endocannabinoids

This page is dedicated to Arizona state legislators who fear that phytocannabinoids might be addictive. We’ve another question: are things that modulate endocannabinoids addictive?

The two main endocannabinoids

The featured image of this page illustrates some possible structures of endocannabinoids anandamide and 2-arachidonyl glycerol. The ambiguity of endocannabinoids arises from rotational freedom about many of the carbon-carbon bonds. . The double bonds, seen as a double line in the structures, are pretty inflexible. The single carbon-carbon bonds are free to rotate. This gives us a variety of structural possibilities. The structures that resemble a paddle are from PubChem. The snake like structure is arachidonic acid all stretched out. The folded in on themselves structures resemble arachidonic acid on its way to becoming prostaglandins and other eicosanoids. Many will recognize the little green icon as the basic structure of a phytocannabinoid. This page will give two examples that address concerns that Arizona state legislatures have that phytocannabinoids might be addicted. Well, if they are, they are are not nearly addictive as endocannabinoids!

Components of the Western diet and exercise release endocannabinoids in mice and humans.

Running and Western diet junk food are very addictive via endocannabinoid pathways.

The runner’s high is all about anandamide

Fuss and coworkers (2015) questioned previous reports that the runner’s high is due to a combination of endorphins and the endocannabinoid anandamide.  Anandamide is derived from a Sanskrit word for joy.  Unlike the lipophilic anandamide, β-endorphin cannot cross the blood–brain barrier.  Fuss and coworkers used a combination of

  1. pharmaceuticals that block endorphin  and endocannabinoid receptors (CB1 and CB2)
  2. a genetically engineered mouse that lacks cannabinoid receptors

in an attempt to dissect the mechanism of a runner’s high. 

Prolonged running is known to increase blood levels of β-endorphin and anandamide  in mice and in men.  There are three components of the runners’ high in humans (1) euphoria, (2) axiolysis, and (3) analgesia.  The authors were unable to measure euphoria in mice, but they were able to measure a decrease in mouse anxiety in stressful situations as well as pain relief. 








β-endorphin is a 31 amino acid neuropeptide. This image was obtained from PubChem.

The mice were allowed to run on a wheel until the day they were split into groups.  The running group was allowed to continue running.  The control group had their wheels taken away. Some human runners may joke that there were no runners on the Institutional Review Board that approved this study. Taking away that wheel constitutes animal torture!

Except for 2-AG and PEA, plasma levels of all endocannabinoids and related compounds increased with wheel running. When p=0.05 we are 95% sure that the observed difference is not due to chance alone.  P<0.05 is generally considered significant.  We can be 99.9% sure that running produces endocannabinnoids.  More amazing is that these little mice can run over 6 km per day without massive inflammation, i.e. the arachidonic acid being used to produce prostaglandins and leukotrienes.

From reference {1}
The benefits of running according to Fuss and coworkers.

For a little mouse, going from a nice, dark, secure chamber to a brightly lit (600 lux) chamber might cause some anxiety (graph B and image below).  The runner mice were able to spend more time exploring the well lit chamber. 

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From Fuss ref [1] Figure 1. Their data plus some images to illustrate what is being measured.

For a human, and probably a little mouse, 53oC is right at the threshold of too hot to handle.    The mouse equivalent was sitting on a hot plate set at 53 oC (± 0.3 oC ). Time of first licking hind paws or jumping was assessed with a cutoff at 45 s to prevent injury. 

Endocannabinoids versus endorphins in the runners’ high

If running induced behavioral changes are elicited by endo cannabinoids binding to cannabinoid recptors CB1, CB2, or both, pharmaceutical inhibitors should prevent these changes.  It should be remembered that there are central and peripheral CB receptors.  Some inhibitors can pass the blood brain barrier, others cannot.

Running releases β-dendorphins and endocannabinoids.  The receptors for these compounds were blocked with small molecules.

