Fasciola hepatica infection in sheep: changes in liver metabolism

Research in VeterinaryScience1996, 61, 152-156

Fasciola hepatica infection in sheep: changes in liver metabolism L. M. LENTON, F. L. BYGRAVE, C. A. BEHM, Division of Biochemistry and Molecular Biology, School of Life Sciences, Australian National University, Canberra, ACT 0200, Australia, J. C. BORAY, NSW Agriculture, Elizabeth Macarthur Agricultural Institute, Camden, NSW 2570, Australia

SUMMARY Several aspects of liver function during infection with Fasciola hepatica were examined in sheep four weeks after infection and compared with the changes observed in infected rats. Previouslyreported respiratory abnormalities in mitochondriaisolated from the left lobe of the liver of infected sheep were characterised further. Evidence is presented that the respiratory lesion is located in the mitochondrial electron transport chain and that the aberrant respiratory behaviour is not associated with an increase in nonesterified fatty acids and the depletion of mitochondrial phospholipids, as is the case in the rat. Microsomal membranes, which have also been shown to be depleted of phospholipids in the fluke-infected rat liver, showed no such changes in the sheep. However, in commonwith the rat, a substantial loss of cytochromeP450 was recorded in microsomesprepared from the left lobe, and the glycogen content of the left lobe was found to be less than 50 per cent of control values. No change was observed in . glucose 6-phosphatase activity. All these changes were localised effects, confinedto areas of fluke infiltration.

CHANGES in hepatic function during the liver migratory phase of infection by Fasciola hepatica have been reported in several species. The changes are diverse and include alterations in mitochondrial bioenergetics, drug metabolism and carbohydrate metabolism (Behm 1996). Most of these studies have been carried out in rats, with few having been extended to an economically important host such as sheep. The aim of the present study was to investigate some aspects of these three changes in liver metabolism in sheep. Previous studies of liver mitochondrial bioenergetics in infected sheep have revealed reduced acceptor control ratios (ACR) in the presence of both Site I (pyruvate/malate) and Site II (succinate) respiratory substrates during the period four to 15 weeks after infection (Rule et al 1991). The most marked changes were reported in samples prepared from the left lobes of infected livers in the presence of ADP (State III respiration). As State III respiration is under the control of several rate-limiting steps, it was important to investigate this change in more detail. It was also necessary to study the change in the context of recent findings of a role for non-esterified fatty acids (NEFA)and changes in the phospholipid composition in the aberrant respiration of mitochondria isolated from the livers of F hepatica-infected rats (Lenton et al 1995). In rats increased phospholipase activity was implicated as the cause of these changes. Similar changes in phospholipid composition have also been documented for microsomes isolated from flukeinfected rat livers (Lenton et al 1995). There is no evidence for a causative role at this stage, but these changes occur at a time when in vitro there is a loss of activity of the integral membrane proteins of the endoplasmic reticulum, such as glucose 6-phosphatase (G6Pase) (Lenton et al 1995) and calcium-ATPase (Galtier et al 1994) as well as a decrease in cytochrome P450 content (Maffei Facino et al 1981, Galtier et al 1983). The latter change has also been reported to occur from four weeks after infection in lambs (Galtier et al 1986). Such a decrease is diagnostic for a general impairment of xenobiotic metabolism (Tekwani et al 1988). As

phospholipase activity has been shown to convert cytochrome P450 to P420 in vitro (Omura and Sato 1964), one of the aims of this study was to determine whether the association between a decrease in cytochrome P450 content and the changes indicative of phospholipase activation in the rat is also present in sheep. A third change in hepatic function during fluke infection affects glucose metabolism. Decreased rates of glycogenolysis and gluconeogenesis have been observed in infected rat livers (Hanisch et al 1991, Millard et al, unpublished observations). The glycogen content of fluke-infected rat livers has also been shown to be significantly lower throughout an infection than that of pair-fed control rats (Millard et al, unpublished observations). Ruminants are highly dependent on gluconeogenesis or glycogen stores for the maintenance of blood glucose. The glycogen stores and G6Pase activity in infected sheep were therefore investigated.

