Forskolin and anti-aging -
Forskolin is a diterpene found in coleus that inhibits the enzyme adenylate cyclase. Adenylate cyclase regulates the formation of cAMP, a compound that controls many cellular activities.
Forskolin-induced elevation of cAMP levels has been shown to cause blood vessel dilation, inhibition of mast cells hence the herb is a powerful agent for reducing inflammation caused by allergies , an increase in thyroid hormone secretion, and the stimulation of fat release from fat cells.
Research carried out in by Agarwal and Parks suggested that forskolin was able to inhibit the spread of cancer cells. Direct application of forskolin to the eyes has consistently been shown to lower the pressure inside the eye, therefore the herb can be useful for treating glaucoma. One study on humans has shown that forskolin can reduce blood pressure and improve heart function in people with cardiomyopathy.
Fluid extract can be taken in the amount of 2 to 4 ml three times per day. The majority of clinical studies have used injected forskolin, so it is unclear if oral ingestion of coleus extracts will provide similar benefits in the amounts recommended above.
Bars represent the means and line its standard error. The bars represent the mean and line its standard error. Each bar represents the mean for each group and the lines represent standard error. It was previously shown that forskolin stimulated insulin release via elevation of intracellular cAMP in pancreatic beta cells [ 4 , 19 , 20 ], that it conferred cytoprotection by decreasing glucotoxicity [ 6 ]and that it increased the expression of the insulin gene [ 21 ].
However, previously published results regarding the effects of forskolin on glucose levels are controversial. Ammon and Müller found that acute, intravenous administration of forskolin induced insulin release, increased cAMP in pancreatic beta cells, but elevated plasma glucose.
However, when an intravenous glucose tolerance test was performed, glucose was not raised further than that observed in controls [ 4 ]. Ahmad et al. They delivered subchronic dosing and found that insulin and cAMP levels were elevated.
They also reported an increase in glucose levels and an elevation in glucagon [ 19 ]. The effects of forskolin delivered in acute or subchronic doses were explained by: 1 elevation of glucagon, 2 increased gluconeogenesis, and 3 increased glycogenolysis [ 22 ].
Moreover, cAMP acutely increased insulin release by two mechanisms: 1 through PKA and a number of substrates, including the inositol phosphate-3 receptor in the endoplasmic reticulum and the glucose transporter, GLUT2; and 2 the ATP-dependent K channel KATP.
A PKA-independent pathway also contributed to insulin release through an exchange protein that was directly activated by cAMP EPAC. This pathway appeared to be activated when the PKA pathway was saturated, which is important for high performance in beta cells [ 20 ].
Here, we studied the chronic effects of forskolin. These effects may be due to a permanent elevation of cAMP in residual beta cells. Studies in other cells showed that, after systemic forskolin administration, the levels of cAMP were high compared to control cells from untreated animals [ 23 ].
It is also known that diabetic rats display a disorder in the AC-cAMP system that responds to glucose [ 24 ]. Thus, chronic administration of forskolin may induce a change in the balance of cAMP in response to glucose, which could enhance insulin release.
This may be another mechanism for reducing glucotoxicity, in addition to the reduction of oxidative stress [ 6 ]. Chronic administration of GLP-1 agonists was found to induce insulin transcription and stabilize insulin levels [ 20 ].
Thus, in the present study, the effects on glucose levels, observed both in healthy and diabetic rats, may be explained by the effect of chronic forskolin on insulin transcription [ 21 ]. However, in healthy rats, the sensitivity to insulin did not change. This lack of response to insulin may be explained by the inhibitory effect of forskolin on glucose transporters, mainly Glut 4, which has a high affinity for glucose [ 25 - 30 ].
Our experimental findings showed no statistically significant difference in oxidative stress levels after forskolin treatment. However, we did observe a trend, similar to that observed in blood glucose levels, where the 8-OHdG values were lower in treated rats than in control rats at the end of the treatment period.
These results were consistent with results described by Balkis Budin et al [ 31 ] and Ihara et al [ 32 ], who showed that oxidative DNA damage was negatively correlated with metabolic control in diabetic rats.
Several metabolic pathways are known to mediate the effects of chronic hyperglycemia on the production of ROS. One pathway is non-enzymatic glycosylation, which leads to the formation of advanced glycation end products, through intermediate products Amadori ; this pathway leads to the generation of ROS.
Another pathway involves the electron transfer system, which becomes a major source of ROS. Therefore, we would expect these pathways to be activated in our chronically hyperglycemic rats [ 33 ].
