Role of Mitochondria in the Phenotypic Expression of Feed Efficiency

2004 Poultry Science Association, Inc.

Role of Mitochondria in the Phenotypic Expression of Feed Efficiency W. G. Bottje,1 M. Iqbal, N. R. Pumford, C. Ojano-Dirain, and K. Lassiter

Primary Audience: Commercial Breeders, University Researchers SUMMARY Recent investigations were conducted on mitochondria obtained from broilers with low and high feed efficiency (FE) within the same genetic line that were fed the same diet, thereby allowing relationships between mitochondrial function and the phenotypic expression of FE to be clearly established. In these studies, breast muscle, liver and duodenal mitochondria obtained from broilers with high FE exhibited a more tightly coupled respiratory chain along with lower electron leak and reactive oxygen species (ROS) production compared with broilers with low FE. Elevated electron leak in low FE was due to site-specific defects in electron transport at Complex I, III, or both of the respiratory chains in breast and leg muscles and at Complex I, II, or III (depending on energy substrate) in duodenal mitochondria. Increased ROS production was presumably responsible for increased protein oxidation in low FE muscle mitochondria. Of 14 respiratory chain proteins investigated in breast muscle, the expression of 4 (3 in Complex III and 1 in Complex IV) was higher in low FE mitochondria. Interestingly, low FE was associated with a general suppression in respiratory chain complex activities (I, II, III, and IV) in breast muscle and liver. Elucidation of the mechanisms responsible for lower ROS production and tighter coupling of the respiratory chain in high FE mitochondria will greatly facilitate our understanding of the cellular basis for the phenotypic expression of FE. Key words: broiler, feed efficiency, mitochondria, electron leak, respiratory chain complex 2004 J. Appl. Poult. Res. 13:94–105

DESCRIPTION OF PROBLEM As feed represents 50 to 70% of the cost of production, feed efficiency (FE) remains a most important trait in commercial animal production agriculture. Feed efficiency in this review will be defined as the amount of body weight gain per unit of feed consumed (gain to feed), the inverse of the feed conversion ratio that is typically used by industry (feed to gain). Numerous studies have provided evidence of a relationship between mito1

chondrial function and efficiency [1, 2, 3, 4, 5, 6, 7, 8, 9, 10]. However, in all cases, differences in mitochondrial function and FE were due to breed or dietary factors. Consequently, this review will focus primarily on a series of recent studies [11, 12, 13, 14, 15] that used a different experimental paradigm in which broiler breeder males were fed the same diet, maintained under identical environmental conditions, and were all from within a highly selected genetic line (i.e., not a breed com-

To whom correspondence should be addressed: [email protected].

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Department of Poultry Science, Center of Excellence for Poultry Science, University of Arkansas, Fayetteville, Arkansas 72701

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parison). Results of these studies conducted in muscle, hepatic, and duodenal mitochondria provide insight into the cellular basis of FE and indicate that mitochondrial function is indeed inextricably linked to the phenotypic expression of FE.

MITOCHONDRIAL FUNCTION AND BIOCHEMISTRY OVERVIEW The Electron Transport Chain and Energy Production The respiratory chain and oxidative phosphorylation system of the inner mitochondrial membrane, consists of 5 multiprotein complexes: Complex I (NADH: ubiquinone reductase), Complex II (succinate: ubiquinone reductase), Complex III (ubiquinol: cytochrome c reductase), Complex IV (ferrocytochrome c: oxygen oxidoreductase), and Complex V [adenosine triphosphate, (ATP) synthase, Figure 1]. Electron movement down the respiratory chain (Complex I to IV) to the terminal electron acceptor, O2, is coupled to proton (H+) pumping from the matrix to the intermembrane space. The resulting protonmotive force drives ATP synthesis [from adenosine diphosphate (ADP) and PI] as protons move back through the

