect against thoracic aortic dissection formation by attenuating oxidative stress, apoptosis, and inflammation in vascular smooth muscle cells (34).
FOXO1 overexpressed by 15 fold. Overexpression regulates many substrates (35), including IRS2, GLUT4, PGC-1α, and SOD2. Previously, we reported that both IRS proteins and GLUT4 exhibit increased expression after treatment of stem cells with Metadichol® (36). These substrates are involved in various cellular processes, such as glucose metabolism, lipid metabolism, oxidative stress, cell cycle arrest, and apoptosis. FOXO1 has been implicated (37–40) in the pathogenesis and prevention of various diseases, such as diabetes, obesity, cardiovascular diseases, neurodegenerative diseases, and cancers. For example, FOXO1 can improve insulin sensitivity and glucose homeostasis by regulating gluconeogenic enzymes, and GLUT4 expression can inhibit adipogenesis and lipogenesis by suppressing PPARγ and SREBP-1c expression. FOXO1 can protect against cardiac hypertrophy.
There are nuclear receptors that activate NAD+. These transcription factors regulate the expression of genes involved in NAD+ biosynthesis or consumption (41–42). NAD+ is a cofactor for various enzymatic reactions and a substrate for NAD+-dependent enzymes such as sirtuins, poly-ADP-ribose polymerases, and cyclic ADP-ribose (cADPR) synthases. NAD+ levels are influenced by circadian rhythms, nutrient availability, and cellular stress. The nuclear receptors (5) that activate NAD+ are shown in Table 5
Table 5
; Nuclear receptor expression induced by Metadichol treatment of HMSC highlighted in yellow are the highest foil increases for each of the nuclear receptors.
Nuclear receptor | Common name | Control | 1 picogram | 100 picogram | 1 nanogram | 100 ng |
NR1D1 | Rev-ErbAα | 1 | 1.5 | 0.64 | 0.07 | 0.75 |
NR1D2 | Rev-ErbAβ | 1 | 2.19 | 1.18 | 1.8 | 1.82 |
NR1H2 | LXRa | 1 | 4.95 | 1.03 | 0.03 | 1.71 |
NR1H3 | LXRB | 1 | 1.63 | 1.79 | 3.84 | 0.71 |
NR2E1 | TLX | 1 | 3.38 | 1.49 | 1.69 | 0.88 |
NR5A1 | SF1 | 1 | 3.2 | 2.56 | 1.92 | 5.09 |
NR3B1 | Err alpha | 1 | 10.31 | 8.3 | 6.58 | 11.1 |
NR1C1 | PPAR alpha | 1 | 1.9 | 1.24 | 0.72 | 1.8 |
REV-ERBα and REV-ERBβ are repressive components of the molecular circadian clock and regulate metabolism and inflammation (43). REV-ERBs repress the expression of E4BP4, a transcriptional repressor of NAMPT, the rate-limiting enzyme in the NAD+ salvage pathway. By repressing E4BP4, REV-ERBs activate NAMPT expression and NAD+ production in a circadian manner
TLX is a transcriptional repressor that promotes neural precursor cell proliferation and self-renewal. TLX directly binds to the promoter of SIRT1 and activates its transcription (44).
LXR regulates cholesterol and lipid metabolism, inflammation, and immunity. LXR upregulates (45) the expression of SIRT1, which can deacetylate and activate LXR, enhancing its transcriptional activity. LXR activation also induces the expression of SIRT6, which modulates lipid metabolism and inflammation. (46)
NR5A1 encodes a steroidogenic factor 1 (SF-1) protein, a nuclear receptor and transcription factor that regulates the expression of genes involved in the development and function of the adrenal glands, gonads, and hypothalamus. There is some evidence that SF-1 and SIRTs interact with each other in different ways (47). For example, SF-1 can regulate the expression of SIRT1 in the adrenal glands and gonads. SF-1 binds to the promoter region of the SIRT1 gene and activates its transcription (48). In addition, SIRT1 and SF-1 can cooperate to regulate fetal and adult Leydig cell development. Leydig cells are the primary source of testosterone in males (48) .
Other nuclear receptors, such as estrogen-related receptor α (ERRα), peroxisome proliferator-activated receptor α (PPARα), and nuclear respiratory factor 2 (NRF2), can control the expression of NAD+ biosynthetic enzymes (49–52)). It can induce the expression of nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1), which is a nuclear enzyme that catalyzes the conversion of nicotinamide mononucleotide (NMN) to NAD+. ERRα, PPARα, and NRF2 can induce the expression of NMNAT1
In addition, thyroid receptors, such as THRA and THRB, interact with SIRTs (53–56). For example, the thyroid can regulate the expression and activity of (SIRT6). SIRT1 can affect TH signaling by deacetylating some target genes or coregulators. Metadichol® binds to and interacts with both THRA and THRB (57).
Our work demonstrates for the first time that all genes of the sirtuin family can be activated directly by upstream genes involved in transcription, that is, nuclear receptors. Given the many nuclear receptors involved, it is likely that sirtuin gene regulation is regulated in a complex manner. These nuclear receptors produce NAD+, which is needed for Sirtuins to function. The kingpin is the activation of nuclear receptors by Metadichol®. For sirtuins to be therapeutically valuable, one needs to control the expression of the entire family, leading to a tightly set of highly connected genes. These genes interact more than expected for a gene set of the same size and distribution degree randomly selected from the genome. This enrichment indicates that this set of genes shares a significant biological connection (57). Analysis of gene networks using Pathway Studio and protein–protein interaction maps indicated the formation of a loop feedback network, as shown in Fig. 6.
We have shown that Metadichol® can increase the expression of all Sirtuin family genes. This can be exploited to modulate multiple targets to enhance therapeutic effects, as ligands today are focused on a single target and limited by their efficacy. Metadichol ® has no known toxicity (58–60).
Metadichol targets MRGs (master regulatory genes_ (61) that control hundreds of other genes. Some examples of MRGs are the IL (interleukin) Family (62), genes for the three transcription factors Sox2, Oct3/4. and Nanog, which have been identified as the MRGs that regulate mammalian embryogenesis, embryonic stem cell self-renewal, and pluripotency (63). Metadichol targets these genes as well (64). Whole-family MRGs, like sirtuins, regulate multiple downstream genes' expression MRGs play a significant role in various human diseases, such as cancer, cardiovascular diseases, and neurological diseases. but also play a role in other signal pathways by regulating additional MRGs. The development of omics technologies (genomics, proteomics, metabolomics, metagenomics, phenomics, and transcriptomics) has led to increased interest in MRGs as they play a crucial role in organism development and have potential as targets for gene therapies and drugs. The use of MRGs for gene therapy and targeted drugs remains a challenge, and clinical application requires ongoing medical research efforts.
Metadichol has all the characteristics needed to reverse aging-related diseases. Based on our previous work (64–67). Metadichol® represents the first of a class of safe therapeutics that target MRGs affecting multiple genes, pathways, and diseases