Gluconeogenic and lipogenic properties of isolated kidney tubules from the domestic fowl (Gallus domesticus).

Isolated kidney tubules synthesize glucose actively from fructose, lactate, glycerol and pyruvate and, to a lesser extent, from a variety of amino acids. Ethanol stimulated gluconeogenesis from pyruvate and inhibited it from lactate. The aminotransferase inhibitor, aminooxyacetate, greatly reduced synthesis from lactate but not from pyruvate. Quinolinate inhibited gluconeogenesis from both precursors, indicating an active role for cytosolic phosphoenolpyruvate carboxykinase (PEPCK) in the gluconeogenic pathway. Incorporation of lactate or glucose into triglycerides was relatively low, and since no fatty acid synthase (FAS) activity could be detected, probably represented chain elongation or reesterification.

Biotin deficiency and susceptibility to fatty liver and kidney syndrome in broiler chicks: reduced 6-phosphofructokinase (EC 2.7.1.11) activity but normal fructose 2,6-bisphosphate content in birds with hepatomegaly.

In two separate experiments, using different strains, broiler chicks were reared on either a commercial-type chick mash (control) or a fatty liver and kidney syndrome (FLKS)-inducing diet. In Expt a, chicks were killed on day 29 and in Expt b, on day 32. Body-weights and liver weights were measured, and values from those given the control ration used to construct a hepatomegaly index by employing a variant of linear discriminant analysis. Application of the index to FLKS birds revealed a statistically significant bimodal distribution of liver size. The birds with enlarged livers (high index) also possessed metabolic abnormalities in that 6-phosphofructokinase (EC 2.7.1.11; PFK-1) activity (measured at low substrate concentration) was depressed despite the presence of normal, or even slightly elevated fructose 2,6-bisphosphate concentration. This indicates the presence of an uncharacterized regulatory mechanism for PFK-1 in FLKS-susceptible birds.

Activation of phosphofructokinase and pyruvate kinase by 6 phosphogluconate in chicken liver (Gallus domesticus): no evidence for a regulatory role.

Phosphofructokinase and pyruvate kinase in crude extracts of chick liver were activated by 6 phosphogluconate at subsaturating concentrations of their respective substrates. The apparent Ka for phosphofructokinase was 0.79 mM and for pyruvate kinase 0.44 mM. Total pentose phosphate dehydrogenase activity was low in the livers of both fed and starved chicks. Hepatic 6 phosphogluconate concentration was too low to be measured accurately by a conventional spectrophotometric method. It is concluded that the concentration of 6 phosphogluconate is too low for it to have a role in the regulation of phosphofructokinase and pyruvate kinase activities in chicken liver.

Lipogenic enzyme activity and fructose 2,6-bisphosphate concentration in livers of two lines of domestic fowl (Gallus domesticus) selected for different body fat content.

Livers were obtained from two lines of domestic broiler which had been selected for low (lean) and high (fat) plasma very-low-density lipoprotein concentration over three generations. The fat line possessed significantly higher hepatic specific activities of malate dehydrogenase (NADP), ATP citrate lyase and fatty acid synthase, and lower glucose bisphosphatase than the lean line. The glycolytic enzymes, pyruvate kinase and phosphofructokinase, were similar and so was the concentration of fructose 2,6-bisphosphate. This recently discovered metabolic regulator was present at somewhat higher concentrations than previously reported in rats or mice. It exhibited a positive correlation with phosphofructokinase activity (only data for the fat line are shown), and stimulated enzyme activity when added to crude preparations.

The effects of oxalate and glucose on lipogenesis by isolated hepatocytes from normal and biotin-deficient chicks (Gallus domesticus).

Isolated hepatocytes synthesize fatty acids and cholesterol from lactate and acetate with lactate being the more effective substrate. Biotin deficiency decreased fatty acid synthesis from both substrates but stimulated cholesterogenesis. Exposure of intact hepatocytes to oxalate inhibited fatty acid and cholesterol synthesis from lactate, this effect was enhanced in biotin-deficient chicks. A similar effect was not observed when acetate was the substrate. Synthesis of fatty acids from lactate and acetate was stimulated by glucose, biotin deficiency increased this response. Cholesterogenesis was reduced in control but not biotin-deficient chicks.

The effect of biotin deficiency and dietary protein content on lipogenesis, gluconeogenesis and related enzyme activities in chick liver.