ATP synthase [16]. Electrons enter the respiratory chain from NADH- or FADH-linked substrates, such as glutamate and succinate at Complex I and II, respectively. Mitochondrial function can be assessed by polarographic measurement of oxygen consumption [17]. In the presence of energy substrates, isolated mitochondria exhibit an initial slow rate of oxygen consumption designated State 2 respiration. The addition of ADP stimulates electron transport chain activity and initiates rapid oxygen consumption (State 3 respiration) that is followed by a slower rate of oxygen consumption (State 4 respiration), when ADP levels decline following oxidative phosphorylation to ATP. Functional indices calculated from these rates of oxygen consumption include the respiratory control ratio (RCR) and ADP:O ratio [17]. The RCR represents the degree of coupling or efficiency of electron transport chain activity and is calculated as State 3 divided by State 4 respiration rate. The ADP:O ratio is the amount of ADP phosphorylated per nanoatom of monomeric oxygen consumed during State 3 respiration and is an index of oxidative phosphorylation. An acceptor control ratio (ACR), similar to the RCR, can also be calculated as State 3 divided by State 2 respiration rate. In some experiments, repeated additions of ADP have been used, which results in 2 sets of respiration measurements (1 and 2) in sequence. Mitochondrial Reactive Oxygen Species Production Mitochondrial inefficiency may occur as a result of electron leak from the respiratory chain. Rather than being completely reduced to water, 2 to 4% of oxygen consumed by mitochondria may be incompletely reduced to reactive oxygen species (ROS), such as superoxide (O2(•−) and hydrogen peroxide (H2O2) due to univalent reduction of oxygen by electrons [18, 19] (Figure 2). These electrons are released or “leak” from the respiratory chain before they reach the terminal electron acceptor (O2). The mitochondrial formation of ROS makes this organelle a major source of oxidative stress in the cell. If not metabolized by antioxidants, oxidation of critical biomolecules (e.g., lipids, proteins, and DNA) in the mitochondrion, cell, or both can lead to further inefficiencies that accentuate additional ROS production.

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FIGURE 1. Diagrammatic representation of the electron transport chain adapted from Lehninger et al. [16]. The electron transport chain consists of 4 protein complexes (Complex I, II, III, and IV). Electrons (e-) that enter the electron transport chain from energy substrates, such as malate (Complex I) and succinate (Complex II) are passed down the electron transport chain (solid arrows) to the terminal electron acceptor, oxygen that is reduced to water. Coenzyme Q (CoQ, ubiquinone) accepts electrons from both Complex I and II and passes them to Complex III. Associated with the movement of electrons along the electron transport chain is the movement of protons (H+, dashed arrows) from the mitochondrial matrix into the intramembranous space, creating a proton motive force. The movement of protons through the adenosine triphosphate synthase (ATPase) provides the energy to support ATP synthesis. Cyt c = cytochrome C.

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TABLE 1. Encoding of respiratory chain complexes (I to IV) and adenosine triphosphate (ATP) synthase (V) by nuclear (n) and mitochondrial (mt) DNAA Complex

nDNA mtDNA

I

II

III

IV

V

35 7

4 0

10 1

10 3

12 2

A

From Sue and Schon [36].

Antioxidant Protection

Increased mitochondrial ROS production has been linked to various metabolic diseases [20, 21, 22, 23, 24, 25, 26, 27]. The use of respiratory chain inhibitors can be employed to identify sitespecific defects in electron transport within mitochondria. Whereas electron leak occurs mainly within Complex I or III of the respiratory chain [23 ,28, 29], Kwong and Sohal [30] demonstrated that sites of H2O2 production are tissue dependent. For example, in broilers with pulmonary hypertension syndrome, increased ROS production was associated with Complex I and III in heart, skeletal muscle, and lung mitochondria [26, 27] and Complex II in liver mitochondria [25]. Inefficiencies of function may also be hypothesized to occur from insufficient activity or expression of respiratory chain proteins. Oxidation of respiratory chain proteins would decrease their activity and, in turn, the overall efficiency (coupling) of the electron transport by the respiratory chain. In addition, free radicals cause oxidantmediated repression of mitochondrial transcription [31] that exacerbates mitochondrial dysfunction by inhibiting synthesis of respiratory chain proteins [22].

