NAFLD and copper deficiency

For you cooks out there, have you ever been cooking a pot of something and and added a second ingredient to compensate for the first? Soon the second ingredient becomes too much and needs to be dealt with. The Harder 2022 study started with the premise that hepatic copper is regulated by fat accumulation.  It makes sense right? If we actually want to burn fat, we must have copper in the cytochrome C oxidase of our mitochondria. Mitochondria tend to be a source of superoxide. We might want to have some Cu/Zn super oxide dismutase (SOD) in addition to the Mn SOD of the mitochondria. When you add one ingredient to the pot, you have to add another…

Daniel Harder and others in Marie Heffern’s laboratory at UC Davis addressed this problem in a cell culture model of fatty liver disease. These coauthors observed that copper is sequestered in a manner that mimics copper deficiency when we the added too much fat to the hepatocyte pot.  DepG2, liver hepatocarcinoma cells, were fed palmitic acid, a 16 carbon saturated fatty acid. Palm oil is a good source of this fatty acid.Figure 1  small, significant in copper handling proteins. Human hepatic carcinoma cells, HepG2, were grown in a standard medium with growth factor containing fetal bovine serum and antibiotics to control the growth of microorganisms. 

HepG2 cells were stimulated with

• 250 µM palmitic (PA)  
• 200 µM fatty acid free bovine serum albumin (BSA) control media.

Ctr1 and intercellular Cu, not measured.

Harder and coworkers included small molecule transport SLC46A3, but not the Cu⁺ transporter Ctr1 in their studies. They also did not measure intracellular copper in response to the PA. The Ctr1 gene, SLC31A1. This site has covered the selectivity of Ctr1 but not whether or not energy sources that require copper enzymes increase its expression. As a general note, the Harder study also did not examine Cu binding to the transcription factor Sp1 that turns off the transcription of CTR1/SLC31A1.

Figure 1D pumping out Cu with ATP7B

Let’s take a look at panel D of Figure 1 first. Harder and others used a technique called immunocytochemistry to visualize two proteins at one: Green is ATP7B, a membrane protein that uses ATP to put Cu⁺ into vesicle that are transported from the trans Golgi network (TGN) to the surface of the cell. Red in 1D is TGN46. a protein maker of Golgi vesicles. The Hepatocyte image on the right was modified from this public access site.

Hepatocyte copper handling proteins in response to PA

At the designated time periods cells were lysed and proteins dissolved in a detergent that gives them a negative charge.  The proteins were separated on a “gel” based on their size as they move through the gel in response to a potential difference.  Proteins in the gel were transferred to a membrane that was incubated with primary antibodies against the copper handling proteins of interest.  These antibodies tend to be produced by injecting rabbits with purified proteins.  The rabbit primary antibodies were detected by antibodies against the invariable parts of rabbit antibodies raised in another animal such a goat.  These goat antibodies are tagged with a reporter.  This is what we see here.  Scientists always perform experiments many times.  Nine times is abbreviated as ”n=9”    

Figure 1A pumping Cu⁺ out of the cell

ATP7B is the copper export protein that uses ATP to pump Cu⁺ out of the cell.  PA in the cell culture medium causes a slight increase in this protein relative to the control.  First protein levels are normalized to a cytoskeletal protein called β-actin.  Then the normalization to the control is computed.

ATP7B is the copper export protein that uses ATP to pump Cu⁺ out of the cell.  PA in the cell culture medium causes a slight increase in this protein relative to the control.  First protein levels are normalized to a cytoskeletal protein called β-actin.  Then the normalization to the control is computed.

The mean is another word for the average.  The SEM is the standard error of the mean.  It is simply an indicator of how much the replicates resemble each other.  The smaller the SEM, the tighter the data.  The “p” value indicates how confident we are that the results are not due to random chance.  The smaller the p value,the more confident we are in the results.  The general cutoff for “statistical significance” is p<0.05. 

Figure 1B, copper chaperone superoxide dismutase

CCS, the copper chaperone for Cu/Zn superoxide dismutase We’ve discussed CCS and Cu/Zn on this site.  If mitochondria are a major source of superoxide, and if they use electrons from fatty acid β-oxidation making more Cu/Zn SOD would sort of be a good idea when taking in more PA.  This is what we see. 

Figure 1C, a transcription factor for Cu/Zn SOD3

ATOX1 is next on the list as it is the chaperone for the export pump ATP7B. Note that unlike ATP7B in panel 1A, ATOX1 increases in response to PA. This site has touched on Atox1 as a transcription factor for Cu/Zn SOD3.

.  COMMD1 is an accessory protein to ATP7B whose exact function, according to Harder and coauthors, is somewhat obscure.  [1]

Figure 2 Other ways to export Cu

The liver makes ceruloplasmin.  Ceruloploasmin is also a protein that transports copper and iron in the blood stream.  Ferroxidase activity happens when ceruloplasmin (Cp) is replete with both copper and iron.  As shown in figure 2, PA has not bearing on Cp secretion and activity.

