Copper metabolism at the cellular level

Most recent fundamental knowledge generated on Cu biology has been produced with models such as cell culture, Caenorhabditis elegans, laboratory animals and human subjects^{(Reference Suttle17)}. These selected species concentrate on a medical or nutritional perspective. The lack of emphasis on ruminants, and the limited overlap with human-focused sciences, has prevented dissemination of this new understanding, resulting in a lack of progress from the classic ideas on Cu in ruminants.

The Cu chaperones and enzymes which exist in ruminants are the same as those studied in other mammalian species^{(Reference Suttle17)}. At the cellular level, basic Cu metabolism appears to be consistent throughout eukaryotic life and can be traced from laboratory animals to man through their shared evolution^{(Reference Nevitt, Öhrvik and Thiele18)}, demonstrating that Cu in the systemic circulation is trafficked in the same manner in mammalian cells, thus providing opportunities to expand our understanding of Cu metabolism in ruminants^{(Reference Suttle17)}.

Since 1966 radiolabelled Cu, cell fractionation and isolation of intracellular membrane components have been used to develop mathematical models to describe Cu movement in rat liver^{(Reference Hazelrig, Owen and Jane19,Reference Bingham, Sargeson and McArdle20)} . This led to the concept that separate pools, of varying availability, existed^{(Reference Bremner21)}. Initially, the pools were designated as ‘storage’, ‘synthetic’ and ‘excretory’^{(Reference Hazelrig, Owen and Jane19)}. The relationship between the pools appeared complex, with no evidence of reversible movement between them. It was suggested that the Cu pools were able to become saturated, and the regulation or exchange between the pools was not determined^{(Reference Bremner21,Reference McArdle, Gross and Creaser22)} . The number and function of the pools were not easily apparent. Most studies agreed that hepatocyte Cu could be divided into at least two pools: one a readily available, extractable Cu pool accounting for the majority of Cu, the second, a less readily available pool containing the remainder of soluble Cu, and potentially a third, non-extractable, insoluble pool which could be considered a potential subset of the second pool^{(Reference Bingham, Sargeson and McArdle20,Reference McArdle, Gross and Creaser22)} . By 1987, it was proposed that three separate pools existed within the liver representing bile, caeruloplasmin and ‘storage’ which was not further defined^{(Reference Bremner21)}.

Subsequent research has mapped the intracellular movement of Cu and improved our understanding of Cu distribution in cells^{(Reference Rubino and Franz23–Reference Puig and Thiele27)}. Fundamentally, this new knowledge does not contradict the description of Cu as cellular pools, but it illustrates Cu physiology in terms of Cu trafficking. Free Cu ions rarely exist within cells, thus Cu is kept complexed to prevent intracellular damage^{(Reference La Fontaine and Mercer28)}. Distinct intracellular pathways exist where Cu is bound to chaperones and channelled across membranes rather than a series of storage compartments as the older model suggests. However, the persistence of the term ‘pool’, even in current literature, conjures images of discrete areas. It is perhaps better to update our terminology, and start discussing the ‘pathways’ of Cu trafficking, rather than its ‘pools’ of availability to better reflect the process and improve understanding of the process as a continuous regulation instead of discrete compartments of varying lability.

Overview of copper trafficking in enterocytes

The one aspect of Cu metabolism that differentiates ruminants from other species is their unique digestive system. Cu availability in the ruminant gastrointestinal tract presents peculiarities that have been extensively reviewed elsewhere^{(Reference Gould and Kendall16,Reference Spears29,Reference Suttle30)} . However, the process of absorption is well preserved across the animal kingdom^{(Reference Peña, Lee and Thiele31–Reference van den Berghe and Klomp33)}. In order for Cu to be absorbed, it must be reduced into its most reactive state (Cu⁺). At the intestinal brush border a Cu specific transporter (Ctr1) is responsible for about 70 % of Cu uptake into the enterocyte; the remainder is taken up by the non-specific transporter divalent metal transporter 1 (DMT1)⁽³⁴⁾. Where Cu is trafficked through the DMT1 route direct competition for the transporter with dietary elements such as Fe and Zn may be more biologically relevant^{(Reference Espinoza, Le Blanc and Olivares35)}. Once inside the cell, Cu chaperone proteins bind Cu and transport it to other specific proteins or incorporate it into enzymes. The pathway via the Golgi is known as the secretory pathway. Cu in excess of cellular requirements enters the secretory pathway to be bound to metallothionein by the Golgi and is stored in the lysosome, which acts as a buffer restricting free cellular Cu. Once the metallothionein reaches its saturation capacity Cu continues through the secretory pathway from the Golgi via its chaperone to the basolateral membrane for efflux from the cell.