On the other hand, in previous studies, forskolin was reported to have anti-inflammatory effects [ 9 , 15 ]. In this study, the results showed that forskolin affected oxidative stress. It is known that these two phenomena interact in several cardiovascular and metabolic pathologies, including diabetes [ 34 , 35 ].
In summary, our results indicated that chronic administration of forskolin: 1 reduced serum levels of fasting glucose in healthy rats, 2 decreased the severity of fasting hyperglycemia in diabetic male Wistar rats, and 3 caused no statistically significant decrease in the h urine levels of 8-OHdG.
We note that the STZ method induces diabetes in rats on a faster timescale than the time it takes to develop diabetes in humans.
Nevertheless, forskolin may be potentially useful in patients due to its mechanisms of action. It is possible that forskolin administration may have effects in humans that are qualitatively similar to those observed in the rat model of diabetes.
However, further studies are needed to determine the proper dosages. In addition, previous studies have reported that the main adverse effect of forskolin was that it contributed to a reduction in blood pressure [ 2 ].
Susuki et al. also reported that forskolin was associated with tachycardia and cephalic pain [ 36 ]. Forskolin may also cause bleeding, due to its inhibitory effects on platelet adhesion [ 37 , 38 ].
Future research in humans is needed to study potential adverse effects in patients with diabetes. AC, adenylate cyclase; cAMP, cyclic adenosine monophosphate; PKA, protein kinase A; 8- OHdG, 8,- Hydroxydeoxyguanosine; STZ, streptozotocine; CTOG, oral glucose tolerance curve; ROS, reactive oxygen species; KATP, ATP-dependent K channel; EPAC, exchange protein activated by cAMP; GLP-1, glucagon-like peptide This work was partially funded by CONACyT-Mexico-MH Monica Rios- Silva had a PhD fellowship from CONACyT-Mexico.
The authors wish to thank Jesus Dueñas for her technical assistance. Mónica Ríos-Silva, Benjamín Trujillo-Hernández, Miguel Huerta, Zorayda Urzúa, and Evelyn Mancilla designed and performed the experiments; Monica Ríos-Silva, Benjamín Trujillo-Hernández, Miguel Huerta, Zorayda Urzúa, Xóchitl Trujillo, and Enrique Sánchez-Pastor participated in the discussion and analysis of results; Mónica Ríos-Silva, Benjamín Trujillo-Hernández, Xóchitl Trujillo, Miguel Huerta, and Enrique Sánchez-Pastor wrote the manuscript.
All authors are responsible for the content of the article. The authors declare no conflict of interest. Insel PA, Ostrom RS. Forskolin as a tool for examining adenylyl cyclase expression, regulation, and G protein signaling. Cell Mol Neurobiol. Bhat SV, Dohadwalla AN, Bajwa BS, Balbir S.
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Activation and inhibition of adenylyl cyclase isoforms by forskolin analogs. J Pharmacol Exp Ther. Ammon HP, Müller AB. Effect of forskolin on isle cyclic AMP, insulin secretion, blood glucose an intravenous tolerance in rats.
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Shigenaga MK, Gimeno CJ, Ames BN. Urinary 8-hydroxy-2'-deoxyguanosine as a biological marker of in vivo oxidative DNA damage. F Top 20 GO processes of genes downregulated by senescence.
Red and blue bars indicate upregulation or downregulation by OSK, respectively. G Schematics of the Tet-On OSK system integrated in NCC reporter system fibroblasts. Scale bar, 50 μm. I Fluorescence intensity profiles corresponding to the arrow in H.
J EGFP intensities in the cytoplasm. Gene ontology GO analysis indicated that the top 20 GO biological processes of upregulated genes encompassed key features of aging, including dysregulation of development, localization, and transport [ 7 ], eleven of which were reversed by OSK Figure 2E. Despite the absence of cell division in all conditions, senescence caused subtle but significant changes in cell cycle gene mRNA levels, including p21 Supplementary Figure 2C [ 49 ].
Numerous cell cycle- related processes were enriched with downregulated genes by senescence, and 19 of the top 20 were reversed by OSK expression Figure 2F. The net outcome of this was the demonstration that induction of OSK partially counteracts the aging related changes resulting from senescence.
Using the NCC system, we examined the deterioration of nucleocytoplasmic integrity as cells transitioned from quiescence to senescence and the rejuvenative effects of OSK treatment on those senescent cells Figure 2G , 2H. Cross-sectional intensity profiles of the cells were used to assess the correlation between distributions of fluorescent molecules Figure 2I.
Compared to quiescent cells, senescent cells had a significant increase in the aggregation of mCherry and eGFP, indicating disruption of nucleocytoplasmic integrity Figure 2J. After four days of OSK treatment, NCC integrity was significantly restored in senescent cells, comparable to the quiescent, non-senescent cell population Figure 2J.
