Category: fatty liver disease

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 (in the TCA cycle) and H₂O.

What separates healthy eaters from the NAFLD pack?

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). The take home study was that these participants were eating wonderful Persian food. Their problem was they were eating too much of it and not getting enough exercise. The food cluster that proved to be protective is counter intuitive for those eating a Western diet.  Four major nutrient patterns. Were identified:

Yogurt and beef along with rice and vegetables are a common part of the Persian diet.

Persians like to enjoy dried fruits with their nuts. Oddly dried fruits were not part of this group.

So what is a healthy diet?

This is a table of correlation coefficients. A value close to zero means no association. A value close to -1.0 is a strong negative correlation. For example Nutrient pattern 4 were eating large amounts of fruit (R=0.81), drinking their share of natural fruit juices (R=0.385), and kind of avoiding red meat (R= -0.105).

	Nutrient pattern 1	Nutrient pattern 2	Nutrient pattern 3	Nutrient pattern 4
Whole grain(g/d)	−0.026	0.141a	0.752a	−0.087b
Refined grain(g/d)	−0.239a	0.427a	−0.194a	−0.360a
Legume(g/d)	−0.006	0.155a	0.071	0.008
Nuts(g/d)	−0.012	−0.152a	0.131b	0.216a
Red and processed meat(g/d)	−0.017	−0.093b	−0.011	−0.105b
White meats(g/d)	−0.035	−0.052	−0.007	0.07
Low fat dairy(g/d)	0.705a	−0.172a	−0.099b	0.046
High fat dairy(g/d)	0.230a	−0.113b	−0.113b	−0.123b
Fruits(g/d)	0.047	−0.073	−0.115b	0.810a
Vegetables(g/d)	0.097b	0.035	−0.051	0.369a
Egg (serving/d)	0.091b	0.034	0.002	−0.091b
Fruit juice (serving/d)	0.047	−0.082b	−0.034	0.385a
Snacks (serving/d)	−0.129b	−0.300a	−0.120b	−0.080b
Artificial bevarages (serving/d)	−0.111b	−0.086b	−0.092b	0.085b
Plant oil (serving/d)	0.095b	−0.014	0.047	0.101b

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. Neither copper nor niacin were different between the healthy controls and those with NAFLD. Physical activity and BMI are the most significant differences between healthy Iranians and those with NAFLD.

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. Copper is absolutely needed to make sure that that exercising muscle is NOT dependent on glycolysis for ATP. For the Iranians participating in this study, access to healthy food was not the problem. Cultural taboos against scantily clade males and females exercising in the heat of the summer might be a factor.

Perhaps a copper and niacin supplement can be part of transitioning to healthy eating and an exercise plan.

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

Song, M., Li, X., Zhang, X., Shi, H., Vos, M. B., Wei, X., Wang, Y., Gao, H., Rouchka, E. C., Yin, X., Zhou, Z., Prough, R. A., Cave, M. C., & McClain, C. J. (2018). Dietary copper-fructose interactions alter gut microbial activity in male rats. American journal of physiology. Gastrointestinal and liver physiology, 314(1), G119–G130. PMC free article

These authors started with the knowledge that too little and perhaps too much copper can lead to non-alcoholic fatty liver diseae (NAFLD). Fructose was also considered a contributor to NAFLD. What we at CopperOne think is really cool is that these investigators also looked at the intestinal bacteria.

Male weanling Sprague-Dawley rats (35–45 g) were fed a purified AIN-76 diet with Cu(II)CO₃. A purified AIN-76-based diet containing nearly 40% sucrose (wt/wt).

• 1.6, marginal Cu
• 6.0 adequate Cu
• 20 ppm supplemental Cu
• The mice were given distilled water to drink ± 30% weight/volume fructose.of copper as marginal, adequate, or supplemental doses, respectively, for 4 wk. Control animals

How much sugar, presumably fructose, is in Coca Cola? This site says that there are 25 g sugar in 7.5 oz. Using the online conversion, this is 213 mL. This comes out to 12% w/v! These little animals were drinking water that had almost 3x as much sugar as Coca Cola. Does this measure the human condition? Humans who are consuming high fructose corn syrup sweetened beverages are probably also consuming foods sweetened with the same.

Copper and copper proteins

• Ceruloplasmin…The Cu carrier protein in the plasma saw a big reduction in the Cu marginal rats. Drinking fructose resulted in a ~25% reduction in the Cu adequate rats.
• Plasma Cu, The plasma is the portion of blood not containing cells and clotting factors. Fructose in the drinking water increased the serum Cu in the Cu supplemented group.
• Liver Cu, Both marginal and supplemented Cu diets showed liver Cu than the fructose free Cu adequate rats.
• Duodenal Ctr1 Fructose in the drinking water decreased mRNA levels that code for the protein Ctr1 in Cu marginal and Cu supplemented rats. Fructose has no effect on Ctr1 mRNA in Cu adequate rats
• Liver Ctr1. All treatments decreased liver Ctr1 mRNA compared to the Cu adequate rats that were not drinking fructose.