The process in detail

Fig. 1 below illustrates the process described.

Fig. 1. Copper trafficking pathways using the copper chaperones from the intestinal lumen. Atx1, antioxidant 1; CCO, cytochrome c oxidase; CCS, copper chaperone for superoxide dismutase; Cox17, cyclo-oxygenase 17; Cp, caeruloplasmin; Ctr1, copper transporter 1; Cybrd1, cytochrome B reductase 1; GSH, glutathione; LOX, lysyl oxidase; Mt, metallothionein; SOD, superoxide dismutase.

Upon arrival at the intestinal brush border the membrane reductase Cybrd1 (cytochrome B reductase 1) and ascorbate (vitamin C) reduce any dietary Cu which is present as Cu²⁺ into Cu^{+ (Reference Collins, Prohaska and Knutson36–Reference Tennant, Stansfield and Yamaji38)}. Reduced Cu is carried across the membrane by high-affinity Cu transporter 1 (Ctr1)^{(34,Reference Boal and Rosenzweig39–Reference Lutsenko41)} . Once inside the cell it is immediately incorporated onto its specific chaperones (Cu chaperone for superoxide dismutase (CCS), antioxidant 1 (Atx1) and cyclo-oxygenase 17 (Cox17)) within the cytosol^{(Reference Maryon, Molloy and Kaplan42,Reference Linder, Zerounian and Moriya43)} . CCS transports Cu within the cytosol where the metalloenzyme superoxide dismutase (SOD) is synthesised^{(Reference Suttle17)}. Cox17 transports Cu to proteins in the mitochondria where the metalloenzyme cytochrome c oxidase (CcO) is synthesised^{(Reference Failla44,Reference Spears45)} . Atx1 and ATP7A transport Cu to the Golgi lumen where dopamine β-hydroxylase, peptidylglycine α-amidating mono-oxygenase, lysyl oxidase (LOX), SOD, tyrosinase, caeruloplasmin (Cp) and hephaestin vital for nerve and connective tissue function and for Cu and Fe transport are synthesised^{(Reference Nevitt, Öhrvik and Thiele18,Reference Polishchuk and Lutsenko46)} . Surplus Cu is bound to metallothionein (Mt) and held in the lysosome after processing by the Golgi^{(Reference Nevitt, Öhrvik and Thiele18,Reference Failla44,Reference Dameron and Harrison47,Reference Thiele48)} . Upon reaching the metallothionein-carrying capacity in the lysosome, surplus Cu from the Golgi is transported using the ATP7A secretory pathway and effluxed from the enterocyte into circulation^{(Reference Suttle17,Reference Nevitt, Öhrvik and Thiele18,Reference Spears45)} . At the point of release from the cell membrane the oxygen tension of the interstitial fluid is sufficient to elicit spontaneous oxidation of the Cu⁺ to oxidised Cu²⁺ without the need for an oxidase in the membrane^{(Reference Gulec and Collins49)}.

Copper movement in the blood

Following efflux from the enterocytes Cu is bound to albumin, an abundant plasma protein accounting for 15–20 % of total Cu transport, and transcuprein, a small protein which, in contrast to albumin, is a specific Cu carrier in plasma carrying 10–30 % of total transported Cu^{(34,Reference Stern, Solioz and Krewski50–Reference Weiss and Linder53)} . The concentration of albumin in blood plasma exceeds that of transcuprein, but transcuprein has a higher affinity for Cu. Around a third of the Cu entering the blood from the small intestine is bound to transcuprein^{(Reference Weiss and Linder53)}. These two proteins transport Cu from the intestines through the systemic circulation to the liver. Metabolic studies have demonstrated that absorbed dietary Cu from the portal circulation is cleared by the liver and appears in newly synthesised caeruloplasmin^{(Reference Hellman and Gitlin54)}. Caeruloplasmin is the predominant Cu transporter in the systemic blood and is responsible for distribution of Cu to the tissues after its synthesis in the liver^{(Reference Nemec55,Reference Bremner and Beattie56)} . In ruminants about 88 % (range 86–90 %) of total plasma Cu is present bound to caeruloplasmin^{(Reference Mackenzie, Illingworth, Jackson, Fischer, L’Abbé, Cockell and Gibson57)}.