MATERIALS AND METHODS

Animals Nine merino sheep aged 20 months, reared on pastures free of F hepatica infection were used. The sheep were drenched with ivermectin six weeks before the experiment to eliminate gastrointestinal nematode infections. Viable metacercariae of F hepatica were obtained from laboratoryreared Lymnaea tomentosa. The cysts were suspended in 0.4 per cent (w/v) carboxymethylcellulose and four of the sheep were each inoculated with approximately 400 metacercariae by intraruminal injection. The other five sheep were left uninfected as controls. All the sheep were then 'moved to concrete pens and provided with hay containing lucerne ad libitum. Four weeks after inoculation the infected and age-matched control sheep were killed with a captive bolt gun and exsanguinated by throat cutting. The peritoneal cavity was opened and the liver rapidly removed. Pieces of tissue approximately 30 g in weight were removed from the left and right lobes. In the infected

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Liver metabolism in parasitised sheep

animals the samples taken from heavily infected left lobe had yellow/white fluke tracks to an extent similar to that observed in three-week-infected rat livers. The samples removed from the right lobe showed no macroscopic evidence of fluke infiltration, and the migration of fluke into this lobe was limited to relatively small, discrete areas. Tissue samples were cut from corresponding areas of the livers of the control sheep.

Subcellular fractionation of the liver The tissue samples were placed immediately into icecold isolation medium (pH 7.4) containing 250 mM mannitol, 10 mM potassium chloride, 5 mM Hepes potassium hydroxide and 0-5 mM EGTA, diced and washed three times. The diced liver was then homogenised in a Thomas C homogeniser with three passes of a teflon pestle rotating at 900 revolutions minute -1 to give a suspension of approximately 10 per cent (v/v) in isolation medium. Aliquots of the homogenate were reserved for the assay of glycogen and stored at -70°C until used. Mitochondria and microsomes were then prepared as described by Lenton et al (1995). Protein was determined by a modification of the method of Lowry et al (1951) as described by Hanisch et al (1992), using bovine serum albumin (BSA) as standard.

153

Glycogen determination The glycogen content of the homogenate was determined by the Komuniecki and Saz (1982) modification of the method of Selling and Esmann (1975).

Chemicals ADP, ATP and glucose 6-phosphate were obtained from Boehringer Mannheim, Germany. Deuterated water was obtained from Australian Nuclear Science and Technology Organisation, Lucas Heights, NSW, Australia. Oligomycin, cccP and the chemicals for the analysis of NEFA and phospholipids were obtained from Sigma Chemical Co, St Louis, MO, USA. All other reagents were of analytical grade.

Statistical analysis The results are expressed as means (SEM) for the five control and four infected sheep. The statistical significance of any differences was determined by an unpaired, twotailed Student's t test.

RESULTS

Measurement of oxygen uptake

Mitochondrial respiration

Oxygen uptake was measured polarographically with a Clark-type oxygen electrode in a medium containing 100 mM mannitol, 50 mM potassium chloride, 2 mM magnesium sulphate, 15 mM Hepes potassium hydroxide, 0.5 mM EDTA and 2 mM potassium dihydrogen phosphate (pH 7-4). Sodium succinate (5 raM) was used as substrate. Mitochondria were added at a final concentration of 2 mg protein m1-1 in a final volume of 2.5 ml at 25°C. Fraction V BSA (Sigma) was defatted by the charcoal method and when included was present at a concentration of 0-2 mg m1-1. States III and IV respiration refer to rates obtained in the presence and absence of AOP (100 taM) respectively; +cccP indicates the respiration rate in the presence of the chemical uncoupler carbonyl cyanide m-chlorophenylhydrazone (2 ~tM). The acceptor control ratio (ACR) is the ratio of the rates of State III respiration to State IV respiration. ADP/O indicates the amount of AOP added divided by the amount of oxygen used during the rapid oxidation brought about by the presence of ADP.

Tile results in Table 1 summarise the respiratory behaviour of liver mitochondria isolated from the right and left lobes of the sheep and control sheep four weeks after infection. In agreement with previous findings (Rule et al 1991), the State III respiration rate was significantly reduced in mitochondria isolated from the left lobe of the infected animals. As a result, the ACR of these mitochondria was significantly decreased. No change was observed in the State IV respiration rate. A significant decrease in ADP/O ratio and a decline in respiration rate in the presence of the chemical uncoupler cccP were also evident. The latter

Measurement of NEFA content and membrane phospholipid composition Mitochondria and microsomes were assayed for NEFA content by the method of Nixon and Chan (1979). Major phosphorus-containing species in cholate extracts of mitochondria and mierosomes were detected by 31p-NMR spectroscopy as described by Lenton et al (1995).

