Quantification of liver and kidney phosphofructokinase by radioimmunoassay in fed, starved and alloxan-diabetic rats.

A newly developed specific radioimmunoassay was used to quantify phosphofructokinase protein directly and independently of assayable activity in liver and kidney cytosol of normal fed, starved and alloxan-diabetic rats. In the fed state, liver phosphofructokinase concentration was 0.096 microM and the kidney enzyme was 0.086 microM (mumol/kg of tissue). In the starved state (24h), liver and kidney phosphofructokinase concentrations decreased by 30%. Prolonged starvation up to 72h did not further decrease enzyme concentration. In liver, total enzyme content during starvation declined by more than 50%, secondary also to a decrease in liver weight. In the alloxan-diabetic rats, there was a 22% decrease in enzyme protein concentration in liver and kidney. Total enzyme content per liver actually decreased much more (46%), because diabetes also resulted in a decrease in liver size. In conjunction with assayable activity measurements, the results of the radioimmunoassay allowed us to calculate the apparent specific activity of the enzyme. The specific activity of the kidney enzyme was 2-3 times that of the liver. Little or no change in specific activity of the liver or kidney enzyme occurred as a result of starvation or chemically induced diabetes. Tissue enzyme concentrations of phosphofructokinase unequivocally reconcile the ultimate results of changing rates of synthesis and degradation and are useful data in the design of spectrophotometric, kinetic, aggregation-disaggregation and other studies.     

Bicarbonate-activated ATPase activity in renal cortex of chronically acidotic rats.

The effect of chronic NH4Cl-induced acidosis on the activity of a bicarbonate-activated component of ATPase was studied in homogenates of renal tissue from Wistar rats. This particular component of ATPase, which is maximally stimulated by 50 mM bicarbonate, and is insensitive to the action of ouabain, has been implicated in the active transport of bicarbonate in various tissues. The activity of this enzyme in cortical homogenates from an acidotic group of animals was 4.3 +/- 0.4 mumol Pi/mg protein per hour compared with 5.8 +/- 0.3 mumol Pi/mg protein per hour in a control group (p less than 0.02). No significant change in bicarbonate ATPase activity was observed in medullary homogenates, and NaK-ATPase activity remained the same in cortex and medulla of both groups. Subcellular fractionation of the cortical tissue homogenates revealed that bicarbonate ATPase activity in a microsomal fraction from acidotic animals was 6.5 +/- 1.1 mumol Pi/mg protein per hour compared with 9.4 +/- 1.2 mumol Pi/mg protein per hour in control animals (p less than 0.02). Bicarbonate ATPase activity in other subcellular fractions was not different in the two groups of animals. These findings are compatible with the hypothesis that a certain percentage of bicarbonate reabsorption in the nephron is mediated by a bicarbonate-activated component of ATPase.     

The content of pentose-cycle intermediates in liver in starved, fed ad libitum and meal-fed rats.

Liver content of pentose-cycle intermediates and the activity of the three major cytoplasmic NADPH-producing enzymes and pentose-cycle enzymes were measured in three dietary states: 48 h-starved rats, rats fed on a standard diet ad libitum, and rats meal-fed with a low-fat high-carbohydrate diet. Measured tissue contents of pentose-cycle intermediates in starved liver were: 6-phosphogluconate, 4.7 +/- 0.5 nmol/g; ribulose 5-P, 3.7 +/- 0.5 nmol/g; xylulose 5-P, 4.3 +/- 0.4 nmol/g; sedoheptulose 7-P, 25.5 +/- 1.3 nmol/g; and combined sedoheptulose 7-P and ribose 5-P, 30.6 +/- 0.7 nmol/g. These values were in good agreement with values calculated from fructose 6-P and free glyceraldehyde 3-P, assuming the major transketolase, transaldolase, ribulose-5-P 3-epimerase and ribose-5-P isomerase reactions were all in near-equilibrium. Similar results were found in animals fed ad libitum. These relationships were not valid in animals fed on a low-fat high-carbohydrate diet, with tissue contents of metabolites in some cases being more than an order of magnitude higher than the calculated values. Measured tissue contents of pentose-cycle intermediates in these animals were: 6-phosphogluconate, 124.2 +/- 13.9 nmol/g; ribulose 5-P, 44.8 +/- 7.1 nmol/g; xylulose 5-P, 77.2 +/- 9.4 nmol/g; sedoheptulose 7-P, 129.9 +/- 10.1 nmol/g; and combined sedoheptulose 7-P and ribose 5-P, 157.0 +/- 11.3 nmol/g. In all animals, regardless of dietary state, tissue content of erythrose 4-P was less than 2 nmol/ml. Liver activities of glucose-6-P dehydrogenase and 6-phosphogluconate dehydrogenase were increased from 3.5 +/- 0.9 mumol/g and 7.3 +/- 0.5 mumol/min per g in starved animals to 13.2 +/- 1.1 and 10.5 +/- 0.7 mumol/min per g in low-fat high-carbohydrate-fed animals. Despite these changes, the activities of transaldolase (3.4 +/- 0.3 mumol/min per g), transketolase (7.8 +/- 0.2 mumol/min per g) and ribulose-5-P 3-epimerase (7.5 +/- 0.4 mumol/min per g) were not increased in meal-fed animals above those observed in starved animals (3.4 +/- 0.2, 7.1 +/- 0.3 and 8.6 +/- 0.4 mumol/min per g respectively). The increase in the activity of oxidative pentose-cycle enzymes in the absence of any change in the non-oxidative pentose cycle appeared to contribute to the observed disequilibrium in the pentose cycle in animals meal fed on a low-fat high-carbohydrate diet.     