Overview of hepatic copper trafficking

The liver has a major role in the regulation of Cu^{(Reference La Fontaine and Mercer28)}. This homeostatic control acts primarily through regulating the secretion of Cu into bile^{(Reference Collins, Prohaska and Knutson36,Reference Linder, Zerounian and Moriya43,Reference Stern, Solioz and Krewski50,Reference Harris58)} . Cu reaching the liver is transported in a similar mechanism to the enterocytes. At the membrane the arriving Cu is reduced and trafficked into the cell by the same Cu transporter (Ctr1). Once inside the hepatocyte the chaperones fulfil their respective roles with one notable difference. The secretory pathway for efflux via the Golgi has a unique chaperone (ATP7B) which directs the majority of Cu to be incorporated into caeruloplasmin which is then effluxed into circulation for distribution to other tissues. However, when caeruloplasmin-bound Cu from the peripheral tissues re-enters the circulation and returns to the liver the whole molecule of caeruloplasmin is absorbed for destruction and excretion through the biliary route.

The process in detail

Fig. 2 illustrates the process described below.

Fig. 2. Copper trafficking pathways using the copper chaperones into hepatocytes and out into the systemic circulation. Atx1, antioxidant 1; CCO, cytochrome c oxidase; CCS, copper chaperone for superoxide dismutase; COMMD1, copper metabolism MURR1 domain; Cox17, cyclo-oxygenase 17; Cp, caeruloplasmin; Ctr1, copper transporter 1; GSH, glutathione; LOX, lysyl oxidase; Mt, metallothionein; SOD, superoxide dismutase.

Cu reaches the liver bound to either transcuprein or albumin which are reduced on arrival by NADH oxidase^{(Reference Crisponi, Nurchi and Fanni52)}. Uptake of the reduced Cu into the hepatocyte is mediated by Ctr1^{(Reference Kim, Son and Bailey59)}. Once inside, CCS and Cox17 traffic their Cu payload to the cytosol and mitochondria respectively and Atx1 delivers Cu to the Golgi body via ATP7B^{(Reference De Bie, Van de Sluis and Klomp60)}. ATP7A is not expressed in the liver; instead, hepatocytes express a unique version ATP7B^{(Reference Failla44)}. ATP7B directs the majority of Cu to be incorporated into caeruloplasmin to be subsequently returned to the circulation for distribution to other tissues^{(Reference Suttle17,Reference La Fontaine and Mercer28,Reference Prohaska40,Reference Failla44,Reference De Bie, Van de Sluis and Klomp60)} . When caeruloplasmin returns from the systemic circulation to the hepatocytes the whole molecule is absorbed. The endothelial hepatocytes must first remove sialic acid residues from the caeruloplasmin to allow the underlying hepatocytes to absorb the caeruloplasmin molecule for proteolysis and destruction through the biliary route^{(Reference Harris58)}. The excess hepatic Cu is exported into the bile using the chaperones COMMD1 (Cu metabolism MURR1 domain) and potentially also XIAP (X-linked inhibitor of apoptosis protein)^{(Reference Collins, Prohaska and Knutson36,Reference Prohaska40,Reference De Bie, Van de Sluis and Klomp60)} . COMMD1 binds to the N-terminal region of ATP7B but not to ATP7A, explaining the difference in ATPase channel expression between hepatocytes and other cells^{(Reference De Bie, Van de Sluis and Klomp60,Reference Tao and Gitlin61)} .