Taken together, these data show that OSK-mediated epigenetic reprogramming substantially reverses senescence-associated pathology and transcriptomic changes and that the NCC reporter system can detect rejuvenation of senescent cells by OSK.
To identify small molecules that rejuvenate old and senescent cells, we curated a list of molecules that have successfully reprogrammed human and mouse somatic cells into chemically induced pluripotent stem cells CiPSCs [ 30 , 31 ] and tested them using the NCC assay. Again, we used fully senescent cells to avoid detecting changes due to the cell cycle or transition to senescence.
Epigenetic age reversal is known to occur within a week of OSK M -mediated reprogramming, while the epigenetic age continuously decreases until pluripotency, reaching an approximate age of zero [ 50 — 52 ]. To ensure consistency, we initially tested small molecule combinations on cells within the same four-day period required for OSK to rejuvenate cells safely and consistently.
To achieve age reduction without altering cell identity, we focused on small molecules that were likely to work in the early stages of CiPSC formation, including valproic acid V , CHIR C , E 6 , tranylcypromine T and forskolin F.
Previous studies of reprogramming efficiency with small molecules demonstrated that either OCT4 alone or SKM, when combined with VC6T or F, respectively, can generate iPSCs, and VC6TF facilitates a mesenchymal-to-epithelial transition, an early stage of reprogramming in mouse cells [ 31 , 53 ].
Because of the known differences in differentiation between mice and humans, we also investigated molecules that have been reported for the initiation states of generating human CiPSCs including CHIR C , E 6 , TTNPB N , Y Y , Smoothened Agonist S , and ABT A [ 30 ].
The molecules VC6TF Cocktail 1: C1 and C6NYSA Cocktail 4: C4 were used as basal reprogramming cocktails and supplemented with other boosters known to increase iPSC efficiency, including sodium butyrate, basic fibroblast growth factor bFGF , and alpha ketoglutarate α-KG Figure 3A , 3B , Supplementary Tables 1 and 2 [ 54 ].
Figure 3. Reprogramming small molecule cocktails restore NCC alterations in senescent cells. A Chemical structures of small molecules of basal cocktails used to generate induced pluripotent stem cells iPSCs from mouse left or human right somatic cells.
C , D Validation of six selected cocktails through independent experiments, showing colocalization C and representative images D of eGFP and mCherry signals. Based on the fact that iPSCs can also be generated using SKM or O alone [ 55 , 56 ], we evaluated the effect of the boosters on VC6T SKM alternative and F O alternative.
We also assessed the effect of combinations including C6N, because it has been reported that the removal of Y, S, or A from Cocktail 4 C6NYSA did not reduce the CiPSC efficiency [ 30 ]. Among 80 cocktails tested in the NCC assay, the VC6TF basal cocktail was the most effective at restoring the integrity of nucleocytoplasmic compartmentalization, a key sign of age-reversal Figure 3B.
A recent, unpublished study reported that 6T pre-treatment prevents senescence in human fibroblasts, and 6, T, or 6T extends the lifespan of Caenorhabditis elegans by up to We, however, saw no benefit of F alone or the VC6T cocktail on reversing senescence phenotypes in our system Figure 3B.
Next, we chose six cocktails of small molecules for further investigation, three of which were based on Cocktail 1 as well as two additives referred to as Cocktail 2 and 3 and the other three based on Cocktail 4 plus additional additives referred to as Cocktail 5 and 6 Supplementary Table 2.
Sodium butyrate, a histone deacetylase inhibitor, was one of the most effective additives in both human and mouse cocktails C2 and C5.
Basic fibroblast growth factor bFGF was used for Cocktail 3, while α-KG was included in Cocktail 6. The six cocktails statistically improved compartmentalization in senescent cells, both in terms of correlation analysis Figure 3C and imaging of NCC signaling Figure 3D.
For nearly two decades, the writing and maintenance of chromatin marks have been known to be critical for reprogramming [ 58 ]. For this reason, we incorporated inhibitors of established chromatin remodeling factors in our screen to investigate whether these factors represented barriers or essential drivers of rejuvenation.
The rejuvenation pathway s initiated by C1 and C4 were both blocked by the inhibition of H3K9 methyltransferase G9a BIX, 0. Based on the improvement in NCC integrity, we performed RNA-seq to test the effect of these six cocktails on transcriptomic age.
After treatments with the chemicals, we observed a strong overlap between genes affected by the chemical treatments and the switch from quiescence to senescence Supplementary Figure 3A.
