Sabinene

Sabinene may be found in black pepper and carrot seed oil. A Korean group that included members of the the Department of Cosmetic Science at Hoseo University were more interested in Chrysanthemum boreale Makino essential oil (CBMEO) as a source of sabinene. An Internet image revealed many images of Chrysanthemum boreale The authors claim that sabinene protects muscles from “aging” by acting as a reactive oxygen scavenger. With the help of a review on the muscle ageing enzyme MuRF1 this post presents the argument that sabinene may be acting on the PPARα transcription. A paper showing that an agonist of PPARα prevents arthritis associated muscle wasting [3] is also presented on this post.

Young skin and young muscle?

If CBMEO is good for keeping skin young, would it also be good for keeping muscles young? The authors used male rats on a two day fast and M6 myoblasts and myotubules subjected to serum deprivation.

Ryu Fig 1. CBEO is good for serum deprived myoblasts

XTT is a colorimetric agent that changes color in response to reducing equivalents that are generally associated with cell viability. Crystal violet is a hydrophobic stain with a positive charge. It tends to be associated with proteins and sometimes DNA.

Ryu Fig. 2 Honing in on the active terpene in CBMEO

These are the structures of the terpenes detected CBMEO

Note the differences in number of double bonds in these compounds. The authors will make the argument that the active ingredient is acting as a scavenger of reactive oxygen species.

Note that serum starvation decreases the size of M6 cells no matter what the terpene treatment (300 μM). If the protective mechanism of CBMEO was simply as an ROS scavenger, β-myrcene would be the better protector.

Figure 3 Moving on to serum starved myotubules

This protocol was basically a repeat of the M6 myoblast experiment. An interesting nuance is that no amount of zooming in on the PMC image yields what appears to be thick filaments in the myotubules. Myosin heavy chain is part of the thick filament, red in the cartoon from the Perez-Moreno review [2] The ubiquitin ligase action of Murf1 attaches ubiquitin groups to lysines that targets the protein for degradation. [2] MuRF1 has many domains and may use these domains to occupy many localities in the cell. One of these localities is the M-line protein titin. [2] Destabilizing the M-line may target thin and thick filaments for attachment of a ubiquitin group and proteosomal degradation.[2]

These results are extremely exciting that lead to more questions.

1. Did lack of insulin decrease cell surface expression of glucose transporters?
2. Do myotubules store glycogen?
3. Is the decrease in myotubule diameter due to the tubules tapping into glycogen stores and/or decreased thin and thick filaments?
4. Are other thin and thick filament proteins degraded?

Ryu Fig 4, MuRF1 and cell signalling

Sabinene decreased the amount of MuRF1 in myotubules. [1] Erk and p38 are kinases that are generally activated by growth factors. Kinases attach phosphate groups to down stream proteins that are often other kinases. Many times the end target is a transcription factor that regulates the transcription of genes like MuRF1..

This image is a composite of Figure 4 of [1] and the MuRF1 review [2]. Left panel MuRF1 is only seen in the starved control myotubules. β-Actin is considered a “house keeping” protein in non-muscle cells. Housekeeping means it is always expressed in about the same amount. It is used to control for variations in protein loading in Western blots. Note that there is about the same amount of β-actin in all three samples. MuRF1 is only seen in the starved cells not treated with sabinene. What if the investigators had also looked at the most abundant isoform of actin seen in thin filaments, α-actin? The center panel from reference [3] proposes that MuRF1 prevents pro-apoptotic, anti-hypertrophy in response to cardiac injury. Mitogen activated protein kinase (MAPK) pathways include ERK and p38. Phosphorylated kinases are activated kinases that attach phosphate groups to down stream proteins, like other kinases. Panel A shows Western blots with antibodies that recognize the specified proteins. Antibodies against p-p38 and p-ERK1/2 recognize specific phosphorylation sites of these kinases. This review did not discuss if muscle wasting, sarcopenia, is a protective mechanism when nutrient levels are low. The other disconnect is whether the ubiquitin ligase function of MuRF1 is part of this process. Finally, the 3rd panel from the MuRF1 review has been edited to show insulin and other growth factors inhibit the FoxO transcription factor. Also note that reactive oxygen species may activate MuRF1 transcription via activation of NFκB

In the Ryu cell culture model there is plenty of oxygen but glucose deprivation. In cardiac repurfusion injury there are plenty of nutrients but oxygen deprivation. In both cases the mitochondria are not making enough ATP. The ratio AMP to ATP increases. AMP activated protein kinase has to be taken into consideration.

Ryu Figure 5 Is sabinene merely a scavenger of oxygen radicals?

Figure 5A-B of the Ryu report asked the question of whether sabinene could exert its effect simply by reacting with reactive oxygen species. Partial reduction of O2 to superoxide rather than H2O can lead to the production of super oxide.

Ryu and coworkers tried to reproduce the sabinene results with another free radical scavengre N-acetyl cysteine. On the surface, some of the positive effects of sabinene were reproduced. Note that the sabinene decrease in ROS is rather minor and N-acetyl cyteine reduction in MuRF1 was not as great as sabinene in figure 4. Myotubule size sparing in the NAC model was not reported.