Adaptations to changing dietary copper supply

Under Cu-limiting conditions the movement of Cu into the secretory pathway (Atx1-ATP7A) is diminished in all tissues^{(Reference Lutsenko25,Reference Stern, Solioz and Krewski50)} . Cu bound to metallothionein is mobilised using the acidic pH of the lysosome to partially degrade the metallothionein held within the lysosome and release its Cu into the cytosol^{(Reference Nevitt, Öhrvik and Thiele18,Reference Klaassen, Choudhuri and McKim62,Reference Suzuki, Someya and Komada63)} . The released Cu is delivered, probably by glutathione (GSH), to the Cu chaperones (cytosolic CCS and mitochondria targeting Cox17) equally, but not into the secretory pathway (Atx1)^{(Reference Lutsenko25,Reference Suzuki, Someya and Komada63,Reference Vulpe and Packman64)} . This redirection diminishes Cu supply to the secretory pathway resulting in the production and secretion into the bloodstream of the Cu-empty apo-caeruloplasmin, rather than its Cu-containing holo form^{(Reference Suzuki, Someya and Komada63)}. This process inhibits excretion and retains Cu for intracellular use^{(Reference Hamza, Gitlin, Trauner and Jansen65)}.

Under Cu-replete conditions in the tissues each of the Cu transporters and proteins are down-regulated^{(Reference Lutsenko25,Reference Thiele48)} . The down-regulation of Cu transporter (Ctr1) in the membrane prevents any further Cu uptake into the cell^{(Reference Nose, Wood and Kim66–Reference Leary, Winge and Cobine68)}. ATP7A (a chaperone in the secretory pathway) moves out of the trans-Golgi network into vesicles that move towards the membrane. These vesicles accumulate Cu and intermittently fuse with the membrane to efflux the remaining excess Cu from the cell into the blood before returning to the cytoplasm^{(Reference Nyasae, Bustos and Braiterman69)}. Increased metallothionein expression (regulated by metal transcription factor MTF1) exerts intracellular homeostatic control through binding excess Cu and acting as a storage buffer protecting the cell^{(Reference Nevitt, Öhrvik and Thiele18,Reference Hamza, Gitlin, Trauner and Jansen65)} .

When hepatocytes are exposed to increasing Cu concentrations they behave similarly to other cells with one exception; ATP7B (from the hepatocyte secretory pathway) leaves the trans-Golgi network but instead of moving towards the membrane it moves towards the lysosome at the canalicular membrane^{(Reference Stern, Solioz and Krewski50,Reference Hamza, Gitlin, Trauner and Jansen65)} . Here, the ATP7B imports Cu into the lysosomal lumen for temporary storage. Increasing intracellular Cu concentrations induce exocytosis of the lysosome, releasing the excess Cu into the biliary canal (mediated by the secretory chaperones ATP7B and COMMD1)^{(Reference Lutsenko25,Reference Collins, Prohaska and Knutson36,Reference De Bie, Van de Sluis and Klomp60,Reference Polishchuk, Concilli and Iacobacci70,Reference Petris, Mercer and Culvenor71)} .