  • The Vehicle is the control.  The runner mice were able to spend more time in the bright lights.
  • The opiod receptor antagonis Naloxon failed to oblate the running induced light tolerance.  So much for endorphins binding to the opiod receptor. 
  • AM630, the CB2 receptor antagonist, failed to reverse the running induced light tolerance.

The failure of AM6545, the peripheral CB1 antagonist, to reduce the running induced bright light tolerance tells us endocannabinoids are not eliciting a peripheral effect that is working its way to the brain. AM251, the brain CB1 antagonist, also reduced the running induced light tolerance.

Exploring the lighted box is also about motivation

Fuss and coworkers established that endocannabinoids work on central CB1 receptors.  The time spent in the light box is about

  1. feeling motivated to enter a strange place in case there might be a treat in there and
  2. feeling chilled out enough to explore the place that might induce anxiety.

Both the  VTA and NAcc are involved with processing motivation and reward.  The PFC is involved with decision making, planning, and complex “cognitive” behavior.  Images similar to this one is used to explain adjective behavior.   What does running induced endocannabinoids mean for a mouse exploring a well light box where there might be a treat?  

Presynaptic GABA releasing neurons put the brakes on the dopamine releasing neurons at pre-synaptic terminals. Anandamide binding to CB1 of GABA releasing neurons takes the neurochemical foot off the bake and prevents GABA release into the synaptic cleft..
From Fuss et al. ref [1 In mice lacking the CB1 receptor running does not make a difference in courage to B enter the light box and C stay in it to explore]

The authors dissected what was going on in the light box.  The running mice that have the CB1 receptor take less time to get up the nerve to explore the lighted box than the genetically engineered mice that like the CB1 receptor.  Running makes no difference in the mice that lack the CB1 receptor.   In mice with CB1 receptors, running makes them “chill” enough to spend almost twice as much time in the lighted box.   In mice lacking the CB1 receptor, running makes no difference. 

Fuss and coworkers were able to address

  1. lowered anxiety
  2. lowered pain threshold in their mouse model
  3. but failed to address the sense of well being in mice. 
Does running lower the threshold of other reward seeking behaviors?

The Science Daily blog about the work of the French team of Francis Chaouloff and coworkers simply looked at a way of getting addicted to exercise as a means of weight control and overall health.  The study of Fuss and coworkers opens the possibility that running or other forms of exercise that release endocannabinoids can empower humans to overcome fears and push past their comfort zones to obtain a potential reward. Is this a bad addiction?

2-AG and why you cannot eat just one potato chip

Urban legend has it that high fat and high sucrose foods that have become a part of the Western diet are engineered to make us want to eat more.  This site suggests that some of our favorite junk food companies are also owned by Phillip Morris, then junk food makers may be the new cigarette companies deliberately engineering foods to make them additive. Donovan Argueta and Nicholas V. DiPatrizio of the University of California River addressed the role of endocanabinoid system in Western diet induced obesity. [2] They showed an endocannabinoid response when mice are fed a standard Western diet. [2]

A quick inventory of the enteric nervous system…

Dr Depatrizeo has two decades experience in the CB1 receptor and behavior.  His work and that of other laboratories was summarized in the introduction of this feeding study. In rats, fasting up to 24 hours was shown to be associated with an increase in the release of the endogenous cannabinoid (eCB)  2-arachidonoyl-sn-glycerol (2-AG) from the upper part of the small intestine.  This release was dependent on the release of cholinergic neurotransmitters, presumably from the  vagus nerve. [2] Fasting induced  2-AG biosynthesis in the jejunum was inhibited by

  1. full subdiaphragmatic vagotomy
  2. local intraduodenal infusionof the subtype 3 muscarinic acetylcholine receptor (m3 mAChR) antagonist,DAU5884.
  3. DAU5884 blocked  fasting-induced refeeding.

Gut-brain eCB signaling is a proposed orexigenic signal that may promote feeding under several distinct behavioral and metabolic conditions. The literature suggested peripheral eCB levels are increased under conditions of obesity. The authors tested the hypothesis that components of a  a western-style diet (WD) promote eCB release from the the upper small intestinal epithelium of mice and the resulting hyperphagia associated with a WD.  The authors also reported increased expression of genes encoding key eCB system components in the small intestine.