Mitochondrial antioxidant protection from oxygen radicals includes reduced glutathione (GSH), vitamin E, Mn superoxide dismutase, GSH peroxidase, and GSH reductase [32]. The GSH is vital to mitochondria by interacting with radicals directly or indirectly through the action of GSH peroxidase. Vitamin E is an important chainbreaking antioxidant in membranes. The Mn superoxide dismutase converts superoxide to H2O2. In turn, GSH peroxidase uses reducing equivalents of GSH to convert hydro- and lipid peroxides to water or lipid alcohols. Metabolism of H2O2 is particularly important due to the propensity to be converted to the highly reactive hydroxyl radical in the presence of transition metals (e.g., Fe2+, Cu2+) via the Haber-Weiss and Fenton reactions. The GSH reductase is vital in reducing oxidized GSH (GSSG), formed by GSH peroxidase, back to GSH to prevent thiol toxicity in mitochondria [33]. Mitochondrial Proteins The mitochondrion is unique among organelles in that it contains its own genome: a 16.8kb double-stranded circular DNA molecule in chickens. Mitochondrial DNA encodes 22 tRNA, 2 rRNA, and 13 respiratory chain proteins [34, 35]. Thus, expression of respiratory chain proteins is under control of both nuclear (n) and mitochondrial (mt)DNA (Table 1) [36]. The genome is found in every nucleated cell with 2 to 10 copies per mitochondrion and as many as 800 mitochondria found within a single liver cell [37]. Mitochondrial function depends upon proper importation of hundreds of proteins, including respiratory chain proteins synthesized by nDNA [38]. Some proteins are part of the mitochondrial import machinery, whereas others are needed for expression of the mitochondrial genome and mito-

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FIGURE 2. An illustration of electron leakage from the respiratory chain. It has been estimated that 2 to 4% of electrons (e–) leak from the electron transport chain [19]. Rather than being passed all the way down the chain to oxygen, with subsequent reduction to water, electrons leak from the chain and result in univalent reduction of oxygen to form reactive oxygen species (ROS), such as superoxide. The ROS can produce oxidative damage to critical structures in the mitochondria, proteins, and lipids. The presence of numerous antioxidants help to combat and prevent oxidative damage caused by the production of ROS within mitochondria. H+ = protons; ADP = adenosine diphosphate; ATP = adenosine triphosphate; ATPase = ATP synthase.

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Other Important Areas Other aspects of mitochondrial physiology and biochemistry may also be important in contributing to the relationship of mitochondrial function with FE that will not be addressed in this review. One of these mechanisms is proton leak, which is the movement of protons across the inner mitochondrial membrane other than through ATP synthase. This movement dissipates the proton motive force and represents an energetic inefficiency for the cell. Proton leak can account for as much as 15% of active metabolic rate and 20% of resting metabolic rate in mammals [43]. Properties of mitochondria that might influence proton conductance include an increase in membrane surface area and alterations in fatty acid composition of phospholipids in the mitochondrial membrane [44]. The fatty acid composition may also affect the behavior of uncoupling protein homologues (UCP1, 2 and 3) [45], but this remains controversial as no studies have demonstrated that UCP play a role in proton leak under physiological conditions [46]. Ubiquinone (UQ), also called coenzyme Q (CoQn), transfers electrons from Complex I and II to Complex III (Figure 1). Autooxidation of CoQ is believed to be the major source of mitochondrial radical production [19, 47]. Lass and Sohal [48] presented evidence that there was an inverse relationship between CoQn bound to proteins and mitochondrial ROS formation and that animals with relatively more CoQ10 had less ROS production than animals with higher CoQ9 levels. Thus, differences in ROS production in low and high FE mitochondria described below may be related to the CoQ9:CoQ10 ratio or relative amounts of CoQ9and CoQ10 bound to proteins. Ubiquinone content was highly correlated with

FIGURE 3. Relationships between total feed intake (feed intake, g) and body weight gain (gain, g) between 6 and 7 wk of age in male broiler breeders with high and low feed efficiency (FE) [11]. Regression equations shown were significant (P < 0.05).

activity of Complex I and II of the respiratory chain, and UQ oxidation made it unable to participate in electron transport [49, 50]. Therefore, decreased Complex I and II activity in low FE mitochondria [11] could also be due to lower levels of UQ or increased UQ oxidation. Cardiolipin (tetra-acyl-diphophatidyl-glycerol) is found in high amounts and is essential in membranes that carry out coupled phosphorylation [51]. Cardiolipin is a “double” phosphoglyceride; i.e., it has 4 long-chain fatty acids compared with 2 side chains in other phospholipids. Full activity of respiratory chain complexes requires the interaction with cardiolipin. Mitochondrial function was impaired in yeast that lacked cardiolipin [52], whereas Bobyleva et al. [53] indicated that exogenously added cardiolipin depressed the RCR but increased ATP synthase activity in liver mitochondria. As the effects of cardiolipin on mitochondrial function [52, 53] were similar to differences observed in low and high FE mitochondria [11], cardiolipin may play a role in the relationship of mitochondrial function or respiratory chain activity with FE described below. A final compound that should also be mentioned with regard to mitochondrial function and FE is the adenine nucleotide transporter protein (ANT1). The ANT1, located on the mitochondrial inner membrane, is responsible for exchange of ADP and ATP between the mitochondrial matrix and the cytosol [54]. As such, ANT1 is important for energy production since cell membranes are impermeable to adenine nucleotides. Indeed, decreased mitochondrial function was observed in

