**4. Copper misbalance and lipid metabolism in human disease**

### **4.1. Lipid metabolism in Menkes disease**

The studies discussed above were focused on the effects of dietary copper deficiency. In humans and in animals, genetic inactivation of the copper transporting ATPase ATP7A impairs copper export from the intestine, effectively limiting copper supply to many tissues and causing lethal pathology known as Menkes disease, MND (72). Depending on the type of mutation in ATP7A, severity of copper deficiency as well as disease manifestations vary between MND patients. Lipid profiles have been explored in some patients and found affected, but inconsistently, with the exception of a higher neutral lipid content of VLDL in all tested MND patients compared to controls. Interestingly, although ApoB in patients appeared normal, it degraded faster during storage suggesting lower stability (73). Variations in lipid profiles could also be age-related. Studies in animals (including an animal model for Menkes disease) demonstrate that unlike adults, copper deficient young animals do not show elevated cholesterol in the serum (74). The resistance of young mice and rats to hypercholesterolemia could be due to insufficient depletion of copper in the liver, which serves as a major store of copper in neonatal animals, or/and insufficiently progressed functional defects in the liver at young age (74).

The Role of Copper as a Modifier of Lipid Metabolism 49

genetically engineered knockouts of Atp7b (*Atp7b-/-* mice) accurately reproduce these key features of WD (77). Consequently, these animals have been used to investigate

**Figure 2. Elevated copper in** *Atp7b-/-* **hepatocytes inhibits activities of nuclear receptors and dysregulates lipid metabolism.** In Wilson's disease, genetic inactivation of the copper transporting ATPase ATP7B results in an impaired copper export from hepatocytes, loss of copper incorporation into ceruloplasmin, CP, (and a secretion of Apo-CP into the serum) along with massive copper accumulation in hepatocytes. Copper concentrates preferentially in the cytosol and nuclei. Cytosolic copper does not prevent sensing of cholesterol levels, allowing SREBP-2 cleavage and entry into the nucleus in response

to low cholesterol. However, high copper in the nucleus blocks activity of nuclear receptors, presumably through binding or oxidation. Impaired function of nuclear proteins is associated with a decrease in the mRNA levels for several enzymes, including HMG-CoA reductase (HMGCR) and

Studies at the early stage of pathology development in *Atp7b-/-* mice (prior to the onset of visible morphologic changes in the liver) were particularly informative. The analysis of liver transcriptome revealed that copper accumulation is associated with distinct metabolic changes: upregulation of genes involved in cell cycle and chromosome maintenance and down-regulation of lipid metabolism, especially cholesterol biosynthesis (78, 79). The mRNA studies were complemented by the analysis of metabolites, which confirmed significant (30-40%) decrease of cholesterol in the liver, and the lower levels of VLDL cholesterol in the serum (79). These early changes in lipid metabolism are maintained throughout the course of the disease as indicated by the analysis of the mRNA levels and serum lipid profiles at the later stages of the disease (80). Follow-up studies revealed that the sterol metabolism in a brain of young *Atp7b-/-* mice was also dysregulated, with cholesterol, 8-dehydrocholesterol, desmosterol, 7-dehydrocholesterol, and lathosterol all

HMG-CoA synthase, and low total cholesterol in the liver.

consequences of copper overload at the molecular level.

ATP7A plays an important role in the development and maintenance of vasculature by supplying copper for functional maturation of lysyl oxidase. Recent studies also suggested the important role for ATP7A in the pathogenesis of atherosclerosis (75). ATP7A was detected in atherosclerotic lesions of mice with genetically inactivated LDL receptor where it colocalized with macrophages. Down-regulation of ATP7A in a macrophage-derived cell culture by siRNA resulted in decreased expression and enzymatic activity of cytosolic phospholipase A(2) alpha, an important enzyme involved in LDL oxidation (75). Furthermore, only cell-mediated LDL oxidation was reduced following down-regulation of ATP7A, whereas conditioned medium from either control or ATP7A down-regulated cells was without such effect (75). This result indicates that the reduced LDL oxidation is not simply due to a diminished copper export from cells with down-regulated ATP7A, but rather due to complex metabolic interactions between copper misbalance and lipid metabolism

#### **4.2. Copper overload in Wilson's disease markedly alters lipid metabolism.**

Direct evidence for the important role of copper in modulating hepatic lipid metabolism and serum lipid profiles has been produced by studies using *Atp7b-/-* mice (an animal model of Wilson's disease, WD) and, subsequently, samples from WD patients. Wilson's disease is a severe genetic disorder of copper overload, caused by inactivating mutations of copper transporting ATPase ATP7B and inability to excrete excess copper from hepatocytes (76). Despite massive accumulation of copper in a cytosol, copper incorporation into ceruloplasmin is impaired and serum levels and activity of ceruloplasmin are greatly diminished (phenotypically resembling consequences of copper deficiency, Figure 2). The genetically engineered knockouts of Atp7b (*Atp7b-/-* mice) accurately reproduce these key features of WD (77). Consequently, these animals have been used to investigate consequences of copper overload at the molecular level.