Role of fructose 2,6-bisphosphate in mammary gland of fed, starved and re-fed lactating rats.

The fructose 2,6-bisphosphate (Fru-2,6-P2) content and intracellular concentration of lactating mammary gland was measured in fed, starved and re-fed rats. There was little or no change on starvation, and about 1.5-fold rise on re-feeding, contrasting with estimated glycolytic changes of about 10-fold. The 6-phosphofructokinase (PFK-1) activity of mammary extracts was highly sensitive to added Fru-2,6-P2 under all conditions examined, and appeared to approach saturation at physiological concentrations of this effector. The activity of mammary PFK-1 measured under optimal and 'physiological' conditions suggested that this enzyme operates in vivo at about 24% of maximal rate, and is likely to be an important rate-limiting factor in mammary glycolysis.     

Differential inhibition of ketogenesis by malonyl-CoA in mitochondria from fed and starved rats.

Rates of ketogenesis in mitochondria from fed or starved rats were identical at optimal substrate concentrations, but responded differently to inhibition by malonyl-CoA. Kinetic data suggest that the K1 for malonyl-CoA is greater in the starved animal. These results indicate that, for the regulation of ketogenesis in the starved state, the lower sensitivity of carnitine palmitoyltransferase to inhibition by malonyl-CoA may be more important than the concentration of malonyl-CoA.     

Liver and kidney cortex gluconeogenesis from L-alanine in fed and starved rats

Circulating [14C]glucose 2, 5 and 10 min after intravenous injection of [U-14C]-L-alanine was greater in 24 hr starved than in fed rats. In vitro uptake of [14C]alanine by liver and kidney cortex slices from 24 hr starved and fed rats rose in parallel with increased medium substrate concentration. Formation of [14C]glucose from 1mM [14C]alanine was similar in liver and kidney cortex slices and increased in tissues from 24 hr starved compared with fed rats. With 5 mM [14C]alanine more [14C]glucose was produced by liver than by kidney cortex slices from 24 hr starved rats. Liver slices always produced more [14C]lactate and less [14C]-CO2 from [14C]alanine than kidney cortex slices. It is proposed that under physiological conditions, the kidneys cortex actively participates in glucose production from alanine.     

The effects of inhibition of gluconeogenesis on ketogenesis in starved and diabetic rats.

Experiments were performed in which the effects of inhibiting gluconeogenesis on ketone-body formation were examined in vivo in starved and severely streptozotocin-diabetic rats. The infusion of 3-mercaptopicolinate, an inhibitor of gluconeogenesis (DiTullio et al., 1974), caused decreases in blood [glucose] and increases in blood [lactate] and [pyruvate] in both normal and ketoacidotic rats. Patterns of liver gluconeogenic intermediates after 3-mercaptopicolinate infusion suggested inhibition at the level of phosphoenolpyruvate carboxykinase. This was confirmed by measurement of hepatic oxaloacetate concentrations which were increased 5-fold after 3-mercaptopicolinate administration. The infusion of 3-mercaptopicolinate caused a decrease in total ketone-body concentrations of 30% in starved rats and 73% in the diabetic animals. Blood glycerol and hepatic triglyceride concentrations remained unchanged. The decreases in ketone-body concentrations were associated with increases in the calculated hepatic cytosolic and mitochondrial [NADH]/[NAD+] ratios. The decrease in ketogenesis seen after inhibition of gluconeogenesis may have resulted from an inhibition of hepatic fatty acid oxidation by the more reduced mitochondrial redox state. It was concluded that gluconeogenesis may stimulate ketogenesis by as much as 30% in severe diabetic ketoacidosis.     

