Comp. Biochem. Physiol., 1976. Vol. 53B, pp. 575 to 579. Per qamon Press. Printed in Great Britain

HEPATIC GLUCONEOGENESIS IN CHICKS: EFFECT OF BIOTIN O N GLUCONEOGENESIS IN BIOTIN-DEFICIENCY A N D FATTY LIVER A N D KIDNEY SYNDROME D. W. BANNISTER Agricultural Research Council's Poultry Research Centre, King's Buildings, West Mains Road, Edinburgh, EH9 3JS, Scotland (Received 4 October 1974)

Abstract--l. Gluconeogenesis was measured in liver slices from healthy, biotin-deficient- and fatty liver and kidney syndrome (FLKS)-affected chicks. 2. There was little difference between healthy and biotin-deficient chicks (the latter were not severely deficient) but gluconeogenesis was virtually absent from FLKS liver. 3. Biotin was without effect on healthy liver in vitro. 4. The vitamin had a slightly beneficial effect on the deficient chicks and a substantial one on FLKS-affeeted birds in vivo and in vitro.

INTRODUCTION cluded. A number of other cofactors were ineffective. FATTY liver and kidney syndrome (FLKS) is the cause A possible explanation of the failure of gluconeoof significant mortality amongst flocks of broiler and genesis and the beneficial effects of dietary biotin is pullet replacement chicks (Gallus domesticus) in the the function of the latter as a coenzyme in pyruvate United Kingdom and elsewhere; consequently it has carboxylase (pyruvate: CO2 ligase (ADP), E.C. become a subject for considerable research. Diets of 6.4.1.1.). It is known that the activity of this enzyme low fat and protein and high carbohydrate content is reduced in experimentally-induced biotin deficiency have been formulated that consistently cause a high in chicks (Atwal et al., 1971) and that activity is reincidence of mortality (20Vo or more) during the stored rapidly to chick liver both in vivo and in vitro period up to 5 weeks after hatching (Whitehead & (Madappally & Mistry, 1970), to rat liver in vivo Blair, 1974a). Death from the syndrome does occur (Deodhar & Mistry, 1969) and to a strain of thermoafter this time although, under experimental condi- philic bacillus (Cazzulo et al., 1969). The mechanism tions, this appears to be the period of maximum sus- of action, apparently, is the synthesis of holoenzyme ceptibility. from apoenzyme and biotin by a holoenzyme syntheThe development of these diets has enabled the un- tase, de novo protein synthesis not being required. dertaking of systematic research into the nature and However, there are serious difficulties in accepting causes of FLKS and has led to a description of histo- the above explanation for the failure of hepatic glulogical and ultrastructural changes occurring within coneogenesis in FLKS. The classical symptoms of the tissues (Wight & Siller, 1975; Siller & Wight, in experimentally-induced biotin deficiency in no way preparation). Also much has been learnt about effects resemble those of FLKS (Whitehead et al., 1974b). of environmental and dietary manipulation on the in- In a rare inborn error of gluconeogenesis in man (incidence of mortality (Whitehead et al., 1974a, 1975; fantile subacute necrotizing encephalomyelopathy) Blairetal., 1975). However, the most significant discovery the virtual absence of hepatic pyruvate carboxylase made so far has been that supplementation of an was reported (Hommes et al., 1968) but neither severe FLKS-inducing diet with biotin drastically reduces hypoglycaemia nor other symptoms resembling mortality from the syndrome (Whitehead & Blair, FLKS (apart from elevated plasma pyruvate levels) 1974b; Whitehead, et al., 1976). were reported. In parallel with the nutritional and histological In an effort to understand and resolve these prostudies, the biochemistry of FLKS has been studied blems a first step is to establish whether or not biotin and some aspects of lipid metabolism were discussed can restore significant hepatic gluconeogenic activity by Evans et al. (1975). Bannister et al. (1975) noted to FLKS-affected birds and to compare its effects that marked hypoglycaemia was present in all birds with those on gluconeogenesis in biotin deficiency. examined, together with the virtual absence of hepatic glycogen. In vitro studies using liver and kidney slices showed the ability of the former tissue to synthesize glucose from a variety of precursors was drastically M A T E R I A L S A N D METHODS reduced or even abolished, whereas the kidney of affected birds exhibited somewhat greater activity B i r d s than normal (Bannister et al., 1975). These workers The chicks used were 3- to 4-week old female Ross 1 also reported a slight though significant restoration broilers obtained either from a commercial hatchery or of activity in their in vitro system if biotin was in- from the Centre's outstation. 575

