Increased Fucosylation of Chick Brain Proteins Following Training: Effects of Cycloheximide

When chicks are trained to avoid pecking a bead coated with methylanthranilate in a one-trial passive avoidance task there is an increase in fucose incorporation in vivo and in vitro in the right forebrain base of methylanthranilate (M)-trained compared to water (W)-trained chicks. The relation of this increase to de novo protein synthesis in vivo and in vitro has been examined. Cycloheximide (Cx), 1 mM, inhibited in vitro fucosylation of chick brain slices by 60% after 3 h. However, the training-related increase in in vitro fucosylation still persisted. When Cx was injected intraventricularly 10 min before training, the subsequent increase in in vitro fucosylation due to training was still apparent. When Cx was injected and [14C]leucine and [3H]fucose incorporation studied in vivo in M-trained and W-trained chicks, there was no increase in fucosylation due to training in the Cx-treated M-trained over the W-trained chicks. These results are taken to indicate that in vitro fucosylation and its increase subsequent to training is not protein synthesis-dependent, but that both in vivo and in vitro there are interactions between Cx and fucosylation steps that are independent of Cx's effects on protein synthesis.     

Effect of Passive Avoidance Training on In Vitro Protein Synthesis in Forebrain Slices of Day-Old Chicks

Slices from the forebrains of day-old chicks represent a highly active in vitro protein-synthesising system. The in vitro incorporation of L-[14C]leucine into protein of slices was estimated to be 2.5 mmol/mg protein/h. Incorporation was linear over 90 min of incubation and was suppressed by 92% by 1 mM cycloheximide. The highest incorporation was into microsomal and cell-soluble fractions. Under the electron microscope, slices appeared vacuolated near the cut surfaces, but well preserved internally (greater than 40 micron from the edge). Autoradiography showed that radioactivity was incorporated evenly across the slice with no decrease in label in the central part of the tissue. The rate of incorporation was only weakly dependent on leucine concentration in the medium (0.04-1 mM). Addition of a mixture of unlabelled amino acids (1 mM) produced a 20-50% inhibition of incorporation of radioactive L-leucine depending on the amino acids involved. In slices prepared from chicks 1 h after training on a one-trial passive avoidance paradigm, L-[14C]leucine incorporation was 23% higher (p less than 0.01) in the forebrain roof than in slices from control chicks. This figure is comparable to the one previously reported in vivo. Subcellular fractionation of incubated slices from the forebrain roof of trained and control birds revealed that the increased protein synthesis was due mainly to an elevated leucine incorporation into the soluble fraction.     

Increased Incorporation of [3H]Fucose into Chick Brain Glycoproteins Following Training on a Passive Avoidance Task

Abstract: Incorporation of [¹H]fucose into the TCA-precipitable material from the particulate and soluble fractions of different regions of the brain in the 1-day-old chick was measured following training on a passive avoidance task. A significant increase in the level of incorporation of [³H]fucose into the particulate fraction of the anterior forebrain roof was observed in the trained birds as compared with untrained controls. The percentage increases in radioactivity in the particulate fraction of the anterior forebrain roof were 29% ( p < 0.01), 16% ( p < 0.01), and 26% ( p < 0.01) in the trained birds as compared with controls, 30 min, 3 h, and 24 h following learning, respectively, but had returned to control levels after 48 h. These results suggest either increased production of glycoproteins or increased fucosylation of preexisting proteins following training, and suggest one of the possible localized neurochemical changes associated with the learning of the passive avoidance response.     

Effects of imprinting on lysine uptake and incorporation into protein in chick brain

One-day old chicks were exposed for either 30, 60, or 120 min to an imprinting stimulus or kept in darkness in similar conditions. At the end of this time they were injected peripherally with ¹⁴C-lysine and killed 20 min later. The radioactivity of free lysine and that incorporated into protein was measured; incorporation was found to differ between exposed and dark birds only in the anterior part of the forebrain roof after 60-min treatment (E/D = 1.25). However, more free radioactive lysine was found in all brain regions of exposed birds at this time. When the specific radioactivity of the free lysine (dpm/nmol lysine) was measured there were no differences between the two types of birds, indicating that the incorporation difference was not due to a change in precursor radioactivity. The use of ¹⁴C-2-aminoisobutyrate confirmed that even with a nonincorporated amino acid pool size changes still occurred. The greater lysine incorporation in anterior forebrain roof was largely restricted to the cytoplasmic soluble fraction.     