Ryu Fig 6, MuRF1 in muscle

The reader is implored to read the public access PDF version of this publication at the link given in reference [1]. Quite often PubMed Central has high resolution of the images that allow the reader to see things with just a little more clarity. This link will take the reader to the high resolution image from which this image was derived. The H&E stained myofibers were subjected to a “Gaussian blur” in the image manipulation program Gimp2 to avoid the pixelated look. Gimp2 was used to manipulate the light levels of the MuRF1 staining. The only goal was to detect sabinene changes in the distribution of MuRF1.

Questions:

1. Are the holes in the fibers in panel A (arrow) due to loss of glycogen, fat deposits, or protein?
2. Where is MuRF1 localized in panel C? Does this have anything to do with which targets are being ubiquitinating? Are the bar shaped structures (arrow) in the untreated fasted muscles MuRF1 binding to titin in the Z-line?
3. Does having more MuRF1 translate into having less major muscle proteins like MHC, skeletal muscle actin, or full length titin?
4. The big unknown in this fasting study is where fuel for muscle coming from in the fasttwitch fibers. Wikipedia glycogen contributors remind us that muscles store glycogen and that fasting coupled with low intensity exercise promotes switching to fat reserves as a source of energy.

 Is sabinene’s target PPARα ?

Arthritis can cause muscle wasting.  [3]  Fenofibrate is a small molecule activator of PPARα that was shown to mitigate muscle wasting in an arthritis model in which   joints were injected with adjuvant (heat inactivated mycobacterium) in the right paw. [3] Presenting images from the Castillero study would be too much for this post and a distraction from the case that PPARα is a target of sabinene. The figures are summarized in the numbered points:

1. Fenofibrate prevented some of the increase in paw volume, spleen weight, and arthritis score.  In the liver, the TNFα mRNA was more than halved compared to the AA treatment alone. [3]
2. Feno, 300mg/kg, increased PPARα mRNA in the gastrocnemius in control rats and in the AA model rats.  No change was seen in PPARα mRNA in the liver.
3. The adjunct arthritis treatment resulted in a slight decrease in gastrocnemius weight and a larger decrease in white adipose tissue.  A decrease was also seen in food intake.  Feno improved these parameters by small but significant amounts.
4. Myofiber diameter decreased by almost 50% in the AA model.  Fast fibers took the biggest hit.  Feno mitigated the decrease in fast fibers but had no effect in the slow fibers.
5. Feno prevented the AA induced increase in atrogenin mRNA that increased ~3.5x over the control.  Feno reduced MuRF1 mRNA to only 2x the control in the AA model compared to a 4.5x increase in untreated AA.
6. No change was seen in phosphorylation of the growth related Akt kinase.  A slight decrease was seen in phosphorylation of the Foxo3 transcription in the AA + Feno group.
7. The AA treatment caused a slight (~20%) increase over control in myostatin mRNA.  The Feno decreased myostatin mRNA to ~20% less than the control. Myostatin is a “myokine” released by and acts on neighboring myocytes to inhibit muscle growth. 
8. PCNA, myogenin and myoD mRNA were both increased by the AA treatment and then decreased by the Feno intervention.  Proliferating cell nuclear antigen (PCNA) is involved in DNA polymerization. Myogenin is a transcription factor that promotes muscle growth/differentiation and repair.   MyoD is a muscle specific transcription factor that promotes muscle differentiation and renewal.

Tying things together

This cartoon was taken from the Peris-Moreno review [2] with the numbered findings (circled) from the Castillero study. [3] Another study addressed the role of hypoxia in fetal muscle development, NAD⁺, and sirtuin. [4] Via upstream pathways that this post will not get into, PPARα is increased by activators of fasting and ageing enzyme sirtuin. [4]

What is the target of sabinene? It seems to behave like the agonist PPARα fenofrimbrate. [3] Targets upstream of PPARα could explain the data of the Ryu fasting study. [1] The Ryu study was very thought provoking. This post humbly proposes that sabinene may be targeting much more than reactive oxygen species. Perhaps terpenes with more oxygen scavenging double bonds contribute to the medicinal attributes of CBMEO.

References

1. Ryu, Y., Lee, D., Jung, S. H., Lee, K. J., Jin, H., Kim, S. J., Lee, H. M., Kim, B., & Won, K. J. (2019). Sabinene Prevents Skeletal Muscle Atrophy by Inhibiting the MAPK-MuRF-1 Pathway in Rats. International journal of molecular sciences, 20(19), 4955. PMC free article
2. Peris-Moreno D, Taillandier D, Polge C. (2020) MuRF1/TRIM63, Master Regulator of Muscle Mass. Int J Mol Sci. 2020 Sep 11;21(18):6663 PMC free article
3. Castillero E, Nieto-Bona MP, Fernández-Galaz C, Martín AI, López-Menduiña M, Granado M, Villanúa MA, López-Calderón A. (2011) Fenofibrate, a PPAR{alpha} agonist, decreases atrogenes and myostatin expression and improves arthritis-induced skeletal muscle atrophy. Am J Physiol Endocrinol Metab. 2011 May;300(5):E790-9. free article
4. Regnault TR, Zhao L, Chiu JS, Gottheil SK, Foran A, Yee SP.(2010) Peroxisome Proliferator-Activated Receptor -β/δ, -γ Agonists and Resveratrol Modulate Hypoxia Induced Changes in Nuclear Receptor Activators of Muscle Oxidative Metabolism. PPAR Res. 2010;2010:129173. PMC free article