576

D . W . BANNISniR

FLKS-affectcd chicks were taken from a large-scale experiment designed to assess dietary biotin requirement sufficient to prevent the syndrome. The husbandry conditions were similar to those already reported (Whitehead & Blair. 1974a: Whitehead et al., 1974b). FLKS diet contained 01 mg total biotin/kg diet of which about 0.06 mg would be available. It gave no signs of typical biotin deficiency. Biotin-deficient chicks were reared under laboratory conditions on a synthetic diet containing adequate protein. fat, carbohydrate and minor nutrients with the exception of biotin and by 3 weeks showed the classical symptonrs of deficiency (McElroy & Jukes. 1940; Jukes & Bird. 1942; Whitehead et al., I974h). The deficient diet contained about 0.02 mg/kg biotin, most of which would be available. Control chicks were housed under similar conditions to the biotin-deficient ones but were fed a diet adequate in all known nutrients. Control- and biotin-deficient chicks were starved overnight before being used, otherwise all birds had unrestricted access to [ood and aater. GhwolleocleHesis by li~'er sliee.s The method of Krebs et al. [1963) was used, as described prc~iously (Bannister et al., 1975}.The precursors employed were malate ( L ( - I malic acid, monosodium salt, Sigma Chemical Corp., St. Louis, U.S.A., lot 62C-5050) and lactate ( L ( + ) l a c t i c acid, lithium salt, Sigma. lot 43C-02001. Glucose was measured by an automated enzymic method (Morley et al., 1968). E.,,lk, rimeJttal desi~lJ~ Three procedures were used: Experimem 1 Tissue slices were assayed for gluconeogenic activity in the presence or absence of biotin (final concentration 50 l~mol/1) in a final volume of 5.25 ml. ExperimeHt 2 Tissue slices were incubated for 3 hr at 38 C m Eagle's Minimum Essential Medium (Wellcome Reagents Ltd.. Bechenham, Kent, U.K, Cat. No. TC 29) supplemented with glucose to a final concentration of 2.5 mg/ml. All flasks contained 0-6 0.8 g (wet weight) in a final volume of 26.25 ml and were gassed continuously with 95'~; O , 5!~; CO2. Experimental flasks contained biotin. concentration 50#mol/h After pre-incubation, slices were washed in Krebs Ringer solution at room temp and assayed for gluconeogenic activity in the absence of biotin.

Experiment 3 Birds were injected intravenously with either 0"9'}, saline or biotin in saline (0.i mg/100g body weight) and after 3hr were killed. Liver slices were then assayed for gluconeogenic activity in the absence of biotin. Other experimental details Birds were killed by intravenous injection of veterinary Nembutal [Abbott Laboratories Ltd., Kent, U.K.) except the cases of biotin deficiency which, because of their small size, were decapitatcd. Liver was removed immediately after death and chilled in ice-cold saline until slices were prepared (not longer than 5 rain). Slices (approx 0.5 ram) were prepared as described previously (Bannister et al., 1975). After each experiment appropriate birds were examined clinically to establish the presence of biotin deficiency, or samples of liver, kidney, heart and proventriculus were removed tot histological examination to confirm the diagnosis of FLKS. All chemicals used were of Analar or Reagent Grade and were used without further purification. RES U LTS Healthy birds T h e effect o f biotin on gluconeogenesis in vitro is given in Table I. O n l y E x p e r i m e n t s 1 a n d 2 were p e r f o r m e d a n d in neither case did the vitamin have a significant effect. L a c t a t e was a satisfactory precursor but there was little net glucose f o r m e d from malate. G l u c o s e release in the a b s e n c e o f p r e c u r s o r was rather higher and m o r e variable than in an earlier e x p e r i m e n t (Bannister et al., 1975 r e p o r t e d a figure o f 52.1 _+ 6 . 6 # m o l / g dry weight/hr) u n d e r similar conditions, the difference, however, was not significant. Biotin-d@cient birds In E x p e r i m e n t 1 (Table 2) there was a rise in glucose p r o d u c t i o n from lactate in the presence o f biotin t h o u g h it c a n n o t be r e g a r d e d as statistically significant. T h e r e was no effect o f biotin o n gluconeogenesis