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chondrial metabolism. Additional proteins are important for other mitochondrial roles, including apoptosis [39] and redox cell signaling and homeostasis [40, 41, 42]. Consequently, “... mitochondrial function in general, and mitochondrial protein synthesis in particular, depend on the conjugated and coordinated expression of both mitochondrial and nuclear genomes” [38]. Thus, a deficiency in these proteins due to lowered expression or oxidative damage could compromise respiratory chain function and possibly the synthesis of ATP by mitochondria.

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ANT1 knockout mice that lacked the ability to exchange ADP and ATP between the mitochondria and cytosol [55]. Thus, differences in expression of ANT1 could be hypothesized to play a role in the phenotypic expression of FE as well.

MITOCHONDRIAL FUNCTION AND FEED EFFICIENCY Background Feed efficiency remains a most important trait in commercial animal breeding programs as feed represents 50 to 70% of the cost of raising an animal to market weight. A 250 to 300% improvement in body weight and FE was observed in a 1991 broiler strain compared with a 1957 random bred control population [56]. Recently, Havenstein et al. [57] conducted a follow-up study and concluded that not only have broiler growth rates and efficiency continued to increase, but genetic progress in this area may actually have been faster during the last 10-yr period (from 1991 to 2001). It was also concluded that genetic selection by commercial breeding companies was responsible for 85 to 90% of the improvement in growth and FE in commercial broilers [57]. Despite marked improvement in growth and FE traits, however, there remains significant within-

Procedures In each of the studies described below from our lab, birds with the lowest or highest FE (FE, 6 to 8 per group) were identified within a group of 100 breeder male replacement stock [11]. The group designated as low feed efficient, would still be quite superior in FE compared with commercial broilers. So in this case, low is a relative term only. Birds were randomly chosen and portions of breast muscle (pectoralis superficialis), leg muscle (quadriceps femoris), liver, or the upper duodenum were obtained for isolation of mitochondria or immediately frozen in liquid nitrogen for biochemical analyses. Differential centrifugation was

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FIGURE 4. Mitochondrial function in breast and leg muscle isolated from broilers with high (shaded bars) and low (open bars) feed efficiency (FE) [11]. Functional measurements include the respiratory control ratio (RCR), the acceptor control ratio (ACR), and the adenosine diphosphate (ADP)/O ratio for breast and leg muscle mitochondria provided glutamate and malate as the energy source. Values represent the mean ± SE for high FE (n = 6) and low FE (n = 7). *Mean value for low FE is lower than high FE mitochondria (P < 0.05). +Mean value for low FE is lower than high FE mitochondria (P < 0.06). A main effect of leg muscle mitochondrial function is lower than breast muscle value (P < 0.05).

and between-strain variations in growth and FE that include a 10% variation in broiler crosses for FE [58]. As mitochondria are responsible for producing 90% of the energy for the cell, some of the variations in broiler growth performance and phenotypic expression of FE may be due to differences or inefficiencies in mitochondrial function. Indeed, differences in rates of oxygen utilization between breeds of chicken [1, 2, 7] and swine [5] have been reported. Mitochondria have been hypothesized to be part of the basis for heterosis observed in plants [59, 60], sheep and swine [4, 5], and chicken [7]. Dietary manipulations of fat and protein levels have also been documented to affect mitochondrial function [3, 6, 8, 9, 10]. For example, Toyomizu et al. [8] reported that decreased FE due to the feeding of 400 mg/kg diet of 2,4-dinitrophenol to broiler chicks had no affect on the ADP:O ratio (oxidative phosphorylation) in muscle and liver mitochondria. Reduced FE associated with feeding different sources of fat had no effect on liver mitochondrial function in mice [9] but lowered the RCR and ADP:O in White Plymouth Rock chicken heart mitochondria [3] and in liver mitochondria in rats [6]. Increasing the dietary protein to metabolizable energy from 25 to 61% reduced the ADP:O ratio but had no effect on the RCR or State 3 respiration in heart and liver mitochondria in Arbor Acre broiler chicks [10]. However, to our knowledge, other than recent reports in broilers [11, 15] and in rats [60], no other studies have investigated relationships of mitochondrial function and FE within a single breed of animals fed the same diet.