A comparison of lipid metabolism in hepatocytes isolated from fed and starved neonatal and adult rats.

1. The utilization of [1-14C]palmitate by hepatocytes prepared from fed and starved neonatal and adult rats has been examined by measuring isotopic incorporation into various products. 2. In cells from fed adult rats the principal products were esters (triglycerides and phospholipids) but ketone bodies were the main metabolic end products in cells from starved adult and fed and starved neonatal rats. Production of triglycerides exceeded that of phospholipids in fed adult cells whereas phospholipid formation always predominated in neonatal cells. 3. The high rate of fatty acid oxidation and hence NADH formation by neonatal cells is reflected by a lower acetoacetate--3-hydroxybutyrate ratio at the earlier stages of incubation of neonatal cells. 4. The addition of glycerol modified quantitatively the products of palmitate metabolism by adult hepatocytes but no such effects were observed with neonatal cells. 5. Compared with adult cells, neonatal hepatocytes showed very low rates of lipogenesis that were only enhanced a little by addition of lactate/pyruvate and did not show any effects of glucose concentration upon incorporation of tritium from 3H2O into lipids.     

The effect of phenobarbitone on protein synthesis by liver polyribosomes in fed and starved rats.

1. The effect of phenobarbitone on the rate of protein synthesis and on the sedimentation patterns of various liver subcellular fractions containing ribosomes was studied in rats. 2. Phenobarbitone treatment increased the incorporation of [114C]leucine into protein by all preparations, provided they had not been subjected to preliminary treatment with Sephadex G-25. The phenobarbitone-induced effect on incorporation was associated with a gain in liver weight and a higher degree of polyribosomal aggregation. 3. Preparations that were treated with Sephadex G-25 incorporated more radioactivity into protein, but did not show the response to phenobarbitone treatment. 4. When the influence of starvation and phenobarbitone was studied separately on membrane-bound and membrane-free polyribosomes, it was shown that whereas both classes of polyribosomes were affected by starvation, apparently only the former class was susceptible to phenobarbitone stimulation of protein synthesis. 5. The decreased capacity for protein synthesis of polyribosomes from starved rats was independent of their association with the membranes of the endoplasmic reticulum, but resulted from polyribosomal disaggregation, from an intrinsic defect of the polyribosomes themselves and from changes in composition of the cell cap. 6. The results are discussed in relation to the problem of the control of protein biosynthesis and of the functional separation of membrane-bound and membrane-free polyribosomes.     

Pyridine nucleotide distributions and enzyme mass action ratios in hepatocytes from fed and starved rats.

Hepatocytes isolated from fed or starved rats were rapidly lysed using the recently described technique of turbulent flow (M. E. Tischler, P. Hecht, and J. R. Williamson, 1977, Arch. Biochem. Biophys., 181, 278–292). Pyridine nucleotide and metabolite contents were measured in the particulate fraction of both whole and disrupted cells after centrifugation through silicone oil. Lactate/pyruvate, β-hydroxybutyrate/acetoacetate, isocitrate/α-ketoglutarate, and malate/pyruvate ratios were determined for calculation of the free $\text{NADH}\text{NAD}{}^{\mathrm{+}}$ and $\text{NADPH}\text{NADP}{}^{\mathrm{+}}$ ratios in the cytosol and mitochondria. Lactate/pyruvate ratios measured in the extracellular and cytosolic compartments were in good agreement. Ratios of β-hydroxybutyrate/acetoacetate measured in the extracellular, cytosolic, and mitochondrial compartments also agreed well. Addition of ammonia to fed or starved rat liver cells incubated with lactate, pyruvate, β-hydroxybutyrate, and acetoacetate caused an oxidation of both the NAD and NADP redox states in the mitochondria and cytosol, although the NADP system was oxidized to a greater extent. Calculation of the free NADH and NAD concentrations in the cytosol provided values of about 1 and 400 to 500 μm, respectively, under control conditions. The concentrations of free NADH and NAD in the mitochondria were considerably higher, being 300 to 400 μm and 4 to 6 mm, respectively. The free andm bound NAD systems in both the cytosol and mitochondria were more oxidized in the presence of ammonia. NAD and NADP redox potential differences across the mitochondrial membrane (ΔE_h) were not significantly affected by ammonia addition and were generally similar in cells from both fed and starved rats: ?52 and ?56 mV for the NAD system and ?19 to ?29 mV for the NADP system. For the NAD system the cytosolic potential was ?260 mV in the absence of ammonia and ?250 mV in its presence, the mitochondrial values being ?315 and ?303 mV, respectively. The average cytosolic NADP potential, on the other hand, was ?400 mV in the absence and ?384 mV in the presence of ammonia. The mitochondrial fractions yielded NADP potentials of ?420 mV in the absence of ammonia with both fed and starved rats. Ammonia decreased the mitochondrial NADP potential to ?404 mV in fed rats and to ?415 mV in starved rats. The calculated free $\text{NADH}\text{NAD}{}^{\mathrm{+}}$ and $\text{NADPH}\text{NADP}{}^{\mathrm{+}}$ ratios as well as metabolite concentrations were used to evaluate the mass action ratios of both cytosolic and mitochondrial enzymes. Cytosolic alanine aminotransferase remained near equilibrium in the absence and presence of ammonia, while cytosolic and mitochondrial aspartate aminotransferase reactions deviated up to fivefold. The glutamate dehydrogenase reaction was in near equilibrium with the NAD system, but deviated by three to four orders of magnitude from equilibrium with the NADP system in the direction favoring glutamate synthesis rather than deaminatión. Cytosolic malate dehydrogenase deviated from equilibrium by about one order of magnitude, while mitochondrial malate dehydrogenase and citrate synthase deviated by two to six orders of magnitude. These data emphasize the importance of regulation of the citric acid cycle at the citrate synthase step.     