Table 1. Hepatic gluconeogenesis by liver slices from healthy chicks Glucose production (net} ltmol/g dry wt/hr Experiment 1" Experiment 2+ No Precursor Control Biotin added Lactate (10 m mol/l final concn) Control Biotin added Malate (10 m mol/l final conch) Control Biotin added

78.3 _+ 225 (5)

366 + 7.8 (5) 3 I-5.4-_ 4.6 (5j

61.3 _+ 11.{,)(5) 41.8 _+ 20-8(5)

1423 ,+ 34'0(5t 146.6 ,+ 35.1 (5/

-0-8 ,+ 12.3 (5) 1.8 _+ 10.2(5)

7.9 ,+ 9-7 (5) 12.6 ,+ 4.1 (5)

* Tissue sliccs were incubated in 4"25 ml of Krebs Ringer solution at 38 ( after gassing with 95~; 02 5°~ CO:. Reaction was started by adding I ml of precursor in Krebs Ringer and glucose assayed in the medium by an automated method (Morley et al., 1968) after 2 hr incubation. Biotin (50 #mol/1) was included in appropriate flasks. + Tissue slices (0"6 0"Sg wet wt) were incubated in Eagles Minimum Essential Medium, supplemented with glucose to 2.5 mg/ml, for 3 hr at 38:C. The vessel was gassed continuously with 9 5o/ o 02 5'~i, COz. Appropriate vessels were supplemented with biotin (50/zmol/l) and the final volume was 26.25 ml. After incubation, slices were washed in Krebs-Ringer solution at room temp and assayed for gluconeogenic capacity as described under Experiment 1 except that no biotin was included Itinal volume 5 ml). Results arc exprcssed as thc mean + S.E.M. of the number of obserxations in parenthesis.

Gluconeogenesis and biotin

577

Table 2. Hepatic gluconeogenesis by liver slices from biotin-deficient chicks Glucose production (net) #mol/g dry wt/hr Experiment 1" Experiment 2t Experiment 3{ No precursors Control Biotin added Lactate (10 m mol/1 final concn) Control Biotin added Malate (10m mol/1 final concn) Control Biotin added

46.0 + 5.3 (6) 74.9 -t- 21.3 (6) 99.1 _+ 23"9(6)~ 22"6 _+ 5'2 (6) 16.5 _+ 7.7(6)

49.7 _+ 5.4 (6) 47.4 _+ 4.4 (6)

34.4 _+ 3.4 (4) 44.0 _+ 15.4 (4)

62.5 _+ 5.7 (6) 118.7 _+ 17.4(6)11

28.7 _+ 12.8 (4) 68"1 _+ 14.2(4)~

0.3 _+ 5-9 (6) 13.4 _+ 7.0(6)

5"8 _+ 2.5 (4) 18-1 _ 7-3(4)