Materials and Methods in Brief

Lab Diet 5001

Dehulled soybean meal, ground corn, dried beet pulp, fish meal, ground oats, dehydrated alfalfa meal, can molasses, brewers dried yeast, wheat germ, whey, porcine animal fat preserved with BHA, citric acid, wheat middlings, porcine meat, bone meal, and other vitamins and minerals.

Calories provided by:

  • protein 29.8%
  • fat        13.4%
  • carbohydrates 56.7%
Western diet Research Diets D1270B

Casein, methionine, cornstarch , maltodextrin, sucrose, cellulose, milk fat, cholesterol, corn oil, choline bitartrate, ethoxyquin (food preservative)

Calories provided by:

  • protein 17%
  • fat  41%
  • carbohydrate  43%

Dr Dipatrizio has a long history of studying animal behavior in  response to CB receptor signaling.  Some of the feeding parameters they examined included

  • total caloric intake
  • average meal size
  • average rate of intake (kcals from food per minute)
  • average number of meals
  • average meal duration
  • average post-meal interval
Mice on the Western Diet put on more weight
Ref [2] Fig.1.Chronic consumption of a Western diet is associated with incread weight gain. Male mice maintained for 60 days on a western diet(Western) become obese (a, cumulative change in body weight; b,gross body weight .Repeated measures two-way ANOVA,with Sidak’s multiple comparison pos thoc test,***=p<0.001( a); unpaired Student’s t-test (two tailed), ***=p<0.001 between St and and Western. Results are expressed as means ± SEM; n=16/condition(a,b)

At the end of this period, the Western diet mice had put on about 15g.  When total body weight, the Western diet mice were substantially bigger than the control (healthy) diet mice.  What was going on with these mice?

Reference [2] Fig.1.Chronic consumption of a Western diet is as associated with hyperphagia. Male mice main tained for 60 days on a western diet(Western) ate more in 24 h, ate bigger meals, and ate faster when compared to mice on standard chow. Repeated measures two-way ANOVA,with unpaired Student’s t-test (two tailed), ***=p<0.001 between St and and Western. Results are expressed as means ± SEMn=8/condition(c-e)

In a 24 hour period the mice were consuming more calories (Fig 1c),  their meal size had increased in terms of caloric content (Fig 1d), and the rate that they consumed their food in terms of kcal (food calories) per minute.  One has to immediately start wondering about the caloric density of the two diets.  This is what we see in Figures 1f-h

From reference [2] Fig.1.Chronic consumption of a Western diet does not change meal timing. Male mice maintained for 60 days on a western diet(Western) Meal duration,frequency,and post meal interval do not significantly differ between diets (f-h) .Repeated measures two-way ANOVA, with ; unpaired Student’s t-test (two tailed), ***=p<0.001 between St and and Western. Results are expressed as means ± SEM; n=8/condition(f-h)
From reference [2] Fig.2. Mice fed a Western diet display increases in levels of 2-AG and anandamide in jejunum mucosa and plasma. Two-way ANOVA with Student-Newman-Keuls multiple comparison post hoc test.=pb0.05,**= p<0.001, ns= not signigicant. Results are expressed as means±SEM; n=7/condition for jejunum,n=9–10/condition for plasma.

Blockade of peripheral CB1 receptors with AM6545 reduces

  • total caloric intake
  • meal size
  • rate of intake

in WD mice to levels indistinguishable from SD control mice.

What about humans?

An Australian followup study examined blood endocannabinoid levels in lean, overweight, and obese men. These men had lipids directly infused into their duodenum. [3] Obese men were found to have greater fasting levels of anandamide. [3] It gets worse. Some North Americans were asked to consume aspartame or high fructose corn syrup sweetened beverages (Kool Aid)

In this study [5] participants consumed not diet cola versus HFCs sweetened colas but Kool Aid sweetened with high fructose corn syrup or Aspartame.