Measurement of microsomaI P45o content Cytochrome P450 content was measured by the speetrophotometric method of Omura and Sato (1964).

Enzyme assays FoF1-ATPase activity was assayed as described by Lenton et al (1994). Microsomal G6Pase was assayed by the method of Bygrave and Tranter (1978).

TABLE 1: Respiratory behaviour of mitochondria isolated from the livers of uninfected sheep and sheep infected with Fasciola hepatica Control Right lobe Left lobe State IV respiration rate State IV respiration rate (+BSA) State lll respiration rate State lll respiration rate (+BSA) Accepter control ratio Accepter control ratio (+BSA) ADP/O +CCCP

Infected Right lobe Left lobe

10.8 (0.7)

10-9 (0-8)

10.5 (0.4)

11-6 (0.5)

8,1 (0.7)

8.2 (0.5)

9.2 (0.4)

8-9 (0.5)

29.9 (2.0)

28.4 (1.8)

30.6 (1.3)

23-0 (1.9)*

30.1 (1-7)

28.9 (1-6)

35.8 (1.2)

23-4 (1-9)**

2.80 (0.10)

2-62 (0-15)

2.93 (0.05)

1.98 (0.08)***c

3.8 (0-2)

3.6 (0.9)

3.6 (0-1)

2.7 (0-9)**cc

1-59 (0-09) 1.55 (0,05) 65.9 (6.9) 60.9 (4,3)

1-73 (0.14 1-15 (0-07)*cc 65-0 (1.8) 46-7 (3-7)**c

Respiration rates are expressed as nmol 0 2 min-1 mg protein -1. Results are mean (SEM) of five control and four infected sheep, Significant differences between the infected left lobe and infected right lobe are shown as * P<0.05, • * P<0.01, *** P<0.001. Significant differences between the infected and control values are shown as c P<0-05 and c c P<0.01 BSA Bovine serum albumin, ADP/O Amount of ADP added divided by the oxygen used during the rapid oxidation induced by ADP, CCCP Carbonyl cyanide m-chlorophenylhydrazone

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L. M. Lenton, F. L. Bygrave, C. A. Behm, J. C. Boray

observation indicates that there was a reduction in respiration, similar to that detected in the presence of ADP, under conditions when the activities of the ADP/ATP translocase, phosphate carrier or FoF1-ATPase could not have been ratelimiting factors. Furthermore, no change was detected in the latent FoF1-ATPase activity in mitochondria from the infected livers and there was no change in its sensitivity to the inhibitor oligomycin (results not shown). It therefore appears unlikely that the observed inefficiency in ATP synthesis was due to a problem at the level of the ATP-synthase. The rate of succinate-supported respiration in freezethawed mitochondria from the infected left lobes was only 58 per cent of the corresponding control values, suggesting that an inhibition of entry of substrate to the matrix was not a cause of the attenuated respiration in freshly prepared mitochondria. For all the parameters considered, the respiratory behaviour of mitochondria isolated from the right lobes of the infected sheep was not significantly different from the behaviour of mitochondria isolated from either the left or fight lobes of the uninfected sheep. Previous findings in the rat have shown that mitochondrial respiration was reduced three to four weeks after infection (Rule et al 1989). The reduction was subsequently shown to be associated with the presence of very high levels of NEFA and a depletion of membrane phospholipids (Lenton et al 1995). The addition of defatted BSA, which binds NEFA (Racker 1963), to the oxygen electrode incubation medium reduced State IV respiration rates and consequently increased the ACR in preparations from both control and infected sheep (Table 1). No difference was detected between State IV respiration rates in control and infected mitochondria in the presence of BSA. That is, in the infected state, the inner membranes of isolated mitochondria were no more permeable to protons than control mitochondria, either as a result of damage to the membranes inducing leakiness or as a result of the presence of a proton-translocating agent such as NEFA. BSA had no effect on the attenuated State III respiration rate of infected mitochondria, or the corresponding control rates. In corroboration of these findings, the mean concentration of mitochondrial NEFA in the left lobes of the infected livers (41.3 [5.3] nmol mg protein -1) was similar to the mean control value (48.3 [2.7] nmol mg protein-l). The major phosphorus-containing species observed by 31p-NMR in cholate extracts of control sheep liver mitochondria isolated from the left lobe are shown in Table 2,