* Tissue slices were incubated in 4'25 ml of Krebs-Ringer solution at 38°C after gassing with 95~o 02 5~o CO2. Reaction was started by adding 1 ml of precursor in Krebs-Ringer and glucose assayed in the medium by an automated method (Morley et al.. 1968) after 2 hr incubation. Biotin (50 #mol/l) was included in appropriate flasks. t Tissue slices (0.64).8 g wet wt) were incubated in Eagles Minimum Essential Medium supplemented with glucose to 2.5 mg/ml, for 3 hr at 38°C. The vessel was gassed continuously with 95~o 02 5~o CO2. Appropriate vessels were supplemented with biotin (50 #mol/l) and the final volume was 26'25 ml. After incubation, slices were washed in KrebsRinger solution at room temp and assayed for gluconeogenic capacity as described under Experiment 1 except that no biotin was included [final volume 5 ml). { Chicks were injected intravenously with either saline or biotin in saline (0.1 mg/100 g body wt) 3 hr before killing. Liver slices were prepared and assayed for gluconeogenic capacity, as described under Experiment 1 except that no biotin was included (final volume 5 ml). Results are expressed as the mean + S.E.M. of the number of observations in parenthesis. §0.1 > P > 0.05. I]P < 0"05. from malate though the values were higher than those given by the healthy birds and the difference was significant (P < 0-05). Glucose production in the absence of precursor was significantly lower than the controls used in this experiment (P < 0.01) though not when compared with earlier work (see above). In Experiment 2 biotin increased glucose synthesis from lactate significantly (Table 2). A comparison with control birds with lactate as precursor (Table

1) revealed that in the absence of biotin, the deficient chicks synthesized less glucose ("probably" significant, P = 0"05) whereas in the presence of biotin there was no difference. Gluconeogenesis from malate was low, and was similar in the presence and absence of the vitamin. Also, there was no difference between control and biotin-deficient birds in this respect. Experiment 3 (Table 2) revealed some increase in gluconeogenesis from lactate though as in Experiment

Table 3. Hepatic gluconeogenesis by liver slices from FLKS-affected chicks Glucose production (net) pmol/g dry wt/hr Experiment 1" Experiment 2~ Experiment 3{ No Precursor Control Biotin added Lactate (10 m mol/l final concn) Control Biotin added Malate (10 m mol/1 final concn) Control Biotin added

12.0 + 8.0 (5)2 - 1'5 + 1.7 (5)2 7.4 + 3'6 (5)3 0 + 0"3 (5)2 0.5 + 0.9 (5)

0 (5) 0 (5)

0 (5) 36.1 + 27.2 (4) 3

8.4 + 8.4 (5) 1 81.2 _ 9.5 (5)§

0 (5) 40.8 + 5.9 (4)rl

0 (5) 0 (5)

0 (5) 15-5 + 6.1 (4)34

* Tissue slices were incubated in 4.25 ml of Krebs-Ringer solution at 38°C after gassing with 95~o O2-5~o CO2. Reaction was started by adding 1 ml of precursor in Krebs-Ringer and glucose assayed in the medium by an automated method (Morley et al.. 1968) after 2 hr incubation. Biotin (50 #mol/l) was included in appropriate flasks. t Tissue slices (0.6-0.8 g wet wt) were incubated in Eagles Minimum Essential Medium supplemented with glucose to 2.5 mg/ml, for 3 hr at 38°C. The vessel was gassed continuously with 95~o O2-5~o CO2. Appropriate vessels were supplemented with biotin (50 #mol/l) and the final volume was 26.25 ml. After incubation, slices were washed in Kreb~ Ringer solution at room temp and assayed for gluconeogenic capacity as described under Experiment 1 except that no biotin was included (final volume 5 ml). } Chicks were injected intravenously with either saline or biotin in saline (0.1 mg/100g body wt) 3 hr before killing. Liver slices were prepared and assayed for gluconeogenic capacity, as described under Experiment 1 except that no biotin was included (final volume 5 ml). Results are expressed as the mean ___S.E.M. of the number of observations in parenthesis. Superscripts after the number of observations represent the number of chicks from which measurable values were obtained. ¶ P < 0'05. §P < 0.01. ]r P < 0.001.

