MD, PhD KMEB Michael Gaster
email: michael.gaster@ouh.fyns-amt.dk

Insulin resistance and the mitochondrial link. Lessons from cultured human myotubes

Keywords: glucose metabolism, insulin resistance, lipid metabolism, metabolic inhibition, mitochondrial function, myotubes, skeletal muscle, Type 2 diabetes AA antimycin A, BSA, bovine serum albumin, DNP 2,4-Dinitrophenol, FCS, foetal calf serum

In order to better understand the impact of reduced mitochondrial function for the development of insulin resistance and cellular metabolism, human myotubes were established from lean, obese, and T2D subjects exposed to mitochondrial inhibitors either affecting the electron transport chain (Antimycin A), the ATP synthase (oligomycin) or respiratory uncoupling (2,4-dinitrophenol). Direct inhibition of the electron transport chain or the ATP synthase was followed by increased glucose uptake and lactate production, reduced glycogen synthesis, reduced lipid and glucose oxidation and unchanged lipid uptake. The metabolic phenotype during respiratory uncoupling resembled the above picture, except for an increase in glucose and palmitate oxidation. Antimycin A and oligomycin treatment induced insulin resistance at the level of glucose and palmitate uptake in all three study groups while, at the level of glycogen synthesis, insulin resistance was only seen in lean myotubes. Primary insulin resistance in diabetic myotubes was significantly worsened at the level of glucose and lipid uptake. The present study is the first convincing data linking functional mitochondrial impairment per se and insulin resistance. Taken together functional mitochondrial impairment could be part of the pathophysiology of insulin resistance in vivo.

Introduction

Type 2 diabetes (T2D) mellitus is characterized by alterations in both lipid and glucose metabolism, and an increasing body of evidence suggests an association between insulin resistance, T2D, and mitochondrial dysfunction. Initial in vivo studies reported a diminished citrate synthase (CS)/hexokinase (HK) ratio in diabetic skeletal muscles, showing that the oxidative/glycolytic ratio correlated with insulin sensitivity [START_REF] Simoneau | Altered glycolytic and oxidative capacities of skeletal muscle contribute to insulin resistance in NIDDM[END_REF]. Mitochondrial size and activity were also shown to be reduced in obese and T2D subjects and to correlate with the degree of insulin resistance [START_REF] Kelley | Dysfunction of mitochondria in human skeletal muscle in type 2 diabetes[END_REF]. Stump et al [START_REF] Stump | Effect of insulin on human skeletal muscle mitochondrial ATP production, protein synthesis, and mRNA transcripts[END_REF] showed that insulin has a direct, stimulatory effect on mitochondrial ATP production and mRNA transcripts of mitochondrial enzymes in skeletal muscle of non-diabetic subjects, whereas no change in mitochondrial ATP production was observed in T2D subjects. Moreover, recent analyses of gene expression patterns demonstrated a coordinated reduction in mRNA transcripts of nuclearencoded genes involved in mitochondrial oxidative phosphorylation including the catalytic betasubunit of the ATP synthase in both prediabetic and T2D subjects [START_REF] Mootha | PGC-1alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes[END_REF][START_REF] Patti | Coordinated reduction of genes of oxidative metabolism in humans with insulin resistance and diabetes: Potential role of PGC1 and NRF1[END_REF][START_REF] Sreekumar | Gene expression profile in skeletal muscle of type 2 diabetes and the effect of insulin treatment[END_REF]. Petersen et al [START_REF] Petersen | GI: Impaired mitochondrial activity in the insulinresistant offspring of patients with type 2 diabetes[END_REF] provided evidence for a possible, inherited defect in mitochondrial oxidative phosphorylation in skeletal muscle of insulin-resistant offspring of T2D patients. This defect was associated with dysregulation of intracellular fatty acid metabolism. In myotubes established from T2D patients, lipid oxidation, and insulin-mediated glucose oxidation and insulin-mediated increase in citrate synthase activity were reduced. In addition, palmitate (PA) impaired insulin-mediated glucose oxidation and insulinmediated increase in citrate synthase activity in myotubes of obese non-diabetic subjects [START_REF] Gaster | Reduced lipid oxidation in skeletal muscle from type 2 diabetic subjects may be of genetic origin: evidence from cultured myotubes[END_REF][START_REF] Gaster | The Reduced Insulin Mediated Glucose Oxidation in Skeletal Muscle from Type 2 Diabetic Subjects may be of Genetic Origin -Evidence from Cultured Myotubes[END_REF][START_REF] Ortenblad | Reduced insulin-mediated citrate synthase activity in cultured skeletal muscle cells from patients with type 2 diabetes: evidence for an intrinsic oxidative enzyme defect[END_REF].

Taken together, these data suggest that abnormalities in oxidative function may be associated with insulin resistance. Whether a direct link exists between reduced mitochondrial function and insulin resistance in skeletal muscles, can be questioned. However, our current knowledge of oxidative enzyme activities in the tricarboxylic acid cycle, mitochondrial beta-oxidation, and electron transport chain in relation to insulin resistance and T2D originates mainly from in vivo studies. Both the mitochondrial oxidative capacity and insulin resistance of skeletal muscle are highly influenced by physical activity, ageing, and fiber type composition rendering it difficult to clarify whether a direct link exists between mitochondrial dysfunction and insulin resistance in T2D. Cultured myotubes offer a unique model to separate the genetic influence on insulin resistance and T2D from environmental factors [START_REF] Gaster | The diabetic phenotype is conserved in myotubes established from diabetic subjects: evidence for primary defects in glucose transport and glycogen synthase activity[END_REF][START_REF] Henry | Glycogen synthase activity is reduced in cultured skeletal muscle cells of non-insulin-dependent diabetes mellitus subjects. Biochemical and molecular mechanisms[END_REF][START_REF] Henry | Acquired defects of glycogen synthase activity in cultured human skeletal muscle cells: influence of high glucose and insulin levels[END_REF], but also the impact of single factors on the metabolism on various genetic backgrounds. In order to better understand the impact of reduced mitochondrial function for the development of insulin resistance and cellular metabolism, human myotubes were established from lean, obese, and T2D subjects exposed to mitochondrial inhibitors either affecting the electron transport chain (Antimycin A (AA), Complex III inhibitor), the ATP synthase (oligomycin (OL), F0-F1 inhibitor) or by respiratory uncoupling (2,4-dinitrophenol, ionophor) in order to evaluate

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if exposure to these inhibitors changes the intermediary metabolism and whether they may induce insulin resistance in exposed myotubes.

Methods

Human study subjects. Ten lean, ten obese control subjects, and ten obese T2D patients participated in the study (Table 1), and clinical characteristics have previously been published [START_REF] Ortenblad | Reduced insulin-mediated citrate synthase activity in cultured skeletal muscle cells from patients with type 2 diabetes: evidence for an intrinsic oxidative enzyme defect[END_REF]. Cell culture. Cell cultures were established as previously described (14;15). In brief, muscle tissue was minced, washed, and dissociated for 60 min. by three treatments with 0.05 % trypsin-EDTA.

The cells harvested were pooled and FCS was added to stop trypsination. The cells obtained were seeded for up-scaling on ECM-gel coated dishes after 30 min. of preplating. Cell cultures were established in DMEM medium supplemented with 10% FCS, 50 U/ml penicillin, 50 µg/ml streptomycin, and 1.25 µg/ml amphotericin B. After 24 h cell debris and non-adherent cells were removed by change of growth medium to DMEM supplemented with 2% FCS, 2% Ultroser G, 50 U/ml penicillin, 50 µg/ml streptomycin, and 1.25 µg/ml amphotericin B. Cells were subcultured twice before final seeding. At 75% confluence the growth medium was replaced by basal medium (DMEM supplemented with 2% FCS, 50 U/ml penicillin, 50 µg/ml streptomycin, 1.25 µg/ml amphotericin B, and 25 pmol/l insulin) in order to induce differentiation. The cells were cultured in humidified 5 % CO 2 atmosphere at 37°C, and medium was changed every 2-3 days.

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Experimental design. Human myotubes established from lean, obese, and T2D subjects were allowed to differentiate under physiological conditions of insulin (25pmol/l) and glucose (5.5 mmol/l) for eight days. All myotube cultures were used for analysis day eight after onset of differentiation.

At day eight, myotubes were exposed to four different protocols: a) basal medium (5.5 mmol/l Glucose, 25 pmol/l Insulin); b) basal medium supplemented with 1.0 µg/ml AA, c) basal medium supplemented with 5.0 µg/ml OL; d) basal medium supplemented with 1.0 mmol/l 2,4-Dinitrophenol, and subsequently basal and insulin-stimulated glucose uptake, glucose oxidation, glycogen synthesis (GS), lipid uptake and lipid oxidation were determined. Furthermore, the lactate concentration was determined in the media after terminating the experiments.

Mitochondrial inhibition:

In order to verify that the inhibitors were inhibiting the mitochondria the O 2 -utilisation rate of trypsinated myotubes in suspension were measured with a Clark-type electrode (Hansatech Instruments DW1) in a water-jacket glass chamber maintained at 25°C and equipped with magnetic stirring in DMEM containing 5.5mmol/l glucose [START_REF] Ortenblad | Reduced insulin-mediated citrate synthase activity in cultured skeletal muscle cells from patients with type 2 diabetes: evidence for an intrinsic oxidative enzyme defect[END_REF]. Myotube cultures were used for analysis day eight after onset of differentiation. When the O 2 -respiration reached the stable rate, the uncoupled respiration was measured after adding increased concentrations of DNP

(1 pM to 1mM). The maximal uncoupled respiration was measured after adding 0.1 mmol/l DNP (final konc) without further increase, thereby immediately (30-60 s) enhancing the respiration rate 2.8 times. In parallel the effect of AA (1pg/ml to 1µg/ml) and OA (5 pg/ml to 5µg/ml) on the respiration of myotubes were studied after a steady state respiration was achieved. Maximal inhibition of the respiration of myotubes was achieved at a concentration of 0.5mg/ml and 1.25 mg/ml (final conc.) respective for AA and OA blocking the respiration rate to 5% and 3% of basal respiration rate (Data not shown, N=2). Furthermore, the total ATP content in cell extract was measured with the ATP monitoring reagent (ATPlite from PerkinElmer, Turku, Finland). Cells were grown and differentiated in 12 well plates, as described above. At day eight, myotubes were exposed to four different protocols: a) basal medium (5.5 mmol/l Glucose, 25 pmol/l Insulin); b) basal medium supplemented with 0.01, 0.1 and 1.0 µg/ml AA, c) basal medium supplemented with 0.05, 0.5 and 5.0 µg/ml OL; d) basal medium supplemented with 0.01, 0.1 and 1.0 mmol/l 2,4-Dinitrophenol, for four hours and subsequently acid soluble metabolites were extracted with tricloroacetic acid (TCA). TCA extracts were neutralized in Tris buffer (0.1mol/l) [START_REF] Gaster | A cellular model system of differentiated human myotubes[END_REF] and ATP determined on a VICTOR Plate Reader model 1420-050 (PerkinElmer, Turku, Finland). Fig. 1 shows the fractional ATP content in myotubes exposed to increasing concentrations of used inhibitor compared to baseline condition. The fractional ATP content tended to be lowered at the maximal used concentrations investigated i.e. 0.92 ± 0.03, 0.91 ± 0.05 and 0.90 ± 0.02 (n=3) for AA, OA and DNP respectively. The inhibitors were used at following final concentrations 5.0 µg/ml

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OL, 1.0 µg/ml AA and 1.0 mmol/l DNP in the presented experiments to ensure maximal inhibition of mitochondrial function and equal fractional ATP content.