48 Lipid Metabolism

metabolism

**4. Copper misbalance and lipid metabolism in human disease** 

The studies discussed above were focused on the effects of dietary copper deficiency. In humans and in animals, genetic inactivation of the copper transporting ATPase ATP7A impairs copper export from the intestine, effectively limiting copper supply to many tissues and causing lethal pathology known as Menkes disease, MND (72). Depending on the type of mutation in ATP7A, severity of copper deficiency as well as disease manifestations vary between MND patients. Lipid profiles have been explored in some patients and found affected, but inconsistently, with the exception of a higher neutral lipid content of VLDL in all tested MND patients compared to controls. Interestingly, although ApoB in patients appeared normal, it degraded faster during storage suggesting lower stability (73). Variations in lipid profiles could also be age-related. Studies in animals (including an animal model for Menkes disease) demonstrate that unlike adults, copper deficient young animals do not show elevated cholesterol in the serum (74). The resistance of young mice and rats to hypercholesterolemia could be due to insufficient depletion of copper in the liver, which serves as a major store of copper in neonatal animals, or/and insufficiently progressed

ATP7A plays an important role in the development and maintenance of vasculature by supplying copper for functional maturation of lysyl oxidase. Recent studies also suggested the important role for ATP7A in the pathogenesis of atherosclerosis (75). ATP7A was detected in atherosclerotic lesions of mice with genetically inactivated LDL receptor where it colocalized with macrophages. Down-regulation of ATP7A in a macrophage-derived cell culture by siRNA resulted in decreased expression and enzymatic activity of cytosolic phospholipase A(2) alpha, an important enzyme involved in LDL oxidation (75). Furthermore, only cell-mediated LDL oxidation was reduced following down-regulation of ATP7A, whereas conditioned medium from either control or ATP7A down-regulated cells was without such effect (75). This result indicates that the reduced LDL oxidation is not simply due to a diminished copper export from cells with down-regulated ATP7A, but rather due to complex metabolic interactions between copper misbalance and lipid

**4.2. Copper overload in Wilson's disease markedly alters lipid metabolism.** 

Direct evidence for the important role of copper in modulating hepatic lipid metabolism and serum lipid profiles has been produced by studies using *Atp7b-/-* mice (an animal model of Wilson's disease, WD) and, subsequently, samples from WD patients. Wilson's disease is a severe genetic disorder of copper overload, caused by inactivating mutations of copper transporting ATPase ATP7B and inability to excrete excess copper from hepatocytes (76). Despite massive accumulation of copper in a cytosol, copper incorporation into ceruloplasmin is impaired and serum levels and activity of ceruloplasmin are greatly diminished (phenotypically resembling consequences of copper deficiency, Figure 2). The

**4.1. Lipid metabolism in Menkes disease** 

functional defects in the liver at young age (74).

**Figure 2. Elevated copper in** *Atp7b-/-* **hepatocytes inhibits activities of nuclear receptors and dysregulates lipid metabolism.** In Wilson's disease, genetic inactivation of the copper transporting ATPase ATP7B results in an impaired copper export from hepatocytes, loss of copper incorporation into ceruloplasmin, CP, (and a secretion of Apo-CP into the serum) along with massive copper accumulation in hepatocytes. Copper concentrates preferentially in the cytosol and nuclei. Cytosolic copper does not prevent sensing of cholesterol levels, allowing SREBP-2 cleavage and entry into the nucleus in response to low cholesterol. However, high copper in the nucleus blocks activity of nuclear receptors, presumably through binding or oxidation. Impaired function of nuclear proteins is associated with a decrease in the mRNA levels for several enzymes, including HMG-CoA reductase (HMGCR) and HMG-CoA synthase, and low total cholesterol in the liver.