TABLE 2: Major phosphorus-containing species observed by 31p-NMR in cholate extracts of mitochondria and microsomes isolated from livers of control sheep

Phosphatidylcholine Phosphatidylethanolamine Phospliatidylinositol Phosphatidylsedne Cardiolipin Lysophosphatidylcheline Lysophosphatidylethanolamine Glycerophosphocholine Glycerophosphoethanolamine Phosphocholine Phosphoethano]amine Sphingomyelin PC:PE

Mitochondria

Microsomes

69-0 (11-7) 64.8 (16.2) 15-0 (1.7) 6.2 (1.5) 27.6 (7.3) 2.0 (2.0) 2.8 (1.4) 23-9 (7-9) 16-6 (4.0) 0.8 (0.8) 7-8 (2-7) 12-9 (1.6) 1.21 (0-32)

279-6 (36.5) 133.9 (17.7) 69.g (8.9) 24.3 (2.7) 0 0 0 31.3 (5.4) 17.9 (5.4) 0 0 34.6 (7.1) 2.06 (0.12)

Units are nmol mg protein-1. The results are expressed as mean (SEM) (n_>3). Chemical shifts were determined by using authentic compounds. Methylene diphosphonate was used as internal shift standard (16.4 ppm) and for quantification. PC Phatidylcholine, PE Phosphatidylcholine

and the results from the infected material were not significantly different. The phospholipid composition of the mitochondria varied considerably between individual sheep and differed from previously published data (Getz et al 1968) in that the ratio of the two major phospholipid species phosphatidylcholine:pho sphatidylethanolamine (PC:PE) was lower in this study. This difference may be associated with differences in the breed of sheep used, their diet and the experimental methods. The concentrations of lysophospholipids, phosphodiesters and phosphomonoesters present in isolated sheep liver mitochondria have not been reported previously. Glycerophosphocholine (GPC) and glycerophosphoethanolamine (GPE) were present at high levels relative to the rat (Table 2) (Lenton et al 1995). In fact, the concentrations approached those observed in mitochondria from infected rat livers, in which there is evidence that they arise from increased phospholipase activity (Lenton et al 1995). This result agrees with the findings of Schmidt et al (1952) who found a high level of GPC in lamb liver and considered that it was not an artefact of the preparation procedure. Unlike mitochondria from infected rat liver, however, there was no increase in lysophosphatides or NEFA in the sheep mitochondria, suggesting that the accumulation of GPC and GPE may be the result of their decreased degradation rather than increased synthesis. This appears likely, because the phosphodiesterase responsible for the catabolism of GPC is almost non-existent in sheep liver but is high in rat liver; this enzyme also hydrolyses GPE (Dawson 1956). There appears to be no published information on phospholipase activities in sheep liver. Microsomes

An increase in NEFA content and a depletion of phospholipids has been reported in liver microsomes isolated from infected rats three weeks after infection (Lenton et al 1995). These changes did not occur in the sheep four weeks after infection, and there was no increase in lysophosphatides or phosphodiesters that would have indicated phospholipase activity (results not shown). The levels of phospholipids observed in the control animals are shown in Table 2. Consistent with the results of the mitochondrial extracts, the cholate extracts of isolated microsomes had a lower PC:PE ratio than that previously reported by Getz et al (1968) (although their results were from only one animal), with GPC and GPE again being high relative to the rat (Lenton et al 1995). However, there was a significant decline in cytochrome P450 content (Table 3). A loss of cytochrome P450 has been reported previously in lambs, but in samples representative of the whole liver and randomly combined (Galtier et al 1986). In the present study there was a more substantial loss of cytochrome P450, similar in magnitude to that observed in rats and mice (Galtier et al 1983, Somerville et al 1995), together with a coi-responding rise in the degrada-

TABLE 3: The cytochrome P450/P420 content (nmol mg protein -1) of microsomes isolated from control and infected sheep Control

Infected

Lobe

P450

P420

P450

P420

Left Right

0.70 (0-08) 0-65 (0-08)

ND ND

0.37 (0.07)* 0-59 (0.04)

0.17 (0,15)* ND

Results are expressed as mean (SEM) for five control and four infected sheep. Significant differences between infected and control values are shown as * P<0-05. ND None detected

Liver metabolism in parasitised sheep

tive product cytochrome P420" These changes were confined to the fluke-infiltrated regions of the left lobe of the liver. The concentration of cytochrome P450 in the right lobe of the infected livers was not significantly different from corresponding control values and no cytochrome P420 was detected in these samples.