Glucose uptake: Cells were grown and differentiated in 12 well plates, as described above.

Cultures were exposed to DMEM supplemented with 0.24 mmol/l fat free albumin, 50 U/ml penicillin, 50 µg/ml streptomycin, 1.25 µg/ml amphotericin B, 2-deoxyglucose(DOG)/2-[ 3 H]DOG (0.2µCi/well), and 25 pmol/l or 1 µmol/l insulin, respectively, in order to study basal and insulinmediated glucose uptake [START_REF] Gaster | The Reduced Insulin Mediated Glucose Oxidation in Skeletal Muscle from Type 2 Diabetic Subjects may be of Genetic Origin -Evidence from Cultured Myotubes[END_REF]. The reaction was stopped after four hours by aspirating the reaction mixture and rapidly rinsing each well four times with PBS at 4ºC. Cells were solubilized by addition of 0.5 ml 0.1 M NaOH. An aliquot of 50 µl was removed for protein determination. The remaining fluid was placed in a scintillation vial, and scintillation fluid was added. Glucose transport activity was expressed as moles of 2-deoxy-glucose taken up per minute per absorbance of total protein or per mg protein.

Glycogen synthesis: Cells were grown and differentiated in 12 well plates, as described above.

Cultures were exposed to DMEM supplemented with 0.24 mmol/l fat free albumin, 50 U/ml penicillin, 50 µg/ml streptomycin, 1.25 µg/ml amphotericin B, D-[ 14 C(U)]glucose (1.0µCi/well), and 25 pmol/l or 1 µmol/l insulin, respectively, in order to study basal and insulin-stimulated GS. The reaction was stopped after four hours by aspirating the reaction mixture and rapidly rinsing each well four times with PBS at 4ºC. Cells were solubilized by adding 0.5 ml 1.0 M KOH and heated at 70 °C for 20 min. 100 µl saturated Na 2 SO 4 , 100 µl 25 mg/ml glycogen were added to the sample in distilled water (freshly made) and 9 ml ice-cold absolute ethanol and then left at -70 °C for 48 hours for glycogen precipitation. The tubes were centrifuged (2000 G, 20 min, 4°C) and the supernatant immediately removed and discarded. The glycogen precipitate was redissolved in 500 µl distilled water by heating at 70°C for 10 min, 9 ml ice-cold absolute ethanol were added and then left at -20 °C for minimum 24 hours. Again, the tubes were centrifuged, the supernatants removed and 500 µl distilled water were added and the glycogen dissolved. 500 µl were taken out for scintillation counting [START_REF] Gaster | Reduced lipid oxidation in skeletal muscle from type 2 diabetic subjects may be of genetic origin: evidence from cultured myotubes[END_REF].

Glucose oxidation: Cells were cultured on 12.5 cm 2 flasks and differentiated, as described above.

Cultures were exposed to DMEM supplemented with 0.24 mmol/l fat free albumin, 50 U/ml penicillin, 50 µg/ml streptomycin, 1.25 µg/ml amphotericin B, D-[ 14 C]glucose ( 2.0µCi/ml), and 25 pmol/l or 1 µmol/l insulin, respectively, in order to study basal and insulin-mediated glucose

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To study the impact of acute exposure to PA, sister cultures were incubated under the above-mentioned conditions supplemented by 0.6 mmol/l PA. Flasks were air-tightened with a rubber stopper. After four hours, 300 µl phenyl ethylamine-methanol (1:1, v/v) were added with a syringe to a centre well containing a folded filter paper. 300 µl 1 M perchloric acid were subsequently added to the cells through the stopper tops by means of a syringe. The flasks were placed for a minimum of one hour at room temperature to trap labeled CO 2 . Cell-free flasks (no cell controls) went through the same procedure to correct for unspecific CO 2 trapping.

Lactate concentration:

The lactate content in the collected media during the various incubation conditions was determined spectrophotometrically in accordance with the method described by

Passonneau et al [START_REF] Passonneau | Enzymatic Analysis. A practical guide[END_REF].

PA uptake. Cells were cultured in 12 well plates and differentiated, as described above. Myotubes were exposed to DMEM supplemented with 0.24 mmol/l fat free albumin (BSA), 0.5 mmol/l Lcarnitine, 20 mmol/l HEPES, either [1-14 C] oleic acid (2.0 µCi/ml, 0.6 mM) or [1-14 C] palmitic acid (2.0 µCi/ml, 0.6 mM) with 5.5 mmol/l and 25 pmol/l or 1 µmol/l insulin and analyzed, as previously described in [START_REF] Gaster | Reduced lipid oxidation in skeletal muscle from type 2 diabetic subjects may be of genetic origin: evidence from cultured myotubes[END_REF]. The reaction was stopped after four hours by aspirating the reaction mixture and rapidly rinsing each well four times with PBS at 4ºC. Cells were solubilized by adding 0.5 ml 0.1 M

NaOH. An aliquot of 50 µl was removed for protein determination [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF]. The remaining fluid was placed in a scintillation vial, and scintillation fluid was added. The incubation media was transferred into two new tubes and assayed for labeled β-oxidation products (acid soluble metabolites (ASM)) [START_REF] Skrede | Stimulation of fatty acid oxidation by a 3-thia fatty acid reduces triacylglycerol secretion in cultured rat hepatocytes[END_REF]. Lipid uptake was determined as the sum of intracellular lipids and ASM. PA:BSA ratio was Eq (1)

Fmet = Gox(inh)/(GT(inh)-GS(inh))
where (inh) indicates rates under conditions of inhibition and where GT(inh)-GS(inh) corrects for glucose which is not available for pentose phosphate shunt as it is mainly incorporated in glycogen.

The activity of pentose phosphate shunt:

Eq (2)
Pentose phosphate shunt = Fmet (GT-GS) = Gox(inh) (GT-GS)/(GT(inh)-GS(inh))

Statistical analysis. Data in text, tables, and figures are given as mean +/-SE. Statistical analyses were performed with INSTAT 2.01 (GraphPad, USA). Kruskal-Wallis test or Friedman's test with Dunn comparison test were used to assess non-paired and paired significant differences between multiple treatments. Pearson correlation coefficient was used for covariance analysis. P ≤ 0.05 was considered to be significant.

Results

Subject characteristics. Clinical characteristics of the lean and obese controls and T2D patients are shown in Table 1. Fasting plasma glucose, serum insulin, and HbA1c levels were significantly higher in the diabetic group compared with both the lean and obese controls. Fasting HDL cholesterol concentrations were lower in T2D patients compared with lean controls. The obese controls showed higher fasting serum insulin levels compared with lean controls. During the steady state of the hyperinsulinemic euglycemic clamp period, the glucose infusion rates (GIR) were significantly lower in T2D patients compared with both lean and obese control subjects, and GIR were significantly lower in obese controls compared with lean control subjects.

Cell culture. Established cultures form many myotubes as shown by phase contrast microscope (not shown). We could not differentiate between study groups by visual inspection. The various treatments used in the present study proved not to be toxic for the cells, as verified by measurements of cellular protein content (not shown).

Glucose uptake is increased in myotubes inhibited with OL, AA, and 2,4-Dinitrophenol (DNP), and insulin resistance is developed in myotubes exposed to OL and AA: Diabetic myotubes expressed an increased basal glucose uptake compared with myotubes from lean controls (p<0.05) (Fig. 1a), while the insulin response was significantly reduced compared with lean myotubes when precultured under normophysiological conditions (p<0.05) (Fig. 2b). Basal and insulin-stimulated glucose uptake increased significantly in myotubes of all three groups (p<0.05) when exposed to OL, AA and DNP, mostly for the latter (Fig. 2a). The insulin response (fold change) was significantly reduced (p<0.05) in myotubes exposed to OL and AA, while only obese myotubes seem to respond with reduced insulin effect during DNP inhibition (Fig. 2b).

Glucose oxidation is reduced in myotubes inhibited with OL, AA and increased by DNP, but they did not induce insulin resistance at the level of glucose oxidation: Diabetic myotubes precultured under baseline conditions expressed an increased basal and insulin-stimulated glucose oxidation compared with lean myotubes (p<0.05) (Fig. 3a), while the insulin response was significantly reduced compared with lean myotubes (p<0.05) (Fig. 3b). Basal and insulin-stimulated glucose uptake decreased significantly in myotubes of all three groups when exposed to OL and AA compared with baseline condition (p<0.05). In contrast, DNP significantly increased both basal and insulin-stimulated glucose oxidation (p<0.05) (Fig. 3a). All myotubes were insulin sensitive, but OL, AA, and DNP did not alter the insulin response significantly (Fig. 3b).

Glycogen synthesis is reduced in myotubes inhibited with OL, AA, and DNP, while Insulin resistance was only induced in lean myotubes: The insulin response on GS was significantly reduced in diabetic myotubes compared with lean myotubes precultured under baseline conditions (Fig. 4b). Both basal and insulin-stimulated GS were decreased significantly in myotubes of all three groups when exposed to OL, AA, and DNP compared with baseline condition, mostly during DNP (Fig. 4a). All myotubes were insulin sensitive, but OL, AA, and DNP only reduced the insulin response (fold change) significantly in lean myotubes (Fig. 4b).

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Lactate production is increased in myotubes inhibited with OL, AA, and DNP: We measured the lactate accumulation in the media of myotubes exposed to baseline conditions and various inhibitors. Lactate production was increased in obese and diabetic myotubes precultured under baseline conditions. Both basal and insulin-mediated lactate production was significantly increased in all three groups when exposed to OL, AA, and DNP. Significant differences between groups were only detected at the basal level of DNP exposure (Fig. 5).

The insulin response on PA uptake in human myotubes is reduced by OL, AA, and DNP: We studied the effect of oligomycin A (OL), AA, and DNP inhibition on basal and acute insulinstimulated PA uptake in myotubes established from lean, obese, and T2D subjects (Fig. 6a,b).

Basal and insulin-stimulated PA uptake was not significantly different between groups (p>0.40). PA uptake was insulin sensitive in all myotubes (p<0.05). Exposure to OL, AA or DNP did not significantly change basal or insulin-stimulated PA uptake (Fig. 7a). However, all inhibitors decreased the insulin response (fold change) in lean, obese, and diabetic myotubes (Fig. 7b).

. PA oxidation is reduced in myotubes inhibited with OL, AA while increased by DNP: PA oxidation was not significantly different between groups in the present study under various experimental conditions, although diabetic and obese myotubes PA oxidation tended to be lower.

Myotubes exposed to OL and AA expressed a reduced ASM and complete PA oxidation compared with baseline conditions, mostly pronounced for AA (Fig. 7a). The complete oxidation is reduced to 11% of baseline by AA and to 21% by OL. ASM is reduced to 65% and 47% of baseline by OL and AA, respectively. In contrast DNP increased PA oxidation in all myotubes based on a significant increase of the complete oxidation (Fig. 7). The complete PA oxidation increased by a factor of 3.2 by DNP, while beta oxidation was reduced to 75% of baseline level. Overall, the PA oxidation was reduced to 51% by OL and to 36% by AA, while DNP increased PA oxidation by 50% compared with baseline level. A measurement of the oxidation in peroxisomes can be obtained under studies blocking mitochondrial oxidation. No significant differences in the fractional distribution of PA between peroxisomes and mitochondrial were detected between groups (data not shown).