Chicks were given biotin-deficient diets containing either suboptimal (low) or supraoptimal (high) concentrations 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 deficiency signs. Two other groups, as controls, received biotin-supplemented rations. Low dietary protein increased lipogenesis by isolated hepatocytes but had little effect on gluconeogenesis compared to high dietary protein. Low dietary protein decreased activities of hepatic isocitrate dehydrogenase (EC 1.1.1.42), fructose-1,6-bisphosphatase (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). 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. 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.

The effect of oxalate on gluconeogenesis by isolated chicken hepatocytes. Increased sensitivity to inhibition as a result of biotin deficiency.

Addition of varying concentrations of oxalate to isolated chicken hepatocytes reduced gluconeogenesis from lactate in a manner indicating that pyruvate carboxylase was not the rate-limiting step. With hepatocytes from biotin-deficient chicks, sensitivity to inhibition was increased, and was consistent with pyruvate carboxylase being rate-limiting. Administration of biotin to deficient chicks overnight restores sensitivity to oxalate to normal.

Application and evaluation of an NADH-linked spectrophotometric assay for pyruvate dehydrogenase complex using chicken liver homogenates.

1. A method for measuring the activity of the pyruvate dehydrogenase complex [EC 1.2.4.1] (Hinman and Blass, 1981) has been adapted for use with crude chicken liver homogenates. 2. It was found necessary to remove cytoplasmic lactate dehydrogenase by centrifugation before releasing the complex from the mitochondria by treatment with sodium deoxycholate. 3. Lipoamide dehydrogenase was preferable to phenazine methosulphate as the electron carrier between the complex and NADH. 4. The assay was not suitable for use at 40 degrees C (approximate body temperature) but was satisfactory at 25 degrees C. 5. The assay exhibited the properties expected of pyruvate dehydrogenase complex i.e. a complete dependence on the presence of NAD, coenzyme A and pyruvate and a partial dependence on MgCl2, dithiothreitol and thiamine pyrophosphate. The pH optimum was between 7.8 and 8.0. 6. The mean activity for a group of normally fed young chickens was 654 mumol/g dry weight h and 88 for birds starved overnight.

The post mortem diagnosis of fatal hypoglycaemia using the fatty liver and kidney syndrome of chicks as a model.

1. The concentrations of vitreous humour and plasma glucose were closely correlated in both healthy and fatty liver and kidney syndrome-affected chicks at time of death. 2. The values of vitreous humour glucose and lactate decreased rapidly after death, such that they were not reliable indicators of the presence of hypoglycaemia immediately ante mortem. 3. Hepatic glycogen was extremely low in fatty liver and kidney syndrome-affected birds, whereas significant quantities remained in healthy birds up to at least 24 h post mortem.

Aspects of metabolism related to the occurrence of skin lesions in biotin-deficient chicks.

1. Chicks were fed on biotin-deficient low- and high-protein diets supplemented with increasing concentrations of biotin. 2. Biotin deficiency decreased hepatic activity of pyruvate carboxylase [EC 6.4.1.1] but activity of acetyl-CoA carboxylase [EC 6.4.1.2] was comparatively unaffected. 3. Increasing dietary protein increased the severity of biotin deficiency as assessed by skin lesions and decreased plasma biotin concentrations. 4. The severity of the skin lesions over all the treatments was most closely related to plasma biotin concentration.

Blood pyruvate carboxylase activity as a criterion of biotin status in young pigs.

A basal diet containing isolated soybean protein was supplemented with 100 and 500 microgram biotin/kg and another basal diet containing hen-egg albumen was supplemented with 1300 microgram biotin/kg. The five diets were fed to groups of young pigs from four to 12 weeks old. Growth depression or skin or foot lesions characteristic of biotin deficiency were not seen but the undersurfaces of the hooves of pigs given the basal albumen diet were unusually soft. Blood pyruvate carboxylase activity showed a maximum response to dietary biotin at six weeks but by 12 weeks there was no response to supplemental levels greater than 100 microgram/kg. Plasma biotin concentration gave a good indication of biotin intake at all ages.

Biotin status, blood pyruvate carboxylase (EC 6.4.1.1) activity and performance in broilers under different conditions of bird husbandry and diet processing.