Stay tuned. The results of this study [4] will be posted shortly. The results were not dramatic, but large enough to give pause to Arizonans who consume HFCS sweetened beverages by the liter during the summer time. In addition to anandamide and 2-AG these authors also measured oleoylethanolamide (OEA), docosahexaenoyl ethanolamide (DHEA), and docosahexaenoyl glycerol (DHG). Fasting levels of minor endocannabinoids. More important, these investigators measured markers of cardiovascular disease (CVD). [4]

Some mionor endocannabinoids measured in the HFCS study. [4]
  • OEA is the monounsaturated analogue of the endocannabinoid anandamide It has a role as a PPARalpha agonist and an EC 3.5.1.23 (ceramidase) inhibitor. It derives from an oleic acid rather than arachidonic acid.
  • DHEA is a bit different from anandamide. It derives from an all-cis-docosa-4,7,10,13,16,19-hexaenoic acid. Arachidonic acid has only four double bonds.
  • DHG has an indefinite structure

Study subjects included 18–40 year old adults with a body mass index (BMI) of 18–35 kg/m2 with no major weight gains or losses in the previous six months. Diabetic and bariatric surgery individuals were excluded. the experimental groups were matched for sex, BMI, In this study the participants were rigorously matched based on: fasting triglyceride concentration, cholesterol, high-density lipoprotein cholesterol, and plasma insulin. Over the course of two weeks significant decreases were seen in anandamide, DHEA, and DEA. [4]. When expressed in terms of percent change, there were significant increases in plasma anandamide and DHEA in the HFCS group. [4]

Assorted data from reference [4]. Table 1 was rearranged so that the entries aligned with Table 3. For instance, participants started the study with statistically similar BMI. The HFCS group gained on average 0.8 kg. Figures 3-4 are regression plots of change in CVD marker as a function of endocannabinoid change. The r2/p values in which p<0.05 were copied and pasted into the table shown.

Making sense of these results came in a later review publication. We are going to take a summary image from the publication [5] and use it as a guide to do our own thinking. Given how our bodies respond to the deleterious things in the Western diet, are phytocannabinoids really that scary if one uses common sense and is monitored by a health care provider?

From Ref [5]
  1. For starters, the apical membrane is of epithelial cells of our gut is the site facing the lumen that sees digested food stuff. The free fatty acid receptor 1 (FFAR1) UniProt confirms that FFAR1 is a GPCR just like CB1Rs. We are told that FFAR4 is another GPCR involved in the secretion of CCk and Let’s pick on linolineic acid, a component of the canola oil used to fry potato chips. This fatty acid may become conjugated. We also learn from UniProt that linolineic acid is a strong stimulant of FFAR1. FFAR4 is also activated by linolineic of our potato chip conspiracy theory. Proteinatlas.com mRNA expression data tells us that there is good expression of FFAR1 in the gastrointestinal tract but it is particularly high in the pancreas. FFAR4 mRNA is more in the rectum and colon. There is no comment on whether linolineic acid conjugation affects either receptor activation.
  2. The Steinert review is all most of us want to know about nutrient signalling in the gut. [6] FFAR1 expressing enteroendocrine cells secrete hunger suppressant CCK. [6] FFAR4 expressing enteroendocrine cells secrete CCK, GLP1, and the hunger peptide ghrelin that is secreted mostly by stomach EC in response to stretch. [6] CB
  3. We are reminded of endocrine signalling by secretion of hormones into the blood that may travel to the brain as well as neurocrine signalling as described by the DiPatrizio review. {5]
  4. It should be pointed out that endocannabinoid feedback inhibition of hormone release theoretically applies to endocrine, neurocrine and paracrine mechanisms. Where do endocannabinoids come from? “Tasting dietary fats—but not sucrose or protein—triggered production of eCBs in the upper small-intestinal epithelium, but not in other peripheral organs tested” [5] “This effect was also specific for mono- and di-unsaturated fats (oleic acid and linoleic acid), but not saturated (stearic acid) or polyunsaturated fats (linolenic acid)” Lay’s potato chips are fried in sunflower ( 84%, 4% ) corn (27%, 58%), and/or safflower oil ( 75%, 13%) Values in parentheses come from a Wikipedia page on corn oil. Percentages are for oleic and linoleic acids, respectively. back on how this impacts the DiPatrizio review [5]
  5. CB1 receptors may also control Vagus afferent neurons by controlling ghrelin release. [5] Ghrelin release and action is discussed in greater depth in another review. [6]
  6. CB1 receptors may also control Vagus afferent neurons. [5]