The activity of the pentose phosphate shunt is insulin sensitive and increased in diabetic myotubes: Based on the above data it is possible to calculate the activity of the pentose phosphate shunt in myotubes as described under metabolic considerations. The activity of the pentose phosphate shunt was insulin sensitive in all myotubes and was significantly increased both at basal level and during insulin stimulation in diabetic myotubes compared with lean myotubes

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(Fig. 8a). The difference between groups was still present when correcting for differences in glucose uptake (Fig. 8b), while the insulin effect seems based on an increased glucose uptake.

Discussion

The impetus for this study was the paradigm that mitochondrial dysfunction is associated with insulin resistance in obese and T2D subjects. Glucose and lipid metabolism were studied in human myotubes established from lean, obese, and T2D subjects during exposure to AA, OL, and DNP in order to reduce ATP production from oxidative phosphorylation, either directly by affecting ATP synthase or the electron transport chain or indirectly by disturbing the proton gradient across the mitochondrial membrane by the ionophor DNP. Experiments were done under normoglycemic conditions, in order to ensure substrates for ATP production from glycolysis. The advantages of using human myotubes are that the extracellular environment can be controlled precisely and kept constant over time, thus allowing us to study the importance of the genetic background or single exogenous factors without systemic, homeostatic regulatory components from the nervous and endocrine systems.

Direct inhibition of the electron transport chain or the ATP synthase was followed by increased glucose uptake and lactate production, reduced glycogen synthesis, reduced lipid and glucose oxidation and unchanged lipid uptake, thus changing the glucose metabolism in the direction of anaerobe metabolism with only minimal changes in lipid metabolism. Glucose metabolism still allows ATP production in the absence of oxygen through oxidation of glucose to pyruvate and the conversion of pyruvate to lactate. In contrast, fatty acids cannot be completely oxidized by other organelles than mitochondria, although the beta oxidation in peroxisomes may partly metabolize fatty acids. Lipid and glucose oxidation were reduced in accordance with the mode of action of these inhibitors. Indirect inhibition of mitochondrial ATP production expresses the same picture, except for an increase in glucose and PA oxidation during uncoupling.

Antimycin A and oligomycin treatment was followed by insulin resistance at the level of glucose and PA uptake in all three study groups, while glycogen synthesis was only affected in lean myotubes, suggesting that impaired mitochondrial function has a role in the pathophysiology of insulin resistance. The present study is the first convincing data linking functional mitochondrial impairment per se and insulin resistance. Rabøl et al [START_REF] Rabol | Mitochondrial oxidative function and type 2 diabetes[END_REF] have very recently reviewed mitochondrial oxidative function and type 2 diabetes and concluded, that the reduced oxidative capacity in most studies are explained by decreased mitochondrial content. We have previously tried to verify a reduced mitochondrial content in diabetic myotubes by three different parameters, but showed no significant differences in mitochondrial content between groups [START_REF] Ortenblad | Reduced insulin-mediated citrate synthase activity in cultured skeletal muscle cells from patients with type 2 diabetes: evidence for an intrinsic oxidative enzyme defect[END_REF]. Although a

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reduced mitochondrial content not could be verified in diabetic myotubes imply our findings, that also a reduced mitochondrial content is linked to insulin resistance, supporting previous in vivo studies describing an association between reduced mitochondrial content and insulin resistance (reviewed ( 19), (2;20). Further studies have to clarify the magnitude of reduction in mitochondrial content necessary for or the degree of functional impairment, which can induce insulin resistance.

Both the mitochondrial oxidative capacity and insulin sensitivity of skeletal muscle are highly influenced by physical activity, ageing, and fiber type composition, rendering it difficult to clarify whether a direct link exists between mitochondrial dysfunction and insulin resistance in T2D.

Cultures of human myotubes are not influenced by these parameters, as myotubes in culture all express fast heavy myosin, seldom contract spontaneous [START_REF] Gaster | A cellular model system of differentiated human myotubes[END_REF] and in the present study are established from subjects at comparable age and used in the same passage. The diabetic phenotype was conserved in diabetic myotubes, as verified by a reduced, insulin-stimulated increment in glucose uptake, glucose oxidation, and glycogen synthesis. The question arises whether the mitochondria in diabetic myotubes already express functional mitochondrial defects.

The insulin resistance in diabetic myotubes could be worsened by the metabolic inhibitors, indicating that possible defects in the mitochondria in diabetic myotubes are only partial. In line, we showed previously that diabetic myotubes express a 14% reduction in basal activity of citrate synthase compared to lean myotubes, implying a mild mitochondrial impairment [START_REF] Ortenblad | Reduced insulin-mediated citrate synthase activity in cultured skeletal muscle cells from patients with type 2 diabetes: evidence for an intrinsic oxidative enzyme defect[END_REF]. Increased accumulation of intracellular triglyceride (TAG) has been associated with insulin resistance and linked to mitochondrial dysfunction (1;2;21-27). We have recently shown that induced inhibition of oxidative phosphorylation by AA and OA was followed by TAG accumulation [START_REF] Gaster | Triacylglycerol accumulation is not primarily affected in myotubes established from type 2 diabetic subjects[END_REF]. Thus above myotubes studies indicate, that induced mitochondrial impairment is linked both t o TAG accumulation and insulin resistance. Taken together functional mitochondrial impairment could be part of the pathophysiology of insulin resistance and TAG accumulation in vivo.

In the present study, a new finding is that the activity of the pentose phosphate shunt was increased twice in diabetic myotubes compared with lean myotubes. The shunt activity seems insulin sensitive, but this effect disappeared when corrected for changes in the insulin-mediated glucose uptake. The pentose phosphate shunt metabolizes 2 % of glucose taken up for glycolysis in human myotubes, which is in line with previous in vitro and in vivo studies. The major function of the pentose phosphate shunt is to provide NADPH for reductive synthesis of fatty acids and amino acids outside the mitochondria and to provide ribose for nucleotide and nucleic acids. It could be speculated whether the increased activity of the pentose phosphate shunt may contribute to the increased triacylglycereol content seen in obese and T2D patients. Further studies are needed to test this hypothesis. The pathway has not been shown to participate directly in the pathogenesis of insulin resistance.

  Muscle biopsies were obtained from the vastus lateralis muscle by needle biopsy under local anesthesia. Diabetic patients were treated with either diet alone or in combination with sulfonylurea, metformin or insulin withdrawn one week before the study. The patients suffered from no diabetic complications except for simplex retinopathy. The control subjects had normal glucose tolerance and no family history of diabetes. All subjects gave written informed consent, and the local ethics committee of Funen and Vejle County approved the study. Materials. Dulbecco`s modified Eagle`s medium, fetal calf serum (FCS), penicillin-streptomycinamphotericin B, and trypsin-EDTA were obtained from Invitrogen (Invitrogen, Scotland, UK). Ultroser G was purchased from Pall Biosepra (Cergy-Saint-Christophe, France). Protein assay kit was purchased from B ioRad (Copenhagen, DK). Palmitic acid, L -carnitine, AA, OL, KN 93, GF109203X, 1,2-bis(2-aminophenoxy)ethane.-N,N,N,N-tetraacetic acid (BAPTA-AM), N-acetyl-Lcysteine (NAC) and ECM-gel were purchased from Sigma Chemical Co. (St. Louis, USA). Bovine serum albumin (BSA) (essentially FA free), compound C, LY 294002, and SB 203580 were from Calbiochem (VWR, Roskilde, DK). Insulin Actrapid was from Novo Nordisk (Bagsvaerd, DK).

2. 5 : 1 .

 51 Control and treated myotubes were exposed for equal amount of fatty acid free BSA PA oxidation: Cells were cultured on 12.5 cm 2 flasks and differentiated and exposed, as described above. Myotubes were exposed to DMEM supplemented with 0.24 mmol/l fat free albumin (BSA), 0.5 mmol/l L-carnitine, 20 mmol/l HEPES, [1-14 C]palmitic acid (2.0 µCi/ml, 0.6 mmol/l), and 25 pmol/l or 1 µmol/l of insulin to study basal PA oxidation. Flasks were air-tightened with stopper tops. After four hours, 300 µl phenyl ethylamine-methanol (1:1, v/v) were added with a syringe to a centre well containing a folded filter paper. 300 µl 1 mol/l perchloric acid were subsequently added to the cells through the stopper tops using a syringe. The flasks were placed for a minimum of one hour at room temperature to trap labeled CO 2 . Cell-free flasks (no-cell controls) went through the same procedure to correct for unspecific CO 2 trapping. PA:BSA ratio was 2.5:1. Control and treated myotubes were exposed for equal amount of fatty acid free BSAA C C E P T E D M A N U S C R I P TMethodological considerations: Pentose phosphate shunt: In the present study we determined glucose uptake (GT), glucose oxidation (Gox), and the rate of GS under normophysiological conditions and during inhibited mitochondrial oxidation. CO2 generation from glucose is mainly explained by mitochondrial oxidation and by the activity of the pentose phosphate shunt. By inhibiting mitochondrial oxidation we can estimate the activity of the pentose phosphate shunt by collecting released CO2. The fraction of glucose metabolized (Fmet) to CO2 by the pentose phosphate pathway can be determined as:

Figure 1 .

 1 Figure 1. The fractional ATP content in myotubes exposed for increasing concentrations of AA, OL and DNP. Human satellite cell cultures were established from lean, grown and differentiated for eight days under physiological conditions (5.5 mmol/l glucose, 25 pmol/l insulin) and exposed to the following media conditions for four hours: a) basal medium (5.5 mmol/l Glucose, 25 pmol/l Insulin); b) basal medium supplemented with 0.01, 0.1 and 1.0 µg/ml AA, c) basal medium supplemented with 0.05, 0.5 and 5.0 µg/ml OL; d) basal medium supplemented with 0.01, 0.1 and 1.0 mmol/l 2,4-Dinitrophenol, for four hours and ATP content was determined as described in "methods". Fig.1 shows the fractional ATP content in myotubes exposed to increasing concentrations of used inhibitor compared to baseline condition. The inhibitor concentration is expressed as percent of maximal used inhibitor. AA (open bars), OL (hatched bars) and DNP (black bars). N=3.

Figure 2 .

