Downloaded from https://doi.org/10.1079/BJN19830097 British Journal of Nutrition (1983), 50, 291-302 291 https://www.cambridge.org/core The effect of biotin deficiency and dietary protein content on lipogenesis, gluconeogenesis and related enzyme activities in chick liver BY D. W. BANNISTER, IRIS E. ONEILL AND C. C. WHITEHEAD Agricultural Research Council’s Poultry Research Centre, Roslin, . IP address: Midlothian EH25 9PS, Scotland (Received 23 August 1982 - Accepted 21 March 1983) 170.106.34.90 1. Chicks were given biotin-deficient diets containing either suboptimal (low) or supraoptimal (high) concen- trations of protein from 1-d-old until they were used during their fourth week of life. The low-protein diet predisposed chicks to develop fatty liver and kidney syndrome and the high-protein diet to develop classical biotin , on deficiency signs. Two other groups, as controls, received biotin-supplemented rations. 24 Sep 2021 at 20:09:39 2. Low dietary protein increased lipogenesis by isolated hepatocytes but had little effect on gluconeogenesis compared to high dietary protein. 3. Low dietary protein decreased activities of hepatic isocitrate dehydrogenase (EC 1 . 1 . 1 .42), fructose- 1,Qbisphosphatase(EC 3.1 .3.11) and glucose-6-phosphatase(EC 3.1 .3.9; GP) and increased activities of fatty acid synthase (FAS), citrate cleavage enzyme (EC 4.1 .3.8; CCE) and malate dehydrogenase (decarboxylating) (EC 1.1.1.39). 4. When biotin deficiency was superimposed, the rate of lipogenesis by isolated hepatocytes (from fed birds) was decreased. Gluconeogenesis from lactate and glycerol was also depressed. , subject to the Cambridge Core terms of use, available at 5. Activity of GP was further decreased by biotin deficiency on the low-protein regimen and FAS and CCE were further increased. PK activity was increased by biotin deficiency. Classical biotin deficiency in chicks is expressed as slow growth, poor feathering, perosis and dermatitis (for a review of the condition, see Whitehead, 1978). Althoughclassical biotin deficiency is largely an experimental curiosity, the condition has been reported under commercial circumstances, particularly amongst turkey poults (Robblee 8z Clandinin, 1953). A number of years ago an apparently-unrelated condition was described in commercial broiler flocks to which the name fatty liver and kidney syndrome (FLKS) was given (Marthedal & Velling, 1958). Signs of the syndrome differ from those of classical biotin deficiency: the onset is sudden, the chick becomes progressively weaker and usually dies within 24 h. FLKS affects chicks most commonly between 3 and 5 weeks old and may cause mortality as high as 20%; with experimental diets mortalities of this level are routinely obtainable (Whitehead 8z Blair, 1976). https://www.cambridge.org/core/terms Despite the dissimilarity between the signs of FLKS and biotin deficiency, there is much evidence that the former is responsive to the vitamin (Payne et al. 1974; Whitehead et al. 19766) although the incidence is greatly influenced by the major dietary constituents (Blair et al. 1975; Whitehead 8z Blair, 1974) and the environment (Whitehead & Blair, 1974; Whitehead et al. 1975). The interrelationship between the way biotin deficiency is manifested and major dietary constituents (protein and fat) was clearly demonstrated (Whitehead et al. 1976a) by using a series of biotin-deficient diets with varying protein and fat compositions. These produced a range of expressions from severe classical biotin deficiency signs on a high-protein, high-fat diet tomodest signs but almost complete susceptibility to FLKS on a low-protein,low-fat diet. The observation of an interaction between biotin deficiency and major dietary constituents which radically alters the expression of that deficiency is of scientific interest because it implies that such an interaction distorts either normal development, or control mechanisms Downloaded from https://doi.org/10.1079/BJN19830097 292 D. w.BANNISTER, IRIS E. O’NEILL AND c. c. WHITEHEAD of major metabolic pathways. At present, no similar phenomenon has been reported in any other species except the turkey, in which FLKS has been induced under laboratory https://www.cambridge.org/core conditions (Whitehead & Siller, 1983). In order to learn more about the nature of the interaction at a biochemical level two semi-purified biotin-deficient diets were employed. One contained suboptimum protein (low-protein) whilst the other was supraoptimal (high-protein) because it is known that rations of the former type predispose chicks to FLKS, whereas the latter increases severity of classical deficiency signs (Whitehead et al. 1976~). MATERIALS AND METHODS . IP address: Procedure The birds were female I-d-old Ross broilers obtained in groups of about twenty at weekly intervals. On arrival the chicks were assigned at random