The potato chip conspiracy theory

  • Linolineic acid, a healthy fatty acid, shuts down the feeding response by binding to FFAR1 in enteroendocrine cells. These cells secrete the satiety hormone CCK.
  • Before the potato chip even gets to the duodenum, endocannabinoids are released into the duodenum.
  • What small percentage of linolineic acid and other polyunsaturated fatty acids were used in the potato chip frying do little to elicit the release of CCK.

Since this site is about terpenes and entourage effects, what terpenes might be in those flavors that make potato chips so addictive? The BBQ flavor chips contain paprika. Depending on how it is processed, paprika contains terpenes also found in cannabis. [7]

Table 2 from reference [7] Note that terpenes found in paprika are also found in cannabis flowers.

Do these terpenes in BBQ flavored potato chips have an entourage effect? Would they have an entourage effect in recovery foods that runners consume? This site is not endorsing any sort of cannabis product for minors without medical supervision. Are our children receiving medical supervision when they consume potato chips? Certainly medical supervision is sometimes encouraged when a person starts an exercise program that will result in the release of endocannabinoids. The state legislator is concerned about the psychological effects of phytocannabinoids. Perhaps we should also be concerned about how the Western Diet is affecting our endocannabinoid systems.

References

  1. Fuss J, Steinle J, Bindila L, Auer MK, Kirchherr H, Lutz B, Gass P.(2015) A runner’s high depends on cannabinoid receptors in mice.Proc Natl Acad Sci U S A.112(42):13105-8. PubMed
  2. Argueta, D. A., & DiPatrizio, N. V. (2017). Peripheral endocannabinoid signaling controls hyperphagia in western diet-induced obesity. Physiology & behavior, 171, 32–39. PMC free article
  3. Little, T. J., Cvijanovic, N., DiPatrizio, N. V., Argueta, D. A., Rayner, C. K., Feinle-Bisset, C., & Young, R. L. (2018). Plasma endocannabinoid levels in lean, overweight, and obese humans: relationships to intestinal permeability markers, inflammation, and incretin secretion. American journal of physiology. Endocrinology and metabolism, 315(4), E489–E495. PMC free article
  4. Price, C. A., Argueta, D. A., Medici, V., Bremer, A. A., Lee, V., Nunez, M. V., Chen, G. X., Keim, N. L., Havel, P. J., Stanhope, K. L., & DiPatrizio, N. V. (2018). Plasma fatty acid ethanolamides are associated with postprandial triglycerides, ApoCIII, and ApoE in humans consuming a high-fructose corn syrup-sweetened beverage American journal of physiology. Endocrinology and metabolism, 315(2), E141–E149. PMC free article
  5. DiPatrizio N. V. (2021). Endocannabinoids and the Gut-Brain Control of Food Intake and Obesity. Nutrients, 13(4), 1214. PMC free article
  6. Steinert, R. E., Feinle-Bisset, C., Asarian, L., Horowitz, M., Beglinger, C., & Geary, N. (2017). Ghrelin, CCK, GLP-1, and PYY(3-36): Secretory Controls and Physiological Roles in Eating and Glycemia in Health, Obesity, and After RYGB. Physiological reviews, 97(1), 411–463. PMC free article
  7. Kevrešana ŽS, JMastilovića JS, Sinadinović-FišerbS, Hrabovskic nČ ,Radusin TI(2013) Spice paprika volatiles as affected by different post harvest ripening treatments of red pepper (Capsicum annum L.) variety aleva NK. APTEFF, 44, 1-321 free article

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