 2 Figure 2. Glucose uptake in myotubes established from lean and obese and T2D subjects after exposure to mitochondrial respiratory inhibitors. Human satellite cell cultures were established from lean (open bars), obese (hatched bars) and T2D subjects (black bars), grown and differentiated for eight days under physiological conditions (5.5 mmol/l glucose, 25 pmol/l insulin) and exposed to the following media conditions for four hours: a) basal medium (5.5 mmol/l Glucose, 25 pmol/l Insulin); b) basal medium supplemented with 1.0 µg/ml antimycin A (AA), and c) basal medium supplemented with 5.0 µgm/l oligomycin (OL); and d) basal medium supplemented with 1.0 mmol/l 2,4-dinitrophenol (DNP), and subsequently basal (bas) and insulinstimulated (stim) glucose uptake was determined as described in "methods". A) Absolute values. * p < 0.05 insulin-stim vs. corresponding basal. ^p < 0.05 vs. lean. # p < 0.08 vs. lean. a p < 0.05 vs. corresponding control. b p < 0.05 vs corresponding OL. c p < 0.05 vs. corresponding AA. B) Glucose uptake increment during acute insulin stimulation expressed as fold change. *p < 0.05 insulin-stim vs. corresponding basal. p < 0.05 vs. lean. # p < 0.08 vs lean. a p < 0.05 vs. corresponding control. b p < 0.05 vs. corresponding OL. c p < 0.05 vs. corresponding AA. Data are shown as mean ± SEM. N=8.

Figure 3 .

 3 Figure 3. Glucose oxidation in myotubes established from lean and obese and T2D subjects after exposure to mitochondrial respiratory inhibitors. Human satellite cell cultures were established from lean (open bars), obese (hatched bars) and T2D subjects (black bars), grown and differentiated for eight days under physiological conditions (5.5 mmol/l glucose, 25 pmol/l insulin)

Figure 4 .

 4 Figure 4. Glycogen synthesis (GS) in myotubes established from lean and obese and T2D subjects after exposure to mitochondrial respiratory inhibitors. Human satellite cell cultures were established from lean (open bars), obese (hatched bars) and T2D subjects (black bars), grown and differentiated for eight days under physiological conditions (5.5 mmol/l glucose, 25 pmol/l insulin) and exposed to the following media conditions for four hours: a) basal medium (5.5 mmol/l Glucose, 25 pmol/l Insulin); b) basal medium supplemented with 1.0 µg/ml AA; and c) basal medium supplemented with 5.0 µgm/l oligomycin (OL); and d) basal medium supplemented with 1.0 mmol/l 2-dinitrophenol (DNP), and subsequently basal (bas) and insulin-stimulated (stim) GS was determined as described in "methods". A) Absolute values. *p < 0.05 insulin-stim vs. corresponding basal. ^p < 0.05 vs. lean. # p < 0.08 vs. lean. a p < 0.05 vs. corresponding control. b p < 0.05 vs. corresponding OL. c p < 0.05 vs. corresponding AA. B) GS increment during acute insulin stimulation expressed as fold change. * p < 0.05 insulin-stim vs. corresponding basal. ^p < 0.05 vs. lean. # p < 0.08 vs. lean control. a p < 0.05 vs. corresponding control. b p < 0.05 vs corresponding OL. c p < 0.05 vs. corresponding AA. Data are shown as mean ± SEM. N=8.

Figure 5 .

 5 Figure 5. Lactate production in myotubes established from lean and obese and T2D subjects after exposure to mitochondrial respiratory inhibitors. Human satellite cell cultures were established from lean (open bars), obese (hatched bars) and T2D subjects (black bars), grown and differentiated for eight days under physiological conditions (5.5 mmol/l glucose, 25 pmol/l insulin) and exposed to the following media conditions for four hours: a) basal medium (5.5 mmol/l Glucose, 25 pmol/l Insulin); b) basal medium supplemented with 1.0 µg/ml AA; and c) basal

Figure 6 .

 6 Figure 6. PA uptake in myotubes established from lean and obese and T2D subjects after exposure to mitochondrial respiratory inhibitors. Human satellite cell cultures were established from lean (open bars), obese (hatched bars) and T2D subjects (black bars), grown and differentiated for eight days under physiological conditions (5.5 mmol/l glucose, 25 pmol/l insulin) and exposed to the following media conditions for four hours: a) basal medium (5.5 mmol/l Glucose, 25 pmol/l Insulin); b) basal medium supplemented with 1.0 µg/ml AA; and c) basal medium supplemented with 5.0 µgm/l oligomycin (OL); and d) basal medium supplemented with 1.0 mmol/l 2,4-dinitrophenol (DNP), and subsequently basal (bas) and insulin-stimulated (stim) PA uptake was determined as described in "methods". A) Absolute values. * p < 0.05 insulin-stim vs. corresponding basal. ^p < 0.05 vs. lean. a p < 0.05 vs. corresponding control. b p < 0.05 vs. corresponding OL. c p < 0.05 vs. corresponding AA. B) PA uptake increment during acute insulin stimulation expressed as fold change. a p < 0.05 vs. corresponding control. b p < 0.05 vs. corresponding OL. c p < 0.05 vs. corresponding AA. Data are shown as mean ± SEM. N=8.

Figure 7 :

 7 Figure 7: PA oxidation in myotubes established from lean and obese and T2D subjects after exposure to mitochondrial respiratory inhibitors. Human satellite cell cultures were established from lean (open bars), obese (hatched bars) and T2D subjects (black bars), grown and differentiated for eight days under physiological conditions (5.5 mmol/l glucose, 25 pmol/l insulin) and exposed to the following media conditions for four hours: a) basal medium (5.5 mmol/l Glucose, 25 pmol/l Insulin); b) basal medium supplemented with 1.0 µg/ml AA; and c) basal medium supplemented with 5.0 µgm/l oligomycin (OL); and d) basal medium supplemented with 1.0 mmol/l 2,4-dinitrophenol (DNP), and subsequently basal PA oxidation was determined as described in "methods". Absolute values: ASM, acid soluble metabolites, CO2, complete oxidation and total oxidation is the sum of ASM and CO2. a p < 0.05 vs. corresponding control. b p < 0.05 vs. corresponding OL. c p < 0.05 vs. corresponding AA. Data are shown as mean ± SEM. N=8.

Figure 8 .

 8 Figure 8. Activity of the pentose phosphate shunt in myotubes established from lean and obese and T2D subjects. Human satellite cell cultures were established from lean, obese and T2D subjects, grown and differentiated for eight days under physiological conditions (5.5 mmol/l glucose, 25 pmol/l insulin) and the pentose phosphate shunt was determined as described in methods. A) Basal (open bars) and insulin-stimulated rates of the pentose phosphate shunt (black bars). * p < 0.05 Insulin-stim vs. corresponding basal. a p < 0.05 vs. lean. B) Activity rates of the pentose phosphate shunt corrected for glucose uptake. a p < 0.05 vs. lean. Data are shown as mean ± SEM. N=8.

Fig. 1 0

 1 Fig.1

  Fig.3a

  Fig.5

  uptake (Fold change)

Fig. 7

 7 Fig.7

  Fig.8a

Table 1 .

 1 Clinical characteristics of the study subject Data are means ± SE. * Significant different from the lean controls (P<0.05), # significant different from the lean and obese controls (P<0.05).

	Control, lean	Control, obese	T2D

Acknowledgements: Irene Lynfort and Karin Dyrgaard provided excellent technical assistance. Kurt Højlund and Klaus Levin are thanked for muscle biopsies. Martin Mogensen is thanked for assisting in measurement of myotubes respiration. The Danish Medical Research Council, the Novo Nordisk Foundation and the Danish Diabetes Association are thanked for financial support.

Referance list

A C C E P T E D

M A N U S C R I P T


Nora Hunter
email: nora.hunter@bbsrc.ac.uk

Scrapie -uncertainties, biology and molecular approaches

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Summary

The study of the biology of scrapie in sheep is irretrievably associated with the genetics of the PrP gene in sheep. Control of susceptibility and resistance is so closely linked to certain alleles of the sheep PrP gene that no review on scrapie can avoid PrP genetics. Before the importance of PrP protein was discovered and before the influence of the gene itself on disease incidence was understood, it was clear there were some sheep which were more susceptible to natural scrapie than others and that this feature was heritable. These early observations have led to the development and use of PrP genotyping in sheep in what is probably the biggest genetic selection process ever attempted. The accompanying increase in surveillance has also discovered a novel type of scrapie, the subject of much speculation about its origin.

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Scrapie susceptibility studies before the discovery of the PrP gene Studies of natural scrapie in sheep field conditions do not have the luxury of pathogenesis experiments in laboratory rodents where every aspect is controlled, particularly the time of initial infection. However from the earliest reports of the disease in sheep, it was noticed that family lines had a strong influence on the occurrence of scrapie and the measures put in place to control the disease relied heavily on this fact. In the history of sheep breeds and scrapie written by H.B.

Parry [START_REF] Parry | Scrapie[END_REF] and published after his death, details of these attempts at disease control are described. In the 18th and early 19th centuries in Britain, scrapie was a severe problem in certain breeds of sheep such as Dorset Horns and Hampshires in the county of Wessex. It was also noticed that using outbred rams could reduce or control the disease. Parry and others believed that scrapie was a simple genetic disease because the familial link was so strong, however this is now considered unlikely.

Both a susceptible genotype and an infection are required before disease develops [START_REF] Hunter | Is scrapie a genetic disease? Evidence from scrapie-free countries[END_REF][START_REF] Bossers | PrP genotype frequencies of the most dominant sheep breed in a country free from scrapie[END_REF] When in more recent times scientists began to study natural scrapie in sheep, the familiar influence on its occurrence was put to the test.

Early experimental studies of transmission of scrapie using sheep often gave confusing and contradictory results. One of the first attempts at a research programme (in England in 1952) used Welsh Mountain sheep because of the breed's small size and low cost. However after three experiments, only 5 of 156 inoculated sheep developed scrapie, an infection rate of 3%. A group of South Country Cheviots was a little better giving 14% and 35% affected sheep in two separate experiments however these rates would render experiments on scrapie in sheep prohibitively

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expensive [START_REF] Nussbaum | The establishment of sheep flocks of predictable susceptibility to expirimental scrapie[END_REF]. It was thought that some breeds would be naturally more susceptible than others and so a large study of British sheep was set up to find a suitable breed to use for pathogenesis studies [START_REF] Gordon | Variation in susceptibility of sheep to scrapie and genetic implications[END_REF]. Twentyfour different breeds of sheep, over 40 animals per breed on average and a total of 1,027 animals were injected intracerebrally (ic) with SSBP/1 scrapie [START_REF] Dickinson | Scrapie in sheep and goats[END_REF] (Table 1). There was a range of levels of susceptibility running from over 70% in Herdwicks and Dalesbreds to zero (apparently complete resistance) in Dorset Downs. Swaledales were 54% susceptible but all other breeds gave much lower disease rates with, for example, Scottish

Blackface at 8% and Welsh Mountains at 4 %. Incubation periods ranged from 3.5 months to 23 months and the experiment was brought to an end in July 1959, two years (24 months) after the scrapie injections were carried out. Later studies gave different results with Herdwicks at 30% susceptible [START_REF] Pattison | The relative susceptibility of sheep, goats and mice to two types of the goat scrapie agent[END_REF] and in the original group of "resistant" Dorset Downs which were left alive after the experiment was supposedly finished, some did actually go on to develop scrapie at later dates [START_REF] Dickinson | Scrapie in sheep and goats[END_REF]. It became obvious that there was as much variation between rates of susceptibility between individual animals as there had been thought to have been between breeds and it became clear that flocks of sheep of predictable susceptibility and resistance were needed for useful pathogenesis studies.

Development of sheep lines selected for resistance and suscepibility to scrapie.