Influence of ethanol on the liver metabolism of fed and starved rats

1. The influence of ethanol on the metabolism of livers from fed and starved rats has been studied in liver-perfusion experiments. Results have been obtained on oxygen consumption and carbon dioxide production, on glucose release and uptake by the liver and on changes in the concentrations of lactate and pyruvate and of β-hydroxybutyrate and acetoacetate in the perfusion medium. 2. Oxygen consumption and carbon dioxide production were lower in livers from starved rats than in livers from fed rats. Ethanol had no effect on the oxygen consumption of either type of liver. After the addition of ethanol to the perfusion medium carbon dioxide production ceased almost completely, the change being faster in livers from starved rats. 3. With livers from fed rats glucose was released from the liver into the perfusion medium. This release was slightly greater when ethanol was present. With livers from starved rats no release of glucose was observed, and when ethanol was added a marked uptake of glucose from the medium was found. A simultaneous release of glycolytic end products, lactate and pyruvate, into the medium occurred. 4. Acetate was the main metabolite accumulating in the perfusion medium when ethanol was oxidized. With livers from starved rats a slightly increased formation of ketone bodies was found when ethanol was present. 5. The lactate/pyruvate concentration ratio in the perfusion medium increased from 10 to 87 with livers from fed rats and from 20 to 171 with livers from starved rats when the livers were perfused with ethanol in the medium. The β-hydroxybutyrate/acetoacetate concentration ratio increased from 0·8 to 7·6 with livers from fed rats and from 1·0 to 9·5 with livers from starved rats when ethanol was added to the medium. 6. The effects of ethanol are discussed and related to changes in the redox state of the liver that produce new conditions for some metabolic pathways.     

Lipogenesis in response to an oral glucose load in fed and starved rats

Lipogenesis in livers of fed but not of starved rats is increased after intragastric feeding with glucose. In contrast, lipogenesis in brown adipose tissue increases in both fed and starved animals. These observations suggest that lipogenesis in brown adipose tissue is regulated by mechanisms in addition to, or other than, those operating in liver. The fate of newly synthesized lipid in brown adipose tissue is not known. However, the formation of palmitoyl-carnitine from palmitoyl-CoA and carnitine by mitochondria from brown fat was inhibited by malonyl-CoA. Although inhibition was not 100%, it is implied that mitochondrial uptake of the newly synthesized fat by the carnitine acyltransferase system is restricted under conditions of increased lipogenesis.     

The reversibility of cytosolic dehydrogenase reactions in hepatocytes from starved and fed rats. Effect of fructose.