Studies at the early stage of pathology development in *Atp7b-/-* mice (prior to the onset of visible morphologic changes in the liver) were particularly informative. The analysis of liver transcriptome revealed that copper accumulation is associated with distinct metabolic changes: upregulation of genes involved in cell cycle and chromosome maintenance and down-regulation of lipid metabolism, especially cholesterol biosynthesis (78, 79). The mRNA studies were complemented by the analysis of metabolites, which confirmed significant (30-40%) decrease of cholesterol in the liver, and the lower levels of VLDL cholesterol in the serum (79). These early changes in lipid metabolism are maintained throughout the course of the disease as indicated by the analysis of the mRNA levels and serum lipid profiles at the later stages of the disease (80). Follow-up studies revealed that the sterol metabolism in a brain of young *Atp7b-/-* mice was also dysregulated, with cholesterol, 8-dehydrocholesterol, desmosterol, 7-dehydrocholesterol, and lathosterol all being highly increased (81, 82). It should be noted that the main cholesterol-sensing pathway that involves proteolysis and nuclear localization of SREBP2 transcription factor is not impaired in *Atp7b-/-* hepatocytes (79).

The Role of Copper as a Modifier of Lipid Metabolism 51

content of triglycerides, free cholesterol and cholesteryl ester when compared to controls. The effect is actually opposite to that reported in *Atp7b-/-* mice. At present, the reason for this discrepancy is not clear. In LEC rat, liver disease is more severe compared to *Atp7b-/* mice, invariably leading to animals death, which is not the case in mice. The mitochondrial function in these rats is more significantly affected (87), perhaps contributing to metabolic differences. Nevertheless, similarly to *Atp7b-/-* mice, the LEC serum is characterized by hypotriglyceridemia, hypocholesterolemia and abnormalities in the composition and size of circulating lipoproteins. Also similarly to *Atp7b-/-* and human liver, the activity of hepatic 3 hydroxy-3-methylglutaryl coenzyme A reductase is reduced in LEC rats, along with other

The earlier studies of copper misbalance were focused mostly on the effects of copper deficiency and resulting dyslipidemia on cardiovascular disease. The liver, however, is the central organ of copper homeostasis and, as discussed above, it is greatly affected in WD. It may also be functionally affected in copper deficiency (88). Caloric excess associated with the modern Western diet is implicated in NAFLD, however caloric excess does not compensate for copper deficiency. Furthermore, copper deficiency can still be experienced by the liver, even when serum copper levels are maintained or increased due to factors such

Although numerous studies clearly link copper deficiency to altered lipid metabolism in animal models (12, 22, 24, 89-92) and human volunteers (29), only recently has low dietary copper been implicated in liver dyslipidemia pathology, including non-alcoholic fatty-liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). In a recent groundbreaking study, hepatic copper content in biopsy specimens was inversely correlated with the severity of fatty liver disease, and copper deficiency in a rodent model was found sufficient to induce NAFLD and metabolic syndrome (12). Hepatic iron accumulation, a known consequence of copper deficiency, is also observed in NAFLD (93). Iron accumulation likely results from the loss of holoceruloplasmin, a copper-dependent ferroxidase instrumental in iron distribution (94). This, in turn, results in lower levels of ferroportin in copper deficient rats; coincidentally, NAFLD patients show less ferroportin expression than controls (93). Copper supplementation has been suggested as a therapy for NAFLD based on study in which a diet-induced (high carbohydrate fat-free diet) NAFLD in rats was improved by

Studies of NAFLD also reflect the intersection of copper deficiency, hepatic lipid metabolism and consumption of fructose as causative agents in NAFLD. High dietary sugars, particularly fructose, have been implicated in development of NAFLD and NASH (96, 97). As discussed above, there is evidence that dietary fructose contributes to copper deficiency (66, 69), indicating cross talk between these dietary factors. Sucrose and fructose may have similar effects, as the enzyme sucrase acts in the digestive system to convert the disaccharide sucrose into fructose and glucose for transport in the

important enzyme involved in lipid biosynthesis (Figure 2) (86).

**4.3. Copper and NAFLD** 

as dietary cholesterol.

treatment with a Cu(I)-nicotinate complex (95).