Three lines of sheep in the UK were produced using challenge with scrapie as the means of selection. Breeding lines were set up using animals proven to be susceptible and/or resistant to this challenge (Cheviots, Herdwicks and Swaledales) [START_REF] Nussbaum | The establishment of sheep flocks of predictable susceptibility to expirimental scrapie[END_REF][START_REF] Dickinson | Some factors controlling the incidence of scrapie in Cheviot sheep injected with a Cheviotpassaged scrapie agent[END_REF][START_REF] Davies | Selection of Swaledale sheep of reduced susceptibility to experimental scrapie[END_REF] . It is possible now to do this using PrP genotype selection very much more

PrP gene and genetics

In mice the PrP gene was known to have polymorphic variants which could be linked with control of scrapie incubation time. [START_REF] Westaway | Distinct prion proteins in short and long scrapie incubation period mice[END_REF]. Mice have been used for many decades to study sheep scrapie strains as scrapie from many field cases will transmit to mice. Mouse passaged strains with varying incubation period and pathology have been immensely useful in providing information about which cells are important in scrapie transmission and in studies of susceptiblity and resistance [START_REF] Bruce | The disease characteristics of different strains of scrapie in Sinc congenic mouse lines: implications for the nature of the agent and host control of pathogenesis[END_REF]. The subsequent development of transgenic lines of mice expressing PrP genes from other species or with specific mutations has in more recent years transformed the way we understand these diseases. Rodent models are outwith the scope of this review but there are many other sources of information which can be consulted on the subject ( for example [START_REF] Manson | A single amino acide alteration (101L) introduced into murine PrP dramatically alters incubation time of transmissible spongiform encephalopathy[END_REF][START_REF] Nazor | Transgenic models of prion diseases, in Prions and prion diseases, current perspectives[END_REF] ). Because the mouse PrP gene showed amino acid variation linked to disease control, it seemed an obvious step to examine DNA from the selection line of the NPU Cheviots for a similar marker. The initial studies were carried out using restriction enzymes and generating RFLPs (restriction fragment length polymorphisms). Two of these showed variation which was associated with

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the susceptible or resistant line, EcoRI and HindIII. Nine other enzymes were also tested and failed to show any variation.

In a study of 32 sheep, 10 negative line and 22 positive line, these RFLPs were reliable in identifying which sheep belonged to which line [START_REF] Hunter | Linkage of the gene for the scrapie-associated fibril protein (PrP) to the Sip gene in Cheviot sheep[END_REF] (Table 2) , however when the study was extended to natural scrapie samples from throughout the UK, only the EcoRI RFLP remained reliable [START_REF] Hunter | Restriction fragment length polymorphisms of the scrapie-associated fibril protein (PrP) gene and their association with susceptibility to natural scrapie in British sheep[END_REF]. From subsequent [START_REF] Hunter | Swaledale sheep affected by natural scrapie differ significantly in PrP genotype frequencies from healthy sheep and those selected for reduced incidence of scrapie[END_REF]studies we have demonstrated that the polymorphism which alters the EcoRI recognition sites lies within the 3' untranslated region of the sheep PrP gene and does not affect the protein sequence (Figure 1). Despite this, the RFLP remains a reasonably good marker for susceptibility in a wide range of breeds of sheep (Goldmann, personal communication).

For predicting which animals would succumb to natural scrapie infection in the normal farm situation, the RFLPs were not 100% accurate and as it was thought likely that a polymorphism which affected the amino acid sequence of the gene would have more profound effects on pathogenesis, a DNA sequencing search of thousands of sheep PrP gene alleles started and continues to this day, new polymorphic codons being described regularly. The first codon known to be polymorphic was codon 171 [START_REF] Goldmann | Two alleles of a neural protein gene linked to scrapie in sheep[END_REF] where either Arginine (R) or Glutamine (Q) could occur. Subsequently two other codons were found to be important: 136 with Valine (V) or Alanine (A) and 154

with Histidine (H) or Arginine (R) [START_REF] Goldmann | Different scrapieassociated fibril proteins (PrP) are encoded by lines of sheep selected for different alleles of the Sip gene[END_REF] (Figure 1). Many other codons are now known to be polymorphic but these three codons remain of major importance. The first studies of incidence of these polymorphisms in scrapie sheep looked at each codon individually and in turn. Experimental challenge studies in NPU Cheviots suggested that with SSBP/1, V at codon 136 (V 136 ) on at least one allele was needed in order to transmit scrapie, whereas A 136 was associated with resistance [START_REF] Goldmann | Different scrapieassociated fibril proteins (PrP) are encoded by lines of sheep selected for different alleles of the Sip gene[END_REF] This

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was supported in a study of natural scrapie in some breeds in France [START_REF] Laplanche | PrP polymorphisms associated with natural scrapie discovered by denaturing gradient gel-electrophoresis[END_REF] but the V 136 allele clearly did not occur in some breeds [START_REF] Laplanche | French autochthonous scrapied sheep without the 136Val PrP polymorphism[END_REF] [START_REF] Elsen | Genetic susceptibility and transmission factors in scrapie: detailed analysis of an epidemic in a closed flock of Romanov[END_REF]. In breeds which do not encode V 136 (including Suffolks) it is not know what distinguishes susceptible QQ 171 animals from resistant QQ 171 animals. To explain further it is helpful to use the now conventional terminology to describe sheep PrP genotypes using the 3 codon format which gives each codon in turn for each allele in turn. In breeds such as Cheviots, Swaledales and Shetlands, it is the presence of V at codon 136 (the VRQ allele) which is linked to scrapie. [START_REF] Hunter | Swaledale sheep affected by natural scrapie differ significantly in PrP genotype frequencies from healthy sheep and those selected for reduced incidence of scrapie[END_REF][START_REF] Hunter | The association of a codon 136 PrP gene variant with the occurrence of natural scrapie[END_REF][START_REF] Hunter | Natural scrapie in British sheep: breeds, ages and PrP gene polymorphisms[END_REF].

Susceptible sheep are likely to be VRQ/VRQ or VRQ/ARQ genotypes. These are still QQ at codon 171 of course, but ARQ/ARQ animals in these flocks will survive. In some breeds such as Suffolks, the VRQ allele is so rare as to be non-existent and the most susceptible sheep are ARQ/ARQ genotype as described previously [START_REF] Westaway | Homozygosity for prion protein alleles encoding glutamine-171 renders sheep susceptible to natural scrapie[END_REF][START_REF] Hunter | Natural scrapie in a flock of Suffolk sheep in Scotland is associated with PrP genotype[END_REF] however as stated above, not all animals of this genotype group will develop disease The link is so strong that it could be interpreted as being a genetic disease, without any infection being involved. This idea is an old one, suggested by the very obvious familial occurrence of scrapie in sheep lines [START_REF] Parry | Scrapie[END_REF]. However it has now been refuted.

Not only are VRQ/VRQ sheep found in countries such as Australia and New Zealand, which do not have endemic natural scrapie [START_REF] Hunter | Is scrapie a genetic disease? Evidence from scrapie-free countries[END_REF][START_REF] Bossers | PrP genotype frequencies of the most dominant sheep breed in a country free from scrapie[END_REF], but it has recently been shown that VRQ/VRQ sheep from the NPU Cheviot flock can be reared to a healthy old age free of scrapie if they are born in clean surroundings and maintained there [START_REF] Foster | Derivation of a scrapie-free sheep flock from the progeny of a flock affected by scrapie[END_REF] . For sheep without the VRQ allele, such as Suffolks, it is also true that the susceptible genotype ARQ/ARQ is common in sheep in scrapie free countries [START_REF] Hunter | Is scrapie a genetic disease? Evidence from scrapie-free countries[END_REF] . Perhaps, as some suggested, sheep from New Zealand are simply not susceptible to scrapie, even with the genotypes (eg VRQ/VRQ and ARQ/ARQ) which develop scrapie in the UK.

By direct challenge, infecting sheep experimentally with SSBP/1 scrapie, it was first shown that Cheviot and Poll Dorset sheep from New Zealand and encoding the VRQ allele were indeed susceptible to scrapie. Incubation periods were however slightly different from those in UK sheep, for example VRQ/ARQ and VRQ/ARR sheep having similar incubation periods rather than the 100 days difference seen with NPU Cheviots [START_REF] Houston | New Zealand sheep with scrapie-susceptible PrP genotypes succumb to experimental challenge with a sheep-passaged scrapie isolate (SSBP/1)[END_REF] (compare Tables 3 and4). In the UK, DEFRA has built up a flock of

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sheep originating in New Zealand and therefore free of endemic scrapie. These animals have been used in many studies from many laboratories and are indeed fully able to develop both scrapie and BSE from inoculationed infected material.

It is now absolutely clear that in order to develop scrapie, an animal has to have both a susceptible PrP genotype AND be exposed to an infecting agent.

Use of PrP genotype information in sheep breeding.

Although the sheep PrP gene is complex in terms of how many codons are polymorphic, the genotype which is usually used is the three codon genotype (136 154 and 171). Using these codons, there are five commonly occurring alleles: VRQ, ARQ, ARR, AHQ and ARH. [START_REF] Belt | Identification of five allelic variants of the sheep PrP gene and their association with natural scrapie[END_REF] Many studies have been carried out in sheep from different countries which confirm this information is not limited to UK sheep.

In the USA, the great majority of reported scrapie cases are in sheep of Suffolk origin and, like all Suffolks, these tend to have only three genotypes ARQ/ARQ, ARQ/ARR and ARR/ARR [START_REF] Westaway | Homozygosity for prion protein alleles encoding glutamine-171 renders sheep susceptible to natural scrapie[END_REF]. This makes breeding against scrapie susceptibility very easy. ARQ/ARQ is the susceptible genotype and in the US, no cases have been found in ARQ/ARR [START_REF] O'rourke | Allelic frequencies of an ovine scrapie susceptibility gene[END_REF]although some sheep with this genotype do develop scrapie in Suffolk sheep in Europe [START_REF] Hunter | Natural scrapie in a flock of Suffolk sheep in Scotland is associated with PrP genotype[END_REF]. There are however in the USA, some breeds of sheep encoding the VRQ allele [START_REF] Desilva | Allelic variants of ovine prion protein gene (PRNP) in Oklahoma sheep[END_REF] and these would be expected to be scrapie susceptible if exposed to infection. Within Europe there are breeds with genetics similar to Suffolks but the sheep breeds frequently have more complicated

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genetics, for example in breeds such as Texels, there are 15 different genotypes [START_REF] Dawson | Guidance on the use of PrP genotyping as an aid to the control of clinical scrapie[END_REF]and as a result selection is more difficult and takes longer.

In [START_REF] Elsen | Genetic susceptibility and transmission factors in scrapie: detailed analysis of an epidemic in a closed flock of Romanov[END_REF], Spain [START_REF] Molina | Merino sheep breed's genetic resistance to scrapie: genetic structure and comparison of five eradication strategies[END_REF], Portugal [START_REF] Orge | Scrapie genetic susceptibility in Portugese sheep breeds[END_REF], Greece [START_REF] Billinis | Prion protein gene polymorphisms in healthy and scrapie-affected sheep in Greece[END_REF], Italy [START_REF] Ligios | PrPSc deposition in nervous tissues without lymphoid tissue involvment is frequently found in ARQ/ARQ Sarda breed sheep preclinically affected with natural scrapie[END_REF],

Slovakia [START_REF] Holko | PrP genotyping of sheep breeds in Slovakia[END_REF] and Germany [START_REF] Luhken | Prion protein allele A136H154Q171 is associated with high susceptibility to scrapie in purebred and crossbred German Marinoland sheep[END_REF]. There is also similar information from Iceland [START_REF] Thorgeirsdottir | PrP gene polymorphism and natural scrapie in Icelandic sheep[END_REF] A C C E P T E D M A N U S C R I P T

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where there is the added difficulty of the apparent complete lack of ARR/ARR sheep for resistance breeding -something which is also a problem for breeds in Britain known as "rare breeds" in which total numbers are small [START_REF] Townsend | PrP genotypes of rare breeds of sheep in Great Britain[END_REF].