Ruminant copper sensitivity

When discussing the unique characteristics of ruminant Cu handling it is important to first note that metallothionein knock-out animals, even from single-stomached species, are hypersensitive to Cu^{(Reference Tapia, González-Agüero and Cisternas72)}. Sheep have a limited ability to synthesise metallothionein in response to rising Cu concentration and they appear to have a restricted capacity to accumulate Cu bound to metallothionein in the liver^{(Reference Bremner and Beattie56,Reference Saylor, Morrow and Leach73)} . In comparison with rats, sheep reach a point where metallothionein synthesis is unable to keep up with rising Cu at a much lower dietary inclusion, resulting in less Cu sequestering by the lysosome^{(Reference Saylor, Morrow and Leach73)}. Additionally, sheep have a limited ability to increase biliary Cu excretion in response to Cu intake^{(Reference López-Alonso, Prieto and Miranda74)}. Cattle also have a lower capacity to store Cu bound to metallothionein in comparison with single-stomached species and a limited capacity to induce metallothionein in response to Cu intake^{(Reference Bremner and Beattie56,Reference Saylor and Leach75)} . Furthermore, in cattle and sheep the Cu-buffering capacity decreases as hepatic Cu loading increases alongside the Cu:Zn ratio^{(Reference López-Alonso, Carbajales and Miranda76)}. If the influx of Cu exceeds the capacity of the metallothionein and lysosomal uptake, unbound Cu will occur in the cytosol and begin to enter the nucleus, causing severe cell damage^{(Reference López-Alonso, Carbajales and Miranda76,Reference López-Alonso, Prieto and Miranda77)} . While, pigs and dogs have about 500–600 mg/kg, sheep and cattle have only about 200 mg/kg metallothionein in their livers^{(Reference López-Alonso, Prieto and Miranda77)}. Additionally, the metallothionein transcription in the lysosome of cattle and sheep does not effectively respond to rapid increases in Cu^{(Reference Saylor and Leach75,Reference Corbett, Saylor and Long78)} , seemingly reaching a plateau of total Cu concentration about 1607 mg/kg DM (25 347 µmol/kg DM) in cattle and about 571–643 mg/kg DM (9006–10 142 µmol/kg DM) in sheep^{(Reference López-Alonso, Prieto and Miranda74,Reference Saylor and Leach75,Reference López-Alonso, Prieto and Miranda77,Reference Corbett, Saylor and Long78)} . Potentially this plateau is linked to the limited production of metallothionein and an inhibited biliary Cu excretion^{(Reference López-Alonso, Prieto and Miranda74)}, theoretically explaining why cattle appear to be more Cu tolerant than sheep and why both species appear sensitive in comparison with single-stomached species such as pigs.

Further to species differences, breed differences among ruminants have also been documented. Texel sheep are more sensitive to Cu than Landrace breeds^{(Reference Menzies, Boermans and Hoff79,Reference van der Berg, Levels and van der Schee80)} . In cattle, Holstein and Angus breeds are more Cu tolerant than Jersey, Charolais and Simmental^{(Reference Du, Hemken and Harmon81–Reference Gengelbach, Ward and Spears83)}. In cattle, the more Cu-tolerant breeds exhibit a greater expression of duodenal Ctr1 and ATP7A, and a higher hepatic expression of Ctr1, Cox17, ATP7B, CCS and SOD where Cu supply is inadequate^{(Reference Fry, Spears and Lloyd84,Reference Dermauw, De Cuyper and Duchateau85)} . These suggest that the ability to increase expression of Cu transporters and chaperones allows more effective uptake and utilisation where Cu supply is insufficient, reducing the susceptibility of these breeds to deficiency in comparison with their counterparts^{(Reference Fry, Spears and Lloyd84,Reference Dermauw, De Cuyper and Duchateau85)} . This research highlights a potential mechanism for the observed breed differences, but further studies in a wider range of breeds and in sheep, under elevated and Cu-replete conditions, would further clarify the role of transporter expression in Cu sensitivity.

Thiomolybdate disruption

Thiomolybdate is known to interact with Cu. It naturally forms in the reducing environment of the rumen between dietary S and Mo. Thiomolybdate poses a problem for Cu availability and post-absorptive utilisation^{(Reference Spears29,Reference Galbraith, Chigwada and Scaife86–Reference Price, Will and Paschaleris88)} . Thiomolybdates interact with available Cu in the digestive tract, forming an insoluble precipitate and greatly reducing Cu availability^{(Reference Spears29,Reference Galbraith, Chigwada and Scaife86–Reference Essilfie-Dughan89)} . If there is insufficient Cu where thiomolybdates form to ‘de-toxify’ them they can be absorbed into the systemic circulation, where they exert their affinity for Cu by complexing with Cu contained in biological compounds, rendering them biologically inactive^{(Reference Gould and Kendall16,Reference Ogra, Komada and Suzuki90)} . Thiomolybdates are able to cross cell membranes but the mechanism by which this takes place is unknown. However, once inside the cell they have the potential to disrupt Cu transport through binding to Cu located on the Cu chaperones, transporters and enzymes^{(Reference Suttle17)}.