These important observations necessitated further mechanistic studies. Such work has recently been done using systems biology approach and biochemical/biophysical measurements. Direct *in situ* imaging of copper using X-fluorescence revealed the nonuniform distribution of accumulating copper in *Atp7b-/-* hepatocytes with a predominant increase in the cytosol and nuclei (80). Mass-spectrometry in combination with generation of protein networks provided strong evidence that accumulating copper had a significant and specific functional impact on *Atp7b-/-* nuclei, where it remodeled the RNA-processing machinery (83) and altered abundance and activity of nuclear receptors (84). Specifically, using quantitative Multidimensional Protein Identification Technology (MuDPIT), Wilmarth and colleagues found that the ligand-activated nuclear receptors FXR/NR1H4 and GR/NR3C1 were less abundant in nuclear preparations from *Atp7b-/-* liver, whereas the DNA repair machinery and the nucleus-localized glutathione peroxidase SelH were more abundant, consistent with the earlier transcriptome studies (79).

These findings provide important mechanistic insights into a copper-dependent dysregulation of lipid metabolism (summarized in Figure 2). It seems that hepatic nuclei are the primary sites of action for elevated copper. In WD, the local copper concentration in the nuclei can increase up to 50-100 fold (80). Copper is a redox active metal, and such marked elevation is likely to alter the nuclear redox environment, as also suggested by the upregulation of nuclear glutathione peroxidase SelH. Oxidation of sensitive cysteine residues and/or competition between copper and zinc in zinc fingers (which are common structural features in nuclear proteins) are likely mechanisms that impair activity of nuclear factors, such as transcription factors and/or components of the RNA splicing machinery (Figure 2). Current data suggest that neither potential cysteine oxidation nor copper-zinc competition are wide spread (83) and that nuclear proteins regulating lipid metabolism are preferentially affected by elevated copper. Thus, further studies are needed to understand the molecular basis of this increased sensitivity to copper levels.

The identification of FXR/LXR/RXR as important players in pathology development of WD (85) opens a new avenue for studies aimed on better understanding of the role of a diet (especially cholesterol and fat components) in the time-of-onset and severity of WD. It is important to emphasize that the effect of high copper on lipid metabolism is conserved between species, as evidenced by down-regulation of the same key enzymes in the lipid biosynthesis pathways in mice and human liver in response to copper overload (79). Recent studies of serum samples in the cohort of WD patient revealed differences in cholesterol metabolism that diminished with a copper-chelation therapy (82). These observations further supports high metabolic significance of copper:lipid interactions.

A marked effect of elevated copper on lipid metabolism was also reported in Long-Evans Cinnamon (LEC) rat, another murine model of WD (86). Analysis of the liver and serum lipid profiles in these animals showed that copper overload was associated with a higher content of triglycerides, free cholesterol and cholesteryl ester when compared to controls. The effect is actually opposite to that reported in *Atp7b-/-* mice. At present, the reason for this discrepancy is not clear. In LEC rat, liver disease is more severe compared to *Atp7b-/* mice, invariably leading to animals death, which is not the case in mice. The mitochondrial function in these rats is more significantly affected (87), perhaps contributing to metabolic differences. Nevertheless, similarly to *Atp7b-/-* mice, the LEC serum is characterized by hypotriglyceridemia, hypocholesterolemia and abnormalities in the composition and size of circulating lipoproteins. Also similarly to *Atp7b-/-* and human liver, the activity of hepatic 3 hydroxy-3-methylglutaryl coenzyme A reductase is reduced in LEC rats, along with other important enzyme involved in lipid biosynthesis (Figure 2) (86).

#### **4.3. Copper and NAFLD**

50 Lipid Metabolism

not impaired in *Atp7b-/-* hepatocytes (79).

abundant, consistent with the earlier transcriptome studies (79).

the molecular basis of this increased sensitivity to copper levels.

further supports high metabolic significance of copper:lipid interactions.

being highly increased (81, 82). It should be noted that the main cholesterol-sensing pathway that involves proteolysis and nuclear localization of SREBP2 transcription factor is

These important observations necessitated further mechanistic studies. Such work has recently been done using systems biology approach and biochemical/biophysical measurements. Direct *in situ* imaging of copper using X-fluorescence revealed the nonuniform distribution of accumulating copper in *Atp7b-/-* hepatocytes with a predominant increase in the cytosol and nuclei (80). Mass-spectrometry in combination with generation of protein networks provided strong evidence that accumulating copper had a significant and specific functional impact on *Atp7b-/-* nuclei, where it remodeled the RNA-processing machinery (83) and altered abundance and activity of nuclear receptors (84). Specifically, using quantitative Multidimensional Protein Identification Technology (MuDPIT), Wilmarth and colleagues found that the ligand-activated nuclear receptors FXR/NR1H4 and GR/NR3C1 were less abundant in nuclear preparations from *Atp7b-/-* liver, whereas the DNA repair machinery and the nucleus-localized glutathione peroxidase SelH were more