In conjunction with the genotype information, there is also EU legislation relating to fallen stock and to sheep and goats at abattoirs which advocates searching for any sign of TSE infection using EU approved rapid testing methods which include those of Biorad TeSeE, Prionics, Enfer, and InPro [43]. All this testing of many thousands of animals has resulted in the discovery not only of the expected preclinical scrapie cases, but also an apparently different form of scrapie known as atypical scrapie which will be discussed below in detail. The original familiar scrapie is now known as classical scrapie.

BSE in small ruminants

Part of the reason for the great interest in TSEs in small ruminants and the enormous amount of work and tax payers' money involved, is because of bovine spongiform encephalopathy (BSE) which, as a disease of cattle, entered the human foodchain and resulted in the devastating disease of vCJD [START_REF] Bruce | Transmissions to mice indicate that 'new variant' CJD is caused by the BSE agent[END_REF][START_REF] Hill | The same prion strain causes vCJD and BSE[END_REF]. The most widely accepted theory on the origin of BSE is that infection was recycled by feeding ruminant derived meat and bone meal protein supplements to cattle [START_REF] Wilesmith | Bovine spongiform encephalopathy -epidemiologic studies on the origin[END_REF]. It is thought likely that sheep and goats were also exposed to the source of BSE infection in their food and it is known that small ruminants are susceptible to BSE infection when experimentally challenged, either by injection or by the oral route using only 0.5g bovine brain [START_REF] Foster | Transmission of bovine spongiform encephalopathy to sheep and goats[END_REF][START_REF] Foster | Transmission of bovine spongiform encephalopathy to sheep, goats, and mice[END_REF]. The use of animals whose PrP genotype was known, allowed

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the genetics of BSE susceptibility in sheep to be determined -the genotype ARQ/ARQ has the shortest incubation periods for example [START_REF] Goldmann | PrP genotype and agent effects in scrapie -change in allelic interaction with different isolates of agent in sheep, a natural host of scrapie[END_REF][START_REF] Foster | Clinical signs, histopathology and genetics of experimental transmission of BSE and natural scrapie to sheep and goats[END_REF]. However in several studies it has been found that not every sheep of ARQ/ARQ genotype is actually susceptible to BSE infection [START_REF] Foster | Maternal transmission studies of BSE in sheep[END_REF]. Recently more detailed analysis has suggested that in experimental challenges with BSE, sheep of genotypes encoding the ARQ allele but also with leucine (L) at codon 168 (ARLQ) have increased resistance to infection, in other words genetic linkage with TSE disease is not always straightforward [START_REF] Goldmann | Ovine prion protein variant A136R154L168Q171 increases resistance to experimental challenge with bovine spongiform encephalopathy[END_REF][START_REF] Kirby | A novel, resistance-linked ovine PrP variant and its equivalent mouse variant modulate the in vitro cell-free conversion of rPrP to PrPres[END_REF] ARQ/ARQ is a genotype which is also linked to susceptibility to Classical Scrapie [START_REF] Belt | Identification of five allelic variants of the sheep PrP gene and their association with natural scrapie[END_REF]and to experimental challenge with CH1641 scrapie [START_REF] Goldmann | PrP genotype and agent effects in scrapie -change in allelic interaction with different isolates of agent in sheep, a natural host of scrapie[END_REF]. However BSE exhibits characteristics which allow (in controlled conditions) its discrimination from scrapie. The BSE phenotype includes the patterns of incubation periods when injected into a specific panel of mouse strains [START_REF] Bruce | Transmissions to mice indicate that 'new variant' CJD is caused by the BSE agent[END_REF][START_REF] Bruce | Transmission of bovine spongiform encephalopathy and scrapie to micestrain variation and the species barrier[END_REF], the Western blot signal from

PrP SC [START_REF] Hill | The same prion strain causes vCJD and BSE[END_REF]and immunohistochemistry (IHC) detection of PrP d patterns in the brain and using antibodies with different epitopes on the PrP molecule [START_REF] Gonzalez | Distinct profiles of PrPd immunoreactivity in the brain of scrapie-and BSE-infected sheep: implications for differential cell targeting and PrP processing[END_REF][START_REF] Martin | Immunohistochemical characteristics of disease-associated PrP are not altered by host genotype or route of inoculation following infection of sheep with bovine spongiform encephalopathy[END_REF] . (PrP d is a term used when the disease associated form of PrP protein is detected using methods without a protease digestion step.) Mouse transmissions are both costly and time consuming and for most research labs the Western blot pattern is the first choice, although if BSE is suspected, a battery of other tests is required for confirmation [START_REF] Efsa | Opinion on classification of atypical transmissible spongiform encephalopathy (TSE) cases in small ruminants[END_REF].

For Western blotting, PrP SC is extracted using protease K and visualised by polyacrylamide gel electrophoresis and autoradiography producing a pattern of three bands or glycoforms which correspond to diglycosylated, monoglycosylated and unglycosylated forms of the protein [START_REF] Hope | Molecular pathology of scrapie-associated fibril protein (PrP) in mouse-brain affected by the ME7 strain of scrapie[END_REF]. Differences in the apparent molecular

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weight of the unglycosylated band can give a preliminary identification of BSE as in this case the band runs to a position slightly lower than with most forms of scrapie [START_REF] Stack | Differentiation of prion protein glycoforms from naturally occurring sheep scrapie, sheep passaged scrapie strains (CH1641 and SSBP/1), bovine spongiform encephalopathy (BSE) cases and Romney and Cheviot breed sheep experimentally inoculated with BSE using two monoclonal antibodies[END_REF]. BSE also tends to have higher amounts of the diglycosylated band than PrP SC from scrapie brains. This is illustrated in Figure 2. In panel 2(a) natural scrapie (NS)

is compared with BSE and with a scrapie source CH1641. In these preparations the lowest molecular weight (unglycosylated) band is not visible in the BSE cases and so an enzyme PNGase (Peptide:N-glycosidase) can be used to cleave the carbohydrates from the protein and leave a single clear unglycosylated band on the gel (Figure 2 b).

The fact that CH1641 scrapie looks sinilar to BSE adds to the caution with which this technique should be used when attempting to diagnose BSE but CH1641 is quite different from BSE in biology and in patterns of PrP deposition in clinically affected brain. [START_REF] Jeffrey | Ovine infection with the agents of scrapie (CH1641 isolate) and bovine spongiform encephalopathy: immunochemical similarities can be resolved by immunohistochemistry[END_REF]. An additional method of BSE detection by Western blot is related to differential binding to antibodies (Figure 3) . BSE PrP SC is detectable using the antibody 6H4 but reacts much less positively with P4 (compare lane 1 in Figure 3a with lane 1 in Figure 3b) which has an N-terminal epitope probably cleaved by proteinase K in the BSE samples. [START_REF] Stack | Differentiation of prion protein glycoforms from naturally occurring sheep scrapie, sheep passaged scrapie strains (CH1641 and SSBP/1), bovine spongiform encephalopathy (BSE) cases and Romney and Cheviot breed sheep experimentally inoculated with BSE using two monoclonal antibodies[END_REF] Using such techniques backed up with mouse transmissions, BSE was identified in a French goat sample [61] although no further cases have been detected despite increased surveillance. In a very small retrospective IHC study of goats in the UK, four animals which had naturally occurring TSE were compared with experimentally challenged goats which had either scrapie or BSE. Of the four natural TSE cases, two resembled natural scrapie in that brain sections stained with all antibodies tested, N-terminal and C-terminal epitopes. One goat however presented antibody intra-neuronal staining patterns identical to those of experimental BSE from goats and sheep in that the N-terminal antibodies did not stain the sections. The fourth goat was intermediate. [62] Transmissions to mice have

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been set up to further test the BSE-like goat but the extent of variation in just four goat samples is remarkable, when compared with sheep. It is possible that natural TSE strains in goats are more variable than in sheep and it also raises the intriguing and highly speculative possibility that the origin of BSE may have not have been in

sheep after all but in goats, although there is no evidence for this.

There have been various attempts to find signs of BSE occurring naturally in the British sheep population. Mathematical modelling and epidemiological studies have looked at various possible scenarios and cannot rule out the chances of finding BSE, albeit at low levels [63]. An estimate of the maximum frequency of cases of BSE mis-identified as scrapie was 5% in one study [64]. This means of course that BSE could be present at any level below 5%, including zero. However a more recent estimate is based on detection of PrP d by IHC and of PrP SC by Western blot in a total of 2,368 scrapie sheep samples from 1998-2004 in Britain. The sheep came from 450 flocks and over half of them came from just 34 flocks but, although some samples gave unusual patterns, none were exactly the same as BSE. With a 95% confidence limit this gives a new estimate for BSE to be hidden amongst scrapie sheep at a maximum rate of 0.66% [65].

Atypical scrapie

The first cases of what are now known as atypical scrapie were discovered in Norway in 1998 and were sufficiently distinct from classical scrapie to be given the name Nor98 [66]. Unlike the pathology found with classical scrapie, there was little or no vacuolation in the obex of the brain, nor was there any accumulated PrP SC in immunohistochemistry of the obex. Instead there was most sheep exhibited

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vacuolation and PrP SC deposition in the cerebellar region. An additional feature was the Western blot pattern of PrP Sc which exhibited a very small molecular weight band at about 12kD, not seen with classical scrapie. When mouse transmissions were attempted, the disease did not transmit to normal mice but did transmit to transgenic mice expressing the VRQ allele of the sheep PrP gene [67]. The genetics of sheep with Nor 98 were also interestingly different from classical scrapie in that Nor98 tends to occur in genotypes more usually associated with some resistance to classical scrapie, particularly with the AHQ allele. In addition an association with another codon outside the "3-codon" genotype, that of codon 141 was found, with phenylalanine (F) being more commonly found than Leucine (L) so that Nor98 sheep were more likely to encode ARFQ than ARLQ [68].