Passive Avoidance Training Increases Fucokinase Activity in Right Forebrain Base of Day-Old Chicks

Fucokinase (EC 2.7.1.52) activity was estimated in supernatants of homogenate from day-old chick forebrain. Enzyme kinetic studies gave a Km of 4.5 X 10(-6) M and Vmax of 3.72 nmol fucose converted into fucose-1-phosphate/mg prot/h. The pH optimum was 7.5. The enzyme is thus considerably more active than was reported for other species and tissues. There were no differences in enzyme activity between the four forebrain regions studied. One hour after chicks were trained on a one-trial passive avoidance learning paradigm, enzyme activity in the right forebrain base increased 14% over control values (p less than 0.02). The 11.3% increase in activity in the left forebrain base and 10.3% increase in the left roof were not statistically significant. The relationship of this change to the increased fucose incorporation into glycoproteins known to occur over a similar time period and the significance of the lateralization of the increase are discussed.     

Characterisation and Regional Localisation of Pre- and Postsynaptic Glycoproteins of the Chick Forebrain Showing Changed Fucose Incorporation Following Passive Avoidance Training

To identify those glycoproteins whose synthesis or modification is necessary for memory formation, we have studied the uptake of radiolabelled fucose into synaptic plasma membranes (SPMs) and postsynaptic densities (PSDs) derived from two specific left and right forebrain loci, at two different times after training of 1-day-old chicks on a one-trial passive avoidance learning task. To increase the reliability of the comparison, a double-labelling method was used. Tissue samples from intermediate medial hyperstriatum ventrale (IMHV) and lobus parolfactorius (LPO) were isolated at 6 and 24 h after training. At both times, training resulted in region-specific changes, both increases and decreases, in incorporated radioactivity into pre- and postsynaptic glycoproteins. After 6 h, there was a relative decline in incorporation into both SPMs and PSDs of the right IMHV of trained chicks, a decline that persisted in the PSDs until 24 h. A small decline in incorporation in SPMs from the right LPO of trained chicks at 6 h was reversed by 24 h, by which time there was a 64% increase in incorporation into SPMs and a 24% increase into PSDs of the left LPO. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis of left and right hemisphere samples containing LPO revealed that 6 h after training the main effect was presynaptic, including a reduction of incorporation into high molecular mass glycoproteins, of 150-180 kDa, and an increase in a lower molecular mass (41 kDa) fraction. By 24 h after training, a left hemisphere presynaptic glycoprotein of molecular mass approximately 50 kDa showed the biggest increase in fucosylation. In addition, a wide group of postsynaptic glycoproteins of both hemispheres, in the ranges 150-180, 100-120, and 33 kDa now showed increases in incorporation. Some other fractions showed decreases. These results are in accord with previous data on incorporation obtained using the amnesic agent 2-deoxygalactose. They also support the hypothesis that memory formation involves the strengthening of connections between pre- and postsynaptic neurons of the LPO by growth or modulation of pre- and postsynaptic structures.     

Effect of naloxone and D-Met2-Pro5-enkephalinamide treatment on the DNA synthesis in the developing rat brain

Effects of a single dose of naloxone and of D-Met²-Pro⁵-enkephalinamide on the DNA synthesis in the forebrain, hypothalamus and cerebellum of 11 day old female rats were studied. As an index of DNA synthesis the rate of incorporation of ³H-thymidine into DNA was measured 30 min after a sc. injection of 40 μCi/100 g b.w.. A time dependent effect of naloxone administration on cerebral DNA synthesis was observed. In the forebrain at 1 and 3 hrs after naloxone injection an increased rate of ³H-thy-midine incorporation into DNA was found followed by a marked decrease at 9 and 12 hrs. The effect in the hypothalamus was similar but the initial increase at 1 hr was absent. On cerebellar DNA synthesis naloxone had no effect. The administration of D-Met²-Pro⁵-enkephalinamide resulted in a marked reduction in the labelling of cerebral and hypothalamic DNA between 1 to 12 hrs. Except a decrease at 1 hr no effect was found in the cerebellum.     