The metabolism of [2-3H]lactate was studied in isolated hepatocytes from fed and starved rats metabolizing ethanol and lactate in the absence and presence of fructose. The yields of 3H in ethanol, water, glucose and glycerol were determined. The rate of ethanol oxidation (3 mumol/min per g wet wt.) was the same for fed and starved rats with and without fructose. From the detritiation of labelled lactate and the labelling pattern of ethanol and glucose, we calculated the rate of reoxidation of NADH catalysed by lactate dehydrogenase, alcohol dehydrogenase and triosephosphate dehydrogenase. The calculated flux of reducing equivalents from NADH to pyruvate was of the same order of magnitude as previously found with [3H]ethanol or [3H]xylitol as the labelled substrate [Vind & Grunnet (1982) Biochim. Biophys. Acta 720, 295-302]. The results suggest that the cytoplasm can be regarded as a single compartment with respect to NAD(H). The rate of reduction of acetaldehyde and pyruvate was correlated with the concentration of these metabolites and NADH, and was highest in fed rats and during fructose metabolism. The rate of reoxidation of NADH catalysed by lactate dehydrogenase was only a few per cent of the maximal activity of the enzymes, but the rate of reoxidation of NADH catalysed by alcohol dehydrogenase was equal to or higher than the maximal activity as measured in vitro, suggesting that the dissociation of enzyme-bound NAD+ as well as NADH may be rate-limiting steps in the alcohol dehydrogenase reaction.     

Differences in trehalase activity in the intestine of fed and starved larvae of Tenebrio molitor L.

Studies made on trehalase activity in some tissues of Tenebrio molitor larvae have shown that enzyme activity is highest in the intestine, with a low level of activity in the fat body and muscles and traces of activity in the haemolymph. Higher intestinal trehalase activity was found in larvae fed on flaked oats with the addition of brewers yeast and fresh carrot (diet A) than in larvae fed the same diet without carrot (diet B).Starvation of larvae previously reared on diet A caused a decrease in trehalase activity in all the examined tissues except the haemolymph where traces remained. Refeeding starved larvae on diet A caused an increase in intestinal trehalase activity after as little as 2 hr.     

gamma-butyrobetaine in tissues and serum of fed and starved rats determined by an enzymic radioisotopic procedure.

A method for the determination of picomole quantities of gamma-butyrobetaine and its application for the determination of gamma-butyrobetaine distribution in tissues are described. The method is based on the quantitative conversion of gamma-butyrobetaine into carnitine by using a 50-60%-satd.-(NH4)2SO4 fraction of rat liver supernatant as the source of gamma-butyrobetaine hydroxylase [4-trimethylaminobutyrate,2-oxoglutarate:oxygen oxidoreductase (3-hydroxylating), EC 1.14.11.1]; the carnitine formed is then measured enzymically. The mean gamma-butyrobetaine content, as nmol/g wet wt. of tissue, ranged from a low of 4.6 in livers to a high of 12.3 in hearts of normal fed male adult rats. Starvation for 48 h did not affect the gamma-butyrobetaine concentration in serum, liver and brain, but that in skeletal muscles, kidney and heart was increased. These data are in line with the present views that most tissues are able to produce gamma-butyrobetaine, and show that starvation enhances the synthesis and/or the retention of this compound in many tissues. The observed high affinity of gamma-butyrobetaine hydroxylase for gamma-butyrobetaine (Km 7 microM), the high activity of this enzyme and the low concentration of gamma-butyrobetaine in liver indicate that gamma-butyrobetaine availability is one of the factors that normally limit carnitine synthesis.     

Changes in the properties of cytosolic acetyl-CoA carboxylase studied in cold-clamped liver samples from fed, starved and starved-refed rats.

The mechanism responsible for the insulin resistance described in vivo in brown adipose tissue (BAT) of lactating rats was investigated. The effect of insulin on glucose metabolism was studied on isolated brown adipocytes of non-lactating and lactating rats. Insulin stimulation of total glucose metabolism is 50% less in brown adipocytes from lactating than from non-lactating rats. This reflects a decreased effect of insulin on glucose oxidation and lipogenesis. However, the effect of noradrenaline (8 microM) on glucose metabolism was preserved in brown adipocytes from lactating rats as compared with non-lactating rats. The number of insulin receptors is similar in BAT of lactating and non-lactating rats. The insulin-receptor tyrosine kinase activity is not altered during lactation, for receptor autophosphorylation as well as tyrosine kinase activity towards the synthetic peptide poly(Glu4-Tyr1). The defect in the action of insulin is thus localized at a post-receptor level. The insulin stimulation of pyruvate dehydrogenase activity during euglycaemic/hyperinsulinaemic clamps is 2-fold lower in BAT from lactating than from non-lactating rats. However, the percentage of active form of pyruvate dehydrogenase is similar in non-lactating and lactating rats (8.6% versus 8.9% in the basal state, and 37.0% versus 32.3% during the clamp). A decrease in the amount of pyruvate dehydrogenase is likely to be involved in the insulin resistance described in BAT during lactation.