These findings provide important mechanistic insights into a copper-dependent dysregulation of lipid metabolism (summarized in Figure 2). It seems that hepatic nuclei are the primary sites of action for elevated copper. In WD, the local copper concentration in the nuclei can increase up to 50-100 fold (80). Copper is a redox active metal, and such marked elevation is likely to alter the nuclear redox environment, as also suggested by the upregulation of nuclear glutathione peroxidase SelH. Oxidation of sensitive cysteine residues and/or competition between copper and zinc in zinc fingers (which are common structural features in nuclear proteins) are likely mechanisms that impair activity of nuclear factors, such as transcription factors and/or components of the RNA splicing machinery (Figure 2). Current data suggest that neither potential cysteine oxidation nor copper-zinc competition are wide spread (83) and that nuclear proteins regulating lipid metabolism are preferentially affected by elevated copper. Thus, further studies are needed to understand

The identification of FXR/LXR/RXR as important players in pathology development of WD (85) opens a new avenue for studies aimed on better understanding of the role of a diet (especially cholesterol and fat components) in the time-of-onset and severity of WD. It is important to emphasize that the effect of high copper on lipid metabolism is conserved between species, as evidenced by down-regulation of the same key enzymes in the lipid biosynthesis pathways in mice and human liver in response to copper overload (79). Recent studies of serum samples in the cohort of WD patient revealed differences in cholesterol metabolism that diminished with a copper-chelation therapy (82). These observations

A marked effect of elevated copper on lipid metabolism was also reported in Long-Evans Cinnamon (LEC) rat, another murine model of WD (86). Analysis of the liver and serum lipid profiles in these animals showed that copper overload was associated with a higher The earlier studies of copper misbalance were focused mostly on the effects of copper deficiency and resulting dyslipidemia on cardiovascular disease. The liver, however, is the central organ of copper homeostasis and, as discussed above, it is greatly affected in WD. It may also be functionally affected in copper deficiency (88). Caloric excess associated with the modern Western diet is implicated in NAFLD, however caloric excess does not compensate for copper deficiency. Furthermore, copper deficiency can still be experienced by the liver, even when serum copper levels are maintained or increased due to factors such as dietary cholesterol.

Although numerous studies clearly link copper deficiency to altered lipid metabolism in animal models (12, 22, 24, 89-92) and human volunteers (29), only recently has low dietary copper been implicated in liver dyslipidemia pathology, including non-alcoholic fatty-liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). In a recent groundbreaking study, hepatic copper content in biopsy specimens was inversely correlated with the severity of fatty liver disease, and copper deficiency in a rodent model was found sufficient to induce NAFLD and metabolic syndrome (12). Hepatic iron accumulation, a known consequence of copper deficiency, is also observed in NAFLD (93). Iron accumulation likely results from the loss of holoceruloplasmin, a copper-dependent ferroxidase instrumental in iron distribution (94). This, in turn, results in lower levels of ferroportin in copper deficient rats; coincidentally, NAFLD patients show less ferroportin expression than controls (93). Copper supplementation has been suggested as a therapy for NAFLD based on study in which a diet-induced (high carbohydrate fat-free diet) NAFLD in rats was improved by treatment with a Cu(I)-nicotinate complex (95).

Studies of NAFLD also reflect the intersection of copper deficiency, hepatic lipid metabolism and consumption of fructose as causative agents in NAFLD. High dietary sugars, particularly fructose, have been implicated in development of NAFLD and NASH (96, 97). As discussed above, there is evidence that dietary fructose contributes to copper deficiency (66, 69), indicating cross talk between these dietary factors. Sucrose and fructose may have similar effects, as the enzyme sucrase acts in the digestive system to convert the disaccharide sucrose into fructose and glucose for transport in the

#### 52 Lipid Metabolism

bloodstream. High dietary fructose results in decreased CuZnSOD expression (98), lowering resistance of oxidative damage. A diet of 60% fructose affects lipid metabolism as well as antioxidant status, including CuZnSOD, in the liver of rats after 13 weeks of treatment (68). It is clear these high fructose diets induce NAFLD in rodent models, however these diets are typically 60-70% fructose and may not accurately reflect human fructose consumption (for review see (99)).

