Oleuropein

We at CopperOne have been telling customers to add cuprous niacin to an oil to keep it in the +1 oxidation state. Why not olive oil? Oleuropein is “the most important phenolic compound present in olive cultivars. It has a role as a plant metabolite, a radical scavenger, an anti-inflammatory agent, an antineoplastic agent, an antihypertensive agent, a NF-kappaB inhibitor, an apoptosis inducer, an antioxidant and a nutraceutical.”

While olive leaves are the main source of oleuropein sold by neutraceutical sorts, olive oil is a common dietary source. We at CopperOne will not go there. We are just mentioning this because customers are intrested in the use of oils to keep their CopperOne in the plus +1 oxidation state.

A high fat diet study and copper handling proteins

Santini, S. J., Tarantino, G., Iezzi, A., Alisi, A., & Balsano, C. (2022). Copper-catalyzed dicarbonyl stress in NAFLD mice: protective effects of Oleuropein treatment on liver damage. Nutrition & metabolism, 19(1), 9. https://doi.org/10.1186/s12986-022-00641-z

Mice were fed a normal or high fat diet for 8 weeks. At the end of this time mice were split into four different groups with four females and four males in each group

• normal protein/carbohydrate/fat 24/58/18
• high fat protein/carbohydrate/fat 15/43/42
• normal diet plus 5.6 mg/kg body weight oleuropein
• high fat plus 5.6 mg/kg body weight oleuropein

About a third of the carbohydrates in the high fat diet came from sucrose. Values are in percent total food calories. The gap in the logic of this rather fascinating publication is the focus on methylglyoxylal, a byproduct of glycolysis.

Note the three blue nitrogens (Ns) on the side chain of arginine. They carry a positive charge. When methylglyoxal reacts with them, there is no longer a positive charge on the arginine side chain and whatever biochemical interactions that come from it.

1. Why Cu²⁺ and a high fat diet are toxic

What is frustrating is that we cannot easily determine what the oxidation state is of the copper is in the diet of these mice. Only small portions of the much larger H&E stained images are shown in the panel 1a presented in this post. The take home are the large fat droplets in the liver slices in the liver from a high fat diet (HFD) mouse.

The authors think that oleuropein is chelating Cu²⁺. Why does Cu²⁺ need to be chelated? For starters, Cu²⁺ may oxidize glutathione (GSH), a small molecule needed to maintain redox balance. Oxidized glutathione, GSSG, might also form mixed thiols with protein thiols, i.e. PSSG.

Glyoxylases are enzymes that detoxify methylglyoxal and other aldehydes before they have a chance to form adducts on proteins. They rely on reduced GSH for detoxification.

The Code Before moving on to looking at bar graphs, this is the code the authors used to make comparisons:

• * is a comparison between the ND (normal diet) and the HFD (high fat diet)
• # is a comparison between HFD and HFD with oleuropein
• § is a comparison between males and females
• 3 symbols in a row, for example ####, means significant at the p<0.001 level
• 2 symbols in a row, ##, means significant at the p<0.01 level
• just one symbol, #, means significant at the p<0.05 level, or 95% certain that the observed difference is not due to random chance. The lower the p value, the more sure we are the results are not due to chance.

2. Oleuropein blunts Cu accumulation

Here the authors are only looking at hepatic and serum Cu. Other tissues were not examined, probably for practical reasons. Small sections of the larger H&E stains are shown only to make the point that a high fat diet results in lipid accumulation in the liver. Male female differences (§, 2c) are not seen in liver Cu content but are seen in the the serum (§, 2b). Oleuopein in the HFD( #, 2b) seems seems to be specific to the female serum but seen in both genders in determining hepatic Cu content (2c).

The male mice on the HFD seem to have the greatest elevation of serum copper. Oleuropein seems to blunt this increase. We do not know if this increase in serum copper is protective or detrimental. The changes in hepatic copper (2c) are really not that great.

3 Ramping up of Cu⁺ channel translation

Many times scientists look only at mRNA transcripts of a given gene because it is easy to perform quick and relatively inexpensive analysis of these transcripts. Not every protein for which transcript are produced in the nucleus get translated into proteins. The scientist has to use a technique known as Western blotting to quantify the levels of a given protein. This post will not show the Western blots. Oleuropein increases the Ctr1 protein levels in male and female mice fed a high fat diet at the p<0.05 level of significance. (#, 3b, second panel.