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used to isolate mitochondria from muscle [62], liver [33, 63], and duodenum [64]. Mitochondrial function was determined by measuring oxygen consumption polarographically, following the addition of energy substrates that donate electrons to the respiratory chain at either Complex I (NADH ubiquinone:oxidoreductase) (e.g., glutamate, pyruvate, and malate, alone or in combination) or Complex II (Succinate: ubiquinone oxidoreductase) (e.g., succinate) according to Estabrook [17]. Mitochondrial hydrogen peroxide (H2O2) (expressed as nmol H2O2/ min per mg of mitochondrial protein) was monitored in freshly isolated mitochondria according to [26] to assess electron leak and to identify sitespecific defects in electron transport. Mitochondrial H2O2 generation was monitored with and without chemical inhibitors that block electron transfer at specific sites in the respiratory chain: rotenone (Complex I); 4,4,4-trifluoro-1-[2-thienyl]-1,3-butanedione (TTFA) and malonate (Complex II); myxothiazol (inhibition of Complex III, Q cycle); and antimycin A (inhibition of cytochrome b562 within Complex III). Respiratory chain complex activities were assessed spectrophotometrically [65, 66]. Levels of protein carbonyls (aldehydes and ketones) were assessed as an index of protein oxidation according to Keller

FIGURE 6. The H2O2 production [nmol/min per mg mitochondrial protein (P)] in A) breast and B) leg muscle of mitochondria obtained from broilers with high (shaded bars) and low (open bars) feed efficiency (FE) [ 11]. Mitochondria (provided glutamate as an energy substrate) were treated with no inhibitor (NI) or treated with rotenone (Rot), malonate (Mal), thenoyltrifluroacetone (TTFA), antimycin A (AA), and myxothiazol (Myx), which inhibit electron transport at Complexes (C) I, II, and III of the respiratory chain. Each bar represents the mean ± SE for high FE (n = 6) and low FE (n = 7). +Mean value in the low FE group is higher than high FE (P < 0.06). *Within group treatment values are higher than no inhibitor (NI) value (P < 0.05). ◆Within group treatment value for rotenone is higher than no inhibitor (NI) value in leg muscle mitochondria (P < 0.07).

et al. [67]. Standard Western blot methods were used for the detection of protein subunits in the mitochondrial respiratory chain using primary mono- or polyclonal antibodies that were either a gift from other labs or purchased (Molecular Probes, Inc. Eugene, OR).

RESULTS AND DISCUSSION Typical differences in FE between broilers with low and high FE in these studies is provided in Figure 3 taken from Bottje et al. [11]. The dependency of body weight gain on feed intake is clearly indicated. Feed efficiency (FE, g of gain/ g of feed) in this study was 0.64 ± 0.01 and 0.83 ± 0.01 for low and high FE groups, respectively. Inefficiency in Electron Transport with Low FE Mitochondria Relationships in muscle mitochondrial function and FE in broilers were presented recently [11]. Although there were no differences in mitochondrial function in muscle and liver mitochondria provided succinate (FADH linked substrate) (not shown), the RCR was higher in breast and

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FIGURE 5. Relative levels of basal H2O2 production in mitochondria obtained from broilers with high feed efficiency (FE, solid bars) and low FE (open bars). The data was obtained from Bottje et al. [11] for breast and leg muscle and from Ojano-Dirain et al. [15] for duodenum (Duod) or liver [14]. Comparisons were made between low FE mitochondrial H2O2 production expressed as a percentage of values obtained in high FE mitochondria. Energy sources used in these studies shown in parentheses below each tissue were glutamate (Glut), succinate (Succ), and malate (Mal). Each of the values represent the mean ± SE of 5 to 8 observations.