to one of four separate 170.106.34.90 compartments on the bottom tier of a four-tier heated brooder. Each compartment was supplied with one of the following four diets: (1) biotin-deficient, low-protein (FLKS- inducing), (2) supplemented control, (3) biotin-deficient, high-protein (enhanced signs of , on classical biotin deficiency), (4) supplemented control. The composition of these diets is given 24 Sep 2021 at 20:09:39 in Table 1. After 1 week, the group was moved up to the next tier of the brooder in order to make room for the subsequent group. This procedure was repeated so that the oldest chicks (from week 4 to week 5) were housed on the top tier. For experiments using isolated hepatocytes and for determination of enzyme activities, only chicks from the top tier were used. For a separate experiment, in which live weight and blood pyruvate carboxylase activity (EC 6.4.1 . 1; PC) were measured, the chicks were , subject to the Cambridge Core terms of use, available at used when 3 weeks old (see p. 293) and were returned to the brooder thereafter. This Table 1. Composition (glkg)of experimental diets (The estimated biotin content of the basal diet was IlOpglkg of which approximately 4Opg was biologically available. The supplemented diet was estimated to contain 240 pg biologically-available biotin/kg) Diet no. Ingredient 1 2 3 4 Wheat 717 717 717 717 Starch 100 100 - - Low-vitamin casein* 30 30 130 130 Isolated soya-bean protein 100 100 100 100 https://www.cambridge.org/core/terms Limestone 15 15 15 15 Dicalcium phosphate 25 25 25 25 Methionine 3 3 3 3 Lysine 2 2 2 2 Salt 3 3 3 3 Vitamin and mineral mix? 5 5 5 5 Rovimix H2$ 0.01 - 0.01 Chemical analysis CP 192 192 274 274 Metabolizable energy (MJ/kg) 12.6 12.6 12.8 12.8 CP, crude protein (nitrogen x 6.25). * FPD 950; Food Production Developments Ltd, London. t Supplied (mg/kg diet): retinol2, cholecalferol20pg, a-tocopherol 17, menadione 1.3, thiamin 16, riboflavin 4, nicotinic acid 28, pantothenic acid 10, pyridoxine 9, folic acid 5, choline 0.8 g, cyanocobalamin 40 pg, copper 3.5, iodide 0.4, iron 80, magnesium 300, manganese 100, zinc 50. Containing 20 g D-biotin/kg; Roche Products Ltd, Dunstable. Downloaded from https://doi.org/10.1079/BJN19830097 Interaction of dietary protein and biotin 293 procedure was chosen because enzyme activity is high at this age (Whitehead & Bannister, 1978) and it was assumed that they would recover in the following 7-14 d. https://www.cambridge.org/core Thus, the chicks were maintained at a density of five or six per compartment until their fifth week. In those experiments where the effect of starvation was to be studied they were removed to a separate compartment and deprived of food for a period of 18-24 h. Water was available at all times. Individual experiments were replicated using chicks from several groups. Preparation of samples Blood was withdrawn into a heparinized syringe for determination of PC activity, which . IP address: was performed on 3-week-old birds that were weighed at the same time. For determination of enzyme activities, chicks were killed by cervical dislocation and portions of liver removed, homogenized in the appropriate buffer and assayed by the 170.106.34.90 methods described below. A sample of liver was taken at the same time for determination of wet weight: dry weight. In order to minimize post-mortem changes in activity, enzyme assays were divided into , on small groups such that the liver samples could be processed quickly. 24 Sep 2021 at 20:09:39 Isolated hepatocytes were prepared as described previously (Bannister & O'Neill, 198 1). Enzyme assays Blood PC activity was measured by the method of Bannister & Whitehead (1976). Enzymes associated with lipogenesis. Fatty acid synthase (FAS) was assayed according to Arslanian & Wakil (1975) in the supernatant fraction (lOOOOOg for 1 h at 4') from a , subject to the Cambridge Core terms of use, available at homogenate (200 g/l) prepared in 0-05 M-potassium phosphate, pH 7.4, 1 mM-dithiothreitol (DTT) and 1 mM-EDTA. Malate dehydrogenase (decarboxylating) (EC 1 . 1 . 1 .39; MDH) was assayed according to Wise & Ball (1964) as described previously (Whitehead et al. 1978). Isocitrate dehydrogenase (EC1 . 1 . 1 .42; IDH) was assayed according to Balnave & Pearce (1969) except that the homogenate (200 g/l) prepared in 0.1 M-potassium phosphate, 7 m~-2-mercaptoethanol,pH 7.0, was centrifuged for 2 min in a Beckman Microfuge (approximately 12000g) at room temperature. Citrate cleavage enzyme (EC 4.1 .3.8; CCE) was assayed according to Osterlund & Bridger (1977) in the supernatant fraction (100000 g for 30 min at 2O) from a homogenate (250 g/l) prepared in 0.25 M-sucrose, 0.05 M-potassium phosphate, 0.05 M-sodium fluoride, 2.5 mM-DTT and 2.5 mM-EDTA, pH 7.0. Enzymes associated with gluconeogenesis. Phosphoenolpyruvate carboxykinase (EC 4.1 .