Phosphorylation of Synaptic Proteins in Chick Forebrain: Changes with Development and Passive Avoidance Training

We have used synaptic plasma membranes (SPMs) and postsynaptic densities (PSDs) to study protein phosphorylation at the synapse in the developing chick forebrain and in 1-day-old chick forebrain following training on a passive avoidance task. Endogenous phosphorylation patterns in SPMs and PSDs prepared by extraction with n-octylglucoside isolated from chick forebrain were investigated by labelling with [32P]ATP. The phosphoprotein components of the SPM and PSD fractions were separated using sodium dodecyl sulphate gradient polyacrylamide gel electrophoresis. Autoradiography and densitometry of the Coomassie Blue protein staining pattern revealed phosphate incorporation into several SPM components including those of molecular mass 52, 37, and 29 kilodaltons (kDa). Bands of similar molecular mass were not phosphorylated in PSD fractions. This difference in phosphorylation between SPMs and PSDs was not due to the detergent n-octylglucoside. In a developmental study in which SPM and PSD fractions were prepared from 1-day-old, 14-day-old, and 21-day-old chickens, the phosphorylation patterns of SPMs were similar throughout, but striking differences occurred in PSDs, both in the level of phosphorylation and in the components phosphorylated. A time-course study was carried out in which phosphorylation of SPMs and PSDs from 1-day-old chicks trained on a passive avoidance task was compared with patterns from control chicks trained on a water-coated bead and untrained chicks. In SPMs prepared from forebrains removed 10 mins following training, a consistent but nonsignificant decrease (-21%) in phosphorylation of a 52 kDa band occurred in chicks with passive avoidance training compared with water-trained and untrained chicks.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of age and visual experience on potentiation of responses in slices from the chick forebrain.

A single coronal slice, containing the intermediate part of the medial hyperstriatum ventrale (IMHV) was cut from the left forebrain of a series of domestic chicks and maintained in vitro. Records were made of field responses evoked in the IMHV by local electrical stimuli. Two 1-min periods of 5 Hz stimulation, separated by 10 min, were used in attempts to induce persistent potentiation of the responses (PPR) to test stimulation at 0.1 Hz. In dark-hatched chicks the probability of producing PPR is much higher in slices from chicks aged 2-5 days post-hatch than in those from either younger or older birds. As an independent measure of plasticity in dark-hatched chicks, the probability of eliciting unit responses to repeated stimulation of remote sites in the slice at 3.3 Hz was analysed. This probability was greater in slices from chicks aged 2-5 days than in those from either younger or older birds. In light-hatched chicks the probability of inducing PPR is significantly higher during the first day post-hatch, than in dark-hatched chicks of this age. The probability of producing PPR in slices from light-hatched chicks aged 2-3 days is less than that in slices from either younger or older birds. It is clear that both the age and past experience of the domestic chick affect the neurophysiological properties of slices of brain, tested in vitro.     

Effects of the Amnesic Agent 2-Deoxygalactose on Incorporation of Fucose into Chick Brain Glycoproteins

The interaction of the amnesic agent 2-deoxygalactose with fucose incorporation into glycoproteins in day-old chick forebrain has been studied with the aim of identifying glycoproteins whose synthesis is modified during memory formation. 2-Deoxygalactose inhibited total exogenous [14C]fucose incorporation into the forebrain glycoproteins by 26%. Sodium dodecyl sulphate-polyacrylamide gradient gel analysis revealed that intracerebrally injected 2-[3H]deoxygalactose labelled the same eight major glycoprotein bands as were identified using [14C]fucose labelling. Subsequent investigations focussed on these selected components. Subcellular fractionation showed that between 4 and 24 h after administration of the deoxy-sugar, the incorporated radioactivity was found predominantly at the synaptic sites, some glycoproteins being more abundant in synaptic plasma membranes and others in postsynaptic densities. This distribution pattern varied according to the time after injection. The effect of passive avoidance training, using a methylanthranilate-coated bead, on [14C]fucose incorporation into forebrain was to decrease fucose uptake into components of molecular mass 150-180 kilodaltons but to increase significantly labelling of glycoproteins of molecular mass 33 and 28 kilodaltons. The possible implications of these training-induced changes are discussed.     