Two liver enzymes and a cytokine

When liver enzymes end up in the blood, it is a sign of liver damage. These authors looked at ALT and AST. These enzymes did not track each other from one diet to the next. The high fructose/Cu supplement diet seemed to be most consistently associated with liver damge. MCP-1, macrophage chemoattractant protein 1, recruits immune cells to he site of an infection. Cu marginal and supplemented diets tended to increase MCP-1 in their Cu adequate counterparts.

Bacteria and leaky guts

Endotoxin/lipopolysaccharide is a lipid component of the cell membranes of Gram negative bacteria. Marginal Cu increased the amount of LPS in the blood. LPS binding protein LBP is produced in the intestine, liver, and adipose tissue. In each of the three diet groups, fructose increased LBP relative to unadulterated drinking water. FD-4, 100uL of FITC-dextran (molecular weight 4,000, FD-4, 40 mg/ml) were injected into the lumen before the gut was ligated to form a sac. The gut sac was then placed in Krebs-Henseleit bicarbonate buffer and incubated at 37°C for 20 min. The FD-4 that penetrated from the lumen into the incubation buffer was measured spectrofluorometrically with an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Only the combination of Cu supplementation and fructose adulterated drinking water caused leaky gut.

Of mucin secreting goblet cells, tight junctions….

Goblet cells are mucin secreting cells of the GI tract. The PAS stain, periodic acid is reacting with mucin

In copper adequate and copper supplemented, the presence of fructose in the drinking water seems to decrease the length of the villi.

Counting bugs

Song and coworkers got quite sophisticated in their counting that we will not get into. Instead we will only present a bar graph in figure 4.

The one thing that stands out is the down sizing of Verrucomicrobia, that Wikipedia authors have little to say about. Given the high sugar content of these rat’s diets, any difference needs to be taken with a certain level of skepticism.

We’d like to make note of a second time, Cu used in this study was Cu(II)CO₃. We at CopperOne would tend to think that the results would have been different with Cu in the +1 oxidation state.

This is just a reminder that the interior mesenteric vein drains blood from the descending colon. If Cu(II) has deleterious effects on our gut bacteria, it will be felt in our livers. Changing the makeup of intestinal bacteria will change short chain fatty acids, polyamines, metabolites will affect our livers. If Cu(II) makes our guts more permeable, the LPS will be seen by the liver. It would be interesting to repeat this study with a diet that is a bit lower in sucrose.

Lessons from a Wilson’s Disease mouse model

Wilson’s Disease is an affliction of not being to export copper due to mutations in the ATP7B Cu⁺ transporter.  This study asked the question of how copper in the proper oxidation state affect the health hazards of a high fat Western Diet (WD) versus the normal control (NC) diet.  This study utilized wild type mice and those same C57BL/6 mice with the ATP7B gene knocked out (ko)

The Western Diet from Research Diets (D12079B ) contained, per kg, 350 g sucrose, 50 g corn starch, 200g butter,

Figure 1A-D

After 9 weeks on the WD vs NC diet, the mouse weight gain was statistically the same regardless of the diet, ATP7B status.  The male mice gained weight faster.  The percentage body fat was a different matter.  When on the Western Diet, both male and female wildtype mice accumulated more fat than their ATP7B knockout counterparts. 

Figure 2

The Western diet increased serum glucose and  cholesterol, but not serum triglycerides.  Liver triglyceride content was increased by the Western diet.  Serum glucose was lower in the TAP7B knockout mice than the wildtypes on the control diet.  The Oil red O staining of fat in the liver was telling.  

Stearoyl CoA desaturase (SCD) is an enzyme that uses O₂ and electrons from reduced cytochrome b5 to introduce double bonds in the delta-9 of  acyl-CoA substrate.  Î’-hydroxy-β-glutaryl CoA is an intermediate in the mevalonate pathway that ultimately leads to cholesterol synthesis. These pathways share NADPH as an intermediate.

Data are from Gottleib 2022 Figures 4 and 5 are not shown in this post.  Much of this post is skipping the metabolic pathway ontology of Gottleib 2022 just because it highlights global clusters of enzymes and not ones that specifically use NAD derivatives. We’ve shown one. The take home theme does seem to be that fats accumulate  in the liver because they are not used to produce cholesterol, a process that requires NADH.

Obviously too much of anything is toxic. While Cu⁺ entered the cells of the ATP7B knock out (ko) mice in this study via normal pathways, it built up to toxic levels. [1] Int is interesting to note that copper toxicity cancelled Western diet toxicity. How much could Cu⁺ improve the toxicity of the Western diet with functioning ATP7B?

References

1. Gottlieb A, Dev S, DeVine L, Gabrielson KL, Cole RN, Hamilton JP, Lutsenko S. (2022) Hepatic Steatosis in the Mouse Model of Wilson Disease Coincides with a Muted Inflammatory Response. Am J Pathol. 2022 Jan;192(1):146-159.