The Role of Copper as a Modifier of Lipid Metabolism 53

Copper deficiency and copper overload have multiple and significant effects on systemic and cellular lipid metabolism. Recent studies indicate that copper misbalance is an emerging factor in dyslipidemia and/or fatty-liver disease. In turn, lipid metabolism could be an important modifier of the time-of-onset and severity in Wilson's disease. Work over several decades has yielded physiological and biochemical data on the consequences of copper deficiency, particularly with respect to lipid metabolism, in rodent models. Understanding of human disease would greatly benefit from further analysis of copper levels and lipid profiles in human clinical specimens, as well as assessment of the influence of other nutrients at the molecular level. Animal models will remain important. As much as has already been learned, further mechanistic studies are bound to yield molecular level understanding of the important copper:lipid relationship. It is still not clear how copper deficiency alters gene expression and protein expression/function to produce observed pathologies. Transcript profiling, proteomic analysis, and metabolite profiling, in both datadriven and targeted formats, promise to provide more mechanistic details in animal models

*Department of Biological Sciences, University of Alaska Anchorage, Anchorage, AK, USA* 

[1] Nose Y, Kim BE, Thiele DJ. Ctr1 drives intestinal copper absorption and is essential for growth, iron metabolism, and neonatal cardiac function. Cell Metab. 2006 Sep;4(3):235-

[4] Kuo YM, Zhou B, Cosco D, Gitschier J. The copper transporter CTR1 provides an essential function in mammalian embryonic development. Proc Natl Acad Sci U S A.

[5] Mercer JF, Ambrosini L, Horton S, Gazeas S, Grimes A. Animal models of Menkes

[2] Vulpe CD, Packman S. Cellular copper transport. Annu Rev Nutr. 1995;15:293-322. [3] Lee J, Prohaska JR, Thiele DJ. Essential role for mammalian copper transporter Ctr1 in copper homeostasis and embryonic development. Proc Natl Acad Sci U S A. 2001 Jun

*Department of Physiology, Johns Hopkins University, Baltimore, MD, USA* 

**5. Conclusions** 

**Author details** 

Jason L. Burkhead

Svetlana Lutsenko

**6. References** 

5;98(12):6842-7.

2001 Jun 5;98(12):6836-41.

disease. Adv Exp Med Biol. 1999;448:97-108.

44.

that can be tested in human pathology.

It is proposed that fructose metabolism induces oxidative stress, and this may trigger NAFLD. A copper-fructose feeding study indicated that lipid peroxidation due to copper deficiency and a 62% fructose diet could be reduced by supplementing vitamin E to 1 g/kg, however copper deficiency remained, indicating that copper deficiency with fructose feeding may not be entirely a result of oxidative stress (100). Nevertheless, hepatic lipid peroxidation is enhanced significantly in copper deficiency with fructose feeding, supporting the role for oxidative stress in liver disease through the impairment of hepatic antioxidant systems (66).


**Table 1.** Tissue-specific effects of copper deficiency on lipid metabolism

#### **5. Conclusions**

52 Lipid Metabolism

fructose consumption (for review see (99)).

cholesterol, phospholipid, stearic acid, docosadienoic acid

triglycerides, phospholipids and cholesterol in lipoproteins; apoA, apoB, apoE; plasma volume

apolipoprotein secretion, cholesterol synthesis and secretion, fatty acid syntase expression

stearic acid; docosahexaenoic acid; total phospholipid

> transcripts in cholesterol transport

Kidney NA triacylglycrol and

**Table 1.** Tissue-specific effects of copper deficiency on lipid metabolism

antioxidant systems (66).

Erythrocytes

Plasma and serum

Liver

Heart

Small intestine

bloodstream. High dietary fructose results in decreased CuZnSOD expression (98), lowering resistance of oxidative damage. A diet of 60% fructose affects lipid metabolism as well as antioxidant status, including CuZnSOD, in the liver of rats after 13 weeks of treatment (68). It is clear these high fructose diets induce NAFLD in rodent models, however these diets are typically 60-70% fructose and may not accurately reflect human

It is proposed that fructose metabolism induces oxidative stress, and this may trigger NAFLD. A copper-fructose feeding study indicated that lipid peroxidation due to copper deficiency and a 62% fructose diet could be reduced by supplementing vitamin E to 1 g/kg, however copper deficiency remained, indicating that copper deficiency with fructose feeding may not be entirely a result of oxidative stress (100). Nevertheless, hepatic lipid peroxidation is enhanced significantly in copper deficiency with fructose feeding, supporting the role for oxidative stress in liver disease through the impairment of hepatic