99

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leg muscle mitochondria in high FE compared with low FE mitochondria, when NADH-linked energy substrates were provided (Figure 4). The ACR was also higher (P < 0.07) in high FE breast muscle mitochondria. These results suggest more efficient coupling of electron transport in high FE than in low FE muscle mitochondria and provide indirect evidence that functional differences (i.e., differences in respiratory chain coupling) in muscle mitochondria between the 2 groups might be due to differences associated with electron transport associated with Complex I. Regression analysis revealed that breast mitochondria RCR values were highly correlated with FE (y = 11.3 (FE) − 1.20, r2 = 0.72, P < 0.001). A similar relationship between mitochondrial RCR and FE in rats has also been observed [61]. As there were no differences in the ADP:O with either energy substrate, there apparently is no problem in the ability of low FE mitochondria to carry out oxidative phosphorylation.

Studies were also conducted to determine relationships of intestinal mitochondrial function and FE in broilers [15]. Duodenal mitochondrial function was assessed following repeated additions of ADP, a paradigm of repeated energy demand that revealed mitochondrial dysfunction in pulmonary hypertension syndrome in broilers [63, 68]. In contrast to muscle mitochondria, there were no differences in the initial RCR in duodenal mitochondria obtained from broilers with low and high FE provided either NADH- or FADH-linked energy substrates. With a second addition of ADP, however, tighter coupling (i.e., higher RCR values) were observed in high FE duodenal mitochondria provided succinate but not when NADHlinked energy substrates were provided. These findings suggest that there could be a defect in electron transport chain coupling associated with Complex II in low FE mitochondria. Interestingly, low FE duodenal mitochondria provided NADHlinked energy substrates actually exhibited a significantly higher ADP:O ratio following the second addition of ADP compared with high FE mitochondria and was numerically higher with succinate as well. Thus, at least in these in vitro experiments, the ability to synthesize ATP may actually be superior in low FE duodenal mitochondria to that observed in high FE mitochondria under some conditions. Site-Specific Defects in Electron Transport To determine if the lower RCR values in low FE muscle mitochondria might be associated with increased electron leak from the respiratory chain, H2O2 production in muscle, liver, and intestinal mitochondria were determined with and without various inhibitors of the electron transport chain [11, 13, 14, 15]. In these studies, basal electron leak is represented by the level of H2O2 production in mitochondria that were not treated with any inhibitor of the electron transport. A summary of relative differences in basal ROS production in high and low FE breast, leg, duodenal, and liver mitochondria provided either NADH- or FADHlinked energy substrates is shown in Figure 5. With the exception of leg muscle, these findings indicate a generalized inefficiency in electron transport that results in increased ROS production and the inherent greater oxidative stress in low FE mitochondria.

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FIGURE 7. Relative activities of respiratory chain complexes (C) (I, II, III, and IV) in muscle (A) and liver (B) obtained from broilers with high feed efficiency (solid bars) and low feed efficiency (open bars) [11,14]. Values represent the mean ± SE of 5 to 7 observations. *Mean low feed efficiency values are significantly lower compared with high feed efficiency values (P < 0.05).

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TABLE 2. A summary of site-specific defect analysis of breast, leg, and intestinal mitochondria obtained from broilers with high and low feed efficiency (FE) from reports by Bottje et al. [11] and Ojano-Dirain et al. [14] Tissue (substrate)

Chemical agent Group

Rot

Mal

TTFA

AA

Myx

B

Low FE High FE

X –

– –

– –

X* –

– –

Leg (glutamate)

Low FE High FE

+C –D

– –

– –

– –

– –

Duodenum (succinate)

Low FE High FE

– –

– –

X* –

– –

– –

duodenum (malate)

Low FE High FE

– X

–* –

–* –

– X

– X

Duodenum (pyr-mal)

Low FE High FE

X X

– –

X* –

X X

X X

A Mitochondria were provided either NADH-linked energy substrates [glutamate, malate, or pyruvate-malate(pyr-mal) combination] and treated with chemicals that inhibit electron transport at Complex I (Rot), Complex II inhibitor [Malonate (Mal), thenoyltrifluroacetone (TTFA)], and Complex III inhibitors [antimycin A (AA), at cytB562 and myxothiazol (Myx), Q cycle]. B X = mean values were different (P < 0.05) from within group basal values (no inhibitor) of hydrogen peroxide production (i.e., electron leak). C + = mean values were different (P < 0.07) from within group basal values (no inhibitor) of hydrogen peroxide production (i.e., electron leak). D – = mean values were not different (P > 0.10) from within group basal values (no inhibitor) of hydrogen peroxide production (i.e., electron leak). * = Hydrogen peroxide values were higher (P < 0.05) in low FE compared with high FE values.