Figure 3 binding up excess Cu

Metallothionein 2A is a putative Cu²⁺ storage protein.  The level of this protein initially increases with PA and then returns to a level statistically less than the BSA control.

Figure 3 Redox balance

The Cu/Zn SOD1 initially increases yet returns to a level indistinguishable from the control.    Figure 3C pertained to the ratio of oxidized to reduced glutathione.  Oxidized glutathione is spent glutathione that can no longer reduced oxidized protein thiols or buffer metal ions like Cu⁺

PA results in an initial loss of redox capacity.

This is a nice, thought provoking study with a lot of unanswered questions.

1. Did intracellular Cu ever increase in response to PA?
2. What about Cu transporter Ctr1?
3. What energy demands of these HepG2 cells could possibly be driving PA conversion to ATP? In other words, if there are no energy demands, what could possibly drive beta oxidation of PA and hence the need for copper?
4. Would the same scenario in their summary Figure 5 also exist in cells that actually burn fatty acid to ATP, CO₂ and H₂O?

It just seems that the end game is to stop the accumulation of excess dietary fats in the liver.

Copper, niacin, other lifestyle factors, and NAFLD

Perhaps the biggest issue we have is to also be finding a reservoir for dumping fat where it will be turned to CO2, H2O, and ideally, ATP. Brown adipose turns fat into CO2, H2O, and heat instead of ATP. An Iranian study points to exercising muscles. This introduction figure shows some ground beef with obvious fat deposits along with some beef fat. Beta oxidation of fatty acids from the triglycerides will enter the TCA (citric acid) cycle in two carbon units. These are released as CO2, which is starred. Cu+ is essential for completion of burning the fatty acids to CO₂ ( started in the TCA cycle) and H₂O.

This study took place at the  Metabolic Liver Disease Research Center at Isfahan University of Medical Sciences in 2019. [2]The study included 405 controls and 225 newly diagnosed cases of NAFLD.  Participants were given a validated   semi-quantitative food frequency questionnaire (FFQ).  Four major nutrient patterns. Were identified:

1. The first nutrient pattern was high in consumption of lactose, animal protein, vitamin D, riboflavin, pantothenic acid, vitamin B12, calcium, phosphorus, zinc, and potassium.
2. The second nutrient pattern included fiber, plant protein, vitamin A, thiamine,niacin, copper, and selenium,
3. The third featured plant protein, zinc, copper, magnesium, manganese, chromium, and selenium.
4. The fourth was characterized by fructose, vitamin A, pyridoxine, vitamin C, and potassium.

After adjusting for confounders, individuals in the highest tertile of NP4 had lower odds of NAFLD (OR: 0.56, 95% CI: 0.32–0.98, P_trend = 0.042);compared to those who were in the lowest tertile

What really is important ,and not important ,between healthy controls and Iranians with NAFLD is somewhat counter intuitive by North American standards. With the exception of copper and niacin, non significant factors have been edited out of Table 2. The counter intuitive demographic variables are that healthy controls consume more sucrose and less plant protein. [2] Physical activity and BMI are the most significant differences between healthy Iranians and those with NAFLD.

The clustering of copper and niacin in the four nutrient patterns may simply be a factor of which foods consumed in bulk contain which nutrients. This is addressed in Table 3 of the study. [2]

Concluding thoughts

Does a similar feedback loop exist in muscle in which palmitic or other fatty acids can influence copper chaperone and transport proteins?

1. BMI is still a very significant predictor of NAFLD. Is there any room to argue that is is possible to kick fat out of the liver to be deposited somewhere more “healthy”? Is there a back and forth between fat in the liver, visceral fat, subcutaneous fat, and intramuscular fat? Certainly the latter two are more healthy than the first two.
2. Lack of exercise is a predictor of NAFLD. This argues that burning dietary fats to CO₂ and H₂0 may be even more important than not consuming the fats in the first place and avoiding things like fructose that may come from fruits or high fructose corn syrup sweeteners popular in the west. It seems ludicrous to think there is a drug that can make liver accumulated fat healthy. A drug would be nice to expedite the mobilization of fat from the liver to the exercising muscle. Copper is absolutely needed to make sure that that exercising muscle is NOT dependent on glycolysis for ATP.

References

1. Harder NHO, Lee HP, Flood VJ, San Juan JA, Gillette SK, Heffern MC. Fatty Acid Uptake in Liver Hepatocytes Induces Relocalization and Sequestration of Intracellular Copper. Front Mol Biosci. 2022 Apr 11;9:863296. PMC free article
2. Salehi-Sahlabadi A, Teymoori F, Ahmadirad H, Mokhtari E, Azadi M, Seraj SS, Hekmatdoost A. Nutrient patterns and non-alcoholic fatty liver disease in Iranian Adul: A case-control study. Front Nutr. 2022 Sep 6;9:977403. PMC free article