We also observed that the two groups of cocktails generally perturbed the same populations of genes Supplementary Figure 3A. Treatment with the chemical cocktails did not lead to fibroblasts taking on non-specific cell identity markers Supplementary Figure 3B. Finally, we did not observe the expression of iPSC specific genes or gene modules in the RNA-seq datasets Supplementary Figure 3C , 3D.
Additionally, we performed immunofluorescence looking for signs of expression of pluripotency-related genes such as NANOG and EPCAM following all cocktail treatments but could not see any expression Supplementary Figure 4.
Collectively, these data indicate that chemical-mediated treatments are only partially reprogrammed and not fully reset to pluripotency. We then tested the effect of these six cocktails on the transcriptomic age tAge of the cells using clocks trained on mouse, human, and a combined training data set Relative transcriptional age was assessed using a rodent transcriptomic clock as well as a combined human and rodent transcriptomic clock Figure 4A , 4B.
The change in years of age was determined using a human-specific chronological clock Figure 4C. Compared to quiescent cells, senescent cells had a significant increase in transcriptomic age, based on the transcriptomic clocks, consistent with previous findings assessing DNA methylation age [ 46 , 47 , 59 ].
Treatment of NCC cells with each of the six chemical cocktails C resulted in statistically significant reduction of the transcriptomic age of senescent cells, with those originating from mouse studies C generally producing a greater decrease in transcriptional age relative to the human derived cocktails Figure 4A , 4B.
The reported magnitude of the effect of all six cocktails differed between the hybrid and rodent transcriptional clocks, with the hybrid clock indicating a greater decrease in age by all six cocktails, with the rodent clock showing less variability between treatments.
Figure 4. Transcriptomic rejuvenation by reprogramming small molecule cocktails. A , B Delta transcriptomic ages tAgeΔ , as measured by a biological transcriptomic clock built on rodent and human transcriptomic data A , or rodent data alone B.
C Delta ages, as measured by a chronological transcriptomic clock built using human data. Normalized Enrichment Score NES 0. All six reprogramming cocktails also significantly decreased the estimated chronological age of NCC senescent cells by several years Figure 4C.
As observed with clock-based transcriptional age estimates, C1, C2, and C3 produced the greatest effect, reducing the measured age by more than three years after only four days of treatment. For reference, the effect of this four day treatment is comparable to the total change seen after a year of a regenerative treatment described in a landmark study from , which also focused on restoring epigenetic information [ 60 ].
To understand the effect of the chemical cocktails on cell identity and function, we assessed overall gene expression patterns of chemically-treated cells and compared them to old human cells [ 61 ] and OSK M -induced mouse and human induced pluripotent stem cells iPSC [ 52 ].
Despite having different chemical components, the transcriptomic profile of all six cocktails grouped most closely together, with human C and mouse Cderived cocktails grouping more closely within their groups Figure 4D. All six of the chemical treatments were positively correlated with the induced pluripotent stem cell iPSC populations and were negatively associated with mammalian age-related changes occurring in specific organs, such as kidney and brain, as well as across multiple tissues of mice, rats, and humans.
In agreement with the transcriptomic clock analysis, mouse Cderived cocktails produced a more consistent and stronger anti-aging effect on the cellular transcriptome than the human cocktails C Next, we performed gene set enrichment analysis GSEA to identify which pathways might be responsible for the similarities and differences between the chemical treatments, signatures of aging, and OSK M -induced iPSCs.
The KEGG genes database, HALLMARK gene set collection, and Reactome pathways database were included in this analysis Figure 4E. The anti-aging effects of chemical cocktails, especially mouse-derived ones, were associated with the upregulation of respiration associated pathways, such as oxidative phosphorylation and mitochondrial translation, as well as downregulation of hypoxia and multiple inflammation-associated pathways, such as interferon and JAK-STAT signaling, which are known to be involved in the SASP.
The activation of JAK-STAT signaling by interferons and other SASP factors, for example, contributes to the complex interplay between senescent cells and their microenvironment. Together, these data show that the chemical cocktails identified in this study not only reverse the effects of senescence on NCC and make them transcriptionally younger, but they also reverse key transcriptional signatures of senescence Figure 4F.
In this study, we provide evidence, based on protein compartmentalization and gene expression patterns in young and senescent cells, that small molecules can reverse the transcriptomic age of cells without erasing cell identity or inducing iPSC-like states.
We refer to this approach as the EPOCH method. The effectiveness of the NCC system as an apparent surrogate biomarker for biological age reversal, with young, old, senescent, HGPS, and OSK-treated cell lines serving as controls, should set the stage for larger, more expansive screens for rejuvenation factors.