**Tissue Increase Decrease Other observation**

cholesterol:phospholipid ratio, cholesterol: mebmbrane protein ratio, linoleic acid, oleic acid

apoE-free HDL binding; monounsaturated:saturate d fatty acids ratio

elastic fibers; palmitic acid; oleic acid

transcripts in fatty-acid

beta-oxidation NA

phospholipid NA

membrane fluidity?

no change in apolipoprotein transcript levels

hypertrophy; inflammation; distorted elastic fibers

N/A hematocrit

Copper deficiency and copper overload have multiple and significant effects on systemic and cellular lipid metabolism. Recent studies indicate that copper misbalance is an emerging factor in dyslipidemia and/or fatty-liver disease. In turn, lipid metabolism could be an important modifier of the time-of-onset and severity in Wilson's disease. Work over several decades has yielded physiological and biochemical data on the consequences of copper deficiency, particularly with respect to lipid metabolism, in rodent models. Understanding of human disease would greatly benefit from further analysis of copper levels and lipid profiles in human clinical specimens, as well as assessment of the influence of other nutrients at the molecular level. Animal models will remain important. As much as has already been learned, further mechanistic studies are bound to yield molecular level understanding of the important copper:lipid relationship. It is still not clear how copper deficiency alters gene expression and protein expression/function to produce observed pathologies. Transcript profiling, proteomic analysis, and metabolite profiling, in both datadriven and targeted formats, promise to provide more mechanistic details in animal models that can be tested in human pathology.

#### **Author details**

Jason L. Burkhead *Department of Biological Sciences, University of Alaska Anchorage, Anchorage, AK, USA* 

Svetlana Lutsenko *Department of Physiology, Johns Hopkins University, Baltimore, MD, USA* 

#### **6. References**


[6] Lutsenko S, Gupta A, Burkhead JL, Zuzel V. Cellular multitasking: the dual role of human Cu-ATPases in cofactor delivery and intracellular copper balance. Arch Biochem Biophys. 2008 Aug 1;476(1):22-32.

The Role of Copper as a Modifier of Lipid Metabolism 55

[21] Reiser S, Smith JJ, Mertz W, Holbrook J, Scholfield D, Powell A, et al. Indices of copper status in humans consuming a typical American diet containing either fructose or

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[23] Saari J. Copper deficiency and cardiovascular disease: role of peroxidation, glycation,

[24] Tang Z, Gasperkova D, Xu J, Baillie R, Lee J, Clarke S. Copper deficiency induces hepatic fatty acid synthase gene transcription in rats by increasing the nuclear content of mature sterol regulatory element binding protein 1. J Nutr. 2000 Dec;130(12):2915-

[25] Klevay L. Is the Western diet adequate in copper? J Trace Elem Med Biol. 2011

[26] Klevay L. Ischemic heart disease as deficiency disease. Cell Mol Biol (Noisy-le-grand).

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[28] Wapnir R, Devas G. Copper deficiency: interaction with high-fructose and high-fat diets

[29] Klevay L, Inman L, Johnson L, Lawler M, Mahalko J, Milne D, et al. Increased cholesterol in plasma in a young man during experimental copper depletion.

[30] Disilvestro R, Joseph E, Zhang W, Raimo A, Kim Y. A randomized trial of copper supplementation effects on blood copper enzyme activities and parameters related to

[31] Allen K, Klevay L. Cholesterolemia and cardiovascular abnormalities in rats caused by

[32] Carr T, Lei K. High-density lipoprotein cholesteryl ester and protein catabolism in hypercholesterolemic rats induced by copper deficiency. Metabolism. 1990

[33] al-Othman A, Rosenstein F, Lei K. Pool size and concentration of plasma cholesterol are increased and tissue copper levels are reduced during early stages of copper deficiency

[34] Carr T, Lei K. In vivo apoprotein catabolism of high density lipoproteins in copper-deficient, hypercholesterolemic rats. Proc Soc Exp Biol Med. 1989

[35] Hoogeveen R, Reaves S, Lei K. Copper deficiency increases hepatic apolipoprotein A-I synthesis and secretion but does not alter hepatic total cellular apolipoprotein A-I

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copper-deficient rats. J Nutr. 1983 Nov;113(11):2178-83.

and nitration. Can J Physiol Pharmacol. 2000 Oct;78(10):848-55.