According to Barja [69], an increase in radical production following inhibition of electron transport chain activity is indicative of increased electron leak between the site of inhibition and entry of substrate into the electron transport chain. Thus, an increase in radical production in mitochondria following addition of rotenone (Complex I inhibitor), TTFA and malonate (Complex II inhibitors), myxothiazol (inhibitor of Complex III at the Q cycle); and antimycin A (an inhibitor of cytochrome b562 within Complex III) would indicate site-specific defects of electron transport at these sites on the electron transport chain. Bottje et al. [11] reported increased electron leak (H2O2 production) in low FE breast muscle mitochondria following inhibition of electron transport at Complex I with rotenone and Complex III (cytochrome B562) with antimycin A (Figure 6), indicating that these are likely areas of site-specific defects in electron transport that contributed to the higher basal H2O2 production in low FE breast muscle mitochondria. Importantly, high FE muscle mitochondria did not exhibit significant increases in H2O2 production following inhibition of electron transport activity at Complex I and III (Figure 6), suggesting mitochondria from high FE birds would exhibit very low elec-

tron leak in vivo. No differences were observed when electron transport was inhibited at Complex II (with malonate and TTFA) or the Q cycle of Complex III (with myxothiazol). A rotenone-induced elevation (P < 0.07) in low FE leg muscle mitochondria suggests that Complex I may be a potential site of electron leak in low FE leg muscle mitochondria also [11]. Additional detailed studies on site-specific defects in electron transport have been investigated in duodenal mitochondria [15]. Similar to muscle, as indicated above, basal radical production was higher in duodenal mitochondria provided NADH- or FADH-linked substrates. However, it is with chemical inhibitor treatment that the picture appears to be somewhat different. Compared with high FE mitochondria, low FE duodenal mitochondria provided with succinate or pyruvatemalate as energy sources exhibited a site-specific defect in electron transport at Complex II following treatment with TTFA. Low FE duodenal mitochondria also exhibited defects at Complex I following treatment with rotenone and at Complex III with both antimycin A and myxothiazol, when pyruvate-malate was provided as an energy source. In addition, significant elevations in H2O2 production above basal values were also observed

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Breast (glutamate)

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TABLE 3. Breast muscle mitochondrial protein subunits detected using Western blot analysis in low and high feed efficiency (FE) broilers [13] Mitochondrial respiratory chain proteinsA Subunitsa

High FE (n = 7)

Low FE (n = 5)

P-value

± ± ± ± ± ±

4 5 11 6 9 3

0.90 0.90 0.90 0.68 0.50 0.10

99 ± 11

0.70

± ± ± ± ±

0.006 0.22 0.48 0.06 0.03

Band intensity 98 119 145 118 158 89

± ± ± ± ± ±

5 4 9 3 6 3

105 ± 9 158 86 130 140 129

± ± ± ± ±

3 4 4 7 3

100 118 142 115 151 88

173 95 126 160 141

3 6 5 6 4

151 ± 4

163 ± 4

0.06

122 ± 8

116 ± 9

0.61

Values represent the mean ± SE. ND = nicotinamide adenine dinucleotide subunits 3 through 7; URF = unidentified reading frame; 70 S = 70 subunit in complex II; NAD = nicotinamide adenine dinucleotide; ISP = iron-sulfur protein; FP = flavoprotein; FMN = flavin mononucleotide; COX = cytochrome c oxidase subunit; cyt c1 = cytochrome c1; cyt b = cytochrome b; ATPase = adenosine triphosphate synthase.