4 Getting Cu out and more GSH

Figure 1d,e speculated that the increase in Cu²⁺ with a high fat diet could cause GSH to decrease and methylglyoxylate to increase. This next figure combines Figures 4 and 6 from the study. Recall that ATP7B is the Cu⁺ efflux pump.

Oeuropein increases the protein levels of the Cu efflux pump (6b) in males and females, but more so in females. Similar improvements are seen in the increased ratio of GSH to GSSG (6a) and decreased MG-H1 levels.

5. Three Cu chaperones

This image was taken from Figure 5 of the Santini 2022 publication. Some images have been added to document the Cu cofactor enzymes that receive the Cu.

Let’s concentrate on protein levels because not every mRNA transcript for a protein produced in the nucleus gets translated into a protein. Oleuropein really increases the transcripts for the Copper Chaperone for Superoxide dismutase (CCS) without a similar increase in actual protein levels (5b). Cu/Zn Superoxide dismutase scavenges the reactive oxygen species superoxide. Cox17 transfers Cu⁺ to cytochrome C oxidase in the mitochondria. We need this transfer for production of ATP. This transfer needs to occur to burn fat too! Finally, Atox1 transfers Cu+ to both Cu+ ATPases: ATP7A transports Cu into the cerebral spinal fluid for the brain and into the golgi for making secreted copper based enayzmes. ATP7B exports extra Cu into the bile.

7 detoxifying methylglyoxal

Figure 6 of the Santini publication was presented with Figure 4 because the authors propose that Cu²⁺ can catalyze the depletion of GSH that is used as a substrate for glyoxylate enzymes that detoxify aldehyde groups that are the precursors to advanced glycation end products. Panels 7a and 7b follow a logic similar to seen in previous figures from the Santini publication.

1. An mRNA transcript is produced in the nucleus… doesn’t mean it gets translated into a protein
2. Just because the ribosomes translate mRNAs into proteins… doesn’t mean they are active enzymes.
3. Just because a protein is made, doesn’t mean it is active. It might require a post translational modification like attaching a phosphate group to a key amino acid. It might also require a cofactor, such as GSH in our case.

Click here to go up to the cartoon of Glo1 and Glo2 enzyme action. Note that only Glo1 uses GSH. The authors added exogenous GSH in their assays so that lack of reduced GSH in the liver homogenate would probably not have been a factor.

Santini and coathors presented one last piece of data suggesting which transcription factor oleuropein works via to produce more Glo1 and Glo2. Let’s first summarize what has been presented so far.

Summary

1. In a mouse model, a high fat diet decreases reduced glutathione that is needed for detoxifying protein modifying methylglyoxal. Toxic Cu²⁺ may be the reason.
2. Oleuropein, a compound n olive oil, blunts Cu accumulation in the liver and serum. We don’t know if the Cu is getting to other tissues.
3. Oleuropein increases mRNA transcripts for Cu⁺ channels Ctr1 and Ctr2. We at CopperOne think that this is a good thing.
4. In mice fed a high fat diet, oleuropine increases the transcripts for the Cu+ efflux pump too. We don’t’ know about the Cu+ pump ATP7A that delivers Cu+ for making proteins that are exported from the cell.
5. Oleuropein increases mRNA and Cu⁺ chaperone proteins themselves that deliver Cu+ to enzymes we need to make ATP, combat reactive oxygen species, and making critical Cu enzymes that are exported from the cell.
6. Oeleuropein increases reduced GSH in mice on a high fat diet. The toxic glycolysis byproduct methylglyoxal is decreased too.
7. Oleuropein increases the activity of enzymes that metabolize this toxic byproduct of too much sugar.

Santini and coauthors have given us some insight as to why Cu²⁺ is toxic, particularly when we eat way too much sugar. We’ve been thinking of oils for keeping CopperOne in the +1 redox state. Why not olive oil with oleuropein to increase the expression of Ctr1 an copper chaperones? Naturally more studies will be required to prove this one.