AUTORADIOGRAPHIC STUDIES OF SYNTHESIS AND INTRACELLULAR MIGRATION OF GLYCOPROTEINS IN THE RAT ANTERIOR PITUITARY GLAND

The incorporation of [³H]fucose in the somatotrophic and gonadotrophic cells of the rat adenohypophysis has been studied by electron microscope autoradiography to determine the site of synthesis of glycoproteins and to follow the migration of newly synthesized glycoproteins. The pituitaries were fixed 5 min, 20 min, 1 h, and 4 h after the in vivo injection of [³H]fucose and autoradiographs analyzed quantitatively. At 5 min after [³H]fucose administration, 80–90% of the silver grains were localized over the Golgi apparatus in both somatotrophs and gonadotrophs. By 20 min, the Golgi apparatus was still labeled and some radioactivity appeared over granules. At 1 h and 4 h, silver grains were found predominantly over secretory granules. The kinetic analysis showed that in both protein-secreting cells (somatotrophs) and glycoprotein-secreting cells (gonadotrophs), the glycoproteins have their synthesis completed in the Golgi apparatus and migrate subsequently to the secretory granules. It is concluded from these in vivo studies that glycoproteins which are not hormones are utilized for the formation of the matrix and/or of the membrane of the secretory granules. The incorporation of [³H]fucose in gonadectomy cells (hyperstimulated gonadotrophs) was also studied in vitro after pulse labeling of pituitary fragments in medium containing [³H]fucose. The incorporation of [³H]fucose was localized in both the rough endoplasmic reticulum (ER) and the Golgi apparatus. Later, the radioactivity over granules increased while that over the Golgi apparatus decreased. The concentration of silver grains over the dilated cisternae of the rough ER was not found to be modified at the longest time intervals studied.     

Action of aspartate on the32Pi incorporation into phospholipids of cerebral cortex

The effect ofl-aspartate on the³²Pi incorporation of phospholipids, was studied on slices of rat cerebral cortex. This amino acid produced an inhibitory effect in concentrations 0.01–10 mM, which was more evident at 120 min. This effect was not stereospecific and did not imply a change in Pi uptake and in nucleotides P precursors. The inhibition was present in PS, PC, PE and to a lesser extent in PI. On liver slices 1 mMl-aspartate had the opposite effect, stimulating the incorporation of³²Pi into total phospholipids. Our results suggest that the effect ofl-aspartate is by a non-specific mechanism, probably not mediated by a receptor.     

Ontogeny of magnetoreception in chickens (Gallus gallus domesticus)

Chickens are precocial birds, capable of moving independently from within a few hours of hatching, and thus provide a useful model to investigate the development of magnetoreception in young birds. Chicks show some sharply-timed behavioural changes at around 11 days of age that may be indicative of improvements in navigation ability around this age. We trained Young (<10 days, n = 7) and Old (11 days or older, n = 6) chicks to find a hidden imprinting stimulus behind one of four screens in a square arena. Once criterion was reached, the directional choices of chicks were recorded in unrewarded tests in the geomagnetic field and in an experimental field shifted by 90° clockwise. These tests were separated with rewarded training trials to avoid extinction. In the first, and the first 3 tests, no indication was found that chicks chose the correct unimodal magnetic direction. Instead, in all tests, Old chicks preferred the screens in the same magnetic axis as the training direction in tests in the geomagnetic and shifted experimental fields significantly more than Young chicks (P < 0.05). Choices of Young chicks were no different to chance in tests, whereas choices by Old chicks were significantly different from chance (P < 0.05). Our findings support the hypothesis that magnetoreception appears in the second week of life in the chicken. It is, however, unclear when a truly directional magnetic response (i.e. an unimodal, rather than axial, response in our test) develops that could be used by the chicks for accurate navigation.     

Ontogenetic and Imprinting-Induced Changes in Chick Brain Protein Metabolism and Muscarinic Receptor Binding Activity

Abstract— Over the 20-min period following exposure of young chicks to a flashing light as an imprinting stimulus there is an increased incorporation of [¹⁴C]leucine into an acidic (tubulin-enriched) protein fraction of the anterior dorsal forebrain in birds which have learnt the characteristics of the stimulus as compared with, either birds which have been exposed to an imprinting stimulus but learn poorly, or chicks kept in the dark. This brain region has been implicated in several studies as the locus for a number of biochemical modulations that accompany learning. The amount of [¹⁴C]leucine incorporated does not seem to be determined by precursor pool availability; it does, however, correlate with a well-validated measure of the extent to which birds have learnt to recognise the characteristics of the stimulus, as shown by a two-choice discrimination test. There is no change in the total content of tubulin dimer as assayed by colchicine binding under these conditions. Additionally, in birds which show evidence of learning, the binding of quinuclidinyl benzilate, an irreversible muscarinic ligand, is altered in both the posterior dorsal forebrain and midbrain regions. None of these effects could be simply the result of visual stimulation. The meaning of these changes is discussed.     