578

D.W. BANNISTER

1 the difference only bordered on statistical significance. F a t t y liver and kidney syndrome

The results given in Table 3 demonstrate a dramatic loss in the ability of liver slices to synthesize glucose, in many cases none could be detected in the medium. The effect of biotin in Experiment I was to cause a small increase in gluconeogenesis from lactate. The increase, however, was not statistically significant, and in this respect is at variance with an earlier report (Bannister et al., 1975). This will be discussed later. Following pre-incubation in a nutrient medium containing biotin (Experiment 2) gluconeogenesis from lactate was just over 55°; of the corresponding control value. In the absence of the vitamin some activity was measured in tissue from one bird but was not detected in the others. This response to the vitamin was statistically significant. Injection of biotin (Experiment 3) produced significant improvements in gluconeogenesis from both lactate and malate, though with the latter precursor 1 bird failed to respond.

do not show any of the symptoms of biotin deficiency (Whitehead et al., 1974h) nor is there evidence of the occurrence of these symptoms in the field (for a review see Laursen-Jones, 1971). Furthermore, the relatively mild degree of biotin deficiency used in these experiments demonstrates quite clearly that depletion of hepatic biotin per se is not the cause of F L K S since reduction of gluconeogenic activity should have been greater with the biotin-deficientthan with the FLKS-inducing diet. rather than the other way round. Thus, it is clear that F L K S is quite distinct from biotin deficiency despite the fact that both respond to the vitamin. Ackm,wh,dgements The author would like to thank Drs W. G. Siller and P.A.L. Wight for conducting diagnoses of biotin deficiency and FLKS and for making known the results of their work before publication. Thanks are due to Drs A. J. Evans, R. Blair and C. C. Whitehead for the discussion of their results before publication and to Dr D. Langslow for his helpful advice.

REFERENCES

DISCUSSION AS anticipated, biotin was without effect on hepatic gluconeogenesis in control birds as measured by either of the in vitro techniques. With biotin deficiency, however, there was some enhancement of gluconeogenic activity though only in Experiment 2 was it statistically significant. A possible reason for the indecisive results is that the conditions were chosen as part of a research project concerned with the relationship between F L K S and biotin deficiency. Accordingly, birds were used in their 4th week after hatching (the age when the incidence of F L K S in increasing) and were fed a synthetic diet. Production of more severe deficiency would require inclusion of avidin in the diet to bind biotin synthesized by intestinal flora and a prolonged depletion period (4~5 weeks). Thus, although clinical symptoms of deficiency were obvious, it is likely that the degree of depletion of hepatic biotin was insufficient to reduce the activity of pyruvate carboxylase to low levels. In contrast, livers from chicks suffering from F L K S presented a completely different picture in that gluconeogenesis was not measurable in many cases. Response to biotin in vitro (Experiment 2) and in vivo was both consistent and substantial. However, there was considerable variation in other respects. For example two birds failed to respond to biotin in Experiment 1 which resulted in a failure to show a statistically significant effect. Not all preparations were inactive in the absence of biotin though activity was usually much lower than in the healthy birds (see Table 3 for details). This variability stems from the fact that it is not yet possible to select birds for study in a closely comparable metabolic state because the onset of F L K S is sudden and its progression frequently rapid (Evans et al., in preparation) and is further complicated by the fact that a minority of chicks recover from the syndrome (Whitehead, 1974). Chicks affected by experimentally-induced F L K S