A B

in high FE duodenal mitochondria with rotenone, antimycin A, and myxothiazol. This would suggest that there are site-specific defects in electron transport at Complex I and III in both high and low duodenal mitochondria, whereas low FE duodenal mitochondria exhibit defects at Complex I, II, and III depending upon the energy substrate. The reason for these differences between groups is not apparent at this time. A summary of the sitespecific defects in muscle and intestinal tissue is provided in Table 2. Respiratory Chain Complex Activities and Protein Expression From the previous section, it is apparent that low FE mitochondria exhibit greater electron leak and therefore are likely to exhibit increased oxidative stress. Indeed, increased protein carbonyl levels (an indicator of protein oxidation) were present in low FE muscle mitochondria [12]. As oxidative damage of proteins could affect enzyme activity, studies were conducted to determine the activities of the various respiratory chain complexes. Bottje et al. [11] reported that activities of Complex I

and II in breast and leg muscle mitochondria from broilers with low FE were 63 to 79% of the levels of activity observed in high FE broilers. Additional studies by Iqbal et al. [13, 14] indicate that complex activities in low FE muscle and liver mitochondria were all significantly lower than in high FE mitochondria (Figure 7, A and B). The biggest difference in both groups was in Complex IV activity with values in the low FE mitochondria that were 44 ± 8 and 59 ± 4 % compared with high FE values in muscle and liver, respectively. These data suggest that low FE is associated with a generalized decrease in respiratory chain complex activity in muscle mitochondria and liver mitochondria. As the activities of the various respiratory chain complexes may depend on the amount of protein present within the respiratory complexes, breast muscle mitochondria from low and high FE broilers were probed with antibodies for specific proteins, and their expression was determined by Western blot analysis. The results shown in Table 3 taken from Iqbal et al. [13] indicate that the expression of cyt b, cyt c1, and core I proteins

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Complex I NAD3 NAD4 NAD5 NAD6 URFC NAD6 URFL NAD7 URFC Complex II 70 S (FP) Complex III core I core II ISP cyt b cytc1 Complex IV COX II Complex V α-ATPase

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associated with Complex III [70]. If these individual proteins were particularly sensitive to oxidative damage, then the increased expression of these proteins might represent a compensatory response to oxidative stress. We also have evidence for increased expression of ubiquitin in low FE tissue [13]. As ubiquitin is a protein that tags damaged proteins and targets them for recycling, higher levels of ubiquitin could indicate greater protein turnover in low FE tissue. Additionally, preliminary results indicate that the expression 70S subunit of Complex II and the alpha subunit of ATP synthase in liver mitochondria may be higher in high FE compared with low FE values [14]. This contrasts with what was observed in the muscle and suggests that tissues differ with regard to relationships between FE and the expression of respiratory chain proteins.

CONCLUSIONS AND APPLICATIONS 1. From recent studies [11, 12, 13 14, 15] in which breed, feed, and environmental effects were removed, a unifying observation was that low FE is associated with increased reactive oxygen species generation in muscle, liver, and intestines. 2. Oxygen radical generation represents an inefficiency in mitochondrial function and can have negative consequences as a result of oxidative damage to critical structures in the cell; e.g., proteins, lipids, and DNA. Increased protein oxidation in muscle mitochondria and liver homogenate was observed in low FE. Further evidence that this oxidation leads to inefficiency was indicated by higher levels of ubiquitin in low FE tissue, a protein associated with increased protein turnover. 3. It should also be recognized that the birds designated as being low feed efficient would be superior in this trait when compared with commercial broilers, even accounting for any differences that would be expected to occur between laboratory and field conditions. Thus, differences in mitochondrial function and biochemistry that were detected in these studies actually reflect differences within groups of very efficient birds. Even more dramatic differences might be obtained if a greater range of FE had been examined. 4. Whereas some respiratory chain proteins were apparently upregulated in broilers with low FE, many other proteins were not. As there are many other proteins that are important for function of mitochondria and the respiratory chain (e.g., transport proteins, scaffolding proteins), it is possible that the differences we seek may lie in this direction (i.e., proteins other than those associated with the respiratory chain). 5. Further studies are currently being conducted to help in understanding the cellular basis of FE. The long-term goals of such studies will help in developing methods of selecting animals with superior FE in poultry.

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Acknowledgments Appreciation is extended to H. Brandenburger for technical editing. The authors are grateful for the generous donation of antibodies from R. Doolittle (University of California, San Diego) and L. Yu (Oklahoma State University, Stillwater, OK). The research is published with approval of the Director of the Arkansas Agricultural Experiment Station and supported in part by grants from USDA-NRI (#2001-03443) and from Cobb-Vantress, Inc., Siloam Springs, AR.

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