Validation of the doubly labeled water method in Japanese Quail Coturnix c. japonica chicks: is there an effect of growth rate?

The Doubly Labeled Water (DLW) method was validated against respiration gas analysis in growing Japanese Quail chicks (between 1 week and 3 weeks of age) as well as in birds after having achieved sexual maturity (7 weeks of age). A comparison was made between a strain selected for high growth rates (P-strain, n=18), and a non-selected strain (C-strain, n=18). Relative growth rates of individual chicks during the measurement ranged from −13.8% day⁻¹ to 23.1% day⁻¹. When employing a single-pool model (eq. 34, Lifson and McClintock 1966), it was found that the relative error of the DLW method was sensitive to assumptions concerning fractional evaporative water loss. The best fit was obtained after taking a fractional evaporative water loss value of 0.33. When applying this value for all chicks, it was found that neither strain, relative growth rate of the chick during measurement, nor age significantly contributed to the explained variance. When employing two-pool models, it was found that the DLW method significantly underestimated the true rates of CO₂ production at all assumed levels of fractional evaporative water loss. Based on an evaluation of DLW validation studies in growing shorebirds, terns, and quail we recommend Speakman's Eq. 7.17 (Speakman 1997) for general use in young birds. Accepted: 14 April 2000     

Effects of nutritional restriction on nitrogen and carbon stable isotopes in growing seabirds

When using stable isotopes as dietary tracers it is essential to consider effects of nutritional state on isotopic fractionation. While starvation is known to induce enrichment of ¹⁵N in body tissues, effects of moderate food restriction on isotope signatures have rarely been tested. We conducted two experiments to investigate effects of a 50–55% reduction in food intake on δ¹⁵N and δ¹³C values in blood cells and whole blood of tufted puffin chicks, a species that exhibits a variety of adaptive responses to nutritional deficits. We found that blood from puffin chicks fed ad libitum became enriched in ¹⁵N and ¹³C compared to food-restricted chicks. Our results show that ¹⁵N enrichment is not always associated with food deprivation and argue effects of growth on diet–tissue fractionation of nitrogen stable isotopes (Δ¹⁵N) need to be considered in stable isotope studies. The decrease in δ¹³C of whole blood and blood cells in restricted birds is likely due to incorporation of carbon from ¹³C-depleted lipids into proteins. Effects of nutritional restriction on δ¹⁵N and δ¹³C values were relatively small in both experiments (δ¹⁵N: 0.77 and 0.41‰, δ¹³C: 0.20 and 0.25‰) compared to effects of ecological processes, indicating physiological effects do not preclude the use of carbon and nitrogen stable isotopes in studies of seabird ecology. Nevertheless, our results demonstrate that physiological processes affect nitrogen and carbon stable isotopes in growing birds and we caution isotope ecologists to consider these effects to avoid drawing spurious conclusions.     

Passive Avoidance Learning Results in Region-Specific Changes in Concentration of and Incorporation into Colchicine-Binding Proteins in the Chick Forebrain

Abstract: Incorporation of [¹⁴T]leucine into trichloracetic acid-precipitable material and tubulin-enriched fractions, and total tubulin levels as determined by colchicine-binding activity and retention on DE81 filter discs, were measured in various regions of the chick brain following training on a one-trial passive avoidance task, suppression of pecking at a chromed bead as a consequence of the aversive taste of methylanthranilate. Radioactive pulse time was 0.5 h. The only brain region in which changes were found was the anterior forebrain roof, the same area in which biochemical changes in response to exposure of the birds to an imprinting stimulus have been observed previously. In the anterior forebrain roof the changes observed as a consequence of training were detectable at 0.5 and 24 h after the 10-s training experience but not 48 h subsequently. One-half hour after training, there were increases of the order of 20 or 30% in [¹⁴T]leucine incorporation into particulate and postmitochondrial TCA-precipitable material and a tubulin-enriched fraction purified as above. There were comparable increases in the total amount of colchicine-binding activity. By 48 h, none of these increases were detectable. Subcellular fractionation of the particulate fraction showed that most of the increase of incorporation into the tubulin-enriched fraction and in colchicine-binding activity was present in the soluble content of the synaptosomes; there were no increases in either measure in the synaptic membrane fraction. The possible role of changed levels and turnover of tubulin in the plastic responses of the brain to learning experiences is discussed.     

