Camphene

Banana Kush is a strain rich in in camphene. This is not something you’d want to smoke according to PubChem “Emits flammable vapors when heated. Emits acrid smoke and irritating fumes at high temperature. ” Camphene is found in ginger, turmeric, and rosemary. [1]

This study came out of Yonsei University in South Korea. Taesun Park was the corresponding author. These authors were interested in high fat diet induced insulin resistant diabetes. According to their review of the literature camphene is found in rosemary, turmeric, and ginger. The dosage used in this study was set lower than the Council of Europe Committee of Experts on Flavoring Substances upper limit of camphene in humans. This is 20 mg/kg body weight.

Figure 1 Camphene and high fat diet weight gain

Male C57BL/6N mice (5-week-old)  were fed a commercial diet for 1 week for acclimation. The mice were then randomly divided into three weight-matched groups (n=8 )

• Normal Diet ND was a purified diet based on the AIN-76 rodent diet composition.
• The  High Fat Diet HFD was the ND plus 200 g fat/kg.  The fat was supplied by 170 g lard and 30 g corn oil. The cholesterol was and 1%  of the added fat.
• The Camphene Diet CPND was the HFD plus 0.2% (w/w) camphene.

Note that the mice on the high fat plus camphene diet (CPND) were statistically the same as the mice on the normal diet (ND) in terms of body weight and body weight gain. All mice were eating the same amount of food in terms of grams, not necessarily in terms of calories.

Where are these calories going?

The food efficiency ratio is also known as the food conversion ratio. A high ratio says that food calories are efficiently converted to a gain in body weight. If mice are running their hearts out on their wheels or burning brown fat to stay warm, this ratio will decrease.

The authors also presented changes in triglycerides, fatty acids, and cholesterol. These graphs have been pasted upon a backdrop of metabolic pathways. The starred enzymes appear in Figure 5A.

They ate more fat but did not accumulate more fat

The data from the Korean study [1] have been pasted on a diagram of metabolic pathways involved in the synthesis of the three forms of fat that they measured.

Keep reading. Kim and coauthors found that camphene affects the starred enzymes. But wait! A high fat diet can really mess up a mouse or human’s liver.

Figure 2 Camphene reduces liver damage

Figure 2 examined triglycerides, fatty acids, and total cholesterol in the livers. Liver weights and steatosis scores were also computed. ALT and AST liver enzymes in the blood were also elevated by the high fat diet. Camphene brought down all of these parameters to the control levels. These liver enzymes in the blood suggest that liver damage accompanies the HFD increases in triglycerides, fatty acids, and cholesterol. These parameters were the same as the normal diet in the high plus 0.02% camphene diet. Images of this figure are being omitted from this post. The reader is invited to read the original public access publication. [1]

Figure 3 Camphene slows making fat cells from fibroblasts

Fibroblast like 3T3-L1 cells, were grown in a medium to keep them in the fiborbalst state in  100-mm dishes (see below) until confluent for 2 days. The cells are considered pre-adipocytes.  They were differentiated into adipocytes by adding what we will consider in this post the “magic sauce” and grown some more. Cells were incubated with medium containing 0.1, 1, 10, 50, and 100 μM for 8 days during differentiation. On day 8, cells were fixed and stained with oil red O to visualize the lipids  Stained oil droplets were dissolved in isopropanol (rubbing alcohol) and quantified by absorbance at 600 nm.

Camphene decreased lipid accumulation as measured by oil Red. 

Fat gene transcription factors PPARγ2 and C/EBPα synergy

RT PCR was used to measure the amount of two fat gene transcription factor transcipts and a mutual target adipocyte protein 2, aka fatty acid binding protein 4 FABP4. C/EBPα and PPARγ2 transcription factors can act as transcription factors for each other’s genes to make more of each transcription factor. [2] Both transcription factors can bind to the same promoter of fat handing genes to really turn things on. [2] As a reminder, p<0.05 means we are 95% certain that the observed differences are not due to chance.

• C/EBPα (228%), p<0.05
• PPARγ2 (269%), p<0.005
• aP2 (245%), p<0.05 

1. ref [1] Note that camphene decreases these fat handling transcripts.
2. ref [2] Why is camphene decreasing the transcripts of these three genes? For starters, the transcription factor PPARγ binds to the promoters of the EBPα and vice versa. [2] and FABP4 genes. A model has been proposed in which EBPα and PPARγ work together to load the promoters of fatty acid metabolism related genes to increase transcription. [2]. The spool like objects in this image are histones. They are what keep our chromosomal tightly compacted. Partial unwinding is required to make mRNA transcripts that are later translated into proteins.
3. ref [3] Many upstream response elements exist in the gene for FABP4 that codes for adipocyte protein 2. These protmoters were compared in five species of mammals. [3] Sites for C/EBPα and PPARγ in all five species. [3] The authors didn’t get into conserved sites 2 and 4. SINE are short interspersed nuclear elements that are important in gene transcription.

A positive feedback loop?