AIaNALA. S.. ROBLEI-A. R. & MIIA.IGANL. P. (1971) Effect of dietary biotin on liver pyruvate carboxylase and 32p incorporation into nucleic acids in livers of chicks. J. Nutr. 101, 1555 1562. BANNISTERD. W.. EVANSA. J. & WHrIEHEADC. Q'. (1975) Evidence lor a lesion in carbohydrate metabolism in fatty liver and kidney syndrome in chicks. Res. rot. Sci. 18. 149 156. BLAIR R., WHITEHEADC. ('. & TIAGUI P. W. (1975) The effect of dietary fat and protein levels, form and cereal type on fatty liver and kidney syndrome in chicks. Res. vet. Sci. 18. 76 81. CAZZULOJ. J., SUNI)ARAMT. K. ~:~ KORNBERGH. L. [1969) Regulation of pyruvate carboxylase formation for the apo-enzyme and biotin in a thermophillic bacillus. Nature, Lond. 223, 1137 1138. DEODHAR A. D. & M1STRY S. P. (1969) Gluconeogenesis in biotin deficiency: m civo synthesis of pyruvate holocarboxylase in biotin deficient rat liver. Biochem. hiophys. Res. Commzm. 34, 755 759. EVANSA. J., BANNISTERD. W. & WHITEIIEADC. C. (1975) Some aspects of lipid metabolism in fatty liver and kidney syndrome in chicks. Res. t:et. Sci. 18, 26,31. HOMMES F. A., POLMAN M. A. 84 REERINK J. D. (1968) Leigh's Encephalomyelopathy: an inborn error of gluconeogenesis. Archs Dis. Childh. 43, 423 426. JUKES T. H. 84 BIRD F. H. 11942) Prevention of perosis by biotin. Prof. Soc. exp, Biol. Med. 49, 231 232. KREBSH. A.. BENNETTD. A. H., DEGASQUETP., GASCOYNE T. & YOSHIDAT. (1963) Renal gluconeogenesis: the effect of diet on the gluconeogenic capacity of rat-kidney-cortex slices. Biochem. d. 86, 22 27. LAURSEN-JONESA. P. (1971) Fatty liver and kidney syndrome in chickens--a review. Prof. X I X t h Vgld. ~,l. Congr. 3, 1165 1168. MADAPPALLY M. M. & MISTRV S. P. (1970) Synthesis of chicken liver pyruvate holocarboxylase in vivo and m vitro. Biochim. hiophys. Acta. 215, 316 322. MCELROY L. W. & JUKEST. H. (1940) Formation of the anti egg-white-injury factor (biotin) in the rumen of the cow. Prof'. Soc. exp. Biol. Med. 45, 29(~297. MORLEY G., DAWSONA. & MARKS V. (1968) Manual and Auto Analyzer methods for measuring glucose using guaiacum and glucose oxidase. Prof. Assoc. clin. Biochem. 5, 42 45.

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WHITEHEAD C. C. (1974) Tissue lipid composition in fatty WHITEHEAD C. C. t~ BLAIR R. (1974b) The involvement liver and kidney syndrome in chicks. Res. vet. Sci. 18. of biotin in the fatty liver and kidney syndrome in 32 35. broiler chicks. Wlds Poult. Sci. J. 30, 23 I. WHITEHEAD C. C., BANNISTER D. W., BLAIR R. & EVANS WHITEHEAD C. C., BLAIR R., BANNISTER D. W. d~z EVANS A. J. (1974a) Fatty liver and kidney syndrome in chicks: A. J. (1975) The involvement of dietary fat and vitamins, effects on liver and kidney of diets causing the syndrome. stress, litter and starvation on the incidence of the fatty Res. vet. Sci. 17, 22~225. liver and kidney syndrome in chicks. Res. vet. Sci. 18, WHITEHEAD C. C., BANNISTER D. W, WIGHT P. A. L. 100~104. WEISER H. (1974b) Studies on biotin requirements and WHITEHEAD C. C., BLAIR R., BANNISTER D. W., EVANS A. deficiency in chicks. X Vth Wlds Poult. Cong. 70-72. J. & MORLEY JONES R. (1976) The involvement of biotin WHITEHEAD C. C. & BLAIR R. (1974a) Fatty liver and kidin preventing the fatty liver and kidney syndrome in ney syndrome in chicks: the involvement of dietary broilers. Res. vet. Sci. (In press). energy-protein ratio and house temperature. Res. vet. WIGHT P. A. L. • S1LLER W. G. (1975) The histopathology Sci. 17, 86-90. of the fatty liver and kidney syndrome in chicks. Res. vet. Sci. (In press.)

Hepatic gluconeogenesis in chicks: effect of biotin on gluconeogenesis in biotin-deficiency and fatty liver and kidney syndrome.

Comp. Biochem. Physiol., 1976. Vol. 53B, pp. 575 to 579. Per qamon Press. Printed in Great Britain HEPATIC GLUCONEOGENESIS IN CHICKS: EFFECT OF BIOTI...
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