Disruption of NCC is a well-established effect of aging across species and is directly associated with other diseases, including amyotrophic lateral sclerosis ALS and frontotemporal dementia FTD.
This study shows that the expression of OSK results in a noticeable improvement in the integrity of nucleocytoplasmic compartmentalization in replicatively senescent cells.
Further study into how EPOCH chemical cocktails restore NCC integrity and the partitioning of proteins may therefore offer therapeutic avenues for improving the health of older individuals and patients with age-related diseases of specific cell types and tissues. The nervous system is one example where the presence of healthy NCC is crucial for the proper functioning of tissue, and it is often affected in diseases related to aging [ 34 — 36 , 62 , 63 ].
Other methods of age control, such as the recently published Inducible Changes in the Epigenome ICE , which has the ability to accelerate normal epigenetic aging both in vitro and in vivo , should aid in such studies [ 5 , 7 ]. Transcriptomic analyses of epigenetic reprogramming by OSK and rejuvenation cocktails indicate that these interventions broadly ameliorate features of senescence, as illustrated by the striking changes in senescence-associated gene expression patterns involving inflammation, mitochondrial metabolism, lysosomal function, apoptosis, p53, and growth signaling.
Furthermore, the observation from the transcriptomic clocks that all six chemical cocktails, C1-C6, decreased both biological and chronological age below that of even the non-senescent cell populations, indicates that the cocktails are potent and capable of reversing senescence-associated cellular dysfunction.
Despite the differences in the composition of mouse- and human-based chemical cocktails, both affected mostly the same grouping of genes, suggesting that the effects may be operating through shared pathways. Experiments are in progress to understand the effect of the cocktails on various cell types from young and old individuals, the results of which will inform us about the extent to which they parallel the broadly beneficial effects of OSK M on cells and tissues.
The chemical cocktail that induced the most potent rejuvenation was VC6TF. Given that VC6TF has not been reported to be capable of fully reprogramming human cells to CiPSCs, and the maximum duration of any chemical treatment was limited to only four days, this study substantiates the notion that the rejuvenation is inherent to early phases of reprogramming and is at least partially separable from pluripotency programs [ 7 , 8 ].
To fully understand how chemical epigenetic age reversal works, it will be important to identify the factors and interactions responsible and compare them to those triggered by expression of OSK. Do they work via transcription factors, OCT4, SOX2 and KLF4, or are they initiating an independent program?
The results from this study, and those in progress, suggest that some, but not all, of the rejuvenation mechanisms are shared between the two modes of partial reprogramming. G9a has not been extensively studied in the context of aging, except for a report citing an age-related decrease in its associated marks in certain tissues [ 65 ].
E, also known as RepSox, is a TGF-β inhibitor that has been used in experiments to replace SOX2 during epigenetic reprogramming [ 68 , 69 ].
All the efficacious reprogramming chemical cocktails included these compounds, suggesting that these components together are potent contributors to the cellular rejuvenation in the treated cell populations.
Various research groups have observed that chemical cocktails containing CHIR and E can induce direct reprogramming between differentiated cell states [ 70 , 71 ].
This is important because it suggests that the processes involved in both rewriting and replacing cellular epigenetic identity are affected by the additive effects of these chemical compounds.
Moreover, independent studies have found associations with individual chemicals and reprogramming in various contexts, indicating that each component likely contributes to rejuvenation through a broad range of mechanisms [ 54 , 72 ].
Valproic acid is a well-known broad-spectrum histone deacetylase inhibitor that leads to a rapid and dramatic spread of histone acetylation marks across the genome [ 73 ]. The fact that valproic acid is a critical component of many of the successful cocktails indicates that the spread of euchromatin may be an important component of partial epigenetic reprogramming [ 73 ].
Sodium butyrate is another histone deacetylase inhibitor that was effective in both human and mouse cocktails. It has been reported to improve the expression of genes associated with reprogramming, supporting the model that the regulation of histone acetylation marks is crucial for rejuvenation via reprogramming [ 54 ].
Coleus Forskohlii is Environment-Friendly Energy member of the mint Forskolin and anti-aging family native anti--aging India. The root is used medicinally. Ancient Xnti-aging Forskolin and anti-aging show that coleus was commonly used to treat heart and lung diseases, intestinal spasms, insomnia, and convulsions. Today it is employed in the treatment of glaucoma. Forskolin is a diterpene found in coleus that inhibits the enzyme adenylate cyclase. Adenylate cyclase regulates the formation of cAMP, a compound that controls many cellular activities.
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