Since the discovery of Nor98, more unusual cases have come to light, picked up during the rapid testing being applied for PrP SC detection in sheep and goats throughout Europe. Such large numbers go through this precautionary testing that various commercial kits have been approved after evaluation by the European Food Safety Authority. The kits vary in which method is used to extract protein from brain samples and it is not clear what exactly is in each kit due to commercial confidentiality however it is clear that for example the method used for the BioRad TeSeE extraction uses a lower level of proteinase K (low stringency) than does for example the Prionics Check kit (high stringency). Labs using the BioRad method started to find positive results from groups of sheep of PrP genotypes regarded as scrapie resistant. This group of animals has been difficult to work with as samples are taken from apparently healthy sheep at slaughter and it is not then possible to go back and examine the animal after the result comes through. However, with the

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Biorad TeSeE detection system, positive signals were being picked up on tests which were negative with other detection systems such as Prionics Check [START_REF] Efsa | Opinion on classification of atypical transmissible spongiform encephalopathy (TSE) cases in small ruminants[END_REF]69]. These were investigated carefully and it was found that this group of atypical scrapie cases had abnormal PrP extracted from their brains which was detectable with methods using low levels of proteinase K (such as BioRad TeSeE) and not detectable with the levels of proteinase K which are normally used in the preparation of PrP Sc from classical scrapie cases (such as Prionics Check). Further analysis indicated similarities with Nor98 in the presence of a 12kD band on Western blots and in transmission characteristics to transgenice mice [67]. This additional band is shown in Figure 3b, lane 3, compared with a classical scrapie case (lane 2 and 4). Figure 3 also shows the differential binding of antibodies to this atypical PrP protein -6H4 does not bind (Figure 3a, lane 3), whereas P4 does (Figure 3b, lane 3). The higher molecular weight bands in atypical cases are highly variable in appearance -this is a single example to illustrate the distinct 12kD band (Hunter and Chong, unpublished). PrP genetics in these sheep were again surprising with even the previously thought highly resistant ARR/ARR genotype being reported as showing such cases [70] (Table 7).

Similar cases are now being reported from all over Europe including Great Britain. [71].

The European Food Safety Authority (EFSA) has produced a very useful set of definitions of Classical Scrapie, Atypical Scrapie and BSE for small ruminants which is summarised in Table 6 [START_REF] Efsa | Opinion on classification of atypical transmissible spongiform encephalopathy (TSE) cases in small ruminants[END_REF]69]. This has allowed individual countries to file reports of case numbers. In the DEFRA report on TSEs in Great Britain for 2005 [72] figures are given for the incidence of both classical and atypical scrapie which are surprising (Table 7). In the abattoir survey in 2005, 11,106 sheep were tested, 12

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were found to have classical scrapie and 16 had atypcal scrapie signs. These sheep are apparently healthy, destined for the foodchain, so any obviously sick animals would have been removed so perhaps this ratio is not too surprising. However in the fallen stock survey 8,271 sheep were tested, 26 had clasical scrapie and 6 had atypical scrapie. This gives an overall figure for scrapie in GB in 2005 of 60 cases, of which 22 (37%) were atypical. Why was this not recognized previously? The answer to this question is straightforward: before the rapid testing methods were introduced the standard detection for PrP Sc was to use high levels of proteinase K which would have destroyed the protein signal from the atypical scrapie cases.

With the increased number of cases being reported, the genotypes of animals affected by atypical scrapie have been compared with classical scrapie animals. For 2005 in GB, these results are shown in Table 7. It is clear that this new form of scrapie is appearing in genotypes previously thought to be scrapie resistant and which remain resistant to classical scrapie (Table 8) . It is also being

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genotype selection being carried out in Europe however this is unlikely in the short timescale. It is more likely that it has been around for much longer and only discovered due to the increased surveillance. In an archive search of sheep samples, a recent atypical scrapie case has been found in a sheep which died in Scotland in 1989 [START_REF] Bruce | Nor-98 like scrapie sheep in the United Kingdom in 1989[END_REF].

Remaining questions

Why do not all sheep of susceptible genotypes succumb to disease -is this because of genotypes defined by codons outside the "three codon" range? Many sheep PrP gene codons are known to vary and more polymorphisms are found every year [START_REF] Baylis | The genetics of scrapie in sheep and goats[END_REF]. Perhaps some of these have an influence on disease progression, such as the increased reistance to experimental BSE linked to codon 168 [START_REF] Goldmann | Ovine prion protein variant A136R154L168Q171 increases resistance to experimental challenge with bovine spongiform encephalopathy[END_REF][START_REF] Kirby | A novel, resistance-linked ovine PrP variant and its equivalent mouse variant modulate the in vitro cell-free conversion of rPrP to PrPres[END_REF]. The possibility is also being investigated that there is DNA variation in regions of the PrP gene which control levels of gene expression and hence protein levels [START_REF] O'neill | New ovine PrP gene haplotypes as a result of single nucleotide polymorphisms in the PrP gene promoter[END_REF] -mouse studies strongly suggest differences in PrP C levels are inversely related to length of incubation period so this is a reasonable suggestion [START_REF] Bueler | Mice devoid of PrP are resistant to scrapie[END_REF][START_REF] Manson | PrP gene dosage determines the timing but not the final intensity or distribution of lesions in scrapie pathology[END_REF].

Development of reliable pre-clinical diagnostic tests in the live animal is a major focus of research. Lymphoid tissues are often accessible for biopsy and tonsil [START_REF] Schreuder | Tonsillar biopsy and PrPSc detection in the preclinical diagnosis of scrapie[END_REF]and third eyelid [START_REF] O'rourke | Preclinical diagnosis of scrapie by immunohistochemistry of third eyelid lymphoid tissue[END_REF]have been proposed as sites for detection of PrP SC . However

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sheep are variable with some reported as having widespread PrP SC and some which do not. [START_REF] Ligios | PrPSc deposition in nervous tissues without lymphoid tissue involvment is frequently found in ARQ/ARQ Sarda breed sheep preclinically affected with natural scrapie[END_REF]. Therefore, using PrP SC as a marker for infection, which most tests do, a positive result gives a good indication of disease being present but a negative result is unreliable. Recently however a very promising new technique has been described which samples lymphoid tissue from the rectal mucosa of sheep and is reported to be genotype independent [START_REF] Gonzalez | Postmortem diagnosis of preclinical and clinical scrapie in sheep by the detection of disease-associated PrP in their rectal mucosa[END_REF].

There are still remaining questions about the origins of atypical scrapie and how it transmits between sheep, if indeed it does -the old idea of "genetic disease" has arisen again due to the sporadic nature of the occurrence of Nor-98 [66]. Even with classical scrapie, thought to infect animals by the oral route and most likely in the perinatal phase of life, the etiology is uncertain and it may be that many possible routes of infection exist in sheep. Intriguing hints are found occasionally -most recently the association of mastitis in sheep with increased PrP SC deposition in the inflamed tissues has raised the issue of whether milk from animals affected by mastitis can pass infection to their suckling lambs [START_REF] Ligios | PrPSC in mammary glands of sheep affected by scrapie and mastitis[END_REF]. True maternal (in utero) transmission has been difficult to demonstrate unequivocally, however it is known that placental tissues can harbour and transmit infection by inoculation [START_REF] Pattison | Spread of scrapie to sheep and goats by oral dosing with foetal membranes from scrapie-affected sheep[END_REF] and recent more detailed studies of PrP SC deposits have found that in scrapie affected ewes, PrP SC is found in placental tissues only if the foetus is of a susceptible genotype [START_REF] Andreoletti | PrPSC accumulation in placentas of ewes exposed to natural scrapie: influence of foetal PrP genotype and effect on ewe-to-lamn transmission[END_REF][START_REF] Tuo | PrPC and PrPSC at the fetal-maternal inferface[END_REF][START_REF] Tuo | Pregnancy status and fetal prion genetics determine PrPSC accumulation in placentomes of scrapie-infected sheep[END_REF].

This implies some interaction between mother and foetus the molecular and cellular basis of which is again unknown.

Although a great deal of progress has been made in our understanding of the interaction between scrapie and its natural host the sheep, it seems that more research and surveillance, whilst answering some questions, also reveal further unknowns in A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T

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A

  C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT quickly but the immense amount of work which went into producing these flocks is in danger of being forgotten and is important as the selection lines allowed us to develop the PrP genetics with solid biological information to underpin the work. The Herdwick flock is now reduced to minimal numbers and is held on the NPU farm (see below). The Swaledale flock has been very recently returned to normal commercial farming and is effectively lost for scientific study (Merrell, personal communication). The Cheviot sheep flock (held on the farm of the Neuropathogenesis Unit (NPU) Edinburgh) was started in 1961 from a foundation flock of 303 ewes and 15rams with no record of exposure to scrapie. They were split into two lines (positive and negative) based on their reponses to injection with SSBP/1 scrapie[START_REF] Dickinson | Scrapie in sheep and goats[END_REF][START_REF] Dickinson | Some factors controlling the incidence of scrapie in Cheviot sheep injected with a Cheviotpassaged scrapie agent[END_REF]. All foundation animals were injected (usually subcutaneously, sc) with SSBP/1 after mating (males) and after lamb weaning (females). The policy of challenging every animal was continued for many years to consolidate the selection lines and to study the genetics of disease control. Animals were mated up to three times prior to challenge but were never mated after scrapie challenge and careful records were maintained to avoid inbreeding as much as possible. Animals developing scrapie by around 500 days post infection formed the basis of the positive line and were assigned to the negative line if experimental scrapie did not develop within 2 years of sc challenge[START_REF] Hunter | Natural scrapie in a closed flock of Cheviot sheep occurs only in specific PrP genotypes[END_REF] From line cross and back cross segregation experiments it became clear that a single gene (given the name Sip, standing for scrapie incubation time) controlled susceptibility to SSBP/1. Sip had two alleles, sA and pA[START_REF] Dickinson | Genetic aspects of unconventional virus infections: the basis of the virino hypothesis[END_REF]. The negative line were all Sip pApA and the positive line were mostly Sip sAsA or sApA, albeit with varying low numbers of Sip pApA in the positive line as a result of heterozygote matings. The A C C E P T E D M A N U S C R I P T

  and it is not known what differentiates susceptible ARQ/ARQ sheep from resistant ARQ/ARQ sheep. The ARQ allele is not a single entity but can be divided up into subgroups based on polymorphisms at other codons. None of these so far, including A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT also polymorphisms in the DNA in the promoter region has so far shown universal links with disease (Goldmann, personnal communication). The genetic disease hypothesis In flocks in the UK which have sheep encoding the VRQ allele, any sheep with a homozygote genotype (VRQ/VRQ) is in some outbreaks virtually guaranteed develop natural scrapie. In the NPU Cheviot flock which has endemic natural scrapie, 100% of VRQ/VRQ animals succumb to natural scrapie at about 2 years of age [10].

  found in closed flocks of sheep used for research, such as the DEFRA flock of New Zealand derived animals (announced in a press release in November 2006 www.defra.gov.uk/news/2006/06114a.htm) and in the NPU Cheviot flock which has been closed to new introductions of sheep since 1962 (Foster and Hunter, unpublished). The source of the disease in these latter cases is far from clear. Atypical cases have been reported from all over Europe giving rise to serious questions about the continuing relevance of the National Scrapie Plan and its European counterparts which are selecting apparently for genotypes of sheep which are susceptible to this new and unknown risk of disease. It is not clear whether atypical scrapie is a novel strain of scrapie which has arisen as a result of all the A C C E P T E D M A N U S C R I P T

  the pathogenesis of scrapie in sheep is variable and linked to PrP genotype. Sheep of VRQ/VRQ genotype have widespread distribution of PrP SC in their peripheral lymphoid tissues however sheep of other genotypes, with clinical such as VRQ/ARQ and VRQ/ARR have very little PrP SC in peripheral tissues [29]. Even ARQ/ARQ A C C E P T E D M A N U S C R I P T

Figure 1 :

 1 Figure legends;

Figure 2 :

 2 Figure 2: Western blots of experimental BSE and scrapie in sheep using the antibody 6H4. (a) PrP SC prepared from natural (classical) scrapie (NS) , CH1641 scrapie and BSE using proteinase K. (b) PNGase treatment to remove the carbohydrates from PrPSC molecules thus showing a single unglysylated band in natural (classical) scrapie (NS), CH1641 scrapie, BSE and SSBP/1 scrapie.