Incorporation of fucose and leucine into PNS myelin proteins in nerves undergoing early Wallerian degeneration

The simultaneous incorporation of [³H]fucose and [1-¹⁴C]leucine into normal rat sciatic nerve was examined using an in vitro incubation model. A linear rate of protein precursor uptake was found in purified myelin protein over 1/2–6 hr of incubation utilizing a supplemented medium containing amino acids. This model was then used to examine myelin protein synthesis in nerves undergoing degeneration at 1–4 days following a crush injury. Data showed a statistically significant decrease in the ratio of fucose to leucine at 2, 3, and 4 days of degeneration, which was the consequence of a significant increase in leucine uptake. These results, plus substantial protein recovery in axotomized nerves, are indicative of active synthesis of proteins that purify with myelin during early Wallerian degeneration.     

Kinetics,nutrition and inhibitor properties of basidiospore germination in schizophyllum commune

Summary The kinetics, nutritional requirements and inhibitor properties of basidiospore germination in the wood-rotting mushroom Schizophyllum commune were investigated. Measurements of changes in absorbancy and dry weight showed a lag period of approximately 15–20 hrs, followed by an abrupt increase in the rate of both processes. Individual basidiospore elongation also showed a lag phase and population changes were heterogenous in this regard.Carbohydrates active for basidiospore germination were grouped into four categories. Those sugars active between 15 and 20 hrs included glycogen, turanose, cellobiose, maltose, sucrose, glucose, fructose, mannose, galactose and xylose. Several sugar alcohols were only active between 30 and 60 hrs incubation and these included mannitol, sorbitol, ribitol, xylitol, arabitol, erythritol and glycerol. A third category of carbohydrates active for germination required prolonged incubation between 30 hrs and 7 days and included lactose, sorbose, raffinose, melezitose, trehalose, ribose and melibiose. Compounds without activity after 7 days included galactitol, inositol, acetate, succinate, gluconate, citrate, fumarate, rhamnose, fucose and inulin.Nitrogen sources active in basidiospore germination included complex organic nitrogenous substrates, asparagine, glutamine, arginine, urea and various ammonium salts.Germination was inhibited by cycloheximide, l-ethionine, p-fluoro-dl-phenylalanine, sodium azide, 2,4-dinitrophenol, phenylmercuric acetate and 2-deoxy-d-glucose. Alkali as a trapping agent arrested germination in glucose-(NH₄)₂SO₄ medium but was without ill-effect in glucose peptone broth.     

The effect of starvation and starvation followed by feeding on enzyme activity and the metabolism of [U-14C]glucose in liver from growing chicks

1. The conversion of [U-(14)C]glucose into carbon dioxide, cholesterol and fatty acids in liver slices and the activities of ;malic' enzyme, citrate-cleavage enzyme, NADP-linked isocitrate dehydrogenase and hexose monophosphate-shunt dehydrogenases in the soluble fraction of homogenates of liver were measured in chicks that were starved or starved then fed. 2. In newly hatched chicks the incorporation of [U-(14)C]glucose and the activity of ;malic' enzyme did not increase unless the birds were fed. The response to feeding of [U-(14)C]glucose incorporation into fatty acids increased as the starved chicks grew older. 3. Citrate-cleavage enzyme activity increased slowly even when the newly hatched chicks were unfed. On feeding, citrate-cleavage enzyme activity increased at a much faster rate. 4. In normally fed 20-day-old chicks starvation decreased the incorporation of [U-(14)C]glucose into all three end products and depressed the activities of ;malic' enzyme and citrate-cleavage enzyme. Re-feeding increased all of these processes to normal or higher-than-normal levels. 5. In both newly hatched and 20-day-old chicks starvation increased the activity of isocitrate dehydrogenase and feeding or re-feeding decreased it. 6. Very little change in hexose monophosphate-shunt dehydrogenase activity was observed during the dietary manipulations. 7. The results indicate that increased substrate delivery to the liver is the principal stimulus to the increased rate of glucose metabolism observed in newly hatched chicks. The results also suggest that changes in the activities of ;malic' enzyme and citrate-cleavage enzyme are secondary to an increased flow of metabolites through the glucose-to-fatty acid pathway and that the dehydrogenases of the hexose monophosphate shunt play a minor role in NADPH production for fatty acid synthesis.