The literature that has been reviewed thus far suggests that if camphene is an activator of PPARγ it should increase the transcripts of C/EBPα and FABP4 aka aP2, not decrease them. Wikipedia has a good page on positive feed back loops. Many physicians will remember the oxytocin positive feed back loop involved in child birth. This positive feed back loop terminates when the child is born and no longer exerting pressure on the cervix. Could camphene be causing premature termination of the fat positive feedback loop? As we shall see, the terminating event seems to be adiponectin.

Figure 4 Adiponectin, the master regulator

mRNA message for adiponectin as well as the translated protein were measured in the 3T3-L1 adipocytes as well as the epidymal fat of the mice. [1]

In 3t3 adipocytes

In mouse epidymal fat

Camphene increased the transcription of the adiponectin gene as well as the translation and release of the protein regulator. Adiponectin binds to a G protein coupled receptor that ultimately activates AMP kinase, see Figure 1f-H. Adiponectin is one way to explain the decrease in fatty acids, triglyceride, and cholesterol.

Figure 5, In the liver, genes involved in lipid metabolism

Panel A tells us that camphene increases transcripts that code for proteins that work against fat synthesis. See Figure 1 F-H for an overview. The adiponectin receptors R1 and R2 bind adiponectin and ultimately result in activation of adenylyl cyclase and phosphorylation of AMP kinase (Figure 5B). CPT1 transports fatty acids into the mitochondria for oxidation and ATP production. The p70-6S kinase 1 is a kinase that is activated by phosphorylation and in turn phosphorylates the ribosome S6 protein. S6 phosphorylation activates translation of mRNA into proteins.

Note that cpamphene decreases fatty acid synthase (FAS) transcripts in mice fed the high fat diet to levels below the mice on the normal diet.

Figure 6 Camphene and glucose clearance in high fat diet mice

The mice were given glucose tolerance tests. Camphene brought the rate of glucose clearance and blood glucose levels back to the level of that of mice on the normal diet. Blood insulin concentrations were also returned to normal.

Figure 7 Camphene, the liver and insulin

The Insulin Receptor Substrate 1 (IRS-1) protein is phosphorlated on tyrosine 465 when insulin bids its receptor. This activates a signallingcacde of phosphorylation that leads to glucose transporters being loaded into the cell membrane. These authors used antibodies against IRS-1 Ser307 of the mouse sequence. ,Phosphorylation of this site is thought to interfere with the IRS-1 activation of PI3K leading to insulin resistance. Not only did camphene reduce this deleterious phosphorylation to normal diet levels but it also restored Akt threonine 308 phosphorylation. [1] While total glucose transporter 2 (Glut2) was not altered by camphene, the amount in the cell membrane GLUT2(M) was significantly increased over normal diet levels. Glucose transported into the liver can be used to make fat or glycogen. The authors demonstrated it was not the former. Two gluconeogenesis enzymes were found to be elevated in mice on the high fat diet. This suggests the livers of the high fat diet mice might have been tapping into the glycerol backbone of triglycerides to make glucose. Not only did camphene reduce the transcripts of G6Pase transcripts to normal diet levels but it also decreased PEP carboxylase kinase transcripts to a level significantly lower than that of the normal diet. [1]

A potential cannabinoid entourage effect?

This image was adapted from reference [4] and authored by a group of investigators with much experience in cannabis research. This review also notes that ∆9-THC also activates PPARγ. [4] It is noteworthy that the CB1 receptor “inverse agonist” Rimonabant was used for eight loss but discontinued for psychological side effects. Perhaps ∆9-THC also activation of PPARγ is part of why ∆9-THC does not induce obesity. Could we infuse camphene in marijuana edibles like brownies for an additional lipid lowing effect? Since we need heat for the THCA to THC conversion we will probably need to cook the edicble first. The described camphene flavors of “moth ball” or “woodsy” might not work in pure form. Perhaps a ginger extract for application to our cannabis brownies? In a related study camphen was shown to affect SREBP1. [5]

References

1. Kim S, Choi Y, Choi S, Choi Y, Park T. (2014) Dietary camphene attenuates hepatic steatosis and insulin resistance in mice. Obesity (Silver Spring). 2014 Feb;22(2):408-17.  PMC free article
2. Madsen, M. S., Siersbæk, R., Boergesen, M., Nielsen, R., & Mandrup, S. (2014). Peroxisome proliferator-activated receptor γ and C/EBPα synergistically activate key metabolic adipocyte genes by assisted loading. Molecular and cellular biology, 34(6), 939–954. PMC free article
3. Shin J, Li B, Davis ME, Suh Y, Lee K. Comparative analysis of fatty acid-binding protein 4 promoters: conservation of peroxisome proliferator-activated receptor binding sites. J Anim Sci. 2009 Dec;87(12):3923-34.
4. Matias I, Di Marzo V, Köfalvi A Chapter 14 Endocannabinoids in Energy Homeostasis and Metabolic Disorders Cannabinoids and the Brain Attila Köfalvi Editor Springer Science Business Media
5. Vallianou, Ioanna, and Margarita Hadzopoulou-Cladaras. “Camphene, a Plant Derived Monoterpene, Exerts Its Hypolipidemic Action by Affecting SREBP-1 and MTP Expression.” PloS one vol. 11,1 e0147117. 19 Jan. 2016, PMC free article