1. Three experiments were conducted to investigate the relationships between blood pyruvate carboxylase (pyruvate: carbon dioxide ligase (ADP-forming), EC 6.4.1.1; PC) activity, growth performance and dietary biotin level in broilers under different husbandry conditions. Blood PC activity was also used to gain information on the ingestion of biotin faecal origin by birds housed on wood shavings and the effect of steam-pelleting on the biotin content of diets. 2. Blood PC activity and relationship to dietary biotin level were similar in birds kept under different husbandry conditions. 3. Biotin requirement for growth was higher in birds kept under conditions that allowed them to reach their growth potential more fully. 4. The contribution of biotin of faecal origin to the biotin intake of birds housed on wood shavings was negligible at 3 weeks of age but by 7 weeks was equivalent to approximately 0.01 mg/kg diet. 5. Steam-pelleting did not affect the stability or availability of biotin of natural or synthetic origin in diets. 6. It is concluded that blood PC activity is a good criterion of biotin status and requirement in fast-growing broilers.

Effects of storage conditions in vitro on the assay of chick liver glycogen.

1. The reproducibility of an enzymic method for assaying chick liver glycogen was found to be +/- 1.6%. 2. Storage of liver at either -21 or -70 degrees C for up to 4 weeks, after freezing in liquid nitrogen, had no significant effect on glycogen concentration. However, the concentration of free glucose increased by about 50% during this time. 3. Storage of liver in ice-cold saline for up to 3 h proved unsatisfactory because of a significant loss of glycogen. 4. There was no significant difference in immediate glycogen concentration in liver tissue that was freeze-clamped, frozen in liquid nitrogen or collected into ice-cold saline. The free glucose concentration was higher, when estimated immediately after, with the latter two methods of collection of liver tissue.

Metabolic changes associated with the occurrence of fatty liver and kidney syndrome in chicks.

1. The changes in a number of metabolic measurements brought about by low-biotin diets associated with high and low incidences of fatty liver and kidney syndrome (FLKS) were studied in healthy 4-week-old broiler chicks. 2. Liver pyruvate carboxylase (pyruvate: CO2 ligase (ADP); EC 6.4.1.1) activity was low in birds fed on a diet causing a high incidence FLKS but the addition of fat or protein to this diet, to decrease the incidence of FLKS, increased enzyme activity. 3. Liver weights, blood lactate concentrations, plasma lactate dehydrogenase (L-lactate: NAD oxidoreductase; EC 1.1.1.27) activitvities and values for C16:1 : C18:0 fatty acid in liver, adipose tissue and plasma triglyceride were highest in birds fed on the high-FLKS diet and all measurements were negatively correlated with pyruvate carboxylase activity. 4. Birds with high plasma lactate dehydrogenase activity or triglyceride C16:1 : C18:0 values were the most likely to develop FLKS when fasted. 5. There was no evidence that increased liver weight was associated with increase activities of certain other liver enzymes. 6. It is concluded that FLKS occurs in birds with little or no hepatic gluconeogenic capacity via pyruvate carboxylase as a result of a dietary insufficiency of biotin but that the initiation of the syndrome in probably associated with the inhibition of other pathways of gluconeogenesis.

Blood pyruvate carboxylase (EC 6.4.1.1) activity as a criterion of biotin status in chickens and turkeys.

1. Blood pyruvate carboxylase (pyruvate-CO2 ligase (ADP-forming); EC 6.4.1.1; PC) activities in young chickens and turkeys given low-biotin diets supplemented with biotin at graded levels were studied in three experiments. 2. In both species PC activity was related positively to the supplemental biotin level. The relationship was sigmoid and maximum activity was attained with supplemental levels above those required to give maximal growth response. 3. Enzyme activity decreased between 2 and 4 weeks of age but remained almost constant thereafter. 4. Activity in chicks was not affected by alterations in the fat or protein content of the diet. 5. Changing poults from high to low and from low to high supplemental biotin levels resulted in reversals in the levels of enzyme activity. 6. It is concluded that blood PC activity is a promising new criterion for assessing the biotin status of young chickens and turkeys.

Deep pectoral myopathy: an experimental simulation in the fowl.

Surgical occlusion of the subclavian artery of domestic fowls has been found to induce a condition closely resembling, in location and histopathology, the deep pectoral myopathy of turkeys. The operative procedure is described and the significance of the collateral blood supply in the pathogenesis of the lesion is briefly discussed.