Thiomolybdates can bind to Cu in cuproenzymes including caeruloplasmin, metallothionein, cytochrome c oxidase (CcO), SOD^{(Reference Ogra, Komada and Suzuki90–Reference Suzuki, Ogra and Ohmichi93)} and Atx1^{(Reference Alvarez, Xue and Robinson94)}. Binding does not remove the Cu component but renders it unable to perform redox reactions (vital to its biological function) through the formation of a stable complex^{(Reference Gould and Kendall16,Reference Spears29,Reference Van Ryssen, Van Malsen and Barrowman95,Reference Hynes, Woods and Poole96)} . Superoxide dismutase has been shown to differ and Cu may be partially stripped from this enzyme^{(Reference Juarez, Betancourt and Pirie-Shepherd97,Reference Juarez, Manuia and Burnett98)} . In the case of the chaperone Atx1, thiomolybdate suppresses the incorporation of Cu into the products of the secretory pathway, disrupting the activity of the Atx1^{(Reference Alvarez, Xue and Robinson94)}. Thiomolybdates have a high affinity for Cu and they have no effect on other trace metals with similar properties such as Fe, Zn or Cd^{(Reference Ogra, Ohmichi and Suzuki99,Reference Ogra, Ohmichi and Suzuki100)} .

Practical implications

Cu provision in ruminants requires a careful balance between intake and availability. The inhibited capacity of these species to adapt to Cu influx explains their sensitivity to overloading. Routine calculation of Cu intake at farm level is not routinely undertaken, which can lead to oversupply^{(Reference Sinclair and Mackenzie11,Reference Sinclair and Atkins101)} . Calculation of Cu supply in combination with monitoring of biological parameters as part of routine management allows a more accurate assessment of Cu status across the entire flock or herd to be made^{(Reference Kendall and Bone102)}. At present, liver sampling is under-utilised as a measure of herd or flock Cu status, especially where there is a history of oversupply. Annual monitoring of a representative sample, from cull animals or from biopsy, allows more effective long-term decisions to be made for Cu provision. It has been recently demonstrated that a significant linear relationship exists between increasing hepatic Cu concentrations and the abundance of rhodamine-stained granules in hepatic tissue histology^{(Reference Strickland, Herdt and Sledge15)}. This staining technique detects the Cu-filled lysosomes which occur as the cellular mechanism for Cu storage becomes overwhelmed^{(Reference Strickland, Herdt and Sledge15)}. In effect, their presence has the potential to be used as an indicator that Cu concentrations are in excess; although this technique is not yet used in practice. Little correlation exists between hepatic Cu concentrations and Cu concentrations in blood parameters^{(Reference Suttle30,Reference Laven, Livesey and Harmon103)} . It is useful to bear this in mind and employ both techniques in conjunction with each other to establish animal status^{(Reference Suttle30,Reference Laven, Livesey and Harmon103)} .

The potential danger posed through absorption of thiomolybdate causing disruption to systemic Cu chaperones and cuproenzymes should also not be neglected. The use of blood assays is of importance to help monitor changes in shorter-term Cu status. Decreases in caeruloplasmin activity can be a useful indicator of systemic thiomolybdate presence or Cu deficiency over and above the use of caeruloplasmin concentration^{(Reference Chidambaram, Barnes and Frieden91)}. Since the apo-protein will continue to be synthesised in the absence of adequate available hepatic Cu its activity can be reduced to nil^{(Reference Gitlin, Schroeder and Lee-Ambrose104)}. This measure is not without flaws, as caeruloplasmin is an acute-phase protein and can be elevated by infection or stress, leading to falsely elevated measures of Cu status^{(Reference Suttle30,Reference Blakley and Hamilton105,Reference Arthington, Martin and Blecha106)} . Unfortunately, a single, reliable measure for Cu status does not yet exist. Therefore, it is important to use both blood and hepatic measures in monitoring ruminant Cu status in addition to monitoring nutritional input^{(Reference Sinclair and Mackenzie11,Reference Sinclair and Atkins101)} . Furthermore, it is important in practice to provide an appropriate Cu source, or combination of sources, which will be sufficient to ‘de-toxify’ thiomolybdate before it is absorbed and retain a sufficient supply of labile Cu for absorption which does not provide an excess or exceed legal restriction^{(Reference Sinclair and Atkins101)}.