[21] Reiser S, Smith JJ, Mertz W, Holbrook J, Scholfield D, Powell A, et al. Indices of copper status in humans consuming a typical American diet containing either fructose or starch. Am J Clin Nutr. 1985 Aug;42(2):242-51.

54 Lipid Metabolism

[6] Lutsenko S, Gupta A, Burkhead JL, Zuzel V. Cellular multitasking: the dual role of human Cu-ATPases in cofactor delivery and intracellular copper balance. Arch

[7] Gupta A, Lutsenko S. Human copper transporters: mechanism, role in human diseases

[8] Lutsenko S, Barnes NL, Bartee MY, Dmitriev OY. Function and regulation of human

[9] Gerbasi V, Lutsenko S, Lewis EJ. A mutation in the ATP7B copper transporter causes reduced dopamine beta-hydroxylase and norepinephrine in mouse adrenal.

[10] Kim B, Turski M, Nose Y, Casad M, Rockman H, Thiele D. Cardiac copper deficiency activates a systemic signaling mechanism that communicates with the copper

[11] Kegley KM, Sellers MA, Ferber MJ, Johnson MW, Joelson DW, Shrestha R. Fulminant Wilson's disease requiring liver transplantation in one monozygotic twin despite

[12] Aigner E, Strasser M, Haufe H, Sonnweber T, Hohla F, Stadlmayr A, et al. A role for low hepatic copper concentrations in nonalcoholic Fatty liver disease. Am J

[13] Lampon N, Tutor JC. Effect of valproic acid treatment on copper availability in adult

[14] Lampon N, Tutor JC. A preliminary investigation on the possible association between diminished copper availability and non-alcoholic fatty liver disease in epileptic patients

[15] Stemmer KL, Petering HG, Murthy L, Finelli VN, Menden EE. Copper deficiency effects on cardiovascular system and lipid metabolism in the rat; the role of dietary proteins

[16] Ernst B, Thurnheer M, Schultes B. Copper deficiency after gastric bypass surgery.

[17] Gletsu-Miller N, Broderius M, Frediani J, Zhao V, Griffith D, Davis SJ, et al. Incidence and prevalence of copper deficiency following roux-en-y gastric bypass surgery. Int J

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**Chapter 4** 

© 2013 Grønning-Wang et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

**The Role of Liver X Receptor in** 

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51357

**1. Introduction** 

**Hepatic** *de novo* **Lipogenesis and** 

**Cross-Talk with Insulin and Glucose Signaling** 

Regulation of nutrient balance by the liver is important to ensure whole body metabolic control. Hepatic expression of genes involved in lipid and glucose metabolism is tightly regulated in response to nutritional cues, such as glucose and insulin. In response to dietary carbohydrates, the liver converts excess glucose into fat for storage through *de novo* lipogenesis. The liver X receptors (LXRα and LXRβ) are important transcriptional regulators of this process. LXRs are classically known as oxysterol sensing nuclear receptors that heterodimerize with the retinoic X receptor (RXR) family and activate transcription of nutrient sensing transcription factors such as sterol regulatory element-binding protein 1c (SREBP1c) (Repa et al., 2000; Yoshikawa et al., 2002; Liang et al., 2002) and carbohydrate response element-binding protein (ChREBP) (Cha & Repa, 2007). LXR also induces the transcription of the lipogenic enzyme genes fatty acid synthase (FAS), stearoyl-Coenzyme A desaturase (SCD1) and Acetyl CoA carboxylase (ACC), alone or in concert with SREBP1c and/or ChREBP (Chu et al., 2006; Joseph et al., 2002; Talukdar & Hillgartner, 2006). LXR activate transcription of hepatic lipogenic genes in response to feeding, which is believed to be mediated by insulin (Tobin et al., 2002). The mechanisms by which insulin activates LXRmediated gene expression is not clearly understood, but may involve production of endogenous ligand for LXRs and/or act by signal transduction mechanisms downstream of the insulin receptor (IR). Both glucose and insulin regulate *de novo* lipogenesis, however, some lipogenic genes can be regulated by glucose without the need of insulin which has been shown for SREBP1c (Hasty et al., 2000; Matsuzaka et al., 2004). A well known glucosemediator in liver is ChREBP, an important regulator of *de novo* lipogenesis in response to glucose (Yamashita et al., 2001). ChREBP is activated by glucose via hexose- and pentosephosphate-dependent mechanisms involving dephosphorylation of ChREBP and

Line M. Grønning-Wang, Christian Bindesbøll and Hilde I. Nebb