Glycogen metabolism The glycogen content of the left lobe of the infected livers was less than 50 per cent of the control value. The control and infected right lobes had similar levels (Table 4). G6Pase catalyses the terminal reaction common to both gluconeogenesis and glycogenolysis. The activity of this enzyme is undetectable in vitro in the fluke-infected rat liver (Lenton et al 1995). This was not the case in the sheep, however, because the mean specific activity of G6Pase in microsomes prepared from the left lobe of infected sheep liver (0.09 [0.02] ~amol min -1 mg protein -1) was similar to that in microsomes from the left lobe of the uninfected sheep (0-11 [0-01] lamol rain -1 mg protein-I).

155

dent upon glycogenolysis for the maintenance of blood glucose concentration. F hepatica infection in sheep has been shown to have no effect on blood glucose levels under laboratory conditions (Rowlands and Clampitt 1979), but no information is available for animals under field conditions. The livers of fluke-infected sheep exhibit some features in common with the livers of infected rats, such as a reduction in cytochrome P450 and glycogen content, but they do not appear to be subject to damage by the products of the apparent phospholipase activation that is a feature of the liver of infected rats. This change, together with associated changes in mitochondrial respiration, have been shown to be precipitated by the host's immune response (Hanisch et al 1992, Lenton et al 1995), as has the decrease in cytochrome P450 content (Topfer et al 1995). As a result, despite the metabolic lesions in sheep liver being restricted to tissue that has macroscopic evidence of fluke infiltration, they may not necessarily be the result of direct physical damage by the parasite. It is known that sheep and rats differ in their immunological response to F hepatica (Rickard and Howell 1982), and it is possible that the differences observed between infected rats and sheep are due to differences at this level.

DISCUSSION Several changes have been demonstrated in the hepatic function of fluke-infected sheep. The results show that mitochondria isolated from the left liver lobes of fourweek-infected sheep were defective in their ability to synthesise ATP and that this defect was confined to areas of the liver that had suffered direct fluke-induced damage. The results also imply that the location of the lesion(s) is in the mitochondrial electron transport chain. This result is different from the aberrant mitochondrial respiration reported in infected rats, in which there appears to be a role for increased phospholipase activity, leading to the release of NEFA and the depletion of phospholipids from mitochondrial membranes (Lenton et al 1995). As with isolated mitochondria, the NEFA content and phospholipid composition of the microsomes isolated from the livers of four-week-infected sheep were not changed. At the same time there was a decline in the cytochrome P450 content of areas showing evidence of recent fluke damage. It therefore appears, at least in the sheep host, that the decline in cytochrome P450 content during fluke infection is not associated with increased phospholipase activity. Furthermore, the effect of fluke infection on cytochrome P450 content in the sheep was localised. In contrast, an apparently systemic effect leading to a similar decrease in liver cytochrome P450 content in rats infected with the intestinal nematode Nippostrongylus brasiliensis has been reported by Tekwani et al (1986). The observation that the glycogen content of the left lobe of the livers of infected sheep was substantially depleted suggests that the health of infected sheep could be compro mised under stress conditions, when the animals are depenTABLE 4: Glycogen content (mg glycogen mg homogenate protein -1) of the livers of sheep infected with Fasciola hepatica Lobe

Control

Infected

Left Right

110.5 (16.6) 83.3 (10.0)

48.7 (8-1)* 76.0 (21.8)

Results are expressed as mean (SEM) for five control and four infected sheep. Significant differences between infected and control values are shown as * P
ACKNOWLEDGEMENT The authors are grateful to the Australian Research Council for financial support.

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Received August 14, 1995 Accepted February 26, 1996