Figure 3 :

 3 Figure 3: Wester blots of PrP SC from BSE in cattle compared with classical and aytpical scrapie in sheep. (a) antibody 6H4 (b) the same blot stripped and analysed again using the antibody P4.

  ACCEPTED MANUSCRIPTcontinued challenge with SSBP/1 injection revealed these resistant sheep but this was a very long drawn out process. The flock, now known as the NPU Cheviot flock, is still in existence and being used for pathogenesis, PrP genetics and epidemiology studies but we now understand that the Sip gene encodes the PrP protein and the older terminology is no longer in use. It does help however in the understanding of the older papers to realise that the Sip sA allele is equivalent to the sheep PrP VRQ allele (see below). The Sip pA allele group includes every other sheep PrP allele, for example ARQ, ARR, AHQ.

  genotype frequencies were 51% QQ 171 , 43% QR 171 and 6%RR 171 . The result is highly significant and it remains true that QQ 171 animals are more likely to develop

	including Suffolks and in those cases
	scrapie occurred in animals homozygous for Q at codon 171 (QQ 171 ). The clear
	association of QQ 171 with natural scrapie in Suffolk sheep was first shown by
	Westaway [23] in a study which indicated that 100% of 31 unrelated scrapie-affected
	Suffolk sheep in the USA were of QQ 171 genotype whereas in the healthy controls
	(n=69)

scrapie than the other genotypes, many animals of QQ 171 genotypes remain healthy and live long lives. It is clear that it is a subgroup of QQ 171 sheep which is most susceptible and that this subgroup has V at codon 136 -but only in breeds which encode V 136

  the UK in 2001 the National Scrapie Plan (NSP) was put in place to assist in sheep breeding to reduce the frequencies of the susceptible genotypes. This was partly due to worries that BSE may have infected the national flock as, if it had done, it would represent a danger to the human foodchain. Within the NSP, there are 15 genotypes listed in 5 different groups relating to the known susceptibility to natural

scrapie, for example NSP type 1 is ARR/ARR, which has high resistance and NSP type 5 contains VRQ/VRQ, ARQ/VRQ, ARH/VRQ, AHQ/VRQ which have high susceptibility themselves and will also pass on the high risk allele VRQ to their offspring (Table

5

). The aim of the NSP is to reduce the number of sheep with the most susceptible genotype to scrapie (VRQ/VRQ) and to increase the number of sheep with the more resistant genotypes. The scheme has run through various programmes including the Compulsory Ram Genotype Scheme (CRGS) which was designed according to European legislation which required all member states to establish such genotyping and breeding plans for TSE resistance. The CRGS aims to remove rams with the most susceptible genotypes and is a legal requirement for all sheep farmers who own purebred breeding flocks. With this and other schemes in progress, the NSP has tested over 2 million sheep to date.[34] With the similar programmes taking place within the European Union countries, this has to be the largest genetic selection programme ever put in place and is generating a huge amount of data on genotype frequencies in different sheep breeds from each country and region including France

Table 1 :

 1 Breed study of scrapie susceptibility initiated in 1957

	Breed	Number inoculated* % affected with scrapie
	Herdwick		36	78
	Dalesbred		43	72
	Swaledale		46	54
	South Country Cheviot	45	36
	Derby Gritstone	46	35
	Exmoor Horn		41	34
	Border Leicester	42	26
	Scottish Blackface	44	18
	South Devon		35	17
	Romney Marsh	43	16
	Welsh Cheviot		40	15
	Ryeland		34	15
	Dorset Horn		45	13
	Suffolk		51	12
	Leicester		42	12
	Welsh Mountain	42	10
	Hampshire Down	30	10
	North Country Cheviot	45	9
	Southdown		38	8
	Wiltshire Horn	57	7
	Shropshire		41	5
	Kerry Hill		41	2
	Clun Forest		52	2
	Dorset Down		48	0
	Sheep inoculated with SSBP/1 scrapie, 10% brain homogenate.
	Taken from reference [5] WS Gordon, 1966

Table 2 .

 2 Association of PrP gene EcoRI restriction site polymorphism with scrapie susceptibility in NPU Cheviot sheep

	_____________________________________________________________________
	Fragments	Sip genotype predicted from breeding records
	hybridising to	(PrP genotype subsequently assigned)
	PrPcDNA
	_____________________________________________________________________
	EcoRI
	6.8 kb DNA fragment only	sAsA (PrP VRQ/VRQ)
	4.4 kb DNA fragment only	pApA (PrP AXX/AXX*)
	Both fragments	sApA (PrP VRQ/AXX)
	____________________________________________________________________
	Data adapted from reference [13]
	X = any of the possible amino acids at these codon positions

Table 3 :

 3 Association of PrP gene coding region polymorphisms with scrapie in NPU Cheviot sheep following sub-cutaneous injection with SSBP/1

	___________________________________________________________________
	PrP Genotype	Incubation period	Survival, no scrapie
		in days (SD)	(days)
	__________________________________________________________________
	VRQ/VRQ	180 (20)	-
	VRQ/ARQ	269 (76)	-
	VRQ/ARR	352 (63)	-
	ARQ/ARQ	-	998-2330
	ARQ/ARR	-	820-4250
	ARR/ARR	-	1150-2720
	____________________________________________________________________
	Data adapted from reference [29]

Table 4 :

 4 Association of PrP gene coding region polymorphisms with scrapie in New Zealand-derived Cheviot sheep following sub-cutaneous injection with SSBP/1

	___________________________________________________________________
	PrP Genotype	Incubation period	Survival, no scrapie
		in days (SD)	(days)
	__________________________________________________________________
	VRQ/VRQ	150 (11)	-
	VRQ/ARQ	256 (39)	-
	VRQ/ARR	259 (9)	-
	ARQ/ARQ	-	>1,000*
	ARQ/ARR	-	>1,000
	ARR/ARR	-	>1,000
	___________________________________________________________________
	* clinical cases have occurred in this genotype of these sheep at over 1,000 days post
	inoculation but still have to be confirmed by pathology and biochemistry
	Data adapted from reference [29]

Table 5 :

 5 National Scrapie Plan, Britain

	PrP genotype	NSP* Type	Degree of resistance
	ARR/ARR	1	Sheep that are most resistant to scrapie
	_______________________________________________________________
	ARR/AHQ	2	Sheep genetically resistant to scrapie but need
	ARR/ARH		careful selection when used for breeding
	ARR/ARQ
	_______________________________________________________________
	ARQ/ARQ	3	Sheep genetically have little resistance to
	AHQ/ARH		scrapie and need careful selection when
	AHQ/ARQ		used for breeding
	ARH/ARH
	ARH/ARQ
	ARQ/ARQ
	_____________________________________________________________
	ARR/VRQ	4	Sheep genetically susceptible to scrapie and
			should not be used for breeding unless in the
			context of a controlled and approved breeding
			programme
	_____________________________________________________________
	VRQ/AHQ	5	Sheep that are highly susceptible to scrapie
	VRQ/ARH		and should not be used for breeding
	ARQ/ARQ
	VRQ/VRQ
	________________________________________________________________
	* NSP -National Scrapie Plan
	Table adapted from [86] (Crown copyright).

Table 6 :

 6 Criteria for the categorisation of TSEs in small ruminants _______________________________________________________________________________________________________________ IHC-immunohistochemistry, DMNV-dorsal motor nucleus of the vagus. Table adapted from[START_REF] Efsa | Opinion on classification of atypical transmissible spongiform encephalopathy (TSE) cases in small ruminants[END_REF]

	TSE type	Approved rapid	Stringent	Mild PK	Pathology	Genotype
		screen	Western blot	Western blot
	_______________________________________________________________________________________________________________
	Classical	positive	positive	positive	Grey matter vacuolation	Susceptible
	scrapie				IHC labelling of PrP
					in medulla and DMNV
	_______________________________________________________________________________________________________________
	BSE in	positive	positive	positive	Usually grey matter	Likely to be
	small		unglycosylated band lower	vacuolation. IHC	ARQ/ARQ
	ruminants		molecular weight than for	IHC labelling of PrP	but not
			classical scrapie controls	in medulla and usually	genotype
					also DMNV	exclusive
	________________________________________________________________________________________________________________
	Atypical	positive	negative	positive	Vacuolation and IHC	Genotypes include
	scrapie			including	negative or low levels	those resistant
	including			band at <15kDa	in obex, DMNV, medulla. to classical scrapie
	Nor98				Cerebellum is IHC positive often with at least one
					for PrP and has vacuolation AHQ or AF141RQ
						allele
	______________________________________________________________________________________________________________
	PK-proteinase K,

Table 7 :

 7 Results from targeted (Active) surveillance for TSE in sheep and goats in Great Britain during 2005 Sheep abattoir survey: random selection of 10,000 sheep aged over 18 months and slaughtered for human consumption 2. Sheep fallen stock survey: random selection of 10,000 fallen sheep aged over 18 months and notified voluntarily 3. Goat abattoir survey: all goats aged over 18 months and slaughtered for human consumption 4. Goat fallen stock survey: random selection of 1,000 fallen goats aged over 18 months and notified voluntarily.

	_________________________________________________________________________________________________________
	Survey category	Number tested	Number negative	Number Classical Scrapie	Number Atypical Scrapie
	___________________________________________________________________________________________________________
	Sheep abattoir survey 1	11,106	11,078	12	16
	Sheep fallen stock survey 2	8,271	8,239	26	6
	Goat abattoir survey 3	1,282	1,282	0	0
	Goat fallen stock survey 4	1,329	1,325	4	0
	___________________________________________________________________________________________________________
	Total sheep	19,377	19,317	38	22
	Total goats	2,611	2,607	4	0
	____________________________________________________________________________________________________________
	1. Data taken from reference [87] Crown copyright 2006

Table 8 :

 8 Genotypes of classical and atypical scrapie cases detected through active surveillance between 2002 and 2005 in Great Britain.

	_______________________________________________________________
	Genotype	NSP type	Classical	Atypical
			Scrapie	Scrapie
	________________________________________________________________
	Unknown	-----------	1	0
	_________________________________________________________________
	ARR/ARR	1	0	15
	_________________________________________________________________
	ARR/AHQ	2	0	29
	ARR/ARQ		1	13
	ARR/ARH		0	1
	__________________________________________________________________
	AHQ/AHQ	3	1	13
	AHQ/ARH		0	2
	ARQ/ARH		1	0
	ARH/ARH		0	0
	AHQ/ARQ		3	21
	ARQ/ARQ		21	13
	___________________________________________________________________
	ARR/VRQ	4	31	0
	__________________________________________________________________
	AHQ/VRQ	5	0	0
	ARH/VRQ		10	0
	ARQ/VRQ		71	1
	VRQ/VRQ		14	0
	_________________________________________________________________
	Total	--------	154	108
	_______________________________________________________________
	NSP: National Scrapie Plan
	Data from reference [87] Crown copyright 2006